Translation and Its Role in Cancer

Translation and Its Regulation in Cancer
Biology and Medicine

Armen Parsyan
Editor
Translation and Its

Regulation in Cancer
Biology and Medicine
13
Editor
Armen Parsyan
McGill University
Surgery
Montreal
Qubec
Canada
Chapter 10 was created within the capacity of a US governmental employment. US copyright

protection does not apply.
ISBN 978-94-017-9077-2ISBN 978-94-017-9078-9 (eBook)

DOI 10.1007/978-94-017-9078-9
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Springer Science+Business Media Dordrecht 2014

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To my mentors
Wherever I lived or studied, whether it was
in Armenia, the USA, the UK or Canada, I
always felt blessed with having outstanding
mentors who supported me and nurtured
my creativity. I feel forever indebted to them
for having shaped my clinical and scientific
personality.
Amongst them I strive to mention my clini-
cal and research mentor, an outstanding
physician, surgeon, scientist and teacher,
Professor Sarkis Meterissian from McGill
University. His mentorship of my research
and clinical endeavors has been tremendous
and elating.
I am proud to call myself a disciple of another
mentor, a pioneer of research in translation
and cancer, Professor Nahum Sonenberg from
McGill University. He has made numerous
fundamental contributions to the fields of
translation and its regulation in physiology
and disease. His mentorship matured me to
a humble and passionate follower of science,
who could dare to conceive the idea of this
book and be able to materialize it.
I wholeheartedly dedicate this book to you!
v
Preface
If you know the enemy and know yourself you need not fear the
results of a hundred battles.
Sun Tzu
Every year millions of people experience a life-changing event of having been di-
agnosed with cancer and start an often tragic battle. Those of us who are privileged
enough to enter this battle willingly and fight at the frontiers of science will soon
learn the fatiguing complexities of the opponents strategies, its unsurpassed re-
silience and unimaginable diversity of the molecular arsenals. This onslaught can
hopefully be matched by a power of our collective knowledge. The idea of this book
originated on the grounds of this philosophy, and was conceived in order to sum-
marize vast knowledge accumulated in relation to translation and its regulation in
cancer biology and medicine.
It is well established that dysregulated protein synthesis or activity is a corner-
stone of a neoplastic process. A central dogma of molecular biology states that the
protein biosynthetic pathway, with some exceptions, follows the three major events:
replication (DNA to DNA), transcription (DNA to RNA) and translation (RNA to
protein). Hence, the abnormal functioning of any of these processes can nurture cel-
lular transformation to malignant growth.
While other pillars of the central dogma were front-page in cancer research,
translation remained underground for decades. In fact, even in specialized minds of
some outstanding cancer researchers and clinicians, the word translation appears
to associate more often with a bench-to-bedside experience or a linguistic phenom-
enon. Hence, alongside with the aforementioned, one of the objectives of this book
is to proselytize among current and future scholars and students the fundamental
importance of translation in cancer development and progression, and enable them
to explore new battlegrounds and thus build new anticancer weaponry.
vii
Acknowledgements
This publication was a large part of my life for almost two years. From its concep-
tion to its completion, I felt privileged and honored to get to know and to work
alongside so many exceptional and talented individuals. My sincere gratitude goes
to you, everybody who supported me during this trial. Thank you for making this
life experience outstanding!
I would like to thank all the members of the Division of General Surgery of Mc-
Gill University who facilitated my work on this book, especially Dr. Sarkis Meteris-
sian, Dr. Paola Fata and Ms. Rita Piccioni. I also would like to thank my physician
and other health care colleagues and friends at McGill who were very supportive
and understanding of my work.
I am thankful to my friends and colleagues at the Professor Sonenberg laboratory
at McGill University, Department of Biochemistry and The Rosalind and Morris
Goodman Cancer Research Centre who supported me in this endeavor, especially
Drs. Bruno Fonseca and Tommy Alain.
My special gratitude goes also to one of the forefathers of research in translation
and cancer Dr. John Hershey who was always available for help and undoubtedly
raised the bar of some of the chapters of this book.
Special thanks to Springers Melania Ruiz and Marleen Moore for their valuable
guidance and assistance.
I would like to acknowledge invaluable voluntary assistance with individual
chapter linguistic editing at the final stages of the preparation of the book pro-
vided by Ms. Anita Svadzian (Chaps.15, 24 and 2634) and McGill medical stu-
dents Megan Elizabeth Delisle (Chapters 9 to 15 and 34), Alexandra Allard-Coutu
(Chaps.28), Dan Moldoveanu (Chaps.2126), Tiffany Huynh (Chaps.1620) and
David Qi Zhang (Chaps.21 and 22).
While I strived to create a forum of professionals and to be as comprehensive as
possible in the coverage of this book, I realize that, it is an idealistic target in respect
to such a global topic. Hence, I would like to apologize to those whose work is not
cited in this publication.
I would not be able to conceive and complete this project without support and
understanding of my wife and dearest friend Anna, daughter Adele and my parents
who kept me focused, motivated and sane, as they usually do.
ix
Contents
1Introduction 1
Armen Parsyan
Part I Translation Machinery in Cancer
2Mechanism of Translation in Eukaryotes 7

Nancy Villa and Christopher S. Fraser
3Diverse Mechanisms of Translation Regulation and Their

Role in Cancer 39
4eIF4E and Its Binding Proteins 73

Nathaniel Robichaud and Nahum Sonenberg
5RNA Helicases and Their Cofactors 115

David Shahbazian, Jerry Pelletier, Yuri Svitkin, John W. B. Hershey
and Armen Parsyan
6PDCD4 135
Hsin-Sheng Yang, Qing Wang, Magdalena M. Bajer and Tobias Schmid
7eIF4G 163
Simon D. Wagner, Anne E. Willis and Daniel Beck
8eIF3 173
John W. B. Hershey
9 T
he eIF2 Complex and eIF2 195
Bertal H. Aktas and Ting Chen
10eIF5A 223
Myung Hee Park, Swati Mandal, Ajeet Mandal and Edith C Wolff
xi
xii Contents
11eIF6 233
Stefano Biffo, Daniela Brina and Stefania Oliveto
12Translation Elongation 241

Bruna Scaggiante, Barbara Dapas, Rossella Farra, Federica Tonon,
Michela Abrami, Mario Grassi, Francesco Musiani,
Fabrizio Zanconati, Gabriele Pozzato and Gabriele Grassi
13Ribosomes 267
Fabrizio Loreni and Sara Ricciardi
14Current and Emerging Therapies Targeting Translation 279

Gabriela Galicia-Vzquez and Jerry Pelletier
Part II Regulation of Translation by Signaling Pathways in Cancer
15mTOR and Regulation of Translation 307

Yoshinori Tsukumo, Mathieu Laplante, Armen Parsyan,
Davide Ruggero and Bruno Fonseca
16Ribosomal Protein S6 and S6 Kinases 345

Mario Pende and Caroline Treins
17eIF4E Phosphorylation Downstream of MAPK Pathway 363

Luc Furic, Emma Beardsley and Ivan Topisirovic
Part III Cell Fate and Translation in Cancer
18Translational Control of Cell Proliferation and Viability in

Normal and Neoplastic Cells 377
Svetlana Avdulov, Jos R. Gmez-Garca, Peter B. Bitterman
and Vitaly A. Polunovsky
19Translation and Apoptosis in Cancer 395

Martin Holcik
20Translation in Cancer at Hypoxia 421

Tingfang Yi and Gerhard Wagner
Part IV Translation and Its Regulation by Cancer Types
21Melanoma and Non-Melanoma Skin Cancers 435

Armen Parsyan, Ryan J. Sullivan, Ari-Nareg Meguerditchian
and Sarkis Meterissian
Contents xiii
22Sarcomas 453
Armen Parsyan, James L. Chen, Raphael Pollock and
Sarkis Meterissian
23Hematological Malignancies and Premalignant Conditions 467

Markus Reschke, Nina Seitzer, John G. Clohessy and
Pier Paolo Pandolfi
24Brain Tumors 487

Armen Parsyan, Justin G. Meyerowitz and William A. Weiss
25Head and Neck Cancers 499

Cherie-Ann O. Nathan, Oleksandr Ekshyyan and
Arunkumar Anandharaj
26Breast Cancer 513

Armen Parsyan, Ana Maria Gonzalez-Angulo, Dimitrios Zardavas,
Martine Piccart and Sarkis Meterissian
27Cancers of the Respiratory System 557

Armen Parsyan and Karen L. Reckamp
28Gastric and Esophageal Cancers 575

Armen Parsyan and Lorenzo Ferri
29Colorectal Cancers 593

Armen Parsyan, Nathaniel Robichaud and Sarkis Meterissian
30Hepatic, Pancreatic and Biliary Cancers 611

Jennifer A. Sanders and Philip A. Gruppuso
31Pancreatic Neuroendocrine Tumors 631

Mamatha Bhat, Peter Metrakos, Santiago Ramon y Cajal,
Nahum Sonenberg and Tommy Alain
32Gynecologic Cancers 645

Armen Parsyan and Susana Banerjee
33Prostate Cancer 657

Nina Seitzer, Markus Reschke, John G. Clohessy and
Pier Paolo Pandolfi
34Cancers of the Urinary System 673

Armen Parsyan, Emmanuel Seront and Jean-Pascal Machiels
Index 681
Contributors
Michela Abrami Department of Engineering and Architecture, University of

Trieste, Trieste, Italy
Bertal H. Aktas Division of Hematology, Department of Medicine, Brigham and
Womens Hospital and Harvard Medical School, Harvard University, Boston, MA,
USA
Tommy Alain Childrens Hospital of Eastern Ontario Research Institute,
Department of Biochemistry, Microbiology and Immunology, University of Ottawa,
Ottawa, ON, Canada
Arunkumar Anandharaj Department of Otolaryngology, Head and Neck
Surgery and Feist-Weiller Cancer Center, Louisiana State University Health
Sciences CenterShreveport, Shreveport, LA, USA
Svetlana Avdulov Department of Medicine and Masonic Cancer Center,
University of Minnesota, Minneapolis, MN, USA
Magdalena M. Bajer Institute of Biochemistry I, Faculty of Medicine, Goethe-
University Frankfurt, Frankfurt am Main, Germany
Susana Banerjee Gynaecology Unit, The Royal Marsden National Health
Service (NHS) Foundation Trust, London, UK
Emma Beardsley Department of Anatomy and Developmental Biology, Monash
University, Clayton, VIC, Australia
Daniel Beck Department of Cancer Studies and Molecular Medicine, University
of Leicester, Leicester, UK; Medical Research Council (MRC) Toxicology Unit,
Leicester, UK
Mamatha Bhat Department of Biochemistry, Rosalind and Morris Goodman
Cancer Research Centre, McGill University, Montreal, QC, Canada; Faculty
of Medicine, Division of Gastroenterology, Department of Medicine, McGill
University, Montreal, QC, Canada
xv
xvi Contributors
Stefano Biffo Department of Science and Technological Innovation (DISIT),

University of Eastern Piedmont, Alessandria, Italy; National Institute of Molecular
Genetics (INGM), Milan, Italy
Peter B. Bitterman Department of Medicine and Masonic Cancer Center,
Daniela Brina Department of Science and Technological Innovation (DISIT),
University of Eastern Piedmont, Alessandria, Italy
James L. Chen Department of Biomedical Informatics and Division of Medical
Oncology, Department of Internal Medicine, The Ohio State Wexner Medical
Center, Columbus, OH, USA
Ting Chen Division of Hematology, Department of Medicine, Brigham and
Womens Hospital and Harvard Medical School, Harvard University, Boston, MA,
USA
John G. Clohessy Preclinical Murine Pharmacogenetics Facility, Cancer
Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine
and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School,
Harvard University, Boston, MA, USA
Barbara Dapas Department of Life Sciences, University of Trieste, Trieste, Italy
Oleksandr Ekshyyan Department of Otolaryngology, Head and Neck Surgery
and Feist-Weiller Cancer Center, Louisiana State University Health Sciences
CenterShreveport, Shreveport, LA, USA
Rossella Farra Department of Engineering and Architecture, University of
Lorenzo Ferri Division of General Surgery, Department of Surgery, Faculty of
Medicine, McGill University, Montreal, QC, Canada; Department of Oncology,
Faculty of Medicine, McGill University, Montreal, QC, Canada; Division of
Thoracic Surgery, Department of Surgery, Faculty of Medicine, McGill University,
Montreal, QC, Canada
Bruno Fonseca Department of Biochemistry, Rosalind and Morris Goodman
Cancer Research Centre, McGill University, Montreal, QC, Canada
Christopher S. Fraser Section of Molecular and Cellular Biology, University of
California, Davis, CA, USA
Luc Furic Department of Anatomy and Developmental Biology, Monash
University, Clayton, VIC, Australia
Gabriela Galicia-Vzquez Department of Biochemistry, McGill University,
Jos R. Gmez-Garca Department of Medicine and Masonic Cancer Center,
Contributors xvii
Ana Maria Gonzalez-Angulo Department of Breast Medical Oncology and

Department of Systems Biology, The University of Texas MD Anderson Cancer
Center, Houston, TX, USA
Gabriele Grassi Department of Life Sciences, University of Trieste, Trieste, Italy
Mario Grassi Department of Engineering and Architecture, University of Trieste,
Trieste, Italy
Philip A. Gruppuso Department of Pediatrics, Brown University and Rhode
Island Hospital, Providence, RI, USA
John W. B. Hershey Department of Biochemistry and Molecular Medicine,
School of Medicine, University of California, Davis, CA, USA
Martin Holcik Apoptosis Research Centre, Childrens Hospital of Eastern
Ontario Research Institute, Department of Pediatrics, University of Ottawa, Ottawa,
Canada
Mathieu Laplante Centre de Recherche de lInstitut Universitaire de Cardiologie
et de Pneumologie de Qubec (CRIUCPQ), Facult de Mdecine, Universit Laval,
Quebec City, QC, Canada
Fabrizio Loreni Department of Biology, University of Rome Tor Vergata, Rome,
Italy
Jean-Pascal Machiels Medical Oncology, Cliniques Universitaires Saint Luc,
Brussels, Belgium
Ajeet Mandal Oral and Pharyngeal Cancer Branch, National Institute of Dental
and Craniofacial Research (NIDCR), National Institutes of Health, Bethesda, MD,
USA
Swati Mandal Oral and Pharyngeal Cancer Branch, National Institute of Dental
USA
Ari-Nareg Meguerditchian Division of General Surgery, Department of Surgery,
Faculty of Medicine, McGill University, Montreal, QC, Canada; Department of
Oncology, Faculty of Medicine, McGill University, Montreal, QC, Canada
Sarkis Meterissian Division of General Surgery, Department of Surgery, Faculty
of Medicine, McGill University, Montreal, QC, Canada; Department of Oncology,
Faculty of Medicine, McGill University, Montreal, QC, Canada
Peter MetrakosDepartment of Anatomy and Cell Biology and
Hepatopancreatobiliary and Transplant Research Unit, Division of General Surgery,
Department of Surgery, Faculty of Medicine, McGill University, Montreal, QC,
Canada
xviii Contributors
Justin G. Meyerowitz Department of Chemistry and Chemical Biology,

University of California, San Francisco, CA, USA; Department of Neurology,
School of Medicine, University of California, San Francisco, CA, USA
Francesco Musiani Scuola Internazionale Superiore di Studi Avanzati (Sissa/
ISAS), Trieste, Italy
Cherie-Ann O. Nathan Department of Otolaryngology, Head and Neck Surgery
and Feist-Weiller Cancer Center, Louisiana State University Health Sciences
CenterShreveport, Shreveport, LA, USA
Stefania Oliveto Department of Science and Technological Innovation (DISIT),
University of Eastern Piedmont, Alessandria, Italy; National Institute of Molecular
Genetics (INGM), Milan, Italy
Pier Paolo Pandolfi Cancer Research Institute, Beth Israel Deaconess Cancer
Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical
Center, Harvard Medical School, Harvard University, Boston, MA, USA
Myung Hee Park Oral and Pharyngeal Cancer Branch, National Institute of
Dental and Craniofacial Research (NIDCR), National Institutes of Health, Bethesda,
MD, USA
Armen Parsyan Division of General Surgery, Department of Surgery, Faculty of
Medicine, McGill University, Montreal, QC, Canada
Jerry Pelletier Department of Biochemistry and Rosalind and Morris Goodman
Cancer Research Centre, McGill University, Montreal, QC, Canada; Department of
Oncology, McGill University, Montreal, QC, Canada
Mario Pende Institut National de la Sant et de la Recherche Mdicale (Inserm)
and Facult de Mdecine, Universit Paris Descartes, Paris, France
Martine Piccart Institut Jules Bordet, Universit Libre de Bruxelles (ULB),
Brussels, Belgium
Raphael Pollock Division of Surgical Oncology, Department of Surgery, The
Ohio State Wexner Medical Center, Columbus, OH, USA
Vitaly A. Polunovsky Department of Medicine and Masonic Cancer Center,
Gabriele Pozzato Department of Medicine, Surgery and Health Sciences,
Cattinara Hospital, University of Trieste, Trieste, Italy
Santiago Ramon y Cajal Department of Pathology, Vall dHebron University
Hospital, Autonoma University of Barcelona, Barcelona, Spain
Karen L. Reckamp Department of Medical Oncology and Therapeutics
Research, City of Hope Comprehensive Cancer Center, Duarte, CA, USA
Contributors xix
Markus Reschke Cancer Research Institute, Beth Israel Deaconess Cancer

Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical
Center, Harvard Medical School, Harvard University, Boston, MA, USA
Sara Ricciardi DISIT (Dipartimento di Scienze e Innovazione Teconologica),
University of Eastern Piedmont, Alessandria, Italy; Istituto Nazionale Genetica
Molecolare (INGM), Milan, Italy
Nathaniel Robichaud Department of Biochemistry and Rosalind and Morris
Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada
Davide Ruggero Helen Diller Family Comprehensive Cancer Center, Department
of Urology, School of Medicine, University of California San Francisco, San
Francisco, CA, USA
Jennifer A. Sanders Department of Pediatrics, Brown University and Rhode
Island Hospital, Providence, RI, USA
Bruna Scaggiante Department of Life Sciences, University of Trieste, Trieste,
Italy
Tobias Schmid Institute of Biochemistry I, Faculty of Medicine, Goethe-
University Frankfurt, Frankfurt am Main, Germany
Emmanuel Seront Medical Oncology, Cliniques Universitaires Saint Luc,
Brussels, Belgium
Nina Seitzer Cancer Research Institute, Beth Israel Deaconess Cancer Center,
Department of Medicine and Pathology, Beth Israel Deaconess Medical Center,
Harvard Medical School, Harvard University, Boston, MA, USA
David Shahbazian Section of Medical Oncology, Yale Cancer Center and School
of Medicine, Yale University, New Haven, CT, USA
Nahum Sonenberg Department of Biochemistry and Rosalind and Morris
Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada
Ryan J. Sullivan Center for Melanoma, Massachusetts General Hospital Cancer
Center, Boston, MA, USA
Yuri Svitkin Department of Biochemistry and Rosalind and Morris Goodman
Federica Tonon Department of Engineering and Architecture, University of
Ivan Topisirovic Lady Davis Institute for Medical Research, Sir Mortimer B.
Davis Jewish General Hospital, Department of Oncology, McGill University,
Caroline Treins Institut National de la Sant et de la Recherche Mdicale
(Inserm) and Facult de Mdecine, Universit Paris Descartes, Paris, France
xx Contributors
Yoshinori Tsukumo Department of Biochemistry, Rosalind and Morris Goodman

Nancy Villa Section of Molecular and Cellular Biology, University of California,
Davis, CA, USA
Gerhard WagnerDepartment of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Harvard University, Boston, MA, USA
Simon D. Wagner Department of Cancer Studies and Molecular Medicine,
University of Leicester, Leicester, UK; Medical Research Council (MRC)
Toxicology Unit, Leicester, UK; Department of Haematology, University Hospitals
of Leicester, University of Leicester, Leicester, UK
Qing Wang Graduate Center for Toxicology, University of Kentucky, Lexington,
KY, USA
William A. Weiss Department of Neurology, School of Medicine, University of
California, San Francisco, CA, USA
Anne E. Willis Medical Research Council (MRC) Toxicology Unit, Leicester,
UK
Edith C Wolff Oral and Pharyngeal Cancer Branch, National Institute of Dental
USA
Hsin-Sheng Yang Graduate Center for Toxicology, University of Kentucky,
Lexington, KY, USA; Markey Cancer Center, University of Kentucky, Lexington,
KY, USA
Tingfang Yi Department of Biological Chemistry and Molecular Pharmacology,
Fabrizio Zanconati Department of Medicine, Surgery and Health Sciences,
Cattinara Hospital, University of Trieste, Trieste, Italy
Dimitrios Zardavas Institut Jules Bordet, Universit Libre de Bruxelles (ULB),
Brussels, Belgium
List of Abbreviations
Miscellaneous
4E-BP eIF4E-binding protein

4E-T eIF4E transporter
4EASO eIF4E-specific ASO
4EGI eIF4E/eIF4G interaction inhibitor
4EGI-1 Inhibitor of eIF4E/eIF4G interaction
4EHP eIF4E homologous protein
5-FU 5-fluorouracil
A-site Aminoacyl site (binding site for charged tRNA in the ribosome)
aa-tRNA Aminoacyl-tRNA
AARE Amino acid response element
ABCB1 ATP-binding cassette, subfamily B (MDR/TAP)
ABCE1 ATP-binding cassette, subfamily E member 1
ABL Abelson murine leukemia viral oncogene homolog
AFPGC -Fetoprotein-producing gastric carcinoma
AGC Protein kinases A, G and C family
AJCC American Joint Committee on Cancer
AKT v-Akt murine thymoma viral oncogene homolog (also known as
protein kinase B, PKB)
ALT Alanine aminotransferase
AML Acute myeloid leukemia
AMP, ADP, ATP Adenosine mono, di- and triphosphates
AMPK AMP-activated protein kinase
AP-1 Activating protein 1
APAF-1 Apoptotic protease activating factor 1
APC Adenomatous polyposis coli
ARE AU-rich element
xxi
xxii List of Abbreviations
ARNT Aryl hydrocarbon receptor nuclear translocator

ASK1 Apoptosis signaling regulating kinase 1, also known as MAP3K5
ASO Antisense oligonucleotide
AST Aspartate aminotransferase
asTORi Active-site TOR inhibitors
ATF Activating transcription factor
ATG13 Autophagy-related 13
ATM Ataxia telangiectasia mutated
ATRX X-linked mental retardation and -thalassemia syndrome protein
AUF1 AU-rich element RNA-binding protein 1
BAD BCL-2 antagonist of cell death

BCL-2 B-cell lymphoma protein 2
BCL-XL B-cell lymphoma protein-extra large
BCR Breakpoint cluster region
BIRC2 Baculoviral IAP repeat-containing protein 2
BOP1 Block of proliferation 1
-TRCP -Transducin repeat-containing E3 ubiquitin protein ligase
BRAF v-RAF murine sarcoma viral oncogene homolog B
BRAFV600E BRAF mutated from Val to Glu at the position 600
BRCA1/2 Breast cancer, early onset 1 and 2
C-FAG C-terminal cleavage fragment of eIF4G

c-FLIP Cellular FLIP (see FLIP)
c-JUN See JUND
c-KIT Proto-oncogene tyrosine protein kinase KIT (v-kit Hardy-Zucker-
man 4 feline sarcoma viral oncogene homolog)
c-MET Mesenchymal-epithelial transition factor
C-MYB Myeloblastosis, cellular proto-oncogene
c-MYC v-Myc avian myelocytomatosis viral oncogene homolog
C-terminus Carboxy-terminus
C/EBP CCAAT-enhancer-binding proteins
CA II Carbonic anhydrase type II
CAD Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase,
and dihydroorotase
CaMKII Calcium/calmodulin-dependent protein kinase II
CaMKK Calcium/calmodulin-dependent protein kinase kinase
cAMP Cyclic AMP
CAT Chloramphenicol acetyltransferase
CBP Cap-binding protein
List of Abbreviations xxiii
CCA Cholangiocarcinoma
CCI-779 Temsirolimus
CCT Chaperonin containing TCP1, subunit 2
CD Cluster of differentiation
CDC Centers of Disease Control and Prevention, USA
CDC25 Cell division cycle 25
CDK Cyclin-dependent kinase
CDKN2A Cyclin-dependent kinase inhibitor 2A
cDNA Complementary DNA
CDR Common deleted region
CHOP C/EBP homolog protein, see DDIT3
cIAP Cellular IAP
CIN Cervical intraepithelial neoplasia
CK1 Casein kinase 1
cMA3 C-terminal MA3 domain
CML Chronic myeloid leukemia
CMP, CDP, CTP Cytidine mono, di- and triphosphates
COP9 COP9 (constitutive photomorphogenic 9) signalosome
COSMIC Catalogue of Somatic Mutations in Cancer
COX Cyclooxygenase
CPEB Cytoplasmic polyadenylation-element binding proteins
CRC Colorectal cancer
CREB cAMP response element-binding protein
CReP Constitutive reverser of eIF2 phosphorylation
CSFV Classical swine fever virus
CTX Chlorotoxin
CXC CXC chemokine
CXCL Chemokine (CXC motif) ligand
CXCR Chemokine (CXC motif) receptor
CYR61 Cysteine-rich angiogenic inducer 61
DAP5 Death-associated protein

DAXX Death domain-associated protein 6
DBA Diamond-Blackfan anemia
DCIS Ductal carcinoma in situ
dCTP Deoxycytidine triphosphate
DDIT3 DNA-damage-inducible transcript 3, see CHOP
DDR DNA damage response
DDX DEAD box helicase
DEAD Aspartic acid, glutamic acid, alanine and aspartic acid sequence
DEAH Aspartic acid, glutamic acid, alanine and histidine sequence
DENR Density-regulated protein
xxiv List of Abbreviations
DEPTOR DEP domain-containing mTOR-interacting protein

DFS Disease-free survival
DHS Deoxyhypusine synthase
DHT Dihydrotestosterone
DHX DEAH box helicase
DHX29 DEAH box helicase 29
DIABLO Direct IAP binding protein with low pI
DICER Dicer ribonuclease type III
DKC1 Dyskeratosis congenita 1, dyskerin
DKO Double knockout
DLBCL Diffuse large B-cell lymphoma
DMBA 7,12-Dimethylbenz(a)anthracene
DNA Deoxyribonucleic acid
DOHH Deoxyhypusine hydroxylase
DRYG Aspartate, arginine, tyrosine and glycine sequence
dsRNA Double-stranded RNA
dTTP Deoxythymidine triphosphate
E-box Enhancer box

E-site Exit site (third and final binding site for tRNA in ribosome)
eEF Eukaryotic translation elongation factor
eEF2K eEF2 kinase
EF-G Prokaryotic elongation factor G
EF-P Prokaryotic elongation factor P
EF-Tu Prokaryotic elongation factor Tu (thermo unstable)
EF3 Elongation factor 3
EF4 Elongation factor 4
EFL1 Elongation factor-like 1
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
EGR1 Early growth response protein
eIF Eukaryotic translation initiation factor
eIF2-TC eIF2-ternary complex of eIF2, GTP and initiator Met-tRNAi
eIF4Ac eIF4F-associated eIF4A
eIF4Af Free form of eIF4A
ELP1 E74-like factor 1
EMA European Medicines Agency
EMCV Encephalomyocarditis virus
EMT Epithelial-mesenchymal transition
ER Estrogen receptor
ERBB2 v-erb-b2 Avian erythroblastic leukemia viral oncogene homolog 2
(also known as HER2)
List of Abbreviations xxv
ERCC5 Excision repair cross-complementing rodent repair deficiency,

complementation group 5
eRF Eukaryotic translation release factor
ERK Extracellular signal-regulated kinase
E-MYC c-MYC gene driven by the IgH enhancer
FAK Focal adhesion kinase

FAS FAS receptor of TNF superfamily
FAT FRAP, ATM, TRRAP domain
FAT1 FAT tumor suppressor homolog 1
FAT10 Ubiquitin-like modifier FAT10
FATC FRAP, ATM, TRRAP C-terminal
FDA Food and Drug Administration
FGF Fibroblast growth factor
FGFR FGF receptor
FIP200 FAK-interacting protein of 200 kDa
FKBP12 FK506/rapamycin-binding protein
FLIP FLICE-like inhibitory protein
FOLFIRI FOL (folinic acid/leucovorin), F (5-FU), IRI (irinotecan)
FOLFOX FOL (folinic acid/leucovorin), F (5-FU), OX (oxaliplatin)
FOS FinkelBiskisJinkins murine osteogenic sarcoma virus oncogene
homolog
FOSB, C-FOS FOS family proteins
FOXO Forkhead box O
FRB FKBP12rapamycin binding domain
FTI Farnesyltransferase inhibitors
FUS Fused in sarcoma
GADD34 Growth arrest and DNA damage-inducible protein (also known as

PP1 regulatory subunit 15A or a PP1C-interacting protein)
GAP GTPase-activating protein
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GAS2 Growth arrest-specific protein 2
GCN2 General control non-derepressible 2 (also known as eukaryotic
translation initiation factor 2 kinase 4 (EIF2AK4))
GEF Guanine nucleotide exchange factor
GIGYF2 GRB10 interacting GYF protein2
GIST Gastrointestinal stromal tumor
GLI Glioma-associated oncogene homolog
xxvi List of Abbreviations
GMP, GDP, GTP Guanosine mono, di- and triphosphates

GnRH Gonadotropin-releasing hormone
GRP75 See HSPA9
GSK3 Glycogen synthase kinase 3
GSPT1 G1 to S phase transition 1, also known as eRF3a
H-RAS or HRAS Harvey rat sarcoma viral oncogene homolog

HBV Hepatitis B virus
HCC Hepatocellular carcinoma
HCV Hepatitis C virus
HDGF Hepatoma-derived growth factor
HDM2 See MDM2
HEAT Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A
(PP2A), and yeast kinase TOR1 domain
HER2/neu Human epidermal growth factor receptor 2 (also known as
ERBB2)
HGF Hepatocyte growth factor
HHT Homoharringtonine
HIF-1 and -2 Hypoxia-inducible factor 1 and 2
HIV Human immunodeficiency virus
HMEC Human mammary epithelial cell
hMSH2 Human mutS homolog 2 (DNA mismatch repair protein)
hnRNPC heterogeneous nuclear ribonucleoprotein C1/C2
hnRNPK heterogeneous nuclear ribonucleoprotein K
HNSCC Head and neck squamous cell carcinoma
HPB Hepato-pancreato-biliary
HPV Human papilloma virus
HRE Hypoxia response element
HRI Heme-regulated inhibitor (also known as eukaryotic translation
initiation factor 2 kinase 1 (EIF2AK1))
HSP Heat shock protein
HSPA9 Heat shock 70 kDa protein 9
hTERT Human telomerase reverse transcriptase
HTLV Human T-lymphotropic virus
HuR Human antigen R
IAP Inhibitor of apoptosis

IFN Interferon
IFNAR1 Interferon-/ receptor chain
List of Abbreviations xxvii
IGF Insulin-like growth factor

IGF-1R or IGF-2R IGF-1 or IGF-2 receptor
IHC Immunohistochemistry
IKK I B Kinase
IL Interleukin
INK4 Inhibitor of cyclin-dependent kinase 4
iNOS Inducible isoform of NOS
INT6 See eIF3e
IRES Internal ribosome entry site
IRF7 Interferon regulatory factor 7
IRS Insulin receptor substrate
ITAF IRES trans-acting factors
JAK Janus kinase

JNK c-JUN N-terminal kinase
JUN v-Jun sarcoma virus 17 oncogene homolog
JUND, JUNB, c-JUN JUN family of transcription factors, see JUN
K-RAS or KRAS Kirsten rat sarcoma viral oncogene homolog

kDa Kilodalton
Ki-67 Antigen identified by monoclonal antibody Ki-67
KO Knockout
KSHV Kaposis sarcoma-associated herpesvirus
LAMB1 Laminin B1
LARP6 La-related protein 6
LCIS Lobular carcinoma in situ
LDH Lactate dehydrogenase
LKB1 Liver kinase B1, also known as STK11
LRRK2 Leucine-rich repeat kinase 2
M-FAG M-terminal cleavage fragment of eIF4G

MAFbx Muscle-specific ubiquitin ligase
MAP Mitogen-activated protein
xxviii List of Abbreviations
MAP3K5 MAP kinase kinase kinase 5, also see ASK1

MAP4K MAP kinase kinase kinase kinase
MAPK Mitogen-activated protein kinase
MBI-eEF1A Modified form of eEF1A1 protein
MCL-1 Induced myeloid leukemia cell differentiation
protein
MCT-1 Monocarboxylate transporter 1
MCV Merkel cell polyomavirus
MDM2 Mouse double minute 2 homolog, also called
HDM2
MDM4 Mouse double minute 4 homolog
MDS Myelodysplastic syndrome
MEF Mouse embryonic fibroblast
MEK MAP kinase kinase (also known MAPKK)
MEN Multiple endocrine neoplasia
MEN1 Multiple endocrine neoplasia 1 protein
MET Mesenchymal-epithelial transition factor
Met-tRNAiMet or Met-tRNAi Initiator methionyl tRNA
MIF Macrophage migration inhibitory factor
MIF4G Middle domain of eIF4G
MIF4GD MIF4G domain containing
miR microRNA
miRNA microRNA
MLN51 Metastatic lymph node 51 protein
mLST8 Mammalian lethal with sec-13 protein 8
MMP Matrix metalloproteinase
MMTV Mouse mammary tumor virus
MNK Menkes disease-associated protein or MAPK-
interacting kinase (also known as MKNK or
MAPK-interacting serine/threonine kinase)
MOS v-mos Moloney murine sarcoma viral oncogene
homolog
mRNA Messenger ribonucleic acid
mRNP Messenger ribonucleoprotein particles
mSIN1 Mammalian stress-activated MAPK-interacting
protein 1
MT2A Metallothionein 2A
mTOR Mechanistic target of rapamycin (also known as
mammalian target of rapamycin)
mTORC mTOR complex
MUC1-C Mucin 1, cell surface associated, C-terminal
subunit
MYC See c-MYC
List of Abbreviations xxix
N-FAG N-terminal cleavage fragment of eIF4G

N-terminus Amino-terminus
NAB2 NGFI-A binding protein 2
NAT N-acetyltransferase
NDRG1 N-MYC downregulated gene family
NF-B Nuclear factor -light-chain-enhancer of activated B cells
NF2 Neurofibromatosis 2
NF45 Nuclear factor 45
NHS National Health Service, UK
NIH National Institutes of Health, USA
NKX3.1 NK3 Homeobox 1
NMD Nonsense-mediated decay
NMP, NDP, NTP Nucleoside mono-, bi- and triphosphate
NNK 4-(Methylnitrosamino)-I-(3-pyridyl)-1-butanone
NO Nitric oxide
NOS Nitric oxide synthase
NOTCH Notch homolog, translocation-associated (Drosophila)
NOXA 1-Horbol-12-myristate-13-acetate-induced protein 1 (PMAIP1)
NPM1 Nucleophosmin
NRF2 Nuclear factor (erythroid-derived 2)-like 2
NS5A Nonstructural protein 5A
NSCLC Non-small-cell lung carcinoma
NTP Nucleoside triphosphate
OB Oligonucleotide/oligosaccharide binding
OCT4 Octamer-binding transcription factor 4
ODC Ornithine decarboxylase
ONCOMINE Cancer Microarray Database and Integrated Data-Mining
Platform
ORF Open reading frame
OS Overall survival
P-body Processing body

P-site Peptidyl site, the second binding site for tRNA in the ribosome
p16INK4A Protein 16/INK4A, see INK4
p27kip Cyclin-dependent kinase inhibitor p27
p53 Tumor suppressor p53
xxx List of Abbreviations
p70S6K 70 kDa ribosomal protein S6 kinase, see S6K1

PABP poly(A)-binding protein
PAIP PABP-interacting protein
PaNET Pancreatic neuroendocrine tumors
PARP Poly (ADP-ribose) polymerase
PAX3 Paired box 3
PCE Posttranscriptional control element
PCR Polymerase chain reaction
PDAC Pancreatic ductal adenocarcinoma
PDCD4 Programmed cell death 4
PDGF Platelet-derived growth factor
PDGFR Platelet-derived growth factor receptor
PDK1 Phosphoinositide-dependent kinase 1
PEComa Peripheral vascular epithelioid cell tumors
PERK PKR-like endoplasmic reticulum kinase (also known as eukaryotic
translation initiation factor 2 kinase 3 (EIF2AK3))
PFS Progression-free survival
PGC1 PPAR coactivator 1
PGF Placental growth factor
PHLPP PH domain and leucine rich repeat protein phosphatase
PI3K Phosphoinositide 3-kinase (or phosphatidylinositol-4,5-bisphos-
phate 3-kinase)
PIC Preinitiation complex
PIK3CA PI3K catalytic subunit
PIKK PI3K-related kinase
PIM Proviral integration of Moloney murine leukemia virus kinase
piRNA PIWI-interacting RNA
PIWI P-element induced wimpy testis protein
PKA Protein kinase A
PKAC PKA catalytic subunit
PKB Protein kinase B (also known as AKT)
PKC Protein kinase C
PKR Protein kinase R (interferon-induced, dsRNA-activated protein
kinase, also known as eukaryotic translation initiation factor 2
kinase 2 (EIF2AK2))
PML Promyelocytic leukemia protein
poly(A) Polyadenosine
PP1 Protein phosphatase 1
PP1C Protein phosphatase 1, catalytic subunit
PP2A Protein phosphatase 2A
PPAR Peroxisome proliferator-activated receptor
PPP1r15 PP1 regulatory subunit 15
PR Progesterone receptor
PRAS40 Proline-rich AKT substrate 40 kDa
pre-mRNA Precursor mRNA
List of Abbreviations xxxi
PRMT Protein arginine methyltransferase

PROTOR Protein observed with RICTOR
PSMA Prostate-specific membrane antigen
PTB Polypyrimidine tract-binding protein
PTC Premature termination codons
PTEN Phosphatase and tensin homolog deleted on chromosome 10
PUF Pumilio/FBF
PUM2 Pumilio homolog 2 protein
RACK1 Receptor of activated PKC1

RAD001 Everolimus
RAF RAF (rapidly accelerated fibrosarcoma) oncogene
RAG RAS-related GTPase
RAGULATOR GEF for the RAG
RAPTOR Regulatory-associated protein of mTOR
RAR Retinoic acid receptor
RAS Rat sarcoma viral oncogene homolog
RASSF1A RAS association domain family 1, isoform A
RB Retinoblastoma protein
RBM19 RNA-binding motif protein 19
RBM4 RNA-binding protein 4
RCC Renal cell carcinoma
rDNA Ribosomal DNA
RECK Reversion-inducing cysteine-rich protein with Kazal motifs
REDD1 Regulated in development and DNA damage responses 1
RHA RNA helicase A
RHEB RAS homolog enriched in brain
RHO RAS homolog protein, a family of small GTPases
rHRE RNA hypoxia response element
RICTOR rapamycin-insensitive companion of mTOR
RNA Ribonucleic acid
RNAi RNA interference
rp Ribosomal protein
rpL Large ribosomal subunit protein, capitalization denotes the gene
symbol
rpS Small ribosomal subunit protein, capitalization denotes the gene
symbol
rpS6 Ribosomal protein S6
RRM2 Ribonucleotide reductase M2 subunit
rRNA Ribosomal ribonulcei acid
RSK Ribosomal S6 kinase
RTK Receptor tyrosine kinase
xxxii List of Abbreviations
S6K Ribosomal protein S6 kinase

SAHA Suberoylanilide hydroxamic acid
SAHF Senescence-associated heterochromatic foci
SBDS Swachman-Bodian-Diamond syndrome protein
SCF skp, cullin, F-box containing complex
SCLC Small-cell lung carcinoma
SDF1 Stromal cell-derived factor 1
SDS Swachman-Bodian-Diamond syndrome
SEGA Subependymal giant-cell astrocytoma
SGK1 Serum/glucocorticoid regulated kinase 1
SH2 SRC homology 2
shRNA Small (or short) hairpin RNA
SILAC Stable isotope labeling by amino acids
SIN1 Stress-activated MAPK-interacting protein 1
siRNA Small (or short) interfering RNA
SKAR S6K1 Aly/REF-like target
SLIP1 SLBP-interacting protein 1
SMAC Second mitochondria-derived activator of caspases
SMAD2 Drosophila mothers against decapentaplegic homolog 2 (MAD) and the
C. elegans protein SMA
SNAIL Snail family zinc finger protein
snoRNP Small nucleolar ribonucleoprotein
SOX2 Sex determining region Y-box 2
SRC v-Src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog
SREBP Sterol regulatory element-binding protein
SS18 Synovial sarcoma translocation, chromosome 18
SSX2 Synovial sarcoma, X breakpoint 2
sT Small T antigen of Merkel cell polyomavirus
STAT Signal transducer and activator of transcription
STK11 Serine/threonine kinase 11, also known as LKB1
SUFU Suppressor of fused
T-ALL T-cell acute lymphoblastic leukemia

TAT Transactivator of transcription from HIV
TC Ternary complex
TCL1 T-cell leukemia/lymphoma 1 protein
TEL2 Telomere maintenance 2
TFEB Transcription factor EB
TGF Transforming growth factor
List of Abbreviations xxxiii
Tif1/2 eIF4A yeast homolog

Tif6 eIF6 yeast homolog
TIMP2 Tissue inhibitor of metalloproteinase 2
TKI Tyrosine kinase inhibitor
TLK Tousled-like kinase
TMZ Temozolomide
TNF Tumor necrosis factor
TNM Tumor, lymph node and metastasis staging system
TOP 5 terminal oligopyrimidine
TOR Target of rapamycin
TP53 Tumor suppressor p53 gene or transcript
TPA 12-O-tetradecanoylphorbol-13-acetate
TRAIL TNF-related apoptosis-inducing ligand
TRC8 Translocation in renal cancer from chromosome 8
TREX Transcription-export complex
TRIP-1 TGF--receptor type II interacting protein
TrkA Neurotrophic tyrosine kinase receptor type 1
tRNA Transfer ribonucleic acid
TSC Tuberous sclerosis complex
TTI1 TEL2-interacting protein 1 homolog
TWIST Twist-related protein
UBF Upstream binding factor

UK United Kingdom
ULK1 UNC-51-like kinase 1
UMP, UDP, UTP Uridine mono, di- and triphosphates
UNR Upstream of N-RAS
uORF Upstream open reading frame
uPAR Urokinase-type plasminogen activator receptor
UPF Up-frameshift protein
URI Unconventional prefoldin RPB5 interactor 1
USA United States of America
UTR Untranslated region
UV, UVA, UVB Ultraviolet, A and B
VATPase Vacuolar H+-ATPase

VEGF Vascular endothelial growth factor
VSV Vesicular stomatitis virus
xxxiv List of Abbreviations
WHO World Health Organization

WNT D
rosophila melanogaster wingless gene, human proto-onco-
gene protein
XIAP X-linked inhibitor of apoptosis
YB-1 Y box-binding protein 1

YY1 Ying-Yang 1
ZAP-70 -Chain-associated protein kinase 70

ZBP89 Zinc-finger-binding protein 89
ZEB2 Zinc finger E-box-binding homeobox 2
ZNF217 Zinc finger protein 217
Chapter 1
Introduction
Armen Parsyan
Abstract Translation, a process by which genetic information is transferred from

RNA to produce a polypeptide chain, is a fundamental step of the protein biosyn-
thesis pathway. Since origins of neoplasia are strongly linked to the aberrancies in
protein synthesis, dysregulation of translation would be expected to have a major
impact on the development and progression of the neoplastic process. This book
summarizes and analyzes decades of knowledge regarding the translation machin-
ery and its regulation in cancer biology. It also details the important role that transla-
tion plays in oncogenic signal transduction pathways. Additionally, we extensively
elaborate on the involvement of translation in cancer etiology and pathogenesis by
specific organ systems. Importantly, this publication explores and unveils applica-
tions of this knowledge in cancer medicine and drug development.
According to its most widely accepted modern connotation, the word gene designates
a DNA molecule whose specific self-replicating structure can, through mechanisms
unknown, become translated into the specific structure of a polypeptide chain (Jacob and
Monod 1961).
In the 1950s and 1960s, building on earlier tenacious work, groundbreaking dis-
coveries in biochemistry, genetics and molecular biology followed one another,
shedding light on the mysteries of protein synthesis. Around the same time, Francis
Crick brilliantly envisioned the central dogma of molecular biology (Crick 1958,
1970) describing a sequential transfer of genetic information involving three key
molecules of lifeDNA, RNA and protein. Somewhere in the middle of the twen-
tieth century, Jacob and Monod, describing the genetic transfer of information from
the nucleic acids to protein, appeared to introduce the word translation to the field
of biomedical science (Jacob and Monod 1961).
Translation is a process of transfer of the information from RNA characterized
by a synthesis of a polypeptide chaina protein. It is hard to imagine that such a
fundamental biological process hides in the shadows when a pathological program
hits the cell. On the contrary, hard and dedicated work of so many decades by thou-
sands of scientists has been providing evidence towards a critical role that transla-
tion plays in physiology and disease, including one of the most common and deadly
A.Parsyan()
Division of General Surgery, Department of Surgery, Faculty of Medicine, McGill University,
Montreal, Quebec, Canada
e-mail: armen.parsyan@mail.mcgill.ca
A. Parsyan (ed.), Translation and Its Regulation in Cancer Biology and Medicine, 1
DOI 10.1007/978-94-017-9078-9_1, Springer Science+Business Media Dordrecht 2014
2 A. Parsyan
ailments, cancer. This evidence, especially vis--vis cancer, has, to my knowledge,

never been comprehensively synthesized. Hence, the current publication dared to
systematically assemble and analyze a vast amount of information related to the
process of eukaryotic cellular translation in cancer biology, which was accumulated
since around the times of the first use of the word translation in the context of the
molecular biology.
Translation in eukaryotes is a very complex process that occurs in various stages
and diverse mechanisms involving a plethora of protein and non-protein molecules.
Translation is a subject of regulation by a number of fundamental signaling path-
ways that communicate external and internal messages to control proliferative, nu-
tritional and other states of the cell. In order to efficiently embrace the complex na-
ture of the eukaryotic cellular translation machinery and its regulatory mechanisms
in physiology and human cancer, the book is conceptualized into four parts.
PartI starts with the introduction to the process of translation and various regula-
tory mechanisms observed in higher eukaryotes. It then, factor by factor, dissects
the translation machinery presenting the role of its components in physiology and
then in cancer biology. While some of these factors, such as translation initiation
factor eIF4E, are well-known players in the cancer etiology and pathogenesis, many
others are still under scrutiny in terms of their physiological roles and involvement
in tumorigenesis. Here, translation factors with known or highly suggestive links
to cancer biology are sequentially presented from the standpoints of their function
and regulation in norm; mechanistic, functional and regulatory aberrancies in can-
cer; as well as their utilization as clinical and therapeutic targets. The first part also
addresses our evolving understanding of the role of eukaryotic ribosomes in cancer
and ends with the highlights in the developing field of targeted therapies directed at
the translation machinery.
PartII outlines the role of translational regulation by signal transduction pathways
in physiology and in neoplasia. While the role of the signal transduction in cancer
deserves at least another volume, we attempt to focus on the role of signaling in
cancer vis--vis translation. The signaling pathways are becoming a hot spot for
targeted cancer therapies. However, I believe, relatively few clinicians, researchers
and pharmaceutics developers, appreciate the fact that whatever complexities signal
transduction entails, it eventually and most likely converges on the regulation of the
translation machinery, one of the most downstream hubs of signaling cascades. The
second part of the book unveils the aforementioned connections between oncogenic
signaling and the translation machinery, as well as clinical and pharmacological
aspects of these connections.
PartIII takes a different angle at translation in cancer biology. It discusses transla-
tion and its regulatory mechanisms in regard to cellular processes involved in neo-
plasia, such as apoptosis, senescence, autophagy, cell cycle regulation, hypoxia and
angiogenesis. The information provided in this part of the book emphasizes critical
pathophysiological correlations between cancer and translation.
1Introduction 3
PartIV focuses on translation and its regulation in various types of cancer. This,
more clinically-oriented part, puts available information in the brackets of a specific
oncopathology, hence providing more clinically-minded audience with knowledge
that can be further enhanced and applied in a clinical setting. In this segment of the
book, I tried to provide as much comprehensive coverage as possible in terms of
translation and various types of cancer. However, some subsets of less common or
less morbid cancer types are not well studied in regards to the topic of this publica-
tion. Nevertheless, there is surfacing of first pieces of evidence linking translation
to some of these cancers, such as thyroid cancers (Kouvaraki etal. 2011; Pennelli
etal. 2013; Wang etal. 2001), pediatric neuroblastoma (Parker etal. 2004), penile
squamous cell cancer (Ferrandiz-Pulido etal. 2013) and others. Despite this, the
forth part contains reviews that have never been accomplished before, including
information on fundamental involvements of the translation machinery in cancers
of more than a dozen organ systems, some of which are represented by the most
prevalent and morbid cancer types, such as skin, lung, breast, prostate, colorectal,
gastric and hematological malignancies, to name a few.
I hope this book will be an enjoyable and useful reading experience for students
and scholars interested in biology, clinical medicine, and pharmaceutical develop-
ment who work and study in the field of cancer. I also hope that this publication
will capture the attention of those interested in learning biochemical, genetic and
molecular complexities of translation in physiology and disease.
References
Crick FH (1958) On protein synthesis. Symp Soc Exp Biol 12:138163

Crick F (1970) Central dogma of molecular biology. Nature 227:561563
Ferrandiz-Pulido C, Masferrer E, Toll A, Hernandez-Losa J, Mojal S, Pujol RM, Ramon y Cajal
S, de Torres I, Garcia-Patos V (2013) mTOR signaling pathway in penile squamous cell carci-
noma: pmTOR and peIF4E over expression correlate with aggressive tumor behavior. J Urol
190:22882295
Jacob F, Monod J (1961) Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol
3:318356
Kouvaraki MA, Liakou C, Paraschi A, Dimas K, Patsouris E, Tseleni-Balafouta S, Rassidakis GZ,
Moraitis D (2011) Activation of mTOR signaling in medullary and aggressive papillary thyroid
carcinomas. Surgery 150:12581265
Parker A, Anderson C, Weiss KL, Grimley M, Sorrells D (2004) Eukaryotic initiation factor 4E
staining as a clinical marker in pediatric neuroblastoma. J Pediatr Hematol Oncol 26:484487
Pennelli G, Fassan M, Mian C, Pizzi M, Balistreri M, Barollo S, Galuppini F, Guzzardo V, Pelizzo
M, Rugge M (2013) PDCD4 expression in thyroid neoplasia. Virchows Arch 462:95100
Wang S, Lloyd RV, Hutzler MJ, Rosenwald IB, Safran MS, Patwardhan NA, Khan A (2001) Ex-
pression of eukaryotic translation initiation factors 4E and 2alpha correlates with the progres-
sion of thyroid carcinoma. Thyroid 11:11011107
Part I
Translation Machinery in Cancer
Chapter 2
Mechanism of Translation in Eukaryotes
Contents
2.1Introduction 8
2.2Translation Initiation 11
2.2.1Binding of eIF4F Complex Prepares the mRNA for Translation 15
2.2.2Several Initiation Factors Prepare the 40S Ribosome for mRNA
Recruitment and Form the 43S PIC 17
2.2.3mRNA Recruitment to 43S PIC 19
2.2.45 to 3 Scanning 20
2.2.5Initiation Codon Selection 21
2.2.660S Ribosome Binding and 80S Ribosome Formation 23
2.3Translation Elongation and Termination 23
2.3.1Translation Elongation 24
2.3.2Translation Termination 25
2.4Ribosome Recycling and Reinitiation 25
2.4.1Ribosome Recycling 26
2.4.2Reinitiation 26
2.5Conclusions and Perspectives 27
References 27
Abstract Recent years have seen a tremendous advance in our understanding of

the mechanism of protein synthesis in eukaryotic cells. Furthermore, our under-
standing of the role of translation in cancer development and progression, as well
as its significance in clinical medicine has also greatly increased. The process of
messenger RNA (mRNA) translation is comprised of four main stages: initiation,
elongation, termination and ribosome recycling. Each stage is promoted by many
different protein factors that interact with mRNA, transfer RNA (tRNA) and the
40S and 60S ribosomes to ensure an mRNA is accurately translated into protein.
Here, we will describe the fundamental mechanisms involved in selection, recruit-
ment, and translation of an mRNA by the eukaryotic ribosome with an emphasis on
aspects most relevant to the theme of translation and cancer.
C.S.Fraser() N.Villa
Section of Molecular and Cellular Biology, University of California, Davis, CA, USA
e-mail: csfraser@ucdavis.edu
8 N. Villa and C. S. Fraser
5-7-methyl- Main Open Reading Frame 3 Poly(A) Tail

guanosine cap
AUG UGA AAAAAAAAAAA
5 UTR 3 UTR
a
P
A E
mRNA
Head exit
Neck
Latch
mRNA
entry
Body
b
Fig. 2.1 Schematic diagrams of eukaryotic mRNA and 40S ribosome. a The mRNA 5 end is post-
transcriptionally modified with a 7-methylguanosine cap and the 3 end with a poly(A) tail. The
main open reading frame (ORF) is the region of the mRNA that encodes the protein, and usually
begins with an AUG start codon and ends with one of three possible stop codons (UGA is used as
an example here). Between the cap and the start codon is the 5 UTR. Between the stop codon and
poly(A) tail is the 3 UTR. b The 40S subunit consists of three main regions: head, neck, and body.
The neck is situated between the head and the body and delineates the mRNA-binding channel.
The mRNA entry and exit sites are depicted together with the A-, P- and E-sites, which indicate
respective tRNA-binding sites.
2.1Introduction
In order for the information encoded in an mRNA to be translated into a protein, the
mRNA must be recruited to a ribosome (Fig.2.1). Protein synthesis occurs in four
phases (initiation, elongation, termination, ribosome recycling), of which transla-
tion initiation is the most complex in as much as it requires the greatest number of
protein factors. Initiation is also the stage most often implicated in cancer develop-
ment and progression. During initiation, mRNA is recruited to the 40S ribosome, the
start codon is located, and the 60S ribosome joins to form an elongation-competent
80S ribosome (Fig.2.2). During elongation, the 80S ribosome migrates along the
2 Mechanism of Translation in Eukaryotes 9
eIF4E
AUG UGA AAAAA eIF4G eIF4F Cap Binding Complex
eIF4A
eIF4B
AUG UGA AAAAA
PABP
ATP
Helicase Unwinding
ADP
AUG UGA AAAAA
GTP
mRNA Recruitment
GTP
E P A
43S PIC
AUG UGA AAAAA

GTP
E P A
43SmRNA Initiation Complex
ATP
eIF2-TC
5
to 3
Scanning
ADP
GDP E P A
eIF1A
eIF1
AUG UGA AAAAA Met-tRNAiMet eIF1A GTP
E P A eIF3
GDP GTP
Start Codon Recognition
GDP GTP
eIF5
MFC
eIF4F? eIF2GDP
eIF2B
eIF1, 3, 5?
eIF5BGTP
60S ribosome
E P A
60S ribosome 40S ribosome
GTP eIF5BGTP
AUG UGA AAAAA
E P A
eIF5BGDP Recycling
eIF1A
Elongation
AUG UGA AAAAA Termination
Cycle
E P A
80S Initiation Complex
Fig. 2.2 Pathway of eukaryotic translation initiation. The target mRNA is recognized and bound
by the eIF4F complex and PABP in preparation for recruitment to the 40S subunit. The eIF4F
complex consists of three subunits (eIF4E, eIF4A, and eIF4G) and the auxiliary factor eIF4B,
which together promote unwinding of secondary structure in the 5 UTR by eIF4A. The 40S sub-
unit is prepared for mRNA recruitment by several factors, including eIF1, eIF1A, eIF2, eIF3 and
eIF5. These factors may bind individually, or as a multifactor complex. eIF2 binds as a ternary
complex, eIF2-TC, with GTP and the initiator tRNA. The message is directed to the 40S ribosome
eEF1B
aminoacyl-tRNA GTP GDP
eEF1A-TC GTP GTP
AUG UGA AAAAA
E P A
GDP
Aminoacyl-tRNA Recruitment A Site
tRNA
Binding
AUG UGA AAAAA AUG UGA AAAAA
E P A E P A
GDP
deacylated
tRNA
Elongation
Cycle
E P A E P A
Translocation
GDP
GTP
E P A E P A
eEF2 GTP
Peptide Bond Formation
(Hybrid State)
Fig. 2.3 The translation elongation cycle. During elongation, eEF1A-TC (eEF1A, GTP and aa-
tRNA) recruits the aa-tRNA to the 80S ribosome. The aa-tRNA delivers the next amino acid to be
incorporated into the growing polypeptide by base-pairing the tRNA anticodon with the mRNA
codon in the A-site of the 80S ribosome. Peptide bond formation is catalyzed by the peptidyl
transferase center in the 60S ribosomal subunit, resulting in the new amino acid being added to
the polypeptide chain. This reaction results in spontaneous ratcheting of the ribosomal subunits
forming a hybrid state in which tRNAs have partially translocated into the P/E- and A/P-sites.
eEF2-GTP then binds the 80S ribosome and hydrolyzes its GTP. This promotes full translocation
of the A-site tRNA to the P-site, and P-site tRNA to the E-site. The E-site tRNA and eEF2-GDP are
then released from the ribosome leaving the next codon to be translated in the A-site.
Fig. 2.2 (continued) through the direct interaction of eIF4G and eIF3. Following recruitment, the
40S subunit scans for the initiation codon by traveling along the message in the 5 to 3 direction,
sampling codons on the mRNA with the initiator tRNA anticodon. When the initiation codon is
recognized through base-pairing, eIF5 induces GTP hydrolysis in eIF2-TC, resulting in the release
of the eIF2-GDP complex and other initiation factors from the 40S subunit. eIF2 is recharged
with GTP by eIF2B and recruits another initiator tRNA to reform the eIF2-TC, and the factors are
free to initiate a new round of translation on another mRNA. eIF1A remains on the 40S subunit
until eIF5B-GTP binds and recruits the 60S subunit, after which both are released following GTP
hydrolysis. The newly formed 80S ribosome then enters the elongation cycle, followed by ter-
mination and recycling steps, which result in free 40S and 60S subunits to be prepared for future
rounds of translation.
GTP P
eRF1 GT
Termination
P
GD
eRF3
UGA UGA UGA
Peptide
A
AUG AUG
A
AUG
A
A
Hydrolysis
A
A
A
A
E E
A
P
A
E A P A
A
P A
Stop Codon Recognition Post Termination Complex
60S ribosome
ABCE1 ADP
Recycling
ATP
ATP Subunit
ATP Hydrolysis ADP Dissociation
UGA UGA UGA
A
AUG
A
AUG
A
AUG
A
A
A
A
A
A
A
A
A
E
A
E P A
A
P A E P A
A
40S ribosome
Fig. 2.4 Translation termination and ribosome recycling. a Translation termination occurs when a
stop codon reaches the A-site of the 80S ribosome. Stop codons are not recognized by aa-tRNAs
but by eRF1, which binds in a complex with eRF3 and GTP. This complex hydrolyzes GTP to pro-
mote peptide hydrolysis and release from the P-site tRNA and 80S ribosome. b Following peptide
release, ABCE1 promotes ribosome dissociation and factor release in an ATP dependent reaction.
mRNA translating the information in each nucleotide triplet, or codon, to an amino

acid, which is then incorporated into the growing polypeptide chain (Fig.2.3). Stop
codon recognition marks translation termination, where the newly formed protein
is released (Fig.2.4a). Lastly, ribosome recycling releases the mRNA and the 80S
ribosome is separated into its component 40S and 60S subunits, which can then
begin the translation cycle once again (Fig.2.4b). The protein factors that function
in each of these stages are summarized in Table2.1, with known associations to
the cancer etiology and pathogenesis (reviewed in Silvera etal. 2010; Spilka etal.
2013; Stumpf and Ruggero 2011). Each of these stages will be described in relevant
detail in the following sections. Understanding the molecular mechanism of these
processes is essential to understanding how they are regulated in vivo, and how they
can become dysregulated in a transformed cell.
2.2Translation Initiation
Translation initiation is almost always the rate-limiting step of protein synthesis, and
as such, it is the most highly regulated (reviewed in Aitken and Lorsch 2012; Fraser
2009; Hinnebusch and Lorsch 2012; Jackson etal. 2010; Sonenberg and Hinnebusch
2009). Initiation rates can vary by many orders of magnitude. This variance can be
due to differences in mRNA regulatory features, such as a highly structured 5 un-
translated region (UTR), or regulation of initiation factors by key signaling cascades,
such as the phosphoinositide 3-kinase (PI3K)/v-Akt murine thymoma viral onco-
gene homolog (AKT)/mechanistic target of rapamycin (mTOR) pathway and the mi-
togen-activated protein kinase (MAPK) pathway, which can affect factor availability
and activity to alter the rate of translation. A recent study suggested that initiation
Table 2.1 Eukaryotic translation factors.
12
Initiation Factor Subunits Molecular Mass (kDa) Function Interacting Partners Cancer Links
eIF1 1 12.7 Prepares 40S ribosome for mRNA 40S ribosome, eIF2, eIF3, Chen etal. 2010; Lian etal. 1999
loading and promotes scanning eIF5
with eIF1A, fidelity of start site
recognition
eIF1A 1 16.5 Prepares 40S ribosome for mRNA 40S ribosome, eIF5B
loading and promotes scanning
with eIF1
eIF2 3 36.1, 38.4, 51.1 Binds and recruits initiator tRNA to Initiator tRNA, 40S ribo- Rosenwald etal. 2001, 2003, 2008;
the 40S ribosome some, eIF1, eIF2B, eIF3, Tejada etal. 2009; Wang etal.
eIF5 1999
eIF2B 5 33.7, 39.0, 50.2, 59.7, GTP exchange factor for eIF2, helps eIF2
80.3 regenerate eIF2-TC
eIF3 13 ~800 total Scaffold that organizes the 43S PIC, 40S ribosome, eIF1, eIF1A, Zhang etal. 2007
increases eIF2-TC affinity for the eIF4G, eIF5
40S ribosome, prevents premature
60S ribosome binding, promotes
mRNA recruitment with eIF4G
eIF4A 1 46.2 Member of eIF4F cap-binding RNA, eIF4G, eIF4B Eberle etal. 1997; Harris etal. 2004;
complex; ATP dependent helicase, Shuda etal. 2000
unwinds secondary structure in the
5 UTR of the mRNA
eIF4B 1 69.2 Stimulates helicase activity of eIF4A RNA, eIF4A, eIF3

N. Villa and C. S. Fraser
Table 2.1 (continued)
Initiation Factor Subunits Molecular Mass (kDa) Function Interacting Partners Cancer Links
eIF4E 1 25.1 Member of eIF4F cap-binding 5 7-methylguanosine cap, De Benedetti and Graff 2004; De
complex; Binds to the mRNA 5 eIF4G Benedetti and Rhoads 1990; Flow-
7-methylguanosine cap, stimulates ers etal. 2009; Lazaris-Karatzas
eIF4A helicase activity with eIF4G etal. 1990; Lazaris-Karatzas and
Sonenberg 1992; Rosenwald etal.
2001, 2003, 2008; Ruggero etal.
2004; Tejada etal. 2009; Wang
etal. 1999, 2001
eIF4G 1 175.5 Member of eIF4F cap-binding com- RNA, eIF4E, eIF4A, eIF3, Fukuchi-Shimogori etal. 1997; Har-
plex; acts as molecular scaffold, PABP, MNK1 ris etal. 2004; Silvera etal. 2009
stimulates helicase and ATPase
activity of eIF4A, promotes
2 Mechanism of Translation in Eukaryotes
mRNA recruitment with eIF3

eIF4H 1 27.4 Homologous to the N-terminus of RNA, eIF4A
eIF4B, stimulates helicase activity
of eIF4A
eIF5 1 49.2 GTPase activating protein for eIF2 40S ribosome, eIF1, eIF2, Harris etal. 2004
following start codon recognition eIF3
eIF5B 1 138.9 Ribosome dependent GTPase, pro- 80S ribosome, eIF1A
motes 80S ribosome formation
eIF6 1 26.6 60S ribosome biogenesis, binds 60S 60S ribosome Harris etal. 2004; Miluzio etal.
ribosomes and prevents premature 2011; Sanvito etal. 2000
association of 40S ribosomes
DHX29 1 155.2 Binds the 40S ribosome and pro- 40S ribosome Parsyan etal. 2009; Pisareva etal.
motes scanning on highly struc- 2008
tured mRNAs
PABP 1 70.7 Binds the mRNA poly(A) tail and eIF4G, poly(A) tail Takashima etal. 2006
eIF4G to promote translation
initiation
13
14
Elongation Subunits Molecular Mass (kDa) Function Interacting Partners Cancer Links
Factors
eEF1A 1 50.1 Binds and delivers aa-tRNA to the 80S ribosome, aa-tRNA Harris etal. 2004
A-site as a ternary complex with
GTP
eEF1B 1 24.8 GTP exchange factor for eEF1A, eEF1A
helps regenerate eEF1A ternary
complexes
eEF2 1 95.3 Completes translocation of aa-tRNAs mRNA, tRNA, 80S Nakamura etal. 2009
from the A- to P-site and the P- to ribosome
E-site in the 80S ribosome
Termination Subunits Molecular Mass (kDa) Function Interacting Partners Cancer Links
Factors
eRF1 1 49.0 Binds to 80S ribosomes as a ternary eRF3, 80S ribosome
complex with eRF3 and GTP,
stop codon recognition, promotes
peptide hydrolysis and release,
eRF3 1 55.8 Binds to 80S ribosomes as a ternary eRF1, 80S ribosome
complex with eRF1 and GTP,
hydrolyzes GTP to allow full
accommodation of eRF1 into the
A-site
Recycling Subunits Molecular Mass (kDa) Function Interacting Partners Cancer Links
Factors
ABCE1 1 67.3 Separates 80S ribosomes following 80S ribosome
termination
rates are likely to vary between 4s and 233s on different mRNAs in yeast (Shah etal.
2013). These findings emphasize the impact that translation initiation has on deter-
mining the overall rate of translation for any mRNA. Initiation of protein synthesis
involves five basic steps, which will be described in further detail: (1) mRNA bind-
ing by the eIF4F cap-binding complex; (2) 43S preinitiation complex (PIC) forma-
tion; (3) mRNA recruitment to the ribosome; (4) localization of the initiation codon;
and (5) 60S ribosome joining (Aitken and Lorsch 2012; Fraser 2009; Hinnebusch
and Lorsch 2012; Jackson etal. 2010; Sonenberg and Hinnebusch 2009).
2.2.1Binding of eIF4F Complex Prepares the mRNA

for Translation
Eukaryotic mRNAs contain several key features involved in regulating translation

(Fig. 2.1). Following transcription, a 7-methyl-guanosine cap is added to the 5
end while several adenosine residues are attached to the 3 end to form the poly-
adenosine (poly(A)) tail. Flanking the protein coding sequence of the mRNA are 5
and 3 UTRs, which may contain regulatory structures or sequences, which affect
mRNA translation (see Chap.3).
mRNAs are selected for translation by recognition of two of the aforementioned
features: the 5 7-methyl-guanosine cap (Carroll and Borden 2013; Topisirovic etal.
2011) and the 3 poly(A) tail (Fig.2.1) (Mangus etal. 2003; Sachs etal. 1997). It
has been known for many years that these features help to protect an mRNA from
degradation and act synergistically to promote translation initiation (Gallie 1991;
Searfoss etal. 2001). The eukaryotic translation initiation factor 4F (eIF4F) inter-
acts directly or indirectly with these key features, and consists of three subunits:
eIF4E (cap-binding), eIF4A (DEAD box helicase), and eIF4G (molecular scaffold)
(Gingras etal. 1999; Grifo etal. 1983). The purpose of eIF4F is to locate the 5 end
of the mRNA through cap recognition, then to ensure that the 40S ribosome will
be able to bind to the 5 end of the mRNA by removing any inhibitory secondary
structures in that region.
The cap-binding protein eIF4E specifically recognizes the 5 7-methyl-guanosine
cap by sandwiching it between two conserved tryptophans located in the cap-bind-
ing pocket of eIF4E (Marcotrigiano etal. 1997; Matsuo etal. 1997). Importantly,
eIF4E is the least abundant initiation factor and therefore is generally regarded as
the initiation factor that limits recruitment of mRNA to the ribosome (Duncan etal.
1987). Since cap recognition by eIF4E plays a critical role in mRNA recruitment the
availability of this initiation factor is subject to strict regulation by eIF4E-binding
proteins (4E-BPs), which can sequester eIF4E from eIF4G, thus inhibiting cap-de-
pendent translation (see Chaps.3 and 4). In a similar theme, programmed cell death
4 (PDCD4) can also inhibit cap-dependent translation by inhibiting the unwinding
activity of eIF4A (Lankat-Buttgereit and Goke 2009) (see Chaps.3, 5 and 6).
Consistent with its being the limiting factor, modest overexpression of eIF4E
by 2.5-fold is able to transform immortalized cells and form tumors in mice (De
Benedetti and Rhoads 1990; Lazaris-Karatzas etal. 1990; Ruggero etal. 2004). In
addition to binding the cap, eIF4E forms a high affinity interaction with eIF4G us-
ing a number of conserved residues located on the convex dorsal surface of eIF4E
on the opposite side of the cap-binding pocket (Marcotrigiano etal. 1999). This
interaction is necessary to position eIF4G near the 5 end of the mRNA so that it can
prepare the mRNA for recruitment to the 40S subunit. It is noteworthy to mention
that in addition to its role in mRNA recruitment to the 40S subunit, eIF4E is also
involved in promoting the export of specific mRNAs from the nucleus (Culjkovic
etal. 2005; Rousseau etal. 1996), many of which are linked to cell cycle progres-
sion and survival (Culjkovic etal. 2006).
The largest component of the eIF4F complex is the 175kDa protein named
eIF4G (Gingras etal. 1999; Hentze 1997; Imataka etal. 1998; Keiper etal. 1999;
Prevot etal. 2003; Yan etal. 1992) (see Chap.7). This protein contains binding
domains for RNA (Berset etal. 2003; Goyer etal. 1993; Park etal. 2011), eIF4E
(Lamphear etal. 1995; Mader etal. 1995), eIF4A (Imataka and Sonenberg 1997;
Korneeva etal. 2001), eIF3 (Korneeva etal. 2001; Lamphear etal. 1995), poly
(A)-binding protein (PABP) (Imataka etal. 1998; Tarun and Sachs 1996), and the
MAPK-interacting kinases (MNKs) (Pyronnet etal. 1999). The protein can there-
fore be thought of as a molecular scaffold that recruits and coordinates the activities
of these other initiation components. One function of eIF4G may be to stabilize
the interaction of eIF4E with the cap structure and PABP with the poly(A) tail.
Data to support this concept comes from a study which used crosslinking assays
to show that eIF4E crosslinking to the cap structure is enhanced in the presence of
eIF4G (Yanagiya etal. 2009). However, quantitative binding assays have provided
inconsistent results as to whether eIF4G stimulates the interaction between eIF4E
and the cap structure (Niedzwiecka etal. 2002; Slepenkov etal. 2008; Yanagiya
etal. 2009). Quantitative binding assays show that eIF4G does indeed increase the
affinity of PABP to the poly(A) tail, which may help to circularize the mRNA and
stimulate translation by facilitating reinitiation of a terminating ribosome on the
same mRNA (Le etal. 1997; Wells etal. 1998). It is worth noting, however, that
whether an mRNA can actually form closed circles as a result of this interaction in
vivo is not clear (Amrani etal. 2008; Rajagopal etal. 2012). In addition to stabiliz-
ing these interactions, mammalian eIF4G has two additional pivotal roles in mRNA
recruitment to the ribosome. Through its direct interaction with eIF3, which will be
discussed in the next section, eIF4G helps bridge the eIF4F-mRNA complex and the
43S PIC (Hinton etal. 2007; Lamphear etal. 1995; Villa etal. 2013). Moreover, its
interaction with DEAD box helicase eIF4A is required to recruit this helicase to the
mRNA, which ultimately functions to unwind secondary structures located in the 5
UTR in order to facilitate ribosome recruitment and scanning.
It has been known for some time that the presence of secondary structure in the
5 UTR of eukaryotic mRNAs plays a key role in controlling recruitment to the ribo-
some (reviewed in Mauger etal. 2013; Parsyan etal. 2011). Secondary structures
typically form when complementary regions of the mRNA base-pair to form stable
hairpin structures. These structures can be particularly inhibitory when located near
the cap since they can sterically hinder the recruitment of the mRNA to the 40S
subunit (Lawson etal. 1986). The reason for this is that the mRNA-binding site in
the 40S subunit can only accommodate single-stranded RNA. Hence any structured
mRNA must be unwound before it can enter the 40S subunit (see Chap.3 for further
information on translation regulation of mRNAs with highly structured 5 UTRs).
To deal with this problem, the DEAD box helicase eIF4A unwinds these second-
ary structures in an ATP-dependent manner, thus creating an unstructured region
of mRNA, or landing pad, that can be stably bound in the mRNA-binding site
of the 40S ribosomal subunit (Lawson etal. 1989; Rogers etal. 1999, 2001; Rozen
etal. 1990). Importantly, the ATP hydrolysis and helicase activities of eIF4A are
greatly stimulated by its association with eIF4G, eIF4E and the helicase accessory
protein eIF4B (Feoktistova etal. 2013; Korneeva etal. 2005; Nielsen etal. 2011;
Ozes etal. 2011; Ray etal. 1985; Rosenwald etal. 2001; Schutz etal. 2008). An ad-
ditional cofactor homologous to the N-terminus of eIF4B, named eIF4H (Richter-
Cook etal. 1998), also stimulates helicase activity in vitro, but to a lesser extent than
eIF4B (Ozes etal. 2011; Rogers etal. 2001). While the 43S complex is capable of
scanning in an ATP-independent manner on a completely unstructured 5 UTR, the
eIF4F complex greatly enhances this activity (Pestova and Kolupaeva 2002). This
implies that the helicases are required for ribosomal movement on mRNAs that
contain even weak structures. There are several models for eIF4A duplex unwind-
ing and eIF4B/eIF4H stimulation (Parsyan etal. 2011; Rogers etal. 2001), but the
exact mechanism remains to be determined. It should be noted that it is not clear
if the interactions between the components of the eIF4F complex and all of its
binding partners are maintained throughout initiation. Presumably, eIF4G must re-
main associated with the scanning 40S ribosomal subunit, however a precise kinetic
analysis of when eIF4G enters and exits the pathway has not yet been undertaken
(Pestova and Kolupaeva 2002; Poyry etal. 2004).
2.2.2Several Initiation Factors Prepare the 40S Ribosome

for mRNA Recruitment and Form the 43S PIC
The purpose of 43S PIC formation is to prepare the 40S ribosome mRNA entry
channel and decoding site for mRNA recruitment, scanning, and initiation codon
selection. To form the 43S PIC several factors (eIF1, eIF1A, eIF2, eIF3 and eIF5)
are recruited to the 40S ribosome (Fig.2.2). The individual roles of each factor in
translation initiation will be discussed in further detail below. In contrast to bacteria,
eukaryotic cellular mRNAs are almost exclusively loaded onto the 40S ribosomal
subunit at the cap structure, followed by a linear migration until an initiation codon
is selected. This is known as the scanning model, and is generally thought to be
the mechanism by which translation of most eukaryotic mRNAs is initiated.
It is important to understand the structure of the mRNA decoding site of the 40S
subunit in order to appreciate how initiation factors need to manipulate its confor-
mation during different stages of initiation (reviewed in Fraser and Doudna 2007;
Lorsch and Dever 2010; Voigts-Hoffmann etal. 2012). Essentially, the mRNA de-
coding site runs across the 40S subunit between the head and the body of the sub-
unit (Fig.2.1). An aminoacyl (A-site), a peptidyl (P-site), and an exit (E-site) are
identified as tRNA-binding sites in the mRNA decoding site of the 40S ribosomal
subunit. Two small initiation factors, eIF1 and eIF1A, have been identified to play a
critical role in altering the shape of the mRNA decoding site during different stages
of initiation. Together, these factors promote opening of the mRNA entry channel
and decoding site of the 40S subunit (Fraser etal. 2009; Passmore etal. 2007). This
conformational change is believed to enable the mRNA to enter the 40S subunit
so that productive scanning and initiation can occur (Pestova etal. 1998). These
initiation factors also play a key role in the fidelity of start site selection, which is
discussed in detail later.
The first amino acid of the polypeptide, methionine, is bound to an initiator
tRNA and must be recruited to the P-site as initiator methionyl tRNA (Met-tRNAi-
Met
or Met-tRNAi). This initiator tRNA may bind the 40S subunit independently, as
a ternary complex with eIF2 and GTP (eIF2 ternary complex or eIF2-TC), or as part
of a multifactor complex composed of eIF1, eIF2-TC, eIF3 and eIF5 (Asano etal.
2000; Sokabe etal. 2012). In vitro, initiator tRNA association with the ribosome
is more efficient when delivered by eIF2-TC or the multifactor complex (Sokabe
etal. 2012), although the order of binding in vivo remains to be determined. Never-
theless, the initiator tRNA ultimately resides on the 43S PIC as a ternary complex
with eIF2 and GTP (Hinnebusch and Lorsch 2012). eIF2 contains three subunits,
eIF2, - and -, where the - subunit directly binds GTP, the initiator tRNA, and
the 40S subunit (Kapp and Lorsch 2004; Schmitt etal. 2010; Shin etal. 2011).
The eIF2 and - subunits increase the affinity of eIF2 for the initiator tRNA
(Naveau etal. 2010). Additionally, eIF2 plays a minor role in tRNA binding and
eIF2 makes multiple contacts with the 40S ribosome and the mRNA during trans-
lation initiation (Hashem etal. 2013; Naveau etal. 2010; Pisarev etal. 2006). The
interaction of eIF2-TC with the 40S subunit is accelerated by the presence of eIF1,
eIF1A and mRNA (Algire etal. 2002; Chaudhuri etal. 1999; Fekete etal. 2005;
Majumdar etal. 2003; Olsen etal. 2003; Passmore etal. 2007), but only eIF1A and
mRNA actually stabilize this interaction (Passmore etal. 2007).
The scaffold believed to organize much of the 43S PIC is eIF3. In humans, this
factor consists of 13 nonidentical subunits (eIF3a-eIF3m) with a combined mass of
approximately 800kDa (Damoc etal. 2007; Dong and Zhang 2006; Hinnebusch
2006). It is worth noting that this is in fact equivalent to half of the 40S subunit
mass! eIF3 is involved in almost every step of translation initiation, although varia-
tions exist between yeast and humans. As a member of the 43S PIC, eIF3 has been
shown to increase the affinity of eIF2-TC to the ribosome (Benne and Hershey
1978; Peterson etal. 1979), to bind eIF1 (Karaskova etal. 2012; Sun etal. 2011;
Valasek etal. 2004), eIF1A (Fraser etal. 2007; Sun etal. 2011) and the 40S subunit
(Hashem etal. 2013; Siridechadilok etal. 2005), and to prevent premature associa-
tion of the 60S ribosome in conjunction with eIF1 and eIF2 (Kolupaeva etal. 2005;
Majumdar etal. 2003). Recent cryo-electron microscopy structures indicate that
the bulk of the human eIF3 mass is primarily located on the solvent side of the 40S
subunit (Hashem etal. 2013; Siridechadilok etal. 2005), although large portions
(roughly half) could not be resolved due to flexible regions. At least one subunit,
eIF3j, binds in the A-site region of the decoding site of the 40S ribosome, where
it reduces mRNA affinity for 40S subunit in the absence of eIF2-TC (Fraser etal.
2007). The reason for this interaction is unclear, but may serve to prevent unproduc-
tive complexes from forming prior to eIF2-TC binding. It is likely that other eIF3
subunits will be discovered in the vicinity of the decoding site in the future, perhaps
helping to explain how eIF3 influences almost every aspect of translation initiation
(see Chap.8).
2.2.3mRNA Recruitment to 43S PIC
In mammals, recruitment of an mRNA to the 43S PIC is stabilized by a direct in-

teraction between the eIF4G component of the cap-bound eIF4F complex and the
eIF3 component of the 43S PIC (De Gregorio etal. 1999; Hinton etal. 2007; Lam-
phear etal. 1995; Morino etal. 2000; Villa etal. 2013). Interaction sites of the two
factors have been mapped to an approximately 90 amino acid region in the middle
domain of eIF4G (Korneeva etal. 2000; Villa etal. 2013) and the subunits eIF3e
(LeFebvre etal. 2006), eIF3c and eIF3d (Villa etal. 2013). These interaction sites
are critical for efficient mRNA recruitment to the ribosome (Hinton etal. 2007;
Villa etal. 2013). Interestingly, the eIF3/eIF4G interaction is stimulated in vivo
by the activation of the mTOR complex 1 (mTORC1) (Harris etal. 2006; Thoreen
etal. 2012), a key component of the PI3K/AKT/mTOR signaling pathway (see
Chap.15). However, specific sites regulating this interaction have not been discov-
ered, despite several phosphorylation sites having been identified on both factors
(Damoc etal. 2007; Raught etal. 2000). Additional contacts between eIF4B/eIF3
(Methot etal. 1996), eIF4B/40S (Methot etal. 1996; Rozovsky etal. 2008; Walker
etal. 2013) and eIF4B/PABP (Bushell etal. 2001; Cheng and Gallie 2010; Le etal.
1997) may also help bridge the 43S PIC and the mRNA.
The presence of the cap structure stimulates mRNA recruitment to the 43S PIC
and confers a strong requirement for the presence of the eIF4F complex (Grifo
etal. 1983; Mitchell etal. 2010; Topisirovic etal. 2011). Nevertheless, the exact
molecular details of mRNA attachment to the 40S subunit and the fate of eIF4F
following attachment are unknown. Experiments to determine the binding sites of
initiation factors on the 43S/mRNA complex have used various approaches, includ-
ing electron microscopy, X-ray crystallography, and chemical probing (reviewed in
Fraser and Doudna 2007; Voigts-Hoffmann etal. 2012). Using a nuclease protec-
tion assay, early work discovered that initiation factors bound to the scanning 40S
subunit protect a 30 nucleotide region of mRNA upstream of the AUG initiation
codon (Kozak and Shatkin 1978). This was more recently confirmed using a cross-
linking assay, which found eIF3 to crosslink to the mRNA roughly 20 nucleotides
upstream of the AUG codon that is placed in the P-site of the 40S subunit (Pisarev
etal. 2008). A higher resolution cryo-electron microscopy structure of the 43S
complex was recently published (Hashem etal. 2013). This structure places a sig-
nificant mass of eIF3 at the mRNA exit channel, where the 5 end of the mRNA
leaves the ribosome. This structure also reveals density for the eIF2-TC on the sub-
unit interface in a similar region to that identified in a recently published chemical
probing study (Shin etal. 2011). The exact position of eIF4F in the 43S-mRNA
complex is less clear (Marintchev etal. 2009; Siridechadilok etal. 2005; Yu etal.
2011). An important question that is yet to be resolved is whether the eIF4A heli-
case activity of eIF4F is positioned towards the mRNA entry channel or the mRNA
exit channel (Marintchev etal. 2009; Siridechadilok etal. 2005). Clearly, further
structural and kinetic data of complete 43S/mRNA structures will be required to
unequivocally determine the location and the order of binding of each factor in this
complex.
At the end of the recruitment stage, the mRNA is stably bound in the decoding
site of the 40S ribosome together with associated initiation factors and the initiator
tRNA. The 40S subunit must now locate the translation initiation codon before the
80S ribosome can be formed and elongation to commence.
2.2.4 5 to 3 Scanning
Once the mRNA is stabilized in the 40S subunit-decoding site, the 40S subunit mi-
grates along the mRNA in the 3 direction by an ATP-dependent mechanism gener-
ally referred to as scanning. During scanning, the anticodon of the initiator tRNA is
thought to sample possible base-pairing with each codon of the mRNA as it passes
through the P-site (Cigan etal. 1988). Only single-stranded mRNA can be bound in
the decoding site, so any secondary structure must be unwound as the 40S subunit
searches for an initiation codon. Two general models exist to explain how the scan-
ning 40S ribosome unwinds secondary structure. In the ratchet model, eIF4B is
bound to the mRNA upstream of the ribosome to prevent backwards motion (Spirin
2009). The message is then pulled through the ribosome and secondary structure
is unwound as it is threaded through the narrow mRNA entry channel (Aitken and
Lorsch 2012; Spirin 2009). In another model, eIF4F unwinds mRNA secondary
structure before it enters the decoding site (Aitken and Lorsch 2012; Jackson etal.
2010; Marintchev etal. 2009). Interestingly, an additional helicase protein, DEAH
box 29 (DHX29), has recently been identified as a 40S subunit associated factor
which assists in translation of highly structured mRNAs (Parsyan etal. 2009; Pisa-
reva etal. 2008) (see Chap.5). The exact role of DHX29 is not yet well understood.
This helicase protein binds near the mRNA entry channel side of the ribosome,
inferring that it helps to unwind mRNA secondary structure before it enters the
decoding site (Hashem etal. 2013). It has also been proposed that DHX29 can
promote modest structural rearrangements in the 40S ribosome, which allow it to
overcome secondary structures in the mRNA (Parsyan etal. 2011; Pisareva etal.
2008). Surprisingly, this helicase protein appears to be present in cells at a substoi-
chiometric amount compared with initiating 40S subunits (Pisareva etal. 2008).
This raises a question as to how scanning 40S subunits are selected by DHX29 to
enable scanning through mRNAs with high amounts of secondary structure. From
these examples it also becomes clear that some mRNAs, such as those containing
highly structured 5 UTRs, may be more dependent on initiation factors such as
eIF4A, and require an additional subset of factors, which are not required for global
translation. This theme will be further explored in Chap.5.
While the scanning model is the most generally accepted mechanism for ribosom-
al movement during initiation, alternative models for other types of mRNAs do exist.
These include the shunting model and initiation at internal ribosome entry sites
(IRESs). In the shunting model, the mRNA is still recruited to the ribosome near the
cap but instead of a linear migration the ribosome shunts downstream, thus bypassing
large portions of the 5 UTR and potentially allowing initiation at downstream co-
dons (Spirin 2009). IRES-driven initiation is a cap-independent mechanism whereby
a subset of translation initiation factors (or in some cases, no factors at all) recruits the
mRNA to the 40S ribosome not via an attachment to the cap but by using an internal
region of the mRNA. Both of these alternatives have been well established in viral
mRNAs, but only a few confirmed examples appear to exist in mammals (Jackson
2013). Examples of IRES translation of eukaryotic mRNAs are presented in the chap-
ter related to the role of translation in regulating apoptosis in cancer (see Chap.19).
Regardless of the mechanism by which the 40S subunit selects the initiation co-
don, it appears that the cooperative binding of eIF1A and eIF1 to the 40S subunit
interface stabilizes an open conformation of the mRNA-binding channel. This is
believed to be a critical requirement for mRNA recruitment and mRNA movement
within the decoding site (Maag etal. 2005; Passmore etal. 2007; Pestova etal. 1998).
In the absence of eIF1, the 40S ribosome latch, a connection between the head and
body, closes in a conformation predicted to impede scanning by clamping the mRNA
into the decoding site (Frank etal. 1995; Passmore etal. 2007; Schluenzen etal.
2000). Consistent with these observations, 43S PIC containing eIF1, eIF1A, eIF2-
TC and eIF3 can recruit and recognize the initiation codon in the absence of eIF4F
and ATP provided the 5 UTR is completely unstructured (Pestova and Kolupaeva
2002). However, the cap structure appears to prevent this type of initiation from
occurring by imposing a strong requirement for the eIF4F complex (Mitchell etal.
2010). In either case, it is clear that there is a strong requirement for eIF1 for produc-
tive scanning, even in the presence of the eIF4F complex (Pestova etal. 1998).
2.2.5Initiation Codon Selection
The context, or sequence, directly surrounding a potential initiation site is important

for defining the likelihood of successful initiation at that start codon. The Kozak
sequence is the consensus sequence found to show optimal initiation at a start co-
don in mammals. It is defined as GCC(A/G)CCAUGG, where the A of the AUG is
known as the +1 position, the C preceding it is known as the 1 position. The bases
at the 3 and +4 positions are the most important, and are indicated in bold (Kozak
1986, 1987). The initiation codon is generally thought to be the first AUG codon in
an mRNA, but it has been recently shown that these types of mRNAs may represent
only 25% of the messages in mammalian cells (Ingolia etal. 2011). All other
messages appear to contain at least one additional potential initiation codon, either
upstream or downstream of the main open reading frame (ORF). Several factors
including the context and position of a potential translation start site can regulate
translation efficiency of an mRNA, and these regulatory mechanisms are explored
in more depth in Chap.3. Although the Kozak sequence is not as important in defin-
ing initiation codons in yeast (Cigan etal. 1988), genetic experiments in yeast have
provided data to show important roles of eIF1, eIF1A, eIF2 and eIF5 in regulating
the fidelity of start site recognition (reviewed in Lorsch and Dever 2010).
eIF1 serves as a key regulator of start codon recognition fidelity by preventing
initiation at non-AUG codons and AUG codons in poor context (Pestova etal.
1998; Pestova and Kolupaeva 2002; Pisarev etal. 2006; Yoon and Donahue 1992).
This function is critical since the selection of an incorrect start codon would likely
result in the expression of an aberrant protein product. In the 43S PIC, eIF1 binds
close to the P-site and partially occludes it with its N-terminal tail (Dallas and Nol-
ler 2001; Lomakin etal. 2003; McCutcheon etal. 1999; Rabl etal. 2011). This pre-
vents the initiator tRNA from fully entering the P-site, which may be important for
enabling 40S subunits movement during scanning (Rabl etal. 2011). Consistent
with this, the initiator tRNA is bound in a partially stabilized conformation, which
may help the tRNA anticodon stem to sample codons during scanning without
making a stable base-pairing interaction prior to start codon recognition (Lorsch
and Dever 2010).
Upon base-pairing between the mRNA and the initiator tRNA during start codon
recognition, eIF1 is displaced from its binding site on the 40S subunit (Cheung
etal. 2007; Lomakin etal. 2003; Maag etal. 2005; Unbehaun etal. 2004). This
key event results in the closing of the latch and mRNA-binding channel (Passmore
etal. 2007), thereby helping to stall scanning by locking the mRNA in the decod-
ing site. This conformation change also alters the position of eIF1A and eIF5 on
the 40S subunit (Maag etal. 2006; Nanda etal. 2013). As the GTPase-activating
protein (GAP) for ribosome-bound eIF2-GTP, eIF5 ultimately functions to commit
the 43S/mRNA complex to stop scanning at the selected start codon (Algire etal.
2005; Das etal. 2001; Nanda etal. 2009; Paulin etal. 2001). Specifically, it appears
that phosphate release from the complex following GTP hydrolysis is the irrevers-
ible committed step controlled by initiation codon recognition and eIF1 dissociation
(Algire etal. 2005). To this end, eIF1 appears to be the master regulator of start
codon recognition, as it also impedes premature GTPase activity and phosphate
release by eIF2 (Algire etal. 2005; Unbehaun etal. 2004). This repression is only
removed upon eIF1 dissociation following start codon recognition. Following GTP
hydrolysis, GDP-bound eIF2 has a lower affinity for the initiator tRNA, allowing
its partial release from the 43S/mRNA complex (Kapp and Lorsch 2004; Pisarev
etal. 2006). Upon dissociation, eIF2-GTP is regenerated by the guanine nucleotide
exchange factor (GEF) eIF2B followed by recruitment of a new initiator tRNA,
thus making the eIF2-TC available for subsequent rounds of translation initiation
(Proud 2005). Due to its pivotal role during initiator tRNA recruitment, eIF2 is also
one of the major targets for translation inhibition during cellular stress, as will be
described in Chap.9.
While this complicated pathway is presented in an organized, linear fashion, the

exact order of events in vivo is unclear, besides the eIF1 switch allowing phosphate
release. It is possible that one or more of these events occur simultaneously, or in a
slightly different order than the one presented here. Determining the precise kinetics
and order of binding for the 43S PIC will be an important goal of future work and
will be necessary to fully understand the underlying scanning mechanism.
2.2.660S Ribosome Binding and 80S Ribosome Formation
Before elongation can continue, the 40S ribosome must recruit a 60S ribosome
to form an elongation-competent 80S ribosome. The 60S ribosome is bound by
another factor, eIF6, which is involved in both ribosome biogenesis and prevents
premature association with the 40S subunit (reviewed in Brina etal. 2011; Miluzio
etal. 2009 and Chap.11). Following start codon recognition, any remaining eIF5
and eIF2-GDP complexes must fully dissociate from the 40S ribosome interface
prior to 60S binding. This process is mediated by the ribosome-dependent GTPase
eIF5B, which binds to the intersubunit cleft of the 80S ribosome in a manner analo-
gous to IF2 in bacteria (Allen etal. 2005; Pestova etal. 2000; Simonetti etal. 2008;
Unbehaun etal. 2004; Unbehaun etal. 2007). The C-terminal tail of eIF1A, which
was previously bound in the P-site, is displaced upon start codon recognition to en-
able its interaction with eIF5B (Nanda etal. 2013). This is an important step in pro-
moting subunit joining and eIF5B-GTP hydrolysis (Acker etal. 2006; Marintchev
etal. 2003; Olsen etal. 2003). Hydrolysis results in a reduced affinity for eIF5B,
allowing it to dissociate from the newly formed 80S ribosome (Shin etal. 2002).
After participating in almost every facet of initiation including mRNA recruitment,
start codon recognition, and 80S ribosome formation, eIF1A is the last factor to dis-
sociate from the ribosome interface (Acker etal. 2009). Factors associated mainly
with the solvent side of the ribosome, such as eIF3, may remain bound following
initiation to promote reinitiation, as described in the following sections.
2.3Translation Elongation and Termination
Translation initiation culminates in the formation of an 80S ribosome with the

initiator tRNA positioned in the P-site and base paired to the initiation codon of the
mRNA. The next aminoacyl-tRNA (aa-tRNA) to be incorporated is determined by
the codon in the newly vacated A-site. Unlike translation initiation and termina-
tion, elongation is highly conserved between eukaryotes and bacteria. This is due
to the fact that decoding by the small ribosomal subunit and the peptidyl transfer-
ase center of the large ribosomal subunit are conserved across all Kingdoms of Life
(Rodnina and Wintermeyer 2009; Voorhees and Ramakrishnan 2013; Wilson and
Doudna Cate 2012). Consequently, many of the mechanistic details of elongation
have been uncovered in bacteria and appear to be largely consistent for eukaryotic
ribosomes.
2.3.1Translation Elongation
Each round of elongation consists of three stages: binding the correct aa-tRNA to
the mRNA codon in the A-site, peptide bond formation, and translocation of the
tRNAs and mRNA by one codon (Dever and Green 2012) (Fig.2.3). GTP-bound
eukaryotic elongation factor (eEF) 1A (eEF1A) binds and delivers aa-tRNA to the
A-site of the 80S ribosome where it forms a meta-stable conformation until codon
recognition through codon:anticodon base-pairing occurs (Dever and Green 2012;
Voorhees and Ramakrishnan 2013). The conformational change that occurs upon
codon recognition activates GTP hydrolysis by coordinating eEF1A and 60S ri-
bosome interactions, as shown by structures of the bacterial ortholog EF-Tu and
the prokaryotic 70S ribosome (Schmeing etal. 2009; Voorhees etal. 2010). GTP
hydrolysis is followed by phosphate and eEF1A/GDP release from the ribosome
(Schuette etal. 2009).
Once the aa-tRNA is stabilized in the A-site, peptide bond formation is catalyzed
by the highly conserved peptidyl transferase center in the 60S ribosome. Peptide
bond formation triggers spontaneous ratcheting of the ribosomal subunits (Zhang
etal. 2009). The reaction involves the shift of the peptidyl portion of the P-site
tRNA to the a-amino group of the aa-tRNA in the A-site. This results in partial
translocation of the tRNAs into hybrid P/E- and A/P-sites as the acceptor stems
alone translocate in the large ribosomal subunit. Full translocation is catalyzed by
GTP-bound eEF2 in eukaryotes and prokaryotic elongation factor G (EF-G) in bac-
teria. EF-G interacts with the mRNA, the P-site tRNA, and the decoding center of
the 30S ribosome, implying a role in preventing tRNAs from returning to the P- and
A-sites from the hybrid state (Gao etal. 2009). GTP hydrolysis and phosphate re-
lease result in conformational changes in eEF2 that induce an open conformation
of the ribosome, allowing for full translocation of the tRNAs to occur, followed by
factor dissociation (Chen etal. 2012).
eEF1B, which serves as GEF for eEF1A, accelerates GDP dissociation and re-
generation of the eEF1A/GTP/aa-tRNA ternary complex for further rounds of elon-
gation. Another factor, eIF5A, was initially attributed a role of an initiation factor
for stimulating formation of the first peptide bond by methionine (Kemper etal.
1976). However, recent work has also shown possible interactions of eIF5A with
eEF2 and its role in elongation (Dias etal. 2012; Gregio etal. 2009; Li etal. 2010;
Saini etal. 2009). Like its prokaryotic elongation factor P (EF-P) ortholog in bac-
teria, eIF5A promotes translation of proteins with polyproline motifs, or stretches
of proline residues that may be conformationally difficult to translate (Doerfel etal.
2013; Gutierrez etal. 2013). Interestingly, eIF5A contains a unique posttransla-
tional modification of a lysine residue called hypusine, which is essential for its
function (Park etal. 2010). See Chap.10 for more information on eIF5A.
Following translocation the A-site becomes vacant of tRNA and now contains
the next codon to be decoded with an appropriate aa-tRNA. The P-site contains the
peptidyl-tRNA and the E-site contains the newly deacylated tRNA that is ready to
be released. This process continues throughout the length of the coding sequence of
the mRNA, incorporating amino acids to the growing polypeptide chain at an aver-
age rate of approximately six amino acids per second (Bostrom etal. 1986; Ingolia
etal. 2011). Elongation in vivo is not an entirely continuous process; it can be inter-
rupted by ribosomal pausing, possibly to allow time for co-translational chaperone
binding and the proper folding of the nascent chain, and commonly occurs upon
reaching a termination codon in the A-site (Ingolia etal. 2011). Interestingly, tRNA
recruitment even for rare codons appears not to be rate-limiting, whereas tRNA
identity and the nascent peptide sequence can affect rates of elongation (Ingolia
etal. 2011).
2.3.2Translation Termination
When one of three stop codons (UAA, UGA, UAG) appears in the A-site, eukary-
otic release factor 1 (eRF1) and eRF3-GTP bind as a ternary complex (Frolova etal.
1998; Ito etal. 1998; Mitkevich etal. 2006; Pisareva etal. 2006) to cooperatively
terminate elongation in a GTP-dependent manner (Alkalaeva etal. 2006; Stans-
field etal. 1995; Zhouravleva etal. 1995). Upon ribosome binding, eRF1 acts as a
tRNA mimic that must be able to specifically recognize any of the three possible
stop codons in the A-site with its N-terminal domain (Song etal. 2000). eRF3 then
hydrolyzes GTP (Frolova etal. 1996), triggering its release and allowing full ac-
commodation of the eRF1 middle domain into the peptidyl transferase center to
promote peptide hydrolysis and release (Song etal. 2000). For a detailed review
of termination mechanisms and structures see (Jackson etal. 2012). The resulting
post-termination complex consists of the 80S ribosome, mRNA, deacylated tRNA
in the P-site, and eRF1 in the A-site. Ribosome recycling ensures that these compo-
nents are released to return to the translating pool for subsequent rounds of protein
synthesis.
2.4Ribosome Recycling and Reinitiation
Post-termination, 80S ribosomes must be separated into their component 40S and
60S subunits in order to be reused in further translation cycles. This process is
known as ribosome recycling and was largely unstudied in eukaryotic systems until
recently. Following termination, the 80S ribosome dissociates, and the 60S subunit
must be prevented from binding back to the 40S subunit before the full complement
of initiation factors can bind and prepare the 40S subunit for a new round of transla-
tion initiation. Alternatively, if a second initiation codon exists within a reasonable
distance of the stop codon, a post-termination ribosome may actually remain bound
to the same mRNA and reinitiate at the downstream AUG start codon without fully
recycling.
2.4.1Ribosome Recycling
Until recently, no eukaryotic ribosome recycling factor analogous to ribosome re-

cycling factor in bacteria had been identified. A major breakthrough in the field
occurred when a eukaryotic ribosome recycling factor was identified as the ATP-
binding cassette subfamily E member 1 (ABCE1) (Dever and Green 2012; Jackson
etal. 2012). This protein can separate 80S ribosomes in an ATP-dependent manner
to regenerate free ribosomal subunits and factors for subsequent rounds of transla-
tion (Pisarev etal. 2010; Shoemaker and Green 2011). As such, this protein appears
to be the functional homolog of the ribosome release factor in bacteria, which func-
tions to destabilize intersubunit bridging interactions (Gao etal. 2005). Following
subunit separation, eIF1 dissociates the tRNA from the P-site of the 40S ribosome,
and eIF1A likely helps to dissociate eRF1 (Jackson etal. 2012). The eIF3j subunit
of eIF3 has also been implicated in helping to promote mRNA dissociation since
it negatively regulates mRNA binding into the mRNA entry channel (Fraser etal.
2007; Pisarev etal. 2007). At this point, eIF3, eIF1 and eIF1A likely remain bound
to the 40S subunit interface to prevent premature association of the 60S subunit and
to prepare for the next round of translation initiation. In yeast, ABCE1 has been
shown to interact with eIF2, eIF3 and eIF5 (Chen etal. 2006; Dong etal. 2004)
to promote 43S PIC formation, and its depletion inhibits translation (Dong etal.
2004). It is currently unclear whether these interactions are conserved in mammals.
ABCE1 may also cooperate with the eRF1 and eRF3 paralogs, Pelota and Hbs1, to
dissociate stalled elongation complexes formed on mRNAs lacking a stop codon
(the non-stop decay pathway) (Pisareva etal. 2011).
2.4.2Reinitiation
Approximately 25% of mRNAs are canonical transcripts that only contain a single
initiation codon (Ingolia etal. 2011). Therefore, most transcripts have more than
one initiation codon from which a ribosome must select to begin translation. This
process is guided by rules of initiation codon selection which take into account se-
quence context and location of the start codon to determine the probability of initia-
tion at a given start site (see above and Chap.3). The expression of many genes is
regulated by expression of upstream open reading frames (uORFs), which are short
ORFs upstream of the main coding sequence. These function to divert ribosomes
from expressing the physiologically relevant downstream product, often lowering
the translation of the main ORF. Reinitiation is the mechanism by which ribosomes
translate a uORF, but only partially recycle the ribosomes and translation factors
after termination. Instead, the 60S ribosomal subunit and termination factors dis-
sociate, leaving an mRNA-bound 40S subunit able to recruit initiation factors and
resume scanning. Retention of eIF3 and eIF4G after translation of a short uORF
may facilitate recruitment of the remaining initiation factors (Poyry etal. 2004).
This corresponds well with the finding that it is the time taken to translate a uORF
that regulates the likelihood of reinitiation (Kozak 2001). Recognition of a second
start codon is regulated by the need to recruit a new eIF2-TC. If eIF2-TC has been
depleted to reduce translation, reinitiating scanning ribosomes will have to wait and
migrate longer before recruiting a new eIF2-TC, making placement of initiation co-
dons critical for translation regulation. See Chap.3 for more information on uORFs
and regulation of eIF2.
2.5Conclusions and Perspectives
The past few years have seen a significant increase in our mechanistic understand-
ing of mRNA translation in eukaryotic cells. Nevertheless, the need for a greater
understanding of the kinetic frameworks for each stage in mRNA translation is
apparent. Emerging techniques, such as single molecule approaches, will likely be
important in achieving this goal (Petrov etal. 2012). Furthermore, it is essential
to relate future thermodynamic and kinetic frameworks to the translation process
in cells so that a complete picture of translation is obtained in normal and disease
states. Continued advances in generating structural models of different translation
intermediates will undoubtedly lead to a greater understanding of eukaryotic trans-
lation at the molecular level. To this end, recent high-resolution structures of the
eukaryotic ribosome have begun to provide much needed structural information
with which to base future genetic and biochemical experiments. Ultimately, one
can hope that a better understanding of the translation mechanism will provide new
insight into potential targets for both therapeutics and diagnostics of disease states.
Acknowledgments Work in the Fraser lab is supported by grant R01GM092927 from the National
Institute of General Medical Sciences. We would like to thank Professor John Hershey for many
helpful discussions.
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Chapter 3
Diverse Mechanisms of Translation Regulation
and Their Role in Cancer
Contents
3.1Introduction 41
3.2mRNA Sequence and Structure Elements that Regulate Translation Efficiency 44
3.2.1 5 7-Methylguanosine Cap and 3 Poly(A) Tail 44
3.2.2 5 UTR Length and Structure 45
3.2.3 Start Codon Selection and Context of Initiation Site 46
3.2.4 Regulation of Translation by uORF 46
3.2.5 5 Terminal Oligopyrimidine Motifs 48
3.2.6 Translation Initiation via IRES 49
3.3 Regulating Translation Through mRNA Availability 51
3.3.1 Nonsense-Mediated Decay 51
3.3.2 Stress Granules and Processing Bodies 51
3.3.3 Translation Control by RNA Interference 52
3.4Regulation of Translation Factor Activity and Availability 53
3.4.1 Competitive Inhibition of eIF4E by 4E-BPs 53
3.4.2Regulation of the eIF4A Helicase Through Activating Factors and Inhibitors 55
3.4.3mRNA Recruitment to the 40S Ribosomal Subunit via eIF4G and eIF3 56
3.4.4 tRNA Recruitment to the 40S Ribosome Subunit 58
3.4.5 Translational Control of Elongation 58
3.5Regulating Translation via Changes in Ribosome Number and Activity 59
3.5.1 Ribosome Biogenesis 59
3.5.2 40S Ribosome Activity and Signaling 60
3.6 Conclusions and Perspectives 61
References 62
AbstractTranslation is a highly regulated multistep process that involves the

recruitment of an mRNA to a ribosome and its translation by aminoacyl-tRNAs
(aa-tRNA) into a polypeptide. Regulation can occur at any of the four stages of
translation (initiation, elongation, termination, and ribosome recycling), and can be
directed at the level of mRNA, translation factors, or the ribosome. In this chapter,
C.S.Fraser() N.Villa
Section of Molecular and Cellular Biology, University of California, Davis, CA, USA
e-mail: csfraser@ucdavis.edu
a Translation Initiation
AUG UGA AAAAA
AUG UGA AAAAA eIF4G eIF4E eIF4A eIF4B PABP
eIF4F Cap Binding Complex

GTP
eIF2-TC
GTP
Ribosome Recruitment ATP
and
ADP
E P A eIF5
GDP
eIF1 eIF1A
AUG UGA AAAAA E P A
E P A
eIF4F?
c Termination and Recycling
eIF5B GTP
ADP
UGA
A
AUG
A
A
A
E P
A
A
GTP eIF5B GTP
AUG UGA AAAAA ABCE1
E P A ATP P
GD
eIF5B GDP
eIF1A
AUG UGA AAAAA UGA

A
AUG
A
A
E P A
A
E P
A
b Elongation Cycle eEF1A-TC

GT
P
GTP
eRF1
GTP
GDP GTP
eEF1A-GDP eEF2 GTP GDP
AUG UGA AAAAA AUG UGA AAAAA AUG UGA AAAAA
E P A E P A E P A
Peptide Bond Formation
Aminoacyl-tRNA Recruitment (Hybrid State)
Fig. 3.1 Overview of the mechanism of translation. a During translation initiation, eukaryotic
mRNAs are bound by the eIF4F cap-binding complex (eIF4E, eIF4G, eIF4A), eIF4B, and PABP.
Together, these factors bind the mRNA cap and unwind secondary structure in the 5 UTR. The
43S PIC, which consists of the 40S ribosome, eIF1, eIF1A, eIF2-TC, eIF3 and eIF5, prepares the
ribosome for mRNA recruitment through eIF4G/eIF3 binding. The newly formed 43S PIC then
scans until the start codon is recognized, the 60S ribosome binds, and the initiation factors are
released. b During elongation, aa-tRNAs are recruited to the 80S ribosome by eEF1A as a ternary
complex with GTP. Peptide bond formation induces a hybrid state conformation in the ribosome.
3 Diverse Mechanisms of Translation Regulation and Their Role in Cancer 41
we will review the diverse approaches the cell uses to regulate protein synthesis.
Translation efficiency can be affected by mRNA sequence and structure elements,
mRNA availability to the translation machinery, regulation of translation factor
activity and availability, and regulation through changes in ribosome number and
activity. We will also discuss how the translation machinery can be manipulated by
cancer cells to promote tumorigenesis.
3.1Introduction
The mechanism of translation can be divided into 4 distinct phases: initiation,

elongation, termination, and ribosome recycling (see Chap.2). During transla-
tion initiation, the mRNA is recognized and bound by the eIF4F cap-binding com-
plex in preparation for recruitment to the 40S ribosome. This complex consists of
eIF4E, eIF4A and eIF4G. The 40S ribosome is prepared for mRNA recruitment
by several factors including eIF1, eIF1A, eIF2/Met-tRNAi, eIF3 and eIF5, which
together form the 43S PIC. The message is directed to the ribosome through the
interaction of eIF4G and eIF3. According to the most widely accepted model, the
ribosome then scans along the 5 UTR of the mRNA in the 5 to 3 direction un-
til the initiation codon is located. 60S subunit-binding is promoted by eIF5B to
form the e longation-competent 80S ribosome, and the initiation factors are released
(Fig. 3.1a). During elongation, eukaryotic elongation factor eEF1A directs aa-
tRNAs in a ternary complex with GTP (eEF1A-TC) to the 80S ribosome to translate
the message (Fig.3.1b). When a stop codon is reached, eukaryotic release factors
eRF1 and eRF3 catalyze the release of the newly formed polypeptide, and ABCE1
promotes release of the 40S and 60S ribosomal subunits to make them available for
further rounds of translation (Fig.3.1c).
In Chap.2, we focused on providing an overview of the detailed mechanism
for each phase of translation. We discussed all of the canonical factors involved
in converting the information contained within an mRNA into a functional protein
(see Chap.2, Table2.1). In this chapter, we will outline how translation factors and
elements of the mRNA can affect translation efficiency of both specific messages
and global translation rates. We will also discuss how modest changes in either the
mRNA or translation factors can result in translation dysregulation and promote
malignancy. See Table3.1 for the overview of the translation regulatory mecha-
nisms discussed below.
Fig. 3.1 (continued) eEF2 promotes translocation of tRNAs from the A- to P-site and P- to E-site
of the ribosome. The elongation cycle continues until a stop codon is reached. c Upon stop codon
recognition, eRF3 induces peptide hydrolysis to release the newly formed protein, and ABCE1
promotes ribosome recycling by releasing the 40S and 60S ribosomal subunits for further rounds
of translation.
Table 3.1 Summary of targets of translation regulation. Translation can be regulated by mRNA-specific modes, such as sequence and structure elements within
42
the mRNA and by regulating mRNA availability to the translation machinery. Alternatively, the cell can modulate translation factor and ribosome activity and
availability to control translation rates.
Mode of regulation Target Description Effect References
mRNA sequence 5 cap and Posttranscriptional modification of Cooperatively increase translation effi- Gallie (1991), Haghighat and Sonen-
and structure poly(A) tail mRNA adds a 5 7-methylguano- ciency and are bound by the eIF4E cap berg (1997), Imataka etal. (1998)
elements sine cap and 3 poly(A) tail binding protein and PABP and Safaee etal. (2012)
5 UTR length Length and amount of secondary Generally decrease translation efficiency Koromilas etal. (1992), Kozak,
and second- structure of mRNA between the due to difficulty in unwinding and ribo- (1986a, 1989) and Svitkin etal.
ary structure 5 cap and the initiation codon some recruitment (2001)
Context of Kozak consensus sequence: Defines the optimal translation initiation Kozak (1986a, b, 1987b)
translation GCC(A/G)CCAUGG site, and deviation particularly in the
initiation site 3 and +4 positions decrease transla-
tion efficiency
Upstream Short ORFs of about 530 amino Generally decrease translation efficiency Calvo etal. (2009), Kozak (2001) and
open read- acids upstream of the main ORF by diverting ribosomes from the initia- Somers etal. (2013)
ing frames tion codon of the main ORF
(uORFs)
Internal Complex secondary and tertiary Recruit translation factors and/or ribo- Holcik (2004), Jackson (2013) and
ribosome structure in the 5 UTR capable somes to the mRNA independently of Kozak (2005b)
entry sites of promoting cap-independent the 5 cap to promote translation
(IRESes) translation
5 TOP motifs 612 pyrimidines in the 5 UTR Regulate translation of these messages in Damgaard and Lykke-Andersen (2011)
of all ribosomal proteins and reponse to cellular signalling pathways. and Hamilton etal. (2006)
several translation factors The exact mechansim is unknown
mRNA availability Stress granules mRNP particles containing mRNAs Sequester specific mRNAs to down- Anderson and Kedersha (2009),
stalled at translation initiation regulate translation. Messages may be Decker and Parker (2012) and
released when mRNA translation is Stoecklin and Kedersha (2013)
needed again
P bodies mRNP particles containing mRNAs Sequester specific mRNAs to downregu- Anderson and Kedersha (2009),
and mRNA decay enzymes late translation, eventually through Decker and Parker (2012), Eulalio
and final destination of many mRNA decay etal. (2007) and Stoecklin and
miRNA targets Kedersha (2013)
Mode of regulation Target Description Effect References
Translation factor eIF4E 4E-BP family of inhibitors can Globally downregulate cap-dependent Mader etal. (1995), Marcotrigiano
activity and reversibly sequester eIF4E from translation etal. (1999) and Martineau etal.
availability eIF4G binding (2013)
eIF4A eIF4A activity is modulated by eIF4A activity is upregulated by interac- Feoktistova etal. (2013), Lankat- Butt-
interacting proteins tions with eIF4B, eIF4G and eIF4E. gereit and Goke (2009), Loh etal.
PDCD4 sequesters eIF4A from eIF4G (2009), Marintchev (2013) and
and inhibits ATP and RNA binding to Suzuki etal. (2008)
downregulate translation in apoptosis
eIF4G eIF4G promotes eIF4A activity, Interactions with eIF4A and eIF3 promote Bushell etal. (2000), Clemens
and eIF3-eIF4G binding is translation. Cleavage during apop- etal. (1998), Harris etal. (2006),
upregulated by mTOR. eIF4G tosis downregulates cap-dependent Marintchev (2013), Marissen and
is targeted by proteases during translation but allows IRES-mediated Lloyd (1998) and Morley etal.
apoptosis translation (1998)
eIF2 eIF2 subunit is posphorylated at Sequesters eIF2B and prevents eIF2-GTP Donnelly etal. (2013) and Pavitt and
Ser 51 by GCN2, PERK, PKR regeneration by increasing eIF2eIF2B Ron (2012)
and heme-regulated kinase affinity, globally downregulating
translation
eEF2 and eEF2 is phosphorylated by eEF2K Phosphorylation inhibits eEF2 ribo- Carlberg etal. (1990), Redpath etal.
eEF2K some translocation activity, inhibiting (1996) and Wang etal. (2001b)
translation. eEF2K phosphorylation
by S6K1 inhibits eEF2K and promotes
translation
Ribosome number Ribosome Regulation of rRNA transcription Cell growth and transformation is sup- Henras etal. (2008) and Kressler etal.
3 Diverse Mechanisms of Translation Regulation and Their Role in Cancer
and activity biogenesis and ribosomal protein synthesis ported by upregulation of ribosome (2010)
biogenesis, which in turn increases
translation capacity of the cell
40S ribosome Ribosome activity may be modu- Role and effect of rps6 phosphorylation Jastrzebski etal. (2007), Pende etal.
activity lated by signaling pathways by S6K1 on cell growth and translation (2004), Ruvinsky etal. (2005) and
of TOP mRNAs is controversial Ruvinsky and Meyuhas (2006)
43
guanosine cap
AUG GCCACC AUGG AUG UGA AAAAAAAA
Kozak Sequence
GCCACC AUGG AUG
Fig. 3.2 Anatomy of an mRNA. Eukaryotic mRNAs are posttranscriptionally modified to add a
5 cap and 3 poly(A) tail. The main open reading frame (ORF) is outlined in green. The 5 UTR
is located between the cap and the main ORF, and the 3 UTR is located between the stop codon
and the poly(A) tail. The initiation codon is shown in the optimal context, the Kozak sequence.
Alternative initiation codons and potential ORFs are also shown.
3.2mRNA Sequence and Structure Elements

that Regulate Translation Efficiency
Several features of mammalian mRNAs can regulate how efficiently a transcript

will be translated into protein (Fig.3.2) (Kozak 1991b, 2005a). These features
typically reside outside of the main open reading frame (ORF) and include the 5
7-methylguanosine cap, 3 poly(A) tail, and both primary sequence and second-
ary structure elements located in the 5 and 3 UTRs. Importantly, sequence and
structure elements within the 5 and 3 UTRs of mRNAs often constitute regulatory
elements that are unique to a single mRNA or to a family of mRNAs that are coor-
dinately regulated.
3.2.1 5 7-Methylguanosine Cap and 3 Poly(A) Tail
The mRNA cap and poly(A) tail are important in mRNA stability and act syner-
gistically to promote translation (Gallie 1991). The mRNA cap is bound by eIF4E
while the poly(A) tail is bound by PABP. Both of these factors interact with eIF4G
during translation initiation (Haghighat and Sonenberg 1997; Imataka etal. 1998;
Safaee etal. 2012). It is presumed that these interactions result in circularization
of the mRNA during translation, thus ensuring that only intact mature mRNAs are
recruited to ribosomes. This interaction may also facilitate multiple rounds of trans-
lation by reinitiation following termination on the same mRNA (Wells etal. 1998).
Although this is an attractive model, whether mRNA circularization actually occurs
in vivo has not been well established (Park etal. 2011).
Interestingly, PABP likely promotes translation initiation through multiple path-
ways that are both dependent and independent of poly(A) tail binding (Kahvejian
etal. 2005). The activity and availability of PABP is also known to be both posi-
tively and negatively regulated by the PABP-interacting proteins (PAIPs) PAIP1,
PAIP2A and PAIP2B (Derry etal. 2006). PAIP1 stimulates translation, possibly
through interactions with eIF4A and eIF3 in addition to PABP (Craig etal. 1998;
Martineau etal. 2008). In contrast, the homologs PAIP2A and PAIP2B inhibit trans-
lation by decreasing PABP/poly(A) binding (Berlanga etal. 2006; Khaleghpour
etal. 2001a, b; Lee etal. 2013). The variety of functions exhibited by the PAIPs
demonstrates their importance in the mechanism and regulation of translation, both
independently and as regulators of PABP.
3.2.2 5 UTR Length and Structure
Secondary structures form when complementary regions of the mRNA base-pair

with each other. Because G-C base pairs are more energetically favorable than
A-U base pairs, sequences with a high GC content are more likely to form stable
secondary structures. The amount and stability of secondary structures located in
the 5 UTR of an mRNA typically correlates with a decrease in translation efficien-
cy due to the fact that the 43S PIC can only accommodate a single-stranded mRNA
in its decoding site (Koromilas etal. 1992; Kozak 1986b, 1989, 2005a; Svitkin
etal. 2001). The cumulative stability of secondary structures located in the entire 5
UTR is often quoted as the determining factor in translation efficiency. However,
it should be emphasized that the distribution of secondary structure, or structural
landscape, in the 5 UTR is likely to play a key role in governing the unwinding of
the secondary structure (Babendure etal. 2006; Ozes etal. 2011). For example, a 5
UTR may have an overall low GC content, but a stable secondary structure near the
5 end may occlude the cap and reduce the rate of eIF4F recruitment (Kozak 1989;
Lawson etal. 1986). Alternatively, secondary structures further downstream from
the cap may pose barriers for scanning. Thus, reduced translation efficiency may
be due to a reduced rate of recruitment to the 43S PIC, a reduced rate of scanning,
or both.
Importantly, many mRNAs containing structured 5 UTRs encode proto-onco-
genes such as survivin, cyclin D1, ornithine decarboxylase (ODC), B-cell lympho-
ma-extra large (BCL-XL), vascular endothelial growth factor (VEGF) and v-MYC
avian myelocytomatosis viral oncogene homolog, c-MYC (Koromilas etal. 1992;
Kozak 1991a; Svitkin etal. 2001). These mRNAs are often found to be in low
abundance and have been suggested to compete poorly for the limited amount of
cellular eIF4F for their recruitment to the 43S PIC, and are therefore translated at
lower rates.
To alter translation efficiency of these types of transcripts, cells can utilize alter-
native splice and transcription start sites to create distinct 5 UTR isoforms to regu-
late the presence or absence of secondary structure (Hughes 2006). Any mutation
that may alter the degree or position of mRNA secondary structure in the 5 UTR
could thereby affect cell homeostasis and contribute to cell transformation (David
and Manley 2010; Smith 2008). Alternatively, increasing the availability or activity
of the eIF4F complex could also promote translation of these mRNAs, if these fac-
tors are present in limiting concentrations within the cell.
3.2.3 Start Codon Selection and Context of Initiation Site
In addition to canonical transcripts that contain a single AUG start codon, tran-
scripts may include alternative initiation codons that result in N-terminally extend-
ed or truncated forms of the main ORF (Fig.3.2). Alternatively, initiation from an
out-of-frame start codon could result in no protein or a non-functional polypeptide
being produced. A powerful new method for determining translation initiation sites
in vivo on a genome wide scale is the ribosome profiling method (Ingolia etal.
2012). During translation, the 80S ribosome occupies a ~30 nucleotide stretch of
mRNA, known as a ribosome footprint, which can be sequenced to reveal the loca-
tion and density of ribosomes on a given transcript (Ingolia etal. 2009). Using the
drug harringtonin to stall ribosomes at initiation sites, the Weissman group showed
that canonical initiation may only constitute ~25% of translation initiation events in
mouse embryonic stem cells (Ingolia etal. 2011). Although only ~30% of the total
mRNA pool was analyzed in this study, the implication is that the regulation of start
site selection may be much more extensive than previously appreciated.
One of the ways by which the ribosome determines the start codon to initi-
ate translation from is to analyze the context or a sequence directly surrounding
the potential start codon. The Kozak consensus sequence, GCC(A/G)CCAUGG,
promotes optimal AUG recognition and translation initiation in mammals (Kozak
1986c, 1987b). The A of the AUG is defined as the +1 position, and the C preceding
it is known as the 1 position. The most important bases, at 3 and +4 positions,
are indicated in bold and the start codon is underlined. Deviation from this consen-
sus site can result in lower levels of translation from a given initiation codon, and
thus translation levels can be controlled by varying the context of the start codon
(Kozak 1987a).
Translation initiation from alternative start codons can be used as a mode of
regulation. In one example, the mRNAs of the CCAAT-enhancer-binding proteins
(C/EBP) - and - each contain three in-frame initiation codons which produce
transcription factor isoforms that are N-terminally truncated or extended, depending
on the start codon utilized (Wethmar etal. 2010). An upstream open reading frame
(uORF) helps modulate translation levels of each isoform, responding to cellular
cues to determine the ratio of each to be expressed (see the following section for
discussion of uORFs) (Wethmar etal. 2010). Increased translation of the shorter,
inhibitory isoform of C/EBP, and deviation from the appropriate ratios between
primary and truncated isoforms have been linked to cancer, demonstrating the im-
portance of start codon selection in translation regulation (Wethmar etal. 2010).
3.2.4 Regulation of Translation by uORF
Another important subset of transcripts includes those that contain uORFs, which
are ORFs that encode a short (~530 amino acid) peptide upstream of the main
ORF (Fig.3.3). The technique of ribosome profiling has also revealed that uORFs
a uORF Initiation Codons Main ORF Initiation Codons
AUG Other
AUG Other
CUG GUG
b Main ORF
Translation
Canonical Initiation
AUG UGA AAAAAAAA
Main ORF
uORF Downregulation
80S
CUG UGA AUG UGA AAAAAAAA
uORF Main ORF
Leaky Scanning
43S 43S
uORF Main ORF
Reinitiation
80S
43S
uORF Main ORF
Fig. 3.3 Regulation by uORFs. a Breakdown of translation initiation codons for uORFs as
opposed to main ORFs, as discovered by and adapted from Ingolia etal. (2011). b Diagrams illus-
trate the mechanisms and relative translation efficiency of canonical initiation, downregulation by
uORFs, bypassing of uORFs by leaky scanning, and reinitiation following translation of a uORF.
are much more prevalent in 5 UTRs, and translated more often, than previously
imagined (Ingolia etal. 2011).
The majority of mammalian uORFs are not believed to encode functional pep-
tides, but instead regulate the number of ribosomes that are able to reach the physi-
ologically relevant ORF (Kozak 2001). Given that the uORF initiation codon is
closer to the 5 end of the transcript, the ribosome has an opportunity to initiate
translation at the uORF instead of scanning through it to initiate translation at the
main ORF. Following translation of the uORF, many of the ribosomes dissociate
from the mRNA. Nevertheless, some 40S subunits from terminating ribosomes can
re-enter the scanning phase and continue to migrate to the main ORF of the mRNA.
The number of ribosomes that ultimately reach the main ORF will depend on the ef-
ficiency of a terminating ribosome re-entering scanning. Depending on the mRNA,
uORFs typically reduce protein synthesis of the main ORF by 3080% (Fig.3.3)
(Calvo etal. 2009; Somers etal. 2013). Interestingly, uORFs are found to most
often initiate from non-AUG start codons, such as the near cognate CUG. This is in
contrast to main ORFs, which are overwhelmingly found to initiate translation from
AUG start codons (Fig.3.3a) (Ingolia etal. 2011).
To increase translation of the main ORF, the cell can increase selectivity of
start codon recognition to initiate mainly at AUG codons and thus scan through
the uORF initiation codon in a process known as leaky scanning (Kozak 1986a).
Alternatively, the 80S ribosome may translate the main ORF by reinitiation. In addi-
tion to the efficiency at which a 40S subunit can resume scanning after termination,
a new eIF2-TC (eIF2, GTP, initiator Met-tRNAiMet ) must also be recruited prior to
reaching the main ORF start codon (Fig.3.3b).
In one example, the archetypal activating transcription factor 4 (ATF4) mRNA
contains two uORFs, which only allow the main ORF to be translated efficiently
under conditions of stress when general translation is downregulated and eIF2-TC
concentration is limiting (Hinnebusch 2005; Somers etal. 2013). In this case, the
scanning ribosome will generally initiate and translate the first uORF, and if eIF2-
TC is abundant the ribosome will quickly reinitiate at the second uORF following
termination of the first and will rarely translate the main ORF. However, under
conditions in which eIF2-TC has been depleted to downregulate global translation
(see the following sections for this mechanism), eIF2-TC recruitment to the ribo-
some is less quick, the second uORF is bypassed and downstream translation at
the main ORF is upregulated. The general mechanism of translation regulation by
uORFs for classic mRNAs, such as ATF4, is well understood (Hinnebusch 2005).
However, it remains to be determined if other transcripts containing uORFs follow
a similar mechanism.
It is easy to imagine how mutation or deletion of these uORFs could have delete-
rious effects on cell homeostasis through aberrant translation of specific mRNAs.
In one known example, a single base mutation in cyclin-dependent kinase inhibitor
2A (CDKN2A) mRNA introduces an AUG start codon and uORF in the 5 UTR,
which reduces expression of CDKN2A and is associated with a familial predisposi-
tion for melanoma (Liu etal. 1999). Over five hundred polymorphic uORFs, or
uORFs created or deleted by a single polymorphism, have been documented (Calvo
etal. 2009). It is likely that more examples of altered gene expression as a result of
aberrant uORF translation remain to be discovered, and could be linked to various
disease states including cancer.
3.2.5 5 Terminal Oligopyrimidine Motifs
Besides secondary structures and uORFs, 5 UTRs can also contain regulatory se-
quences. The 5 terminal oligopyrimidine (TOP) motif is a span of 612 pyrimidines
at the beginning of an mRNA. TOP mRNAs are upregulated in response to prop-
roliferative signals, and downregulated in response to nutrient starvation via the
mTOR pathway (see Chapter 15) (Damgaard and Lykke-Andersen 2011; Hamilton
etal. 2006). Translation proteins encoded by 5 TOP mRNAs include all ribosomal
ITAFs?
GTP
eIFs?
AUG UGA AAAAAAAA
IRES
Fig. 3.4 Translation initiation by IRES. Translation may be initiated internally (as opposed to
recruitment at the 5 cap) using a subset of canonical translation initiation factors and ITAFs. Fac-
tor requirements differ between different IRESs.
proteins, translation elongation factors, and three subunits of the translation initia-
tion factor eIF3 (Iadevaia etal. 2008; Yamashita etal. 2008). Although mTOR sig-
naling is known to be involved, the exact regulators of TOP mRNA translation are
currently not well understood, but may include eIF4E (Hsieh etal. 2012; Patursky-
Polischuk etal. 2009; Thoreen etal. 2012). Since upregulation of ribosome biogen-
esis is required to keep up with the demands of protein synthesis in a growing cell,
understanding how regulation of this process is bypassed in transformed cells could
be of therapeutic interest (Ruvinsky and Meyuhas 2006).
3.2.6 Translation Initiation via IRES
It is important to note that not all structured 5 UTRs are inhibitory. Internal ribo-
some entry sites (IRESs) are sequences or structures generally in the 5 UTR of
viral or cellular messages that promote mRNA recruitment to the 40S subunit inde-
pendent of the 5 cap. In fact, IRESs typically function under conditions that do not
favor cap-dependent initiation, thereby enabling lowly abundant IRES-dependent
mRNAs to efficiently compete with capped mRNAs for the translation machinery.
The first IRES was discovered in the poliovirus genome and our understanding
of IRES structure and function has come mainly from poliovirus and other viral
genomes (Balvay etal. 2009; Komar etal. 2012; Pelletier and Sonenberg 1988).
Viral IRESs have been classified according to the subset of factors they require for
initiation compared to cap-dependent translation (Fraser and Doudna 2007; Hellen
and Sarnow 2001; Komar etal. 2012). Additionally, many viral IRESs utilize cel-
lular IRES trans-acting factors (ITAFs) to stimulate translation initiation (Fig.3.4)
(Komar and Hatzoglou 2011). All viral IRESs studied to date have high levels of
secondary and tertiary structure that provides binding sites for canonical (eIFs) and
non-canonical (ITAFs) initiation components. This may have created the expecta-
tion that all IRESs, whether viral or cellular, must be structured to function. In fact,
there is evidence to suggest that unstructured sequences possessing poly(A) regions
in mammalian mRNAs can function in recruiting ribosomes internally (Shirokikh
and Spirin 2008). It is also important to note that because most cellular mRNAs are
posttranscriptionally modified with a 5 cap and poly(A) tail, cellular mRNAs con-
taining IRESs may be translated through both cap-dependent and cap-independent
mechanisms (Nanbru etal. 1997; Stoneley etal. 1998).
While the role of IRESs has been well characterized in regulating viral trans-
lation, their role in cellular translation has been more controversial. Identifying
true cellular IRESs has been difficult, in part because they tend to be much weak-
er than viral IRESs at stimulating translation. Identification of IRES-containing
mRNAs is further complicated by the presence of cryptic promoters or splicing
variants in translation assays, which can be confused for IRES-mediated trans-
lation (Baranick etal. 2008). Recently, the methods for establishing presence
of cellular IRESs have come into question and have inspired critical reviews
and guidelines for thorough validation of targets (Jackson 2013; Kozak 2005b;
Thompson 2012).
Many cellular IRESs are predicted to possess structured 5 UTRs to promote
translation during conditions that are not favorable to global translation, such as cel-
lular stress and mitosis (Qin and Sarnow 2004; Spriggs etal. 2008). Interestingly, a
number of oncogenes are encoded by mRNAs that appear to be less cap-dependent,
and may indeed function through an IRES-mediated initiation mechanism. These
include MYC, cyclin-dependent kinase (CDK) 11, B-cell lymphoma 2 (BCL-2) and
VEGF (Cornelis etal. 2000; Sherrill etal. 2004; Stein etal. 1998; Stoneley etal.
2000b). Translation of these molecules promotes tumorigenesis and antagonizes
cellular efforts at self-destruction prior to cell transformation. MYC has been of
particular interest, as it functions as a transcription factor with additional roles in
protein synthesis and DNA replication and is dysregulated in several types of can-
cer (Cole and Cowling 2008; Dang 2012; Luscher and Vervoorts 2012; Shi etal.
2008). Increased levels of MYC have been shown to directly upregulate global
protein synthesis rates, increase cell size and accelerate cell cycle progression in
the E-MYC mouse model (Barna etal. 2008). It also stimulates transcription of
many components required for ribosome biogenesis and all members of the eIF4F
cap-binding complex, which could contribute to translation dysregulation (Gran-
dori etal. 2005).
Considering that highly structured 5 UTRs and IRES-containing mRNAs
seem to be preferentially translated during the process of transformation (Komar
and Hatzoglou 2011; Spriggs etal. 2010), there is a great deal of interest in
identifying mRNAs translated through this mechanism, as well as methods of
inhibition that may be of therapeutic use (Holcik 2004). Study of cellular IRESs
could be further aided by structural data for the true architecture of these IRES
elements, and it would be interesting to see how actual structures correlate with
the currently presumed models and whether any structural conservation exists.
While it is likely that alternative translation initiation mechanisms do occur un-
der specific cellular conditions, more work will be necessary to gain a better
understanding of the mechanism and regulation of IRES-driven translation of
eukaryotic mRNAs.
3.3 Regulating Translation Through mRNA Availability
The cell can regulate translation of specific messages by controlling mRNA avail-
ability. Here, we will only sample the vast array of options the cell has for altering
the availability of mRNA to the translation machinery and limit our discussion to
fates of mRNA once they have been transcribed and exported from the nucleus. If
an mRNA is sequestered from the translation machinery, it cannot be translated into
protein regardless of its abundance. This process can be reversible, as in localiza-
tion of mRNAs to stress granules, or generally irreversible as in processing body
(P-body) localization and decay of aberrant mRNAs.
3.3.1 Nonsense-Mediated Decay
Nonsense mutations create premature termination codons (PTCs) from sequences

that previously coded for an amino acid and as such can result in production of a
potentially deleterious truncated protein. These mutations can be spontaneous, or
result from errors during splicing of pre-mRNA (McGlincy and Smith 2008). The
pioneer, or initial round of translation acts as a quality control mechanism by identi-
fying and targeting PTC-containing mRNAs for degradation in a process known as
nonsense-mediated decay (NMD) (Kervestin and Jacobson 2012; Peltz etal. 2013;
Schoenberg and Maquat 2012). Essentially, up-frameshift protein 1 (UPF1) and the
protein kinase SMG1 interact with eRF1 and eRF3 at PTCs upstream of an exon
junction complex, where the UPF/eRF complex targets the mRNA for degradation
(Maquat etal. 2010). Many genetic disorders including cystic fibrosis, muscular
dystrophy and thalassemia can be caused by mutations that introduce a PTC in
specific transcripts, leading to reduced protein synthesis (Peltz etal. 2013). Interest-
ingly, NMD has also been shown to degrade a number of wild-type, or nonmutated,
transcripts (Gardner 2010). This is important since NMD appears to be inhibited
when cells are deprived of amino acids or made hypoxic, a condition generally
found during tumorigenesis (Gardner 2010). This may therefore promote the stabil-
ity of specific mRNAs that protect against stress, leading to an increase in avail-
ability of mRNA for enhanced protein synthesis.
3.3.2 Stress Granules and Processing Bodies
In both yeast and mammalian cells, stress granules and P-bodies are cytoplasmic
messenger ribonucleoprotein particles (mRNPs) that are dynamically linked mRNA
and protein aggregates that repress translation of specific mRNAs by prevent-
ing their recruitment to the active ribosome pool (Anderson and Kedersha 2009;
Buchan and Parker 2009; Decker and Parker 2012; Eulalio etal. 2007; Kedersha
etal. 2005; Stoecklin and Kedersha 2013). Conditions that inhibit translation
o ften result in mRNA accumulation in mRNP aggregates and mRNA degrada-

tion (Anderson and Kedersha 2009; Decker and Parker 2012). These structures are
mainly distinguished by their components. P-bodies contain mRNA decay enzymes
(Anderson and Kedersha 2009; Decker and Parker 2012; Lian etal. 2009; Liu etal.
2005; Valencia-Sanchez etal. 2006). Conversely, stress granules typically contain
mRNAs stalled at the initiation stage of translation (Anderson and Kedersha 2009;
Buchan and Parker 2009; Decker and Parker 2012). Both structures are dynamic,
and mRNAs may be released from these aggregates to re-enter the ribosome pool
or be degraded (Kedersha etal. 2005). Not surprisingly, localization of mRNAs to
stress granule has been implicated in cancer through inhibition of specific mRNA
targets (Arimoto etal. 2008; Moeller etal. 2004; Thedieck etal. 2013).
3.3.3 Translation Control by RNA Interference
RNA interference (RNAi) encompasses a series of related mechanisms of gene

regulation mediated by short (~2030 nucleotide) noncoding RNAs and their as-
sociated proteins (Wilson and Doudna 2013). These small, inhibitory RNAs can be
naturally occurring, such as microRNA (miRNA), PIWI-interacting RNA (piRNA)
and some small interfering RNA (siRNA), or synthetically produced, such as most
siRNA and small hairpin RNA (shRNA) (Luteijn and Ketting 2013; Moore etal.
2010; Sontheimer and Carthew 2005; Wilson and Doudna 2013). piRNAs are gen-
erally germ line-specific and are the least well characterized, but may also have
some links to cancer (Luteijn and Ketting 2013; Siddiqi and Matushansky 2012). In
general, siRNA and shRNA describe exogenous sources of small RNAs introduced
into cells through transfection or viral vectors, which are then processed through
mechanisms similar to that of miRNA (Moore etal. 2010; Wilson and D oudna
2013). Here, we will describe what is generally known about the mechanism of
miRNA formation and translation inhibition, but recently published reviews offer
more detailed overviews (Jackson and Standart 2007; Wilson and Doudna 2013).
miRNAs are encoded in the genome and initially transcribed as primary miRNA.
They are then processed by the DROSHA ribonuclease and its associated proteins
into precursor miRNA prior to export from the nucleus. In the cytoplasm, the endori-
bonuclease DICER cleaves the precursor miRNA to form a double-stranded RNA
(dsRNA) 2125 nucleotides in length to prepare it for loading onto an Argonaute
protein. Only one strand of the duplex, the mature miRNA, will remain associated
with Argonaute and acts as a guide for targeting mRNAs for inhibition through
base-pairing (Wilson and Doudna 2013).
Binding sites for miRNAs are typically found in the mRNA 3 UTR, and targeting
results in silencing of the mRNA through inhibition of translation followed by
mRNA degradation. Regulation by miRNAs is highly sequence specific, and dys-
regulation of miRNA function by single nucleotide polymorphisms has been linked
to cancer progression (Ryan etal. 2010). In fact, dysregulation of RNAi pathways
has been linked to several disease states (Lu etal. 2008; Siddiqi and Matushansky
2012). The estimate that roughly half of the mRNAs in the human genome may
be targeted by miRNAs makes this pathway an attractive one to target for cancer
therapy. Although the exact mechanism by which a miRNA can inhibit translation
is not known, it appears that an early stage such as initiation is inhibited prior to
sequestration of the mRNA in stress granules and P-bodies (Djuranovic etal. 2012;
Leung etal. 2006; Liu etal. 2005). Recent data has implicated the DEAD box pro-
tein eIF4AII as an important factor in promoting miRNA silencing (Meijer etal.
2013). In light of the fact that miRNA targeted mRNAs are predicted to have highly
structured 5 UTRs it is tempting to speculate that some aspect of mRNA unwind-
ing may play a role in the inhibition of translation by miRNAs (Meijer etal. 2013).
3.4Regulation of Translation Factor Activity

and Availability
When translation regulation is discussed, the typical mechanisms examined are

those that target the activity or availability of the protein factors that mediate this
process. The vast majority of translation regulation mechanisms target the initiation
phase to avoid wasting energy and cellular resources on production of unnecessary
proteins. Besides initiation, the elongation phase of translation is also regulated, but
there are no clear examples of translation control during the termination or recy-
cling phases. There are many examples for dysregulation of translation factor activ-
ity or availability and links to cancer. However, it is important to note that merely
finding factors that are overexpressed in tumors, while an important observation,
does not imply a direct role in cell transformation as the factor concentration may be
increased as a result of transformation rather than being causative. In other words,
dysregulation of translation factor expression or activity in cancer may be related to
the initiation of cancer or be a reflection of an overall tumorigenic state or both. In
this chapter, we will discuss the main strategies the cell uses to regulate translation
through modulation of translation factor activity and availability.
3.4.1 Competitive Inhibition of eIF4E by 4E-BPs
During times of stress, cells may preserve energy and cellular resources by glob-
ally reducing translation rates. One method of doing so is by disrupting the eIF4G/
eIF4E interaction (Fig.3.5a). A family of regulatory eIF4E-binding proteins, the
4E-BPs, sequesters eIF4E by binding competitively to the same site utilized by
eIF4G (Mader etal. 1995; Marcotrigiano etal. 1999; Martineau etal. 2013). This
effectively eliminates cap-dependent translation in a reversible manner by prevent-
ing eIF4F cap-binding complex formation, thereby disrupting the interactions that
bridge the mRNA and the ribosome (Figs.3.1a and 3.5a). Under more favorable
growth conditions, the 4E-BPs are phosphorylated by mTOR, which inhibits
AA
AA
A
A
GDP GTP
eEF1A-GDP eEF2 GTP GDP
AUG UGA AUG UGA

P
AA
AA
eEF2K
AA
E P E P
AA
A A
A
Peptide Bond Formation GTP

(Hybrid State)
Fig. 3.5 Regulation of translation factor activity and availability. a eIF4E directly binds the mRNA
cap and helps recruit the 43S complex to the mRNA through its interaction with eIF4G in the cap-
binding complex. The 4E-BPs, a family of regulatory factors, competitively bind and sequester
eIF4E to prevent eIF4G binding and mRNA recruitment and translation. eIF4E is released upon
4E-BP phosphorylation by mTOR, and translation is upregulated. b Following initiation codon
recognition, eIF2-GDP is released from the ribosome and must be recharged with GTP by eIF2B
in order to participate in further rounds of translation. The subunit of eIF2 is phosphorylated by
any of four different kinases to sequester eIF2B, prevent regeneration of eIF2-GTP and globally
reduce rates of protein synthesis. c During elongation, eEF2 promotes translocation of tRNAs fol-
lowing peptide bond formation from the A- to P-sites and P- to E- sites of the ribosome. Elongation
is inhibited by eEF2K upon eEF2 phosphorylation.
eIF4E/4E-BP binding and promotes cap-dependent translation (as discussed in

Chaps.4 and 15) (Fig.3.5a).
It is generally accepted that eIF4E is the least abundant and therefore the limiting
initiation factor for the entire pathway (Duncan etal. 1987; Hiremath etal. 1985).
eIF4E is overexpressed in many cancers (De Benedetti and Graff 2004) (see Chap.4,
Table4.2 and Part IV), and typically indicates poor clinical outcome (Flowers etal.
2009). eIF4E overexpression has also been shown to cause malignant transforma-
tion of immortalized cells and solid tumor formation in mice (De Benedetti and
Rhoads 1990; Koromilas etal. 1992; Lazaris-Karatzas etal. 1990; Lazaris-Karat-
zas and Sonenberg 1992; Ruggero etal. 2004). In addition, its overexpression in
NIH3T3 cells increases translation of mRNAs with highly structured 5 UTRs in
vivo (Koromilas etal. 1992; Mamane etal. 2007).
Conversely, inhibition or suppression of eIF4E results in decreased translation

of mRNAs implicated in tumorigenesis and metastasis, such as VEGF and cyclin
D1 (Nasr etal. 2013), and suppresses cell growth and migration (Graff etal. 2007;
Zhou etal. 2011). It is possible that 5 UTRs with extensive secondary and tertiary
structures may occlude access to the mRNA cap, and increasing eIF4E concentra-
tion could therefore increase the likelihood of cap-binding and translation of these
messages. Alternatively, a novel role of eIF4E as an activator of eIF4A helicase ac-
tivity was recently discovered and suggests an additional mechanism by which ele-
vated eIF4E levels can increase the translation of highly structured mRNAs through
stimulation eIF4A helicase activity (Feoktistova etal. 2013).
As major inhibitors of protein synthesis, the potential of harnessing the activity
of the 4E-BPs for therapeutic intervention is being actively pursued (Jia etal. 2012;
Martineau etal. 2013). The eIF4E/4E-BP interaction has been targeted using a small
molecule, eIF4E/eIF4G interaction inhibitor 4EGI-1 (Moerke etal. 2007), which
induced apoptosis in multiple myeloma (Chen etal. 2012; Descamps etal. 2012),
although the mechanism of action is controversial (McMahon etal. 2011). The ef-
fectiveness of suppressing eIF4E activity has also come into question, as a sepa-
rate study has shown that cells may reestablish translation following loss of eIF4E
through compensatory degradation of unphosphorylated 4E-BP (Yanagiya etal.
2012). Clearly, inhibiting translation in vivo will require a multifaceted approach to
prevent recuperation of translation activity in cancer cells. The diverse functions,
regulation, and targeting of eIF4E in cancer studies is discussed further in Chap.4
3.4.2Regulation of the eIF4A Helicase Through Activating

Factors and Inhibitors
Because eIF4A is a helicase that is regulated by cofactors (Marintchev 2013), in-

creased expression or activity of eIF4B, eIF4H, eIF4G and eIF4E could also be
suspected of playing a role in cell transformation by increasing the activity of eIF4A
(see Chaps.2 and 5). As mentioned previously, eIF4B (Shahbazian etal. 2010a)
stimulates the helicase and ATPase activity of eIF4A (Ozes etal. 2011; Rozen etal.
1990). In addition, eIF4B interacts with RNA (Naranda etal. 1994), eIF3 (Methot
etal. 1996), and the 40S ribosome (Methot etal. 1996; Rozovsky etal. 2008), pos-
sibly facilitating ribosome recruitment and thereby stimulating translation of highly
structured mRNAs (Dmitriev etal. 2003). Its activity may be modulated by the
ribosomal protein S6 (rpS6) kinase 1 (S6K1) (Raught etal. 2004), as phosphoryla-
tion increases eIF4B association with a PIC-containing eIF3 and eIF4F (Holz etal.
2005). eIF4H is homologous to the N-terminus of eIF4B and is similar in function
as it also stimulates helicase activity of eIF4A, although to a lesser extent than
eIF4B in vitro (Ozes etal. 2011; Rogers etal. 2001). The individual roles of eIF4H
and eIF4B are not well understood, and more work will be necessary to elucidate
independent as well as shared activities of these factors in vivo.
The effects of eIF4B overexpression on translation vary; some reports conclude
that transient overexpression increases translation rates (Holz etal. 2005; van Gorp
etal. 2009), while others suggest that translation is inhibited (Raught etal. 2004).
Since eIF4B has multiple binding partners, it is possible that translation inhibition is
a result of eIF4B binding to RNA, eIF4A, eIF3 and the 40S ribosome in translation-
ally inactive complexes (Shahbazian etal. 2010a). Conversely, RNAi knockdown
of eIF4B results in translation repression of mRNAs with highly structured 5 UTRs
involved in cell proliferation and survival via polysome depletion (Shahbazian etal.
2010b). The mechanism behind changes in translation when eIF4B levels are ma-
nipulated is not well understood, although many theories including those listed here
exist. The effects on translation measured following eIF4B overexpression could be
due to either activation of eIF4A, increased mRNA recruitment, or a combination
of both.
eIF4A itself has been reported to be overexpressed in hepatocellular carcinoma
(HCC) (Shuda etal. 2000) and some melanomas (Eberle etal. 1997). However,
both studies examined only mRNA levels, not protein expression levels of eIF4A.
Additionally, as other initiation factors, elongation factors, and ribosomal proteins
are also overexpressed, it is unclear which factors actually contributed to cell trans-
formation as opposed to increasing concentrations as a secondary effect in response
to cell transformation (Eberle etal. 1997). As mentioned previously, this is a ques-
tion for all overexpressed factors found in cancer cell lines or tumors, but the sig-
nificance of factor overexpression is particularly questionable for already abundant
proteins such as eIF4A (Duncan etal. 1987).
While eIF4A is activated by several factors, few proteins are known to inhibit
its activity in vivo. PDCD4 is an inhibitor of eIF4A, whose level is increased after
apoptosis is induced in cells (Lankat-Buttgereit and Goke 2009). PDCD4 inhibits
ATP and RNA binding by eIF4A (Loh etal. 2009; Suzuki etal. 2008) and competes
for eIF4A binding with eIF4G (Suzuki etal. 2008), thereby inhibiting the helicase
activity in order to reduce translation (Yang etal. 2003). Under more favorable
cellular conditions, PDCD4 is phosphorylated by S6K1, which leads to its ubiqui-
tination and proteasomal degradation (Dorrello etal. 2006; Jastrzebski etal. 2007).
PDCD4 may act as a tumor suppressor (Lankat-Buttgereit and Goke 2009), and has
been found downregulated in several types of cancer including lung primary carci-
nomas (Chen etal. 2003), invasive ductal breast carcinoma (Wen etal. 2007), and
skin cancer (Matsuhashi etal. 2007). For more information on translation control in
apoptosis and the role of PDCD4 in cancer, see Chaps.19 and 6 respectively. The
eIF4A helicase plays a central and essential role in translation initiation, and as such
has also become a promising target for both antiviral and chemotherapeutic drugs
(Bordeleau etal. 2006).
3.4.3mRNA Recruitment to the 40S Ribosomal Subunit

via eIF4G and eIF3
The eIF3/eIF4G interaction is thought to be the molecular scaffold bridging the

eIF4F/mRNA complex and the 43S PIC during mRNA recruitment in mammals (De
Gregorio etal. 1999; Hinton etal. 2007; Lamphear etal. 1995; Morino etal. 2000;
Villa etal. 2013). This interaction is enhanced in vivo by insulin treatment through
mTOR, but specific phosphorylation targets remain unknown (Harris etal. 2006).
However, recent studies have provided a structural model for eIF3/eIF4G binding,
which could help inform elucidation of specific regulation targets (LeFebvre etal.
2006; Villa etal. 2013).
mRNA recruitment to the ribosome may also be regulated or manipulated by
modifying eIF3 and eIF4G in other ways (see Chaps.7 and 8). For example, eIF4G
is targeted for proteolytic cleavage by caspase 3 during apoptosis to reduce cap-
dependent translation (Bushell etal. 2000; Clemens etal. 1998; Marissen and Lloyd
1998; Morley etal. 1998). IRES-dependent translation can continue following this
event by using the apoptotic cleavage fragments of eIF4G to recruit ribosomes to
mRNAs (Henis-Korenblit etal. 2002; Nevins etal. 2003; Stoneley etal. 2000a),
and thus the cell modifies the translation apparatus to adjust to its current needs (see
Chaps.18 and 19 for more information on translation regulation during apoptosis
and cancer). Recent work has also shown that the eIF3d subunit, which forms part
of the surface that interacts with eIF4G, is targeted by the human immunodeficiency
virus type 1 (HIV-1) protease, although it is not yet clear what affect cleavage of this
subunit may have on translation (Jager etal. 2012; Villa etal. 2013).
Aberrant expression of eIF3 and eIF4G, like other factors, could have d eleterious
effects via their diverse roles in the translation pathway. In general, eIF4G overex-
pression may contribute to an increase in global translation rates by merely increas-
ing the number of 40S recruitment events through its interaction with eIF3 (see
Chaps.7 and 8). Alternatively, its role as an activator of eIF4A may help drive the
increase in expression of low abundance oncogenic mRNAs. Lastly, as shown in in-
flammatory breast cancer, eIF4G may be promoting expression of specific mRNAs.
In a rare example where the translational mechanisms promoting malignancy have
been clearly defined, eIF4G overexpression in inflammatory breast cancer is crucial
for disease pathogenesis by promoting IRES-driven expression of p120 catenin,
which anchors E-cadherin to the cell surface and promotes formation of tumor em-
boli (Silvera etal. 2009).
Stable overexpression of eIF4G induces malignant transformation in NIH3T3
cells (Fukuchi-Shimogori etal. 1997), although it should be noted that an eIF4G
construct missing the PABP binding site was used in this study. Additionally, the
overexpression of eIF3 subunits a, b, c, h, or i promotes malignant transformation
of immortalized cells in culture, and several eIF3 subunits have also been found
aberrantly expressed in cancer (Zhang etal. 2007). The eIF3 complex is known to
contain 13 nonidentical subunits in humans, and it is interesting to note that only
select subunits have this effect. This may indicate that these proteins have roles out-
side of the complete eIF3 complex, or that some eIF3 complexes possess different
stoichiometry with regard to the exact number of subunits. In support of this, at least
two different eIF3 complexes with different subunit compositions have been found
in yeast (Zhou etal. 2005). Although subcomplexes of eIF3 subunits can be formed
and stabilized in vitro, whether similar subcomplexes exist in humans and what
their physiological significance may be has yet to be determined (Masutani etal.
2007, 2013; Sun etal. 2011). For more information on eIF3 and cancer, see Chap.8.
3.4.4 tRNA Recruitment to the 40S Ribosome Subunit
The phosphorylation state of eIF2 is critical in regulating both global and specific
mRNA translation. During translation initiation, tRNA is recruited to the ribosome in
the eIF2-TC complex with eIF2 and GTP (Fig.3.1a). Following start codon recogni-
tion, GTP is hydrolyzed and eIF2-GDP dissociates from the 40S subunit. In order to
enter another round of initiation, GDP must be exchanged for GTP by eIF2B. Cellular
stress results in phosphorylation of eIF2 at Ser51 by any of four kinases: heme-reg-
ulated inhibitor (HRI); protein kinase R (PKR); general control non-derepressible 2
(GCN2); or PKR-like endoplasmic reticulum kinase (PERK) (see Chap.9) (Donnelly
etal. 2013; Pavitt and Ron 2012). Once phosphorylated, the dissociation rate of eIF2
from eIF2B is reduced by roughly ten-fold. In other words, eIF2 is stuck on eIF2B,
thus preventing eIF2B from regenerating eIF2-GTP and eIF2-TC for further rounds of
translation initiation. Because eIF2B is significantly less abundant than eIF2, even a
small amount of phosphorylated eIF2 can severely reduce eIF2B activity (Fig.3.5b).
This results in a dramatic decrease in the availability of eIF2-TC and inhibition of
general protein synthesis (Pavitt and Ron 2012).
While eIF2 phosphorylation generally downregulates translation, in certain in-
stances it can promote translation of specific transcripts, such as those containing
uORFs. In a classic example, ATF4 mRNA translation increases with decreasing
availability of eIF2-TC, as this allows certain uORFs to be bypassed and thus in-
creasing translation at the main ORF (see Sect.3.2) (Lu etal. 2004; Vattem and Wek
2004). For more information regarding the regulation of eIF2, see Chap.9.
eIF2 is aberrantly overexpressed, along with eIF4E, in non-Hodgkins lym-
phoma (Wang etal. 1999), Hodgkins lymphoma (Rosenwald etal. 2008),
bronchioloalveolar lung cancer (Rosenwald etal. 2001), thyroid carcinoma (Wang
etal. 2001a), melanocytic and colonic epithelial neoplasms (Rosenwald etal. 2003),
and brain tumors (Tejada etal. 2009). It is also important to note that levels of PKR,
which phosphorylates eIF2 in response to viral double-stranded RNA in the cell,
can be increased (Haines etal. 1996; Kim etal. 2000; Shimada etal. 1998) or de-
creased (Haines etal. 1993a, b, 1998; Terada etal. 2000) in several types of cancer.
Lastly, despite important roles in stabilization of eIF2-TC, scanning and AUG
codon selection, few examples exist of dysregulation of either eIF1 or eIF1A in
cancer. In one recent study, eIF1 (also known as SUI1) was identified as a tumor-
associated antigen in HCC, highlighting its potential as a biomarker for diagnosis
of HCC or as a novel immunotherapy target for cancer (Chen etal. 2010; Lian etal.
1999). Further work will be needed to validate this as a target, and also to deter-
mine the effects of overexpression of eIF1 and eIF1A on eIF2-TC recruitment and
translation in vivo.
3.4.5 Translational Control of Elongation
Cells mainly direct translation regulation mechanisms at the initiation phase, likely
to avoid wasting time and energy. Presumably, this is why there are no established
examples of translation regulation at the termination or recycling phases. Once pro-

tein synthesis is underway however, translation can still be regulated at the elonga-
tion phase (see Chap.12). eEF2 is the elongation factor responsible for completing
tRNA and mRNA translocation from A- to P-sites and P- to E-sites on the ribosome
following aa-tRNA delivery. Under unfavorable growth conditions eEF2 is phos-
phorylated by eEF2 kinase (eEF2K) which impairs the ability of eEF2 to bind the
ribosome and perform translocation (Carlberg etal. 1990) (Fig.3.5c). S6K1 has
been shown to repress eEF2K activity and upregulate translation by phosphorylating
eEF2K (Redpath etal. 1996; Wang etal. 2001b). eEF2 has been found overex-
pressed in gastrointestinal cancers, and knockdown using shRNAs inhibited cell
growth in gastric and colon cancer cell lines. Interestingly, eEF2 knockdown also
resulted in eEF2K activation and G2/M cell cycle arrest. Conversely, induced over-
expression of eEF2 promoted G2/M cell cycle progression (Nakamura etal. 2009).
Elongation may also be targeted for downregulation during mitosis through in-
hibition of the GEF eEF1B. During elongation, eEF1A is charged with GTP by
eEF1B prior to ribosome binding and aa-tRNA recruitment (Fig.3.1b). One study
has shown that eEF1B is phosphorylated during mitosis, reducing eEF1A binding
and thus potentially limiting availability of eEF1A-TC during elongation (Sivan
etal. 2011). The extent to which elongation rates are inhibited by this mechanism,
and whether this inhibition is applicable in other cellular environments remains
to be determined. For more information on the elongation phase and cancer, see
Chaps.2 and 12.
3.5Regulating Translation via Changes in Ribosome

Number and Activity
The human ribosome is a 4.3 megadalton ribonucleoprotein complex consisting of

both ribosomal RNA (rRNA) and ribosomal protein components. This complex is
responsible for binding the mRNA and all translation factors involved in translating
the information in the mRNA into a polypeptide. The 60S ribosome itself catalyzes
peptide bond formation. It has long been recognized that an increased rate of global
protein synthesis is required to sustain cell growth (Ruvinsky and Meyuhas 2006).
This can be accomplished by increasing the number of ribosomes, the activity of
existing ribosomes, or by a combination of both.
3.5.1 Ribosome Biogenesis
During ribosome biogenesis, rRNA components are transcribed, processed

and modified in the nucleolus. Meanwhile, the genes of protein components
are transcribed in the nucleus, translated in the cytoplasm, and returned to the
nucleolus for ribosome assembly. Assembled ribosomes are then transported to
the cytoplasm to begin translation (Henras etal. 2008; Kressler etal. 2010). As
entioned p reviously, this process is regulated in part by the transcription factor

m
MYC, which promotes transcription of rRNA genes (Grandori etal. 2005). S6K1
also regulates rRNA transcription through activation of the nucleolar transcrip-
tion factor upstream binding factor (UBF) (Hannan etal. 2003; Jastrzebski etal.
2007). Dysregulated ribosome biogenesis normally triggers a stress response,
which activates the transcription factor and tumor suppressor p53, which in turn
promotes DNA repair, cell cycle arrest, senescence and apoptosis (Chakraborty
etal. 2011). Interestingly, several ribosomal proteins have been identified in
this signaling pathway (Chakraborty etal. 2011; Dutt etal. 2011; Warner and
McIntosh 2009).
Mutations or deletions of several ribosomal proteins, as well as aberrant ri-
bosome assembly, modification, or export, have been associated with inherited
diseases known as ribosomopathies (Freed etal. 2010; Montanaro etal. 2012;
Stumpf and Ruggero 2011). These diseases are characterized by an increased sus-
ceptibility to cancer and various phenotypic abnormalities (Luft 2010; Narla and
Ebert 2010). DiamondBlackfan anemia (DBA) is amongst the most studied of
these diseases. Approximately 25% of DBA patients have a mutation or deletion
in ribosomal protein rpS19, and mutations in other proteins in both the 40S and
60S ribosome subunits have also been documented (Draptchinskaia etal. 1999;
Lipton and Ellis 2009; Luft 2010; Willig etal. 1999). Interestingly, a recent study
has also shown that variance in the length or sequence of the 5 UTR of rpS19
could affect its translation in DBA (Badhai etal. 2011), once again illustrating
the importance of the 5 UTR as a mechanism for translation control. More work
on these complex regulatory pathways will be needed to elucidate the signal-
ing mechanisms, or failure thereof, underlying the cancer susceptibility of ribo-
somopathy patients. Please refer to Chap.13 for more information on the role of
ribosomes in cancer.
3.5.2 40S Ribosome Activity and Signaling
S6K1 was originally identified as the kinase responsible for rpS6 phosphorylation,
which had been correlated with translation activity and cell growth (Jastrzebski
etal. 2007; Ruvinsky and Meyuhas 2006). However, it appears that stimulation of
cell growth by S6K1 is actually independent of rpS6 phosphorylation since S6K1-
null mice (S6K1/) experience a cell growth defect despite rpS6 phosphorylation
by S6K2 (Pende etal. 2004). Phosphorylation of rpS6 has also been implicated
in translation regulation of 5 TOP mRNAs (see Sect. 3.2). However, this corre-
lation has since been challenged by results showing normal translation of TOP
mRNAs in both RPS6P/ knock-in mice, where the phosphorylation sites of rpS6
have been converted to alanine residues, and in S6K1//S6K2/ double knockout
(DKO) mice (Pende etal. 2004; Ruvinsky etal. 2005). More work will be needed
to discover the regulatory function of ribosomal protein phosphorylation. For more
information on S6K activity, see Chap.15.
3.6 Conclusions and Perspectives
In this chapter, we aimed to convey the variety of mechanisms available to a cell to

precisely regulate global levels of translation and to modulate expression of individ-
ual mRNAs. Translation can be controlled through mRNA sequence and structure
elements, availability of mRNA to the translation apparatus, and through control of
the activity and availability of translation factors and ribosomes. Many of the mech-
anisms discussed in this chapter have not been fully elucidated, and more work in
coming years is likely to expose new translation regulation mechanisms. Although
translation consists of four phases, regulation mechanisms are mainly directed at
the initiation phase in order to conserve cellular resources, time, and energy, and
imbalance in this system can lead to or promote disease states.
Dysregulation of translation can play a critical role in driving malignant transfor-
mation. While global translation rates are only increased by ~20% in transformed
cells, some mRNAs are preferentially more highly upregulated. The reason for this
could stem from dysregulation of the translation machinery, or mutations in an
mRNA that enhance its translation or availability to the translation machinery. Con-
sistent with the importance of mRNA recruitment as a committed step in translation,
it is perhaps not surprising that eIF4E is found to be overexpressed in roughly 30%
of major cancers (Sonenberg 2008). As a result, a considerable amount of effort
is now being made to try and reduce the activity of eIF4E and the cap-binding
complex in cancer cells. To better understand the selective upregulation of onco-
genic mRNAs, it will be important to gain a better understanding of what structural
features these messages may share. New chemical probing techniques that can be
carried out on a genome wide scale are likely to assist in understanding the dys-
regulation of oncogenic mRNAs in the coming years (Ding etal. 2012; Lucks etal.
2011; Wilkinson etal. 2006).
Since major signal transduction pathways that regulate many key ribosome
associated proteins are often dysregulated in cancers, it is not surprising that trans-
lation rates are generally altered as a result. Significant advances have been made in
our understanding of how translation components are regulated by posttranslational
modifications, although we are far from a complete understanding of the breadth
of these mechanisms. For example, some ~30 phosphorylation sites have been re-
ported on human eIF3, although the functional significance of many of these is
unknown (Damoc etal. 2007). To date, only one of these has been shown to be im-
portant in promoting cell growth (Zhang etal. 2008). Since eIF3 plays an important
role throughout the initiation pathway, it is likely that other phosphorylation sites
will be found to regulate specific or global rates of translation.
Lastly, ribosome profiling has provided us with a new technique with which
to take snap shots of the translation machinery at work on a genome wide scale
(Ingolia etal. 2012). This approach has already revealed many important details re-
garding the selection of initiation sites during cell differentiation and the cell cycle
(Ingolia etal. 2011, 2012). It will be interesting to use this approach to determine
how the activity of the translation machinery is altered during cell transformation.
These studies will provide information to help us understand how the translation
apparatus is reprogramed in transformed cells and could provide essential clues in
the search for ways to combat cancer.
Acknowledgments Work in the Fraser lab is supported by grant R01GM092927 from the National
Institute of General Medical Sciences. We would like to thank Professor John Hershey for many
helpful discussions.
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Chapter 4
eIF4E and Its Binding Proteins
Nathaniel Robichaud and Nahum Sonenberg
Contents
4.1eIF4E Binds the Cap 74

4.2eIF4Ea Central Component of the Translation Initiation Machinery 76
4.3eIF4Ea General Initiation Factor with Tumorigenic Properties 77
4.4Regulation of eIF4E Activity by Interacting Proteins 78
4.4.1eIF4G 78
4.4.24E-BPs 79
4.5Regulation of eIF4E Activity by Phosphorylation 81
4.6Transcriptional Regulation of eIF4E 81
4.7Other Mechanisms of eIF4E Regulation 82
4.7.1Posttranscriptional Regulation 82
4.7.2Ubiquitinylation and Proteasomal Degradation 83
4.7.3Cap-Competitive Homologs of eIF4E 83
4.8Viruses, Cancer and eIF4E 84
4.9eIF4E and the Hallmarks of Cancer 85
4.9.1Evading Growth Suppressors 85
4.9.2Sustaining Proliferative Signaling 85
4.9.3Enabling Replicative Immortality 86
4.9.4Resisting Cell Death 87
4.9.5Inducing Angiogenesis 87
4.9.6Activating Invasion and Metastasis 87
4.9.7Emerging Hallmarks and Enabling Characteristics 88
4.10An eIF4E-Centric View of Tumorigenic Signaling 88
4.10.1eIF4E at the Convergence Point of Oncogenic Signaling 89
4.10.2eIF4E Regulates Upstream Signaling 91
4.10.3eIF4E as the Translational Hub of Complex Signaling Networks 92
4.11eIF4E and 4E-BPs in Human Cancers 92
4.11.1eIF4E Overexpression 92
4.11.2Expression of the 4E-BPs 94
4.11.3Phosphorylation of the 4E-BPs 95
4.11.4eIF4E Phosphorylation 95
N.Robichaud() N.Sonenberg
Department of Biochemistry and Rosalind and Morris Goodman Cancer Research Centre,
McGill University, Montreal, QC, Canada
e-mail: nathaniel.robichaud@mail.mcgill.ca
74 N. Robichaud and N. Sonenberg
4.12eIF4E/4E-BPs and Clinical Correlates 95

4.12.1eIF4E and 4E-BPs in Cancer Recurrence and Survival 95
4.12.2eIF4E and 4E-BPs in Cancer Grade and Progression 97
4.13Therapeutic Strategies and Resistance to Therapy 98
4.14Conclusion and Perspectives 99
References 100
AbstractThe link between protein synthesis and cancer was first suggested by
Pianese in 1896, who observed that malignant cells contain larger and more numer-
ous nucleoli than normal cells. Yet the role of translation in cancer biology has
been largely overlooked in comparison with transcription. Nearly one century
elapsed before the first report that ascribed oncogenic properties to a translation
initiation factor was published. That factor is the eukaryotic translation initiation
factor 4E (eIF4E). eIF4E binds the mRNA 5 end and is critical for its translation.
It has received much attention for its important biological functions, as well as for
its involvement in cancer development and progression. Indeed, eIF4E possesses
proto-oncogenic properties as its overexpression or hyperactivation leads to tumori-
genesis. Increased levels of eIF4E are detected in as many as 30% of human can-
cers across a wide variety of sites including head and neck, bladder, colon, breast,
prostate, lung, and blood. In several studies, eIF4E overexpression or activation
has been associated with poor disease prognosis. This chapter reviews the current
knowledge regarding function, activity and regulation of eIF4E, as well as its bind-
ing partners, in relation to cancer etiology and pathogenesis.
4.1eIF4E Binds the Cap
Nuclear transcribed mRNAs possess a cap structure (m7GpppN, where m7G is

7-methylguanosine and N is any nucleotide) at their 5 end, which is critical for
mRNA splicing and polyadenylation, stability and translation. eIF4E was first dis-
covered as a 24kDa protein that could be chemically crosslinked to the cap (Sonen-
berg etal. 1978). It is one of the two well-characterized cap-binding proteins (CBP),
the other being CBP20, which is important for the various steps of nuclear mRNA
metabolism. Though structurally unrelated, both proteins bind to the cap through
similar mechanisms (Topisirovic etal. 2011).
The structure of eIF4E resembles that of a cupped hand pinching the cap be-
tween finger and thumb (Marcotrigiano etal. 1997; Matsuo etal. 1997; Tomoo
etal. 2005). The molecular basis for the specificity of eIF4E for capped mRNAs is
described in Fig.4.1. The most important interaction is the stacking of the guanine
ring between two aromatic tryptophans, which is strengthened by delocalized posi-
tive charge arising from the methyl group of the cap structure. Further interactions
occur between the cap-binding pocket and the guanine, the methyl group, the tri-
phosphate bridge and the second nucleotide (Fig.4.1). Each of these interactions is
important for cap recognition, as demonstrated by the hierarchy of eIF4Es affinity
4 eIF4E and Its Binding Proteins 75
Fig. 4.1 Three-dimensional structure of cap-bound eIF4E and 4E-BP1 peptide. a Aromatic stack-
ing of the purine ring between Trp56 and Trp102 and electrostatic interactions between N1/N2
and Glu103. b Van der Waals interactions between the methyl group and Trp166. c Electrostatic
interactions of the triphosphate bridge with Arg157 and Lys162. d Interactions between the second
nucleotide and residues of the flexible C-terminal loop of eIF4E. Structure from the Protein Data
Bank (reference 1WKW (Tomoo etal. 2005)).
Table 4.1 Relative affinity of eIF4E for various cap analogs.

Interaction Relative binding
Second nucleotide m7GpppA>m7Gppp
Triphosphate bridge m7GpppA>m7GppA
m7Gppp>m7Gpp>>>m7Gp
Methyl group m7Gppp>>>Gppp
m7 methyl group on position N7 of the guanine, G guanosine, A adenosine, p phosphate group.
Adapted and modified from Hu etal. (1999) and Niedzwiecka etal. (2002).
to various cap analogs (Hu etal. 1999; Niedzwiecka etal. 2002) (summarized in
Table4.1).
The activity of eIF4E is intrinsically linked to its cap-binding properties. Indeed,
impairing cap-binding activity by mutating tryptophan 56 to an alanine abrogates its
oncogenic effects in mouse models and human cell lines (Topisirovic etal. 2011).
The exact role of eIF4E in determining the fate of mRNAs is determined by its vari-
ous binding partners in the regulation of mRNA translation.
4.2eIF4Ea Central Component of the Translation

Initiation Machinery
Translation initiation in eukaryotes (see Chap.2) begins under most circumstances

by binding of the eIF4F complex to the cap. eIF4E is an integral component of the
eIF4F hetero-trimeric complex, together with the modulatory scaffolding protein
eIF4G and the DEAD box helicase eIF4A. eIF4G also interacts with PABP that
binds to the poly(A) tail at the 3 end of the mRNA. The eIF4G/PABP interaction
engenders a closed-loop structure or circular mRNP, which promotes translation
(Jackson etal. 2010; Jacobson and Favreau 1983; Mathews etal. 2007; Sonenberg
and Hinnebusch 2009). eIF4G also binds to the eIF3 multisubunit protein, which
is associated with the 43S pre-initiation complex (PIC) to recruit the latter to the
mRNA. The resulting 48S initiation complex traverses the mRNA in a 5 to 3 di-
rection until it encounters the initiation codon, which base-pairs with the initiator
tRNA anticodon. Although the 43S PIC is sufficient to perform this process on very
short, intrinsically unstructured mRNAs in vitro (Pestova and Kolupaeva 2002),
almost all cellular mRNAs possess some degree of secondary structure. Thus, the
eIF4F complex is essential for their scanning as they are dependent on the helicase
activity of eIF4A to unwind secondary structure (Mathews etal. 2007). Since eIF4E
is the least abundant translation initiation factor (Duncan etal. 1987; Hiremath etal.
1985), its levels control formation of the eIF4F complex and therefore eIF4A he-
licase activity. Importantly, eIF4E has recently been shown by Feoktistova etal to
stimulate unwinding of mRNA secondary structure independently of its cap-binding
functions (Feoktistova etal. 2013). They demonstrated that eIF4E binding to eIF4G
relieves autoinhibition, allowing eIF4G to stimulate the helicase activity of eIF4A.
Consistent with these findings, the dependency of mRNA translation on eIF4E
and eIF4A increases with 5 UTR complexity (Pestova and Kolupaeva 2002; Sonen-
berg etal. 1981; Svitkin etal. 2001). In rabbit reticulocyte lysate, substitution of uri-
dines for bromouridines in reovirus mRNA, which leads to enhanced base-pairing
and more stable 5 UTR secondary structures, resulted in an increased dependence
on eIF4E for translation (Sonenberg etal. 1981). The converse experiment, substitu-
tion for inosine resulting in relaxed secondary structure, alleviated the requirement
for eIF4E in the translation of reovirus mRNA (Sonenberg etal. 1981). Similar
conclusions were drawn from studies using a reconstituted in vitro translation sys-
tem. While intrinsically unstructured mRNAs do not depend on eIF4E, even weak
secondary structure (6.6kcal/mole) is sufficient to render an mRNA dependent on
the eIF4F complex (Pestova and Kolupaeva 2002). Furthermore, the requirement of
mRNAs for eIF4A positively correlates with the extent of their 5 UTR secondary
structure (Svitkin etal. 2001). Notably, eIF4A promotes the translation of mRNAs
with structured 5 UTRs mainly as a part of the eIF4F complex (Pause etal. 1994b),
and the formation of this complex is regulated by eIF4E availability (Duncan etal.
1987; Hiremath etal. 1985). Taken together, these data support the idea that eIF4E
preferentially increases the translation of mRNAs with structured 5 UTRs by re-
cruiting the helicase activity of eIF4A.
4.3eIF4Ea General Initiation Factor with Tumorigenic

Properties
eIF4E is a general translation initiation factor that is required for the translation of
the vast majority of cellular mRNAs (Sonenberg and Hinnebusch 2009). However,
modulating its expression only mildly affects global protein synthesis (De Benedetti
and Harris 1999; Graff etal. 2007; Mamane etal. 2007; Rosenwald etal. 1999).
Under normal conditions, there is sufficient eIF4E available for the optimal transla-
tion of most mRNAs, which remain relatively insensitive to additional eIF4E. In
contrast, a subset of mRNAs with extensive secondary structure in their 5 UTR is
highly sensitive to modulations in eIF4E expression and activity (De Benedetti and
Graff 2004; De Benedetti and Harris 1999; Koromilas etal. 1992). These mRNAs
encode a wide variety of proteins promoting proliferation, survival and progression
to metastasis such as the c-MYC oncogene, various cyclins, ODC, the antiapop-
totic factor BCL-XL, VEGF and matrix metalloproteinases (MMPs) (Graff etal.
1995; Kevil etal. 1996; Li etal. 2003; Rosenwald etal. 1995, 1993; Rousseau etal.
1996b; West etal. 1995). Polysome profile experiments have confirmed that these
mRNAs display increased translational efficiency when eIF4E is overexpressed, as
the number of ribosomes per mRNA molecule greatly increases (Graff etal. 1995;
Jiang and Muschel 2002; Kevil etal. 1996; Li etal. 2003; Rosenwald etal. 1995;
Rousseau etal. 1996b; West etal. 1995). Such changes are not observed in house-
keeping mRNAs with short and simple 5 UTRs such as that encoding glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH) (reviewed in De Benedetti and Graff
2004; Mamane etal. 2004). Thus, the tumorigenic properties of eIF4E can at least in
part be ascribed to its aforementioned critical importance in recruiting and stimulat-
ing the helicase activity of the eIF4F complex.
However, this view remains controversial as recent studies using ribosome pro-
filing experiments rather ascribe eIF4E sensitivity to 5-terminal oligopyrimidine
tracts (TOP) (Hsieh etal. 2012; Thoreen etal. 2012). Curiously, such genome-wide
studies of translationally regulated mRNAs have reached widely differing conclu-
sions. Some bioinformatics analyses have provided further evidence for the role of
eIF4E in promoting the translation of structured mRNAs and their involvement in
tumorigenesis (Larsson etal. 2006; Provenzani etal. 2006; Santhanam etal. 2009).
Microarray analysis of translated mRNAs in eIF4E overexpression models has un-
covered several cis-regulatory elements consistent with a requirement for eIF4F
helicase activity. eIF4E-sensitive mRNAs have been reported to have longer 5
UTRs (Provenzani etal. 2006). They also have a significantly higher probability
of forming secondary structures and possessing high G/C content near the cap and
immediately upstream of the start codon (Santhanam etal. 2009). Several studies
have identified other features of eIF4E-sensitive mRNAs, such as TOP (Amaldi
and Pierandrei-Amaldi 1997; Hsieh etal. 2012; Mamane etal. 2007; Thoreen etal.
2012), short 5 and 3 UTRs (Bilanges etal. 2007; Santhanam etal. 2009), avoidance
of miRNA sites (Larsson etal. 2007; Santhanam etal. 2009) and other structural
features in the 3 UTR (Santhanam etal. 2009). Several studies in which 5 UTR
secondary structure was not found as a determinant of eIF4E sensitivity also failed
to identify well-known individual eIF4E-sensitive mRNAs with complex 5 UTRs,
such as cyclin D1, c-MYC and ODC in their screens (Bilanges etal. 2007; Mamane
etal. 2007; Thoreen etal. 2012). This discrepancy between biochemical studies
and various computational analyses may be due to the poor quality of 5 UTR an-
notations and the difficulty of assessing mRNA structural properties based on the
sequence (Fan etal. 2009a; Larsson etal. 2013). Other unknown mechanisms con-
ferring eIF4E-sensitivity may also exist. Aside from these debates on the molecular
mechanisms involved, it is evident that eIF4E-sensitive mRNAs are enriched for
cancer-promoting properties such as ribosome biogenesis, apoptosis resistance, pro-
liferation, angiogenesis and invasion (Bilanges etal. 2007; Hsieh etal. 2012; Kim
etal. 2009; Larsson etal. 2006, 2007; Mamane etal. 2007; Provenzani etal. 2006;
Rajasekhar etal. 2003; Thoreen etal. 2012).
4.4Regulation of eIF4E Activity by Interacting Proteins
The most widely studied mechanism of eIF4E regulation is effected through several
interacting partners, which bind to the dorsal side of eIF4E (see Fig.4.1). eIF4E
interaction with eIF4G promotes translation initiation, and the 4E-BPs inhibit this
process by binding to and sequestering eIF4E.
4.4.1eIF4G
The first protein that was described as a binding partner of eIF4E was eIF4G (Etchi-
son etal. 1982; Grifo etal. 1983; Prevot etal. 2003). The human genome encodes
two eIF4G family members: eIF4G1 and eIF4G2 (Gradi etal. 1998). These proteins
are functionally redundant: they bind to eIF4E, eIF3 and eIF4A, and can restore
cap-dependent translation in rabbit reticulocyte lysates treated with rhinovirus 2Apro
(Gradi etal. 1998; Grifo etal. 1983). eIF4G1 and 2 are ubiquitously expressed,
though their relative expression varies in different tissues (Gradi etal. 1998). eI-
F4G1 was the first identified homolog and is generally used in studies on eIF4E/
eIF4G interactions. Therefore, it is referred to as eIF4G in this chapter and the
numbering of important residues relate to this homolog. Two conserved residues in
eIF4E are critical for its interaction with eIF4G, Val69 and Trp73 (Marcotrigiano
etal. 1999). eIF4G interacts with eIF4E via the sequence YDREFLL, which con-
forms to the canonical eIF4E-binding motif, YXXXXL (where X is any amino
acid and is hydrophobic). In yeast, the eIF4E/eIF4G interaction leads to confor-
mational changes in both proteins, resulting in eIF4G wrapping around the hand-
like structure of eIF4E that leads to the tightening of its grip on the cap. Thus, eIF4G
allosterically increases the affinity of eIF4E for the cap and decreases its disso-
ciation rate (Haghighat and Sonenberg 1997; Marcotrigiano etal. 1999; Ptushkina
etal. 1998; von Der Haar etal. 2000). However, such an allosteric mechanism has
not been described for the mammalian eIF4E/eIF4G interaction, despite a similar
increase in cap-binding affinity. Instead, RNA-binding motifs in mammalian eIF4G
stabilize the interaction of the eIF4F complex with the cap (Yanagiya etal. 2009).
The RNA-binding motifs on eIF4G likely do not affect the on/off binding rates of
eIF4E to the cap structure (Slepenkov etal. 2008). Rather, the interaction of eIF4G
with mRNA maintains eIF4E in the vicinity of the cap, increasing its local con-
centration and favoring the bound state. Yeast eIF4G also possesses RNA-binding
motifs, but these motifs do not appear to be important to stabilize the eIF4E/cap in-
teraction (von Der Haar etal. 2000; Yanagiya etal. 2009). Thus, higher eukaryotes
may have developed a distinct mechanism to stabilize the interaction between the
eIF4F complex and mRNA.
The eIF4E/eIF4G interaction is critical for the translation of mRNAs encoding
prosurvival and proproliferative proteins by promoting the formation of the eIF4F
complex and the unwinding of 5 UTR secondary structures. Indeed, eIF4G over-
expression leads to tumorigenic transformation of fibroblasts (Fukuchi-Shimogo-
ri etal. 1997). Furthermore, blocking eIF4G binding to eIF4E with the inhibitor
4EGI-1 results in reduced translation of c-MYC and BCL-XL with an associated
induction of apoptosis in leukemia cell lines (Moerke etal. 2007).
4.4.2 4E-BPs
The 4E-BPs are small (~1520kDa) proteins that interact with eIF4E (Pause etal.
1994a). There are three known isoforms in mammals (4E-BP 1, 2, 3). Relatively
little is known regarding 4E-BP3, especially in cancer. Most studies focus on 4E-
BP1, and to a lesser extent 4E-BP2. Both isoforms 1 and 2 are ubiquitously ex-
pressed, although the predominant species varies across different tissues (Lin and
Lawrence 1996; Tsukiyama-Kohara etal. 1996). As these isoforms are functionally
redundant in the context of cell growth and cancer (Rousseau etal. 1996a), they are
jointly referred to as the 4E-BPs in this chapter. The 4E-BPs are regulated by the
mTOR complex 1 (mTORC1) (see Chap.15), which phosphorylates several resi-
dues in a hierarchical manner. First, Thr37/Thr46 are phosphorylated by mTOR,
followed by Thr70 and finally Ser65 (Gingras etal. 2001). Thr70 and Ser65 are re-
sponsive to extracellular cues such as serum stimulation (Gingras etal. 2001). Phos-
phorylation of all of these sites (the hyperphosphorylated form) inhibits 4E-BPs
binding to eIF4E. Phosphorylation of only Thr37/Thr46 (the hypophosphorylated
form) does not lead to the disassembly of the eIF4E/4E-BP complex (Gingras etal.
1999; Gingras etal. 2001). In this manner, mTORC1, via the 4E-BPs, regulates
eIF4E and translation.
The 4E-BPs act as inhibitors of eIF4E function by competing with eIF4G for binding
to the dorsal side of eIF4E (Haghighat etal. 1995; Mader etal. 1995) to prevent the for-
mation of the eIF4F complex and subsequent translation initiation (Pause etal. 1994a).
mRNAs possessing extensive 5 UTR structure are particularly sensitive to sequestra-
tion of eIF4E by the 4E-BPs (Cawley and Warwicker 2012; Provenzani etal. 2006),
purportedly due to their stronger dependence on the activity of the eIF4F complex.
eIF4G and the 4E-BPs bind to eIF4E via their conserved eIF4E-binding motif
(YXXXXL) using a similar disorder-to-order transition mechanism, and pos-
sess similar affinities for eIF4E (Fletcher and Wagner 1998; Gosselin etal. 2011;
Lukhele etal. 2013; Marcotrigiano etal. 1999). Despite this, the kinetics of bind-
ing differ widely, as the 4E-BPs display rates of binding and dissociation two
to three orders of magnitude faster (Umenaga etal. 2011). Conceptually, this
difference is consistent with the inhibitory role of the 4E-BPs, requiring rapid
control of eIF4E binding, whereas eIF4G requires longer-lived interactions with
eIF4E to promote translation initiation. Recent studies have discovered the mo-
lecular basis for this important distinction by uncovering a second eIF4E-binding
site that differs between eIF4G and the 4E-BPs (Mizuno etal. 2008; Umenaga
etal. 2011). A recent structural study using full-length 4E-BP2 has confirmed the
importance of the second binding site: while 4E-BP2 is intrinsically disordered,
both eIF4E-binding sites possess significant transient secondary structure and
contribute to 4E-BP2s affinity for eIF4E (Lukhele etal. 2013). The dynamic
nature of the eIF4E/4E-BPs interaction is attributed to the second binding site
(Lukhele etal. 2013).
Because they inhibit eIF4E, the 4E-BPs act as tumor suppressors. Genetic abla-
tion of the 4E-BPs sensitizes mice to carcinogens (Kim etal. 2009) and synergizes
with p53 loss in tumorigenesis (Petroulakis etal. 2009). Furthermore, low levels of
4E-BP1 and its hyperphosphorylation are associated with poor prognosis in mela-
noma, childhood rhabdomyosarcoma, as well as prostate, breast and ovarian can-
cers (Armengol etal. 2007; Graff etal. 2009; OReilly etal. 2009; Petricoin etal.
2007; Rojo etal. 2007). Considering that mTOR regulates eIF4E via phosphoryla-
tion of the 4E-BPs, this kinase plays an important role in tumorigenesis. For more
on mTOR and the 4E-BPs in cancer, see Chap.15.
4.5Regulation of eIF4E Activity by Phosphorylation
eIF4E is phosphorylated on a single site, Ser209, by the mitogen-activated protein

kinase (MAPK)-interacting kinases (MNK) 1 and 2, which are activated in response
to cellular stress and survival signals from the MAPK kinase (MEK)/extracellu-
lar signal-regulated kinase (ERK), and p38 MAPK pathways (Buxade etal. 2008;
Waskiewicz etal. 1999) (see Chap.17). Phosphorylation requires prior binding of
the MNKs to eIF4G, indicating that phosphorylation of eIF4E occurs after the for-
mation of the eIF4F complex (Pyronnet etal. 1999). eIF4E phosphorylation is not
required for global translation as mutation of Ser209 to alanine or genetic abroga-
tion of the MNKs has no deleterious effects in in vivo models (Furic etal. 2010;
Ueda etal. 2010). Rather, eIF4E phosphorylation controls the translation of a spe-
cific subset of mRNAs, although the mechanism by which this occurs is uncertain.
Biophysical studies including surface plasmon resonance, stopped-flow kinetics and
fluorescence titration experiments indicate that eIF4E phosphorylation decreases the
affinity for the cap (Scheper etal. 2002; Slepenkov etal. 2006; Zuberek etal. 2003).
This is due to electrostatic repulsion between the phosphorylated Ser209 and the
intrinsic negative charges within the cap. This hypothesis is supported by the fact
that increasing the number of phosphate groups present in cap analogs exacerbates
the effect of eIF4E phosphorylation (Zuberek etal. 2003). Hence, considering that
phosphorylation by the MNKs occurs after eIF4F complex formation (Pyronnet etal.
1999), it appears that eIF4E phosphorylation promotes dissociation of eIF4E from
the cap. This may facilitate the dissociation of eIF4E from the mRNA and its recy-
cling for further rounds of translation (Scheper and Proud 2002).
There is strong evidence linking eIF4E phosphorylation to tumorigenesis. Muta-
tion of serine 209 to alanine impairs eIF4Es tumorigenic properties when overex-
pressed in cells and in mice (Topisirovic etal. 2004; Wendel etal. 2007). Similar
findings have been obtained using mouse models in which endogenous eIF4E phos-
phorylation is abrogated either by mutation of the phosphorylation site or genetic
ablation of the MNKs (Furic etal. 2010; Ueda etal. 2010). Recent experiments fur-
ther suggest that eIF4E phosphorylation promotes invasion and metastatic progres-
sion (Robichaud et al. 2014). These studies have established that the phosphoryla-
tion of eIF4E exerts its effects through translational upregulation of protumorigenic
transcripts, such as MMP-3, as well as antiapoptotic factors, such as baculoviral
inhibitor of apoptosis repeat-containing protein 2 (BIRC2) and induced myeloid
leukemia cell differentiation protein MCL-1 (Furic etal. 2010; Wendel etal. 2007).
4.6Transcriptional Regulation of eIF4E
Transcriptional regulation of eIF4E plays an important role in determining its

level of expression. The vast majority of studies exploring eIF4E transcription
have focused on the c-MYC proto-oncogene, which is a key regulator of eIF4E
expression. However, heterogeneous nuclear ribonucleoprotein K (hnRNP K)

(Lynch etal. 2005); the p30 isoform of C/EBP (Khanna-Gupta etal. 2012); the
nuclear factor -light-chain-enhancer of activated B cells (NF-B) (Hariri etal.
2013); and the hypoxia-inducible factor 1 (HIF-1) (Yi etal. 2013) have also
been suggested to play a role. The promoter of eIF4E possesses two conserved
enhancer box (E-box) motifs, which recruit c-MYC to upregulate eIF4E transcrip-
tion (Jones etal. 1996). Other c-MYC targets include factors promoting apopto-
sis. Interestingly, eIF4E expression counteracts the apoptotic effects of c-MYC
and allows cells to proliferate unchecked (Ruggero etal. 2004). These effects
are due to translational upregulation of antiapoptotic factors such as MCL-1 and
BCL-XL by eIF4E. Strikingly, the c-MYC mRNA is itself a translational target of
eIF4E, highlighting a regulatory feed-forward loop where c-MYC promotes the
transcription of eIF4E, which in turn promotes the translation of c-MYC mRNA
(Lin etal. 2008). Consequently, eIF4E and c-MYC promote each others expres-
sion and act in combination to induce growth, proliferation and survival. In addi-
tion, c-MYC has recently been suggested to regulate eIF4E activity by promoting
mTOR phosphorylation of the 4E-BPs by an as-of-yet undetermined mechanism
(Pourdehnad etal. 2013).
This mutual dependency of c-MYC and eIF4E could potentially be exploited
to treat c-MYC-driven cancers, as c-MYC itself is notoriously difficult to target
directly (Hopkins and Groom 2002). Indeed, c-MYC-driven lymphomas are highly
dependent on 4E-BP1 hyperphosphorylation and thus are exquisitely sensitive to
active site mTOR inhibitors (Pourdehnad etal. 2013). Thus, the development of in-
hibitors of eIF4E for cancer therapy is critical, as MYC family members are among
the most frequently dysregulated oncogenes in human cancers (Vita and Henriksson
2006).
4.7Other Mechanisms of eIF4E Regulation
4.7.1 Posttranscriptional Regulation
Translational control of eIF4E mRNA by miRNAs has been suggested to play an

important role in melanoma cell lines, where miR-768-3p represses eIF4E transla-
tion (Jiang etal. 2014). Oncogenic activation of the MEK/ERK pathway down-
regulates miR-768-3p, thus promoting high eIF4E expression (Jiang etal. 2014).
Additionally, eIF4E mRNA possesses AU-rich elements (AREs) which regulate its
stability via the opposing effects of the ARE-binding proteins: the human antigen
R (HuR), having stabilizing effects and the ARE RNA-binding protein 1 (AUF1),
possessing destabilizing effects (Topisirovic etal. 2009). It will be interesting to ex-
pand upon these studies, especially considering the important role of HuR in cancer
(Wang etal. 2013a).
4.7.2 Ubiquitinylation and Proteasomal Degradation
A major regulator of protein turnover is the ubiquitin-proteasome pathway

(Ciechanover 2005). eIF4E is ubiquitinylated on Lys159 and degraded by the
proteasome during heat shock or exposure to cadmium (Murata and Shimotohno
2006). Ubiquitinylation of the 4E-BPs has also been observed and is thought to
be a mechanism for the maintenance of an optimal eIF4E/4E-BPs ratio (Yanagiya
etal. 2012). Hypophosphorylated 4E-BP1 binds to and sequesters eIF4E. How-
ever, when it is hypophosphorylated, in excess and unbound to eIF4E, it is rapidly
ubiquitinylated and degraded by the proteasome (Yanagiya etal. 2012). Thus,
proteasome inhibitors may enhance the anticancer effects of antisense oligonucle-
otides (ASOs) targeting eIF4E expression, as this would prevent a concomitant
degradation of the 4E-BPs. As a result, the eIF4E/4E-BPs ratio would be greatly
reduced. The ratio between these factors has been shown to be more important
than their absolute levels of expression in tumorigenesis and drug resistance
(Alain etal. 2012; Coleman etal. 2009). These findings highlight the importance
of targeting proteasomal degradation in cancer, in the context of therapies which
reduce eIF4E expression.
4.7.3 Cap-Competitive Homologs of eIF4E
The human genome encodes three members of the eIF4E family: (1) eIF4E1, the
most widely studied homolog that is generally (and throughout this book) referred
to as eIF4E; (2) eIF4E2, also known as the eIF4E homologous protein or 4EHP;
and (3) eIF4E3. eIF4E1 is the predominant species, being ubiquitously expressed
at levels 510 times higher than 4EHP (Rom etal. 1998). eIF4E3 has not been
documented at the protein level. In vitro synthesized 4EHP and eIF4E3 can com-
pete with eIF4E1 for binding to the mRNA cap (Joshi etal. 2004). However,
they act as translational inhibitors as they do not recruit eIF4G. Considering the
reduced cap-binding properties of 4EHP and eIF4E3 and their low relative expres-
sion (Osborne etal. 2013; Zuberek etal. 2007), it is unlikely that they function
as general inhibitors of cap-dependent translation. It is more probable that they
repress specific mRNAs by recruiting other RNA-binding factors to form transla-
tionally repressed mRNPs. Such a mechanism has been described for Drosophila
4EHP, which binds to Bicoid to inhibit the translation of caudal mRNA (Cho etal.
2005). Recently, this concept has been extended to mammals, where it was found
that 4EHP acts as a translational repressor in conjunction with GIGYF2 (GRB10
interacting GYF protein 2) (Morita etal. 2012). One group has presented data
that are contradictory to these findings. They demonstrate that 4EHP promotes
the translation of specific mRNAs during hypoxia in a cap-dependent manner
(Uniacke etal. 2012, 2014).
4.8Viruses, Cancer and eIF4E
Some viruses are known to cause cancer. For example, the vast majority of cervi-
cal cancers are caused by human papilloma viruses (HPV) (Crosbie etal. 2013).
Viruses have long been known to target the translation machinery to control the syn-
thesis of their own proteins as well as the expression of host proteins (Walsh etal.
2013). This regulation plays a critical role in the oncogenicity of certain viruses that
are dependent on eIF4E. Thus, the HPV E6 oncoprotein induces the transcription of
eIF4E, promoting tumorigenic effects of HPV (Wang etal. 2013b). Further support-
ing the importance of eIF4E in HPV-induced cervical cancer is the finding that E7,
which is essential for HPV replication and transformation, is translationally induced
by the mTOR pathway through phosphorylation of the 4E-BPs (Oh etal. 2006).
Similarly, the small T antigen encoded by Merkel cell polyomavirus, which causes
Merkel cell carcinoma, promotes 4E-BP1 phosphorylation (Shuda etal. 2011), as
does the nonstructural protein 5A of the hepatitis C virus (HCV), which can cause
HCC (George etal. 2012). A particularly interesting example is that of Kaposis
sarcoma-associated herpesvirus (KSHV), which leads to Kaposis sarcoma in im-
munocompromised patients (Chang etal. 1994). KSHV activates translation in
part by promoting the phosphorylation of 4E-BP1 (Arias etal. 2009). Inactiva-
tion of 4E-BP1 promotes the expression of paracrine signaling molecules such as
VEGF-A and interleukin 6 (IL-6) that are important for tumor development (Martin
etal. 2014). Importantly, treatment with the mTOR inhibitor rapamycin leads to
dephosphorylation of 4E-BP1 and counteracts KSHV-related paracrine signaling
and tumorigenesis (Martin etal. 2014). Rapamycin is currently the standard of care
for renal transplant recipients with Kaposis sarcoma (Stallone etal. 2005). Thus,
compounds targeting eIF4E could be used to treat eIF4E-dependent oncoviruses.
This finding has been expanded to a mouse model of Epstein-Barr virus-related
Burkitts lymphoma, where treatment with rapamycin decreased tumor growth and
metastasis (Cen and Longnecker 2011).
Interestingly, the opposite effect of eIF4E inhibition is seen in oncolytic viruses,
which preferentially kill cancer cells. Infection of tumors by these viruses in combi-
nation with mTOR inhibitors has improved the efficacy of viral cancer treatments.
Rapamycin enhances myxoma virus infection in cancer cells (Stanford etal. 2007),
as well as vesicular stomatitis virus (VSV) oncolysis, without toxicity to the host
(Alain etal. 2010). Similarly, oncolytic poliovirus and inhibitors of the PI3K path-
way act synergistically to treat glioblastoma multiforme (Goetz etal. 2010). In these
situations, inhibiting eIF4E-dependent translation is thought to restrict the produc-
tion of type I interferon (IFN), thus enhancing viral spread (Alain etal. 2010).
However, VSV mRNAs are translated in a cap-dependent manner, and, therefore,
inhibiting eIF4E could be counterproductive. While this issue has yet to be experi-
mentally addressed, a possible explanation may come from the intrinsically high
translation of cancer cells, where the balance between eIF4E and the 4E-BPs is
dysregulated. Translational inhibition would occur only for mRNAs that are highly
sensitive to eIF4E, such as the IFN regulatory factor 7 (IRF7), which controls type I
IFN expression (Colina etal. 2008). In contrast, efficiently translated mRNAs, such
as those encoded by viruses, would require only low levels of eIF4E, allowing viral
replication to proceed unhindered.
4.9eIF4E and the Hallmarks of Cancer
The accumulated knowledge on eIF4E and its regulation overwhelmingly points to

its critical role in cancer. Its tumorigenic properties were first described in 1990 by
transforming fibroblasts upon overexpression (Lazaris-Karatzas etal. 1990). There
has since been a plethora of studies establishing eIF4E as an oncoprotein and dis-
secting its role in cancer (Blagden and Willis 2011; Mamane etal. 2004; Martineau
etal. 2013; Sonenberg 2008; Sonenberg and Hinnebusch 2009; Topisirovic etal.
2011; Wendel etal. 2007). A detailed framework of the essence of cancer was in-
troduced in 2000 by Hanahan and Weinberg (Hanahan and Weinberg 2000) and
is known as the Hallmarks of Cancer. This system describes a set of six char-
acteristics that cancer cells must acquire to evolve from normalcy to malignancy,
which are: (1) evading growth suppressors; (2) sustaining proliferative signaling;
(3) enabling replicative immortality; (4) resisting cell death; (5) inducing angio-
genesis and (6) activating invasion and metastasis. This list was later updated to
include more recent findings. The updated version includes two novel hallmarks:
reprogramming energy metabolism and evading immune destruction. In addition,
so-called enabling characteristics are described, which favor the acquisition of the
hallmarks of cancer: genomic instability and tumor-promoting inflammation (Ha-
nahan and Weinberg 2011). The role of eIF4E in cancer biology is presented below
in agreement with the hallmarks of cancer (Fig.4.2). At times, the boundaries be-
tween the roles of eIF4E in each hallmark appear blurred, as certain factors play a
role in multiple hallmarks. Nonetheless, they present a useful framework for the
comprehensive understanding of eIF4E in cancer.
4.9.1Evading Growth Suppressors
The first experiment identifying eIF4E as an oncoprotein established its role in evad-
ing growth suppression. Overexpression of eIF4E conferred on NIH 3T3 cells the
ability to escape contact inhibition and form foci (Lazaris-Karatzas etal. 1990). Sub-
sequently, eIF4E was found to promote the nuclear export of MDM2 (mouse double
minute 2 homolog, also known as HDM2), which degrades the prototypical tumor
suppressor p53 (Phillips and Blaydes 2008). It also was shown to facilitate the transla-
tion of various cyclins (A, D1, D3, E1), which can override growth suppressive signals
(Deffie etal. 1995; Lukas etal. 1997). In contrast, overexpression of 4E-BP1 result-
ed in downregulation of these factors with a concomitant increase in CDK inhibitor
p27kip1, resulting in cell cycle arrest (Jiang etal. 2003). Thus, eIF4E availability causes
the evasion of growth suppressors by promoting the export and translation mRNAs
encoding factors that can either degrade them (MDM2) or bypass them (cyclins).
4.9.2Sustaining Proliferative Signaling
Perhaps the most studied outcome of overexpressing eIF4E is the increase in the
translation of mRNAs encoding multiple factors that promote proliferation inde-
Fig. 4.2 The eIF4E/4E-BPs

Evad
axis and the hallmarks of ing i
cancer. Summary of the role u s tain tive grow ng
S e r a s upp t h
of eIF4E and the 4E-BPs lif
pro naling ress
o
en llul ing
in promoting the different sig rs
ce ulat
hallmarks of cancer. Black
Av mu tion
erg ar
cs
HDM
im truc
oi d n e
de
yc
arrows indicate hallmarks c-M s cycli 2
eti
reg
ing
n ns
cycli
rt
po
promoted by eIF4E. Red bars
De
ns
tra
indicate hallmarks inhibited
i al
?
dr
on
???
ch
by the 4E-BPs. Examples
eIF4E
to
Mi
of mRNAs regulated by Resisting Enabling
MCL1
eIF4E/4E-BPs are indicated cell BCL-xL replicative
next to each hallmark. Black death immortality
boxes indicate emerging
ok es
s
hallmarks. Red boxes indicate
ine
em in
ch ytok
?
enabling characteristics.
mo r-
g
cers
pro umo
Ge tabi tion
tin
VEG
indu
ins uta
(Modified from Hanahan and

no lity
FA
&m
FGF EMT teases

m
2 r o
Weinberg (2011)).
T
p
e
ing
Ind
ang ucing ivat
Act sion &
ioge
nesi inva stasis
s ta
me
pendently of external cues. These factors include the insulin-like growth factor
(IGF), the hepatoma-derived growth factor (HDGF) and the placental growth factor
(PGF) (Larsson etal. 2007; Mamane etal. 2007); cyclins A, D1, D3, E1 and cyclin-
dependent kinases CDK 2 and 4 (Larsson etal. 2007, 2012; Rousseau etal. 1996b);
the transcription factor c-MYC (Darveau etal. 1985; Saito etal. 1983) and others.
eIF4E also engenders RAS (rat sarcoma viral oncogene homolog) hyperactivation
(Lazaris-Karatzas etal. 1992), thus sustaining proliferative signaling independently
of growth factors. The 4E-BPs negatively regulate the proliferative functions of
eIF4E. In fact, eIF4E inhibition by the 4E-BPs is the mechanism by which mTOR
and its inhibitors affect proliferation (Dowling etal. 2010). Thus, similar to evasion
of growth suppression, eIF4E functions to sustain proliferative signals via transla-
tional upregulation of specific sets of mRNAs.
4.9.3Enabling Replicative Immortality
The main barriers to replicative immortality are telomere shortening and senescence
(Hanahan and Weinberg 2011). eIF4E appears to be insufficient to sustain repli-
cative immortality, as its overexpression cannot rescue telomere-dependent crisis
and mortality in primary human mammary epithelial cells (HMECs) (Larsson etal.
2007). In addition, as seen with other proto-oncogenes, eIF4E overexpression or
hyperactivation, via loss or phosphorylation of the 4E-BPs, promotes senescence in
various models (Kolesnichenko etal. 2012; Petroulakis etal. 2009; Ruggero etal.
2004). This effect likely occurs through increased translation of inhibitors of the
cell cycle, such as GAS2 (growth arrest-specific protein 2) and p21 (Kolesnichenko
etal. 2012; Petroulakis etal. 2009). Thus, eIF4E does not cause replicative immor-
tality. However, it can synergize with other oncogenes that do directly enable repli-
cative immortality for the induction of neoplastic transformation. Indeed, eIF4E can
cooperate with overexpression of human telomerase reverse transcriptase (hTERT)
(Larsson etal. 2007) or c-MYC (Ruggero etal. 2004), as well as with the loss of
p53 (Petroulakis etal. 2009) to promote tumorigenesis. Thus, eIF4E-driven cancers
require at least a second hit to enable replicative immortality.
4.9.4 Resisting Cell Death
It is well established that eIF4E promotes resistance to cancer cell death by in-
creasing the translation of survival factors such as BCL-XL, MCL-1, BIRC2, sur-
vivin and others (Furic etal. 2010; Larsson etal. 2006; Mamane etal. 2007). Their
upregulation via eIF4E overexpression promotes resistance to the induction of
both mitochondria-mediated and endoplasmic reticulum-mediated apoptosis (Li
etal. 2003, 2004,). On the other hand, the 4E-BPs are sufficient to block eIF4E
and/or c-MYC-driven transformation, resulting in increased cancer cell death
(Lynch etal. 2004). Thus, increasing the pool of available eIF4E results in cancer
cell survival.
4.9.5Inducing Angiogenesis
The angiogenic program is essential for tumor development, growth and metastasis
(Folkman etal. 1963). To achieve this, cancer cells produce factors which favor the
proliferation of endothelial cells and vascularization of the tumor, including vascu-
lar endothelial growth factor A (VEGF-A) and VEGF-C (a lymphoid-specific mem-
ber of the VEGF family), as well as the fibroblast growth factor (FGF2), among oth-
ers. These factors are under translational control by eIF4E (Furic etal. 2010; Kevil
etal. 1996; Nathan etal. 1997a; Scott etal. 1998). Accordingly, high eIF4E levels
in breast cancers, head and neck cancers and non-Hodgkins lymphomas correlate
with VEGF-A expression and microvessel density (Byrnes etal. 2006; Nathan etal.
1999b; Zhao etal. 2005; Zhou etal. 2006). Thus, dysregulation of eIF4E promotes
angiogenesis and tumor vascularization.
4.9.6 Activating Invasion and Metastasis
One of the most clinically relevant hallmarks of cancer is the ability to invade the
tissue surrounding the primary tumor and eventually form distant metastatic colo-
nies. Recent studies have highlighted the role of eIF4E in this process. Translational
control via eIF4E overexpression, availability and phosphorylation is required for
transforming growth factor (TGF-)-induced epithelial-mesenchymal transition
(EMT) and invasion (Ghosh etal. 2009; Grzmil etal. 2011; Hsieh etal. 2012; Pola
etal. 2013). Accordingly, eIF4E availability through mTOR activation as well as
eIF4E phosphorylation promote metastasis in mouse models of breast cancer (Nasr
etal. 2013; Robichaud et al. 2014). eIF4E promotes the translation of mRNAs en-
coding proteins involved in invasion and metastasis, including the Y box-binding
protein 1 (YB-1), SNAIL, MMPs, SMAD2, vitronectin, integrins and others (Ghosh
etal. 2009; Grzmil etal. 2011; Hsieh etal. 2012; Nasr etal. 2013; Pola etal. 2013).
4.9.7 Emerging Hallmarks and Enabling Characteristics
Additional hallmarks of cancer are emerging as our knowledge of cancer accu-

mulates (Hanahan and Weinberg 2011). For example, the dysregulation of cellular
energetics, a novel hallmark, may be influenced by eIF4E overexpression via in-
creased synthesis of factors involved in mitochondrial transport, the cellular energy
machinery (Larsson etal. 2012). Furthermore, the Warburg effect, which consists
of a rewired energy metabolism to promote the rapid proliferation of cancer cells,
may rely on dysregulated eIF4E (Topisirovic and Sonenberg 2011). With regards to
the enabling characteristics of cancer, eIF4E could be involved in tumor-promoting
inflammation, as evidenced by its important role in the expression of numerous cy-
tokines and chemokines (Joshi etal. 2010; Joshi and Platanias 2012) and guidance
of CD4+T cells towards distinct subpopulations (Bjur etal. 2013). Furthermore,
4E-BP1 has been implicated in maintaining DNA stability during mitosis (Shang
etal. 2012), providing evidence that eIF4E activation could be important in induc-
ing genomic instability. As our knowledge of cancer biology in general and eIF4E
function in particular increases, the multifaceted roles of eIF4E in tumorigenesis
are becoming clearer. Its involvement in almost every facet of cancer development
and progression explains its frequent dysregulation in human tumors. It is therefore
critical to understand how dysregulation of eIF4E and its upstream signaling path-
ways can occur.
4.10An eIF4E-Centric View of Tumorigenic Signaling
Most cancers are caused by mutations that lead to abnormal signaling, proliferation
and survival. According to the Catalogue of Somatic Mutations in Cancer (COS-
MIC) database (Bamford etal. 2004), mutations in the 4E-BPs (<0.1%)1 or in eIF4E
Hereafter, percentages indicate the frequency of mutations in human cancers according to COS-
1
MIC database
itself (0.2%) are rare and generally consist of synonymous substitutions. This is not
surprising considering eIF4Es crucial role in the fundamental process of translation
and its high conservation across eukaryotes (Aravind and Koonin 2000). Hence,
cancer cells develop other means of dysregulating eIF4E. A frequent means is to
dysregulate upstream signaling pathways that feed into eIF4E. Thus, commonly
mutated signal transducers can (1) transcriptionally induce eIF4E expression via
c-MYC activation; (2) increase eIF4E phosphorylation via the MNKs; (3) increase
eIF4E availability via mTOR phosphorylation of the 4E-BPs. For these reasons,
most cancers exhibit abnormal eIF4E levels or activity (Sonenberg and Hinnebusch
2009).
4.10.1eIF4E at the Convergence Point of Oncogenic Signaling
eIF4E activates translation of a subset of mRNAs in response to external cues (Rob-

ert and Pelletier 2009). Signaling pathways that are induced in response to these
cues converge on eIF4E to regulate its availability, expression and activity. Thus,
eIF4E is a key hub integrating signals from the major signaling pathways. In the
context of cancer, fundamental signaling pathways that have been shown to par-
ticipate in tumorigenesis converge on eIF4E and depend on its function in transla-
tion (Fig.4.3) (Hou etal. 2012; Petroulakis etal. 2007; Topisirovic and Sonenberg
2011). The most frequently mutated gene in human cancers encodes the tumor sup-
pressor TP53 (30%). One of the many roles of p53 is binding to c-MYC to prevent
transcriptional activation of eIF4E (Zhu etal. 2005). Hence, mutations in TP53,
that generally result in loss of expression or loss of function, would lead to release
of c-MYC and high eIF4E expression. Moreover, p53 mediates senescence through
a 4E-BP-dependent pathway (Petroulakis etal. 2009), suggesting further interplay
between p53 and the eIF4E/4E-BP axis.
Next to TP53, two of the most frequently mutated genes in human cancers are
KRAS (22%) and the v-RAF (rapidly accelerated fibrosarcoma) murine sarcoma
viral oncogene homolog B (BRAF, 19%), both of which lead to activation of the
MEK/ERK pathway and phosphorylation of eIF4E by the MNKs. In addition, acti-
vated ERK leads to phosphorylation of c-MYC and induction of its transcriptional
targets, including eIF4E (Sears etal. 2000). Cancers driven by these activating mu-
tations are highly dependent on eIF4E for tumorigenesis (Croft etal. 2014; Lazaris-
Karatzas etal. 1992). Thus, oncogenic mutations in the MEK/ERK pathway dys-
regulate eIF4E, which in turn promotes their tumorigenic effects.
Mutations in components of the PI3K/AKT/mTOR pathway are common in tu-
mors. Dysregulation of this pathway leads to hyperphosphorylation of the 4E-BPs
and increased eIF4E availability (Mendez etal. 1996). For example, mutations in
the PI3K catalytic subunit (PIK3CA) gene (11%) activate this pathway (Yuan and
Cantley 2008). The gene encoding the phosphatase and tensin homolog (PTEN), a
well-characterized tumor suppressor that normally inhibits the PI3K/AKT/mTOR
pathway, is mutated in 9% of cancers, leading to aberrantly activated signaling
Fig. 4.3 eIF4E Tumorigenic signaling hub. Top part Examples of external cues initiating tran-
scriptional and/or translational responses. Middle part Major signaling pathways converging on
eIF4E; also shown are translationally regulated mRNAs relevant to cancer and/or that play impor-
tant roles in these pathways. Bottom part Examples of transcriptional activation downstream of
major signaling pathways; examples of transcriptionally induced factors relevant to cancer, that
regulate eIF4E or that are regulated by eIF4E.
and tumorigenesis (Yuan and Cantley 2008). Although less frequent, mutations in
other components, such as AKT and tuberous sclerosis complex 1 and 2 proteins
(TSC1 and TSC2), activate the mTOR/4E-BPs/eIF4E axis and drive cancer devel-
opment (Yuan and Cantley 2008). The critical importance of eIF4E downstream of
dysregulated PI3K/AKT/mTOR is demonstrated by the fact that cancers driven by
mutations in this pathway are exquisitely sensitive to eIF4E activity (Hsieh etal.
2010; Neshat etal. 2001). In addition, overexpression or hyperactivation of eIF4E
commonly confers resistance to PI3K and mTOR inhibitors, further emphasizing
the fundamental importance of eIF4E as an oncogenic effector of PI3K signaling
(Cope etal. 2014; Ilic etal. 2011; Wendel etal. 2004).
NOTCH1 (Notch homolog, translocation-associated (Drosophila)) is mutated
in 8% of cancers, particularly in hematopoietic and lymphoid tissues (17%). It is
thought to dysregulate eIF4E by feeding into the mTOR pathway (Chan etal. 2007;
Graziani etal. 2008; Mungamuri etal. 2006) and by increasing c-MYC expression
(Shepherd etal. 2013). Similarly, WNT (Drosophila melanogaster wingless gene,
human proto-oncogene protein) mutations (11%) lead to activation of the mTOR
pathway by relieving inhibition by glycogen synthase kinase 3 (GSK3)/TSC2
(Inoki etal. 2006; Jin etal. 2008) and promote the transcription of c-MYC via
-catenin (He etal. 1998), thus dysregulating eIF4E expression and availability.
For both pathways, inhibition or downregulation of eIF4E was shown to abrogate
their tumorigenicity (Lim etal. 2013; Mungamuri etal. 2006; Song and Lu 2011).
Taken together, these findings emphasize an important paradigm, that major signal-
ing pathways implicated in cancer biology converge on eIF4E and assert their func-
tion in part via eIF4E-dependent activation of translation.
4.10.2 eIF4E Regulates Upstream Signaling
While eIF4E is generally thought of as a downstream effector of the major signal-

ing pathways, it can regulate these pathways in feedback signaling loops. This was
first shown in NIH 3T3 cells, where overexpression of eIF4E led to RAS activa-
tion, which was required for eIF4Es oncogenic activity (Lazaris-Karatzas etal.
1992). The best-known example is the regulation of c-MYC and eIF4E by a feed-
forward loop, which has been discussed above. Another example is the finding that
-catenin, a transcription factor in the WNT pathway, is translationally regulated by
eIF4E (Karni etal. 2005). As described above, eIF4E is also an effector of WNT
signaling, and thus eIF4E and WNT signaling regulate each other (Fig.4.3). This
interaction has recently been shown to promote leukemia stem cell maintenance
and function (Lim etal. 2013). Furthermore, TGF- signaling activates the mTOR
and MNK kinases, thus increasing eIF4E activity and translational activation of
SMAD2, which in turn promotes TGF-s transcriptional program (Grzmil et al.
2011). In glioblastomas, the TGF-/eIF4E coregulation leads to enhanced EMT and
invasion (Grzmil etal. 2011). Thus, an important concept is emerging that highlights
the importance of the crosstalk between eIF4E and upstream signaling pathways.
4.10.3eIF4E as the Translational Hub of Complex Signaling

Networks
The complex interplay between eIF4E and signaling networks is critical for an inte-
grated regulation of transcription and translation (Fig.4.3). Indeed, major signaling
pathways possess both transcriptional and translational branches that act coopera-
tively to affect gene expression (Bader etal. 2005). Such cooperation is well docu-
mented in bacteria, where transcription and translation are directly coupled (Prosh-
kin etal. 2010). However, in mammals, this coupling is much more subtle, where
transcriptional programs activated by oncogenic signaling include factors which
depend strongly on concomitant translational activation. For example, WNT signal-
ing includes a transcriptional branch through -catenin and a translational branch
through the mTOR/4E-BPs/eIF4E axis (Fig.4.3). While -catenin promotes the
transcription of c-MYC (He etal. 1998), eIF4E is required for its translation (West
etal. 1995). Thus, signaling through eIF4E promotes the translation of mRNAs,
which are transcriptionally induced by the same signaling pathways. Further sup-
port for this notion is provided by the recent finding that TGF- signaling includes
both a transcriptional program via SMAD2 phosphorylation and a translational
program via eIF4E phosphorylation (Robichaud et al. 2014). A subset of mRNAs
transcriptionally induced by SMAD2, including SNAIL and MMP3, require the
phosphorylation of eIF4E for effective translation. Similarly, the NF-B pathway
leads to enhanced expression of PIK3CA and increased mTOR signaling (Yang
etal. 2008) to promote the eIF4E-dependent translation of other NF-B targets such
as BCL-2 and cyclin D1 (Robert and Pelletier 2009). In light of these findings, it
is important to consider the role of translational control via eIF4E in the context of
specific transcriptional programs.
4.11eIF4E and 4E-BPs in Human Cancers
To assess the relevance of the basic research on eIF4E/4E-BPs to cancer patients, it

is necessary to confirm the dysregulation of this axis in human tumors. This section
summarizes the studies performed on human samples. The different means of eIF4E
dysregulation and their associated clinicopathologic features are discussed for vari-
ous cancers. As oncogenic dysregulation of eIF4E was first reported for breast can-
cer (Kerekatte etal. 1995), much of the information on the role of eIF4E in human
cancer presented here will be pertinent to breast cancer biology. Details of eIF4E
involvement in other cancers are described and summarized in Table4.2.
4.11.1eIF4E Overexpression
In breast cancer, eIF4E was found to be overexpressed in the majority of cases,

with levels reaching as high as 30 times that in the adjacent normal tissue (Li etal.
Table 4.2 eIF4E dysregulation in human cancers.

Cancer Dysregulation Clinical features
Breast eIF4E overexpression Increased risk of recurrence and death (Holm etal.
2008; Li etal. 1998, 2002; McClusky etal. 2005);
correlates with microvessel density and expression of
FGF2 and VEGF (Byrnes etal. 2006; Nathan etal.
1997a; Scott etal. 1998); correlates with aggressive
subtypes and resistance to chemotherapy (Heikkinen
etal. 2013)
eIF4E phosphorylation High in early stages of cancer development (Fan etal.
2009b)
4E-BP1 overexpression Inversely correlates with tumor grade (Coleman etal.
2009)
4E-BP1 phosphorylation Correlates with tumor grade and poor prognosis (Cole-
man etal. 2009; Rojo etal. 2007; Zhou etal. 2004)
Bladder eIF4E overexpression Correlates with poor prognosis and increased VEGF
expression (Crew etal. 2000)
Brain eIF4E overexpression Correlates with increased malignancy in meningiomas,
glioblastomas and astrocytomas (Tejada etal. 2009)
Cervix eIF4E overexpression Increases according to histopathologic grades (Lee etal.
2005; Matthews-Greer etal. 2005), correlates with
VEGF and mean vessel density (Mathews-Greer
etal. 2002)
Colon eIF4E overexpression Occurs at early stages of cancer progression due to
dysregulated -catenin (Rosenwald etal. 1999),
associates with histological type (Berkel etal. 2001),
correlates with VEGF expression (Yang etal. 2007)
eIF4E phosphorylation Elevated in early stages of tumor development (Fan
etal. 2009b)
4E-BP1 phosphorylation Elevated in high-grade intraepithelial neoplasias (Zhang
2009)
Liver eIF4E overexpression Correlates with poor prognosis (Wang etal. 2012)
Lung eIF4E overexpression Correlates with aggressive histological subtypes (Ros-
enwald etal. 2001; Seki etal. 2002), correlates with
VEGF expression (Yang etal. 2007)
eIF4E phosphorylation Elevated in early stages of tumor development (Fan
etal. 2009b), poor prognosis marker in non-small-
cell lung cancer (Yoshizawa etal. 2010)
4E-BP1 overexpression Correlates with better survival (Seki etal. 2010)
4E-BP1 phosphorylation Correlates with poor prognosis in adenocarcinomas
(Trigka etal. 2013)
Head and eIF4E overexpression Expression in surgical margins correlates with recur-
neck rence and mortality (Franklin etal. 1999; Nathan
etal. 1997b, 1999a).
4E-BP1 phosphorylation Elevated in malignant squamous cell carcinomas (Fred-
erick etal. 2011)
Lym- eIF4E overexpression Correlates with aggressiveness in non-Hodgkins
phoma lymphomas (Wang etal. 1999), correlates with VEGF
expression (Yang etal. 2007)
eIF4E phosphorylation Correlates with antiapoptotic gene expression in diffuse
large B-cell lymphoma (DLBCL) (Wendel etal.
2007)

Cancer Dysregulation Clinical features
4E-BP1 overexpression High in mature B-cell lymphomas (Kodali etal. 2011)
4E-BP1 phosphorylation Elevated in anaplastic large cell lymphomas (Vega etal.
2006)
Ovary eIF4E overexpression Correlates with VEGF expression (Yang etal. 2007)
eIF4E phosphorylation Correlates with better survival (Noske etal. 2008)
4E-BP1 phosphorylation Poor prognosis marker (Castellvi etal. 2006), associ-
ated with poor differentiation and higher mitotic rate
(Noske etal. 2008)
Esophagus eIF4E overexpression High levels associate with advanced tumor stages
(Salehi and Mashayekhi 2006), correlates with
MDM2 expression (Hsu etal. 2011)
4E-BP1 overexpression Inversely correlates with advanced tumor stages (Salehi
and Mashayekhi 2006)
Pancreas 4E-BP1 loss In 50% of tumors, appears to be responsible for loss of
translational control (Martineau etal. 2014)
Prostate eIF4E overexpression Correlates with poor survival (Graff etal. 2009), cor-
relates with VEGF expression (Yang etal. 2007)
eIF4E phosphorylation Increased in human samples (Graff etal. 2009), corre-
lates with androgen independence (Furic etal. 2010)
4E-BP1 overexpression Uniform expression correlates with better survival
(Graff etal. 2009)
4E-BP1 phosphorylation Correlates with poor survival (Graff etal. 2009)
Skin eIF4E overexpression Levels increase with progression of squamous cell
carcinoma (Salehi etal. 2007), correlates with VEGF
expression in melanoma (Yang etal. 2007)
Stomach eIF4E overexpression Correlates with vascular invasion and poor prognosis
(Chen etal. 2004)
eIF4E phosphorylation High in early tumor stages (Fan etal. 2009b)
4E-BP1 overexpression Correlates with absence of lymph node and distant
metastases (Martin etal. 2000)
1997). Overexpression of eIF4E has since been observed in a variety of cancers

(Table4.2). Two mechanisms to increase eIF4E levels have been described in hu-
mans. Gene amplification has been shown to lead to increased eIF4E mRNA and
protein levels in breast cancer and head and neck cancer (Sorrells etal. 1998, 1999)
as well as early onset colorectal cancer (CRC) (Berg etal. 2010). Transcriptional
upregulation of eIF4E due to oncogenic c-MYC is thought to play a major role in
non-Hodgkins lymphomas (Wang etal. 1999) and CRCs (Melhem etal. 1992;
Rosenwald etal. 1999).
4.11.2 Expression of the 4E-BPs
Another mechanism to dysregulate eIF4E in human tumors is through loss of ex-

pression of the 4E-BPs. Some cancers, such as pancreatic tumors, rely on decreased
4E-BP1 expression to increase the availability of eIF4E (Martineau etal. 2014).

Low 4E-BP1 levels have been reported in other cancers such as breast, lung and
prostate (Coleman etal. 2009; Graff etal. 2009; Seki etal. 2010). It remains unclear
whether the low expression of the 4E-BPs in these cancers is due to regulation at the
genetic, transcriptional, translational or posttranslational levels.
4.11.3 Phosphorylation of the 4E-BPs
Increased eIF4E availability due to hyperactivated mTOR signaling, which results

in increased 4E-BP1 phosphorylation is commonly encountered in various malig-
nancies including breast, colon, and ovarian cancers (Castellvi etal. 2006; Coleman
etal. 2009; Noske etal. 2008; Rojo etal. 2007; Zhang 2009; Zhou etal. 2004). In
an unbiased phosphoproteomic screen, phosphorylated 4E-BP1 was found to be
highly increased in head and neck squamous cell carcinomas (HNSCC) (Frederick
etal. 2011).
4.11.4eIF4E Phosphorylation
Another mechanism of eIF4E activation in human cancer is its phosphorylation via

oncogenic MAPK signaling (see Chap.17). Phosphorylated eIF4E is elevated in
most human cancers including lung, breast, HNSCC and prostate (Fan etal. 2009b;
Frederick etal. 2011; Graff etal. 2009). While mutations in upstream signaling mol-
ecules, such as KRAS are likely candidates, the precise genetic perturbations leading
to increased phosphorylation of eIF4E have yet to be determined.
4.12eIF4E/4E-BPs and Clinical Correlates
4.12.1 e IF4E and 4E-BPs in Cancer Recurrence and

Survival
Perhaps the most important utility of eIF4E in cancer is as a biomarker predicting

poor prognosis. Indeed, studies in breast cancer have revealed that increased expres-
sion of eIF4E correlates with increased risk of recurrence and decreased survival, ir-
respective of the nodal status (Holm etal. 2008; Li etal. 1998, 2002; McClusky etal.
2005). The predictive value of eIF4E in the clinic has since been extended to cancers
of the prostate, head and neck, pharynx, lung, bladder, liver, esophagus and stomach
(Chen etal. 2004; Crew etal. 2000; Graff etal. 2009; Nathan etal. 1999a; Salehi and
Mashayekhi 2006; Seki etal. 2002, 2010; Wang etal. 2012; Wu etal. 2013).
In osteosarcoma and acute myeloid leukemia (AML), eIF4E levels were reported
to be elevated, but did not correlate with poor prognosis (Green etal. 2012; Osborne
etal. 2011). This discrepancy may be explained by other mechanisms to dysregulate
eIF4E such as decreased expression of the 4E-BPs or their increased phosphoryla-
tion. Indeed, various studies have demonstrated that 4E-BP status must be taken
into account when assessing prognosis based on eIF4E (Coleman etal. 2009; Rojo
etal. 2007; Zhou etal. 2004). Thus, phosphorylation of 4E-BP1 predicts poor sur-
vival in breast cancer (Rojo etal. 2007; Zhou etal. 2004), and combined analysis
of eIF4E, 4E-BP1 and their phosphorylation status performs better as a prognostic
tool than eIF4E levels alone (Coleman etal. 2009). In a specific subset of recep-
tor positive breast cancer, where eIF4E did not show prognostic value, analysis of
phosphorylated 4E-BP1 predicted poor outcome (Meric-Bernstam etal. 2012). In
prostate cancer, high eIF4E levels or increased levels of phospho-4E-BP1 inde-
pendently associated with poor survival, whereas high total 4E-BP1 displayed the
opposite association (Graff etal. 2009). Similar findings were obtained in gastroin-
testinal (Martin etal. 2000), ovarian (Castellvi etal. 2006) and esophageal cancers
(Salehi and Mashayekhi 2006).
The phosphorylation of eIF4E has also been investigated for its role as a prog-
nostic marker, with varying results. High phospho-eIF4E correlates with poor sur-
vival in non-small-cell lung carcinoma (NSCLC) (Yoshizawa etal. 2010), and with
recurrence and metastasis in penile squamous cell carcinoma (Ferrandiz-Pulido
etal. 2013). However, the opposite correlation was found in ovarian cancer (No-
ske etal. 2008). Therefore, the prognostic value of eIF4E phosphorylation requires
more investigation and will most likely be more valuable in the context of total
eIF4E levels and 4E-BP status.
The prognostic value of eIF4E is particularly interesting in the case of HNSCC,
which is characterized by high lethality due to local recurrence, rather than metas-
tasis (see Chap.25). Complete resection of tumors is the key to avoiding recurrence
and associated mortality. Therefore, defining the tumor margins is of critical impor-
tance. Levels of eIF4E are high in HNSCC, but remain low in normal tissue and
benign lesions (Nathan etal. 1997b). Most importantly, in tumor margins that ap-
pear histologically normal, as little as 5% of eIF4E-positive cells predict recurrence
and mortality whereas eIF4E-negative margins predict increased survival (Franklin
etal. 1999; Nathan etal. 1999a, 1997b). The presence of eIF4E in surgical margins
has been identified as an independent prognostic factor and can be used to alter
surgical management (Franklin etal. 1999). Furthermore, mTOR is highly activated
in tumor margins (Nathan etal. 2004). Accordingly, high eIF4E/4E-BP1 ratio cor-
relates with disease recurrence better than the analysis of eIF4E expression alone in
HNSCC (Sunavala-Dossabhoy etal. 2011).
It is noteworthy that eIF4E expression has also been associated with the success
of chemotherapeutic treatment. In one study of breast cancer, tumors presenting
relatively low eIF4E levels (<7.5 fold increase over normal tissue) displayed a de-
creased probability of recurrence after neoadjuvant treatment. Interestingly, some
tumors in this study expressed high eIF4E levels (>7.5 fold increase over normal
tissue) before neoadjuvant therapy, but low levels of eIF4E after treatment. Patients
with such tumors had a decreased probability of cancer recurrence (Hiller etal.
2009). In another, much larger, study high eIF4E expression was associated with
failure of anthracycline chemotherapy (Heikkinen etal. 2013). Thus, not only can
the analysis of eIF4E dysregulation predict patient prognosis, it can also be used in
the evaluation of treatment options.
4.12.2 eIF4E and 4E-BPs in Cancer Grade and Progression
An important consideration in cancer progression is the extent of vascularization of

the tumor. One of the features of cancers with dysregulated eIF4E is their propen-
sity to be highly vascularized. A direct correlation between high eIF4E expression
and microvessel density and vascular invasion has been observed in cancers of the
breast, cervix and stomach (Byrnes etal. 2006; Chen etal. 2004; Matthews-Greer
etal. 2005). This is likely due to translational activation of eIF4E-sensitive an-
giogenic factors. In these vascularized cancers, eIF4E overexpression correlated
with high expression of VEGF-A (Byrnes etal. 2006; Matthews-Greer etal. 2005;
Nathan etal. 1997a; Scott etal. 1998). Both high VEGF-A and eIF4E levels were
documented in tumors of the bladder, colon, lung, ovary, prostate and skin cancers,
as well as in lymphomas (Crew etal. 2000; Yang etal. 2007). Another factor favor-
ing tumor vascularization, FGF2 was associated with increased eIF4E expression in
breast cancer (Nathan etal. 1997a). Unfortunately, research into the role of eIF4E
phosphorylation and the 4E-BPs in tumor vascularization is lagging, hindering a
complete analysis of dysregulated eIF4E in assessing tumor vascularization.
In contrast to vascularization, histological grades of certain cancers have been
associated with various aspects of eIF4E regulation other than overexpression. For
example, levels of phospho-eIF4E were observed to be highest at early stages of
tumor progression in lung, gastric and CRCs (Fan etal. 2009b). In prostate cancer,
phosphorylation of eIF4E correlated with the androgen independent stage of the
disease (Furic etal. 2010). With regards to the 4E-BPs, high 4E-BP1 expression
inversely correlated with tumor grade in breast cancer (Coleman etal. 2009) and
esophageal cancer (Salehi and Mashayekhi 2006), as well as with the absence of
lymph node and distant metastases in gastric cancers (Martin etal. 2000). In con-
trast, phosphorylated 4E-BP1 correlated with advancing tumor grade in breast can-
cer (Coleman etal. 2009; Rojo etal. 2007; Zhou etal. 2004) and with high-grade
intraepithelial neoplasia in colon cancer (Zhang 2009). In terms of eIF4E levels,
they have been found to increase with histological grade in cancers of the cervix
(Lee etal. 2005; Matthews-Greer etal. 2005), colon (Berkel etal. 2001), esophagus
(Salehi and Mashayekhi 2006) and skin (Salehi etal. 2007). Overexpression of
eIF4E also correlated with aggressive subtypes of lung cancers (Rosenwald etal.
2001; Seki etal. 2002), lymphomas (Wang etal. 1999) and breast cancer (Heik-
kinen etal. 2013). Additionally, eIF4E has been analyzed in the context of breast
cancer molecular subtypes. In one study, the highest levels of eIF4E mRNA were
detected in the luminal B subtype (Pettersson etal. 2011). The predictive power of
eIF4E overexpression also performed best in this subtype (Pettersson etal. 2011).
Taken together, studies using patient samples demonstrate that eIF4E is a criti-
cal player in human cancers. They support many of the findings originating from
basic research such as the relative roles of eIF4E and its binding partners in cancer.
Furthermore, the significance of cancer-promoting factors, which are controlled by
eIF4E and the 4E-BPs, such as VEGF-A were validated by human studies. Most im-
portantly, the findings derived from patient data highlight the importance of eIF4E
as a biomarker and argue for the development of inhibitors of the eIF4E/4E-BPs
axis for cancer therapy.
4.13Therapeutic Strategies and Resistance to Therapy
There is much interest in therapeutic targeting of eIF4E. Every aspect of its activity
and regulation is under investigation for drug development. Cap analogs competing
with mRNAs for eIF4E binding are the oldest class of eIF4E inhibitors, but have
had limited use in cell culture and animal systems due to permeability and insta-
bility issues in vivo (Wagner etal. 2000). However, a recently developed prodrug
termed 4Ei-1 has been engineered to overcome these limitations and is reported
to function in vivo (Ghosh etal. 2009; Li etal. 2013). Formation of the eIF4F
complex can be targeted either by blocking the interaction between eIF4E and eI-
F4G using a compound such as 4EGI-1 (Moerke etal. 2007) or by using allosteric
or active site inhibitors of mTOR such as rapamycin, PP242, Torin 1 and others
(Apsel etal. 2008; Feldman etal. 2009; Thoreen etal. 2009; Zhang etal. 2011).
The mTOR inhibitors prevent 4E-BP phosphorylation and lead to sequestration of
eIF4E. Inhibitors of upstream signaling pathways can also be used to prevent eIF4E
phosphorylation, such as the MNK inhibitor cercosporamide (Konicek etal. 2011).
Finally, eIF4E levels can be reduced using ASOs (Graff etal. 2007). These various
strategies are discussed in detail in Chap.14.
The importance of developing inhibitors targeting eIF4E is bolstered by the find-
ing that dysregulated eIF4E confers resistance to existing and developing cancer
therapies. In various cancer cell lines, eIF4E overexpression and 4E-BP1 hyper-
phosphorylation promote resistance to ionizing radiation (Hayman etal. 2012; Su-
navala-Dossabhoy etal. 2004). This effect is thought to be mediated by increased
translation of mRNAs encoding survival, DNA repair and radioresistance factors,
such as Tousled-like kinase 1B (TLK1B) (Sunavala-Dossabhoy etal. 2004). eIF4E
has also been shown to promote resistance to the DNA-damaging agents doxorubi-
cin and cisplatin, and the antimitotic microtubule stabilizers paclitaxel and docetax-
el (Dong etal. 2009; Zhou etal. 2011). This effect appears to be due to the role of
eIF4E in promoting survival through translational upregulation of antiapoptotic fac-
tors (Zhou etal. 2011). Furthermore, eIF4E overexpression or amplification confers
resistance to tyrosine kinase receptor inhibitors such as trastuzumab, cetuximab, and
erlotinib (Li etal. 2012; Zindy etal. 2011), as well as PI3K and mTOR inhibitors
such as BEZ235, AZD8055 and rapamycin (Cope etal. 2014; Ilic etal. 2011; Wen-
del etal. 2004). Consequently, there is a need to assess the efficacy of combining
eIF4E-targeting therapies with other therapeutic strategies to prevent the develop-
ment of resistance. More detailed discussions on the role of eIF4E and the 4E-BPs
in resistance to chemo and radiotherapy are presented in other chapters of this book.
4.14Conclusion and Perspectives
The past 20 years of research on the association of eIF4E with cancer have yielded
a vast amount of knowledge and cemented eIF4E as a critical player in cancer biol-
ogy. Its expression and availability represent important biomarkers for recurrence
and mortality in various cancers. Numerous therapies targeting eIF4E are currently
being developed or used in the clinic. Its molecular properties and function are
relatively well understood, as are the various ways in which it can be stimulated
or inhibited. Nonetheless, there is much that requires further investigation. Even at
the molecular level, where research has been ongoing for the past 30 years, there
are surprising new findings. This is exemplified by recent findings such as eIF4Es
cap-independent role in stimulating eIF4A, and the discovery of a secondary eIF4E-
binding site in the 4E-BPs (Feoktistova etal. 2013; Lukhele etal. 2013; Mizuno
etal. 2008; Umenaga etal. 2011).
There remain important gaps in our knowledge of the role of eIF4E in cancer
biology. Importantly, in most cancers, in-depth analysis of activation and expres-
sion of eIF4E and the 4E-BPs remains to be performed, especially with regards to
response to therapy. This is particularly important considering the importance of
the eIF4E/4E-BPs ratio in determining sensitivity to mTOR inhibitors. In the era
of personalized cancer therapy, establishing this eIF4E/4E-BPs ratio will be critical
when assessing treatment options. To achieve the best results for patients, the most
effective compounds and combinations targeting eIF4E will need to be determined.
Furthermore, the mRNAs whose translation is affected by cancer treatments remain
to be explored, and most likely vary among cancers and compounds. Microarray
analysis of polysome profiles along with the recently developed ribosome profiling
techniques will be important in addressing the issues pertaining to the mRNA ele-
ments conferring eIF4E sensitivity at the level of the genome-wide transcriptome or
translatome. Translatome analysis could also be developed to predict clinical out-
come or determine subtypes of cancer, such as has been done with the genome and
transcriptome. Considering that the translatome most closely corresponds to protein
expression (Schwanhausser etal. 2011), we expect that such analysis of translated
mRNAs would exhibit better predictive value than genome-wide techniques previ-
ously used (Larsson etal. 2013; Pradet-Balade etal. 2001).
The gap between the bench and the clinic is starting to narrow. eIF4E-targeting
therapies are now entering clinical trials using basic research. The mTOR inhibi-
tor rapamycin and its derivatives are being tested in numerous drug combinations
for treatment of most cancers; according to clinicaltrials.gov, there have been 1512
studies on these drugs in the clinic. More recent studies are investigating mTOR
inhibitors in combination with inhibitors of upstream signaling (e.g. trastuzumab in
human epidermal growth factor receptor 2 (HER2/neu)-positive metastatic breast
cancer or BEZ235 in advanced solid tumors). Other clinical trials are investigating
these drugs in combination with microtubule stabilizing agents (e.g. abraxane in
advanced solid cancers) or DNA intercalating agents (e.g. doxorubicin in recurrent
sarcoma) or both (e.g. paclitaxel and carboplatin in metastatic melanoma and ovar-
ian cancer). In head and neck cancer with eIF4E positive surgical margins, a phase
I study of everolimus in combination with cisplatin and radiation has just been
published (Fury etal. 2013). The more recently developed ASOs blocking eIF4E
expression have also entered clinical trials in such combinations in NSCLC and
castration resistant prostate cancer (according to clinicaltrials.gov, accessed 2013-
10-29). There is therefore hope that in the near future, the breadth of research under-
taken on eIF4E and cancer will culminate in life-saving therapies.
Acknowledgments We are very grateful to Dr. Nadeem Siddiqui (GCRC-McGill Univer-

sity) for his helpful insights on eIF4E structure and function and Dr. Armen Parsyan for editing
this manuscript. This work was supported by The Susan G. Komen Breast Cancer Foundation
(IIR12224057), the Canadian Cancer Society (2010-700377) and the Canadian Institutes of Health
Research (MOP-7214). N.S. is a Howard Hughes Medical Institute International Scholar. N.R.
was supported by scholarships from the Fonds de la Recherche en Sant du Qubec (20874), the
Canadian Institutes of Health Research (220151) and the Vanier Canada Graduate Scholarship
program (267839).
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Chapter 5
RNA Helicases and Their Cofactors
David Shahbazian, Jerry Pelletier, Yuri Svitkin, John W. B. Hershey

and Armen Parsyan
Contents
5.1Introduction 116
5.2eIF4A and Translation 117
5.3eIF4A and Cancer 120
5.4Pharmacological Targeting of eIF4A in Cancer 120
5.5eIF4B and Translation 122
5.6Regulation of eIF4B by Phosphorylation 122
5.7eIF4B in Cancer 123
5.8eIF4H 124
5.9PDCD4 125
5.10DHX29 125
5.11DDX3 127
5.12RHA (DHX9) 128
References ................................................................................................................................129
David Shahbazian and Jerry Pelletier contributed equally to this work.
A.Parsyan()
Division of General Surgery, Department of Surgery, Faculty of Medicine,
D.Shahbazian
Section of Medical Oncology, Yale Cancer Center and School of Medicine,
Yale University, New Haven, CT, USA
J.Pelletier Y.Svitkin
Department of Biochemistry and Rosalind and Morris Goodman Cancer Research Centre,
J.Pelletier
Department of Oncology, McGill University, Montreal, QC, Canada
J.W.B.Hershey
Department of Biochemistry and Molecular Medicine, School of Medicine,
University of California, Davis, CA, USA
116 D. Shahbazian et al.
AbstractRNA helicases are proteins that function by melting RNA secondary

structures or remodeling ribonucleoprotein complexes. Canonical RNA helicase
eIF4A plays an essential role in translation initiation by resolving secondary struc-
tures in the 5 UTR of mRNAs and preparing the mRNA template for ribosome
recruitment. Interestingly, the majority of mRNAs that encode proteins responsible
for cell survival, proliferation, cell cycle transitions and angiogenesis contain 5
UTRs with significant secondary/tertiary structures, and thus, appear to be more
dependent on the RNA helicase activity of eIF4A. In addition, several other RNA
helicases have been implicated in other steps of translation initiation, as well as in
exerting mRNA-specific regulation. In this chapter, we discuss the RNA helicases
implicated in translational control, as well as their role in cancer biology and their
potential as diagnostic, prognostic and therapeutic targets.
5.1Introduction
The initiation of translation is a multistep process that is described in detail in

Chap.2. Here, we focus on an essential step of translation initiation that involves
resolving or remodeling RNA secondary structures with the aid of RNA helicases.
RNA helicases represent a large group of proteins that participate in diverse pro-
cesses of RNA metabolism (see recent comprehensive reviews (Parsyan etal. 2011;
Robert and Pelletier 2013). Among these helicases, the most studied one is the ca-
nonical RNA helicase, eukaryotic initiation factor eIF4A, which is one of the most
abundant initiation factors. eIF4A is present in the cell as a free form (eIF4Af) or
as a part of the mRNA 5 cap-binding heterotrimeric complex, eIF4F (eIF4Ac). The
latter also includes the cap-binding protein eIF4E and the large scaffolding protein,
eIF4G.
Eukaryotic mRNAs have several common features and elements. At the very 5
terminus, all cellular cytoplasmic mRNAs contain an m7GpppN cap structure (see
Chap.2), a unique nucleotide that is recognized by eIF4E. The distance from the cap
and the initiation codon, the 5 UTR, can vary significantly in length and structural
complexity. Increasing secondary structures in the 5 UTR of mRNAs impairs the
ability of the mRNA to bind ribosomes and, consequently, to be translated (Baben-
dure etal. 2006; Kozak 1986; Pelletier and Sonenberg 1985). To some extent, this
explains the range of variability in translation efficiency among mRNAssome
mRNAs have less secondary structures in their 5 UTRs and behave as strong,
easy-to-translate templates, whereas others are translationally weak and contain
high degrees of secondary structures. Extensively structured 5 UTRs are inhibitory
to translation and these mRNAs are more dependent on eIF4A for their transla-
tion (Svitkin etal. 2001). The mRNA-binding channel of the eukaryotic ribosome
can accommodate only a single-stranded template (reviewed by Marintchev 2013).
Hence, secondary structure within mRNAs selected for initiation must be resolved
or melted during ribosome movement on the 5 UTR. The main factor that is
thought to resolve such structures in mammalian cells is eIF4A (Rogers etal. 2002).
5 RNA Helicases and Their Cofactors 117
It is also thought to unwind mRNA secondary structures, thereby preparing a land-

ing platform for the incoming 43S PIC (Rogers etal. 2002).
There is a range of other RNA helicases that have been implicated in transla-
tion (see Parsyan etal. 2011), however, only a few of them have been described
in higher eukaryotes and even fewer have been linked to cancer. One such RNA
helicase is a recently characterized DEAH box helicase 29 (DHX29) that contains a
DEAH amino acid motif and plays a role in translation initiation in higher eukary-
otes (Parsyan etal. 2009; Pisareva etal. 2008). This protein is hypothesized to func-
tion by remodeling the 40S ribosome, making scanning of more structured mRNAs
more efficient (Marintchev 2013). Other helicases such as DEAD box helicase 3
(DDX3) and RNA helicase A (RHA) will also be discussed herein.
5.2eIF4A and Translation
For the sake of this discussion, helicases can be conceptualized as containing either
DEAD or DEAH (where D is aspartic acid (Asp), E is glutamic acid (Glu), A is
alanine (Ala) and H is histidine (His)) box motifs. These and other motifs within
helicases are able to bind RNA, Mg2+ and/or ATP or provide helicase activity (for
review see (Parsyan etal. 2011)).
eIF4A belongs to the family of DEAD box helicases. Additional domains pres-
ent in eIF4A are responsible for interactions with RNA, ATP, eIF4G, the cofactors
eIF4B and eIF4H, and the translational repressor and tumor suppressor PDCD4
(Chang etal. 2009; Oberer etal. 2005; Rozovsky etal. 2008). Although eIF4G
possesses two potential eIF4A binding sites (in its middle and C-terminal portions),
there is usually a single eIF4A molecule bound to it (Imataka and Sonenberg 1997;
Morino etal. 2000). eIF4A is more active as a component of the eIF4F complex,
which is required for 43S PIC recruitment and scanning (Pause etal. 1994; Rogers
etal. 2002).
There are three isoforms of eIF4A: eIF4AI (DDX2A), eIF4AII (DDX2B) and
eIF4AIII (DDX48). The two highly homologous isoforms, eIF4AI and eIF4AII,
share 90% sequence identity and, until recently, were thought to be functionally
redundant based on in vitro data (Yoder-Hill etal. 1993). Here, we refer to these two
isoforms collectively as eIF4A. However, some evidence suggests that eIF4AI and
eIF4AII have functional differences. For example, eIF4AI silencing induces e IF4AII
transcription and generates protein to levels beyond what should be sufficient to
rescue the ensuing translational and cell proliferation block (Galicia-Vazquez etal.
2012). In addition, eIF4AII has been ascribed a role in miRNA-mediated trans-
lational repression and destabilization of mRNAs possessing structured 5 UTRs
(Meijer etal. 2013). eIF4AIII is ~65% identical to eIF4AI and localizes to the nu-
cleus where it plays a key role in NMD (Ferraiuolo etal. 2004; Shibuya etal. 2004).
Functionally, eIF4A behaves as an RNA-dependent ATPase (Rogers etal. 2002).
Although eIF4A is a relatively abundant protein, its helicase activity and route of
delivery to mRNA templates depends on the eIF4F complex, with eIF4E being the
limiting component (Duncan and Hershey 1983; Duncan etal. 1987). eIF4A cycles
between the eIF4F-bound and free forms (Pause etal. 1994; Yoder-Hill etal. 1993).
The catalytic activity of eIF4Af, albeit very low, is increased approximately 20-fold
upon addition of eIF4E and eIF4G (Feoktistova etal. 2013; Nielsen etal. 2011;
Rogers etal. 1999; Rozen etal. 1990). Dominant-negative mutants of eIF4A, that
strongly suppress translation of mRNA, are defective for recycling through eIF4F
and addition of either eIF4F or eIF4Af relieves translational inhibition, with eIF4F
being sixfold more efficient than eIF4Af in the derepression assay (Pause etal.
1994).
The dependency of weak mRNAs on eIF4A activity has been demonstrated by
a study that showed that the level of inhibition of translation by eIF4A mutants is
proportional to the degree of 5 UTR secondary structure (Svitkin etal. 2001). In
this study, dominant-negative eIF4A (lacking RNA helicase and ATPase activities)
associated with eIF4G with eightfold higher affinity than wild-type eIF4A (Svitkin
etal. 2001). Interestingly, incorporation of dominant-negative eIF4A into the eIF4F
complex also prevented interaction of the mRNA with eIF4F and inhibited 48S
complex formation (Svitkin etal. 2001). The findings from this study suggested
that incorporation of functional eIF4A into the eIF4F complex is necessary for mul-
tiple steps of translation initiation including: (a) eIF4F interaction with mRNA, (b)
mRNA unwinding, (c) ribosome recruitment to mRNA and (d) scanning of the 43S
PIC to the initiation codon. A recently published study provided evidence of direct
involvement of eIF4E in stimulation of eIF4A activity (Feoktistova etal. 2013).
In this work, eIF4E binding to eIF4G was shown to neutralize an eIF4G autoin-
hibitory domain responsible for modulating the ability of eIF4G to stimulate eIF4A
(Feoktistova etal. 2013). This model explains why eIF4E-mediated stimulation of
helicase activity is independent of its cap-binding function.
The activity of eIF4A is also controlled by various cofactors, some of which (e.g.
PDCD4) are strongly linked to the pathobiology of various types of cancers (see
Part IV and Fig.5.1). Together with PDCD4, eIF4A is also regulated by its cofac-
tors eIF4B and eIF4H, which will be discussed below (Dorrello etal. 2006; Richter
etal. 1999; Richter-Cook etal. 1998; Rogers etal. 2001). Finally, eIF4A and its
cofactors are the most downstream targets of signaling cascades, such as the PI3K/
AKT/mTOR/S6K and RAS/MAPK/ribosomal S6 kinase (RSK) pathways (Fig.5.1)
(Raught etal. 2004; Shahbazian etal. 2006). The importance of these pathways in
cancer development and progression is well established and further discussed in
Part IV.
Not only is eIF4A required for events subsequent to the binding of eIF4F to the
cap structure, but a role for it has also been proposed in 43S PIC scanning. There
are several models describing eIF4A participation in mRNA duplex unwinding in
the course of 43S PIC recruitment and scanning. A widely accepted model posits
that eIF4A unwinds mRNA structures in the 5 UTR to create a landing pad for the
incoming ribosome and then travels with the scanning complex while unwinding
RNA structures ahead of the 43 PIC (Marintchev 2013). More recently, a molecular
Brownian ratchet-and-pawl mechanism was introduced into the original scanning
model to explain the one-dimensional diffusion of the scanning ribosomal com-
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Fig. 5.1 Control of eIF4A activity by signaling cascades. Growth factor signaling-sensitive
MAPK/RSK and PI3K/mTORC1/S6K cascades, as well as AKT, activate eIF4B via phosphory-
lation at serine residues depicted in the figure. eIF4B is also phosphorylated by PIM1/2 that are
upregulated in response to the BCR-ABL1-activated STAT (signal transducer and activator of
transcription) pathway (Yang et al. 2013). Phosphorylation of eIF4B leads to increased binding
to eIF3 and stimulates translation. PDCD4, the negative regulator of eIF4A, is phosphorylated by
S6K1 and targeted for ubiquitylation with subsequent proteasomal degradation (Chap. 6). See text
for more details.
plex along the mRNA (Spirin 2009). According to this model, repetitive changes in
the affinity of eIF4A towards eIF4B, mRNA and ATP act to melt mRNA structure
and position single-stranded RNA-binding proteins (such as eIF4B or eIF4H) be-
hind the scanning 43S PIC to prevent ribosomes from sliding in the wrong (35)
direction (Spirin 2009). Thus, ribosome recruitment and scanning are affected by
the presence of secondary structures, with the availability of eIF4A and auxiliary
factors being determinants of how efficiently this process is executed.
Although eIF4A activity is stimulatory for the translation of most cellular
mRNAs, a recent study suggested that its inhibition can indirectly stimulate
translation in some circumstances (Olson etal. 2013). Under stress conditions,
such as starvation, FOXO (forkhead box O) transcription factors were shown to
drive expression of the eIF4E repressor, 4E-BP1, and PDCD4, leading to seques-
tration of eIF4E and eIF4A from the eIF4F complex and decreased translation.
Under these conditions, translation of the insulin receptor mRNA was found to
increase, and this effect was attributed to the presence of the 5 UTR IRES (Ol-
son etal. 2013).
5.3eIF4A and Cancer
The proposition that eIF4A is important in cancer biology stems from the para-
digm that is revisited on multiple occasions in this book. This paradigm originates
from strong evidence demonstrating that the eIF4E protein, a proto-oncogene, as-
serts its tumorigenic effects by facilitating translation of mRNAs with structured 5
UTRs which are generally believed to encode proteins associated with cell survival,
growth, proliferation and angiogenesis. Importantly, this facilitation of translation
of weak mRNAs by eIF4E is thought to involve eIF4A, the only RNA helicase
component of the eIF4F complex. Although direct evidence for a role eIF4A in
cancer is lacking, there are indirect data indicating that eIF4A plays a major role in
cancer biology.
At the mRNA level, eIF4A has been reported to be overexpressed in mela-
noma (Eberle etal. 1997) and HCC (Shuda etal. 2000). However, whether this
overexpression is causative of the neoplastic process or is a mere reflection of the
overall tumorigenic state is still unclear. It is unlikely that overexpression of eIF4A
would lead to significant effects on cell growth and proliferation mainly due to
the fact that the intracellular concentration of eIF4A significantly exceeds that of
the rate-limiting eIF4E (Duncan etal. 1987). As discussed above, eIF4A activity is
highest when it is a part of the eIF4F complex. Thus, an increase in eIF4Af concen-
tration should not result in increased levels of eIF4F complexes unless the levels of
PDCD4, a negative regulator of eIF4A, are also elevated and render eIF4Ac limiting.
On the other hand, increased eIF4E levels are strongly associated with cellular
transformation (see Chap.4). According to the paradigm described above, eIF4Es
role in cancer depends on its ability to increase translation of weak mRNAs (some
of which encode protumorigenic, proproliferative and prosurvival proteins). Trans-
lation of these weak mRNAs is more dependent on RNA helicase activity and
hence overexpression of eIF4E disproportionally increases translation of mRNAs
with structured 5 UTRs (Koromilas etal. 1992). High levels of eIF4E can increase
the level of the eIF4F complex and hence the associated eIF4A helicase activity.
On the other hand, overexpression of eIF4A has to be paralleled by substantial in-
creases in eIF4E concentration in order to exhibit tumorigenic effects. By the same
token, silencing of eIF4A should be significant enough to decrease the expression
of this protein to the levels below eIF4E to reduce levels of the eIF4F complex.
Hence, given the fundamental role of eIF4E in cancer biology (see Chap.4), one
can extrapolate that eIF4A plays a similarly important role in cancer development
and progression.
5.4Pharmacological Targeting of eIF4A in Cancer
Evidence for a role of eIF4A in cancer also comes from the studies using small
molecule inhibitors in cancer cells. Various pharmacological compounds affect-
ing eIF4A activity are discussed in detail in Chap.14. Here they are presented in
the context of providing evidence for a role of eIF4A in tumor cell maintenance.
RNAi or pharmacological inhibition of eIF4A with the small molecule inhibitor,
hippuristanol, sensitizes lymphoma cells to DNA-damaging agents or to BCL-2
family inhibitors both in vitro and in vivo (Cencic etal. 2013). Hippuristanol inhib-
its the interaction of eIF4A with RNA, thus blocking helicase and RNA-dependent
ATPase activities (Bordeleau etal. 2006). An early preclinical study found that hip-
puristanol is toxic for adult T-cell leukemias but does not affect the viability of
normal peripheral blood mononuclear cells (Tsumuraya etal. 2011). In this study,
hippuristanol also suppressed the growth of human T-lymphotropic virus type 1
(HTLV-1)-infected tumor cells in a mouse xenograft model (Tsumuraya etal. 2011).
Hippuristanol has been shown to reduce viability of cells of primary lymphoma
caused by Kaposis sarcoma-associated herpesvirus (KSHV) (Ishikawa etal. 2013).
Another inhibitor of eIF4A, pateamine A, has been shown to exhibit anticancer
effects in various cancer models, including leukemia and melanoma cells (Hood
etal. 2001; Kuznetsov etal. 2009; Northcote 1991). Pateamine A stimulates the
binding of eIF4A to RNA in a non-sequence dependent manner, likely leading to
depletion of the latter from the eIF4F complex (Bordeleau etal. 2005).
Flavaglines or rocaglamides (natural products found in plants of the genus
Aglaia (Meliaceae)) show anticancer and cytoprotective activities mediated in
large part by inhibition of eIF4A (Basmadjian etal. 2013). Yeast genetics identi-
fied eIF4A (Tif1/2) as a target of rocaglamides. As well, mutation of key resi-
dues near the RNA-binding site of eIF4A leads to rocaglamide resistance without
impairing eIF4A activity (Sadlish etal. 2013). Rocaglamides also show antineo-
plastic activity against leukemia cells and Hodgkins lymphoma, as well as other
cancer types (Giaisi etal. 2012; Hwang etal. 2004; Kim etal. 2006; Kim etal.
2007; Lee etal. 1998).
Silvestrol is a member of the rocaglamide family and has a mechanism of ac-
tion quite similar to pateamine A. Both are thought to behave as chemical inducers
of dimerization and cause eIF4A to be depleted from the eIF4F complex (Bor-
deleau etal. 2008; Cencic etal. 2009). Silvestrol inhibits tumor growth in acute
lymphoblastic leukemia, AML, breast and prostate cancers and HCC xenograft
models and a chronic lymphocytic leukemia mouse model, as well as in human
cancer cells (Alachkar etal. 2013; Cencic etal. 2009; Kogure etal. 2013; Lucas
etal. 2009). Silvestrol showed strong anticancer activity against AML both in
vitro and in vivo (Alachkar etal. 2013). In vivo, the median survival of leukemia-
engrafted mice treated with silvestrol was 63 days versus 29 in placebo-treated
controls. In vitro, the drug abolished colony-formation and induced robust apop-
tosis in patient-derived blast cells. By using transgenic and allograft breast cancer
models, it has been shown that disruption of the eIF4F complex by pharmacologic
suppression of eIF4A by silvestrol or hippuristanol inhibited translation, delayed
cancer cell invasion and migration, and decreased pulmonary metastasis (Nasr
etal. 2013). In addition, translation of MUC1-C (mucin 1, cell surface associated,
C-terminal subunit), an oncogene overexpressed in breast cancers is sensitive to
silvestrol (Jin etal. 2013).
5.5eIF4B and Translation
eIF4B, eIF4H and PDCD4 are eIF4A cofactors that modulate its catalytic activity
(Dorrello etal. 2006; Ozes etal. 2011; Rogers etal. 2001). eIF4B was originally
purified as a factor stimulating translation in cell-free systems, and was later found
to augment the RNA helicase and ATPase activity of eIF4Af and eIF4Ac in vitro
(Rozen etal. 1990). Both eIF4B and eIF4G are necessary to synergistically induce
RNA-dependent ATPase activity of eIF4A.
In contrast to other translation initiation factors, there is a very little conservation
among eIF4B homologs from different species. At the sequence level, yeast (Tif3)
and human eIF4B are only ~20% identical. However, the overall domain structure
of eIF4B is preserved among species. Cloning of eIF4B has led to identification of
an N-terminal consensus RNA-binding recognition motif characteristic of many
other RNA-interacting proteins (Milburn etal. 1990). Later studies on eIF4B identi-
fied a distinct C-terminal RNA-binding domain consisting of an arginine-rich motif
responsible for enhancing eIF4A binding to RNA and stimulating RNA helicase
activity (Methot etal. 1994). eIF4B dimerizes and interacts with eIF3 through a re-
gion rich in aspartate, arginine, tyrosine and glycine (DRYG) (Methot etal. 1996).
Domains responsible for the eIF4B cofactor activity were mapped to the N-ter-
minal part of the protein adjacent to the arginine-rich motif. However, the ability
of eIF4B to stimulate eIF4A is also reduced after deletion of the DRYG domain
or mutation of the RNA recognition motif. Although functional cooperation and
copurification of eIF4B and eIF4A have been known for a long time, conditions
resulting in stable complex formation between eIF4A and eIF4B have only recently
been identified (Nielsen etal. 2011; Rozovsky etal. 2008). In one of these studies,
the addition of RNA and the non-hydrolysable ATP analog, AMPPNP, to recombi-
nant eIF4A and eIF4B was necessary for formation of a stable complex, as detected
by the electrophoretic mobility shift assay. Another study, using recombinant trun-
cated forms of eIF4G and eIF4B suggested that the eIF4G-induced conformational
changes in the eIF4A structure were sufficient for eIF4B recognitioneven in the
absence of the ATP analog and RNA (Nielsen etal. 2011). Using a fluorescence-
based RNA duplex unwinding assay, it was shown that eIF4G and eIF4B coopera-
tively stimulate eIF4A activity (Ozes etal. 2011).
5.6Regulation of eIF4B by Phosphorylation
Phosphorylation of eIF4B on Ser422 by several kinases (e.g. S6K, RSK, AKT, PIM1
and PIM2) was proposed to increase its ability to bind eIF3 and stimulate translation
(Peng etal. 2007; Raught etal. 2004; Shahbazian etal. 2006; van Gorp etal. 2009)
(Fig.5.1). A stimulatory role for eIF4B in global translation has been shown using
4E-BP1/2 DKO mice cells expressing a constitutively active S6K mutant (Dennis
etal. 2012). This effect was attributed to S6K-mediated phosphorylation of eIF4B
and PDCD4 since RNAi against PDCD4 and overexpression of the eIF4B phos-
phomimetic mutant (Ser422Asp) were similarly sufficient to alter translation rates

under serum-starved or IGF-1-stimulated conditions.
An early study has shown a direct interaction between ribosome bound eIF3
and eIF4B (Methot etal. 1996). This eIF3/eIF4B interaction is controlled by eIF4B
phosphorylation on Ser422 (Holz etal. 2005; Shahbazian etal. 2006). The rapamy-
cin-sensitive and serum-responsive phosphorylation site of eIF4B, Ser422, was
first found to be S6K-dependent (Raught etal. 2004). Later studies demonstrated
that the MAPK/RSK and PI3K/mTOR/S6K pathways cooperatively phosphorylate
eIF4B on Ser422, and eIF3 binding to eIF4B is augmented by this phosphorylation
event (Holz etal. 2005; Shahbazian etal. 2006). An eIF4B phosphomimetic muta-
tion (Ser422Glu) results in constitutive interaction between eIF4B and eIF3 even in
serum-starved cells, whereas the non-phosphorylatable eIF4B mutant (Ser422Ala)
is unable to bind eIF3 even following serum stimulation. Pathways known to phos-
phorylate eIF4B are shown in Fig.5.1.
The eIF4B interaction with eIF4G also appears to be important for signaling-de-
pendent translation regulatory mechanisms (Dobrikov etal. 2013). eIF4G contains
three HEAT (huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A),
and yeast kinase TOR1) domains (see Chap.7), with a highly regulated eIF4B/eIF3-
binding site within the HEAT-1/2 interdomain wherein resides two ERK1/2 and
casein kinase 2-sensitive phosphorylation sites. Phosphorylation at these sites leads
to the detachment of the eIF4A/eIF4B/eIF3 complex from eIF4G and association of
the MNK1 kinase with the HEAT-3 domain (Dobrikov etal. 2013).
5.7eIF4B in Cancer
Evidence supporting a role for eIF4B in cancer is limited, but could be associ-
ated with its overexpression or activation via signaling pathways. While not ex-
tensively studied in other cancers, overexpression of eIF4B has been reported in
ovarian granulosa cell tumors (Rico etal. 2012) and appears to play an impor-
tant role in diffuse large B-cell lymphoma (DLBCL) (Horvilleur etal. 2014). In
DLBCL, analysis of polysome-associated mRNAs suggested that translation of an-
tiapoptotic and DNA-repair proteins is increased due to the relief of the repression
normally imposed by the presence of structured 5 UTR (Horvilleur etal. 2014).
This stimulation of expression was linked to increased eIF4B synthesis. Reduction
of eIF4B expression was sufficient to downregulate synthesis of proteins associ-
ated with enhanced tumor cell survival, such as DAXX (death domain-associated
protein 6), ERCC5 (excision repair cross-complementing rodent repair deficiency,
complementation group 5)) and BCL-2. In this study, eIF4B was also identified as
a prognostic marker for poor survival in DLBCL (Horvilleur etal. 2014). Simi-
larly, RNAi-mediated silencing of eIF4B leads to selective translational repression
of mRNAs harboring structured 5 UTRs, many of which encode mitogenic (c-
MYC, cell division cycle 25C (CDC25C)) and survival proteins (BCL-2 and XIAP)
(Shahbazian etal. 2010). RNAi -mediated silencing of eIF4B resulted in inhibition
of cell proliferation and survival and sensitized human cervical cancer cells to low
doses of the cytotoxic drug camptothecin (Shahbazian etal. 2010). Thus the role of
eIF4B in cancer, likely in conjunction with eIF4A, appears to facilitate translation
of mRNAs with structured 5 UTRs that encode proteins involved in cell survival,
proliferation and angiogenesis.
The role of eIF4B expression levels and phosphorylation in cell growth and
chemosensitization has also been suggested by another study. PIM1 kinase-me-
diated eIF4B overexpression and Ser422 and Ser406 hyperphosphorylation were
recently reported in Abelson murine leukemia viral oncogene homolog 1 (ABL1)
-transformed and human breakpoint cluster region protein (BCR)-ABL1-positive
cells (Yang etal. 2013). Treatment of BCR-ABL1-positive cells with either ima-
tinib (a BCR-ABL1 inhibitor) or SMI-4a (a PIM1/2 inhibitor) led to eIF4B de-
phosphorylation and decreased eIF4B protein levels. eIF4B silencing sensitized
ABL1-transformed cells to imatinib-induced cytotoxicity, whereas overexpression
of a phosphomimetic eIF4B mutant conferred resistance to SMI-4a. Most impor-
tantly, human leukemic cells stably expressing shRNAs to eIF4B grew much more
slowly in nude mouse xenograft models than their counterparts expressing neutral
control shRNAs, suggesting that eIF4B is required for ABL1-driven tumor growth.
ABL1-induced transformation efficiency was significantly decreased in murine
bone marrow cells derived from transgenic mice with constitutively silenced eIF4B.
The latter effect could be reversed by expressing wild-type eIF4B, but not by the
expression of a nonphosphorylatable eIF4B variant. In concert with previously re-
ported data, eIF4B silencing resulted in a reduction of c-MYC and BCL-2 levels
(Shahbazian etal. 2010; Yang etal. 2013; Zhang etal. 2011).
A long-term, low-dose, arsenite-dependent neoplastic transformation study of
mouse epidermal cells also suggests a role for eIF4B in cancer biology (Zhang
etal. 2011). Concurrent eIF4B Ser422 hyperphosphorylation and overexpression
at both the mRNA and protein levels have been documented in mouse epidermal
cells cultured in the presence of arsenite, and is associated with increased prolif-
eration and neoplastic transformation (Zhang etal. 2011). eIF4B silencing in this
model decreased cellular proliferation rates, anchorage-independent growth and
cap-dependent translation. Overexpression of c-MYC in these cells was also found
to be eIF4B-dependent. Taken together, these studies point to an important role of
eIF4B in cancer biology, although more studies will be required to shed light on its
potential as a therapeutic target.
5.8eIF4H
Another cofactor of eIF4A is eIF4H (Richter etal. 1999; Richter-Cook etal. 1998).
This protein is smaller than eIF4B with significant sequence similarity maintained
between the two proteins. The ability and efficiency of eIF4B and eIF4H to stimu-
late the RNA helicase activity of eIF4A are similar in the absence of eIF4G (Rogers
etal. 1999). However, in the presence of eIF4G, eIF4B is much more potent than
eIF4H in stimulating eIF4A (Ozes etal. 2011), suggesting that eIF4H and eIF4B
may have distinct roles in translation.
eIF4B and eIF4H bind the same domain of eIF4A, and their interactions with
eIF4A are mutually exclusive (Rozovsky etal. 2008). Similar to the eIF4A/eIF4B
interaction, eIF4H binding to eIF4A is enhanced in the presence of ATP (Marintchev
etal. 2009). The topology of the complex containing eIF4G, eIF4A and eIF4H has
been resolved using nuclear magnetic resonance chemical shift perturbation. Like
eIF4B, eIF4H is also able to partially displace eIF4G from eIF4A. An interaction
with the HEAT-1 domain of eIF4G increases the affinity of eIF4A towards ATP
by fourfold, while ATP decreases eIF4As affinity towards the HEAT-2 domain by
threefold. eIF4A binding to RNA is ATP-dependent, and the HEAT-1 and HEAT-2
domains of eIF4G have opposing effects on this interaction. These results implicate
dynamic rearrangement and conformational flexibility of eIF4G in the stimulation
of eIF4A activity.
The role of eIF4H in cancer is poorly studied. eIF4H is one of the genes deleted
within the 7q11.23 chromosomal region in patients with Williams-Beuren syndrome,
a rare neurodevelopmental disorder (Merla etal. 2006). Mice with genetic ablation
of eIF4H are viable but display growth retardation, altered cerebral anatomy, abnor-
mal memory and fear-related associative learning (Capossela etal. 2012). In a recent
study, recombinant overexpression of one of the alternatively spliced isoforms of
eIF4H (isoform 1) transformed mouse fibroblasts and enabled the establishment of
subcutaneous tumors in nude mice (Wu etal. 2011). Overexpression of this eIF4H
splice variant has also been observed in CRC (Wu etal. 2011). Silencing eIF4H
isoform 1 in colon cancer cells inhibited their proliferation ex vivo, as well as their
ability to form tumors in a xenograft model. Cyclin D1 was proposed to be the major
target of isoform 1 since RNAi-mediated silencing of eIF4H decreased cyclin D1
expression levels, while ectopic expression of cyclin D1 was able to neutralize the
antiproliferative effect of eIF4H isoform 1 suppression by RNAi (Wu etal. 2011).
5.9PDCD4
PDCD4 has an inhibitory role in the regulation of eIF4A. It is a bona fide tumor
suppressor and is described in greater detail in Chap.6. Originally discovered as a
protein upregulated during apoptosis, PDCD4 plays a role in the development and
progression of several types of human malignancies (see Part IV of this book).
5.10DHX29
The DEAH box helicase DHX29 was first identified as an RNA helicase associ-
ated with the 40S ribosomal subunit (Pisareva etal. 2008). Toeprinting experiments
showed that DHX29 is indispensible for ribosome positioning at the start codon on
mRNAs harboring even moderately structured 5 UTRs. Further investigation iden-

tified DHX29 as being capable of facilitating translation of mRNAs with structured
5 UTR (Parsyan etal. 2009).
It appears that in the absence of DHX29, stem-loop structures in the mRNA 5
UTR can enter, but not be threaded through, the mRNA-binding channel of the 40S
ribosomal subunit (Abaeva etal. 2011). The helicase activity of unbound DHX29 is
very weak but is strongly enhanced by association with the 40S ribosomal subunit
(Pisareva etal. 2008). This scenario is reminiscent of what is seen with eIF4A
both eIF4Ac and DHX29 attain optimal activity following incorporation into mul-
timolecular complexes.
Analysis of the DHX29 structure/function relationships identified multiple func-
tional units within this helicase (Dhote etal. 2012). DHX29 contains an N-terminal
region, central catalytic RecA1/RecA2 domains, and a C-terminal region, which
has winged-helix, ratchet, and oligonucleotide/oligosaccharide-binding (OB) do-
mains. Both RecA2 and OB domains inhibit the basal NTPase activity of DHX29.
Deletion of the OB domain abrogated the responsiveness of the NTPase activity to
stimulation by 40S ribosomes and abolished activity of DHX29 in translation initia-
tion. Ribosome binding domains of DHX29 were mapped to the N-terminal region
and parts of the winged-helix domain. The N-terminal region responsible for 40S
binding was hypothesized to bind double-stranded RNA.
A follow-up crystallographic study corroborated this notion (Hashem etal.
2013). This study of eIFs within DHX2943S crystal complexes identified multiple
interaction sites but did not detect any major conformational changes elicited by
DHX29 in the small ribosomal subunit (Hashem etal. 2013). A large portion of
DHX29 interacts with eIF3 and binds the h16 loop of rRNA and several ribosomal
proteins in the beak region of the small ribosomal subunit. A smaller domain of
DHX29 was mapped in close proximity to the A-site of the ribosome and the mRNA
entry channel latch. Based on the structure analysis, it was suggested that the small-
er domain of DHX29 may account for the inability of the protein to efficiently bind
80S ribosomal particles (Pisareva etal. 2008), as well as may explain DHX29s role
in dissociating aberrant ribosomal complexes containing intact mRNA hairpins in
the A-site (Abaeva etal. 2011). Hence, DHX29 guards the ribosomal entry site and
prevents intact hairpins from slipping pass it. It was also hypothesized that DHX29
contributes to trapping the mRNA in the mRNA-binding channel and by doing so
acts to increase scanning processivity of the 43S PIC.
The role of DHX29 in tumorigenesis is not well studied. However, it appears
that it is required for translation of structured mRNAs in vivo (Parsyan etal. 2009).
Mining of the ONCOMINE database has shown that DHX29 is upregulated in some
glioblastomas and melanomas. Our unpublished results (Parsyan, Shahbazian and
Sonenberg) indicate that knockdown of DHX29 in various glioblastoma cell lines
induces drastic arrest of cell proliferation and leads to cell death. Further studies
into the role of this protein in cancer are warranted.
5.11DDX3
DDX3 is a mammalian homolog of yeast translation initiation factor Ded1, a heli-

case that is essential for yeast cell viability and that assists in scanning long 5 UTRs
(for reviews, see Parsyan etal. 2011; Robert and Pelletier 2013; Soto-Rifo and
Ohlmann 2013)). There are two DDX3 isoforms in mammals, one on the X chromo-
some (DDX3X) and the other on the Y chromosome (DDX3Y). The two isoforms
are 91% identical and, whereas DDX3X is ubiquitously expressed, DDX3Y is ex-
pressed only in the testes (Chang and Liu 2010). Although DDX3Y expression has
been detected in leukemias (Rosinski etal. 2008), there is no functional evidence
indicating that it plays any significant role in the tumorigenic process. In contrast,
there is much data implicating DDX3X in tumorigenesis.
DDX3X (and its rodent homolog, PL10) appears to promote translation initia-
tion in higher eukaryotes. Although controversy exists suggesting that DDX3X
specifically represses cap-dependent translation by acting as an eIF4E inhibitory
protein (Shih etal. 2008), the majority of studies indicate that DDX3X is a trans-
lation initiation factor that promotes translation (Lai etal. 2008, 2010; Lee etal.
2008; Soto-Rifo etal. 2012). It appears that the helicase activity of DDX3X is
required for its role in translation initiation and in cell growth control (Lai etal.
2010). DDX3X plays a role in cell cycle progression by promoting translation of
cyclin E1 mRNA (Lai etal. 2010). It appears necessary to prime 5 UTR unwinding
of specific mRNAs where secondary structure is present in immediate proximity
(less than 15 nucleotides) of the 5 cap structure (Soto-Rifo etal. 2012). DDX3X
also appears to play important roles in translation of viral RNAs (Shih etal. 2008;
Soto-Rifo etal. 2013). For example, substitution of eIF4E by the DDX3X/PABP/
eIF4G complex selectively enhances translation of HIV-1 genomic RNA (Soto-Rifo
etal. 2013).
Our current understanding of the role of DDX3X in tumorigenesis vis--vis
translation is unclear because of the pleiotropic effects this protein has on transla-
tion, transcription and regulation of apoptosis (Chao etal. 2006; Soto-Rifo etal.
2012; Sun etal. 2011). DDX3X has been found mutated at various frequencies in
a number of cancer types, such as medulloblastoma, head and neck cancer, chronic
lymphocytic leukemia and Burkitts lymphoma (Jones etal. 2012; Richter etal.
2012; Stransky etal. 2011; Wang etal. 2011). However, the role of these mutations
in cancer etiology and pathogenesis in general and vis--vis translational dysregula-
tion is unclear.
Overexpression of DDX3X in breast cancer cells stimulated EMT, increased cell
motility and invasive properties, and lead to colony formation in soft-agar (Botla-
gunta etal. 2008). In other systems, DDX3X was found to support the expression
of the SNAIL transcription factor, which in turn represses expression of cell adhe-
sion proteins, thereby leading to increased cell migration and metastasis character-
istic of numerous types of cancers (Sun etal. 2011). A strong correlation between
DDX3X and SNAIL expression was found in a large panel of glioma specimens.
An additional mechanism through which DDX3X may regulate cancer progression
is DDX3-mediated inhibition of apoptosis (Sun etal. 2008). In this study, DDX3X

was found to counteract caspase 3 activation induced by four different death recep-
tors. A regulatory mechanism was proposed where CDK1/cyclin B phosphorylated
DDX3X at Thr204 and Thr323, leading to loss of function and diminished cyclin
A expression, ribosome biogenesis, and reduced translation in mitosis (Sekiguchi
etal. 2007).
While these data suggest protumorigenic roles of DDX3X in cancer, there
are studies suggesting that this protein can also act to antagonize tumorigenesis.
DDX3X has been reported as downregulated in hepatitis B virus (HBV)-infected
(but not HCV-infected) HCC (Chang etal. 2006). Tumor suppressor-like proper-
ties of DDX3X were proposed when its overexpression in various cancer cell lines
was found to potently inhibit proliferation while increasing expression of p21waf1/cip1
via a transcriptional mechanism (Chao etal. 2006). While the majority of HCC
specimens show downregulation of DDX3, the cutaneous squamous cell carcino-
mas appear to exclude DDX3X from the nuclei and relocalize it to the cytoplasm,
suggesting potential posttranscriptional regulatory functions (Chao etal. 2006). It
is not clear why DDX3X shows both pro-oncogenic and tumor suppressive activity
in different settings, but these may be a reflection of the various dependencies or
oncogene addictions of the cell lines used.
5.12RHA (DHX9)
DHX9 (also known as and RHA) functions in transcription, splicing and nuclear
export (see Parsyan etal. 2009), but has also been shown to facilitate translation of
a specific set of mRNAs (Hartman etal. 2006; Manojlovic and Stefanovic 2012;
Narva etal. 2012). DHX9 differs from the other RNA helicases presented here in
that it appears to be mRNA target-specific. It is required for translation initiation of
mRNAs that contain a specific 5 posttranscriptional control element (PCE) (Hart-
man etal. 2006). It likely tethers to the PCE and rearranges the 5 UTR to facilitate
initiation by resolving structural impediments to ribosome scanning (Ranji etal.
2011). Retroviral (such as HIV-1 and HTLV-1) RNA transcripts containing PCEs
use RHA for their translation and to enhance viral infectivity (Bolinger etal. 2010).
In cellular mRNAs, PCE is present within the mRNA of JUND proto-oncogene
(Ranji etal. 2011; Short and Pfarr 2002).
Relatively recent evidence suggests that RHA participates in the translation of
type 1 collagen chains (Manojlovic and Stefanovic 2012). The 5 UTR of these
mRNAs contains a stem-loop structure that binds specifically to LARP6 (the La-
related protein 6). RHA binds LARP6 and thus facilitates translation of the mRNA
(Manojlovic and Stefanovic 2012). Thus, RHA appears to interact with proteins that
specifically bind to mRNA templates, and in this way becomes recruited to these
templates to stimulate their translation. A similar mechanism appears at work for the
octamer-binding transcription factor 4 (OCT4) mRNA by RHA (Narva etal. 2012).
Here, a complex involving RHA and the stem cell-specific RNA-binding protein
L1TD1 is suggested to promote translation of OCT4 and thus to regulate the stem-
ness phenotype. Thus, one would expect RHA to play a role in tumorigenesis via
dysregulation of its translational targets.
Our understanding of RNA helicases in general, and eIF4A in particular, in cancer

biology is expanding. While limited data are available to implicate eIF4A directly
in tumor initiation or progression, this protein does hold potential as a therapeutic
target. Since eIF4A functions as a component of the eIF4F complex, its physiologi-
cal function should be considered in this context. However, eIF4A is one of the most
abundant translation factors and may have activity outside of the eIF4F complex
that remains to be defined. In addition, eIF4A activity and availability are regu-
lated by eIF4B, eIF4H, and PDCD4proteins that have also been implicated in
the neoplastic process. The relationship of other helicases implicated in translation,
such as DHX29, DDX3 and DHX9, and their relationship to cancer biology is not
well-understood. Further efforts will be required to unravel significance of these
enzymes in translation in normal and transformed cells.
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Chapter 6
PDCD4
Hsin-Sheng Yang, Qing Wang, Magdalena M. Bajer and Tobias Schmid
Contents
6.1Introduction 136
6.2PDCD4, a Binding Partner of eIF4A and an Inhibitor of Translation 137
6.2.1eIF4A 137
6.2.2PDCD4 Physically Interacts with eIF4A 138
6.2.3PDCD4 Inhibits Translation 139
6.3Regulation of PDCD4 Expression 140
6.3.1Epigenetic Regulation 140
6.3.2Transcriptional Regulation 142
6.3.3Posttranscriptional Regulation 143
6.3.4Posttranslational Regulation 143
6.4PDCD4 in Cancer Development and Progression 144
6.4.1PDCD4 Inhibits Tumor Promotion 144
6.4.2PDCD4 Inhibits Tumor Progression 146
6.4.3PDCD4 Regulates Cell Cycle Progression and Cell Proliferation 148
6.4.4PDCD4 Translational Targets are Involved inTumorigenesis 149
6.5PDCD4 as a Tumor Marker and Therapeutic Target 150
6.5.1PDCD4 as a Biomarker 150
6.5.2Loss of PDCD4 in Therapy Resistance 150
6.5.3PDCD4 as a Therapeutic Target 151
References 153
Abstract PDCD4 is a novel tumor suppressor. Its expression is frequently down-

regulated in many types of cancers, and it has been demonstrated to inhibit tumor
promotion and progression. The biochemical function of PDCD4 is the inhibition of
translation through binding to eIF4A helicase. In this review, we will focus on recent
H.-S.Yang() Q.Wang
Graduate Center for Toxicology, University of Kentucky, Lexington, KY, USA
e-mail: Hsin-Sheng.Yang@uky.edu
H.-S.Yang
Markey Cancer Center, University of Kentucky, Lexington, KY, USA
M.M.Bajer T.Schmid
Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt,
Frankfurt am Main, Germany
136 H.-S. Yang et al.

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Fig. 6.1 Structure of PDCD4. The blue arrows indicate the AKT phosphorylation sites, the red
arrow indicates the S6K1 phosphorylation site and the purple arrow indicates the methylation
site. The +++ indicates the positive charge cluster. The MA3 domains are shown in yellow.
advances in understanding the mechanism of inhibiting translation and tumorigen-

esis by PDCD4, the regulation of PDCD4 expression and the potential application
of PDCD4 as a target for cancer diagnostics and therapeutics.
6.1Introduction
Programmed cell death 4 (PDCD4) is a tumor suppressor (initially named MA3)

that was cloned from mouse gene in 1995 (Shibahara etal. 1995). Subsequently,
PDCD4 gene was identified in different species, including human (H731, 197/15)
(Azzoni etal. 1998; Matsuhashi etal. 1997), rat (DUG; Goke etal. 2002) and chick-
en (PDCD4; Schlichter etal. 2001). PDCD4 is comprised of 469 amino acids and is
a highly conserved protein from Drosophila to humans. Mouse and human PDCD4
share approximately 92% identity of an amino acid level. The structure of PDCD4
has several interesting features (Fig.6.1). First, the Ser67 and Ser457 can be phos-
phorylated by AKT or S6K1 (Dorrello etal. 2006; Palamarchuk etal. 2005). Phos-
phorylation of PDCD4 at Ser67 leads to degradation by proteasome, while phos-
phorylation of Ser457 regulates PDCD4 intracellular localization. Second, PDCD4
contains two clusters of positively charged amino acid residues centered at amino
acids 63 and 105 (Wedeken etal. 2010). The function of these two clusters may be
involved in RNA binding. Third, the Arg110 can be methylated by protein arginine
methyltransferase 5 (PRMT5) (Powers etal. 2011). Methylated PDCD4 was sug-
gested to promote tumor cell growth in breast cancer cells. Lastly, PDCD4 has
two MA3 domains located from amino acids 164275 (N-terminal MA3 domain,
nMA3) and amino acids 327440 (C-terminal MA3 domain, cMA3) (Yang etal.
2004). The MA3 domain, playing an important role for binding to eIF4A (Chang
etal. 2009; Loh etal. 2009; Suzuki etal. 2008; Waters etal. 2011; Yang etal. 2004),
is also present in the human eIF4G, plant eIF(iso)4F and eIF4G isoform DAP5/p97/
NAT1, but not in yeast eIF4G (Aravind and Koonin 2000; Ponting 2000).
PDCD4 has been clearly demonstrated to be a tumor suppressor, which inhibits
both early stages (tumor promotion) and late stages (tumor progression) of carci-
nogenesis. Suppression of tumorigenesis by PDCD4 is believed to take place via
binding to eIF4A, and thereby inhibiting translation of a set of mRNAs that require
eIF4A activity (Wedeken etal. 2010, 2011; Yang etal. 2003a). In agreement with its
6PDCD4 137
tumor suppressor function, PDCD4 expression is frequently downregulated in nu-

merous types of cancer including breast (Wen etal. 2007), liver (Zhang etal. 2006),
lung (Chen etal. 2003), ovarian (Wang etal. 2008b; Wei etal. 2009a), pancreas
(Hayashi etal. 2010), colon (Horiuchi etal. 2012; Lim and Hong 2011; Mudduluru
etal. 2007; Wang etal. 2008a), renal (Li etal. 2012) and skin (Matsuhashi etal.
2007) cancers. Clinical data suggest that a higher PDCD4 protein level correlates
with a good prognosis in lung (Chen etal. 2003), colon (Horiuchi etal. 2012; Mud-
duluru etal. 2007) and ovarian (Wei etal. 2009b) cancer patients, indicating that
PDCD4 is an important tumor suppressor for many types of cancer.
Although PDCD4 cDNA was initially identified in cells treated with apoptosis in-
ducers (Shibahara etal. 1995), several controversial results have been reported regard-
ing the role of PDCD4 in apoptosis. For example, overexpression of PDCD4 cDNA in
hepatocellular carcinoma (HCC) Huh7 and breast MDA-MB-231 cancer cells induced
cell death (Afonja etal. 2004; Zhang etal. 2006). In agreement with the positive regu-
lation of apoptosis, knockdown of PDCD4 expression prevented ultraviolet radiation
(UV)-induced apoptosis in cervical carcinoma HeLa cells (Bitomsky etal. 2008)
and promoted cell proliferation in colon HT29 cells (Guo etal. 2011). Contrary to
these findings, Eto etal. showed that loss of PDCD4 expression enhances trans-
lation of procaspase 3 resulting in induction of apoptosis in HeLa and myoblast
C2C12 cells (Eto etal. 2012). Overexpression of PDCD4 had no effect on either
proliferation or apoptosis in kidney embryonic HEK293 cells (Bitomsky etal. 2008;
Shibahara etal. 1995). It is unclear whether the discrepancy in these findings is due
the tissue-specificity of PDCD4 function.
6.2PDCD4, a Binding Partner of eIF4A

and an Inhibitor of Translation
6.2.1eIF4A
eIF4A is a 46-kDa polypeptide that exhibits ATP-dependent RNA helicase activity.

It belongs to the DEAD box protein family that has nine highly conserved motifs
shared with other DEAD box proteins (reviewed in Rogers etal. 2002). Mutations
in these motifs greatly reduce the RNA-binding ability, ATPase activity or RNA
helicase activity of eIF4A, and result in translation inhibition (Pause etal. 1994).
In mammals, three eIF4A isoforms have been identified. The first two isoforms,
eIF4AI and eIF4AII, are encoded by two different genes that are highly (91%)
identical in amino acid sequence (Nielsen and Trachsel 1988). Although eIF4AI and
eIF4AII are functionally exchangeable and indistinguishable, the pattern of expres-
sion in different cell types varies distinctly (Nielsen and Trachsel 1988; Williams-
Hill etal. 1997). eIF4AI is synthesized in actively growing cells whereas eIF4AII is
synthesized under growth-arrested conditions (Williams-Hill etal. 1997). The third
isoform, eIF4AIII, is approximately 65% identical to eIF4AI and eIF4AII in amino
acid sequence. The eIF4AIII, that is functionally distinct from eIF4AI and eIF4AII,
is located in the nucleus and is involved in RNA splicing (Zhang and Krainer 2007).
Svitkin et al., using mRNAs with various stability of 5 UTR secondary structure
and eIF4A mutants, showed that the more stable the secondary structure the lower
the efficiency of translation (Svitkin etal. 2001). These observations suggest that
the requirement for eIF4A for translation is proportional to the stability of the sec-
ondary structure within the 5 UTR. Consistent with these findings, a reconstituted
complex containing 40S ribosome subunit, eIF1, eIF1A, eIF3 and the ternary com-
plex without eIF4A, eIF4B or eIF4F is able to scan an unstructured mRNA, but this
complex cannot scan mRNAs with weak secondary structure (Pestova and Kolu-
paeva 2002). Thus, it is expected that eIF4A activity is required for unwinding the
secondary structure in the 5 UTR of the mRNA, allowing the translation initiation
complex to scan the mRNA and locate the translation initiation codon.
6.2.2 PDCD4 Physically Interacts with eIF4A
Using yeast two-hybrid screen, coimmunoprecipitation, pull-down and immuno-

fluorescent colocalization, PDCD4 was demonstrated to interact with eIF4A (Goke
etal. 2002; Yang etal. 2003a; Zakowicz etal. 2005). Bioinformatics analysis of
the amino acid sequence of PDCD4 indicates that PDCD4 contains two MA3 do-
mains, nMA3 and cMA3, with 8085% consensus -helices structure (Aravind and
Koonin 2000; Ponting 2000). Indeed, the nuclear magnetic resonance and crystal
structure studies showed that nMA3 and cMA3 of PDCD4 form eight and nine
-helices, respectively (Chang et al. 2009; Loh etal. 2009; Suzuki etal. 2008).
PDCD4-eIF4A co-crystal structure analysis revealed that the nMA3 and cMA3 do-
mains of PDCD4 bind to eIF4A in a nearly perpendicular position with each MA3
domain contacting both the N-terminal domain and C-terminal domain of eIF4A.
One molecule of PDCD4 binds to two molecules of eIF4A through both nMA3 and
cMA3 domains in two different modes: the two-MA3-binding mode and the cMA3-
binding mode (Chang etal. 2009; Loh etal. 2009). In the two-MA3-binding mode,
the two MA3 domains of PDCD4 interact with both the N-terminal and C-terminal
domains of eIF4A. At the center of the interface, Glu210, Glu249 and Asp253 in
nMA3 form an ionic interaction with Arg110 and Arg161 of eIF4A. In the cMA3-
binding mode, only cMA3 interacts with N-terminal and C-terminal domains of
eIF4A. The Glu373, Arg414 and Asp418 of cMA3 interact with Arg110 and Arg161
of eIF4A, which is similar to the interaction between nMA3 and eIF4A in the two-
MA3-binding mode. In both binding modes, PDCD4 binds to the N-terminal and
C-terminal domain interface of eIF4A, which blocks RNA binding and prevents
eIF4A from conformational change to be active (Suzuki etal. 2008). In agreement
with the crystal structural analyses, the mutations of Glu249 to Lys or Asp253 to
Ala in the nMA3 of PDCD4, corresponding to the mutations of Asp414 to Lys
and Asp418 to Ala in the cMA3 of PDCD4, loses the binding ability to eIF4A in
mammalian two-hybrid analyses (Yang etal. 2004). In contrast, recent studies by
Waters etal showed that only PDCD4E249K,D235A (Glu249 to Lys and Asp253 to Ala)
mutant loses the binding ability with eIF4A but not the PDCD4D414A,D418A (Asp414
to Lys and Asp418 to Ala) mutant in an immunoprecipitation assay, arguing that
6PDCD4 139
PDCD4 binding to eIF4A is mainly through two-MA3-binding mode (Waters etal.

2011). It is unclear whether the discrepancy of these observations is due to different
experimental approaches (mammalian two-hybrid versus immunoprecipitation) or
under different cellular conditions with different binding modes (two-MA3-binding
mode versus cMA3-binding mode). It is clear that PDCD4 and eIF4A can form the
two-MA3-binding mode and cMA3-binding mode in the crystal. Do these two
binding modes also occur under physiologic condition? Does PDCD4 binding to
eIF4A shift from the two-MA3-binding mode to cMA3-binding mode under cel-
lular condition? Or vice versa? Answering these questions will definitely provide
insights into the mechanism of inhibition of translation by PDCD4.
6.2.3 PDCD4 Inhibits Translation
According to a widely accepted model for cap-dependent translation (Hinnebusch

and Lorsch 2012), the 40S ribosome subunit binds with eIF1, eIF1A, eIF2 (contain-
ing initiator tRNAiMet) and eIF3 to form a 43S PIC (for more details see Chap.2).
The 43S PIC subsequently binds to mRNA near the 5 end cap through eIF3, eIF4F
and other translation initiation factors. The complex then scans mRNA from the 5
to 3 end to form a 48S translation initiation complex at the translation initiation
codon. After recognition of AUG codon, eIF2, eIF3 and other initiation factors dis-
sociate from the translation initiation complex, and the resulting 40S ribosome joins
with the 60S ribosome to start translation. By binding to eIF4A, PDCD4 inhibits
helicase activity of both free eIF4A and eIF4F-bound eIF4A (Yang etal. 2003a).
Thus, suppression of eIF4As helicase activity is expected to inhibit cap-dependent
translation. To support this notion, we compared the effects of PDCD4 on transla-
tion of mRNA with or without a stem-loop structure within the 5 UTR and re-
vealed that PDCD4 preferentially inhibits translation of mRNA with a stem-loop
structure at 5 UTR (Yang et al. 2004). Moreover, mutation analysis of PDCD4
revealed that binding to eIF4A is required for PDCD4 to inhibit cap-dependent
translation since PDCD4 mutants defective in binding to eIF4A lose the ability to
inhibit cap-dependent translation (Chang etal. 2009; Loh etal. 2009; Yang etal.
2004). Therefore, the ability of PDCD4 to inhibit cap-dependent translation is be-
lieved to depend on the interaction with eIF4A and the inhibition of eIF4A helicase
activity. However, eIF4A is one of the most abundant translation initiation factors,
at the concentration of about 0.17mg/ml in the rabbit reticulocyte lysate in vitro
translation system (Pause etal. 1994). Inhibition of cap-dependent translation by
PDCD4 would be very inefficient if PDCD4 just binds to or interacts with eIF4A.
Indeed, in vitro studies showed that addition of native eIF4F, but not eIF4A, can
relieve the inhibitory effects of PDCD4 on cap-dependent translation (Yang etal.
2003a). Moreover, overexpression of eIF4A cDNA only partially relieves the in-
hibitory effect of PDCD4 on translation of stem-loop structured mRNA (Yang etal.
2004). On that end, Kang etal. and Wedeken etal. suggested that PDCD4 might
interact with another component of eIF4F complex, eIF4G, and with 40S ribosomal
protein (Kang etal. 2002; Wedeken etal. 2010). These observations suggest that
PDCD4 does not simply bind to eIF4A and sequesters its activity; rather PDCD4
inactivates and traps eIF4A through binding to additional translation factors and/or
proteins. Consistent with this rationale, PDCD4D418A, an eIF4A binding defective
mutant with Asp418 mutated to Ala, is able to pull down eIF4G. This suggests that
PDCD4D418A pulling down eIF4G does not occur via binding to eIF4A (Yang etal.
2003a). Thus, PDCD4 may bind to eIF4G or other translation initiation factors in
addition to eIF4A. Although PDCD4 is able to pull down or coimmunoprecipitate
with eIF4G, it does not seem to directly interact with eIF4G since no interaction
was detected in the PDCD4-eIF4G mammalian two-hybrid assays (our unpublished
data) and nuclear magnetic resonance binding assays (Suzuki etal. 2008). Thus,
it is possible that PDCD4 binds to ribosome in addition to eIF4A, and traps the
inactivated eIF4A in the translation initiation complex resulting in inhibition of cap-
dependent translation. Further investigation of PDCD4 binding to ribosomal pro-
teins is required for understanding the mechanism of regulation of cap-dependent
translation by PDCD4.
In contrast to cap-dependent translation, relatively little is known about the role
of PDCD4 in IRES-dependent translation. Using in vitro rabbit reticulocyte lysate
system, we initially demonstrated that recombinant PDCD4 inhibits encephalomyo-
carditis virus (EMCV) IRES-dependent translation of the bicistronic CAT/EMCV
luciferase reporter in a dose-dependent manner (Yang etal. 2003a). Recent studies
by Liwak etal. showed that PDCD4 is able to bind to the IRESs of antiapoptotic
proteins, such as XIAP and BCL-XL, and represses their translation by inhibiting
the formation of 48S translation initiation complex at the initiation codon (Liwak
etal. 2012). Upon FGF2 stimulation, the activated S6K2 phosphorylates PDCD4
and thereby leads to PDCD4 degradation. The downregulation of PDCD4 by S6K2
results in de-repression of XIAP and BCL-XL expression. On the other hand, over-
expression of PDCD4 in U373 glioblastoma cells reduces BCL-XL expression and
cell viability (Liwak etal. 2013). These findings are consistent with the apoptotic
role of PDCD4 by inhibiting translation of antiapoptotic proteins.
Taken together, PDCD4 functions as a translation inhibitor, which inhibits cap-
dependent translation and IRES-dependent translation. PDCD4 inhibits eIF4A he-
licase activity and may play a major role in inhibition of cap-dependent translation.
However, the detailed mechanism still needs further investigation.
6.3Regulation of PDCD4 Expression
6.3.1Epigenetic Regulation
Importantly, aside from its translation inhibitory properties, PDCD4 was validated
as a tumor suppressor (Hilliard etal. 2006; Jansen etal. 2005; Schmid etal. 2008;
Wang etal. 2008a, 2013a; Yang etal. 2001, 2003b, 2006). Thus, it is not surprising
that PDCD4 expression is commonly lost in tumors. Interestingly though, no mu-
tational inactivation of PDCD4 has been reported thus far, and a potential epigen-
6PDCD4 141
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Fig. 6.2 Regulation of PDCD4 expression. a PDCD4 expression is regulated at transcriptional,
translational, and posttranslational levels. The expression of PDCD4 is regulated at multiple levels
by inhibiting (red) and inducing (green) factors. The transcription of PDCD4 is not only inhibited
by factors within the tumor microenvironment, such as IL-2, IL-8, IL-15 and prostaglandin E2,
but also by tumor-intrinsic signals such as HER2/neu-mediated signals. Oppositely, IL-12, TGF-
as well as the extracellular matrix component decorin, enhance PDCD4 transcription. At the post-
transcriptional level, numerous miRNAs including miR-21, miR-182, miR-183 and miR-499-5p,
limit either the mRNA stability, or the translation of PDCD4, or both. The expression of miR-21
is regulated via AKT, AP-1, NF-B, but also by TGF--mediated signals. Lastly, PDCD4 level is
further regulated posttranslationally via phosphorylation-dependent proteasomal degradation. This
process can be triggered by activation of S6K1, S6K2 or AKT and is mediated by the E3-ubiquitin
ligase -TRCP1 that is overexpressed in various tumors. Phosphorylation-dependent degradation
of PDCD4 has been shown to be activated by TNF, FGF2, SDF1 (stromal cell-derived factor
1, CXCL12), BCR-ABL mutations, mitogens, and macrophage-generated inflammatory condi-
tions, signals that are commonly transmitted via mTOR. The tumor suppressive activity of PDCD4
can further be inhibited by PRMT5-mediated methylation. b Feedback regulation of miR-21 by
PDCD4. miR-21 inhibits PDCD4 expression. Yet, PDCD4 can also suppress miR-21 expression
through inactivation of AP-1.
etic inactivation by DNA methylation is controversially discussed. Indeed, while

chromatin remodeling and DNA methylation were excluded for the inactivation of
PDCD4 in breast tumor cell lines (Wen etal. 2007), methylation of CpG islands ap-
peared to contribute to PDCD4 inactivation in HCC and gliomas (Fan etal. 2007;
Gao etal. 2009).
6.3.2Transcriptional Regulation
Initially, the attempts to characterize the regulation of PDCD4 expression focused

on transcriptional changes. PDCD4 transcription was affected by a multitude of
extracellular signals, especially by a number of cytokines (Fig.6.2a). Along these
lines, IL-15 and IL-2 attenuated PDCD4 transcription in natural killer and T cells
(Azzoni etal. 1998). Similarly, the proinflammatory cytokine IL-8 reduced PDCD4
mRNA expression in breast tumor cells (Nieves-Alicea etal. 2009). In contrast,
IL-12 appeared to stimulate PDCD4 expression (Azzoni etal. 1998). Furthermore,
the lipid mediator prostaglandin E2, which is a typical component of the tumor mi-
croenvironment, also had a repressive impact on PDCD4 expression (Nieves-Alicea
etal. 2009). This finding corroborated a previous study demonstrating enhanced
expression of PDCD4 in colon carcinoma cells after treatment with the selective
cyclooxygenase 2 (COX-2) inhibitor NS-398 (Zhang and DuBois 2001). In the con-
text of inflammatory conditions, decorin, a small leucine-rich proteoglycan of the
extracellular matrix, was reported to induce PDCD4 transcription in macrophages
via toll-like receptor 2 and 4-mediated signaling (Merline etal. 2011). Decorin
further enhanced PDCD4 expression by repressing TGF-1-dependent production
of the PDCD4-inhibitory miRNA 21 (miR-21; see below). This observation was
in contrast to a previous report that TGF-1 enhanced the expression of PDCD4
(Zhang etal. 2006). Interestingly, PDCD4 transcription was also elevated by the
activation of the retinoic acid receptor in both AML cells (Ozpolat etal. 2007) and
retinoid-sensitive breast tumor cells (Afonja etal. 2004). In the latter case, PDCD4
expression was enhanced by the antiestrogen fulvestrant (ICI-182780), as well as
the HER2/neu antagonist trastuzumab (herceptin). In the case of myeloid cells,
PDCD4 was critical for terminal granulocyte differentiation, whereas it contributed
to proliferation inhibition in breast tumor cells. Thus, PDCD4 expression appeared
tumor repressive in either case.
While many stimuli affect PDCD4 transcription, the exact mechanisms of regu-
lation (i.e. involved transcription factors) remain largely elusive. Allgayer and col-
leagues recently provided a detailed analysis of the PDCD4 promoter, identifying
binding sites for zinc-finger-binding protein 89 (ZBP89) and various Sp transcrip-
tion factors (Sp1, Sp3, Sp4; Leupold etal. 2012). They further showed that while the
Sp transcription factors appeared indispensable for basal promoter activity, ZBP89
overexpression further induced PDCD4 expression. Others provided evidence for
stress-induced binding of the transcription factor FOXO to the PDCD4 promoter,
resulting in increased PDCD4 transcription (Olson etal. 2013). As an interesting
side note, when characterizing the connection between COX-2 and PDCD4, Zhang
and coworkers found that the expression of the transcription factor FAT1 increased
in parallel to PDCD4 upon COX-2 inhibition (Zhang and DuBois 2001). Surpris-
6PDCD4 143
ingly, FAT1 was recently proposed to specifically inhibit PDCD4 expression in

glioblastoma cells (Dikshit etal. 2013). Thus, FAT1 could be envisioned to act as a
safeguard to prevent a potentially deleterious overexpression of PDCD4.
In conclusion, while many PDCD4 transcription stimulating factors and con-
ditions have been identified and first attempts towards the characterization of
individual transcription factors have been made, a comprehensive picture of the
transcription factors regulating PDCD4 remains to be elucidated.
6.3.3Posttranscriptional Regulation
Marked differences between PDCD4 mRNA and protein levels were observed
in a panel of tumor cell lines and primary lung tumors (Jansen etal. 2004;
Kalinichenko etal. 2008), suggesting that PDCD4 expression might also be con-
trolled posttranscriptionally (Fig.6.2a). Asangani etal. first described that miR-21
binds to PDCD4 mRNA in colorectal cells, thereby attenuating the translation of
PDCD4 protein (Asangani etal. 2008). In subsequent work, miR-21-dependent at-
tenuation of PDCD4 expression was further shown for breast cancer cells (Frankel
etal. 2008; Lu etal. 2008), bladder carcinoma (Baffa etal. 2009), cholangiocar-
cinoma (Selaru etal. 2009), esophageal carcinoma (Hiyoshi etal. 2009), cervical
cancer (Yao etal. 2009), pancreatic ductal adenocarcinoma (Nagao etal. 2012),
urothelial carcinoma (Fischer etal. 2012), peripheral nerve sheath tumors (Itani
etal. 2012), and glioblastoma (Chen etal. 2008; Gaur etal. 2011). Moreover, miR-
21 was found to play an important role in the regulation of PDCD4 in response to a
number of inflammatory and/or tumor-associated stimuli. Along these lines, DNA
damage and bacteria-derived lipopolysaccharide were shown to induce miR-21 in
an NF-B -dependent manner resulting in the loss of PDCD4 expression (Niu etal.
2012; Sheedy etal. 2010). Furthermore, the interaction of hyaluronan-CD44 with
protein kinase C (PKC) was found to enhance miR-21 and consequently de-
crease PDCD4 expression (Bourguignon etal. 2009). Although TGF- was shown
to induce PDCD4 transcription, Davis etal. showed that TGF-1 also enhanced the
expression of miR-21, eventually limiting the expression of PDCD4 (Davis etal.
2008). Similarly, miR-183 was recently found to inhibit TGF--dependent apop-
tosis by downregulating PDCD4 (Li etal. 2010a). In addition, miR-499-5p and
miR-182 were described to target PDCD4 during tumorigenesis (Liu etal. 2011;
Wang etal. 2013b). Furthermore, bioinformatics predictions of more than 80 puta-
tive PDCD4 targeting miRs (Dweep etal. 2011) indicate that posttranscriptional
regulation of PDCD4 via miRNAs likely is even more complex.
6.3.4Posttranslational Regulation
PDCD4 has also been described to be regulated posttranslationally (Fig.6.2a). The

first evidence of posttranslational modifications of the PDCD4 protein was provided
by Palamarchuk and coworkers, who characterized two putative phosphorylation
sites in PDCD4. Specifically, they found that AKT phosphorylates PDCD4 at Ser67
and Ser457, and noticed enhanced nuclear accumulation of PDCD4 in response to
AKT-mediated phosphorylation at Ser457 (Palamarchuk etal. 2005). In contrast,

Ser67 appears to be a part of a phosphodegron motif, which is recognized and bound
by the ubiquitin ligase -TRCP1 (-transducin repeat-containing 1) upon phosphory-
lation by S6K1 or AKT. Upon binding, -TRCP1 facilitates the ubiquitylation of
PDCD4, thereby marking the protein for proteasomal degradation (Dorrello etal.
2006). Mitogens, and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate
(TPA), were shown to limit PDCD4 protein expression in tumor cells by activat-
ing the PI3K/AKT/S6K axis and consequently enhance proteasomal degradation of
PDCD4 (Nakashima etal. 2010; Schmid etal. 2008). Consistent with this notion,
overactivation of mTOR/S6K in BCR-ABL-transformed cells resulted in enhanced
degradation of PDCD4 (Carayol etal. 2008). Interestingly, stimuli provided by the
tumor microenvironment also affected PDCD4 protein stability. For example, in-
flammatory conditions supplied by activated macrophages, as well as the proinflam-
matory cytokine tumor necrosis factor (TNF) , were found to enhance the deg-
radation of PDCD4 (Lee etal. 2013; Schmid etal. 2011; Yasuda etal. 2010). Along
these lines, PDCD4 was identified as part of the phosphoproteome in response to
CXC chemokine ligand 12 (CXCL12) /chemokine receptor 4 (CXCR4) signal-
ing in chronic lymphocytic leukemia (OHayre etal. 2010). Liwak etal. recently
demonstrated that FGF2-induced activation of S6K2 enhances phosphorylation and
degradation of PDCD4 (Liwak etal. 2012). They further showed that the prosur-
vival effect of FGF2/S6K2 signaling is mediated in part by the loss of PDCD4 and
the resulting de-repression of the translation of the antiapoptotic proteins XIAP and
BCL-XL. Importantly, as the PI3K signaling cascade is commonly activated in tu-
mors, attenuated protein stability might explain low PDCD4 levels in tumors. In fact,
PDCD4 expression was shown to inversely correlate with enhanced activation of the
PI3K/AKT/S6K axis in an animal tumor model (Schmid etal. 2008). Moreover, loss
of PDCD4 correlating with enhanced presence of phospho-AKT was put forward as
a prognostic marker for disease-associated survival in CRC (Mudduluru etal. 2007).
While phosphorylation-dependent degradation of PDCD4 emerged as a central
mechanism of PDCD4 regulation, the different layers of PDCD4 regulation appear
to be highly interconnected. The complex interplay is not only highlighted by the
aforementioned connections of TGF- and PDCD4, but also by an autoregulatory
loop in which PDCD4 inhibits the transcription factor activating protein 1 (AP-1),
thereby limiting the expression of miR-21 (Talotta etal. 2009; Fig.6.2b). As miR-
21 in turn represses PDCD4 expression, the PDCD4/AP-1/miR-21 circuit appears
as a self-enhancing regulatory cycle. In addition, enhancement of miR-21 expres-
sion is also regulated by activation of AKT signaling that impacts PDCD4 stability
but also PDCD4 localization (Fassan etal. 2012; Polytarchou etal. 2011).
Taken together, the tight regulation of PDCD4 expression is an indicator for
its importance as a tumor suppressor. This is further underscored by recent obser-
vations in breast cancer patients, where in tumors that express high levels of the
methyltransferase PRMT5 unexpectedly elevated PDCD4 protein expression cor-
related with poor prognosis (Powers etal. 2011). This was explained by the fact that
PRMT-mediated arginine methylation of PDCD4 changed the tumor suppressive
function of PDCD4 to a tumor promoting one. Thus, inactivation of PDCD4, either
by attenuating its expression or by preventing its tumor suppressive functionality,
appears crucial for tumorigenesis.
6PDCD4 145
6.4PDCD4 in Cancer Development and Progression
6.4.1 PDCD4 Inhibits Tumor Promotion
Tumorigenesis is a multistep genetic process that can often be divided into initia-
tion, promotion and progression. Promotion is a low-frequency event required for
chronic exposure to tumor promoters, such as phorbolester or growth factors result-
ing in formation of benign tumor. Using differential display of mRNA, Cmarik etal.
identified that PDCD4 mRNA as one of several genes whose expression is high
in the JB6 promotion-resistant but low in the promotion-susceptible cells (Cmarik
etal. 1999). The JB6 mouse epidermal clonal genetic variant cell system is a unique
cell culture model for studying tumor promoter-dependent biochemical events oc-
curring during preneoplastic progression (Colburn etal. 1979; Zhang etal. 2011).
Promotion-susceptible JB6 cells undergo neoplastic transformation in response to
tumor promoters such as TPA and TNF, forming anchorage-independent colonies
in soft agar (Colburn etal. 1979) and tumors when injected into mice (Colburn
1980; Takahashi etal. 1986). Promotion-resistant cells are resistant to promoter-
induced anchorage-independent neoplastic transformation. To test the tumor sup-
pressor function of PDCD4, Cmarik etal. lowered PDCD4 protein levels in promo-
tion-resistant cells by expressing antisense PDCD4 and found that downregulation
of PDCD4 expression rendered cells sensitive to TPA-induced neoplastic transfor-
mation (Cmarik etal. 1999). Compatible with this finding, promotion-susceptible
cells with elevated PDCD4 protein levels via overexpression of PDCD4 cDNA gain
the phenotype of resistance to TPA-induced neoplastic transformation (Yang etal.
2001). In addition to cell culture system, overexpression of PDCD4 in the epidermis
significantly reduces 7,12-dimethylbenz(a)anthracene (DMBA)/TPA-induced skin
papilloma formation and carcinoma incidence in PDCD4 transgenic mice (Jansen
etal. 2005). Conversely, knockout (KO) of PDCD4 in mice results in an increase in
DMBA/TPA-induced skin papilloma multiplicity and carcinoma incidence (Schmid
etal. 2008). These in vitro and in vivo results indicate that PDCD4 suppresses the
tumor promotion stage.
Elevation of PDCD4 protein levels in JB6 promotion-susceptible cells has been
shown to inhibit basal and TPA -induced AP-1-dependent transcription (Yang etal.
2001). Similar results were obtained from expressing AP-1 luciferase reporter in the
keratinocytes of PDCD4 overexpressed transgenic mice (Jansen etal. 2005) and de-
livering PDCD4 cDNA into the lung of AP-1 reporter mice (Hwang etal. 2007).
AP-1 is a complex of transcription factors comprised of homodimers of the JUN
family proteins (c-JUN, JUNB and JUND) or heterodimers of JUN and FOS family
proteins (c-FOS, FRA-1, FRA-2, and FOSB), which belong to the basic leucine zip-
per domain family. The JUN/JUN or JUN/FOS dimers bind with higher affinity to the
TGA(G/C)TCA TPA response element, and with lower affinity to the TGACGTCA
cAMP (cyclic AMP) response element (Chinenov and Kerppola 2001). The down-
stream targets of AP-1-dependent transcription are frequently involved in regulation
of cell proliferation, transformation and cell invasion (Ozanne etal. 2000; Shaulian
and Karin 2002). Inactivation of AP-1 activity by expressing dominant-negative
c-JUN reverses the transformation phenotype to a normal phenotype (Dong etal.

1994). Thus, inhibition of AP-1-dependent transcription by PDCD4 likely contributes
to suppression of TPA -induced tumor promotion. Using a transactivation luciferase
system, we initially suggested that PDCD4 suppresses c-JUN transactivation result-
ing in inactivation of AP-1-dependent transcription in JB6 cells (Yang etal. 2003a).
Bitomsky etal. (2004) similarly observed that elevated PDCD4 protein levels reduce
phosphorylation of c-JUN by inactivation of the c-JUN N-terminal kinase (JNK) in
QT6 quail fibroblasts cells. A more detailed analysis of the activation of c-JUN in
colon RKO cells revealed that PDCD4 inhibits the expression of mitogen-activate
protein (MAP) kinase kinase kinase kinase 1 (MAP4K1), an upstream kinase of JNK,
leading to inactivation of JNK and c-JUN (Yang etal. 2006; Fig.6.3). Consistent
with these results, we recently found that knockdown of PDCD4 in colon HT29 cells
upregulates MAP4K1 expression and thereby activates JNK, c-JUN and AP-1-de-
pendent transcription (Wang etal. 2012; Fig.6.3).
6.4.2 PDCD4 Inhibits Tumor Progression
Tumor progression describes the transformation from a benign tumor to a malignant

tumor followed by invasion, metastasis, and angiogenesis. Tumor metastasis is a
process to spread tumor cells from the primary tumor to the secondary site within
the human body, which includes: (1) detachment of tumor cells from primary tu-
mor; (2) intravasation into blood or lymphatic vessels; (3) extravasation from blood
or lymphatic vessels; and (4) colonization and outgrowth at a secondary site (Thiery
2002). A number of studies have demonstrated that PDCD4 regulates tumor inva-
sion and metastasis. For instance, ectopic expression of PDCD4 cDNA suppressed
invasion in colon carcinoma RKO (Leupold etal. 2007; Yang etal. 2006), breast
MCF-7 (Gonzalez-Villasana etal. 2012; Nieves-Alicea etal. 2009), HCC MHCC-
97L (Zhang etal. 2009) and ovarian carcinoma OVCA433 and SKOV3 cells (Wei
etal. 2012). Conversely, knockdown of PDCD4 expression promoted invasion in
colon HT29 and GEO cells (Wang etal. 2008a, 2010a) as well as in breast cancer
MCF-7 and T47D cells (Santhanam etal. 2010). In agreement with these in vi-
tro studies, knockdown of PDCD4 in colon tumor cells also promotes metastasis
when injected into nude mice (Wang etal. 2013a). In addition, KO of PDCD4 in
mice induces lymphomas with frequent metastasis (Hilliard etal. 2006). A detailed
mechanism of inhibiting invasion and metastasis by PDCD4 was studied in colon
tumor cells. Knockdown of PDCD4 in the colon HT29 and GEO cells was shown
to decrease expression of epithelial proteins (-catenin, -catenin, and E-cadherin)
and increase expression of mesenchymal proteins (N-cadherin and fibronectin) ,
suggesting that knockdown of PDCD4 leads to EMT (Wang etal. 2013a).
The EMT is an important process associated with development of metastatic
tumors and increase in tumor aggressiveness. One of the hallmarks of EMT is
the functional loss of E-cadherin, a transmembrane glycoprotein on the cell sur-
face. Downregulation of E-cadherin expression by PDCD4 knockdown is mainly
achieved via upregulation of SNAIL, a transcription repressor that binds to the E-
6PDCD4 147
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Fig. 6.3 PDCD4 suppresses tumorigenesis through different pathways. PDCD4 regulates cell pro-
liferation, tumor promotion, and tumor progression via various pathways. The different pathways
are shown in different colors. PDCD4 inhibits cell proliferation or cell cycle progression through
repression of CDK1/CDK2 via the upregulation of p21Waf1/Cip1 expression, or by inhibition of
cyclin D1 expression by the attenuation of AKT, or by the inhibition of CA II translation. PDCD4
regulates tumor promotion mainly via inhibition of AP-1-dependent transcription that is controlled
by the JNK signaling pathway. Suppression of tumor progression by PDCD4 is mediated via the
inhibition of the expression of transcription repressor SNAIL. SNAIL negatively regulates E-cad-
herin expression resulting in activation of -catenin-dependent transcription and increased c-MYC
and uPAR expression. Then c-MYC subsequently stimulates MAP4K1 expression and thereby
activates both JNK signaling pathway and AP-1-dependent transcription. PDCD4 may also elevate
TIMP2 (tissue inhibitor of metalloproteinase 2) expression that inhibits tumor invasion. In addi-
tion, knockdown of PDCD4 may increase cell survival by induction of p53 translation in response
to UV irradiation or treatment with DNA-damaging agents.
cadherin promoter and inhibits E-cadherin transcription (Wang etal. 2010a). As a

consequence of decreased E-cadherin expression, -catenin translocates into the
nucleus to activate -catenin-dependent transcription and thereby enhances expres-
sion of c-MYC and a urokinase-type plasminogen activator receptor (uPAR) (Wang
etal. 2008a, 2010a). Then, c-MYC stimulates MAP4K1 expression leading to acti-
vation of the JNK signaling pathway and AP-1-dependent transcription (Wang etal.
2012; Fig.6.3). Consistent with these findings, overexpression of PDCD4 in colon
RKO cells suppresses MAP4K1 expression, JNK signaling pathway activation, and
AP-1-dependent transcription (Yang etal. 2006). uPAR is a 4560kDa glycosylat-
ed membrane protein. By binding to its ligand, uPA, uPAR leads to the degradation
of extracellular matrix components and promotion of invasion and metastasis. Both
c-MYC and uPAR contribute to the promotion of tumor cell invasion since knock-
down of c-MYC or uPAR inhibits invasion induced by PDCD4 knockdown (Wang
etal. 2010a). In addition, studies from breast cancer cells have shown that PDCD4
increases tissue inhibitor of metalloproteinase 2 (TIMP2) expression, resulting in
inhibition of tumor invasion (Gonzalez-Villasana etal. 2012; Nieves-Alicea etal.
2009; Fig.6.3).
How does PDCD4 regulate SNAIL expression? One possible mechanism for
controlling SNAIL expression is through AKT, whose activity is regulated by
PDCD4 (Guo etal. 2011; Lankat-Buttgereit etal. 2008; Wei etal. 2012). Cytoplas-
mic translocation and degradation of SNAIL require phosphorylation by GSK3
(Zhou etal. 2004). Phosphorylation of GSK3 by AKT leads to inactivation of
GSK3, resulting in stabilization of SNAIL (Zhou etal. 2004). The other possible
mechanism is translational regulation. Evdokimova etal. showed that the 5 UTR of
SNAIL mRNA forms a stable secondary structure and translation of SNAIL mRNA
is regulated by Y-box binding protein 1 (YB-1) (Evdokimova etal. 2009). PDCD4
has been demonstrated to preferentially inhibit translation of mRNA with a second-
ary structure within the 5 UTR (Yang etal. 2004). PDCD4 may thus directly inhibit
translation of SNAIL mRNA. Alternatively, PDCD4 may interact with the helix-
loop-helix-loop domain of TWIST1 and inhibit expression of YB-1 (Shiota etal.
2009), resulting in suppression of SNAIL expression.
In addition to the regulation of tumor invasion and metastasis, Krug etal. found
that knockdown of PDCD4 increases tube formation in an in vitro angiogenesis
model (Krug etal. 2012). Stimulation of angiogenesis by PDCD4 knockdown might
be through upregulation of angiopoietin-2, whose expression is regulated by PI3K/
AKT signaling pathway and/or AP-1 activation (Tsigkos etal. 2006; Ye etal. 2007).
6.4.3 P
DCD4 Regulates Cell Cycle Progression
and Cell Proliferation
There is further evidence that PDCD4 regulates cell cycle progression and cell pro-
liferation. Goke etal. showed that overexpression of PDCD4 inhibits CDK1 expres-
sion through the upregulation of CDK inhibitor p21Waf1/Cip1 in human carcinoid
Bon-1 cells (Goke etal. 2004). CDK1 binds with cyclin B to control G2/M tran-
sition during a cell cycle. Similarly, overexpression of PDCD4 in ovarian cancer
cells causes cell cycle arrest in the S and G2 phases (Wei etal. 2009b). However,
Bitomsky etal. showed that knockdown of PDCD4 upregulates p21Waf1/Cip1 and
several other p53-regulated genes to resist DNA damage-induced apoptosis in HeLa
6PDCD4 149
cells (Bitomsky etal. 2008). p21Waf1/Cip1 was shown to contain proprolifera-

tive and antiproliferative function depending on the cell type and cellular context
(Liu etal. 2003). The discrepancy in these findings may be due to specific cell
types. In agreement with the negative regulation of cell proliferation and cell cycle
progression, knockdown of PDCD4 in colon HT29 cells enhances G1/S progres-
sion and increases cell proliferation (Guo etal. 2011). Moreover, immunohisto-
chemistry (IHC) showed that expression of PDCD4 is inversely correlated with the
proliferation marker Ki-67 in human intraductal papillary mucinous carcinoma tis-
sues (Hayashi etal. 2010). In addition to tissue culture and IHC studies, aerosol de-
livery of PDCD4 cDNA into murine lung inhibits cell proliferation in K-RAS-null
mice and AP-1 reporter mice (Hwang etal. 2007; Jin etal. 2006). Furthermore, we
recently showed that the proliferation index of colon tumors in nude mice derived
from injected PDCD4 knockdown cells is three fold higher than that of control cells
(Wang etal. 2013a). Another line of evidence is that PDCD4 is a bona fide target of
miR-21. Several reports have shown that posttranscriptional inhibition of PDCD4
expression by miR-21 leads to apoptosis suppression and proliferation promotion in
tumor cells (Bhatti etal. 2011; Ma etal. 2011; Niu etal. 2012; Yang etal. 2011). In
addition to cancer cells, it has been reported that overexpression of PDCD4 increas-
es apoptosis and inhibits proliferation of vascular smooth muscle cells (Liu etal.
2010). Promotion of cell proliferation by PDCD4 knockdown in colon HT29 cells,
at least in part, takes place through elevation of cyclin D1 expression (Guo etal.
2011). The increased cyclin D1 expression is partially due to the activation of NF-
B regulated by the IkB kinases (IKK and IKK) through AKT (Guo etal. 2011).
Moreover, since PDCD4 also has been reported to regulate AP-1- and -catenin-
dependent transcription (Wang etal. 2008a, 2010a; Yang etal. 2001, 2006), it is
possible that downstream targets function as effectors on modulating proliferation
in some types of cells. All of these findings suggest that PDCD4 inhibits cell prolif-
eration in vitro and in vivo.
6.4.4 P
DCD4 Translational Targets are Involved
inTumorigenesis
It has been suggested that CA II (carbonic anhydrase type II) is translationally regu-
lated by PDCD4. Lankat-Buttgereit etal. showed that overexpression of PDCD4 in
HEK293 cells inhibited CA II expression at the protein level but not at the mRNA
level (Lankat-Buttgereit etal. 2004). CA II catalyzes the hydration of CO2 to bicar-
bonate required for fast growth in tumor cells. In addition, a recent study suggested
that PDCD4 directly binds to C-MYB (myeloblastosis, cellular proto-oncogene)
mRNA in the coding region, and thereby inhibits cap-dependent translation but not
HCV IRES-dependent translation (Singh etal. 2011). C-MYB is a transcription
factor which has been shown to be involved in leukemia, colon cancer and breast
cancer development (Ramsay and Gonda 2008). A more detailed study by Wedeken
etal. (2011) using sucrose gradient fractionation analysis demonstrated that knock-
down of PDCD4 in HepG2 cells shifts TP53 mRNA distribution into polysomal
factions, suggesting that PDCD4 regulates TP53 mRNA translation. Further analy-
sis showed that wild-type PDCD4, but not eIF4A binding defective PDCD4 mutant,
is able to inhibit translation of TP53 5 UTR luciferase, in which TP53 5 UTR
was fused with the luciferase coding region. This observation suggests that PDCD4
inhibits p53 translation through binding to eIF4A (Wedeken etal. 2011). Although
downregulation of PDCD4 is correlated with upregulation of p53 in response to
UV irradiation or DNA-damaging agents, the physiological role of inhibiting p53
translation by PDCD4 is still unclear.
6.5PDCD4 as a Tumor Marker and Therapeutic Target
6.5.1 PDCD4 as a Biomarker
Loss of PDCD4 mRNA expression was suggested as an indicator for tumor pro-
gression and prognostic marker in lung (Chen etal. 2003), colon (Mudduluru etal.
2007), and ovarian cancer (Wang etal. 2008b). Low PDCD4 expression was also
associated with poor prognosis in glioma patients, where PDCD4 silencing resulted
from CpG island methylation (Gao etal. 2009). Further reports showed an inverse
correlation of PDCD4 protein expression with advancing tumor stages and proposed
loss of PDCD4 protein as a negative prognostic markers for renal cell carcinoma
(RCC) (Li etal. 2012) and an independent risk factor for salivary adenoid cystic
carcinoma (Qi etal. 2013). Importantly, the predictive value of PDCD4 has strongly
been improved since the PDCD4 regulatory circuits were included in the analyses.
For example, decreased PDCD4 mRNA expression in association with enhanced
miR-21 expression appeared as a negative prognostic marker in CRC (Horiuchi
etal. 2012). Furthermore, Fassan etal. (2011) showed a correlation between en-
hanced miR-21 expression with repressed PDCD4 mRNA and protein expression
in colon cancer, putting forward low nuclear levels of PDCD4 protein as an early
diagnostic marker. Similarly, in urothelial carcinoma, elevated miR-21 expression
was confirmed to downregulate PDCD4, which emerged as a diagnostic marker
for transitional cell carcinoma (Fischer etal. 2012). Apart from its connection with
miR-21, increased activation of PI3K/AKT signaling has been associated with low
PDCD4 protein levels as a tumor marker. Allgayer and coworkers established that
loss of nuclear PDCD4 expression correlated with elevated AKT phosphorylation
as a supportive diagnostic tool for the transition between normal and tumor tissues
in colon cancer (Mudduluru etal. 2007). In a recent study, Fassan etal. (2012) even
provided evidence for a correlation between attenuated PDCD4 protein expression
and enhanced phosphorylation of AKT or elevated expression of miR-21 in esopha-
geal tumor lesions, the latter being a reliable biomarker of early-stage squamous
cell esophageal neoplasia.
Thus, loss of PDCD4 emerges as a promising diagnostic marker of tumor pro-
gression and appears to contain prognostic value for various tumor types.
6PDCD4 151
6.5.2 Loss of PDCD4 in Therapy Resistance
There is increasing evidence that loss of PDCD4 may be a useful biomarker for
tumor progression, and that low PDCD4 levels could be predictive of the develop-
ment of resistance to various tumor therapies. Bourguignon etal. were the first to
suggest that PDCD4 expression could be functionally important to sustain tumor
cell sensitivity against several chemotherapeutics (e.g. paclitaxel, doxorubicin)
(Bourguignon etal. 2009). Similarly, loss of PDCD4 appeared to be critical for the
development of miR-21-dependent resistances against docetaxel (Shi etal. 2010)
and hypoxia (Polytarchou etal. 2011). Moreover, the miR-21/PDCD4 axis emerged
as being differentially expressed in tamoxifen-resistant as compared to sensitive
MCF-7 cells (Manavalan etal. 2011). Mechanistically, the role of PDCD4 in thera-
py sensitization was attributed to its repressive effect on a number of antiapoptotic
proteins such as BCL-XL (Liwak etal. 2013) and FLIP (FLICE-like inhibitory pro-
tein; Wang etal. 2010b). Interestingly, several studies have aimed at resensitizing
tumors to chemotherapeutics by inhibiting miR-21 and/or elevating PDCD4 protein
expression (Li etal. 2010b; Liu etal. 2013; Lu etal. 2012).
Aside from its tumor prognostic properties, whether PDCD4 can be established
as an indicator for therapeutic response remains an interesting topic for future study.
6.5.3 PDCD4 as a Therapeutic Target
Since transgenic overexpression of PDCD4 was shown to limit tumor formation

in a mouse model (Jansen etal. 2005), increasing PDCD4 protein expression in a
tumor setting is increasingly investigated as potential therapeutic target. Consistent
with this observation, administration of PDCD4 in an aerosol-inhalation model
proved effective in suppressing the activity of the transformation marker AP-1 and
controlling cell cycle progression in lungs of treated animals (Hwang etal. 2007).
Similarly, delivering PDCD4 expression vectors into tumor xenografts suppressed
tumor growth (Kim etal. 2010). Thus, rescuing PDCD4 protein expression in tu-
mors indeed appeared as an appealing therapeutic concept. Based on the multi-
tude of regulatory mechanisms contributing to the elimination of tumor suppressor
PDCD4, a broad spectrum of therapeutic approaches can be envisioned to increase
PDCD4 expression indirectly by counteracting negative regulators. Therapeutics
preventing enhanced, tumor-associated degradation of PDCD4, i.e. stabilizing
PDCD4 protein, by inhibiting PI3K/mTOR/S6K signaling or interfering with the
proteasomal degradation can be predicted to elicit antitumorigenic effects. In sup-
port of this concept, the effect of fluvastatin against RCC was shown to rely on the
inhibition of AKT/S6K, which was at least partially attributed to the stabilization
of PDCD4 (Woodard etal. 2008). Along similar lines, in BCR-ABL-transformed
leukemic cells the use of the second generation BCR-ABL kinase inhibitors
imatinib mesylate or nilotinib efficiently blocked S6K activation and increased
PDCD4 protein expression. Importantly, PDCD4 stabilization appeared essential
for the inhibition of leukemic progenitor colony formation by these compounds
(Carayol etal. 2008). In an attempt to further explore PDCD4 stabilization thera-

peutically, screening tools have been developed to identify compounds interfer-
ing with the PI3K-signaling cascade and consequently phosphorylation-dependent
degradation of PDCD4 (Blees etal. 2010; Carlson etal. 2009). The use of such an
approach revealed a number of novel stabilizers of PDCD4 (Grkovic etal. 2011;
Zhao etal. 2011; Bajer et al. 2014). Interestingly, one natural product-derived com-
pound, the sesquiterpene lactone erioflorin, did not affect the phosphorylation of
PDCD4, but rather inhibited the interaction between phosphorylated PDCD4 and
the E3-ubiquitin ligase -TRCP1 to stabilize PDCD4 (Blees etal. 2012). Thus, in
addition to inhibitors of the kinases responsible to mark PDCD4 for degradation,
interference with the proteasomal degradation machinery could emerge as promis-
ing therapeutic approach, either by globally inhibiting the proteasome or by selec-
tively inhibiting the specific E3-ubiquitin ligase -TRCP1.
In addition, PDCD4 expression is predicted to increase when miR-21 is inhibited,
at least in tumors where PDCD4 is primarily controlled by this miR. For instance,
delivery of anti-miR-21 oligonucleotides or the inhibition of miR-21 expression
with resveratrol elevated PDCD4 levels (Bourguignon etal. 2009; Kim etal. 2011;
Tili etal. 2010). Similarly, Mudduluru etal. (2011) showed that curcumin interferes
with the AP-1/miR-21/PDCD4 circuit by inhibiting AP-1 activity, thereby prevent-
ing miR-21 expression and consequently allowing for increased production of
PDCD4 and further inhibition of AP-1.
Therapeutically increasing PDCD4 levels thus appears to be a promising ap-
proach. Nevertheless, based on the tumor-specific mechanism of PDCD4 regula-
tion, a thorough evaluation of the PDCD4 limiting factors would be advisable. This
notion is corroborated by a recent report that resveratrol enhanced PDCD4 expres-
sion by inhibiting miR-21 only in some prostate cancer cells, while in other cell
lines resveratrol increased PDCD4 protein exclusively by inhibiting AKT-signaling,
independent of miR-21 (Sheth etal. 2012).
It is clear that PDCD4 suppresses both early (tumor promotion) and late stages (tu-
mor invasion, metastasis, and angiogenesis) of carcinogenesis (Jansen etal. 2005;
Santhanam etal. 2010; Schmid etal. 2008; Wang etal. 2008a, 2010a, 2013a; Wei
etal. 2009a, 2012; Yang etal. 2003b, 2006). The biochemical function of PDCD4
is a translation inhibitor. PDCD4 is one of the only two known factors that function
as a tumor suppressor as well as a translation inhibitor, the others represented by
4E-BPs. Thus, PDCD4 provides a perfect platform for understanding how transla-
tional inhibition of certain mRNAs can suppress tumorigenesis. Although PDCD4
was shown to bind with eIF4A and thereby inhibit translation a decade ago, the
PDCD4 translational targets important for coordination of tumorigenesis and the
mechanism(s) underlying this process are still unclear. It is believed that inhibiting
eIF4A helicase activity is a key action for PDCD4 to inhibit translation. Further-
more, recent studies suggested that in addition to eIF4A, PDCD4 might bind with
6PDCD4 153
other translation factor(s). This finding suggests that PDCD4 may not simply se-
quester eIF4A activity. Investigation of PDCD4 binding to eIF4A and other transla-
tion factor(s) will reveal mechanistic insights of regulation translation by PDCD4.
PDCD4 is known to inhibit AP-1-dependent transcription. AP-1 is a transcription
factor, which regulates expression of numerous genes involved in cell proliferation,
survival, migration, and invasion. In addition, recent studies suggest that PDCD4
regulates AKT activation and phosphorylation. AKT, one of the most frequently
activated protein kinases in human cancer, mediates a large spectrum of cellular
functions including proliferation, survival, and invasion. Thus, elevation of PDCD4
expression is a promising strategy for cancer therapeutics. Since PDCD4 expression
is frequently downregulated in cancer cells through miRNA-mediated inhibition or
proteasomal degradation, the compounds to suppress these actions will be valuable
agents for cancer therapeutics.
Acknowledgments We are very grateful to Dr. Nancy Colburn (NCI-Frederick) for reading and
discussion of this manuscript. We would like to thank Mrs. Heather Russell-Simmons and Donna
Gilbreath for editing this manuscript. This work was supported by the National Institute of Health
Grant RO1CA129015 (to H.-S.Y.), the LOEWE-Schwerpunkt OSF [III L 4518/55.004 (2009)]
funded by the Hessian Ministry of Higher Education, Research and Arts (to T.S.), and the DFG
GRK1172 (to M.M.B.) and SCHM2663/3 (to T.S.).
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Chapter 7
eIF4G
Simon D. Wagner, Anne E. Willis and Daniel Beck
Contents
7.1eIF4G and its Regulation 164

7.2Genomic Organization, Expression and Structure of eIF4G 165
7.3Involvement of eIF4G in Transformation and Cancer 166
7.4eIF4G and eIF4E Interaction as a Therapeutic Target 168
References 169
Abstract eIF4G is an essential component of the cap-binding complex, acting as

a scaffold for the assembly of mRNA, ribosomal subunits and cap-binding pro-
teins during translation. The most highly expressed human isoform, eIF4G1, is also
functionally the most important. eIF4G is implicated in the biology of breast and
lung cancers, where it is found overexpressed. eIF4G locus is also amplified in lung
cancer. Systematic attempts to abrogate eIF4G function have included the design
of peptidomimetics and small molecules to prevent the interaction between eIF4E
and eIF4G.
S.D.Wagner() D.Beck
Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK
e-mail: sw227@le.ac.uk
S.D.Wagner A.E.Willis D.Beck
Medical Research Council (MRC) Toxicology Unit, Leicester, UK
S.D.Wagner
Department of Haematology, University Hospitals of Leicester, University of Leicester,
Leicester, UK
164 S. D. Wagner et al.
3$%3 (,)( (,)$ (,) (,)$ 0.1.

DD (,)*
0,)* 0$
a
DD (,)*
b DD (,)*
Fig. 7.1 The domain structure of the three human eIF4G protein isoforms. a The domain struc-
ture of the main eIF4G isoform, eIF4G1. Binding sites of the main interacting proteins are indi-
cated: PABP, eIF4E, eIF4A, eIF3 and MNK1. The dark grey-shaded rectangles represent HEAT
domains. Other conserved domains (Ponting 2000), MIF4G (middle domain of eIF4G) and MA3
are also indicated. b The relative sizes of eIF4G2 and eIF4G3 are illustrated. eIF4G2 lacks PABP
and eIF4E-binding sites.
7.1eIF4G and its Regulation
Protein synthesis is a process comprised of initiation, elongation, termination and ribo-

some recycling (see Chap.2). Initiation is thought to be the rate-limiting step of the
process and, in agreement with this hypothesis, dysregulation of eukaryotic initiation
factors, either through their overexpression or changes in their phosphorylation status, is
associated with tumorigenesis (Blagden and Willis 2011; Le Quesne etal. 2010).
Initiation of mRNA translation requires the recruitment of the cap-binding pro-
tein complex, eIF4F, to the 5 end of an mRNA (see Chap.2). This complex consists
of eukaryotic initiation factor eIF4G, a scaffold protein, eIF4E, which binds the
7-methyl GpppN cap of mRNA, and eIF4A a DEAD box helicase (Sonenberg and
Hinnebusch 2009). Recruitment of a 40S ribosomal subunit containing a ternary
complex, comprising eIF2, Met-tRNAiMet and GTP, to the eIF4F complex is fol-
lowed by start codon recognition and association with the 60S ribosomal subunit
to form a translation-competent 80S ribosome, which accomplishes the elongation
phase of protein synthesis.
In more detail, eIF4G binds to eIF4E (and therefore the mRNA cap structure)
through a domain at its N-terminal whilst the C-terminal contains two binding do-
mains for eIF4A (Dever 2002; Preiss and W Hentze 2003). eIF4G is able to enhance
association of eIF4E with the cap structure through allosteric mechanisms (Gross
etal. 2003); it has binding sites for PABP also at its N-terminal (Fig.7.1) allow-
ing PABP to interact with both the poly(A) tail of the mRNA and eIF4G. This both
stabilizes and circularizes the complex and stimulates translation (Sonenberg and
Hinnebusch 2009). eIF4G also possesses a binding site for eIF3, a multisubunit
polypeptide that is required to bring the 40S ribosomal subunit to the complex (see
Chaps.7 and 8). Through interactions with these additional proteins and their inter-
acting partners translation can be regulated in an eIF4G-dependent manner.
The translational repressor, PAIP2, inhibits translation by promoting the dissoci-
ation of PABP from poly(A), thereby disrupting PABP/poly(A)/eIF4G interactions.
In addition, PAIP2 has been shown to inhibit the activity of PABP, independent of
its ability to bind to eIF4G (Karim etal. 2006).
The translation machinery is very responsive to changes in the external environ-
ment, which allows both stimulation and repression of this process. A major route
7eIF4G 165
to achieve this is through activation of the PI3K/AKT/mTOR pathway, which al-

ters the phosphorylation status of the eIF4E-binding proteins, 4E-BPs. When phos-
phorylated these proteins dissociate from eIF4E and increase its bioavailability for
eIF4G, thereby allowing formation of the eIF4F complex. As discussed in Chap.15
this pathway also participates in direct phosphorylation of eIF4G, although the
physiological relevance of this is not clear.
The MAPK pathway is an alternative route by which changes in the external en-
vironment influence translation (see Chap.17). MNK1 and MNK2 are phosphorylat-
ed through the MAPK pathway, which in turn is activated in response to exogenous
growth factors and contributes to driving cellular proliferation. Following phosphoryla-
tion MNK1/2 associate with eIF4G and phosphorylate eIF4E, also bound to eIF4G, to
promote cap-binding (Scheper and Proud 2002). The binding of MNK1/2 to eIF4G is
further regulated by protein kinase C (PKC), with eIF4G being phosphorylated at
Ser1185 to promote binding to MNK1/2 (Dobrikov etal. 2011).
eIF4G is also regulated by proteolysis under a range of different pathophysi-
ological situations. For example, following infection by picornaviruses including
enteroviruses, rhinoviruses and aphthoviruses, eIF4G1 is cleaved by the viral pro-
tease 2A causing repression of cap-dependent translation (Glaser and Skern 2000;
Lamphear etal. 1993; Ventoso etal. 1998). Cleavage by the viral protease sepa-
rates the cap-binding and PABP regions of eIF4G from the eIF3 and eIF4A binding
sites (Lamphear etal. 1993). The overall effect is that cap-dependent translation is
inhibited. However, translation of viral mRNAs (which is dependent on complex
RNA structural elements termed IRES) continues (see Chap.3). IRESs are able to
recruit the ribosome to the mRNA in a cap-independent manner and in this case the
ribosome is deposited on the mRNA at a site that can be a considerable distance
downstream of the 5 end of the message. eIF4G can also be proteolytically cleaved
during apoptosis (Bushell etal. 2000; Marissen and Lloyd 1998; Marissen etal.
2000), specifically by caspase 3 (Bushell etal. 1999). As a result of cleavage dur-
ing apoptosis three eIF4G fragments (N-terminal (N-FAG), middle (M-FAG) and
C-terminal (C-FAG)) are produced. Interestingly the fragments produced during
apoptosis are functionally distinct from those produced following viral infection.
M-FAG contains binding sites for eIF4E, eIF4A and eIF3, thereby allowing contin-
ued linkage between the cap structure and the 40S ribosomal subunit (Bushell etal.
2000). However, despite this it has been determined that under these conditions
there is a shut down of cap-dependent translation and much of the synthesis of the
key proteins required for this process to proceed is mediated by cellular IRES-
mediated translation (Bushell etal. 2006).
7.2Genomic Organization, Expression and Structure

of eIF4G
There are three eIF4G paralogs in the human genome: eIF4G1 is located at chromo-
some 3q27, eIFG2 at 11p15 and eIFG3 at 1p36. eIFG1 and eIFG3 are proteins of
1600 and 1621 amino acid residues respectively, whereas eIFG2 is 907 amino acids
long. eIFG2 lacks the N-terminal portion, which contains both the PABP and eIF4E-
binding sites and is, therefore, non-functional (Fig.7.1) and likely to be a repressor
of cap-dependent translation (Imataka etal. 1997).
The N-terminal 600-residues contains interaction sites for PABP and eIF4E
(Marintchev and Wagner 2005) (Fig.7.1). The structure of yeast eIF4G has been
solved (Gross etal. 2003) to demonstrate that the middle portion (amino acids 745
1003), which binds eIF4A and contacts mRNA, forms a crescent shape consist-
ing of five antiparallel -helical hairpins (HEAT repeats) forming a helical stack
(Marcotrigiano etal. 2001). The HEAT domain is named for the proteins in which
it was first identifiedhuntingtin, EF3, PP2A and the yeast kinase TOR1. The C-
terminal region (amino acids 12001600) contains binding sites for the second
eIF4A-binding and for MNK1 and MNK2 (Marintchev and Wagner 2005; Prvt
etal. 2003). The crystal structure of the C-terminal region of eIF4G1 has also been
solved and consists of two further HEAT domains.
Sequence comparisons have suggested similarity between the mid-portion of
eIF4G and yeast protein MIF4G domains, and similarly a region of eIF4G distal
to this has sequence similarity to domains within the product of the mouse MA3/
PDCD4 gene (Ponting 2000). Therefore, the C-terminal 1000-residues consists of
three consecutive HEAT domains, the first two of which interact with eIF4A. Com-
bined structural and functional analysis of mammalian eIF4G have demonstrated
a decrease in ribosome binding on mutation of the C-terminal eIF4A-binding site
(Morino etal. 2000). Others, utilizing picornavirus cleavage products of eIF4G
have demonstrated that the eIF4E-binding site is not absolutely required for transla-
tion although protein lacking this site is three- or fourfold less efficient that one that
possesses this site (Ali etal. 2001).
eIF4G1 expression has been investigated in detail with a number of different transla-
tional regulatory mechanisms being identified. eIF4G1 mRNA is produced from three
alternative promoters in HeLa cells and there are two alternative polyadenylation cleav-
age sites leading to 3 UTRs of different lengths (Byrd etal. 2002). There are several
alternatively spliced variants of eIF4G mRNAs in HeLa cells, which may produce a
range of different proteins. Additionally there are a number of AUG sites from which
up to five different eIF4G proteins are translated from full length eIF4G mRNA. Trans-
lation is also modified by the presence of IRES within the 5 UTR (Byrd etal. 2005).
Experiments in which knockdown of eIF4G1 by RNA interference was followed
by expression of either eIF4G1 or eIF4G3 suggest that eIF4G3 is less able to res-
cue translation than eIF4G1 (Ramrez-Valle etal. 2008). These two proteins may,
therefore, not be functionally equivalent (Coldwell etal. 2012). eIF4G1 and eIF4G3
also differ in their sensitivity to HIV protease (Ohlmann etal. 2002). eIF4G1 but not
eIF4G3, is a substrate for HIV protease, and cleavage of eIF4G1 by this protease
impairs cap-dependent and cap-independent translation.
7.3Involvement of eIF4G in Transformation and Cancer
Overexpression of eIF4E promotes malignant transformation alone and in coop-

eration with proto-oncogenes such as c-MYC (Lazaris-Karatzas etal. 1990; Rug-
gero etal. 2004; Wendel etal. 2004). eIF4G also causes transformation of mouse
7eIF4G 167
fibroblast NIH 3T3 cells independent of eIF4E expression (Fukuchi-Shimogori

etal. 1997). Supportive evidence for the role of eIF4G in breast cancer derives from
an immunohistochemistry study showing an association between overexpression of
eIF4G and 4E-BP1 and advanced breast cancer (Braunstein etal. 2007). Coexpres-
sion of phosphorylated 4E-BP1 and eIF4G correlates with high proliferation rates
in breast cancer (Rojo etal. 2007) suggesting that this may be a route to promote
carcinogenesis.
Increased expression of eIF4G selectively increases translation of mRNAs in-
volved in cell survival, growth arrest and DNA damage response (Badura etal.
2012), a pattern of responses that is likely to reflect increased global translation, but
which may play a part in maintaining cancers.
Reprogramming of translation occurs in normal cells to allow responses to the
external environment. For example, translation is reprogrammed following a vari-
ety of cellular insults including irradiation, hypoxia and the induction of apoptosis
(Bushell etal. 2004; Spriggs etal. 2010). Hypoxia within a tumor setting drives
gene expression changes that are associated with enhanced cell survival and resis-
tance to chemotherapy. During the hypoxia that occurs in large advanced breast can-
cers both 4E-BP1 and eIF4G are overexpressed, a combination that in this disease
is associated with a switch from cap-dependent to cap-independent cellular-IRES-
mediated translation (Braunstein etal. 2007). Similarly, in inflammatory breast
cancer, a subtype of a clinically aggressive and highly metastatic disease, eIF4G1
is overexpressed. Increased expression of eIF4G in this context was found to be
associated with enhanced translation of IRES-containing mRNAs which encode
proteins that promotes inflammatory breast cancer cell survival and the formation
of tumor emboli and metastasis (Silvera etal. 2009).
Prominent roles for eIF4G have also been suggested in lung cancer. eIF4G1
protein expression has been determined in 33 cases of squamous cell lung carci-
noma. As compared to normal lung tissue overexpression of eIF4G1 protein was
observed in 61% of the tumors by Western blotting and in 72% of tissue sections
of squamous cell lung carcinoma by IHC (Bauer etal. 2002). Further support for
the importance of eIF4G derives from an analysis of 61 cases of small-cell lung
cancer (SCLC). The proportion of cases demonstrating a 3q27 amplification (the
eIF4G1 locus) associates with more malignant tumors as defined by growth frac-
tion. Noninvasive (T1) and minimally-invasive (T2) tumor stages showed simi-
lar low fractions of amplified and nonamplified tumors, but amplifications of the
locus occurred in an increased proportion of locally-invasive (T3) tumors. Gene
expression profiling was carried out in order to confirm that eIF4G was indeed
overexpressed in cases that contain amplifications of 3q27 (Bauer etal. 2002;
Comtesse etal. 2007).
In a novel approach to determining cancer-relevant gene expression one group
has defined anti-eIF4G antibodies in 33 heterologous sera from lung carcinoma
patients. Antibodies against eIF4G were demonstrated in five serum samples (15%)
but no immune responses to this protein were observed in 17 sera from patients with
squamous cell carcinoma tissues of the head and neck or in 17 samples from normal
subjects. This study again associated tumor tissue, as compared to normal tissue,
with increased expression of eIF4G (Bauer etal. 2002).
7.4eIF4G and eIF4E Interaction as a Therapeutic Target
Conventional chemotherapy for cancers, of course, include agents that cause apop-
tosis and reduced translation to disrupt the cap-binding complex. For example, the
interruption of cell signaling, to reduce cancer cell proliferation, has been shown to
decrease the formation of the eIF4F complex. Trastuzumab, a monoclonal antibody
that interferes with the HER2/neu receptor in breast cancer, cetuximab, an epider-
mal growth factor receptor (EGFR) inhibitor utilised in colon cancer, and erlotinib,
a tyrosine kinase inhibitor (TKI) acting on EGFR, which is employed in head and
neck cancers all inhibit eIF4G binding to eIF4E (Zindy etal. 2011).
Autophagy is a specific response to endoplasmic reticulum stress and the in-
duction of autophagy is considered a route to treatment for some cancers. Protein
synthesis is repressed as a consequence of the induction of autophagy, but eIF4G
enhances resistance to this induction following irradiation-mediated genotoxic
DNA damage (Badura etal. 2012). Increased translation is a feature of cancers
(Bjornsti and Houghton 2004; Bordeleau etal. 2008; Wendel etal. 2007) and
repression of translation is an attractive therapeutic approach (Blagden and Wil-
lis 2011; Hagner etal. 2010). Whereas several natural products have been shown
to inhibit translation (Chen etal. 2011; Lucas etal. 2009) these do not act spe-
cifically. Efforts to produce specific cap-dependent translation inhibitors have
focused on perturbing the interaction between eIF4E and the cap structure (As-
souline etal. 2009; Fischer 2009; Kentsis etal. 2005) and the interaction between
eIF4G and eIF4E.
One approach to the interruption of the eIF4G/eIF4E interaction has been the
production of a peptidomimetic in which an -helix derived from the eIF4E-binding
peptide of eIF4G was stabilized by the inclusion of -helix inducers. A cell-perme-
able version of this peptide, produced by the addition of the TAT peptide, was found
to cause apoptosis (Brown etal. 2011).
However, small molecules are more likely leads for drug discovery, and, accord-
ingly, a high-throughput screening assay for identifying small-molecule inhibitors
of the eIF4E/eIF4G interaction was carried out. The most potent inhibitor, 4EGI-1,
binds eIF4E, disrupting the eIF4E/eIF4G association and therefore inhibiting cap-
dependent translation. As anticipated from its mechanism of action, 4EGI-1 dis-
places eIF4G from eIF4E, and enhances 4E-BP1 association with eIF4E both in
vitro and in vivo. 4EGI-1 showed activity against multiple cancer cell lines (Moerke
etal. 2007) but it is likely to have off-target effects that are independent of inhibi-
tion of cap-dependent protein translation. 4EGI-1 enhanced tumor necrosis factor
(TNF) -related apopotosis-incuding ligand (TRAIL) -induced apoptosis through in-
duction of DR5 and repression of c-FLIP (Fan etal. 2010). 4EGI-1 also has effects
on the BCL-2-family proapoptotic protein NOXA, possibly through induction of
the endoplasmic reticulum stress response (Descamps etal. 2012; Willimott etal.
2013).
7eIF4G 169
eIF4G is a scaffold protein around which mRNA, ribosomal subunits and the cap-
binding complex assemble in order to accomplish translation. There is evidence for
the involvement of eIF4G in maintaining breast and lung cancers. It is overexpressed
in these diseases with the level of expression correlating with clinical aggression.
The cause of overexpression is not always clear but in a subgroup of cases of lung
cancer there is amplification of the eIF4G1 locus. Peptidomimetics and small mol-
ecule inhibitors to interrupt the eIF4E/eIF4G interaction are able to slow prolifera-
tion of cancer cells and suggest that this may be a useful therapeutic route.
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Chapter 8
eIF3
John W. B. Hershey
Contents
8.1Introduction 174
8.2Pathway of Initiation 174
8.3eIF3 Structure 176
8.4eIF3 Subunit Expression and Modification 180
8.5Role of eIF3 in the Initiation Pathway 180
8.6Evidence Linking eIF3 to Cancer 182
8.6.1eIF3a 183
8.6.2eIF3b 184
8.6.3eIF3c 184
8.6.4eIF3e 184
8.6.5eIF3f 185
8.6.6eIF3h 186
8.6.7eIF3i 186
8.6.8eIF3m 187
References 188
Abstract The initiation phase of protein synthesis is promoted by at least a dozen

initiation factors, which play key roles in its regulation. The largest of these, eIF3,
is an 804-kDa protein complex comprising 13 nonidentical subunits. eIF3 interacts
with many other translational components and is involved in most of the steps in the
initiation pathway, suggesting an organizing and regulatory role on the ribosome.
Recent work has led to low-resolution models of its structure, although a clear
mechanism of action for this factor is not yet developed. It is widely reported that a
number of the eIF3 subunits are elevated in tumor cells, suggesting that the factor
may play an important role in tumorigenesis. Furthermore, individual overexpres-
sion of five of the eIF3 subunits results in the malignant transformation of immortal
cells in culture. Knockdown of such subunits reverses the malignant phenotypes,
J.W.B.Hershey()
Department of Biochemistry and Molecular Medicine, School of Medicine,
University of California, Davis, CA, USA
e-mail: jwhershey@ucdavis.edu
174 J. W. B. Hershey
providing additional support for their pro-oncogenic involvement. A number of

other eIF3 subunits exhibit low levels in cancerous cells, implying a possible role
as tumor suppressors. This chapter examines the evidence that implicates eIF3 in
cancer biology and identifies areas where further work is needed.
8.1Introduction
Cancer may result from the step-wise accumulation of mutations in genes that regu-
late cell proliferation and survival, such as growth factors and their receptors, sig-
nal transduction protein kinases, transcription factors etc. Early studies in cancer
biology focused on the transcriptome, which was thought to be the major target of
signal transduction. However, it is currently realized that the regulation of cell pro-
liferation is much more complex and involves the coordination of both transcription
and translation. Enhanced rates of protein synthesis in cancer cells have long been
recognized, but could be a consequence rather than a cause of malignancy. Here, we
examine recent evidence that establishes protein synthesis as a major factor in the
etiology of cancer, with a focus on the role of eIF3 in this process.
Cancer cells are known to display high rates of protein synthesis (Ruggero 2012;
Silvera etal. 2010). That signal transduction pathways target protein synthesis as
well as transcription indicates that changes in translational control also may be im-
portant in establishing cell homeostasis. In addition, the recently developed ribo-
some profiling methodology (Ingolia etal. 2009) has established that translational
control is a major player in the regulation of gene expression in normal cells, com-
parable in importance to transcriptional regulation. The observations that many of
the components of the translation machinery are overexpressed in tumors supports
the notion that translation may be a key factor in the regulation of cell proliferation.
Specifically, as discussed in other chapters of this book, high levels of eIF2, eIF3
subunits, eIF4A, eIF4E, eIF4G or eIF5AII have been seen in various tumors, sug-
gesting that the initiation phase of protein synthesis is particularly relevant from the
standpoint of cancer etiology and pathogenesis.
8.2Pathway of Initiation
The major pathway of initiation of protein synthesis in mammalian cells is the so-
called scanning mechanism (Hinnebusch 2011), although other mechanisms such
as IRES-directed initiation (Jackson 2012) are found as well. Scanning involves
recognition of the m7G cap at the 5-terminus of mRNAs, recruitment to the mRNA
of the 40S ribosomal subunit carrying the initiator Met-tRNAi, subsequent scan-
ning along the mRNA to the initiation codon, and junction with the 60S ribosomal
subunit to form the 80S initiation complex capable of entering the elongation phase
of protein synthesis. This pathway is promoted by about a dozen known initia-
tion factors comprising more than 30 individual proteins (for a detailed review, see
8eIF3 175
Chap.2). Of major importance is the selection of the mRNA to be translated, the

recognition of the initiation codon, which thereby establishes the reading frame, and
the rate by which these reactions proceed.
Briefly, the mRNA is selected for translation by its interaction with the cap-
binding complex, eIF4F, comprising eIF4A, eIF4E and eIF4G, which also binds to
PABP at the tail of the mRNA. It has been proposed that the simultaneous interac-
tion with both the m7G cap and the poly(A) tail assures that the mRNA being select-
ed is full-length (Wells etal. 1996). eIF4F, together with eIF4B and ATP hydrolysis,
removes mRNA secondary structure proximal to the m7G cap, enabling the 40S
PIC to bind there. In a parallel pathway, the 40S ribosomal subunit binds stably to
eIF3, a large multiprotein complex comprising 13 nonidentical subunits, and to the
ternary complex, eIF2/GTP/Met-tRNAi, which carries the initiator tRNA. This PIC
also contains eIF1, eIF1A and eIF5. Recruitment of the PIC to the mRNA occurs
through an interaction between eIF3 bound to the PIC and eIF4G on the mRNA.
Once the PIC is bound near the 5-terminus of the mRNA, it is thought to scan in
a 5 to 3 direction along the mRNA (Hinnebusch and Lorsch 2012). Scanning is
dependent on eIFl that holds the 40S ribosomal subunit in an open conformation
required for movement, and on the melting of downstream secondary structures
in the 5 UTR of the mRNA, promoted by the RNA helicase activity of eIF4A and
possibly other RNA helicases. Scanning ceases when the initiation codon is recog-
nized through its interaction with the anticodon of the Met-tRNAi. Initiation codon
recognition involves the hydrolysis of GTP bound to eIF2, followed by the release
of eIF1 and dissociation of inorganic orthophosphate, resulting in accommodation
of Met-tRNAi in the ribosomal P-site and a closed conformation of the 40S initia-
tion complex. eIF2/GDP dissociates, allowing the 60S ribosomal subunit to bind
in a reaction involving eIF5B/GTP tethered to the 40S-bound eIF1A. GTP is then
hydrolyzed, enabling the Met-tRNAi to enter the peptidyl transferase site in the 60S
ribosomal subunit, and the remaining initiation factors are thought to dissociate.
The 80S initiation complex is then prepared to enter the elongation phase of protein
synthesis.
The generally accepted pathway described here is not yet firmly established in
mechanistic and kinetic detail. Although the structures of the eukaryotic 80S ribo-
some and a number of protein factors have been determined at the atomic level
(Voigts-Hoffmann etal. 2012), we still lack an atomic-resolution structure of com-
plete 40S initiation complexes (but see below). The order of binding of the initia-
tion factors to mRNA and the 40S ribosomal subunit is not firmly established in
the mammalian system, although many sophisticated kinetic studies have been per-
formed with the yeast system (Aitken and Lorsch 2012). Similarly, precisely when
each of the initiation factors dissociates from the initiation complexes is not known
for all proteins. Even how Met-tRNAi is recruited to the ribosome is uncertain, as
it is possible that the Met-tRNAi binds, not as the ternary complex with eIF2-GTP,
but as a multifactor complex containing eIF1, eIF2, eIF3 and eIF5 (Sokabe etal.
2012). A number of slightly different pathways for assembly of the initiation com-
plexes are possible, and a number of these may function in a cell simultaneously,
as has been reported for bacterial initiation (Tsai etal. 2012). To better elucidate
the pathway, rigorous kinetic approaches and single molecule methods are needed.
Such a detailed kinetic understanding of initiation is required to explain the various

mechanisms of translation control (reviewed in Chap.3), some of which may cause
only subtle effects on the process. Such studies also are needed to explain how eIF3
functions during initiation, a topic discussed in greater detail below.
8.3eIF3 Structure
This chapter focuses on the largest and most complex of the initiation factors, eIF3.
Mammalian eIF3 is an 804kDa protein complex comprising 13 nonidentical sub-
units with masses ranging from 166.525.0kDa (Table8.1) (Hinnebusch 2006).
Similar eIF3 subunits are found in other species, e.g. Drosophila melanogaster,
Caenorhabditis elegans and Schizosaccharomyces pombe, whereas in Saccharomy-
ces cerevisiae, only five core submits (3a, 3b, 3c, 3g, 3i) and a sub-stoichiometric
3j are present. Sequence comparisons among different species suggest that eIF3 is
highly conserved. This view is supported by the fact that yeast eIF3 with only its
five core subunits stimulates methionyl-puromycin synthesis in an in vitro initia-
tion assay based on mammalian components (Naranda etal. 1994). Such stimulation
by yeast eIF3 suggests that the same five mammalian eIF3 subunits may provide the
major functions of the factor, with the remaining subunits playing regulatory roles.
Detailed reviews of mammalian and yeast eIF3 are available (Dong and Zhang
2006; Hinnebusch 2006; Valasek 2012).
Of the 13 subunits in human eIF3, six (3a, 3c, 3e, 3k, 3l and 3m) contain a con-
served structural motif called the PCI domain (Pick etal. 2009) that also is shared
with six subunits in the proteasome lid and COP9 (constitutive photomorphogenic
9) signalosome. Two other eIF3 subunits, 3f and 3h, contain the MPN domain also
found in two related proteins present in the proteasome lid and COP9 (Pick etal.
2009). Sharing six PCI and two MPN proteins among the three complexes suggests
that their structures may be similar, and that regulation of protein synthesis, protein
degradation and transcription might be coordinated through their related complex-
es. However, this is yet to be demonstrated.
No atomic resolution structure of the human eIF3 complex has been generated,
although crystal structures have been reported for human eIF3k (Shen etal. 2004a),
the RNA recognition motif of human eIF3g (Couchalova etal. 2010), yeast eIF3i
complexed with a fragment of eIF3b (Herrmannova etal. 2012) and a complex of
fragments of human eIF3j and eIF3b (ElAntak etal. 2010). A 30 resolution model
for human eIF3 was proposed based on cryo-electron microscopy (Siridechadilok
etal. 2005). The cryo-electron microscopy studies also indicate that eIF3 binds
to the solvent side of the 40S ribosomal subunit. The shape of eIF3 in the model
resembles a starfish, with the five arms extending from a central core, sometimes
labeled a head and two right and left arms and legs. Recently, a cryo-electron mi-
croscopy study of the 43S PIC reported a similar shape for rabbit eIF3, in this case
while bound to the 40S PIC (Hashem etal. 2013). This exciting study, at a resolu-
tion of 11.6, provides the best structural information about how eIF3 and some
8eIF3 177
Table 8.1 Characterization of eIF3 subunits and their involvement in cancer.

Sub-unit Mass (kDa) eIF3 binding to other proteins/ Naturally elevated () or
molecules reduced () levels in tumors
and cancer cells
3a 166.5 UPF1 (Isken etal. 2008), cytokeratin Breast (Bachmann etal.
7 (Lin etal. 2001), TrkA (MacDon- 1997), cervix (Dellas etal.
ald etal. 1999), WILI (Unhavai- 1998), esophagus (Chen and
thaya etal. 2009), actin (Pincheira Burger 1999), lung (Pin-
etal. 2001), RNA (Block etal. cheira etal. 2001), stomach
1998) (Chen and Burger 2004)
3b 92.5 Not available Breast (Lin etal. 2001), blad-
der (Wang etal. 2013a)
3c 105.3 eIF1 (Fletcher etal. 1999), eIF4G Meningioma (Scoles etal.
(Villa etal. 2013), eIF5 (Phan etal. 2006), testicular seminoma
1998), schwannomin (Scoles etal. (Rothe etal. 2000)
2006) HSP 90 (Ujino etal. 2012)
3d 64.1 eIF4G (Villa etal. 2013) Not available
3e 52.2 eIF4G (LeFebvre etal. 2006), p56 Breast (Marchetti etal.
(Hui etal. 2003), MIF4GD/SLIP1 2001), lung (Buttitta etal.
(Neusiedler etal. 2012), ATM 2005), prostate (Marchetti
(Morris etal. 2006), etal. 2001)
3f 37.5 TRC8 (Lee etal. 2010), CDK11p46 Breast (Shi etal. 2006),
(Shi etal. 2003), mTOR and S6K1 colon (Shi etal. 2006), small
(Harris etal. 2006; Holz etal. intestine (Shi etal. 2006),
2005), MAFbx (Lagirand-Canta- melanoma (Doldan etal.
loube etal. 2008), hnRNP K (Wen 2008b), ovary (Shi etal.
etal. 2012) 2006), pancreas (Doldan
etal. 2008a), vulva (Shi
etal. 2006)
3g 35.7 PAIP1 (Martineau etal. 2008), eIF4B Not available
(Park etal. 2004; Vornlocher etal.
1999)
3h 40.0 TRC8 (Lee etal. 2010) Breast (Nupponen etal.
1999), colon (Tomlinson
etal. 2008), liver (Okamoto
etal. 2003), prostate (Nup-
ponen etal. 1999; Saramaki
etal. 2001)
3i 36.5 TRIP-1 (Choy and Derynck 1998), Breast (Matsuda etal. 2000),
AKT1 (Wang etal. 2013b) head/neck (Rauch etal.
2004), liver (Gray etal.
2000), melanoma (Bernar-
dini etal. 1997), neuroblas-
toma (Bernardini etal. 1997)
3j 29.0 Not available Not available
3k 25.0 cyclin D3 (Shen etal. 2004b) Not available
3l 66.7 Not available Not available
3m 42.6 MIF (Goh etal. 2011), MT2A (Goh Colon (Goh etal. 2011)
etal. 2011)
Fig. 8.1 eIF3 subunits and

their interaction. A modified
model of human eIF3 subunit
interactions determined by
mass spectrometric analysis
of subcomplexes gener-
ated by solution disruption.
Adapted and modified from
Zhou etal. 2008.
of the other initiation factors bind to the ribosome. However, when this complex is
compared with the one proposed earlier, the orientations of eIF3 on the 40S ribo-
somal surface differ substantially.
Surprisingly, a reconstituted eight-subunit core consisting of subunits 3a*,
3c*, 3e, 3f, 3h, 3k, 3l and 3m (the * designates a truncated protein) generated
from recombinant proteins expressed in E. coli (see below) exhibits a similar
five-arm structure although its mass is only half that of the native complex (Sun
etal. 2011). Furthermore, incorporation of the 3b, 3g and 3i subunits into the
reconstituted 8-mer complex does not greatly alter the cryo-electron microscopy
image. These results indicate that much of the mass of the native eIF3 subunit is
not seen in the cryo-electron microscopy model of native eIF3, and also of 40S-
bound eIF3, likely due to flexibility of much of the mass (Sun etal. 2011). The
findings suggest that eIF3 has flexible extensions capable of wrapping around the
40S subunit and entering the interface surface to interact with other components
of initiation. The higher-resolution 40S PIC model supports this view, as non-
ribosomal density, presumably due to eIF3, is found on the interface side of the
ribosome as well (Hashem etal. 2013).
A model for how the different proteins interact with one another in the eIF3
complex has been proposed based on mass spectrometric analysis of subcom-
plexes generated from intact native eIF3 by solution disruption (Zhou etal. 2008)
(Fig. 8.1). It has been convincingly shown that human eIF3 can contain all 13
subunits in the same complex (Damoc etal. 2007), but it remains possible that
8eIF3 179
complexes also exist in mammalian cells that lack one or more of its subunits,
thereby generating a heterogeneous population of eIF3 complexes. Such hetero-
geneity has been reported for eIF3 in S. pombe, where two distinctly different
complexes are found (Zhou etal. 2005), called eIF3e (lacking 3h and 3m) and
eIF3m (lacking 3d and 3e). Evidence discussed below indicates that the 3a, 3e
and 3f subunits may be sub-stoichiometric in some human cells. It is prudent to
consider the possibility that some of the subunits may exist partly outside the eIF3
complex and function independently as well.
Other attempts to elucidate how the different subunits interact within the eIF3
structure have been made by generating subcomplexes through the synthesis of
recombinant human eIF3 subunits, initially in baculovirus-infected insect cells
(Fraser etal. 2004; Masutani etal. 2007), and more recently in E. coli (Querol-
Audi etal. 2013; Sun etal. 2011) and in mammalian cell lysates (Masutani etal.
2013). Each of these approaches offers advantages and disadvantages: bacterial
expression can generate large amounts of complexes suitable for attempts to crys-
talize them, but relies on significant truncations of the 3a and 3c subunits (called
3a* and 3c*); the mammalian cell lysate system produces limited yields of pro-
teins, but all of the subunits are full-length. All three approaches are capable of
generating a 13-subunit eIF3 complex that is active in in vitro initiation assays.
The insect cell system can generate a stable complex containing only subunits 3a,
3b, 3c, 3e, 3f and 3h, whereas a basic stable core from the bacterial expression
system requires eight subunits: 3a*, 3c*, 3e, 3f, 3h, 3k, 3l and 3m. Of the eight
subunits in this core, six contain the PCI domain and two (3f, 3h) carry the MPN
domain. Interestingly, the cryo-electron microscopic structures of the eIF3 8-mer
and the proteasome lid are very similar in overall shape and homologous subunit
organization (Querol-Audi etal. 2013). Mapping the N-terminal regions of the
subunits to the cryo-electron microscopy structural model also enabled the authors
to identify specific subunits in each of the arms of eIF3. The 8-mer formed with
the bacterial system is active in binding to 40S ribosomal subunits and to eIF1 and
eIF1A, even though the 3a* and 3c* subunits lack regions thought to be important
for such binding (Sun etal. 2011). However, eIF4G does not bind to this 8-mer,
but binds if subunits 3b and 3d are added. The insect cell and mammalian cell ly-
sate expression systems, which employ full-length 3a and 3c subunits, can gener-
ate a stable complex lacking the 3j, 3l and 3m subunits; however, omitting either
the 3e or 3h subunit results in no stable complex. Recently, the spectrin domain
in eIF3a has been shown to be the docking site for the 3b and 3i subunits (Dong
etal. 2013). Although the different systems produce somewhat different results,
it is anticipated that further work will help to elucidate some of the activities of
individual subunits. The reader should keep in mind that current models showing
eIF3 subunit interactions use circles or spheres to depict subunits, although it is
quite possible, if not likely, that many of these proteins are not globular, but rather
have long extensions capable of interacting with many different proteins. Thus, a
high-resolution structure showing precisely how the different subunits interact to
form the 13-subunit complex is still to be achieved.
8.4eIF3 Subunit Expression and Modification
Little is known about how eIF3 subunits are synthesized and assembled into the
complex. Based on the assembly of recombinant subunits expressed in E. coli (Sun
etal. 2011), it appears that a complex of 3a and 3c is critical, to which all other sub-
units can bind. However, the order of assembly of eIF3 subunits in cells is yet to be
studied. It is noteworthy that when a subunit is overexpressed in transfected cells,
the protein only very modestly increases in concentration (Zhang etal. 2007), sug-
gesting that subunits not incorporated into the eIF3 complex are rapidly degraded.
It has been reported that eIF3a is synthesized primarily during the S phase of the
cell cycle (Dong etal. 2004). Curiously, eIF3e, eIF3f and eIF3h are synthesized from
5 TOP mRNAs (Iadevaia etal. 2008), which typically encode ribosomal proteins and
elongation factors. Therefore these three eIF3 subunits appear to be unique among
the initiation factors with respect to their 5 TOP mRNAs. However, whether or how
expression of individual eIF3 subunits is coordinated remains to be elucidated.
When individual eIF3 subunits are overexpressed in stably transfected mammalian
cells, only modest (less than 50%), if any, effects are seen on the rate of global protein
synthesis (Zhang etal. 2007). In the case of subunits 3a, 3b or 3c, the level of the
whole eIF3 complex is enhanced, perhaps causing the modest stimulation of activity.
Finding an elevated eIF3 when only one subunit is overexpressed suggests that the
overexpressed subunit enables a more efficient assembly of the whole eIF3 complex,
although this has never been studied directly. Also pertinent are the findings from
lowering individual eIF3 subunits through the action of siRNAs, as described in the
sections for individual subunits below. However, a comprehensive study of the effects
of such lowering on other eIF3 subunits has not yet been performed.
eIF3 is posttranslationally modified by phosphorylation of Ser and Thr residues
and by N-terminal acetylation; still other modifications are possible. Mass spectro-
metric analysis identified 29 sites of phosphorylation affecting subunits 3a (4 sites),
3b (7 sites), 3c (10 sites), 3f (1 site), 3g (2 sites), 3h (1 site) and 3j (4 sites) (Damoc
etal. 2007); additional sites not detected in this study may be phosphorylated under
different physiological conditions (e.g., see eIF3f below). The numerous sites found
in 3a, 3b and 3c suggest likely regulation, especially in the case of 3c where many
of the sites are found in its N-terminal region that binds eIF1 and eIF5; however,
regulation by phosphorylation at any of these sites has yet to be demonstrated. In
contrast, phosphorylation of Ser183 in eIF3h is required for the malignant trans-
formation activity of this protein (see below). Phosphorylation during apoptosis of
eIF3f at Ser46 and Thr119 by CDK11p46 (a modified form of caspase 3) enhances
the proteins ability to inhibit protein synthesis (Shi etal. 2009).
8.5Role of eIF3 in the Initiation Pathway
Although this chapter focuses on human eIF3 and its role in cancer, it is useful to
consider insights into its function derived from studies of yeast eIF3. The func-
tion of yeast eIF3 has been reviewed recently (Chiu etal. 2010; Hinnebusch 2011;
8eIF3 181
Valasek 2012), so major concepts are discussed here only briefly. eIF3 stimulates
mRNA binding to 43S ribosomes, in part through interactions of the N- and C-
terminal domains of eIF3a with the mRNA exit and entry channels, respectively.
A module consisting of the C-terminal domain of eIF3a, the N-terminal domain of
eIF3b and eIF3j is thought to reside near the mRNA entry channel in the 40S ribo-
some. The C-terminal region of eIF3b binds to eIF3g and eIF3i and this module
may bind just above the 3abj module near the entry channel. However, yeast eIF3g
and eIF3i are not essential in vitro for mRNA binding to 40S ribosomes. Finally, the
N-terminal domain of eIF3c binds eIF1 and eIF5. Through such interactions, eIF3
affects mRNA and Met-tRNAi binding, initiation codon recognition and the process
of reinitiation after termination at uORFs.
Human eIF3 alone binds to 40S ribosomal subunits and is stabilized there by
the presence of 3j (Fraser etal. 2004). eIF3-40S complexes lacking Met-tRNAi and
mRNA are found in mammalian cell lysates (Smith and Henshaw 1975), indicating
that eIF3 binding may be an early event in the initiation pathway. Models for the
eIF3-40S complex, based on cryo-electron microscopy, place the initiation factor
on the solvent side of the 40S subunit with the five arms embracing the ribosome
(Hashem etal. 2013; Siridechadilok etal. 2005). Indeed, eIF3j bound to the N-
terminal region of eIF3b can enter the mRNA-binding channel which is located
on the interface side of the 40S subunit (Fraser etal. 2007). eIF3j competes with
mRNA for binding in the decoding channel; such competition is relieved when the
ternary complex of eIF2/GTP/Met-tRNAi is present on the 40S subunit (Fraser
etal. 2007). A number of other initiation factors interact with eIF3. Importantly,
eIF3 binds eIF4G (Korneeva etal. 2000), which plays a crucial role in recruiting the
40S subunit to the mRNA. The region of eIF4G that binds eIF3 is located between
amino acid residues 10151118 (Korneeva etal. 2001); the eIF3 subunits involved
are eIF3e, eIF3c and eIF3d (Villa etal. 2013). eIF1 (Fletcher etal. 1999) and eIF5
(Valasek etal. 2004) bind to eIF3c; eIF4B binds to eIF3g (Park etal. 2004; Vornlo-
cher etal. 1999); and eIF5 and eIF2 bind to the eIF3 complex (Sokabe etal. 2012).
It therefore appears that eIF3 helps to organize various components of the transla-
tional apparatus on or near the interface surface of the 40S subunit. Other than in the
case of eIF4G, it is not clear how the binding of eIF3 to the other initiation factors
contributes to the overall process. In addition to its interactions with other initiation
factors, eIF3 (specifically, its 3a and 3d subunits) binds to the region of the mRNA
near the ribosomes mRNA exit site (Pisarev etal. 2008), perhaps preventing or
slowing mRNA dissociation either prematurely or following 80S initiation complex
formation. However, a clear and complete understanding of just how eIF3 promotes
the various steps in the initiation pathway is not yet available.
Besides interacting with other initiation factors, mRNA and the ribosome, eIF3
has been shown to bind to other proteins that affect protein synthesis (Table8.1).
The eIF3e subunit binds to p56, a protein induced by IFN that inhibits translation
(Hui etal. 2003). eIF3g interacts with the PABP-interacting protein PAIP1 to stimu-
late initiation (Martineau etal. 2008). eIF3k interacts with cyclin D3 (Shen etal.
2004b). The 3f and 3h subunits interact with the E3 ligase, TRC8, possibly leading
to proteasome degradation of the factor (Lee etal. 2010). eIF3a binds to UPF1 in
the exon junction complex, affecting the nonsense-mediated mRNA degradation
pathway (Isken etal. 2008). In contrast, the MLN51 (metastatic lymph node 51)
protein, also in the exon junction complex, binds to eIF3a and eIF3d to stimulate
translation (Chazal etal. 2013).
It is also possible that different eIF3 subunits function outside of the eIF3 com-
plex, affecting other metabolic pathways. Small amounts of the 3a, 3e, 3f and 3k
subunits are located in the nucleus (Severin etal. 1997). eIF3i was first identified
as a TGF--receptor-type-II-interacting protein (TRIP-1), implying a role in signal
transduction (Chen etal. 1995). The sharing of PCI and MPN structural domains
between subunits of eIF3 and those of the proteasome lid complex and the tran-
scription factor complex (COP9 signalosome) may indicate coordinated regulation
of protein synthesis, degradation and transcription (Kim etal. 2001). Indeed, it is
claimed that human eIF3e can associate with all three complexes (Hoareau Alves
etal. 2002). Such ideas, while provocative, require further investigation in order to
establish their validity.
8.6Evidence Linking eIF3 to Cancer
As this book amply demonstrates, regulation of protein synthesis plays a critical

role in the etiology and pathogenesis of malignancies. It has been known for over
20 years that high levels of a number of initiation factors are present in specific can-
cers. In particular, a high abundance of eIF4A, eIF4E, eIF4G or eIF5AII has been
reported, as well as decreased levels of the 4E-BPs or extents of eIF2 phosphoryla-
tion (see Chaps.4, 5, 9 and 10). Best studied is eIF4E, where overexpression stimu-
lates the translation of mRNAs possessing highly structured 5 UTRs (Koromilas
etal. 1992; Lazaris-Karatzas etal. 1990) (see also Chaps.3 and 4). Such weak
mRNAs encode oncogenic proteins such as cyclin D1, c-MYC, growth factors and
ODC that presumably are the ultimate cause of the malignant transformation.
Numerous subunits of eIF3 also have altered levels in cancerous cells. The level
of eIF3a, eIF3b, eIF3c, eIF3h, eIF3i or eIF3m is naturally elevated in a number of
specific cancer types, whereas a decreased level of eIF3e or eIF3f is seen in other
cases (Table8.1). The strong correlation of altered levels of specific eIF3 subunits
and cancer suggests that these proteins too play a causative role in malignant trans-
formation.
Individual ectopic overexpression of 12 eIF3 subunits (all except eIF3m were
tested) in stably transfected NIH 3T3 immortal fibroblasts results in malignant
transformation when subunit 3a, 3b, 3c, 3h or 3i is elevated, but not when any
of the other subunits is overexpressed (Zhang etal. 2007). The five subunits are
among those identified as naturally overexpressed in various tumor cells (listed in
Table8.1). This correlation supports the view that each of the five eIF3 subunits is
directly responsible for the malignant phenotype. Their overexpression results in
only a mild stimulation of protein synthesis, but a stronger stimulation of the trans-
lation of oncogenic mRNAs occurs, as seen with eIF4E. In contrast, overexpression
8eIF3 183
of the 3e or 3f subunit in NIH 3T3 cells inhibits protein synthesis and slightly en-
hances apoptosis (Zhang etal. 2007). This finding correlates with the observation
that decreased levels of 3e and 3f are found in cancer cells (Table8.1), and suggests
that these eIF3 subunits might behave like tumor suppressors. It is therefore useful
to review the findings for each of the subunits implicated in cancer, with the aim to
better understand how each may function in regulating cell proliferation.
8.6.1eIF3a
eIF3a levels are elevated in many cancers (Table8.1), and its overexpression in-
duces malignant transformation of immortal cells (Zhang etal. 2007). In contrast,
depletion of eIF3a reduces the malignant characteristics of cancer cells (Dong etal.
2004). Other studies suggest that high eIF3a is often observed during early stages of
cancer development compared to later stage high-grade malignancies (Bachmann
etal. 1997). How different levels of eIF3a affect cell metabolism is not entirely
clear. In the case of ectopic overexpression in NIH 3T3 cells, the modest increase
in eIF3a level is accompanied by a similar increase in the levels of all of the other
eIF3 subunits (Zhang etal. 2007), suggesting that the tumorigenesis may be caused
by the increased abundance of the whole eIF3 complex, not by the action of eIF3a
alone. Unfortunately, most studies involving altered levels of eIF3a have not as-
sessed the abundance of the other eIF3 subunits, or of other initiation factors.
How might eIF3a promote cell malignancy? It is generally assumed that eIF3a
functions as a part of the eIF3 complex, which promotes initiation of protein syn-
thesis. However, siRNA knockdown of eIF3a has only a modest effect on the rate
of translation in vivo (Dong and Zhang 2003; Dong etal. 2009). Furthermore, puri-
fied eIF3 lacking the 3a subunit retains its activity in promoting the formation of
40S and 80S initiation complexes in vitro (Chaudhuri etal. 1997). In contrast, yeast
eIF3a is essential for cell viability and protein synthesis (Vornlocher etal. 1999).
Thus the actual function of eIF3a in human cell protein synthesis remains unclear.
Besides its function in protein synthesis, the 3a subunit has been found to bind to
RNA (Block etal. 1998), cytokeratin 7 (Lin etal. 2001), the -tubulin TrkA (Mac-
Donald etal. 1999) and WILI (Unhavaithaya etal. 2009) (Table8.1). It also is found
attached to membranes through its interaction with actin (Pincheira etal. 2001);
curiously, the membrane-bound form appears not to be phosphorylated, whereas
the soluble form is.
Unexpectedly, the level of eIF3a can affect mRNA-specific translation, as high
levels enhance the synthesis of RRM2 (ribonucleotide reductase M2 subunit) and
inhibit p27kip1 and NDRG1 synthesis, whereas low levels have the opposite effect
(Dong and Zhang 2003; Dong etal. 2004). Such changes are observed even though
global rates of protein synthesis are hardly affected. A detailed molecular mecha-
nism for how eIF3a or its absence affects specific mRNA translation has not yet
been defined.
8.6.2eIF3b
Involvement of eIF3b in cancer is based on its high levels in a few number of can-
cers (Table8.1) and on the effects of its overexpression in immortal NIH 3T3 cells
(Zhang etal. 2007). Furthermore, siRNA knockdown of eIF3b in glioblastoma and
colon cancer cells reduces the rate of proliferation and induces apoptosis (Liang
etal. 2012). The subunit forms an active subcomplex with 3a and 3c in yeast, and
human eIF3b also binds to 3g and 3i at its C-terminal region and to 3j at its N-ter-
minal region. However, how elevated levels alter the regulation of cell proliferation
is not known.
8.6.3eIF3c
The level of eIF3c is found high in only a very few tumors (Table8.1). Ectopic
overexpression of eIF3c in NIH 3T3 cells causes their malignant transformation
(Zhang etal. 2007), supporting the view that eIF3c possesses pro-oncogenic prop-
erties. Cells transformed by eIF3c translate oncogenic mRNAs (e.g., cyclin D1,
c-MYC, ODC and fibroblast growth factor) much more efficiently (Zhang etal.
2007), which may be the mechanism whereby eIF3c and other eIF3 subunits pro-
mote malignancy. Decreasing eIF3c levels by siRNA treatment of a number of dif-
ferent cancer cell lines results in an inhibition of protein synthesis and cell prolifera-
tion (Emmanuel etal. 2013), as expected. The N-terminal region of eIF3c binds to
eIF1 (Fletcher etal. 1999) (and in yeast, to eIF5 (Phan etal. 1998)). This region of
eIF3c is rich in sites of phosphorylation (Damoc etal. 2007), suggesting that such
interactions may be regulated by protein kinases. eIF3c also has been shown to bind
to the anticancer drug schwannomin (Scoles etal. 2006) and to interact with the
heat shock protein 90 (HSP90) (Ujino etal. 2012) (Table8.1). Curiously, mutations
in the eIF3c gene cause the extra-toes spotting phenotype in mice, perhaps through
disruption of hedgehog signaling (Gildea etal. 2011). How these observations may
relate to the role of eIF3c in cancer is not obvious.
8.6.4eIF3e
The involvement of eIF3e in cancer was first proposed when it was discovered that
its gene (then called INT6) is a frequent site of integration by the mouse mammary
tumor virus (MMTV) genome, resulting in a truncated version of eIF3e (Marchetti
etal. 1995). Loss of heterozygosity or reduced levels of eIF3e have been found
in a number of tumors (Table8.1), suggesting that eIF3e may function as a tumor
suppressor. Ectopic expression of truncated eIF3e (Mayeur and Hershey 2002; Ras-
mussen etal. 2001) or knockdown of the protein by siRNA (Gillis and Lewis 2012)
results in malignant transformation. However, high levels of eIF3e are found in
8eIF3 185
some lung tumors (Buttitta etal. 2005) and are found in high-grade human breast
tumors (Grzmil etal. 2010). In the latter study, siRNA knockdown of eIF3e does
not reduce global protein synthesis, but affects the translation of specific mRNAs
either positively or negatively. Clearly, the role of eIF3e in carcinogenesis is more
complicated than first appreciated.
One important role for eIF3e in translation is that it participates in the crucial
step of recruiting the mRNA to the 40S PIC through the binding of eIF4G to eIF3;
the 3e subunit was found to be involved in this interaction (LeFebvre etal. 2006),
although it is now known that subunits 3c and 3d also contribute to the binding
(Villa etal. 2013). Consistent with its involvement in the eIF4G/eIF3 interaction,
overexpression of truncated eIF3e in NIH 3T3 cells results in a reduction in eIF3
binding to eIF4G and an inhibition of global protein synthesis (Chiluiza etal. 2011);
the authors also report that truncated eIF3e actually stimulates the translation of a
subset of mRNAs thought to be driven by the IRES mechanism. eIF3e binds to the
translational inhibitor p56, a stress and interferon-induced protein (Hui etal. 2003).
The subunit also binds to MIF4GD (MIF4G domain containing)/SLIP1 (SLBP-in-
teracting protein 1), proteins involved in the translation of histone mRNAs that lack
a poly(A) tail, and thereby stimulates histone synthesis (Neusiedler etal. 2012).
eIF3e may play a role in nonsense mediated decay (Morris etal. 2007) and in a
cells response to DNA damage through its interaction with ATM (Morris etal.
2006). Nevertheless, a clear understanding of its role in carcinogenesis is yet to be
developed.
8.6.5eIF3f
Numerous cancer cells exhibit reduced levels of eIF3f (Table8.1). Stable knock-
down of eIF3f in normal human pancreatic ductal epithelial cells with siRNA re-
sults in a stimulation of protein synthesis and causes increased cell proliferation
and other characteristics of malignant cells (Shi etal. 2006; Wen etal. 2012). Fur-
thermore, overexpression in cancer cells inhibits translation and cell growth and
induces apoptosis (Shi etal. 2006). eIF3f contains the MPN domain which ap-
pears to be important in establishing the structural core of the eIF3 complex, in part
through its interaction with the other MPN domain subunit, eIF3h (Querol-Audi
etal. 2013; Sun etal. 2011). Therefore the finding that low levels of eIF3f stimu-
late protein synthesis is counterintuitive. A mechanistic explanation for how eIF3f
inhibits translation has not yet been developed. eIF3f is phosphorylated at two sites
by CDK11p46 during apoptosis, and such phosphorylation enhances the factors
inhibition of protein synthesis (Shi etal. 2003, 2009). eIF3f also is known to interact
with a number of other proteins through its Mov34 domain. It interacts with mTOR
and S6K1 (Harris etal. 2006; Holz etal. 2005), key regulators of protein synthesis.
It may contribute to the degradation of 60S subunit rRNA, perhaps through its bind-
ing to hnRNP K (Wen etal. 2012). eIF3f binds to MSS4, a stress-stimulated protein
that reduces the translation inhibitory action of eIF3f and therefore may modulate
its functions (Walter etal. 2012). Curiously, eIF3f appears to affect protein synthe-
sis completely differently in muscle cells; low levels correlate with reduced transla-
tion rates and atrophy whereas high levels or overexpression cause stimulation of
protein synthesis and hypertrophy (reviewed in Marchione etal. 2013; Sanchez
etal. 2013). eIF3f in myotubes interacts with MAFbx (Lagirand-Cantaloube etal.
2008), a muscle-specific ubiquitin ligase, leading to its degradation. Finally, eIF3f
is partly localized in the nucleus, where it joins an eIF3 complex that lacks the 3a
subunit (Shi etal. 2009). Whether or not eIF3 or its 3f subunit has a functional role
in the nucleus has not been determined.
8.6.6eIF3h
A number of tumors contain high levels of eIF3h (Table8.1), due in part to chromo-
some amplifications in the 8q23 region of the chromosome that carries the eIF3h
gene (and also the nearby c-MYC gene) (Pittman etal. 2010; Savinainen etal. 2004).
Ectopic overexpression in NIH 3T3 cells results in their malignant transformation
(Zhang etal. 2007), an outcome dependent on its phosphorylation at Ser183 (Zhang
etal. 2008). Further strengthening its role in tumorigenesis, knockdown of eIF3h
by siRNA reverses the malignant phenotypes of a number of cell types (Savinainen
etal. 2006). Plant eIF3h promotes reinitiation following translation of short uO-
RFs (Roy etal. 2010), a function also dependent on its phosphorylation by TOR
(Schepetilnikov etal. 2013). The subunit binds to the matrix protein of rabies virus,
perhaps enabling matrix protein inhibition of host cell protein synthesis by being
tethered to the ribosome through eIF3h (Komarova etal. 2007). eIF3h also binds
to the E3 ligase, TRC8 (Lee etal. 2010). However, the function of eIF3h in protein
synthesis is not defined.
8.6.7 eIF3i
The subunit is overexpressed in a number of different tumor types (Table8.1), and

ectopic overexpression in NIH 3T3 cells induces a malignant phenotype (Ahlemann
etal. 2006; Zhang etal. 2007). Furthermore, siRNA treatment of transformed HeLa
or PCI-1 cells causes an opposite effect, reducing proliferation rates, cell size and
anchorage-independent growth (Ahlemann etal. 2006). These authors also found
that inhibition of mTOR mitigates the transformation activity of overexpressed
eIF3i, suggesting that eIF3i phosphorylation might be important. Although no
phosphorylation of eIF3i was identified by mass spectrometric analysis of purified
eIF3 (Damoc etal. 2007), yeast eIF3i is known to be phosphorylated (Chen etal.
1995). In addition, deletion of 6 amino acid residues that contain a putative mTOR
phosphorylation site at the C-terminus of human eIF3i results in loss of transforma-
tion activity (Ahlemann etal. 2006). Unfortunately, specific functions for eIF3i in
protein synthesis have not been identified, although it is phosphorylated when it
8eIF3 187
interacts with TRIP-1 (Choy and Derynck 1998). Interestingly, a very recent report
(Wang etal. 2013b) provides evidence that eIF3i interacts with the signaling kinase,
AKT1, thereby preventing AKT1 dephosphorylation by PP2A ; they hypothesize
that the resulting highly active AKT1 drives tumorigenesis.
8.6.8eIF3m
This subunit, the last to be identified in eIF3, has not yet been studied extensively
and generally has not been identified as having altered expression in cancer cells.
However, a recent report (Goh etal. 2011) claims a small increase in eIF3m protein
in a variety of tumors, and provides evidence that high levels of eIF3m promote cell
proliferation, and that low levels induced by siRNA treatment of colon cancer cells
slows cell growth and enhances apoptosis. mRNAs found to coimmunoprecipitate
with tagged eIF3m mostly encode ribosomal proteins and translation factors, but
also two proteins (macrophage migration inhibitory factor (MIF) and metallothio-
nein 2A (MT2A)) implicated in tumorigenesis. It appears that eIF3m may be onco-
genic, but its mode of action remains unknown.
As described above for individual subunits of eIF3, a tumorigenic role for these
subunits is based in part on the findings that overexpression of an eIF3 subunit can
cause malignant transformation and knockdown of the same subunit results in a
loss of the malignant phenotypes. However, there are problems with many of the
experimental findings described here. The enhanced levels may cause other trans-
lational components to increase, making eIF3 only one of many players, yet these
other factor levels frequently are not measured. The knockdown experiments in
cells overexpressing the subunit should reduce the subunit only to its normal levels,
and not more, if the effects of overexpression are being addressed. Yet most often
the level is reduced much more, such that reversal of malignant phenotypes may
be due to a strong inhibition of global protein synthesis resulting in a reduction of
growth rate and of other phenotypes. An appropriate control would be to reduce the
elevated level to that found in normal (nonmalignant) cells. The reader is therefore
cautioned to carefully evaluate the experimental findings and identify those that are
not yet rigorously established.
The relationship of eIF3 to cancer is exceedingly complex. The major ideas devel-
oped in the field are that enhanced levels of five or six of the eIF3 subunits can pro-
mote malignancy, whereas reduced levels of two others are pro-oncogenic. In many
cases, an oncogenic or tumor suppressor role for the eIF3 subunit is confirmed by
knockdown or overexpression of the subunit in cancer cell lines (but consider the
argument in the paragraph above). The numerous examples of changes in a spe-
cific proteins level leading to or reducing malignancy supports the view that these
proteins are direct causes of the transformation. How elevated levels of certain eIF3
subunits enhance translation and promote tumorigenesis is not obvious. Possible
explanations are that they may increase the level of the whole eIF3 complex, may
complete eIF3 complexes deficient in the subunit (see below), or act outside the
eIF3 complex. Clearly, much remains to be learned about how eIF3 and its subunits
function in cells.
eIF3 plays important roles in many of the steps in the initiation pathway, promot-
ing both Met-tRNAi and mRNA binding to the 40S ribosomal subunit. It interacts
with many other initiation factors and regulatory elements, but the lack of an ad-
equate atomic-level structure of the whole eIF3 complex limits our understanding
of its contributions to initiation and translational control. Little is known about how
eIF3 subunits are themselves expressed or assembled into the complex, although
heterozygosity appears responsible for some of the aberrations in abundance. A fur-
ther complication is the possibility that different eIF3 complexes may exist in cells,
some lacking one or more specific subunits, and that such complexes may affect
the translation of specific mRNAs, as seen with eIF3a. It is also apparent that eIF3
interacts with other molecules not normally associated with protein synthesis, sug-
gesting that it or one of its subunits may function outside of translation, perhaps
helping to coordinate the process of protein synthesis with other cellular metabolic
pathways.
To better understand eIF3s contributions to tumorigenesis, we need atomic reso-
lution structures of eIF3 and its initiation complexes. We also need a quantitative
kinetic description of the entire initiation pathway, what limits the translation rate
of specific mRNAs, and how the steps are altered by protein modifications and
trans-acting RNAs or proteins. It is anticipated that such information will enable re-
searchers to better understand the role of eIF3 in cancer and to develop therapeutic
strategies that target this fascinating initiation factor.
Acknowledgments I thank Nancy Villa (UC Davis) for help in preparing Fig.8.1. I also thank
Chris Fraser (UC Davis) for numerous helpful discussions.
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Chapter 9
The eIF2 Complex and eIF2
Bertal H. Aktas and Ting Chen
Contents
9.1Introduction 196
9.2eIF2 197
9.3eIF2 197
9.4eIF2 197
9.5eIF2B 198
9.6Signaling Pathways and Regulation of eIF2B and eIF2 198
9.7The eIF2 Regulation 199
9.7.1PKR 199
9.7.2PERK 200
9.7.3GCN2 200
9.7.4HRI 201
9.7.5Nitric Oxide Signaling 201
9.7.6Dephosphorylation of eIF2 202
9.8eIF2 Regulation of mRNA Translation via Upstream ORFs 203
9.9Role of eIF2 and Its Regulation in Cancer 204
9.9.1Overexpression of eIF2 205
9.9.2Phosphorylation of eIF2 206
9.10eIF2 Kinases in Cancer 207
9.10.1PKR 207
9.10.2PERK 208
9.10.3Other eIF2 Kinases 208
9.11eIF2 and Anticancer Therapy 209
9.12eIF2-Based Therapeutic Approaches 210
References 213
Abstract eIF2 is a three-subunit complex that forms the core of the eIF/GTP/Met-
tRNAi ternary complex, which is essential for translation initiation. eIF2 cycles
between the GTP-bound active and GDP-bound inactive forms. This GTP/GDP
B.H.Aktas() T.Chen
Division of Hematology, Department of Medicine, Brigham and Womens Hospital and Harvard
Medical School, Harvard University, Boston, MA, USA
e-mail: huseyin_aktas@hms.harvard.edu
196 B. H. Aktas and T. Chen
cycle is regulated primarily through reversible phosphorylation of the subunit of

eIF2 (eIF2) by a set of kinases that specifically respond to various stress stimuli.
Phosphorylation of eIF2 locks eIF2 in GDP-bound inactive form, thereby reducing
protein synthesis and favoring metabolic dormancy required for cell survival under
stress. Inversely, nonphosphorylated eIF2 increases protein synthesis and favors
cell proliferation. Unsurprisingly, the eIF2 complex, more specifically eIF2,
appears to be an important hub of translation that participates in cell proliferation
and survival, malignant transformation, tumor initiation, progression, and metas-
tasis. In this chapter, we will review the molecular and chemical biology of eIF2
with particular emphasis on eIF2 and its regulation by phosphorylation. We will
attempt to reconcile seemingly controversial observations related to the involve-
ment of eIF2 and its regulatory kinases in cancer and discuss if and how eIF2 can
be targeted for cancer therapy.
9.1Introduction
Translation is the third step in the central dogma of biology; the synthesis of pro-
teins directed by an mRNA template (Coldwell etal. 2010; Cooper 1981; Crick
1970). Protein translation proceeds in four phases: initiation, elongation, termina-
tion and ribosome recycling (see Chap.2) (Wiessbach and Brot 1974). During eu-
karyotic translation initiation, the mRNA is recruited to the 43S PIC, containing the
small 40S ribosomal subunit and various translation initiation factors. Generally,
this recruitment takes place via interaction of the trimeric eIF4F complex attached
to the cap structure of the mRNA and the eIF3 large multiprotein complex bound
to the 40S subunit. Once the mRNA is recruited, the 43S PIC scans along the 5
UTR to reach the initiation codon (see Chap.2). In a simplified model, once the
43S PIC reaches the initiation codon, the 60S large ribosomal subunit is recruited
to form the 80S ribosomal complex, the translation initiation factors dissociate and
the elongation step commences. Important for this discussion are the late events
of translation initiation where the role of the eIF2 translation initiation complex
comes into play.
As discussed in Chap.2, eIF2 forms a ternary complex with initiator methio-
nine tRNA, Met-tRNAi, and GTP. The start codon recognition is coupled to GTP
hydrolysis (Nanda etal. 2013). The GDP of the eIF2-GDP binary complex, re-
leased from the PIC upon subunit joining, must be exchanged for GTP to enable
a new round of translation. This GDP to GTP exchange reaction is catalyzed by
eIF2B, the eIF2-specific GEF (Kubarenko etal. 2005). Phosphorylation of the
subunit of eIF2 (eIF2) on serine 51 (Ser51 or S51) dramatically increases affinity
of eIF2 for eIF2B while also inhibiting its GTP-GDP exchange activity (Kimball
etal. 1998a). Coupled with a low eIF2B/eIF2 ratio, this phosphorylation signifi-
cantly reduces abundance of the ternary complex. The eIF2 is a heterotrimer of
three subunits, , , and (Kimball 1999; Kimball etal. 1998b; Nika etal. 2001;
Suragani etal. 2006).
9 The eIF2 Complex and eIF2 197
9.2 eIF2
The subunit is at the core of the eIF2 complex, interacting with both and sub-
units. Based on its sequence homology with the structurally characterized prokary-
otic translation elongation factor, EF-Tu, and its crystal structure, eIF2 is thought
to contain three domains and is distinguished by presence of a zinc-binding hinge
(Roll-Mecak etal. 2004). This subunit is essential for GTP binding (Naranda etal.
1995). The structural studies of eIF2 have not provided a full explanation for the
GTP dependence of Met-tRNAi biding to eIF2. However, biochemical studies, mu-
tational analysis, as well as the most recent studies with hybrid subunits indicate
that eIF2 binding to the eIF2 subunit is essential for GTP binding (Naveau etal.
2013; Roll-Mecak etal. 2004).
9.3 eIF2
The C-terminal domain of eIF2 with a C2C2 zinc-binding motif is conserved in

the Archaea and eukaryotes. However, the archaeal eIF2 lacks an N-terminal inter-
action domain found in eukaryotic eIF2 (Thompson etal. 2000). The N-terminal
region of eIF2 is the binding site for the C-terminal domains of eIF5 that catalyzes
GTP hydrolysis. The N-terminal region of eIF2 is also a binding site for eIF2B that
catalyzes GDP-GTP exchange on the eIF2-GDP binary complex (Cho and Hoffman
2002).
9.4 eIF2
Structural studies of eIF2 revealed two domains: an N-terminal oligonucleotide/

oligosaccharide binding fold (OB-fold) domain and a compact -helix domain (Ito
etal. 2004). The latter contains serine 51 (Ser51) that is adjacent to a 310 helix of an
extended loop connecting two strands of the OB-fold domain. This subunit also
contains a S1 motif domain, which is a potential RNA-binding site. Similarly to
its eIF2 counterpart, archaeal IF2, interacts specifically with Met-tRNAi and con-
tributes to its loading to the ribosomal P-site (Dhaliwal and Hoffman 2003). Ser51
phosphorylation by eIF2 kinases appears to be the main mechanism by which
activity of eIF2 is regulated. However, it is not clear if the archaeal IF2-subunit is
posttranslationally modified (Stolboushkina etal. 2008). Perhaps the most important
difference between archaeal and eukaryotic IF2 is that in the Archaea the contribu-
tion of the subunit to GTP binding is dramatically more important than contribu-
tion of the subunit. In contrast, in eukaryotes the contribution of the subunit is
significantly more important in that regard (Naveau etal. 2013). In eukaryotes from
yeast to mammals, the translational control exerted by the phosphorylation of eIF2

is required for proper development and adaptation to cellular stresses encountered
during the life cycle (Joyce etal. 2013; Naveau etal. 2013).
9.5eIF2B
The eIF2 GEF, eIF2B, is a complex of five subunits designated from to , from
the smallest to the largest subunit. eIF2B is the catalytic core of this complex. Its
activity is significantly enhanced by binding to the subunit. The eIF2B assembly
is called the catalytic subcomplex, whose GEF activity is similar to that of the full
five-subunit eIF2B complex (Yang and Hinnebusch 1996). The remaining , and
subunits form the regulatory subcomplex and play a critical role in high-affinity
binding to phosphorylated eIF2 (Pavitt etal. 1998; Reid etal. 2012; Yang and Hin-
nebusch 1996). These high affinity interactions underlie the regulation of protein
synthesis by eIF2 phosphorylation.
9.6Signaling Pathways and Regulation

of eIF2B and eIF2
Control of translation initiation by reversible phosphorylation of the eukaryotic

translation initiation factors eIF2 and eIF2B (Yoshizawa etal. 1997) has been ex-
tensively studied. Regulation of eIF2B activity occurs via eIF2B. The latter is
phosphorylated by GSK3 resulting in inhibition of guanine nucleotide exchange
activity (Welsh etal. 1998). Insulin inhibits GSK3 activity, resulting in the dephos-
phorylation of eIF2B and restoration of the guanine nucleotide exchange (Proud
etal. 2001; Wang etal. 2001b; Welsh etal. 1998).
Phosphorylation of its and subunits plays an important role in the regulation
of the eIF2 activity. eIF2 is phosphorylated by cAMP-dependent protein kinase/
protein kinase A (PKA) that increases the guanine-nucleotide exchange (Welsh
etal. 1994). eIF2 and eIF2 are also phosphorylated by the DNA-dependent pro-
tein kinase, as well as by PKC and casein kinase 2, but the significance of these
modifications is not well understood (Andaya etal. 2011; Llorens etal. 2005).
The subunit of eIF2 is a target of four serine/threonine kinases that phosphory-
late this protein on Ser51. These are protein kinase R (PKR, EIF2AK2), PKR-like
endoplasmic reticulum kinase (PERK, EIF2AK3), general control non-derepress-
ible 2 (GCN2, EIF2AK4), and heme-regulated inhibitor (HRI, EIF2AK1). Each
kinase primarily responds to a distinct type of stimuli or cellular stress: GCN2 re-
sponds to nutritional stress, particularly depletion of branched chain amino acids;
PERK is sensitive to perturbations in the endoplasmic reticulum; HRI responds to
heme deprivation in erythroid cells and cytoplasmic stress in others; and PKR is
activated by double-stranded RNA as part of antiviral defense. Phosphorylation of

eIF2 by these kinases inhibits the guanine nucleotide exchange activity of eIF2B,
thus reducing the abundance of the eIF2/GTP/Met-tRNAi translation initiation
complex. In general, eIF2 phosphorylation represents a response aimed at secur-
ing cell survival in the face of stress or assault. Once the causative stress stimulus is
remedied, the eIF2 phosphorylation is reversed, since sustained eIF2 phosphory-
lation usually leads to cell death. Although PKR, PERK, GCN2, and HRI are best
known for phosphorylating eIF2, additional substrates of these kinases have now
been identified, which broadens the scope of their activity and likely contributes to
the selectivity of their signaling downstream (Baird and Wek 2012).
9.7 The eIF2 Regulation
As mentioned above, four kinases regulate eIF2 phosphorylation and hence trans-
lation initiation. Each of these kinases is responsive to a unique stress stimulus or
a set of stimuli.
9.7.1 PKR
PKR was initially characterized as a kinase that phosphorylates eIF2 in response to

viral infection to block translation of viral mRNAs. Not surprisingly, some viruses
have developed strategies to cope with the effects of PKR. For example, HCV,
which is a major risk factor for HCC, expresses gene products, such as nonstruc-
tural protein NS5A, that interfere with the activation of PKR (Gale etal. 1998).
Reovirus infection of nontransformed cells results in the activation of PKR, and the
resultant phosphorylation of its substrate, eIF2, with subsequent block to transla-
tion (Gale etal. 1998). In some strains, PKR deficiency renders mice highly sensi-
tive to VSV infection (Stojdl etal. 2000). PKR-null, as opposed to PKR-wild-type,
mouse embryonic fibroblasts (MEFs) are highly sensitive to oncolytic viral infec-
tion, for example by VSV (Balachandran and Barber 2007; Baltzis etal. 2004).
Moreover, transformation of mouse and human fibroblasts, either spontaneously or
through forced expression of oncogenes, renders them sensitive to VSV oncolysis
(Balachandran and Barber 2004). Consistently, tumor cells with p53 mutations or
activating RAS and MYC mutations are highly susceptible to lytic viral infection
(Balachandran etal. 2001; Farassati etal. 2001). Earlier studies attributed sensi-
tivity of transformed cells to oncolytic viruses to the MEK/ERK-dependent inac-
tivation of PKR and implicitly, to eIF2 phosphorylation (Farassati et al. 2001).
However, in an elegant series of experiments Balachandran etal. demonstrated that
sensitivity of transformed cells to oncolysis is caused not by defective PKR activa-
tion or eIF2 phosphorylation, but by the high activity of eIF2B that could efficient-
ly catalyze guanine nucleotide exchange even in the presence of phosphorylated
eIF2 (Balachandran etal. 2001). These data demonstrate that defective regulation
of translation initiation downstream of eIF2 accounts for the sensitivity of the trans-
formed cells to oncolysis (Balachandran etal. 2001; Stojdl etal. 2000).
PKR also plays a role in regulating autophagy that involves its direct interactions
with signal transducer and activator of transcription 3 (STAT3). Specifically, the
SRC homology 2 (SH2) domain of STAT3 interacts with the catalytic domain of
PKR. Consistently, PKR depletion inhibits autophagy initiated by chemical STAT3
inhibitors or free fatty acids such as palmitate (Shen etal. 2012).
9.7.2 PERK
PERK is primarily activated by the accumulation of misfolded proteins in the endo-

plasmic reticulum (Harding etal. 2000a, b). Through its phosphorylation of eIF2,
PERK blocks the synthesis of new proteins. In the absence of PERK activity, un-
folded proteins accumulate and clutter the folding and secretion machinery. Patients
with inactivating mutations of this gene develop Wolcott-Rallison syndrome (Julier
and Nicolino 2010). These patients and PERK-deficient mice develop permanent
neonatal diabetes (Cavener etal. 2010). PERK regulates proinsulin trafficking and
quality control in the secretory pathway (Gupta etal. 2010). There is also a crosstalk
between the unfolded protein response and the autophagy pathway, which contrib-
utes to the handling of cellular stress in neurodegenerative diseases where PERK
plays an important role (Avivar-Valderas etal. 2011).
In addition to unfolded proteins, PERK may also be activated by changes in the
endoplasmic reticulum Ca2+ levels or by the oxidative stress independent of protein
misfolding (Wang etal. 2013a). Finally, PERK appears to be directly phosphory-
lated and inhibited by AKT1/2 (Mounir etal. 2011). Given the role of AKT in cell
survival, PERK phosphorylation by AKT, via eIF2, may protect cells from activat-
ing transcription factor 4 (ATF4) and/or C/EBP homolog protein (CHOP)-induced
cell death (Hu etal. 2004; Lin etal. 2009).
9.7.3 GCN2
GCN2 functions as a sensor of amino acid availability by directly detecting the pres-
ence of deacylated or uncharged tRNAs, which accumulate during amino acid de-
pletion (Wek etal. 1995). It is also activated by glucose deprivation in both yeast and
mammalian cells although the exact mechanism remains to be defined (Yang etal.
2000). GCN2 coordinates with the mTOR pathway; these two evolutionarily con-
served and nutrient-sensing signaling pathways promote stress adaptation following
starvation (Chotechuang etal. 2009; Gallinetti etal. 2013; Rodland etal. 2013). The
mTOR signaling is depressed in GCN2-null mice fed a diet devoid of leucine. Leu-
cine deprivation also decreased phosphorylation of the negative regulator of transla-
tion initiation factor eIF4E, 4E-BP1, on Thr37 and Thr46, and positive regulator of
translation S6K1 on Thr389 in GCN2-wild-type but not GCN2-null cells (Anthony

etal. 2004). However, some studies suggest that mTOR activity may not be reduced
in the livers of leucine deprived GCN2-null mice, suggesting that GCN2 may be
only one of the mTOR regulators (Xiao etal. 2011). GCN2 and mTOR cooperatively
regulate lifespan and aging in a nematode Caenorhabditis elegans through PHA-4,
a forkhead transcription factor (Rousakis etal. 2013). This pathway appears to play
a role in early development, survival of first L1 stage larvae under starvation, and
dietary restriction-mediated longevity of adults (Rousakis etal. 2013). Thus, GCN2,
via phosphorylation of eIF2 and likely in concert with the mTOR/4E-BPs/eIF4F
axis, regulates translation initiation in response to amino acid deprivation.
9.7.4 HRI
HRI couples hemoglobin synthesis to heme availability and affects the severity of
hemolytic anemias such as -thalassemia by regulating the abundance of the eIF2/
GTP/Met-tRNAi ternary complex (Bank 2005; Han etal. 2005). Hemoglobin syn-
thesis in the absence of heme or in the presence of improperly folded globin re-
sults in the generation of red blood cells with impaired oxygen carrying capacity
(Chen 2007; Chung etal. 2012; Suragani etal. 2012). Consistently, HRI appears to
be a modifier of hemoglobin disorders (Han etal. 2005).
9.7.5 Nitric Oxide Signaling
While all four eIF2 kinases evolved separately and seem to respond to different
cellular and environmental cues, nitric oxide (NO) signaling appear to be capable
of activating all four kinases albeit through distinct mechanisms (Tong etal. 2011).
For example, by converting arginine to NO, the NO synthases can cause arginine
deficiency that result in activation of GCN2 (Lee etal. 2003). This GCN2 activation
couples stress conditions that increase demand for NO synthesis to cellular physiol-
ogy, for example by reducing protein synthesis in stressed cells. NO activates PERK
indirectly by inducing endoplasmic reticulum stress, likely by partially depleting
endoplasmic reticulum Ca2+ stores or by protein nitrosylation (Uehara etal. 2006;
Yan etal. 2006). Activation of HRI by NO is caused by NO-mediated reduction of
the HRI bound heme-Fe(III) to NO-bound heme-Fe(II), which converts catalyti-
cally inactive HRI to an active enzyme (Igarashi etal. 2004). The mechanistic basis
of PKR activation by NO is poorly understood; it is likely that S-nitrosylation-
mediated PKR dimerization accounts for activation of this kinase by NO (Du etal.
1998). The presence of an auto-regulatory loop involving PKR, NF-B and NO has
also been proposed (Uetani etal. 2000).
Fig. 9.1 Schematic representation of eIF2 regulation including eIF2 phosphorylation and dephos-
phorylation. Various stimuli such as endoplasmic reticulum (ER) stress, amino acid or heme depri-
vation and dsRNA specifically activate eIF2 kinases, which include PKR, PERK, GCN2 or HRI,
leading to phosphorylation of eIF2 and decreased translation. On contrary, dephosphorylation
of eIF2 by phosphatases, such as PP1C (protein phosphatase 1, catalytic subunit), leads to the
activation of the protein synthesis and translation. See text for more details.
9.7.6 Dephosphorylation of eIF2
Reversion of eIF2 phosphorylation is essential for translational recovery and

cell survival once the primary stressor is dealt with. Several regulatory subunits
that target phosphorylated eIF2 to serine/threonine protein phosphatase 1 (PP1)
have been identified (Kloft etal. 2012). Growth arrest and DNA damage-inducible
protein (GADD34), also known as PP1 regulatory subunit 15A (PPP1r15A) or a
PP1C interacting protein, is induced by endoplasmic reticulum stress and plays
an important role in catalyzing eIF2 dephosphorylation, an essential step in re-
covery of protein synthesis once the endoplasmic reticulum stress is ameliorated
(Novoa etal. 2001). Another regulatory subunit, the constitutive reverser of eIF2
phosphorylation (CReP)/PPP1r15B, targets the catalytic subunit of PP1C to phos-
phorylated eIF2 to promote its dephosphorylation and thereby restore translation
initiation (Latreille and Larose 2006). A schematic representation of eIF2 regulation
including eIF2 phosphorylation and dephosphorylation is depicted in Fig.9.1.
9.8 e IF2 Regulation of mRNA Translation

via Upstream ORFs
Phosphorylation of eIF2 leads to a global reduction in protein synthesis, with dif-

ferent functional consequences for various mRNAs. Specifically, expression of
proteins coded for by inefficiently translated mRNAs is generally reduced more
drastically than expression of many housekeeping proteins (Aktas etal. 2011; Aktas
etal. 1998; Aktas and Halperin 2004). Furthermore, as will be apparent from the
discussion below, translation of a small subset of mRNAs may be selectively en-
hanced by eIF2 phosphorylation.
One such example is represented by ATF4, a basic leucine zipper transcrip-
tion factor that is preferentially translated in response to eIF2 phosphorylation
(Harding et al. 2000a). The 5 UTR of ATF4 mRNA contains three uORFs in hu-
mans and two in mice (also see Chap.3). These uORFs orchestrate a mechanism
named leaky scanning (Fig.9.2), by which ATF4 expression is paradoxically en-
hanced during eIF2 phosphorylation (Lee etal. 2009). This results in transcription-
al activation of ATF4 target genes usually involved in the endoplasmic reticulum
stress response, including the C/EBP isoforms, FOS, JUN, NRF2 and CHOP (Baird
and Wek 2012).
CHOP is the most important effector downstream of ATF4 for triggering apopto-
sis during massive or chronic stress. ATF4 induces expression of CHOP by binding
the amino acid response element (AARE) sequence in the CHOP promoter (Aver-
ous etal. 2004). CHOP mRNA is poorly translated under basal conditions as a result
of a single uORF (Palam etal. 2011). Translation of this uORF under non-stressed
conditions serves as a barrier to translation of a bona fide CHOP ORF. However,
upon stress, phosphorylated eIF2 facilitates bypass by the 43S PIC of the uORF
thereby favoring translation of a bona fide CHOP ORF (Palam etal. 2011).
A more intricate example of gene specific regulation by eIF2 phosphorylation
is selection of a start codon in C/EBP/ mRNA (Calkhoven etal. 2000). These
mRNAs contain one uORF and three in-frame start codons that can each initiate
translation of the same ORF. The uORFs translation plays a significant role in the
selection of the translation start site in the bona fide ORF, suggesting that C/EBP
translation is regulated by reinitiation rather than leaky scanning. Specifically,
translation of the uORF coupled with the activity of translation initiation factors
such as eIF2 determines whether the first or the second versus the third translation
start site is utilized. In differentiating cells, C/EBP is translated from the first two
start sites giving rise to an active transcription factor. In proliferating cells, transla-
tion starts at the third start site that produces a protein with the same DNA bind-
ing properties but without a transactivation domain. This truncated protein acts as
dominant-negative over the longer isoforms and promotes cell proliferation rather
than differentiation (Calkhoven etal. 2000; Wethmar etal. 2010). Phosphoryla-
tion of eIF2 favors expression of long isoforms in a uORF-dependent manner
(Calkhoven etal. 2000).
Fig. 9.2 Leaky scanning mechanism of paradoxically elevated ATF4 translation. a In the delayed
translation initiation model, the uORFs (yellow boxes) in the 5 UTR of the ATF4 mRNA direct
preferential translation of uORFs at the expense of bona fide ORF. b The bypass model of trans-
lational control. Stress-induced phosphorylated eIF2 facilitates leaky ribosome scanning through
the inhibitory uORFs and leads to increased translation of bona fide ORF of ATF4 mRNA, which
is suggested to result from the poor Kozak context of the start codons in the uORF.
9.9 Role of eIF2 and Its Regulation in Cancer
Dysregulation of translation is becoming increasingly recognized as a founda-

tion of the pathobiology of cancer (Li etal. 2002). Within the intricate process
of protein biosynthesis, translation initiation is one of the most complex steps
involving numerous translation initiation factors and various mRNA control ele-
ments. It is the rate-limiting step of protein synthesis and tightly regulated by
fundamental signaling cascades. Hence, it is not surprising that dysregulation of
translation initiation could lead to disease in general and to cancer in particular. As
discussed in this book, translation initiation can be dysregulated in various ways:
one of the most studied and notable is the activation of translation via oncogenic
signaling. For example the MAPK and PI3K/AKT/mTOR pathways directly cause
translational dysregulation and participate in cancer development and progres-
sion (Chapuis etal. 2010; Schneider etal. 2008). One of the major mechanism by
which these pathways participate in tumorigenesis involves regulation of proto-
oncogenic eIF4E protein, the mRNA cap-binding component of the eIF4F com-
plex (see Chaps.4, 15 and 17). However, oncogenic signaling from these path-
ways also reduces phosphorylation of eIF2, increasing formation of the ternary
complex and thus promoting translation (Mounir etal. 2011). Importantly, and in
general contrast to other components of the eIF2 complex, it is due to its ability to
be regulated by phosphorylation that eIF2 has garnered attention as an etiologic
and pathogenic factor in cancer. However, as follows from our discussion below,
the role of eIF2 in cancer biology is not well understood and is under intense
study by several groups.
9.9.1 Overexpression of eIF2
The role of eIF2 regulation in cancer is signified by the fact that forced expres-
sion of a nonphosphorylatable eIF2 mutant, eIF2S51A, increases the amount of the
ternary complex, renders the translation initiation unrestricted, and causes transfor-
mation of normal cells (Donze etal. 1995). Consistently, overexpression of initia-
tor Met-tRNA also transforms immortalized cells, as does the overexpression of
dominant-negative PKR (Donze etal. 1995; Marshall etal. 2008). Other studies in-
dicate that even in cell lines where defective eIF2 phosphorylation is not sufficient
to cause malignant transformation by itself, it can cooperate with other oncogenic
stimuli to induce cellular transformation (Perkins and Barber 2004).
In cancer patients, eIF2 expression is upregulated in various tumors, such as
malignant brain tumors (Tejada etal. 2009), sarcomas (Perez-Mancera etal. 2008),
various lymphoma subtypes (Lam etal. 2004), non-Hodgkins lymphoma (Rosen-
wald etal. 2008; Wang etal. 1999), melanomas (Rosenwald etal. 2003), gastro-
intestinal tumors (Lobo etal. 2000), thyroid carcinomas (Wang etal. 2001a), and
bronchioloalveolar carcinomas (Rosenwald etal. 2001). In NSCLC, eIF2 expres-
sion is elevated (He etal. 2011). In some of these malignancies, increased expres-
sion of eIF2 also positively correlates with tumor aggressiveness.
Coordinated overexpression of eIF2 and oncogenic short isoforms of C/EBP
have been reported in human infiltrating breast ductal adenocarcinomas and corre-
lates with progression of mammary epithelial carcinomas in all genetic and induced
models of rodent breast cancers tested (Raught etal. 1996). It is presumed that
inadequate adjustment of C/EBP isoform ratios contributes to neoplastic conver-
sion in tissues that express C/EBP and/or C/EBP. As we have discussed earlier,
reduced phosphorylation of eIF2 favors expression of short isoforms of C/EBP,
which may account for the tight correlation between enhanced eF2 expression
and increased expression of truncated C/EBP in mammary epithelial cancer cells
(Raught etal. 1996). Oncogenic isoform of C/EBP is also highly expressed in nu-
cleoplasmin 1 haploinsufficiency-induced AML, however the role of eIF2 in this

instance is unclear (Khanna-Gupta etal. 2012).
Thus, overall evidence suggests that eIF2 is overexpressed in various cancers,
agreeing with its role in promoting translation with prosurvival and proproliferative
effects.
9.9.2 Phosphorylation of eIF2
As discussed above overexpression of eIF2, observed in many tumor types, is ex-

pected to cause a protumorigenic state by increasing protein synthesis and changing
patterns of mRNA translation. Similarly, the activated state of eIF2 would be an
expected finding in cancer cells and, hence, signaling pathways that would decrease
phosphorylation of eIF2 could also be expected to be activated. However, as evi-
denced from the presentation below, the issue may be much more complex.
As discussed earlier, eIF2 is subject to control by phosphorylation, therefore,
its role in human cancers is likely to be not only limited to the expression levels but
also characterized by inactivating phosphorylation at Ser51. The number of stud-
ies that specifically investigated the phosphorylation of eIF2 in human cancers is
rather limited. In some cancer models phosphorylation of eIF2 leads to a proapop-
totic and tumor suppression phenotype (Tuval-Kochen etal. 2013). He and cowork-
ers observed higher levels of eIF2 phosphorylation in NSCLC (He etal. 2011).
In these patients, levels of eIF2 phosphorylation correlated positively with longer
survival (He etal. 2011). This suggests that eIF2 phosphorylation may be activated
as a reaction to oncogenic transformation and may restrict cancer progression. This
is consistent with in vitro studies demonstrating that transforming mutants of RAS
induce eIF2 phosphorylation (Mounir etal. 2009; Mundschau and Faller 1992).
Similarly, ectopic expression of BRAF with Val to Glu mutation at the position 600
(BRAFV600E) in melanocytes increases levels of phospho-eIF2 (Croft etal. 2013).
The increase in phospho-eIF2 correlates with elevated levels of PKR and PKR
autophosphorylation activity (Kim etal. 2002). Inactivation of PTEN, often found
in various cancers including melanoma (Bello etal. 2013), results in reduced eIF2
phosphorylation (Mounir etal. 2009). On a mechanistic level, phosphorylation of
eIF2 appears to be an essential mediator of tumor suppression by PTEN (Ko-
romilas and Mounir 2013; Mounir etal. 2009). Consistently, antiproliferative and
proapoptotic effects of PTEN are compromised in MEFs lacking PKR or expressing
a phosphorylation-defective eIF2 mutant (Koromilas and Mounir 2013; Mounir
etal. 2009). Based on these findings, we hypothesize that eIF2 phosphorylation
in a milieu of oncogenic programs that do not directly involve eIF2 is a reactive
defense mechanism to oppose activated tumorigenic signaling and repress transla-
tion. On the other hand, when eIF2 lies within an oncogenic program, as in the
case of PTEN mutations described above, eIF2 would be expected to be activated
to provide upregulation of translation. However, evidence towards this hypothesis
needs experimental corroboration.
There are also reports of high levels of eIF2 phosphorylation in human tu-
mors (Lobo etal. 2000) and tumor cell lines including breast cancer cell lines
(Kim etal. 2000). At least in the case of some breast carcinoma cell lines, increased
phosphorylation of eIF2 cosegregates with overexpression of eIF2B which neu-
tralizes the effects of eIF2 phosphorylation on translation initiation (Kim et al.
2000). Consistently, in MCF-7 breast cancer and transformed MEFs, inhibition
of the catalytic subunit of eIF2B expression suppresses transformed phenotypes
(Gallagher etal. 2008).
As outlined above, most studies suggest that eIF2 activity may contribute to
cancer initiation and or progression. In contrast, at least in some experimental
models, reduction of eIF2 activity through eIF2 phosphorylation appears to fa-
vor establishment and/or progression of tumors. Transformed MEFs derived from
eIF2S51A/S51A knock-in mice (where eIF2 cannot be phosphorylated on Ser51) fail
to form robust tumors in nude mice (Blais etal. 2006; Fels and Koumenis 2006).
This has been attributed to inability of eIF2S51A/S51A MEFs to establish tumors un-
der the hypoxic tumor environment (Blais etal. 2006). Consistently, transformed
PERK/ MEFs also fail to form robust tumors compared to their similarly trans-
formed wild-type counterparts (Blais etal. 2006). A recent report suggests that
phosphorylatable eIF2 is not essential for growth of established tumors but plays
a role in survival of treatment-resistant populations (Rouschop etal. 2013). As we
will elaborate below, additional clues on the role of eIF2 phosphorylation in can-
cer can be gleaned from the studies on the role of eIF2 kinases or other genes that
impinge on the activity of eIF2 in cancer.
9.10 eIF2 Kinases in Cancer
9.10.1 PKR
Expression of a dominant-negative PKR mutant causes tumorigenic transformation

of murine fibroblasts (Barber etal. 1995). This observation led to the proposal that
PKR is the product of a tumor suppressor gene, providing a potential explanation
for the antitumor activity of IFN, since PKR is an IFN-inducible gene (Koromilas
etal. 1992). Further proof of the concept of the tumor suppressor role of PKR came
from genetically engineered tumor cells that express double-stranded RNA, which
activates endogenous PKR. These genetically engineered cells fail to proliferate.
Tumors formed by such cells also fail to grow once expression of double-stranded
RNA was turned on (Shir and Levitzki 2002).
In a murine lymphocytic leukemia model, PKR gene is rearranged (Abraham
etal. 1998). Deletions of 2p2122 at the chromosomal location of the PKR gene are
frequently observed in AML (Hanash etal. 1993; Haus 2000). Chronic lymphocytic
leukemia patients express a soluble inhibitor of PKR (Hii etal. 2004). High PKR
levels correlate with a low level of recurrent or residual disease in head and neck
cancer patients (Haines etal. 1993a; Haines etal. 1993b). PKR expression also cor-
relates negatively with the size of NSCLCs and rectal tumors and positively with
relapse free survival (He etal. 2011; Kwon etal. 2005). In colon cancer, expression
of PKR correlates with the degree of cellular differentiation (Singh etal. 1995).
Beneficial effects of PKR expression on chemosensitivity have also been reported

(Bennett etal. 2012; Garcia etal. 2011). At the mechanistic level, increased PKR
activity appears to mediate the antitumor effects of the members of the TNF family.
Preponderance of evidence suggests that PKRs interaction with NF-B mediates
inhibition of cell proliferation and induction of apoptosis by TNF (Zamanian-Dary-
oush etal. 2000). For example, TNF-induced activation of caspase 8 and the down-
stream apoptotic program are abrogated in PKR-null cells (Zamanian-Daryoush
etal. 2000).
Other studies challenge the role of PKR as a tumor suppressor. There is no report
of PKR/ mice being particularly prone to tumor development (Stojdl etal. 2000).
Furthermore, levels and/or activity of PKR are elevated in some breast cancers, mel-
anomas and colon tumors, and cancer cell lines (Kim etal. 2002; Pataer etal. 2009).
The pathophysiological reasons for increased PKR activity or its altered expression
in cancers is unclear. In at least one of the studies, while overexpressed in breast
cancer cells, PKR was inactive due to the presence of a soluble trans-dominant
inhibitor (Kim etal. 2000). This finding indicates that not just the expression level
of PKR but also its activity must be considered. In the final analysis, what matters
is the amount of eIF2/GTP/Met-tRNAi complex; we are however, not aware of any
report comparing the amount eIF2/GTP/Met-tRNAi in cancer versus normal tissue.
9.10.2 PERK
There are small clusters of population with inactivating PERK mutations (Julier and
Nicolino 2010). However, there are no reports of altered cancer incidence in these
populations. As we discussed earlier, deletion of PERK may interfere with tumor
establishment (Blais etal. 2006) while its sustained activation is also antitumori-
genic in xenograft models (Ranganathan etal. 2008). PERK is usually activated as
part of the integrated endoplasmic reticulum stress response by several anticancer
agents (see Vandewynckel etal. 2013 for a recent review). Much of existing lit-
erature indicates that PERK activation is essential for chemosensitivity of tumor
cells (Lan etal. 2013; Niknejad etal. 2007; Zhang etal. 2013). Others however,
suggested that PERK activity is responsible for development of chemoresistance or
survival of chemotherapy or radiotherapy resistant cancer cell populations (Rous-
chop etal. 2013). It is likely that the nature of driver mutations in the tumors as
well as the mechanism of action of antitumor agents will influence whether PERK
contributes to the sensitivity or resistance to antitumor therapy.
9.10.3 Other eIF2 Kinases
The evidence for the involvement of HRI or GCN2 in cancer is rather scant. There is
one report of HRI downregulation in human ovarian cancer cells (Hwang etal. 2000).
HRI may also mediate cytostatic effects of NO on human breast cancer cells (Pervin
etal. 2008). There have been no reports of GCN2 perturbations in human cancers
per se but GCN2 was implicated in amino acid deficiency-triggered tumor angio-
genesis (Wang etal. 2013b).
9.11 eIF2 and Anticancer Therapy
The role of eIF2 phosphorylation in the chemosensitivity and radiosensitivity of

tumors has been extensively studied. Inhibitors of the ubiquitin/proteasome path-
way, used for treatment of multiple myeloma, induce eIF2 phosphorylation. Mul-
tiple myeloma patients treated with ubiquitin/proteasome inhibitors invariably
relapse (Jiang and Wek 2005). In vitro treatment of multiple myeloma cells with
proteasome inhibitors, such as bortezomib, potently induces cell death but a frac-
tion of cells become quiescent and survive. The chemoresistance to bortezomib
appears to be associated with sustained expression of endoplasmic reticulum chap-
erone BiP and repression of eIF2 phosphorylation leading to a reduced expression
of its downstream effector, CHOP (Schewe and Aguirre-Ghiso 2009). Consistently,
inhibition of eIF2 dephosphorylation with eIF2-specific phosphatase inhibitor
salubrinol or expression of phosphomimetic eIF2 mutant eIF2S51D resensitizes
multiple myeloma cells to bortezomib treatment (Schewe and Aguirre-Ghiso 2009).
Phosphorylation of eIF2 may have different impacts on the sensitivity of tumors
to other treatment modalities. For example, phosphorylation of eIF2 is associated
with development of TKI imatinib resistance in chronic myeloid leukemia (CML)
(Kusio-Kobialka etal. 2012). In this instance, eIF2 phosphorylation appears to
mediate the prosurvival activity of BCR-ABL signaling. Consistently, inhibition of
eIF2 phosphorylation resulted in resensitization of imatinib resistant CML cells to
this agent (Kusio-Kobialka etal. 2012). Likewise, phosphorylated eIF2 renders
cancer cells resistant to IFN type 1 treatment. At the mechanistic levels, phosphory-
lated eIF2 accelerates degradation of the IFN-/ receptor chain through p38
kinase (Bhattacharya etal. 2013). Consequently, activation of eIF2 kinases results
in development of resistance to IFN type 1 therapy (Bhattacharya etal. 2013). As
these examples demonstrate, the consequences of eIF2 phosphorylation for che-
motherapy appear to be dependent on the chemotherapeutic agent and the genetics
of the specific cancers.
Radiation therapy causes extensive DNA damage. Cells respond to radiation-in-
duced DNA damage by activating DNA repair machinery or by activating apoptotic
pathways, depending on the extent of the damage (see Selzer and Hebar 2012 for
review). Phosphorylation of eIF2 appears to be part of the early response to radi-
ation-induced DNA damage and at least initially favors cell survival (Deng etal.
2002; Rouschop etal. 2013; Wu etal. 2002). It is also implicated in inducing cancer
radiation resistance (von Holzen etal. 2007) or sensitivity (Koizumi etal. 2012;
Oommen and Prise 2013). For example, Rouschop etal suggest that eIF2 phos-
phorylation contributes to the development of radiotherapy resistance by protecting
cancer cells from radiotherapy-induced reactive oxygen species and by increasing
cysteine uptake and glutathione synthesis (Rouschop etal. 2013). In contrast, some
in vitro studies suggest that induction of eIF2 phosphorylation is associated with

increased radiotherapy sensitivity (Kim etal. 2010; Koizumi etal. 2012). It is likely
that the driver mutations of tumors as well as the degree of oxygenation will have
a profound influence on whether eIF2 phosphorylation will increase or reduce
radiotherapy sensitivity of tumors.
9.12 eIF2-Based Therapeutic Approaches
The first attempts at targeting eIF2 phosphorylation for cancer therapy were con-
ducted with several small molecules that partially deplete endoplasmic reticulum
Ca2+ stores. These include clotrimazole, n-3 polyunsaturated fatty acid eicosapen-
taenoic acid, and certain thiozolidoneindenones, such as troglitazone. These agents
inhibit growth of both normal and cancer cells in vitro by inducing phosphoryla-
tion of eIF2, and sustained inhibition of translation initiation (Aktas etal. 1998;
Palakurthi etal. 2001; Palakurthi etal. 2000). They were later shown to induce
eIF2 phosphorylation in xenograft tumors and in the case of troglitazone, in can-
cer patients (Aktas etal. 2013). These initial observations prompted synthesis and
evaluation of clotrimazole and troglitazone analogs (Chen etal. 2004; Fan etal.
2004; Natarajan etal. 2004). Among these, clotrimazole analog diaryl-oxindoles
such as compound #1181 were shown to possess antitumor activity in xenograft
models (Chen etal. 2012).
Several agents that activate eIF2 kinase PERK by disturbing the endoplasmic
reticulum homeostasis have also been reported. Examples of these are: 2-methoxy-
5-amino-N-hyroxynezamide which prevented inflammatory bowel disease-related
CRC (Stolfi etal. 2010); TZD18, a dual peroxisome proliferator activated / li-
gand that displayed antiproliferative activity against breast cancer cells (Zang etal.
2009); and OSU-03012, which sensitizes breast cancer cells to lapatinib (West etal.
2013). The common property of the above agents is that they are not direct modi-
fiers of eIF2 phosphorylation. These agents likely cause eIF2 phosphorylation in-
directly by modifying yet unknown targets. For example troglitazone, a peroxisome
proliferator-activated receptor (PPAR) ligand, causes eIF2 phosphorylation and
inhibition of cell proliferation to a similar extent in PPAR-null and PPAR-wild-
type cells (Palakurthi etal. 2001). Lack of knowledge of their direct molecular
target responsible for inducing eIF2 phosphorylation renders the task of improving
the potency of the above-mentioned agents by structure-based drug design or even
classical structure activity relationship studies very difficult.
The first crop of what appeared to be direct chemical modifiers of eIF2 phos-
phorylation came from unbiased cell-based screens. Salubrinal was discovered in
a screen for agents that protect PC-12 neuronal cells from the endoplasmic reticu-
lum stress-induced death (Boyce etal. 2005). Studies of the effect of this com-
pound on the endoplasmic reticulum stress revealed that salubrinal is an inhibitor
of eIF2 dephosphorylation (Boyce etal. 2005). It causes accumulation of phos-
phorylated eIF2 by preventing the GADD34/PP1C complex from catalyzing eIF2
dephosphorylation (Boyce etal. 2005). Salubrinal sensitizes SW1353 chondrosar-

coma cells to radiation; inhibits HER2/neu-induced dysregulation of mammary aci-
nar morphogenesis (Sequeira etal. 2009), inhibits leukemia cell proliferation syn-
ergistically with proteosome inhibitors (Drexler 2009), and renders a subpopulation
of bortezomib-resistant multiple myeloma cells sensitive to bortezomib (Drexler
2009).
Other cell-based screens for modifiers of eIF2 phosphorylation identi-
fied N, N-diarylurea analogs as activators of HRI (Chen etal. 2011) and
3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHB-
DC) as a specific dual activator of PKR and PERK (Bai etal. 2013). Both classes of
agents inhibit expression of oncogenic proteins and proliferation of various cancer
cell lines. In a xenograft model, an N, N-diarylurea compound, BTdCPU, induced
eIF2 phosphorylation and inhibited growth of mammary tumors without apparent
toxicity (Chen etal. 2011). Evaluation of a substituted urea library and subsequent
structure activity relationship studies identified N-aryl-N-cyclohexyl urea analogs
as potent and specific activators of HRI that inhibit proliferation of melanoma can-
cer cell lines with BRAF mutation (Chen etal. 2013). The in vivo activity of these
later compounds remains to be determined. Finally a cell based screening for the in-
hibitors of G1/S cell cycle transition identified CCT020312 (Stockwell etal. 2012),
which inhibits cell proliferation by specifically activating PERK.
The appreciation for the role of PERK/eIF2 axis in protecting tumors from hy-
poxia prompted search for the agents that inhibit eIF2 kinases. These studies led to
the identification of one or more inhibitors for each of the four eIF2 kinases with
some level of specificity (see Joshi etal (Joshi etal. 2013) for a recent review). As
best we can ascertain, only one of these compounds, PERK inhibitor GSK2606414,
was tested in xenograft models of cancer and shown to cause a modest (59%) inhi-
bition of xenografted pancreatic tumor growth with no apparent toxic side effects
(Axten etal. 2012).
Extensive molecular and chemical genetic studies unequivocally demonstrate that

sustained phosphorylation of eIF2 at levels sufficient to inhibit translation initiation
abrogates cell proliferation in vitro and xenograft tumor growth in vivo (Axten etal.
2012; Chen etal. 2011). The only exception to this rule appears to be tumors with
compensatory mutations that relieve inhibition of translation initiation by eIF2
phosphorylation. This is highly consistent with the molecular genetic studies dem-
onstrating that deficiency of eIF2 phosphorylation leads to malignant transforma-
tion (Donze etal. 1995; Koromilas etal. 1992). Furthermore, in most cases, sus-
tained induction of eIF2 phosphorylation renders cancer cells or xenograft tumors
more sensitive to radiation- and/or chemotherapy. It is important to emphasize that
the eIF2 phosphorylation inhibits proliferation of cancer cells in vitro and tumor
growth in vivo only if it is a sustained rather than transient phosphorylation (Aktas
etal. 1998). Sustained phosphorylation of eIF2 activates terminal differentiation or

apoptotic cell death by activating downstream effectors such as C/EBPs and CHOP
and/or inhibiting expression of growth stimulatory genes such as cyclin D1 (Aktas
etal. 2013; Aktas etal. 1998; Moreno-Torres etal. 2011; Sequeira etal. 2009).
Spontaneous phosphorylation of eIF2 observed in tumors (Blais et al. 2006;
Shannon etal. 2003) is qualitatively different from the therapeutic eIF2 phos-
phorylation induced by genetic or chemical manipulation. First and foremost,
spontaneous eIF2 phosphorylation in the tumors is localized to hypoxic regions
of tumors (Blais etal. 2006; Shannon etal. 2003), i.e. it is spatially regulated. Sec-
ondly, because hypoxia itself is transient, hypoxia-induced eIF2 phosphorylation
is reversed when hypoxia is relieved, i.e. through recruitment of new blood vessels.
Similarly, radiation- or chemotherapy-induced eIF2 phosphorylation is reversed
once the therapy-induced damage (i.e. DNA damage) is dealt with. The spatially
and temporally regulated eIF2 phosphorylation induces tumor cell dormancy and
survival by activating downstream effectors such as chaperone BiP and stimulating
angiogenesis (Moreno-Torres etal. 2011; Rodland etal. 2013; Sequeira etal. 2009).
Reversion of eIF2 phosphorylation allows for cell proliferation and tumor growth
once the conditions become permissive. The qualitative differences between thera-
peutically induced sustained versus spatially and temporally regulated spontaneous
eIF2 phosphorylation may clarify much of the confusion related to the diverse
observations in alterations of eIF2 functions in cancer.
It is now clear that both activators and inhibitors of eIF2 phosphorylation show
promise in the animal models of human cancers (Chen etal. 2011; Ranganathan
etal. 2008). In some tumors, such as those with inactivating mutations of PTEN,
inducers of eIF2 phosphorylation may serve as direct compensatory treatment.
In other tumors such as those with activating mutations in the RAS/RAF/MEK
pathway, inducers of eIF2 phosphorylation may display anticancer activity due to
synthetic lethal interactions that exploit unique tumor vulnerabilities. In the specific
case of RAS/RAF/MEK driven tumors, dependence of cell proliferation on cyclin
D1 (Aktas etal. 1997; Yu etal. 2001) combined with potent inhibition of cyclin D1
expression by eIF2 phosphorylation (Chen etal. 2013) may account for the ex-
pected synthetic lethality. In the future, these agents may be deployed for treatment
of human cancers either as monotherapy or in combination with other agents. The
choice of inhibitor versus activator of eIF2 phosphorylation must be tailored to the
tumor types, and in the case of combination therapy to other agents in the combina-
tion. For example activators of eIF2 phosphorylation may be utilized in combina-
tion with proteasome inhibitors (Schewe and Aguirre-Ghiso 2009) while inhibitors
may be combined with asparaginase therapy (Moreno-Torres and Murguia 2011;
Richards and Kilberg 2006). Significant differences between the driver mutations
in the tumors as well as the tumor microenvironment will ensure that the same drug
will likely produce different outcomes for different patients or similar patients at
different stages of disease. Tumor heterogeneity and dynamism also necessitates
development of biomarkers to select responsive patient populations and actively
monitor treatment efficacy. Furthermore, these biomarkers should be robust enough
to measure the drug effects in near-real time. In short, both inhibiting and inducing
eIF2 phosphorylation offers potential for cancer therapy, but much work lies ahead.
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Chapter 10
eIF5A
Myung Hee Park, Swati Mandal, Ajeet Mandal and Edith C Wolff
Contents
10.1Introduction 223
10.2eIF5A in Translation: Is There an Analogy with EF-P? 225
10.3Two eIF5A Isoforms: Is There a Role for eIF5A in Cancer? 226
10.4 eIF5A and Apoptosis 228
10.5eIF5A and the Hypusine Modification Pathway as a Potential Target for
Anticancer Therapy 228
References 230
AbstracteIF5A is a small (18kDa) protein involved in the eukaryotic protein

synthesis, in the elongation step. It is the only cellular protein containing the post-
translationally modified lysine, hypusine, which is required for its activity. eIF5A is
essential for cell growth and has been implicated in various types of human cancers.
This protein and the hypusine modification enzymes present new potential targets
for anticancer therapy. Furthermore, an isoform, eIF5A2, has been suggested as an
oncogene and also as a prognostic marker in human cancers. There are several pos-
sible mechanisms for a role for eIF5A in tumor promotion and suppression as well
as in apoptosis. Future investigations are needed to elucidate these relationships.
10.1Introduction
Eukaryotic translation initiation factor 5A (eIF5A) is a small acidic protein, ini-

tially isolated from rabbit reticulocyte ribosomes as a factor that stimulates me-
thionyl-puromycin synthesis in vitro (Benne etal. 1978; Kemper etal. 1976). It
M.H.Park() S. Mandal A.Mandal E.C.Wolff

Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research
(NIDCR), National Institutes of Health, Bethesda, MD, USA
e-mail: mhpark@nih.gov
DOI 10.1007/978-94-017-9078-9_10, US Government 2014
224 M. H. Park et al.
Fig. 10.1 Hypusine modification pathway and proposed role of eIF5A isoforms in cancer. Both
eIF5A-1 and eIF5A-2 isoforms consist of two domains (I and II) and undergo unique posttrans-
lational modification at a specific lysine residue, by two steps involving DHS and DOHH. The
polyamine spermidine serves as a donor for the 4-aminobutyl side chain in hypusine. The inactive
precursor proteins, eIF5A-1(Lys) and eIF5A-2(Lys) are converted to active proteins by deoxyhy-
pusine/hypusine modification. Upon high accumulation, the eIF5A precursors, eIF5A-1(Lys) and
eIF5A-2(Lys) may interfere with the active hypusinated forms (broken red line). The mature eIF5A,
eIF5A(Hpu), may influence translation of a subset of mRNAs encoding various factors involved in
cancer initiation, progression or suppression. eIF5A(Lys), precursor containing unmodified lysine
residue; eIF5A(Dhp), eIF5A intermediate containing deoxyhypusine; eIF5A(Hpu), mature eIF5A
containing hypusine.
is the only cellular protein containing an unusual amino acid, hypusine [N6-(4
amino-2-hydroxy)lysine] (Cooper etal. 1983). Hypusine is formed posttranslation-
ally in two enzymatic steps (for a review see, Park 2006). In the first step, DHS
catalyzes the transfer of the 4-aminobutyl moiety from spermidine to the -amino
group of a specific lysine (Lys50 in the human eIF5A) to form an intermediate
deoxyhypusine. This intermediate is hydroxylated by a specific enzyme, deoxy-
hypusine hydroxylase (DOHH), to complete hypusine synthesis and eIF5A activa-
tion (Fig.10.1). Hypusine plays a key role in eIF5A activity, as the unmodified
eIF5A precursor is inactive in the stimulation of methionyl-puromycin synthesis.
A long basic side chain of hypusine residue may be important in the association of
eIF5A to ribosomes. Disruption of eIF5A genes and the deoxyhypusine synthase
gene causes loss of viability in yeast and in mice (Park etal. 2010). Furthermore,
eIF5A isoforms (eIF5A-1 and eIF5A-2) (Jenkins etal. 2001) have been reported to
be intimately involved in cellular regulation, including proliferation, differentiation,
transformation, apoptosis and tumorigenesis (Caraglia etal. 2013). eIF5A has been
implicated in several diseases including cancer, diabetes (Maier etal. 2010) and
HIV-1 infection (Hoque etal. 2009). In view of their extraordinary specificities,
eIF5A and its two modification enzymes have been proposed as potential targets for
intervention in cancer and other pathological conditions.
10eIF5A 225
10.2eIF5A in Translation: Is There an Analogy

with EF-P?
In spite of the essential nature of eIF5A and its unique modification, the biological
function of eIF5A has remained obscure for decades. Depletion of eIF5A in a yeast
mutant strain UBHY-R with two eIF5A genes disrupted and supplemented with an
ubiquitin-conjugated unstable eIF5A, caused a relatively small decrease in protein
synthesis (Kang and Hershey 1994), leading to a proposal that eIF5A is a translation
factor specific for a subset of mRNAs. Although the C-terminal domain of eIF5A
exhibits an RNA-binding fold, it is not known whether eIF5A specifically asso-
ciates with a subset of mRNAs. UBHY-R and other yeast eIF5A mutants exhibit
pleotropic phenotypes, including defects in mRNA turnover, cell wall integrity, cell
cycle progression and actin cytoskeletal organization, in addition to arrest in cell
growth (Chatterjee etal. 2006; Zanelli and Valentini 2005). The observed defects
may result directly or indirectly from loss of a subset of cellular proteins whose
translation is dependent on eIF5A. Furthermore, depletion of eIF5A in yeast mutant
strains did not cause a reduction in the polysome to monosome ratio (Gregio etal.
2009; Kang and Hershey 1994; Saini etal. 2009), as it would be expected in the case
of a translation initiation factor that has an effect on global mRNA translation. On
the contrary, the ratio of polysome over 80S monosome was consistently higher in
different yeast mutant strains upon depletion of active eIF5A, suggesting a defect
in translation elongation (Gregio etal. 2009; Saini etal. 2009). Furthermore, the
ribosome transit time was estimated to be longer when active eIF5A was depleted,
consistent with the notion that eIF5A is involved in the elongation step rather than
the initiation step of translation.
Regarding the unsolved mystery on the precise mode of eIF5A action in trans-
lation, it is worthwhile to mention its bacterial ortholog, elongation factor P (EF-
P) (Aoki etal. 2008). eIF5A and EF-P share significant sequence similarity and
the crystal structure of Archea eIF5A is super-imposable on that of the first two
domains of EF-P (Hanawa-Suetsugu etal. 2004). Interestingly, the two proteins
undergo similar but distinct basic modifications at the specific corresponding lysine
residue, namely hypusination for eIF5A and -lysylation, plus hydroxylation, for
EF-P (Bullwinkle etal. 2013; Park etal. 2012; Peil etal. 2012). The crystal structure
of Thermus thermophilus EF-P, bound to the 70S ribosome in the presence of the
initiator tRNA and a short mRNA revealed binding of EF-P between the peptidyl
tRNA site and the exiting tRNA site (Blaha etal. 2009), suggesting a role for EF-P
in the first peptide bond formation, without excluding a role on subsequent elonga-
tion. Both factors moderately stimulate methionyl-puromycin synthesis in vitro in
a manner dependent on hypusine or -lysyl-lysine. Recently, EF-P was shown to
enhance translation by preventing stalling of ribosome at sites containing consecu-
tive proline residues, such as PPP, PPG or longer proline stretches (Doerfel etal.
2013; Ude etal. 2013), by acting as a sequence-specific elongation factor in vitro.
Thus, EF-P may affect the translation rate of a subset of mRNAs encoding proteins
containing proline stretches. Alterations in a subgroup of E. coli proteins contain-
ing PPP and PPG motifs may directly or indirectly contribute to the phenotypes of
mutant strains with deletion of the efp gene, or its modification enzyme genes, yjeA
and yjeK, including slow growth, weakened resistance to stress, or loss of virulence
(Navarre etal. 2010). Careful analyses of changes in protein patterns on two-dimen-
sional maps of these mutant cells (Hersch etal. 2013; Navarre etal. 2010) reveal
divergent protein motifs regulated by EF-P and suggest that translational regulation
by EF-P may involve sequences longer than tri-peptide motifs.
A recent paper (Gutierrez etal. 2013) presented evidence that, in fact, eIF5A acts in
a similar manner to EF-P, as has been proposed (Doerfel etal. 2013; Ude etal. 2013).
The presence of eIF5A can relieve stalling of the eukaryotic ribosome when translat-
ing two or three consecutive proline residues and the absence of eIF5A appears to
affect the translation of proteins containing polyproline motifs. However, the in vivo
effects of eIF5A deficiency in the cellular proteome needs to be evaluated as in the
case of efp mutants, to identify the proteins whose translation is dependent on eIF5A
and the eIF5A-dependent peptide motifs. Identification of the full range of proteins
affected by loss of active eIF5A, and their functional categorization, may provide
insights into eIF5A function in in vivo translation and in tumorigenesis (Fig.10.1).
10.3Two eIF5A Isoforms: Is There a Role

for eIF5A in Cancer?
In mammals, there exist two isoforms of eIF5A with high sequence identity (84%
identity in human eIF5A) (Jenkins etal. 2001). Both forms undergo hypusine modi-
fication at the same specific lysine residue (Clement etal. 2003). Whereas eIF5A-1
is expressed in all cells and tissues, and is abundant in rapidly proliferating cells, the
eIF5A-2 isoform, in contrast, normally is hardly detectable and may be expressed in
a tissue-dependent manner (Clement etal. 2006; Jenkins etal. 2001). Although it is
not clear whether the two isoforms have differentiated functions in cells and tumori-
genesis, both have been associated with the proliferation rate in cultured cells and
tissues. The EIF5A2 gene is located at 3q26, the locus frequently amplified in many
human cancers (Guan etal. 2001). Amplification of the EIF5A2 gene has been de-
tected by fluorescence in situ hybridization and comparative genomic hybridization;
increased expression of eIF5A2 isoform mRNA or protein was measured by various
techniques, including the expressed sequence tag analyses, microarray, quantitative
PCR, IHC, two-dimensional difference gel electrophoresis and Western blotting in
various human cancers (Table10.1). Either form has been suggested as a diagnostic
marker in certain cancers where it was overexpressed. In particular, EIF5A2 was
first identified as a candidate oncogene in ovarian cancer, as its gene amplification
and overexpression appeared to be a common occurrence in ovarian cancer tissues
and cell lines (Guan etal. 2001; Yang etal. 2009). Furthermore, ectopic overexpres-
sion of eIF5A-2 in the human liver cell line LOS2 caused its transformation into
a tumorigenic cell line that induces tumors in nude mice (Guan etal. 2004). Over-
expression of eIF5A-2 in p53//MYC liver progenitor cells also enhanced tumor
growth upon injection into mice (Zender etal. 2008). EIF5A2 gene amplification
was often, but not necessarily, associated with overexpression of eIF5A-2 protein
in other cancers, suggesting additional mechanisms of eIF5A-2 upregulation at the
10eIF5A 227
Table 10.1 Overexpression of eIF5A-1 and eIF5A-2 in various human cancers. Overexpression of
eIF5A-1 or eIF5A-2 isoform mRNA or protein was detected in various human cancers. Amplifica-
tion of EIF5A2 gene at 3q26 locus is a common occurrence in human cancers. The two isoforms
are potential diagnostic and prognostic markers. The level of eIF5A-2 protein was often correlated
with tumor staging, metastasis and poor prognosis.
Type of eIF5A Gene Overexpression Clinical References
Cancer Isoform Amplification Correlation
mRNA Protein
CRC (colon) 5A-1 + + + (Lam et al. 2010)
5A-2 + + + + (Xie et al. 2008; Zhu
et al. 2012)
CIN (cervix) 5A-1 + + (Cracchiolo et al. 2004)
Glioblastoma 5A-1, + + (Preukschas et al. 2012)
5A-2
HCC (liver) 5A-2 + + + + (Lee et al. 2010; Shek
et al. 2012; Tang
etal. 2010)
NSCLC 5A-2 + + + + (He et al. 2011)
(lung)
Ovarian 5A-2 + + + + (Guan et al. 2004; Guan
Carcinoma et al. 2001; Yang
etal. 2009)
Urothelial 5A-2 + + + + (Luo et al. 2009)
Carcinoma of
the Bladder
CRC colorectal cancer, CIN - cervical intraepithelial neoplasia, HCC - hepatocellularcarci-
noma, NSCLC non-small-cell lung carcinoma.
transcriptional or translational level. Ectopic overexpression of eIF5A-2 in HCC

cells and CRC cells (Zhu etal. 2012) enhanced cell motility and promoted tumor
metastasis (Tang etal. 2010; Xie etal. 2008). The levels of two epithelial markers,
E-cadherin and -catenin, were downregulated while the two mesenchymal markers,
fibronectin and vimentin, were enhanced upon overexpression of eIF5A-2, suggest-
ing a mechanism involving EMT. Aberrant expression of eIF5A-2 correlated with
advanced tumor stage and poor prognosis in patients with ovarian cancers (Yang
etal. 2009), HCCs (Lee etal. 2010; Shek etal. 2012), colon cancers (Tunca etal.
2013), bladder cancers (Luo etal. 2009) and lung cancers (He etal. 2011).
While there is abundant literature on the positive correlation between eIF5A lev-
el and aberrant growth in various cancers, an opposite view was presented recently,
in the case of lymphoma (Scuoppo etal. 2012). Upon in vivo screening of an shRNA
library targeting genes deleted in human lymphoma, eIF5A-1 and deoxyhypusine
synthase genes, along with S-adenosylmethionine decarboxylase (AMD1) were
identified as tumor suppressor genes in a mouse lymphoma model. It is possible that
eIF5A-1 could function as a tumor suppressor as well as a tumor-promoting factor
in a system/tissue-dependent manner, probably with a biphasic mode depending on
its concentration. Since suppression of eIF5A-1 expression using siRNA in a mouse
liver cell line did not enhance colony formation (Zender etal. 2008), the tumor
suppressive effect of eIF5A appears to be specific for the mouse lymphoma model
rather than a general phenomenon.
10.4 eIF5A and Apoptosis
Endogenous eIF5A exists mainly as the hypusinated form in cells and tissues, since
the hypusine modification of eIF5A occurs readily after translation. However, un-
modified eIF5A can accumulate upon depletion of the polyamine spermidine, upon
inhibition of deoxyhypusine synthesis, or upon ectopic overexpression of eIF5A
alone without its modifying enzymes. eIF5A isoforms may exert different cellular
functions in apoptosis. Thus, under the circumstances mentioned above, we need to
consider not only potentially different roles of the hypusinated eIF5A isoforms, but
also take their unmodified precursors into consideration.
A role for hypusinated eIF5A in apoptosis was first suggested in the hepatoma
cell line, DH23A/b, expressing high levels of stable ODC (Tome etal. 1997). Ex-
cess accumulation of putrescine in this cell line inhibited hypusine formation and
led to an induction of apoptosis. Furthermore, a reduction in hypusinated eIF5A
was correlated with IFN--induced apoptosis in human epidermoid cancer KB cells
(Caraglia etal. 1997). On the contrary, the ectopic overexpression of eIF5A precur-
sor in mammalian cells, using a mammalian vector or adenoviral vector, caused
apoptosis in colon cancer cell lines (Jin etal. 2008). Overexpression of eIF5A pre-
cursors of either isoform, or of the eIF5A/K50R mutant, also induced apoptosis.
The apoptosis induced by excess eIF5A precursor was reported to involve activa-
tion of the intrinsic mitochondrial pathway (Sun etal. 2010). Thus, either a reduc-
tion in hypusinated eIF5A or a high accumulation of eIF5A precursor may lead to
apoptosis. Although it is not clear whether unhypusinated eIF5A precursor exerts
its effect independent of mature eIF5A or by interference with hypusinated eIF5A
activity, the latter is more likely, since a net reduction of eIF5A by siRNA nanopar-
ticles also induced apoptosis in the multiple myeloma model in mice in the absence
of increased accumulation of eIF5A precursor (Taylor etal. 2012).
10.5eIF5A and the Hypusine Modification Pathway

as a Potential Target for Anticancer Therapy
eIF5A and its modification enzymes have been proposed as therapeutic targets in
various cancers. The hypusinated eIF5A level generally correlates with the rates
of growth in mammalian cells and tissues (Park 2006). Since the two modification
enzymes, deoxyhypusine synthase and deoxyhypusine hydroxylase, are totally spe-
cific for eIF5A modification, the enzymes as well as eIF5A can be targeted for inter-
vention in aberrant cell proliferation. Of a number of spermidine analogs developed
as inhibitors of deoxyhypusine synthase, N1-guanyl-diaminoheptane (GC7) is most
potent and effectively inhibits hypusine synthesis and growth of many human can-
cer cell lines (Park etal. 1994; Preukschas etal. 2012) and tumors in animal tumor
models (Jasiulionis etal. 2007). However, the toxicity of GC7 in animals precludes
its use in human clinical trials. The second enzyme, deoxyhypusine hydroxylase, is
10eIF5A 229
an iron-dependent mono-oxygenase and can be effectively inhibited by several iron

chelators including ciclopirox and deferoxamine (Clement etal. 2002; Hanauske-
Abel etal. 1994), although the specificity of their actions has yet to be established.
The two compounds have been approved for clinical use in human for treatment of
fungal infection and iron overload, respectively. Ciclopirox exhibits antiprolifera-
tive and antiangiogenic effects in vitro (Clement etal. 2002), suggesting its poten-
tial utility as an anticancer drug.
As mentioned above, ectopic overexpression of eIF5A-1 or eIF5A-2 precur-
sors using an adenoviral vector caused apoptosis and inhibition of tumor growth
of mouse melanoma or human lung xenograft, suggesting a novel gene therapy ap-
proach (Jin etal. 2008). Since a reduction in hypusinated eIF5A-1 or eIF5A-2 also
inhibits cellular growth, eIF5A was targeted with the use of antisense RNA, siRNA
or shRNA. In a CRC cell line, UACC-1598, which overexpresses eIF5A-2, eIF5A-2
antisense RNA reduced the growth rate of this cell line (Guan etal. 2004) and eI-
F5A-2 siRNA also inhibited the growth of HCC cells (Lee etal. 2010). Knockdown
of EIF5A2 attenuated proliferation and colony formation of human hepatoma cells
harboring deletion of the exportin 4 gene (XPO4) (Zender etal. 2008). In cells not
overexpressing eIF5A-2, eIF5A-1 siRNA, delivered as nanoparticles, inhibited tu-
mor growth in murine models of multiple myeloma (Taylor etal. 2012).
eIF5A is a unique cellular protein containing hypusine that is required for its ac-
tivity. The posttranslational modification pathway leading to hypusine synthesis
by two enzymes, DHS (deoxyhypusine synthase) and DOHH, has been biochemi-
cally characterized and the essential nature of eIF5A and its hypusine modification
have been firmly established from gene disruption studies (Nishimura etal. 2012;
Schnier etal. 1991). However, regarding the cellular function of eIF5A, its mode
of action and role in tumorigenesis, many questions still remain to be answered.
It is unknown whether the two eIF5A isoforms are functionally identical or not in
mammals. It is also unknown whether the unhypusinated eIF5A precursor exerts its
effects in apoptosis induction by interfering with mature eIF5A or independently of
hypusinated eIF5A. There are a number of reports suggesting an important role for
eIF5A isoforms in various human cancers, mainly based on correlation between eI-
F5A isoform levels and tumor staging, metastasis and prognosis. However, a caus-
ative role of eIF5A in tumor development and progression is yet to be established.
Most importantly, future investigation should be directed toward elucidation of the
mode of eIF5A action in translation, identification of a subset of mRNAs whose
translation is regulated by eIF5A and determination of the mechanism as to how the
changes in the eIF5A-dependent proteins contribute to tumor development.
Acknowledgement The research was supported by the Intramural Research Program of the
National Institute of Dental and Craniofacial Research (NIDCR), NIH.
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Chapter 11
eIF6
Stefano Biffo, Daniela Brina and Stefania Oliveto
Contents
11.1Dual Function of eIF6 in Ribosome Biogenesis and Translation 234

11.2Effects of eIF6 on Translation 234
11.3Effects of eIF6 on Ribosome Biogenesis and Antiassociation Activity 235
11.4eIF6 and Cancer 236
11.5Control of eIF6 Activity in Cancer Biology 237
References 238
AbstractGenetic and biochemical studies demonstrate that eIF6 controls the

availability of 60S ribosomal subunits and does it in two ways. First, it regulates
the biogenesis of 60S in the nucleolus through an unknown mechanism. Second, it
prevents improper binding of free 60S to 40S subunits in the cytoplasm, keeping
them available for translation. Total loss of eIF6 is lethal due to loss of 60S biogen-
esis. Fifty percent reduction of eIF6 results in impairment of growth factor-induced
translation, and is accompanied by increased survival in tumor models. It is becom-
ing increasingly clear that eIF6 levels and activity are rate-liming for tumor growth
in various models. However, our understanding of the connection between eIF6
and tumorigenesis is still in its infancy. Uncovering oncogenic pathways regulating
eIF6 and its downstream targets will provide further clues as to the likely role of this
protein in cancer biology.
S.Biffo() D.Brina S.Oliveto

Department of Science and Technological Innovation (DISIT),
University of Eastern Piedmont, Alessandria, Italy
e-mail: biffo.stefano@hsr.it
S.Biffo S.Oliveto
National Institute of Molecular Genetics (INGM), Milan, Italy
e-mail: biffo.stefano@hsr.it
234 S. Biffo et al.
11.1Dual Function of eIF6 in Ribosome Biogenesis

and Translation
eIF6 (eukaryotic initiation factor 6) is an evolutionary conserved protein of 245

amino acids, 77% identical between yeast and human cells (Biffo etal. 1997). The
primary sequence of eIF6 has no homologs or conserved motifs. No evidence for
gene duplication exists, suggesting a strong evolutionary pressure for tight control
of the protein concentration. eIF6 structure has been solved. It is a rigid protein,
organized in a cyclic structure, called pentein, formed by five stretches of quasi-
identical /-subdomains arrayed around a five-fold axis of pseudosymmetry (Groft
etal. 2000). The semi-conserved C-terminal tail was proposed as a candidate region
for eIF6 regulation due to its flexibility (Groft etal. 2000). Recent structural data
have also identified that eIF6 binds to the intersubunit space of the large ribosom-
al subunit (Klinge etal. 2011). This binding site is conserved between biological
kingdoms. The highly conserved helical binding region of eIF6 interacts with the
hydrophobic C-terminal chain of the ribosomal protein L23 (rpL23). rpL24 and the
sarcin-ricine loop also contribute to the interaction of eIF6 with the 60S subunit.
eIF6 and the 40S subunit share a common binding region on the 60S subunit, thus
eIF6 prevents 60S binding to the 40S due to steric hindrance. The phosphorylation
sites Ser174 and Ser175 are located at the accessible surface of eIF6, not involved
in the interaction with the 60S subunit. Also the more flexible C-terminal sequence
that contains the Ser235 phosphorylation site is located at the outer surface of eIF6
(Gartmann etal. 2010).
Several studies have shown that eIF6 has a dual function: (1) it is necessary for
the maturation of the large 60S ribosomal subunit in the nucleus and likely possess-
es a ribosomal antiassociation activity (Miluzio etal. 2009) and (2) it is involved in
translation in the cytoplasm.
11.2Effects of eIF6 on Translation
In vitro, eIF6 is able to strongly repress translation after binding to mammalian

60S subunits (Ceci etal. 2003). Similar observations were made with eIF6 bound
to 50S (Benelli etal. 2009), the Archibacteria large ribosomal subunit. Since the
binding to either 60S or 50S impairs further translational initiation, it is reasonable
to assume that there is a release mechanism leading to dissociation of the protein
from the large subunit. Hence, two models for eIF6 release from 60S have been
proposed. One model postulates that 60S, bound to eIF6, is translocated from the
nucleus to the cytoplasm. Then, the interaction between the SwachmanBodianDi-
amond syndrome protein (SBDS) and the GTPase Elf1p with the 60S subunit leads
to a conformational change of the latter and subsequently mediates release of eIF6
(Wong etal. 2011). This mechanism seems to be particularly relevant in the matura-
tion step of the 60S subunit (Bussiere etal. 2012). In this context it is important to
note that point mutations of eIF6 can rescue the loss of function phenotype of SBDS
11eIF6 235
(Menne et al. 2007). The other model is based on the function of the RACK1 (recep-
tor of activated PKC1)/PKC complex. RACK1 acts as a scaffold receptor for active
PKC and simultaneously binds to the small ribosomal subunit (Ceci etal. 2003;
Volta etal. 2013). In this model, activated PKC translocates from endomembranes
to the 40S subunit, comes in close vicinity with eIF6 bound to the 60S subunit and
catalyzes the phosphorylation of eIF6 on Ser235 and its subsequent release. For a
detailed discussion of this topic please see (Brina etal. 2011).
In vivo evidence that eIF6 is critical for translation initiation comes from the
mouse model of eIF6 haploinsufficiency (Gandin etal. 2008). eIF6 homozygote de-
letion is lethal in mammals, whereas eIF6 heterozygote mice are viable. All mouse
tissues of heterozygote mice have halved levels of the eIF6 protein compared to
the wild-type controls. Interestingly, the reduction of eIF6 levels in this model was
observed only in the cytoplasm and not in the nucleus, providing support to the
notion that it is cytoplasmic, translation-related, but not nuclear, ribosomal biogen-
esis-related, function of the protein that is affected in this model. Consistently, it is
possible to speculate that the biogenesis of the 60S ribosomal subunit is not affected
in the eIF6 heterozygous mice. By analyzing polysome profiles, an increase in the
80S peak and a decrease in polysomes were observed in the eIF6 heterozygous
mice, suggesting the role of eIF6 in translation initiation. Furthermore, while the
basal level of translation is not reduced, insulin-induced stimulation of translation
is blunted in eIF6 heterozygous hepatocytes compared to the wild types. Thus, full
levels of eIF6 are needed to execute the insulin-induced translational program of
the cell in vivo. The precise mechanism by which eIF6 increases translation down-
stream of insulin signaling still remains to be elucidated. It has been reported that in
vitro the 80S ribosome dissociation can be substantially increased by the presence
of eIF6 or eIF3, eIF1 and eIF1A, which have the potential to prevent re-association
of recycled ribosomal subunits (Jackson etal. 2012). Perhaps a similar mechanism
exists in vivo. Intriguingly, changes in translational efficiency due to eIF6 depletion
are accompanied by tissue specific changes that include liver and white adipose tis-
sue weight reduction (Gandin etal. 2008).
11.3Effects of eIF6 on Ribosome Biogenesis

and Antiassociation Activity
In addition to its possible direct role in translation, eIF6 is necessary for the biogen-
esis of a 60S ribosomal subunit. This is supported by several lines of direct and indi-
rect evidence. Firstly, deletion of Tif6, the eIF6 yeast homolog, leads to a loss of the
60S ribosomal subunit that can be rescued by the ectopic expression of human eIF6
(Brina etal. 2011; Sanvito etal. 1999; Si and Maitra 1999). Secondly, eIF6 is found
in molecular complexes ranging from the pre-60S to mature 60S subunits (Volta etal.
2005). Lastly, a pool of eIF6 is found to localize to the nucleolus of mammalian and
yeast cells (Sanvito etal. 2000). However, our understanding of the eIF6 function in
the biogenesis of the 60S subunits is not clear and requires further studies.
236 S. Biffo et al.
Importantly, eIF6 has an observed biochemical activity that prevents 60S to 40S
binding in the absence of the mRNA, thus avoiding an accumulation of inactive 80S
particles. The net result of the ribosomal antiassociation activity is to keep the small
and large subunits available for initiation of translation. The early evidence of the
antiassociation activity of the protein came from in vitro studies using wheat germ
(Russell and Spremulli 1979) and calf liver (Valenzuela etal. 1982) extracts. How-
ever, further in vivo experiments in yeast cells did not provide compelling evidence
in support of the ribosomal antiassociation activity of eIF6, since its depletion did
not alter the balance of free ribosomes (Sanvito etal. 1999; Si and Maitra 1999).
A subsequent study showed that the release of eIF6 from the 60S subunit is neces-
sary for the initiation of translation (Ceci etal. 2003). Consistently, the site of eIF6
binding to 60S was mapped to the 40S60S interface, close to SRL (sarcine ricine
loop 17) and ribosomal proteins rpL23 and rpL24 (Gartmann etal. 2010; Klinge
etal. 2011). Thus direct in vivo evidence in support of an active role of eIF6 in the
ribosomal antiassociation activity is lacking. It is reasonable to speculate that the
antiassociation activity of eIF6, as observed in vitro, is relevant for translational
control in vivo. Although eIF6 is dispensable for translation in vitro, Russell and
Spremulli (1979) showed that low concentrations of eIF6 have a slight stimulatory
effect on translation. However, as mentioned previously, deletion of Tif6 in yeast
has no effects on the phenotype (Miluzio etal. 2009).
11.4eIF6 and Cancer
Dysregulation in translational control is a likely endpoint of oncogenic pathways

that promote cellular transformation and tumor development (Silvera etal. 2011).
In normal cells, these pathways act as sensors of nutrient availability, energy or
stress, adapting ribosome production and gene expression to the cellular conditions.
In tumors, most of these pathways are hyperactivated and pro-oncogenic (Loreni
et al. 2013).
The research into the role of eIF6 in cancer is still in its infancy. eIF6 was found over-
expressed only in selected cancer types, such as CRCs, where the highest levels of eIF6
are seen in metastatic disease (up to ten-fold) (Sanvito etal. 2000), head and neck cancer
(Rosso etal. 2004), lung metastasis (Martin etal. 2008), acute promyelocytic leukemia
(Harris etal. 2004) and malignant mesothelioma (Biffo etal. 1997).
What mechanism leads to eIF6 overexpression in cancer is still unclear. For ex-
ample, c-MYC oncogene was found to regulate the transcription of pro-oncogenic
eIF4E and various translation factors (Ruggero 2009, 2012). However that was not
shown to be the case for eIF6 (Miluzio etal. 2011). Overexpression of eIF6 per se
in the aforementioned cancer types does not necessarily signify its etiologic role in
cancer development but could just reflect an increased demand for this protein by
rapidly proliferating cancer cells. Studies in mice provided answers to the tumori-
genic potential of the protein. Cells which have 50% decrease in eIF6 expression
have a reduction in oncogene-mediated transformation of up to 80%, in spite of
11eIF6 237
being virtually normal in basal conditions (Gandin etal. 2008). Notably, MYC-
induced lymphomagenesis is highly reduced in murine lymphomas with reduced
eIF6 levels. The average survival of eIF6 heterozygous mice crossed to E-MYC
mice is increased to up to 1 year, compared to 4 months in the E-MYC animals
(Miluzio etal. 2011). The molecular mechanism by which a partial reduction of
eIF6 levels reduces tumorigenesis and tumor growth are unknown, but remarkably,
eIF6 heterozygous mice kept in captivity do not show overt negative phenotypes.
Taken together these data suggests that eIF6 is likely not a bona fide oncogene but
a modulator of tumorigenesis and tumor growth.
11.5Control of eIF6 Activity in Cancer Biology
Two pathways converging the growth factor machinery to the translational appara-
tus that are frequently mutated in cancer are the PI3K/AKT/mTOR and the MAPK
signaling pathways. The mTORC1 cascade is the most characterized pathway in-
volved in translational regulation, enabling cap-dependent translation and affecting
tumorigenesis in a 4E-BP/eIF4E dependent fashion (Dowling etal. 2012). If eIF6
takes part in this pathway is not fully clear, but a variety of data indicate that the
eIF6 activity in cancer is likely mTORC1 independent. Since there are large gaps
in our understanding of eIF6 control, we will focus on the most relevant informa-
tion related to the regulation of the eIF6 activity by signaling. First, eIF6 is hyper-
phosphorylated in cancer cells, especially in the nonessential C-terminus at Ser235,
Ser239 and Thr243 (Ceci etal. 2003; Dephoure etal. 2008). Second, at least in
some contexts, mutation of Ser235 to Ala reduces translation, tumorigenesis and
alters the physiological role of eIF6 (De Marco etal. 2010; Gandin etal. 2008;
Miluzio etal. 2011). Third, eIF6 activity is independent from mTORC1 activation
but essential for growth factor/insulin activation (Gandin etal. 2008). Forth, eIF6
interacts with RACK1 (Ceci etal. 2003; Guo etal. 2011) that is both a receptor for
active PKC (Ron etal. 1994) and a structural component of the ribosome (Sen-
gupta etal. 2004) that affects translation (Volta etal. 2013). RACK1 overexpression
induces chemoresistance and phosphorylation of initiation factors such as eIF4E
(Ruan etal. 2012). Thus the RACK1/PKC complex could potentially activate trans-
lation leading to tumorigenesis. The preferred PKC isoforms binding RACK1 is
PKC, whose locus generates two isoforms, I and II, but only the latter retains
a higher affinity for the RACK1 receptor (Stebbins and Mochly-Rosen 2001). Of
importance is that PKC inhibition reduces translation without affecting mTORC1
targets (Grosso etal. 2008), suggesting a role for the PKC axis in the regulation of
translation. Selective PKC inhibitors with antitumor activity, involving the inac-
tivation of AKT-mediated cell survival, have been described (Podar and Anderson
2011; Wick etal. 2011; Witzig and Gupta 2010), but their impact on translation is
not known. However, it is possible that eIF6 activity is affected by mTORC2, which
is upstream of several PKCs (Hagiwara etal. 2012).
238 S. Biffo et al.
In conclusion, existing evidence is highly suggestive of the role of eIF6 in tumor-

igenesis, however the exact mechanisms of its functions and regulation in physiol-
ogy and cancer biology and pharmacology requires further research.
Acknowledgements Work in our laboratory has been generously funded by AIRC, AIRC 5 per
mille, AICR, Telethon Foundation, Fondazione Buzzi, Minister of University and Research. We
thank for critical reading Dr. Nina Offenhaeuser.
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Chapter 12
Translation Elongation
Bruna Scaggiante, Barbara Dapas, Rossella Farra, Federica Tonon,

Michela Abrami, Mario Grassi, Francesco Musiani, Fabrizio Zanconati,
Gabriele Pozzato and Gabriele Grassi
Contents
12.2Elongation Factors and their Role in Translation and Cancer 244
12.2.1eEF1A 244
12.2.2eEF1B 245
12.2.3eEF2 246
12.3Control of Elongation by Signaling Pathways 246
12.3.1eEF1A and eEF1B 247
12.3.2eEF2 249
12.4Elongation Factors and their Regulation in Cancer 250
12.4.1eEF1A1 250
12.4.2eEF1A2 252
12.4.3eEF2 253
12.5Elongation and Targeted Therapies in Cancer 254
12.5.1eEF1A1 and eEF1A2 254
12.5.2eEF2 256
References 259
AbstractThe synthesis of proteins in eukaryotes is typically divided into four

steps, i.e. initiation, elongation, termination and ribosome recycling. This key bio-
logical event is tightly regulated by the cell. Under conditions of increased demand
G.Grassi() B.Scaggiante B.Dapas

Department of Life Sciences, University of Trieste, Trieste, Italy
e-mail: ggrassi@units.it
R.Farra F.Tonon M.Abrami M.Grassi
Department of Engineering and Architecture, University of Trieste, Trieste, Italy
F.Musiani
Scuola Internazionale Superiore di Studi Avanzati (Sissa/ISAS), Trieste, Italy
F.Zanconati G.Pozzato
Department of Medicine, Surgery and Health Sciences, Cattinara Hospital,
University of Trieste, Trieste, Italy
242 B. Scaggiante et al.
Fig. 12.1 Elongation step of translation. During elongation, each additional amino acid is added
to the nascent polypeptide chain in a three-step microcycle, which consists of the: (1) positioning
of the correct aa-tRNA in the A-site of the ribosome, (2) formation of the peptidyl bond, and (3)
shifting, or translocation of the mRNA by one codon relative to the ribosome.
or reduced production of cellular energy the cell can downregulate this process.
While a tight regulation exists for the rate-limiting initiation step, elongation also
appears to be under somewhat strict control. Among key targets of elongation regu-
lation are the eukaryotic elongation factors A1/A2 (eEF1A1/A2) and the eukaryotic
elongation factor 2 (eEF2). In addition to being involved in the regulation of protein
synthesis, evidence indicates that these factors play a relevant role in the genesis
and maintenance of many forms of human cancers. For this reason, elongation fac-
tors have been considered as valuable markers for different forms of cancers, as
well as attractive candidates for targeted anticancer therapeutic approaches. In this
chapter we focus on the description of the factors taking part in the process of
elongation, as well as their involvement in cancer. Particular emphasis is put on the
description of (1) the physiological role of elongation factors in elongation and in
other biological processes; (2) the control of elongation factors by signaling path-
ways; (3) the involvement of elongation factors in cancer and (4) the anticancer
therapeutic approaches based on the targeting of elongation factors.
12.1Introduction
The synthesis of protein in eukaryotes is typically divided into four steps, i.e.
initiation, elongation, termination and ribosome recycling (see Chap.2 and No-
eske and Cate 2012). Components of this pivotal biological process include the
12 Translation Elongation 243
ribosomes, which in eukaryotes are made up of small 40S and large 60S subunits.
The other components are represented by the mRNA, the aminoacyl-tRNA (aa-
tRNA) and by a variety of cytosolic proteins required for the efficient execution
of the process.
Biochemical mechanisms of translation are described in much detail in Chap.2.
Here we will emphasize the most important aspects of translation relevant to the
current topic. Translation starts with the recruitment of the 40S ribosome to mRNA
using translation initiation factors. The initiation step culminates in hydrolysis of
GTP that triggers the release of initiation factors and allows the binding of the 40S
subunit to the 60S subunit forming the elongation competent 80S complex posi-
tioned at the initiation codon of the mRNA. It is then when the elongation step of
translation commences.
Protein elongation starts with the binding of the aa-tRNA to the GTP bind-
ing protein eukaryotic elongation factor eEF1A (Fig.12.1). Three binding sites
for tRNA in the ribosome are known.These include the A-site for aa-tRNA, the
P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribo-
some.Once bound to eEF1A, the aa-tRNA can have access to the ribosome A-site
where the hydrolysis of eEF1A/GTP to eEF1A/GDP allows the dissociation of
eEF1A/GDP from the aa-tRNA. This last event permits, by means of a catalytic
RNA named peptidyl transferase, the formation of a peptidyl binding between the
aa-tRNA present in the A-site and the aa-tRNA present in the P-site. Following
peptide bond formation, the 3 end of the tRNA in the P- and A-sites moves to the
E- and P-sites, respectively. This event is followed by the release of the deacyl-
ated tRNA from the E-site (Rodnina and Wintermeyer 2009). As the eEF1A/GDP
cannot bind a novel aa-tRNA, the eEF1A/GDP is recycled into the active GTP-
bound form by the eukaryotic elongation factor eEF1B. During chain elongation,
each additional amino acid is added to the nascent polypeptide chain in a three-
step micro-cycle, which consists of the: (1) positioning of the correct aa-tRNA in
the A-site of the ribosome, (2) formation of the peptidyl bond and (3) shifting, or
translocation of the mRNA by one codon relative to the ribosome (Rodnina and
Wintermeyer 2009). The translocation step is governed by the eEF2. This GTP
bound protein catalyses the GTP-dependent translocation of the growing peptide
chain. eEF2 binds to the ribosome and, due to the hydrolysis of GTP to GDP, in-
duces a ribosomal conformational change which allows the sliding of the mRNA
along the ribosome by one codon (Rodnina and Wintermeyer 2009). The eEF2-
GDP is released and the free tRNA leaves the ribosome from the P-site while the
peptidyl-tRNA, bearing the nascent polypeptide, translocates from the A-site to
the P-site ; this leaves the A-site free and thus chargeable with a novel aa-tRNA.
Notably, in contrast to eEF1A/GDP, eEF2-GDP does not require a nucleotide-
exchange factor to regenerate the eEF2-GTP. It should be pointed out that in yeast
and higher fungi an additional elongation factor is required (eEF3, for a review
see Dever and Green 2012), however the reasons for the requirement of this pro-
tein in yeast and fungi are not yet well understood. A possibility is that eEF3 may
facilitate the release of deacylated tRNA from the E-site (Andersen etal. 2006).
Additionally, there is another highly conserved elongation factor 4 (EF4), found in
all bacteria and mitochondria whose function is not decoded either but is thought
to help in sustaining translation under suboptimal conditions in vivo, such as those
Table 12.1 Mammalian elongation factors and their function.

Mammalian elongation factors Canonical functions Non-canonical functions
eEF1A1 GTP and aa-tRNA binding; Cell cytoskeleton organization
recruitment of aa-tRNA to Cell cycle progression
the A-site Protein degradation
Apoptosis
eEF1A2 GTP and aa-tRNA binding; Resistance against oxidative
recruitment of aa-tRNA to stress
the A-site Antiapoptotic effect
eEF1B GDP/GTP exchange on eEF1A subunit: acting binding
subunit: keratin binding
subunit: oxidative stress
signaling
eEF2 GTP and aa-tRNA binding; Cell cycle
ribosomal translocation dur-
ing elongation
occurring under high ionic strength or low temperature (Pech etal. 2011). It ap-
pears that EF4 triggers the back-translocation of the elongating ribosome, causing
the translation machinery to move one codon backwards along the mRNA (Zhang
and Qin 2013).
Once the elongating 80S complex reaches the termination codon and positions it
into the A-site, the termination of translation occurs. No tRNA can bind the termi-
nation codons, which, instead, are recognized by proteins called eukaryotic release
factors (eRF) (Chap.2). eRF1 and eRF3 terminate elongation in a GTP-dependent
manner. eRFs induce the hydrolysis of the ester bond in peptidyl-tRNA and the re-
lease of the synthesized polypeptide from the ribosome (Rodnina and Wintermeyer
2009). See Chap. 2 for more details.
12.2Elongation Factors and their Role in Translation

and Cancer
Since the function of other elongation factors present in eukaryotes is largely un-
known, the present discussion will focus on biological features of eEF1A, eEF1B
and eEF2 (Table12.1).
12.2.1eEF1A
Two genes encode for the eEIF1A protein: EEF1A1 that is located on chromosome
6q14.1 and EEF1A2 that is located on chromosome 20q13.3. These proteins, with
a mass of 50.1kDa, share 78% coding sequence and 92% protein homology (Lund
etal. 1996; Timchenko etal. 2013). Both eEF1A1 and eEF1A2 have similar func-
tions in translation with the major differences being a higher affinity of the former
to the eEF1B guanine nucleotide exchange factors (GEF, see below) (Mansilla etal.
2002), and a 7-fold higher dissociation rate constant for GDP shown by eEF1A1
(Kahns etal. 1998). It appears that these proteins could have other differences in
their biophysical properties, since it has been observed that eEF1A1 is more hydro-
phobic, less resistant to urea denaturation and has a better ability to self-associate
compared to eEF1A2 (Timchenko etal. 2013).
eEF1As are very abundantly expressed GTP-binding proteins, representing ap-
proximately 12% of the total cellular protein (Condeelis 1995). eEF1A1 has a
broad expression among tissues, with the exception of the heart, brain and mature
skeletal muscle where only eEF1A2 is expressed (Knudsen etal. 1993). Moreover,
eEF1A2 expression has been observed in human pancreas islets, in the base of the
gut crypts and in specific regions of the lung (Newbery etal. 2007).
The biological significance of eEF1As isoforms is not limited to protein transla-
tion; it extends to other biological processes representing noncanonical functions.
eEF1A1 is implicated in the modulation of cytoskeleton organization (Sasikumar
etal. 2012) likely related to its ability to interact with F-actin via a separate actin-
binding site (Gross and Kinzy 2005; Yang etal. 1990). Using the same F-actin
binding region, eEF1A1, can also interact with microtubules during the cell cycle
(Bektas etal. 1994; Condeelis 1995; Gross and Kinzy 2005; Moore and Cyr 2000).
In contrast to the aa-tRNA binding, the affinity of eEF1A1 for F-actin inversely cor-
relates with the pH suggesting a possible switch of the protein function depending
of the intracellular milieu (Liu etal. 1996). Additionally, eEF1A1 can interact with
microtubules during cell cycle progression (Bektas etal. 1994; Condeelis 1995;
Gross and Kinzy 2005) via the same region involved in F-actin binding (Moore and
Cyr 2000). It still remains unclear how the eEF1A1/F-actin/microtubules interac-
tion is regulated and what its biological function is. Current data strongly point
towards an involvement of eEF1A1 in cell cytoskeleton organization via the modu-
lation of important cellular functions such as molecular trafficking, replication and
mobility (Sasikumar etal. 2012).
Among other noncanonical functions, eEF1A1 appears to play a role in protein
degradation by recognizing damaged proteins and directing them to proteasomes
(Chuang etal. 2005). Notably whereas in some experimental models eEF1A1 has
been proposed as a proapoptotic factor (Ruest etal. 2002) in others it has been
shown to have antiapoptotic effects (Talapatra etal. 2002; Whitlock etal. 2005).
Interestingly, eEF1A2 has been invariably shown to have antiapoptotic properties
(Chang and Wang 2007; Ruest etal. 2002). Additionally, eEF1A2 has a unique
ability to potentiate the resistance to oxidative stress-induced cell death exerted by
peroxiredoxin I, a protein which functionally protects cells from oxidative stress by
reducing the level of reactive oxygen species (Chang and Wang 2007).
12.2.2eEF1B
The eEF1B is a GEF necessary to convert the inactive eEF1A/GDP into the active
eEF1A/GTP form. It consist of three (, / and ) subunits (Browne and Proud
2002). Notably, the -subunit is found in plants while the in metazoans (Sasiku-
mar etal. 2012). The and / subunits are involved in the GDP to GTP exchange
(Rodnina and Wintermeyer 2009). The component is thought to promote the as-
sembly of all the eEF1B subunits thus allowing the interaction between the and
/ subunits and eEF1A/GDP (Browne and Proud 2002).
As for eEF1A, the role of eEF1B subunits is not limited to the elongation pro-
cess. For example, eEF1B has been proposed to modulate the switch of the eEF1A
from its cytoskeletal function to translation (Pedersen etal. 2001; Pittman etal.
2006). eEF1B has the ability to directly bind actin (Furukawa et al. 2001) and
eEF1B has been shown to interact with keratin, a component of the intermediate
filaments in the cytoskeleton (Kim etal. 2006). Finally, eEF1B has been suggested
to play a role in response to the oxidative stress (Olarewaju etal. 2004) and tran-
scription, localization and translation of the vimentin mRNA (Corbi etal. 2010).
12.2.3eEF2
The human EEF2 gene, located on chromosome 19, encodes for a protein whose
amino acid sequence is highly conserved among mammalian species. In contrast to
eEF1A, this monomeric GTP-binding protein does not need any GEF to be regener-
ated into the active GTP-bound form; this is due to the high GDP release rate which
renders GEF unnecessary to generate eEF2-GTP (Kaul etal. 2011). As mentioned
above, eEF2 binds to the ribosome and using the energy of the hydrolysis of GTP to
GDP, induces a conformational change in the whole ribosome that allows the slid-
ing of the mRNA along the elongating ribosome (Kaul etal. 2011).
12.3Control of Elongation by Signaling Pathways
Protein synthesis is a biological process, which requires a considerable amount of

cellular energy to be executed. It is therefore reasonable that under conditions of
transient increased demand or reduced production of cellular energy, the rate of
protein synthesis has to be reduced. By this regulatory mechanism the cell can save
energy and channel it towards other biological processes that need more imme-
diate attention. While, as described in this book, protein synthesis is very tightly
and complexly regulated at the initiation step, some degree of translational control
also exists at the elongation level. In this regard it has been proposed (Browne
and Proud 2002; Kaul etal. 2011) that the reason for the control at the elonga-
tion step most likely springs from the fact that the downregulation of elongation
allows the ribosome, that is already engaged in protein synthesis, to be stalled on
the mRNA. This implies that, upon de-repression of translation, ribosomes are al-
ready in place to quickly resume protein synthesis. Cerebral ischemia is one of
the conditions that require downregulation of protein synthesis. While in cerebral
ischemia the major contribution to the inhibition of the protein synthesis is de-
rived from the downregulation of translation initiation it also involves inhibition of
the elongation step (Althausen etal. 2001; Martin etal. 2001). Another condition
with and increased energy demand where the protein synthesis is downregulated
Fig. 12.2 Phosphorylation sites of elongation factors. Reported are the phosphorylation sites
for the indicated elongation factors; phosphorylation sites are also indicated for the eEF2 kinase
(eEF2K). C-term = C-terminus; N-term = N-terminus. See List of Abbreviations.
is represented by intense muscle contraction and involves downregulation of the

elongation step (Browne and Proud 2002).
Our present knowledge indicates that the phosphorylation/dephosphorylation
mechanism is a prevalent regulatory mechanism in the case of elongation. These as
well as other emerging mechanisms will be described below.
12.3.1eEF1A and eEF1B
Regulation of elongation is achieved via the phosphorylation/dephosphorylation of

elongation factors by various kinases or phosphatases (Browne and Proud 2002;
Kaul etal. 2011; Negrutskii etal. 2012). eEF1As possess phosphorylation sites
and hence could be subject to regulation via signaling pathways. eEF1A/B can be
activated via phosphorylation by the multipotential S6K (Chang and Traugh 1997;
Traugh 2001), which activity is influenced by insulin and serum, providing further
evidence as to how extracellular factors can regulate protein synthesis (Chang and
Traugh 1997; Traugh 2001). PKC can also phosphorylate the eEF1A/B proteins
(Venema etal. 1991a, b) inducing an increased GEF activity (Peters etal. 1995)
which results in a faster elongation process. eEF1B and eEF1B have been report-
ed to be phosphorylated by the kinase activity of the maturation-promoting complex
(MPF) during meiosis (Janssen etal. 1991; Mulner-Lorillon etal. 1994). Although
it is well known that during meiotic maturation protein synthesis is upregulated,
there is no clear evidence that the aforementioned phosphorylation improves the
activity of the eEF1A/B complex. Recently, it has been observed that during mi-
tosis eEF1B is phosphorylated by CDK1 (Sivan etal. 2011). This phosphoryla-
tion significantly decreases the affinity of eEF1B for eEF1A, resulting in a reduced
availability of eEF1A/tRNA complexes and a reduced association of eEF1A with
elongating ribosomes (Sivan etal. 2011).
Worth of mention is the different phosphorylation pattern observed between
eEF1A1 and eEF1A2 (see also Fig.12.2) that in part may explain the distinct
biological behaviours of the two proteins. Sanges etal. have shown that Ser21 and
Thr88 of eEF1A1 are sites phosphorylated by BRAF. In contrast, in eEF1A2 only
Ser21 is phosphorylated by BRAF (Sanges etal. 2012). Phosphorylation at Ser21
was found to sustain protein stability, since phospho-deficient eEF1A1 and eEF1A2
mutants exhibited a reduced stability due to increased proteasome degradation. No-
tably, phosphorylation of Ser21 was strongly enhanced when both eEF1A isoforms
were pre-incubated together prior to the addition of the kinase. This fact suggests
that the eEF1A isoforms may heterodimerize in so increasing the accessibility of
Ser21 to phosphorylation. This observation also opens a possibility that the two
isoforms may, at least under some circumstances, cooperate. Interestingly, binding
of GEF to eEF1A seems to prevent phosphorylation at Ser21, as demonstrated by
the absence of modification at the Ser21 in eEFA1 derived from proliferating cells.
This suggests that the regulation of phosphorylation at Ser21 site could modulate
the switch of eEF1As between translation and others noncanonical functions. In this
respect, a reduced Ser21 phosphorylation on eEF1A1 was found to lead to an in-
crease in early cellular apoptosis, in line with the data indicating a proapoptotic role
for eEF1A1 (Talapatra etal. 2002; Whitlock etal. 2005). In the case of eEF1A2, a
reduced Ser21 phosphorylation induced an increase of the late cellular apoptosis.
Lin etal. recently showed a connection between TGF-, a potent inhibitor of cell
proliferation, and eEF1A1 as a part of a new pathway regulating cellular protein
synthesis demand. TGF- can induce the phosphorylation of eEF1A1 on Ser300
via the TGF- receptor, which has serine/threonine kinase activity (Lin etal. 2010).
This phosphorylation impairs the aa-tRNA binding to eEF1A1 resulting in a reduced
protein synthesis and cell growth both in vitro and in vivo. Thus, TGF--dependent
phosphorylation at Ser300 might represent a novel mechanism for control of cell
proliferation during translation.
Aside from the serine phosphorylation sites described above there are tyrosine
residues in these proteins that can be phosphorylated. In nontransformedcells Tyr29
and Tyr141 of eEF1A1 can be phosphorylated, suggesting that these phosphoryla-
tion sites are used in normal physiological conditions (Huttlin etal. 2010; Negrutskii
etal. 2012). It seems that ZAP-70 kinase signaling is responsible for these modifi-
cations (Negrutskii etal. 2012). No proof of Tyr29 and Tyr141 phosphorylation in
eEF1A2 is available so far (Negrutskii etal. 2012). Other tyrosine residues phos-
phorylated in eEF1A1 are represented by Tyr85 and Tyr86. It has been proposed
that the c-MET (mesenchymal-epithelial transition factor) proto-oncogene pathway
is responsible for the phosphorylation (Negrutskii etal. 2012). Furthermore, in gen-
eral Tyr phosphorylation of eEF1A does not seem to play a major role in regulating
the general process of translation, although it cannot be excluded that it may affect
translation of some specific proteins and/or noncanonical functions of eIF1A1.
In addition to the known modulation of eEF1A functions via phosphorylation,
other regulatory mechanisms are emerging. Acetylation of eEF1As in tumor cells
has been recently reported (Hu etal. 2012). The study suggests that acetylation,
mediated by histone deacetylase class I and II, reduces eEF1A activity. A more
complex type of regulation by PUF (pumilio/FBF) and Argonaute has been ob-
served in the nematode C. elegans (Friend etal. 2012). PUF and Argonaute are
RNA-binding proteins characterized by their ability to regulate a broad spectrum

of mRNAs and different types of small RNAs (Fabian etal. 2010; Wickens etal.
2002). The authors showed that formation of PUF/Argonaute/eEF1A complex re-
presses translation elongation. The study was also extended to the mammalian ho-
molog of PUF, pumilio homolog 2 protein (PUM2), using in vitro translation assay
and human embryonic kidney cells. It was demonstrated that the PUM2/Argonaute/
eEF1A complex did form in the mammalian cell models and attenuated translation
elongation. Another regulatory pathway has been proposed by Lee etal. (2013) who
reported that the antiproliferative protein p16INK4A, a member of the INK4 family of
CDK inhibitors, interacts with eEF1A2. By using in vitro and in vivo approaches,
the authors demonstrated that the interaction between p16INK4A and eEF1A2 results
in the downregulation of both eEF1A2 expression and activity with the subsequent
reduction in protein synthesis and cell proliferation. Last but not least, very recently
a regulatory strategy for eEF1A2 by miRNAs has been described in human cancer
cells. Namely, miR-663 and miR-744 have been shown to be able to downregu-
late eEF1A2 expression resulting in decrease of cell proliferation (Vislovukh etal.
2013).
12.3.2eEF2
eEF2 is a phosphoprotein which has a major phosphorylation site on Thr56 (Ovchin-

nikov etal. 1990) (see Fig.12.2). Thr56 phosphorylation inhibits eEF2 activity
by affecting translocation and preventing the binding to ribosome (Carlberg etal.
1990). The phosphorylation status is regulated by PP2A phosphatase (Kaul etal.
2011) and by a kinase known as eEF2 kinase (eEF2K) (Hincke and Nairn 1992;
Mackie etal. 1989). eEF2K phosphorylates eEF2 during mitosis when protein syn-
thesis is typically downregulated (Celis etal. 1990). To outline the complex regu-
lation system, it should be noted that eEF2K is, in turn, under control of different
kinases, such as PKA and the stress-activated protein kinase 4(SAPK4)/p38 MAP
kinase . Very recently it has been reported that eEF2 is also phosphorylated by
cyclin-dependent kinase 2 (CDK2) on a novel site, Ser595. This phosphorylation
varies during the cell cycle and is required in vivo for the efficient phosphorylation
of the amino acid residue Thr56. It has been proposed that Ser595 phosphorylation
favors the recruitment of eEF2K to eEF2 thus facilitating Thr56 phosphorylation
(Hizli etal. 2013).
The importance of regulation of eEF2 in cell cycle control has been demonstrat-
ed by the finding of interplay between cyclin D3 and eEF2 (Gutzkow etal. 2003). In
human cancer cells the activation of eEF2K caused eEF2 inactivation and cell cycle
arrest in G1 via loss of cyclin D3, a cyclin necessary for progression through G1.
eEF2K regulation also depends on the Ca2+/calmodulin concentration (Mitsui
etal. 1993). When Ca2+/calmodulin levels rise, eEF2K undergoes an autophosphor-
ylation which enhances its ability to phosphorylate eEF2 (Mitsui etal. 1993). A
possible explanation for the Ca2+/calmodulin-dependent activation in the muscle
tissue has been proposed (Rose etal. 2005). Upon Ca2+ release, triggering of muscle
contraction increases the energy demand. Under this condition the cell reduces the
energy consumption by downregulating protein synthesis to favor the usage of en-
ergy for contraction.
Different isoforms of eEF2K have been proposed to exist in different tissues and
other regulators of eEF2K have been described suggesting that the regulation of
eEF2 by Ca2+/calmodulin might be more complex (Hait etal. 1996; McLeod etal.
2001). For example, PKA can phosphorylate eEF2K on Ser500 (see Fig.12.2) that
activates the kinase rendering it partially independent from Ca2+/calmodulin (Mitsui
etal. 1993; Redpath and Proud 1993). In contrast, S6K1 and RSK1 can phosphory-
late eEF2K on Ser366, reducing its activity and thus promoting protein elongation
(Wang etal. 2001). Moreover, SAPK4/p38 MAP kinase , phosphorylates eEF2K
on Ser359 leaving eEF2K inactive and thus promoting translation (Knebel etal.
2001). The fact that all the aforementioned kinases are regulated by a variety of
molecules (glutamate, muscarinic agonist, adrenalin, fosfokolin angiotensin etc.)
and pathophysiological conditions (exercise, ischemia etc.) indicates that a complex
networking governs the control of eEF2 and eventually regulation of the elongation
step (for review see Browne and Proud 2002).
12.4Elongation Factors and their Regulation in Cancer
While fundamental discoveries of the role of translation initiation in cancer biology

somehow distract our attention from elongation as a step that might be also involved
in cancer development and progression, the recent discoveries highly suggest that
eEF1A1/A2 and eEF2 might be important components of tumorigenesis. Relatively
little information is available with regard to eEF1B and its involvement in cancer.
Here we will focus our attention on the role of elongation factors in cancer biology
and cancer medicine (see Table12.2).
12.4.1eEF1A1
Whereas the role of eEF1A1 in tumorigenesis is not completely defined, evidence

supports a possible involvement of this elongation factor in cancer development
and progression. eFF1A1 overexpression can potentiate the transforming ability of
3-methylcholanthrene and UV (Tatsuka etal. 1992) and determine drug resistance
in head and neck cancer cell lines (Johnsson etal. 2000). The differentiation of the
human acute promyelocytic leukemia cell line induced by the all-trans-retinoic acid
has been found to be paralleled by a reduction of eEF1A1 protein levels (Harris
etal. 2004). Microarray and IHC analysis of eEF1A1 indicated the upregulation
of the protein and mRNA levels of eEF1A1at the edges of breast tumor compared
to the tumor center, suggesting a possibility that eEF1A1 may be involved in can-
cer invasion (Zhu etal. 2003). In HCC cell lines we observed an inverse correla-
tion between cell differentiation and eEF1A1 expression level (Grassi etal. 2007).
Table 12.2 Mammalian elongation factors and their role in cancer.

Mammalian Role in cancer References
elongation
factors
eEF1A1 Drug resistance in head and neck Johnsson etal. (2000)
cancer cells
Breast cancer cell invasion? Zhu etal. (2003)
Cell proliferation in HCC cells Grassi etal. (2007) and Yu etal.
(2012)
The MBI-eEF1A1 form promotes Scaggiante etal. (2013b) and Dapas
cell proliferation in acute lym- etal. (2003)
phoblastic leukemia cells
eEF1A2 Cell proliferation in ovarian cancer Lee and Surh (2009), Tomlinson
cells etal. (2007) and Li etal. (2007)
Cell proliferation in breast cancer Vislovukh etal. (2013)
cells
Cell migration in breast cancer cells Jeganathan etal. (2008)
Cell proliferation in HCC cells Grassi etal. (2007) and Pellegrino
etal. (2014)
eEF2 Cell proliferation in gastric cancer Nakamura etal. (2009)
cells
Cell survival in lung Chen etal. (2011a)
adenocarcinoma
Increased eEF1A1 expression was also positively correlated with the ability of the
cells to proliferate under nonoptimal growing conditions, evading cell cycle arrest.
We also have evidenced that eEF1A1 depletion in poorly differentiated HCC cells
strongly impairs cell viability (Grassi etal. unpublished results). Recently eEF1A1
mRNAs and protein levels have been reported to be upregulated in HCC by FAT10
(Yu etal. 2012), an oncogene belonging to the ubiquitin-like modifier (UBL) pro-
tein family frequently amplified in HCC. This observation led to the hypothesis that
the oncogenic potential of FAT10 is, at least in part, dependent on the upregulation
of eEF1A1 in HCC cells.
In the murine pro-B-cell lymphoid cell line derived from fetal liver cells, in-
creased eEF1A1 levels determine the resistance against apoptosis induced by en-
doplasmic reticulum stress (Talapatra etal. 2002). In prostate adenocarcinoma cell
lines we observed increased eEF1A1 protein levels in the cytoskeletal/nuclear frac-
tion compared to noncancerous control cells (Scaggiante etal. 2012). It should be
however noted that the most pronounced increase was observed for the eEF1A2 iso-
form. Upregulation of eEF1A1 was also observed in cardio-esophageal carcinoma
tissues where independent overexpression of at least one of the eEF1 components
(eEF1A1, eEF1A2, eEF1B) was present in 72% of the clinical samples examined
(Veremieva etal. 2011).
Not only the availability and level of expression but also the posttranslational
modifications of eEF1A1 might contribute to its proproliferative properties. It has
been observed that a modified form of eEF1A1 protein, MBI-eEF1A, represent-
ing a more basic variant of eEF1A, was present in acute lymphoblastic leukemia
cells but not in normal lymphocytes (Dapas etal. 2003; Scaggiante etal. 2013b).
Notably, the disappearance of MBI-eEF1A in lymphoblastic leukemia cells and a

reduction in cell growth, were obtained by the treatment with the cell differentiation
and antiproliferative agent phorbol 12-myristate 13-acetate. This suggests a rela-
tion between the presence of MBI-eEF1A and the proliferative status of the cells.
Consistent with this hypothesis, normal lymphocytes (which do not express MBI-
eEF1A and do not proliferate under resting conditions), upon treatment with the
proproliferative agent phytohemagglutinin, were induced to produce MBI-eEF1A
and to increase their proliferation capacity (Dapas etal. 2003; Scaggiante etal.
2013b). Taken together these data support a functional role for MBI-eEF1A in cell
proliferation and transformation.
12.4.2eEF1A2
Even if eEF1A2 overexpression has been detected in many tumor tissues (reviewed
in Lee and Surh 2009), its contribution to tumorigenesis has been most studied in
ovarian cancers. Whereas a low expression level of eEF1A2 has been observed
in normal ovary tissue, increased mRNA and protein levels were evident in ovar-
ian cancer cells (Tomlinson etal. 2007). Interestingly, not all ovarian carcinomas
overexpressing eEF1A2 mRNA display a rise in the eEF1A2 protein content. In
this regard, it is possible that the rise in the eEF1A2 transcript levels may represent
an untranslated pool of mRNA which the cells can use in case of the need for the
rapid production of proteins required for survival or invasion purposes in response
to the surrounding stimuli. The mechanisms responsible for eEF1A2 upregulation
in ovarian cancer are not completely clear. They neither depend on eEF1A2 gene
amplification nor on the methylation status of the gene promoter (Tomlinson etal.
2007). It hence has been hypothesized that trans-acting factors may play a role in
the upregulation of the EEF1A2 gene. Indeed, in the immortalized normal ovarian
surface epithelial cells (IOSE), the overexpression of the transcription factor zinc
finger protein 217(ZNF217), frequently amplified in ovarian cancers, induces the
rise of eEF1A2 levels (Li etal. 2007).
Another set of evidence to the role of eEF1A2 in cancer comes from the studies
in breast cancer (see Lee and Surh 2009 for a review). While the level of eEF1A2
protein in normal breast tissue is barely detectable, it is overexpressed in about two-
thirds of primary breast tumors. The overexpression has also been confirmed in cul-
tured breast cancer cells (Jeganathan etal. 2008; Pinke and Lee 2011; Vislovukh etal.
2013; Yao etal. 2011), thus suggesting a tumorigenic role in breast cancer (Tomlin-
son etal. 2005). Notably, however, in an in vitro study eEF1A2 does not appear to
be necessary for breast cancer growth (Zhang and Tong 2010) and in vivo eEF1A2
overexpression correlated with a better patient survival (Kulkarni etal. 2007). The
molecular mechanisms by which eEF1A2 contributes to breast cancer have not yet
been fully elucidated. An inverse correlation has been observed between the protein
levels of eEF1A2 and the tumor suppressor p53 (Kulkarni etal. 2007) suggesting
that the tumorigenic properties of eEF1A2 may depend on the reduced antitumor
activity of p53. Another possible mechanism may be mediated by a downregulation
of miR-663 and miR-744 expression. In human breast cancer cells a reduction in the
expression levels of these two miRNAs has been observed resulting in an increase of
eEF1A2 protein and mRNA levels; eEF1A2 overexpression was in turn paralleled by
the increase in cell proliferation and protumorigenic effects (Vislovukh etal. 2013).
However, the importance of miR-663 and miR-744 in breast cancer is unclear, hence
the role of its regulation of eIF1A2. eEF1A2 might also play a role in breast cancer
through the regulation of filopodia; these are membrane structures necessary for cell
migration playing a critical role in the process of cancer cell metastasis. In breast
cancer cell lines overexpressing eEF1A2, the degree of filopodia formation was sig-
nificantly higher in comparison with the same cells in which eEF1A2 was down-
regulated by siRNA (Jeganathan etal. 2008). The mechanism by which eEF1A2
stimulates filopodia formation is mediated by the increase of the cytosolic and plas-
ma membrane-bound levels of phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], a
compound required to activate actin nucleation, necessary for filopodia formation.
There were studies showing overexpression of eEF1A2 mRNA and protein in
HCC (Grassi etal. 2007; Schlaeger etal. 2008). Interestingly, eEF1A2 has been
found overexpressed in a HCC cell lines with a low differentiation grade compared
to HCC cell lines with a high differentiation grade and to normal liver tissue (Grassi
etal. 2007). This upregulation neither correlates with apoptosis resistance nor with
proliferation rate in sub-confluent cells. However, in confluent cells, a clear ten-
dency to maintain the cells proliferation was observed. In vivo, that may confer a
more aggressive phenotype facilitating cellular proliferation in the nutrient- and
oxygen-deficient areas of the tumor far from vessels. Very recently, a mechanism
has been proposed by which eEF1A2 may promote HCC (Pellegrino etal. 2014).
The increased levels of eEF1A2 are responsible for the activation of the PI3K/AKT/
mTOR pathway, which in turn induces upregulation of the mouse double minute
4 homolog (MDM4) (Pellegrino etal. 2014). As a consequence, MDM4 blocks
transcriptional activity of p53 by binding to its N-terminal transactivation domain.
Importantly, activation of the aforementioned mechanism is associated with shorter
survival of HCC patients (Pellegrino etal. 2014).
Finally, while studying the role of eEF1A2 in cancers, we observed that, com-
pared to nontumorigenic prostate cells, prostate cancer cell lines had a significant
rise in eEF1A2 mRNA and protein levels (Scaggiante etal. 2012). Pilot evalua-
tion in archive prostate tissues confirmed the observation indicating the presence of
eEF1A2 mRNA in nearly all neoplastic but not in normal peritumoral samples or in
benign prostatic adenoma.
12.4.3eEF2
eEF2 has been implicated in the tumorigenesis of gastrointestinal cancers. Using

IHC, overexpression of eEF2 was observed in more than 90% of the gastric and
CRC samples analyzed (Nakamura etal. 2009). On the other hand, depletion of
eEF2, resulted in a potent inhibition in the growth of two gastric cancer cell lines,
AZ-521 and MKN-28, and one colon cancer cell line, SW620 (Nakamura etal.
2009). This phenomenon was due to a G2/M arrest accompanied by the inactiva-
tion of cell cycle regulatory molecules such as AKT and CDK1 and activation of
negative regulators of eEF2, such as eEF2K. In agreement with depletion experi-

ments, recombinant overexpression of eEF2 resulted in enhanced tumorigenicity in
mouse xenograft models (Nakamura etal. 2009). Increased protein levels of eEF2
have been also described in lung adenocarcinoma tissue, compared to the neighbor-
ing nontumorous lung tissue (Chen etal. 2011a). Patients with the increased levels
of eEF2 had a significantly higher incidence of early tumor recurrence and a sig-
nificantly worse prognosis. In vitro, eEF2 depletion resulted in an increased induc-
tion of cell death and increased sensitivity to cisplatin (Chen etal. 2011a). Finally,
a strong correlation between eEF2K expression and the aggressiveness of tumor
has been observed in medulloblastoma (the most common malignant brain tumor
of childhood) and glioblastoma multiforme (the most malignant adult brain tumor)
(Northcott etal. 2011; Verhaak etal. 2010).
Tumor cells are able to react to acute nutrient depletion by blocking translation
elongation via the activation of eEF2K (reviewed in Kaul etal. 2011). This capac-
ity, which confers to the cells extended survival under unfavorable conditions, is
not quantitatively identical among different tumor cell types. For example, whereas
HeLa cervical carcinoma cells are sensitive to nutrient depletion and die soon of
it, the MG63 osteosarcoma cells are very resistant (Leprivier etal. 2013). This dif-
ferent behavior has been attributed to the eEF2 phosphorylation level, which is
minimal in HeLa and high in MG63. Minimal eEF2 phosphorylation in HeLa cells
permits protein synthesis; this, however, induces the cells to waste energy, a process
that leads to cell death under nutrient depletion. The sensitivity of the HeLa cell to
nutrient depletion can be reversed by downregulation of eEF2, with the consequent
reduction of protein synthesis (Leprivier etal. 2013). Consistently, the overexpres-
sion of eEF2K, which increases of the amount of the nonfunctional eEF2 form (the
phosphorylated one), makes HeLa more resistant to nutrient depletion. Notably, in a
murine animal model, the overexpression of eEF2K renders tumor cells remarkably
resistant to nutrient depletion (Leprivier etal. 2013).
12.5Elongation and Targeted Therapies in Cancer
The aforementioned data indicate the involvement of elongation factors in human

tumors, making them potential targets for anticancer therapies. In the following
chapter some notable examples of drugs affecting elongation factors are reported
(see Table12.3).
12.5.1eEF1A1 and eEF1A2
Resveratrol, a natural polyphenol commonly found in grapes, has proapoptotic,

antiproliferative and anti-inflammatory effects (Tili and Michaille 2011; Vang etal.
2011). These properties make resveratrol a valuable compound for the treatment
of different human tumors. It has been shown that a mechanism by which resvera-
trol exerts its antitumor effects is mediated by the inhibition of eEF1A2 expression
Table 12.3 Targeted therapies affecting mammalian elongation factors.

Mammalian Targeting drugs Cancer type
elongation
factors
eEF1A1 Narciclasine Glioblastoma and melanoma
siRNA
eEF1A2 Resveratrol Ovarian and breast cancer
siRNA
eEF2 Doxorubicin Glioma, colorectal and renal
Taxol cancers
Candidaspongiolide
Temsirolimus
Bouvardin
Ontak
Imidazolium histidine
Kinase inhibitor NH125
siRNA
Targeting eE2F Kinase
Rotllerin, geldanamycin,
siRNA
(Lee etal. 2009). In the human ovarian cancer PA-1 cell line, resveratrol attenuated
cell proliferation and induced cell apoptosis in the presence of insulin or serum
(Lee etal. 2009). Moreover, resveratrol reduced the expression of eEF1A2 and de-
layed growth of PA-1 cells in the mouse xenograft model. In the breast cancer cell
line MCF-7, the effect of resveratrol was shown to be dependent on the upregulation
of miR-663 and miR-744 which in turn reduced the mRNA and protein levels of
eEF1A2 (Vislovukh etal. 2013). Additionally, miR-663 targets and thus inhibits the
tumorigenic capacity of the proto-oncogenes JUNB and JUND, further contributing
to the antitumor effects of resveratrol (Tili and Michaille 2011). These observations
can explain the mechanism of resveratrol action and show the relevance of targeting
eEF1A2 in anticancer therapeutics.
The use of siRNA/miRNA targeting translation factors may represent a valuable
approach to downregulate cancer cell growth. In this regard we have evidence (Gras-
si etal. unpublished data) that eEF1A1 targeting by siRNAs is effective in reducing
the expansion of liver and prostate cancer cell lines in culture. Moreover, targeting
of translation factors by siRNAs may be a strategy to improve cancer cells sensitiv-
ity to chemotherapeutic agents. For example, in different cancer cell lines, such as
HEK293, HeLa, U2OS, A549 and HCT116, siRNA mediated depletion of eEF1A1
potentiated the proapoptotic effect of the anticancer drug cisplatin (Blanch etal.
2013). Similar results were observed with camptothecin and doxorubicin (Blanch
etal. 2013). Notably, this effect occurs in cancer cells with wild-type p53 but not in
p53-null cells, suggesting that effects of eEF1A1 occur via the p53 pathway.
Aptamers can be used to potentiate the action of anticancer drugs. Aptamers are
noncoding single-stranded DNA or RNA molecules that can be selected to bind any
given target by chemical recognitions different from the Watson-Crick base-pairing
(Scaggiante etal. 2013a). We have observed that aptamers that are able to recognize
eEF1A1 can induce cell growth inhibition in human T-lymphoblastic CCRF-CEM
cell lines (Scaggiante etal. 2006) and other leukemic cell lines (Scaggiante etal. un-
published results). Notably, anti eEF1A1 aptamers were able to potentiate the thera-
peutic index of the conventional chemotherapeutics such as doxorubicin and vin-
blastine in human leukemic and colon adenocarcinoma cell lines (Dapas etal. 2002).
In in vivo animal models administration of narciclasine (a RHO/RHO kinase/
LIM kinase/cofilin signaling pathway activator) at the dose of 10mg/kg has been
found to induce apoptosis and cytotoxicity in tumor cells (Van etal. 2013). At a
lower dose of 1mg/kg, the antitumor effect (principally mediated by a cytostatic
activity) can increase the life span of immune-deficient mice orthotopically xeno-
grafted with invasive human glioblastoma cells and with metastatic cells derived
from NSCLCs. These effects, mediated by impairments of actin cytoskeleton orga-
nization and protein synthesis, are exerted via the targeting of GTP-bound proteins,
including eEF1A (Van etal. 2013). Narciclasine has also been shown to be effective
in melanoma cells, where it induces marked actin cytoskeleton disorganization and
protein synthesis impairment (Van etal. 2010). Also in this case the antitumor effect
depends on the eEF1A targeting which in turn allows bypass of the apoptosis resis-
tance of melanoma cells. Notably, targeting eEF1A by narciclasine has been shown
by biochemical and computer prediction approaches (Van etal. 2010).
In conclusion, examples of drugs used for eEF1A targeting are represented by
resveratrol and narciclasine, both active against different forms of tumors. Notably,
the targeting of eEF1A can also potentiate the antitumor effects of different drugs
such as cisplatin, camptothecin and doxorubicin.
12.5.2eEF2
Various canonical chemotherapeutic agents were observed to result in phos-

phorylation of eEF2 (see White-Gilbertson etal. 2009 for a review), however the
specificity of this effect is still questionable and needs to be further studied. A com-
monly employed chemotherapeutic agent doxorubicin can be used to target eEF2
(White etal. 2007). Doxorubicin induces, among other effects, the phosphoryla-
tion of eEF2, which results in the block of protein translation with consequent cell
cycle arrest in G2/M likely via induction of eEF2 phosphorylation by free radicals
(White etal. 2007).
Paclitaxel (taxol) is a drug which exerts its antitumor activity by stabilizing mi-
crotubules thus inducing the block of cell cycling in the G2/M phase (Huizing etal.
1995). One of the molecular effects of paclitaxel includes the phosphorylation of
eEF2. The mechanisms of eEF2 phosphorylation by paclitaxel are not completely
clear. Another example of a drug able to induce eEF2 phosphorylation is Can-
dida spongiolide, a polyketide obtained from a marine sponge (Meragelman etal.
2007). In U251 glioma and HCT116 CRC cells this compound could induce eEF2
phosphorylation together with a potent apoptotic effect in both cell lines (Trisci-
uoglio etal. 2008). As Candida spongiolide also induced the phosphorylation of
eIF2, it was hypothesized that the proapoptotic effect observed is the result of
the combined inhibition of eIF2 and eEF2 (Trisciuoglio etal. 2008). An mTOR
inhibitor temsirolimus (CCI-779) was also reported to induce phosphorylation of

eEF2 (Shor etal. 2008).
Another drug that can target the elongation step is bouvardin. This molecule can
block polypeptide chain elongation by inhibiting both the GTP- and eEF2-depen-
dent translocation of peptidyl-tRNA and the binding of aa-tRNA to the 80S ribo-
some (Zalacain etal. 1982). Results have been reported with regard to its activity
in vitro and in murine xenograft models, where it showed effectiveness in some but
not all tumor models tested (Geran etal. 2013). This partial efficacy discouraged the
use of bouvardin alone and suggested its use in combination with other therapeutics.
In this regard, the ability of bouvardin to enhance the effects of radiotherapy and
chemotherapeutic agents was shown in Drosophila larvae and in human cancer cell
models, where this compound could enhance the cytotoxic effects of X-rays and the
efficacy of paclitaxel (Gladstone etal. 2012).
The drug denileukin diftitox (Ontak), used in the treatment of different hema-
tological tumors, has been designed to target eEF2 (Duvic and Talpur 2008). This
drug contains an IL-2 moiety and an inactive form of the diphtheria toxin. Upon
uptake by the tumor cells that overexpress the IL-2 receptor, the diphtheria toxin is
separated from the IL-2 subunit. Once free in the cell, the toxin inactivates eEF2 via
ADP ribosylation, thus inducing the downregulation of protein synthesis and cell
death (Duvic and Talpur 2008). Ontak represents an example of how the rational
drug design method can generate molecules with improved specificity of action.
Another example of the rational design of a drug is represented by combining
an aptamer with an anti-eEF2 siRNA. Wuller etal. have generated a chimeric mol-
ecule containing an aptamer able to specifically bind to PSMA (prostatic-specific
membrane antigen) and a siRNA targeting eEF2 (Wullner etal. 2008). As PSMA is
a cell surface glycoprotein found in abundance on prostate cancer cells, it confers
tissue specificity for the aptamer. The compound has shown significant cytotoxicity
towards prostate cancer cells.
Of potential therapeutic interest are also molecules able to interfere with eEF2K.
It is known that a dysfunctional eEF2K causes cell cycle arrest in the S phase
(Parmer etal. 1997, 1999). Drugs that directly or indirectly inhibit eEF2K are, for
example, rottlerin, geldanamycin, and the imidazolium histidine kinase inhibitor
NH125 (Arora etal. 2003; Parmer etal. 1997; Yang etal. 2001). In human glioma
cells, targeting of eEF2K by either NH125 or siRNAs enhanced the apoptotic ef-
fect of TRAIL, a potent cancer cellspecific apoptosis-inducing agent (Zhang etal.
2011). However, NH125 was ineffective in other cells such as the breast cancer cell
line MCF-7, the melanoma cell line B16F10 and the colon cancer cell line HCT15
(Chen etal. 2011b). Additionally, in the lung carcinoma cell lines H420 and H1299,
eEF2K targeting by a siRNA, did not downregulate cell growth, despite reducing
the levels of eEF2K (Chen etal. 2011b). In contrast, in an orthotropic xenograft
mouse model of a highly aggressive breast cancer, eEF2K targeting by a siRNA
resulted in a significant inhibition of cell proliferation together with the induction
of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin
(Tekedereli etal. 2012). Together, the data reported above suggest that the targeting
of eEF2K represents a potential antitumor target.
The process of protein synthesis is a key biological event tightly regulated by the
cell and requiring such considerable amount of energy (Browne and Proud 2002;
Kaul etal. 2011). Under conditions of increased demand or reduced production of
cellular energy the rate of protein synthesis is downregulated. This allows the cell
to save energy for biological processes that have higher priority at that particular
point of time. While a tight regulation exists for the rate-limiting initiation step,
elongation also appears to be under somewhat strict control. The control of elonga-
tion allows the ribosome already engaged in protein synthesis to be retained on the
mRNA, which in turn likely permits the cell to promptly resume protein synthesis
upon the restoration of favorable energy conditions. Aforementioned good example
of this regulation is the one for muscle contraction where the increased energy de-
mand determines a downregulation of protein synthesis and elongation (Browne
and Proud 2002). Additionally, in different pathological conditions such as cerebral
ischemia, the cell saves energy by downregulating translation initiation and elonga-
tion (Althausen etal. 2001; Martin etal. 2001).
Key targets of regulation of this step of translation are eEF2 and eEF1A1/
eEF1A2 that mainly via phosphorylation/dephosphorylation regulate the process
of elongation. As presented in this chapter elongation factors are proteins that are
now being implicated in the biology of different human cancers. The mechanisms
responsible for their contribution to the aetiology and pathogenesis of cancers are
likely complex and poorly studied and understood. In the case of eEF1A1, its in-
volvement in the genesis and maintenance of tumors has been suggested. In contrast
to eEF1A1, the roles for eEF1A2 and eEF2 in cancer biology are being more recog-
nized. These proteins can represent valuable prognostic markers for various forms
of malignancies, such as ovarian, breast and gastro-intestinal cancers (reviewed in
Lee and Surh 2009; White-Gilbertson etal. 2009). These elongation factors appear
also to be promising targets for anticancer therapy. A notable example for such an
approach might be a combination of a tissue specific targeting aptamer with a thera-
peutic siRNA. This strategy is particularly relevant for the targeting of tumor cells
within vital organs such as the liver, where specific targeting of tumor cells but not
normal liver cells is essential.
In conclusion, whereas further efforts are still requested to better understand the
role of elongation factors in normal and tumor cells, the present knowledge indi-
cates the pivotal role played by these factors in the cell. Improvements in this field
can lead to the identification of novel molecular pathways that can be targeted by
future anticancer therapeutic agents.
Acknowledgments This work was in part supported by PRIN number 20109PLMH2, by Fon-
dazione Cassa di Risparmio of Trieste and by the Fondazione Benefica Kathleen-Foreman Casali
of Trieste and by 5 per mille Lega Italiana per la Lotta contro i Tumori, Ministero della Salute.
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Chapter 13
Ribosomes
Fabrizio Loreni and Sara Ricciardi
Contents
13.2Alterations of Ribosomal Proteins and Ribosome Biogenesis Factors in
Tumorigenesis: The Role of MYC Proto-Oncogene 269
13.3Alterations of Components of Ribosomal Machinery: Congenital
Ribosomal Abnormalities and Cancer 270
13.4Ribosomal Alterations and p53 Tumor Suppressor 272
References 275
Abstract Ribosomes are fundamental components of the cell that are essential for its
growth and proliferation. It is hard to imagine that such a fundamental physiological
machinery, as ribosomes, does not participate in cancer biology. However, surpris-
ingly, there is a lack of studies towards the role of ribosomal alterations in cancer.
Here, we will attempt to summarize current knowledge regarding the link between
the ribosomal machinery and human cancer. Various malfunctions in ribosomal
activity represented by defects in ribosome biogenesis have been associated with
human disease. Recent studies performed both in yeast and in higher eukaryotes have
linked various aspects of ribosome biogenesis to the control of cell growth and pro-
liferation. It is now clear that disruption of ribosome biogenesis is a cause of several
inherited genetic disorders that have been associated with an increased risk of tumor
development. In this chapter we discuss some recent insights into the mechanisms by
which alterations in ribosome biogenesis contribute to the biology of cancer.
F.Loreni()
Department of Biology, University of Rome Tor Vergata, Rome, Italy
e-mail: loreni@uniroma2.it
S.Ricciardi
DISIT (Dipartimento di Scienze e Innovazione Teconologica), University of Eastern Piedmont,
Alessandria, Italy
Istituto Nazionale Genetica Molecolare (INGM), Milan, Italy
268 F. Loreni and S. Ricciardi
Fig. 13.1 Scheme of ribosome biogenesis. Ribosomal RNAs are transcribed as a polycistronic
transcript known as pre-rRNA. The pre-rRNA is subsequently processed through endonucleolytic
and exonucleolytic cleavage into three rRNA species, 18S, 5.8S and 28S rRNA, which are then
assembled in the nucleolus with ribosomal proteins and with 5S rRNA in the small and large ribo-
somal subunits (40S and 60S). In the cytoplasm, the 40S and 60S subunits assemble with mRNA
to initiate protein synthesis. More than 200 factors are necessary for ribosome biogenesis.
13.1Introduction
Morphological and functional changes of the nucleolus have been documented in

cancer cells decades ago (reviewed in Ruggero 2012a). From that moment on, a se-
ries of studies have been performed to understand whether these nucleolar changes
were a consequence or a cause of a neoplastic transformation. Recent studies sug-
gest that the process of ribosome biogenesis could be linked to cancer etiology and
pathogenesis (Loreni etal. 2013; Ruggero 2012a). Although outside of the scope
of this work, it is, however, important to take a brief look at the physiology of ribo-
somal biogenesis to be able to better understand its relevance in cancer.
Production of ribosomes is a complex and multistep process that requires a sub-
stantial amount of energy, especially in cycling cells (Lempiainen and Shore 2009)
(see Fig.13.1). The synthesis of ribosomal RNA (rRNA) is the first event in ribo-
some biogenesis. The structural RNA components of the ribosome include the 5.8S,
18S and 28S rRNAs (Fig.13.1), that are encoded by ribosomal DNA (rDNA) which
is organized as expanded chromosome loops in the nucleolus (Fatica and Tollervey
2002). RNA polymerase I transcribes a single 47S rRNA precursor (pre-rRNA), that
13Ribosomes 269
is then processed by the endonucleolytic and exonucleolytic cleavage into 18S, 5.8S
and 28S rRNA (Fatica and Tollervey 2002). Since a ribosome is a ribonucleopro-
tein, it also incorporates various intrinsic proteins, called ribosomal proteins. Thus,
together with the production of rRNA, ribosome biogenesis is also dependent on the
RNA polymerase II -mediated transcription of ribosomal protein genes (Ferreira-
Cerca etal. 2005). Ribosomal proteins are synthetized in the cytoplasm and then
transported to the nucleus where they are assembled together with rRNA into the
small and large ribosomal subunits (40S and 60S).
The function of various ribosomal proteins has been a matter of interest for many
years. Ribosomal proteins constitute a functionally active part of the ribosome
(Ferreira-Cerca etal. 2005), although some of them have been shown to also have
extraribosomal functions (Naora etal. 1998; Warner and McIntosh 2009). Altered,
notably increased, expression of a number of ribosomal proteins (rpS8, rpL12,
rpL23a, rpL27 and rpL30) has been associated with tumor growth (Kondoh etal.
2001; van Riggelen etal. 2010).
It is important to emphasize that mammalian ribosomal proteins are encoded
by a particular class of mRNAs that are called 5 terminal oligopyrimidine (TOP)
mRNAs (Avni etal. 1994) (see Chap.3). All TOP mRNAs present a characteristic
sequence of pyrimidines at the 5 end of the mRNA and exhibit a growth-relat-
ed translation regulation (Caldarola etal. 2009). Importantly, translation of TOP
mRNAs is controlled by the PI3K/AKT/mTORC1 pathway (Hsieh etal. 2012; Sto-
lovich etal. 2002; Thoreen etal. 2012) (see Chap.15). Since this pathway is often
implicated in cancer (Laplante and Sabatini 2012), it is plausible to assume that its
activation would result in upregulation of synthesis of the major components of
ribosome biogenesis.
13.2Alterations of Ribosomal Proteins and Ribosome

Biogenesis Factors in Tumorigenesis: The Role
of MYC Proto-Oncogene
It can be hypothesized that the high functional state of the translation apparatus is
contributory to tumorigenesis. This model finds indirect support from the fact that c-
MYC proto-oncogene controls transcription of ribosomal components, hence its ac-
tivation would lead to an increase in production of ribosomal proteins (van Riggelen
etal. 2010). Consistent with this model, loss of one allele of RPL24 reduced MYC-
induced lymphomagenesis in the E-MYC cancer model system (Barna etal. 2008).
Aside from ribosomal proteins, other proteins that participate in ribosome bio-
genesis could contribute to the altered function of ribosomes and hence tumorigen-
esis. Notably, MYC proto-oncogene also controls expression of these ribosome bio-
genesis factors, such as nucleolin and nucleophosmin (NPM1) (van Riggelen etal.
2010). Nucleolin binds to the 47S pre-rRNA and is required for its cleavage into
18S, 5.8S and 28S rRNA in the nucleolus (Ghisolfi-Nieto etal. 1996), while NPM1
is responsible for the regulation of multiple steps of ribosome biogenesis, including
rRNA processing, ribosomal protein stability and transport of ribosomal subunits
into the cytoplasm (Lindstrom 2011). Therefore, a modest increase in nucleolin or

NPM1 expression is sufficient to increase levels of protein synthesis. Indeed, both
proteins have been found overexpressed in a wide array of tumors and might have
direct consequences for tumor progression that is initiated by MYC (Coller etal.
2000). Alterations (such as mutation or aberrant localization) of NPM1 are com-
monly found in a number of hematopoietic malignancies, such as acute promyelo-
cytic leukemia, anaplastic large cell lymphoma, AML, and myelodysplasia (Estey
2013; Sportoletti etal. 2008).
While MYC proto-oncogene asserts its tumorigenic function via various mecha-
nisms, it is important to emphasize that, as discussed in previous chapters, c-MYC
also regulates transcription of translation factors, with the most notorious example
being eIF4E. eIF4E in turn controls c-MYC protein expression at the translational
level (van Riggelen etal. 2010). Thus, in a simple model, an increase of ribosomal
proteins and ribosomal biogenesis would lead to upregulation of translation and to
a protumorigenic state. However, the situation appears to be more complex and, as
described in the following paragraph, it is now becoming evident that at least some
of the components of ribosomal machinery (such as ribosomal proteins, dyskerin
and SBDS) could behave as tumor suppressors.
13.3Alterations of Components of Ribosomal

Machinery: Congenital Ribosomal Abnormalities
and Cancer
An observation that loss of ribosomal proteins increases cancer risk came from
zebrafish studies in which heterozygosity of several ribosomal proteins, such as
rpS7, rpS8, rpS15a, rpS18, rpS29, rpL7, rpL13, rpL23a, rpL35, rpL36 and rpL36a,
resulted in a high incidence of tumor formation (Amsterdam etal. 2004). Similar re-
sults have been obtained from patients with congenital ribosomal abnormalities. For
instance, DiamondBlackfan anemia (DBA) is a rare hematological disease, char-
acterized by a defective maturation of the erythroid progenitor cells (Dianzani and
Loreni 2008; Ellis and Lipton 2008). A recent quantitative analysis of a cancer risk
indicated that DBA is a malignant predisposition syndrome (Vlachos etal. 2012).
In fact, DBA patients have been shown to have significantly increased incidence of
both hematologic malignancies and solid tumors (Vlachos etal. 2012). This genetic
disease is characterized by loss of function mutations in any one of eleven ribosomal
protein genes, such as RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPL5, RPL11,
RPL15, RPL26 and RPL35A (Cmejla etal. 2007; Doherty etal. 2010; Draptchins-
kaia etal. 1999; Farrar etal. 2008; Gazda etal. 2006, 2008, 2012; Landowski etal.
2013). For this reason DBA has been defined as a ribosomopathy (Luft 2010). As
to why only a subset of ribosomal proteins causes this disease is not understood (for
more specific reviews see Ellis and Gleizes 2011; Narla and Ebert 2010).
Erythroid abnormalities similar to DBA can be observed in 5q- syndrome. This
disease is a subtype of myelodysplastic syndrome (MDS) that is a heterogeneous
13Ribosomes 271
group of hematological disorders characterized by defective hematopoiesis and in-

creased susceptibility to AML (Vardiman etal. 2002). Ebert etal. showed that hap-
loinsufficiency of the RPS14 gene is the cause of the erythroid failure observed in
the syndrome (Ebert etal. 2008).
Another link between ribosomal protein alterations and cancer has been recently
reported in a large scale sequencing study of T-cell acute lymphoblastic leukemia
(T-ALL) (De Keersmaecker etal. 2013). In this study, authors identified RPL5 and
RPL11 mutations in about 10% of pediatric T-ALL, further supporting a notion that
ribosomal proteins play an important role in tumorigenesis.
In addition to ribosomal proteins, more than 200 biogenesis factors are required
to complete ribosomal subunit assembly (Henras etal. 2008). In principle, altera-
tion in any of these factors could have a tumorigenic effect similar to ribosomal
protein mutations. Indeed, a number of correlations have been reported between
alterations of nucleolar proteins involved in ribosome synthesis and tumorigenesis
(Boocock etal. 2003; Estey 2013; Kirwan and Dokal 2008; Sportoletti etal. 2008).
Some of these examples stem from studies of ribosomopathies and are summarized
in Table 13.1 One such congenital ribosomal abnormality that is also considered
a cancer susceptibility syndrome is a ribosomopathy called Shwachman-Bodian-
Diamond syndrome (SDS). SDS patients have an increased risk of developing se-
vere aplastic anemia or MDS and also have a predisposition to AML (Raaijmakers
etal. 2010). SBDS gene is found mutated in SDS (Boocock etal. 2003). It has been
shown in the yeast model that the SBDS protein has a role in the 60S ribosomal
subunit maturation, subunit joining and translational activation (Menne etal. 2007).
Interestingly, point mutations of translation initiation factor eIF6 rescue the loss of
function of SBDS in several models (Menne etal. 2007; Wong etal. 2011), and SDS
patients with MDS often have loss of heterozygosity at 20q11, that is the locus of
eIF6 gene (Pressato etal. 2012) (see Chap.11).
Another example is represented by dyskerin (dyskeratosis congenita 1 (DKC1))
mutations associated with dyskeratosis congenita, an X-linked disease character-
ized by premature ageing, bone marrow failure, hyperkeratosis of the skin, and a
high risk of developing bone marrow malignancies (Alter etal. 2009; Kirwan and
Dokal 2008). DKC1 encodes dyskerin, a pseudouridine synthase that converts spe-
cific uridine residues to pseudouridine during the maturation of ribosomal RNAs.
The defect in rRNA modifications impairs translation efficiency of a subset of
mRNAs (Loreni etal. 2013; Ruggero 2012b). These mRNAs are characterized by
the presence of internal ribosome entry sites, or IRES, which facilitate translation in
a cap-independent manner (see Chap.3 ). IRES-driven mRNAs are translated in a
cap-independent way during stress and some of them encode for tumor suppressors
such as p53 or p27 (Bellodi etal. 2010a, b). Therefore, alterations in the physiologi-
cal modifications of rRNA could, as presented in this case, via changes in transla-
tion of a specific set of mRNAs, contribute to tumorigenesis.
While our understanding of the complexities behind the etiology of these ribo-
somopathies is still in its infancy, data from studies in these congenital conditions
suggest a potentially important role of proteins participating in ribosomal biogen-
esis in the control of cell growth and proliferation.
Table 13.1 Correlation between ribosome biogenesis components and tumorigenesis.

Function in
Associated Cancer
Altered Gene Ribosome Clinical Features References
Disease Association
Biogenesis
RPS7, RPS10,
MDS, AML, colon (Da Costa et al.,
RPS17, RPS19,
Diamond- macrocytic anemia, adenocarcinoma, 2010; Narla and
RPS24, RPS26, Ribosomal
Blackfan reticulocytopenia, physical osteogenic Ebert, 2010;
RPL5, RPL11, protein
anemia abnormalities sarcoma, genital Vlachos et al.,
RPL15, RPL26,
cancer 2012)
RPL35A
Ribosomal
RPS14 5q- syndrome macrocytic anemia AML (Ebert et al., 2008)
protein
skin hyperpigmentation,
X-linked AML, head and (Alter et al., 2009;
rRNA nail dystrophy and mucosal
DKC1 dyskeratosis neck tumors Kirwan and Dokal,
processing leucoplakia
congenita 2008)
bone marrow failure
(Boocock et al.,
Shwachman-
pancreatic insuficiency, 2003; Burroughs
Large subunit Bodian-
SBDS impaired hemopoiesis, MDS, AML et al., 2009;
biogenesis Diamond
physical abnormalities Ganapathi and
syndrome
Shimamura, 2008)
Transport of
NPM1 pre- NA NA AML (Falini et al., 2005)
ribosomes
rRNA (Chung et al.,
BOP1 NA NA HCC
processing 2011)
AML - acute myeloid leukemia, HCC - hepatocellular carcinoma, MDS - myelodysplastic syndrome, NA -
not available
13.4Ribosomal Alterations and p53 Tumor Suppressor
A body of evidence accumulated during the last decade shows that alterations in
ribosomal protein expression can induce cell cycle arrest or apoptosis through ac-
tivation of the tumor suppressor p53 (Miliani de Marval and Zhang 2011). It was
suggested that the mechanism of p53 activation after perturbation of ribosome
biogenesis was the consequence of a nucleolar stress that triggers a ribosomal
protein/MDM2/p53 stress response pathway (Fig.13.2). MDM2 is an E3 ubiquitin
ligase that binds p53 and promotes its ubiquitination and degradation. As a conse-
quence of altered ribosome biogenesis, several ribosomal proteins, such as rpL5,
rpL11, rpL23 and rpS7, bind to MDM2 and relieve its inhibitory activity toward
p53, thus inducing p53 stabilization (Deisenroth and Zhang 2010). For instance,
knockdown of RPS6, RPS9 or RPL29 in human cell lines has been shown to lead to
p53 activation (Fumagalli etal. 2009; Lindstrom and Zhang 2008; Liu etal. 2006).
Disruption of rRNA synthesis is also sufficient to activate the ribosomal protein/
MDM2/p53 response. This conclusion was reached by manipulating rRNA produc-
tion experimentally with drugs that inhibit the activity of RNA polymerase I, such
as actinomycin D (Bhat etal. 2004), 5-flourouracil (Sun etal. 2007) or mycophe-
nolic acid (Sun etal. 2008). In all cases, enhanced association of ribosomal proteins
(rpL5, rpL11, rpL23) with MDM2 activates p53 by stabilizing it (Bhat etal. 2004;
Sun etal. 2007, 2008).
13Ribosomes 273
Fig. 13.2 The mechanism of ribosomal stress. Defects of ribosome biogenesis lead to the block
of the cell cycle or apoptosis through p53-dependent and p53-independent pathways. Red crosses
indicate steps that may be affected. See text for details.
Other examples of ribosomal stress involve ribosome biogenesis factors. For

instance the block of proliferation 1 protein (BOP1), that is a nucleolar protein
involved in rRNA processing, has been shown to cooperate with p53 in regulating
the G1/S transition. Indeed, a study observed that the expression of a dominant-
negative form of BOP1 in mouse cells, impairs 28S and 5.8S rRNA synthesis, thus
leading to defective large ribosomal subunit assembly and p53-dependent inhibition
of the cell cycle progression (Strezoska etal. 2000). Additional work shed light on
the effect of mutational inactivation of RNA-binding motif protein 19 (RBM19) on
the activation of the ribosomal protein/MDM2/p53 pathway. Mutations in RBM19,
which encodes for a protein contributing to rRNA processing, has been shown to in-
crease p53 activity in the developing embryo, subsequently blocking development
beyond the morula stage because of increased apoptosis (Zhang etal. 2008). The
significance of p53 activation through disruption of rRNA processing has been sub-
stantiated in a zebrafish model, where deficiency of wdr36, a protein essential for
processing of 18S rRNA, was found to activate a p53-dependent response (Skarie
and Link 2008).
On the other hand, a model of p53-independent response to ribosomal stress
(Iadevaia etal. 2010) have been put forward (Fig.13.2). In fact, Iadevaia and
c olleagues showed that in cultured erythroid cells K562, the depletion of one of
a number of ribosomal proteins causes a block in cell cycle and proliferation even
in the absence of p53 (Iadevaia etal. 2010). This is possibly due to the activation
of a ribosomal stress response which causes increased proteasome degradation of
the PIM1 kinase and stabilization of the cell cycle inhibitor p27kip (Iadevaia etal.
2010). Moreover, it has been shown that inhibition of p53 activity did not rescue the
defect in erythroid progenitors in rpS19-deficient zebrafish, suggesting that the he-
matopoietic phenotype is due to a p53-independent response (Torihara etal. 2011).
Ribosome biogenesis is a pivotal task for the cell that dedicates a considerable
amount of energy to this process. The quantity of ribosomes produced appears to
correlate with cell growth rate. The observation of increased ribosome synthesis
in cancer cells is consistent with this notion. An important question is where, in
the sequence of events that lead to cancer, we can place the increase of ribosome
biogenesis: is it a simple downstream effect of tumorigenic transformation or is it
one of the initial causes or both? Given the variety of tumorigenesis mechanisms
the two models are probably both true. In fact, there are studies that support the hy-
pothesis that an oncogenic factor, such as MYC, generates a series of effects among
which there is an increase in ribosome synthesis. However, a number of reports
on the genetic diseases called ribosomopathies, taught two important lessons: (1)
ribosome synthesis (and possibly function) is supervised by quality control mecha-
nisms, which include p53, and (2) haploinsufficiency of ribosomal proteins or ribo-
some biogenesis factors are associated to (and possibly cause) different kinds of
cancerous or precancerous phenotypes. This seems to indicate that loss of function
mutations in ribosome components (including biogenesis factors) can be causative
in terms of cancer development. This hypothesis could appear to contradict the
above stated requirement of ribosomes for tumor cell growth. However, on the basis
of the scarce but accumulating evidence, at least three general mechanisms can be
envisioned: (1) growth and proliferation impairment caused by ribosomal protein
(or biogenesis factor) mutations favors the selection of additional tumorigenic mu-
tations or alterations. Consistent with this hypothesis, cells derived from zebrafish
tumors caused by ribosomal protein mutations, were not able to synthesize a tumor
suppressor p53 protein (Amsterdam etal. 2004). (2) Defect in ribosome biogenesis
causes alteration in the translation pattern of the cell causing an increased synthesis
of tumorigenic proteins. In addition to the aforementioned case of DKC1 mutations,
changes in the polysomes-associated mRNAs have been detected after depletion
of rpS19 or rpL11 in mouse erythroblasts (Horos etal. 2012). These mRNAs, in-
cluding ones that are coding for Bag1 and Csde1 proteins that are thought to be
translated via IRES, are important for hematopoiesis (Horos etal. 2012). Whether
these alterations can favor tumorigenesis, however, has not been addressed. (3) De-
ficiency of ribosome production or function could be advantageous for tumor cell
13Ribosomes 275
survival. This possible mechanism likely has similarities observed while studying
the function of eEF2K in cell response to nutrient stress (Leprivier etal. 2013).
Leprivier and colleagues showed that survival of cancer cells depends on the pres-
ence of eEF2K that, by phosphorylating eEF2, inhibits translation elongation in
response to nutrient deprivation. Therefore, it appears that inefficient or decreased
translation, that is a physiological cell response to different stress stimuli, can also
facilitate the survival of a cancer cell.
The three models are obviously not necessarily alternative to each other and
could coexist or act sequentially in different cases of cancer development. In sum-
mary, from the aforementioned studies, the relationship between ribosomal function
and cancer biology appears complex and dual: from one side oncogenic factors can
induce an increase in ribosome synthesis; on the other hand defective ribosome pro-
duction can induce oncogenesis through not yet clarified mechanisms. If and how
these two aspects can be reconciled will be the subject of future studies.
Acknowledgements The financial support of Telethon, Italy (Grant No. GGP13177 to FL) is
gratefully acknowledged. Work in our laboratories has been generously funded also by AIRC
(IG14756 to FL) and PRIN (20104AE23N to FL).
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Chapter 14
Current and Emerging Therapies Targeting
Translation
Gabriela Galicia-Vzquez and Jerry Pelletier
Contents
14.2Targeting eIF4F as an Antineoplastic Approach 280
14.3Targeting eIF4F Function 282
14.4Suppression of eIF4E Activity 284
14.5Targeting eIF4E Phosphorylation 285
14.6Interfering with eIF4E/eIF4G Interaction 286
14.7Targeting eIF4EmRNA Recognition 286
14.8Targeting eIF4A Helicase Activity 287
14.8.1Hippuristanol 287
14.8.2Pateamine A 288
14.8.3Rocaglamides 289
14.9Targeting Other Components of Translation Initiation 291
14.10Targeting Translation Elongation as an Antineoplastic Approach 292
14.10.1Aplidine 292
14.10.2Omacetaxine Mepesuccinate 292
14.11Targeting Translation to Sensitize Tumor Cells to Standard-of-Care Therapeutics 294
14.12Perspectives 294
References 295
Abstract The highly regulated process of protein synthesis is frequently dysregu-

lated in cancers. In nontransformed cells, one of the primary control points occurs
when ribosomal subunits are recruited to mRNA templates. This process is lim-
ited by the availability of the trans-acting factor, eIF4F, a heterotrimeric complex
composed of the eIF4E cap-binding protein; the scaffold protein, eIF4G; and the
J.Pelletier() G.Galicia-Vzquez
Department of Biochemistry, McGill University, Montreal, QC, Canada
e-mail: jerry.pelletier@mcgill.ca
J.Pelletier
Rosalind and Morris Goodman Cancer Research Centre, McGill University,
280 G. Galicia-Vzquez and J. Pelletier
ATP-dependent DEAD box helicase eIF4A. As the eIF4F checkpoint is under regu-
lation of the PI3K/AKT/mTOR cascade and its subunits are transcriptional targets
of MYC, this step is often dysregulated in human cancers. In this review, we discuss
the development, mode of action and biological activity of small-molecule inhibi-
tors that interfere with eIF4F activity and their potential for clinical development.
14.1Introduction
A major impetus of current cancer research is to uncover vulnerabilities in gene

networks to which tumor cells have become dependent for their growth and survival
(Felsher 2004). The PI3K/AKT/mTOR pathway is among these, as it is dysregulated
in a large proportion of cancer cells leading to a series of pleiotropic effects includ-
ing dysregulated protein synthesis (Hay and Sonenberg 2004). A second vulnerabil-
ity that is also a major regulator of translation and a high profile anticancer target,
the MYC oncogene, that is frequently amplified in human tumors (Beroukhim etal.
2010) and is a transcriptional regulator of eIF4E, eIF4AI, and eIF4G (Jones etal.
1996; Lin etal. 2008). Here, we discuss therapeutic strategies aimed at targeting
translation as an antineoplastic approach, with an emphasis on the eIF4F complex,
the rate-limiting initiation complex involved in the ribosome recruitment phase of
translation initiation.
14.2Targeting eIF4F as an Antineoplastic Approach
The eIF4F complex consists of eIF4E, an m7GpppN cap-binding protein; eIF4A,

an RNA DEAD box helicase responsible for unwinding proximal RNA secondary
structure; and eIF4G, a large scaffolding protein, which mediates the binding of
eIF4F to the 43S ribosomal complex through its interaction with eIF3 (Hinnebusch
and Lorsch 2012). The role of the eIF4F complex is to recruit small ribosomal
subunits and associated factors (43S PICs) to the 5 end of mRNAs. As it turns out,
eIF4E is the least abundant initiation factor making cap-dependent initiation the
rate-limiting step of translation (Duncan etal. 1987; Galicia-Vazquez etal. 2012).
Features that affect initiation rates include cap accessibility, the degree of cap-prox-
imal secondary structure, and protein-mRNA complexes (Babendure etal. 2006;
Goossen etal. 1990; Lawson etal. 1986; Pelletier and Sonenberg 1985a, b).
The eIF4A helicase subunit exhibits bidirectional unwinding activity and its
delivery to the mRNA template by eIF4F imparts 53 directionality. The eIF4A
helicase is not required for eIF4E cap-recognition but is needed for initiation on
mRNAs with structured 5 UTR (Pestova and Kolupaeva 2002; Sonenberg 1981).
It is thought to prepare a landing pad for the incoming 43S PIC by unwinding RNA
secondary structure and/or disrupting protein-RNA interactions. eIF4A has been
observed to crosslink to RNA up to 50 nucleotides downstream of the cap structure
14 Current and Emerging Therapies Targeting Translation 281
mTOR
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Fig. 14.1 Schematic diagram illustrating regulation of eIF4F assembly by mTOR. Shown are
sites of interdiction of various translation inhibitors described in this chapter (see text for details).
and so may unwind sequences distal to the cap structure (Lindqvist etal. 2008a).
There are two eIF4A isoforms, eIF4AI and eIF4AII (or DDX2A and DDX2B re-
spectively) that share 90% sequence identity at the amino acid level and although in
vitro they appear functionally indistinguishable (Yoder-Hill etal. 1993), in vivo they
harbor some distinct activities (Galicia-Vazquez etal. 2012; Meijer etal. 2013). The
limiting amounts of eIF4F implies that different mRNA templates must compete
for this factoran event where secondary structure within the 5 UTR can have a
significant impact (with increased thermal stability being associated with inefficient
ribosome loading) (Babendure etal. 2006; Lawson etal. 1986; Pelletier and Sonen-
berg 1985a, b; Pestova and Kolupaeva 2002; Shah etal. 2013; Svitkin etal. 2001).
Elevated levels of eIF4E are oncogenic (Lazaris-Karatzas etal. 1990; Ruggero
etal. 2004; Wendel etal. 2004). This is thought to be due to a disproportionate stim-
ulation of translation of inefficient mRNAs, some of which encode for prosurvival
and proliferative functions (Sonenberg and Hinnebusch 2007). eIF4F assembly is
under mTOR regulation via two mechanisms (Fig.14.1). Under conditions of low
translation, eIF4E is prevented from binding to eIF4G due to its association with
one of three eIF4E-binding proteins (4E-BP 1, 2 and 3). Stimulation of PI3K/AKT/
mTOR signaling results in phosphorylation of 4E-BP and in the release of eIF4E,

allowing eIF4E to associate with eIF4G (Gingras etal. 2001). As well, the product
of the tumor suppressor gene, PDCD4, associates with eIF4A and prevents its bind-
ing to eIF4G, leading to suppression of translation (Yang etal. 2003a). Phosphory-
lation of PDCD4 by S6K1, a downstream mTOR effector, leads to degradation of
PDCD4 enabling eIF4A to interact with eIF4G (Dorrello etal. 2006) (Fig.14.1).
The discovery that eIF4F assembly is under mTOR regulation has lead to the idea
that drugging this step may be a viable approach to curtail the oncogenic ef-
fects of translation activation seen upon increased PI3K/AKT/mTOR signaling flux
(Hay and Sonenberg 2004). Since the repertoire of mRNAs translated in a nontrans-
formed cell differ significantly from those of a transformed cell, this approach could
offer a greater therapeutic window compared to what is achievable by using global
inhibitors of translation, since the former would selectively inhibit expression of
mRNAs that encode products involved in cell survival and cycle progression.
14.3Targeting eIF4F Function
One way to block eIF4F activity is to prevent its assembly by targeting the PI3K/
AKT/mTOR signaling pathway. The role of the PI3K/AKT/mTOR pathway in tu-
mor maintenance has been extensively studied and several inhibitors targeting this
pathway are currently being assessed as antineoplastic agents. An overview of this
pathway and the inhibitors under study are beyond the scope of this chapter, but the
reader is referred to excellent recent reviews on this topic (Benjamin etal. 2011;
Burris 2013; Feldman and Shokat 2010; Malina etal. 2012).
There are several shortcomings with strategies aimed at targeting components of
the PI3K/AKT/mTOR pathway. Firstly, it is not always apparent whether the bio-
logical effects seen with inhibitors of this signaling branch are a direct consequence
of interfering with translation or due to the effects on other processes controlled
by these kinases. Secondly, resistance mechanisms to rapamycin and PI3K/mTOR
kinase inhibitors can arise due to elevated eIF4E (and MYC) levels (Ilic etal. 2011;
Wendel etal. 2004, 2006). These results have two implications: they indicate that
the major effector mechanism of these targeting strategies may lie in inhibiting
translation and that a cell can develop resistance to PI3K/AKT/mTOR inhibitors by
elevating eIF4E levels. Lastly, strategies aimed at inhibiting mTOR (i.e. rapamycin
and rapalogs) weaken an mTOR/S6K/IRS-1 (insulin receptor substrate 1) negative-
feedback loop, leading to an unwanted increased signaling flux through the insulin
receptor. Rapamycin treatment failure has been attributed to the dampening of this
negative feedback loop (OReilly etal. 2006). A strategy that directly inhibits trans-
lation initiation by curtailing eIF4F activity would be expected to exhibit less off-
target effects, leave the afore-mentioned feedback loop intact, and avoid potential
eIF4E-based resistance mechanisms.
In this review, we focus on small molecules that have been shown to directly act
on the eIF4F complex (see Table14.1). Early studies provided proof-of-concept
Table 14.1 Development status of compounds targeting eIF4F.

Target/compound Mode of action Stage of development References/notes
eIF4E
4EGI-1 analogs Inhibits eIF4E/eIF4G Preclinical Chen etal. (2012a),
interaction Moerke etal. (2007)
4ERCat1; Inhibits eIF4E/eIF4G Preclinical Cencic etal. (2011b)
4ERCat2 interaction
4EASO4 (aka Antisense oligonucleotide Clinical phase I Graff etal. (2007), Hong
LY2275796) to eIF4E etal. (2011)
4Ei-1 Cap analog prodrug Preclinical Li etal. (2013)
Cmpd 33 Cap analog Tool compound Chen etal. (2012b)
eIF4A
Pateamine A/ Binds to free eIF4A, Preclinical Bordeleau etal. (2005),
Des-amino sequestering it from the Kuznetsov etal. (2009)
pateamine A eIF4F complex Irreversible MOA
Hippuristanol Inhibits eIF4A/RNA Preclinical Bordeleau etal. (2006b),
interaction Cencic etal. (2013)
Silvestrol, Chemical inducer of Preclinical Bordeleau etal. (2008),
rocaglamide dimerization. Engages Cencic etal. (2009),
hydroxamates eIF4A/RNA inter- Lucas etal. (2009),
actions, depleting Rodrigo etal. (2012)
eIF4A from the eIF4F
complex
MNK
CGP57380 MNK inhibitor Tool compound Knauf etal. (2001); Short
half-life in vivo
Cercosporamide MNK Inhibitor, MNK1 Preclinical Konicek etal. (2011);
(IC50=160 nM); JAK3 (IC50=31 nM)
MNK2 (IC50=11 nM)
on the value of blocking eIF4F-dependent initiation to impact tumor cell mainte-

nance and survival. Overexpression of 4E-BP1 in transformed NIH 3T3 cells led
to a significant reversion of the phenotype (Rousseau etal. 1996). Expression of a
nonphosphorylatable mutant of 4E-BP1 in MCF-7 breast cancer (Jiang etal. 2003)
or Rat-1 (Lynch etal. 2004) cells inhibited proliferation ex vivo and curtailed eIF4E
or c-MYC-driven transformation ex vivo and in vivo in nude mice (Lynch etal.
2004). Fusing the eIF4E-binding segment of eIF4G1 to the cell permeable TAT
peptide inhibited cap-dependent translation and triggered apoptosis in several cell
lines (Brown etal. 2011), providing additional validation for eIF4E as an anticancer
target. Coupling a 4E-BP-like peptide to a gonadotropin-receptor agonist showed
impressive antineoplastic activity following intraperitoneal delivery in an ovarian
xenograft model with no detectable toxicity (Ko etal. 2009).
Strategies to curtail oncogenesis by targeting eIF4A have shown similar out-
comes. Antisense RNA directed to eIF4AI or overexpression of PDCD4 blocked
transformation ex vivo (Eberle etal. 2002; Yang etal. 2003b) and delayed tumor
progression in a chemically-induced murine skin tumor model (Jansen etal. 2005).
A recent approach used a folated poly(ethylene glycol)-chitosan-graft-polyethyl-
enimine carrier to deliver the PDCD4 gene into HRAS12V derived liver cancer-
burdened mice and demonstrated a reduction in tumor size (Kim etal. 2013). Such
studies highlight the importance of high throughput screens and medicinal chem-
istry efforts aimed at finding small molecules that could selectively interfere with
eIF4F activity ex vivo and in vivo in preclinical models (Cencic etal. 2007a; Moerke
etal. 2007; Novac etal. 2004).
The mechanism by which reduced eIF4F activity affects transformation is
thought to be due to selective inhibition of initiation of a limited set of mRNAs
required for tumor cell maintenance and/or survival. mRNAs that have been identi-
fied as being eIF4F-responsive encode for pro-oncogenic, proproliferative, proan-
giogenic and antiapoptotic proteins, such as c-MYC, cyclin D1, ODC, MMP-9,
FGF2, VEGF, TGF-, BCL-2, BCL-XL and MCL-1. Several of these transcripts
encode proteins with short half-lives (e.g. MCL-1, cyclin D1 and ODC), placing a
demand on tumor cells to maintain a constant translational output of these mRNAs.
The idea that targeting a general translation initiation factor could have mRNA
class-specific effects is not new and was proposed over 35 years ago (Bergmann
and Lodish 1979; Lodish 1974).
14.4Suppression of eIF4E Activity
Suppressing eIF4E expression is an effective strategy to reduce eIF4F complex lev-

els since the former is the least abundant of all translation factors (0.05 copies per
ribosome) (Duncan etal. 1987; Galicia-Vazquez etal. 2012). Graff and colleagues
have shown impressive antineoplastic activity upon targeting eIF4E expression us-
ing antisense oligonucleotides (4EASO) (Graff etal. 2007). An ~80% knockdown
of eIF4E was obtained with ASOs while only marginally affecting global protein
synthesis (20% change). This produced a dose-dependent decrease in expression
of eIF4E-responsive prosurvival genes, and also suppressed tumor growth in breast
and prostate xenograft models by eliciting apoptosis (Graff etal. 2007). In addi-
tion, the eIF4E ASOs also inhibited endothelial cell tube formation suggesting a
possible antiangiogenic activity (Graff etal. 2007). Recently, the use of 4EASO
in three mesothelioma cells inhibited proliferation and caused apoptosis. Further-
more, the 4EASO was able to synergize with gemcitabine and pemetrexed (Jacob-
son etal. 2013). A phase I dose escalation study demonstrated that one eIF4E ASO,
LY2275796, was well tolerated in patients with minor toxicities documented (fa-
tigue, nausea, fever, vomiting, prolongation of activated partial thromboplastin and
prothrombin time, and thrombocytopenia) (Hong etal. 2011). Expression of two
eIF4E-responsive transcripts, VEGF and cyclin D1, were found reduced in some
(5360%) patients (Hong etal. 2011).
Suppression of eIF4E at the organismal level appears to be well tolerated (Graff
etal. 2007) and has also been modeled in the mouse (Lin etal. 2012). By taking
advantage of a newly developed RNAi platform that mimics pharmacological tar-
geting in vivo, mice in which eIF4E can be inducibly and reversibly suppressed by
shRNAs have been developed (Lin etal. 2012). eIF4E suppression was well toler-
ated in sheIF4E mice, with gastrointestinal degeneration appearing after 34 weeks

of chronic eIF4E suppressionan effect that could be readily reversed by allowing
eIF4E levels to recover. In addition, when bred to mice bearing an E-MYC allele,
a translocation that leads to non-Hodgkins lymphoma in the mouse (Adams etal.
1985), suppression of eIF4E at the organismal level significantly delayed sporadic
tumor onset (Lin etal. 2012). The effects of eIF4E suppression on cell survival
appeared to be lethal with high MYC expression (Lin etal. 2012) indicating that
tumors with elevated MYC levels may be responsive to eIF4E suppression.
14.5Targeting eIF4E Phosphorylation
eIF4E is phosphorylated on a single residue, Ser209, by two kinases: the MAPK-

interacting kinases MNK 1 and 2 (Pyronnet etal. 1999). The consequence of this
phosphorylation event on eIF4E/eIF4F activity appears to be stimulation of activity
(Beretta etal. 1998). The absence of MNK 1 and 2 in the mouse leads to a complete
loss of eIF4E phosphorylation and has no discernible phenotype (Ueda etal. 2004)
although eIF4E phosphorylation appears essential for its oncogenic activity (Furic
etal. 2010; Topisirovic etal. 2004; Wendel etal. 2007). These results have fueled
interest in developing MNK inhibitors as potential antineoplastic agents. A small
molecule inhibitor of the MNK kinases, CGP57380, that prevents phosphorylation
of eIF4E has been described (Knauf etal. 2001), but unfortunately is not effective in
vivo. Subsequently, cercosporamide was identified from a high-throughput screen-
ing and was shown to inhibit MNK1 (IC50=160nM) and MNK2 (IC50=11nM).
Cercosporamide had sufficiently favorable pharmacological properties to allow for
its testing in vivo (Konicek etal. 2011) and in colon HCT116 and B6 melanoma
metastasis models was found to reduce tumor progression and incidence. Cerco-
sporamide has also been used in combination with cytarabine resulting in an en-
hanced antileukemic response in a xenograft mouse model (Altman etal. 2013).
Recently, a series of cercosporamide analogs have been synthesized exhibiting anti-
proliferative effects against two human NSCLC cell lines (Bazin etal. 2013).
To support the notion that the effect of MNK inhibition on tumorigenesis is due
to loss of eIF4E phosphorylation, mice were engineered with a nonphosphorylat-
able allele of eIF4E, Ser209Ala. Cells from these mice are relatively more resistant
to oncogenic transformation by activated RAS than their wild-type counterparts.
When crossed to mice in which PTEN was selectively deleted in the prostate, the
development of invasive prostate neoplasia was significantly attenuated in mice
harboring the Ser209Ala allele (Furic etal. 2010). The MNK-eIF4E axis has also
been proposed as a therapeutic target in CML to inhibit leukemia stem cell func-
tionan effect that was associated with inhibition of -catenin (Lim etal. 2013).
14.6Interfering with eIF4E/eIF4G Interaction
The eIF4E-binding site on eIF4G has been well characterized and consists of a
Y(X)4LF motif (where X is any amino acid and F is hydrophobic) (Marcotrigiano
etal. 1999). This motif is also present in the 4E-BPs and binding of eIF4E to eIF4G
or 4E-BPs is mutually exclusive. A fluorescent polarization-based assay involving a
labeled eIF4G-like peptide and recombinant eIF4E identified 4EGI-1, a compound
capable of inhibiting expression from eIF4E-dependent transcripts and inducing cell
death in several tumor cell lines (Moerke etal. 2007). Whereas 4EGI-1 inhibited
eIF4E/eIF4G interaction, it enhanced eIF4E/4E-BP1 association. 4EGI-1 was also
shown to induce apoptosis in human lung cancer cells and cooperated with TRAIL
in this event, although the effect appeared dependent on an endoplasmic reticulum
stress, CHOPdependent mechanism and not on inhibition of cap-dependent trans-
lation (Fan etal. 2010). In a zebrafish model of engrafted AML tumor cells, 4EGI-1
showed teratogenic activity, precluding its assessment for antineoplastic activity
in vivo (Pruvot etal. 2011). 4EGI-1 was shown effective at inducing apoptosis in
multiple myeloma cells through a NOXA-dependent mechanism (Descamps etal.
2012) and at inhibiting growth of breast and melanoma cancer xenografts without
any apparent overt toxicity (Chen etal. 2012a). The mechanism of action of 4EGI-1
appears complex and experiments demonstrating its ability to restore sensitivity to
the BCL-2 family inhibitor, ABT-737, revealed a cap-dependent and cap-indepen-
dent mode of action (Willimott etal. 2013). Recently, elevated translation rates
have been associated with autism in a murine model and intracerebroventricular
infusions of 4EGI-1 were found to correct the autistic-like behavior (Santini etal.
2013). In another setting, a reduction in the accumulation of disease-associated pri-
on protein PrPSc was noted upon exposure of cells to 4EGI-1 (Allard etal. 2013).
Using a time-resolved fluorescence resonance energy transfer based assay, Cen-
cic etal. (2007b, 2011a, b) also identified eIF4E/eIF4G inhibitors, named 4E1RCat
and 4E2RCat. These blocked cap-dependent translation and inhibited association
between eIF4E and eIF4G or 4E-BP1. In the E-MYC mouse lymphoma mod-
el, 4E1RCat improved chemotherapy response and increased tumor-free survival
when combined with the DNA-damaging agent, doxorubicin (Cencic etal. 2011b).
Although these compounds have proven to be useful tool compounds, medicinal
chemistry programs are now required to improve their selectivity and potency.
14.7Targeting eIF4EmRNA Recognition
The cap-binding properties of eIF4E have been extensively studied (Adams etal.
1978; Cai etal. 1999; Darzynkiewicz etal. 1981, 1987, 1989; Grudzien-Nogalska
etal. 2007a, b; Hickey etal. 1977; Jemielity etal. 2010; Kowalska etal. 2008;
Su etal. 2011) and much information on structure-activity relationships has been
obtained. The greatest limitation of these cap analog compounds is their poor cell
permeability (due to the presence of phosphate groups that are required for potency)
and poor stability in cell culture studies (Jemielity etal. 2010; Wagner etal. 2000).
In an attempt to overcome these shortcomings, Ghosh etal. (2009) synthesized
phosphoramidate derivatives of m7GTP. These showed increased half-life in plas-
ma, were nontoxic and water-soluble. One compound, a tryptamine phosphorami-
date prodrug of 7-benzyl-GMP (4Ei-1), inhibited cap-dependent translation in vitro
and ex vivo upon injection into fertilized zebrafish eggs (Ghosh etal. 2009). 4Ei-1
is a substrate for histidine triad nucleotide binding protein-1, which is responsible
for its conversion to the corresponding nucleoside monophosphate. 4Ei-1 is cell
permeable and has been shown to chemosensitize breast and lung cancer cells to
gemcitabine (Li etal. 2013).
A different series of cap analogs lacking the characteristic ribose, phosphates,
and positive charge on the nucleoside ring have also been generated and shown
to inhibit cap-dependent translation in vitro with an IC50=~2.5M (Chen et al.
2012b). Unfortunately, these compounds showed limited cell permeability and con-
sequently could not be tested ex vivo. One issue that will have to be addressed with
these series of compounds is whether they also affect the activity of other cellular
cap-binding proteins (i.e. CBP20/80 and Dcp2) implicated in nuclear cap function
and mRNA turnover.
14.8Targeting eIF4A Helicase Activity
The RNA helicase eIF4A is the enzymatic subunit of the eIF4F complex. A trans-
lation screen of >300,000 compounds using a bicistronic mRNA containing a
cap- and IRES-dependent reporter led to the identification of three compounds that
inhibited exclusively cap-dependent translation: pateamine A, silvestrol, and hip-
puristanol (Bordeleau etal. 2005, 2006b, 2008). These natural products all perturb
cap-dependent translation by interfering with eIF4A activity and are currently being
explored as chemotherapeutic agents.
14.8.1Hippuristanol
Hippuristanol inhibits the interaction of eIF4A with RNAthus impairing eIF4A

RNA-dependent ATPase activity, its helicase activity, and inhibiting eIF4F activity
(Bordeleau etal. 2006b). The hippuristanol binding site has been mapped to the
eIF4A C-terminal domain and is considered an allosteric inhibitor (Lindqvist etal.
2008b). The hippuristanol-binding site is not conserved among other DEAD box
helicases, thus making this compound a selective inhibitor of eIF4AI and eIF4AII.
eIF4AIII or DDX48, a DEAD box helicase implicated in splicing and nonsense-
mediated decay, has six amino acid mismatches in the hippuristanol-binding site
and shows a tenfold higher IC50 compared to eIF4AI/II (Lindqvist etal. 2008b).
To date, extensive pharmacokinetic studies have not been performed with hip-
puristanol and in vivo studies are limited due to solubility issues. Routes for hip-
puristanol synthesis have been developed which should pave the way for more
potent and soluble derivatives (Li etal. 2009, 2010; Ravindar etal. 2010, 2011).
As a single agent, hippuristanol has been shown to inhibit proliferation of adult
leukemia cells and suppress tumor growth in immunodeficient mice injected with
HTLV-1-infected T cells (Tsumuraya etal. 2011), as well as reduce viability of
primary lymphoma cells derived from KSHV (Ishikawa etal. 2013). We recently
demonstrated that hippuristanol resensitizes E-MYC lymphomas to DNA-damag-
ing agents (Cencic etal. 2013). Since levels of a key prosurvival regulatory pro-
tein, MCL-1, are significantly affected by hippuristanol, combining its use with the
BCL-2 family inhibitor, ABT-737, also leads to a potent synergistic response in trig-
gering cell death in mouse and human lymphoma and leukemia cells (Cencic etal.
2013). Suppressing eIF4AI using RNAi strategies also synergized with ABT-737 in
lymphomas, thus highlighting eIF4AI as a target for modulating tumor cell response
to chemotherapy (Cencic etal. 2013).
Recently, a toxin from the bacteria Burkholderia pseudomallei, BPSL1549, has
been shown to target eIF4A via deamidation of Gln339 (Cruz-Migoni etal. 2011).
This amino acid also experiences significant nuclear magnetic resonance chemical
shifts when eIF4A is exposed to hippuristanol and is located in proximity of residues
that directly interact with hippuristanol (Lindqvist etal. 2008b). The consequence
of Gln339 deamidation by B. pseudomallei toxin is to generate a dominant-negative
mutant of eIF4A (Cruz-Migoni etal. 2011). Whether BPSL1549 can be developed
into an antineoplastic agent remains to be determined.
14.8.2Pateamine A
Pateamine A was first isolated from the marine sponge Mycale sp. and is a chemi-
cal inducer of dimerization that stimulates the binding of eIF4A to RNA in a non-
sequence dependent manner. This leads to its depletion from the eIF4F complex and
results in inhibition of cap-dependent translation (Bordeleau etal. 2005). The effect
of this small molecule on translation initiation is irreversible and cells treated with
pateamine A fail to regain normal translation levels upon withdrawal of the com-
pound (Bordeleau etal. 2005, 2006a; Low etal. 2005). An alternative mechanism
of action for pateamine A has been proposed whereby increased interaction between
eIF4A and eIF4B leads to disruption of the eIF4F complex and/or eIF4B function
(Low etal. 2005). However, the increased interaction of eIF4A with eIF4B was later
shown to be RNA-mediated and not a direct consequence of pateamine A (Borde-
leau etal. 2006a). Pateamine A also appears to only affect eIF4A in its free form
and not when part of the eIF4F complex, indicating that the pateamine A binding
site is not available to pateamine A when eIF4A is in eIF4F (Bordeleau etal. 2005).
Mutations within the N-terminal domain of eIF4A and the linker region affect the
ability of pateamine A to bind eIF4A (Low etal. 2007).
Exposure of cells to pateamine A induces stress granule formation (Mazroui etal.

2006) and can block nonsense mediated decay due to its effects on eIF4AIII (Dang
etal. 2009). As a chemotherapeutic, pateamine A has been shown to exhibit cyto-
toxic effects against P388 murine leukemia cells (Northcote etal. 1991), in addition
to inducing cell death in different cell lines (Hood etal. 2001). DMDA-pateamine
A, a synthetic derivative of pateamine A, is tolerated in animals and as a single
agent inhibits growth of LOX and MDA-MB-435 melanoma xenografts (Kuznetsov
etal. 2009). Pateamine A has also recently been shown to inhibit cachexia-induced
muscle wasting in vivo. Very low doses of pateamine A were found to inhibit muscle
wasting of C2C12 myotubes ex vivo and in mice injected intramuscularly with IFN-
/TNF (Di Marco etal. 2012). In response to IFN-/TNF induced muscle wast-
ing, the levels of iNOS (inducible isoform of nitric oxide synthase) and NO were
found to increase (Di Marco etal. 2005). Pateamine A prevented these responses
and at low doses appears to stimulate recruitment of MyoD mRNA into polysomes
and iNOS mRNA to stress granules (Di Marco etal. 2012).
Recently, a series of pateamine A analogs have been synthesized with the intent
to undertake structure-activity relationships aiming to identify structural features
important for pateamine A activity. These compounds were assessed for their an-
tiproliferative activity against a melanoma cell line and it was found that this was
dependent on the rigidity of the macrolide structure and the functionality of the side
chain (Low etal. 2014).
14.8.3Rocaglamides
Silvestrol is a member of the rocaglamide small molecule family and was isolated
from the plant genus Aglaia found in Malaysia, Indonesia, Taiwan, Fiji, and Viet-
nam. Similar to pateamine A, it behaves as a chemical inducer of dimerization and
exposure of cells to this compound depletes eIF4A from the eIF4F complex (Bor-
deleau etal. 2008; Cencic etal. 2009). Unlike pateamine A, silvestrol is a reversible
inhibitor of translation capable of affecting eIF4A activity whether eIF4A is free or
complexed to eIF4F (Cencic etal. 2009).
A synthetic route to silvestrol has been developed (El Sous etal. 2007; Gerard
etal. 2007) opening the window for the generation and study of new analogs. Sever-
al studies have provided insight into the mechanism of action silvestrol (Chambers
etal. 2013; Sadlish etal. 2013). The use of chemogenomic profiles and mutagenesis
in S. cerevisiae identified the yeast eIF4A homologs, Tif1/2, as the primary target
of a synthetic rocaglamide and of silvestrol (Sadlish etal. 2013). Cell-based screens
lead to the isolation of Tif1 mutants that could impart rocaglamide resistance to
yeast cells. In silico modeling showed that the identified mutations mapped to the
RNA-binding interface within the N-terminal domain of Tif1 (Sadlish etal. 2013).
Whether the corresponding residues will also confer silvestrol resistance to mam-
malian eIF4A remains to be determined. In vitro experiments are consistent with
eIF4A being a direct target of silvestrol (and related compounds) (Chambers etal.
2013). In a different set of affinity pull-down experiments, a rocaglamide lacking a

key functional group of silvestrol (the dioxanyl ring) was immobilized and found to
bind to Prohibitins 1 and 2components of the RAF/MEK/ERK signaling pathway
(Polier etal. 2012). This is unlikely to be the mechanism responsible for inhibition
of translation by rocaglamides since these compounds are active in translation ex-
tracts where the RAF/MEK/ERK signaling pathways are not intact (Bordeleau etal.
2008). Furthermore, the RAF/MEK/ERK pathway signals to MNK 1/2, which in
turn phosphorylates eIF4E, an event that is essential in the mouse (see above). Ad-
ditionally, while the yeast prohibitin homologs share 55% identity with the human
protein, the growth of the haploinsufficient strains was unaffected by silvestrol in
the aforementioned screen performed in S. cerevisiae (Sadlish etal. 2013), suggest-
ing that they are not the primary target in a cellular context.
Rocaglamides exhibit cytotoxic effects on cancer cells (Hwang etal. 2004; Kim
etal. 2006, 2007; Lee etal. 1998) and have been reported to inhibit NF-B activa-
tion (Baumann etal. 2002; Salim etal. 2007). In vivo, silvestrol shows antitumor
activity in a variety of settings. Silvestrol exhibits single-agent activity and inhib-
its tumor growth in breast and prostate cancer xenograft models without causing
immunosuppression, altering activity of ALT (alanine aminotransferase) and AST
(aspartate aminotransferase) liver enzymes, or causing weight loss (Cencic etal.
2009). Silvestrol shows anticancer activity in an acute lymphoblastic leukemia xe-
nograft model, in an E-TCL1 mouse model of chronic lymphocytic leukemia, and
against samples of human chronic lymphocytic leukemia (Lucas etal. 2009). In this
latter study, B cells were found to be more sensitive to silvestrol exposure than T
cells, suggesting a preferential killing for proliferating leukemic cells. Moreover,
inhibition of eIF4F activity using silvestrol and a potent derivative, CR-1-31-B (Ro-
drigo etal. 2012), blunted the overexpression of MUC1-C oncoprotein in breast
cancer cells (Jin etal. 2013). Recently, silvestrol was shown to exhibit antileukemic
activity against AML cells in a xenograft model by downregulating translation of
the FMS-like tyrosine kinase receptor-3, overexpression of which is associated with
poor outcome in AML (Alachkar etal. 2013).
Silvestrol showed no activity as a single agent in the E-MYC mouse model,
maybe the consequence of the low doses that were used and/or the more chemore-
sistant nature of these tumors. In experiments aiming to test the effects of silvestrol
in combination with standard-of-care agents (i.e. doxorubicin), silvestrol was able
to chemosensitize E-MYC/eIF4E, as well as TSC2+//E-MYC tumors, by sen-
sitizing to apoptotic triggers (Bordeleau etal. 2008). The ability of silvestrol to
synergize with different chemotherapeutic agents has been explored in numerous
systems. In the case of AML, silvestrol was shown to effectively synergize with
daunorubicin, etoposide and cytarabine ex vivo and this correlated with a decrease
in levels of the prosurvival factor, MCL-1 (Cencic etal. 2010). Rocaglamides have
also been shown to sensitize HTLV-1-associated adult T-cell leukemia/lymphoma
cells to CD95L- and TRAIL-induced cell death by downregulating c-FLIP transla-
tion (Bleumink etal. 2011). Recently, silvestrol has been shown to increase apopto-
sis of human HCC cells in vitro, showed synergy when combined with sorafenib or
rapamycin, and was effective in orthotopic human HCC xenografts (Kogure etal.
2013). Unfortunately, silvestrol is a substrate for the ABCB1/P-Glycoprotein pro-

tein, a property that might limit its clinical usefulness (Gupta etal. 2011).
14.9Targeting Other Components of Translation

Initiation
A second important regulatory node of translation initiation involves eIF2 phos-

phorylation. eIF2 is a trimeric factor that plays a central role in the control of trans-
lation initiation upon various stresses (Wek etal. 2006). eIF2 recruits Met-tRNAiMet
and GTP to form TC that then associates with the 40S ribosome. Following each
round of initiation, eIF2-GTP is hydrolyzed to eIF2-GDP, which must be recycled
by the guanine nucleotide exchange factor, eIF2B (Hinnebusch 2000). Stress stim-
uli can lead to the phosphorylation of eIF2, resulting in a high-affinity binding of
the latter to eIF2B and effectively decreasing levels of the TC (Krishnamoorthy
etal. 2001; Wek etal. 2006). Since eIF2 is in excess of eIF2B, small increases
in the level of phospho-eIF2 can substantially inhibit translation. Four different
kinases are known to phosphorylate eIF2 on Ser51, with each being activated in
response to specific stresses and environmental stimuli (Wek etal. 2006). These are:
PKR, which is activated by dsRNA, as occurs during infection with RNA viruses
(Barber 2005); PERK, an endoplasmic reticulum-associated kinase that is activated
following accumulation of unfolded proteins in the endoplasmic reticulum (Wek
and Cavener 2007); HRI, a kinase which phosphorylates eIF2 upon heme depri-
vation (Chen 2007); and GCN2, a kinase known to induce eIF2 phosphorylation
upon amino acid starvation (Hinnebusch 2000; Zhang etal. 2002) (see Chap.9).
Cancer cells are generally under tremendous stress to maintain appropriately
high protein outputs and often have constitutively elevated levels of eIF2 phos-
phorylation (Rosenwald etal. 2008; Rosenwald etal. 2003). Compounds that could
alter the phosphorylation status of eIF2 are being sought and tested for potential
therapeutic effects. To this end, a compound (Cmpd #1181) capable of increasing
phospho-eIF2, strongly inhibited growth of human breast and melanoma cancer
xenografts (Chen etal. 2012a; Natarajan etal. 2004). Recently, a group of novel
activators of HRI was also reported (Chen etal. 2011) and one of these, BTdCPU,
caused eIF2 phosphorylation through direct activation of HRI and inhibited tumor
cell growth ex vivo and in xenograft models. BTdCPU was well tolerated in vivo
with little apparent weight loss and little effect on the hematopoietic system. It
will be interesting to determine whether targeting TC assembly (Robert etal. 2006)
shows antineoplastic activity similar to what has been observed with increased
eIF2 phosphorylation.
14.10Targeting Translation Elongation as an

Antineoplastic Approach
Inhibitors of translation elongation have been extensively studied for potential anti-
neoplastic activity in cell lines and xenograft models with much of the primary data
publicly available (http://dtp.nci.nih.gov). Several inhibitors of translation elonga-
tion have even been tested in clinical trials.
14.10.1Aplidine
Aplidine, a didemnim family member inhibits translation by preventing EF2 from

binding to the 60S ribosome (Ahuja etal. 2000; Crews etal. 1994). Aplidine showed
minor activity and prolonged tumor stabilization in patients with advanced medul-
lar thyroid carcinoma, in one patient with SCLC, and one patient with CRC (Faivre
etal. 2005; Maroun etal. 2006). Dose-limiting myotoxicity was observed, although
no cardiac toxicity was noted (Faivre etal. 2005). Other reported toxicities were
mild hematological toxicity, nausea and vomiting, asthenia, fatigue, skin rash, and
diarrhea (Faivre etal. 2005; Maroun etal. 2006). Multiple clinical trials phase I/II
have been developed since then (Baudin etal. 2010; Dumez etal. 2009; Eisen etal.
2009a, b; Geoerger etal. 2012; Izquierdo etal. 2008; Mateos etal. 2010; Peschel
etal. 2008; Ribrag etal. 2013; Salazar etal. 2011; Schoffski etal. 2009) and at the
time of writing, one current phase III trial is recruiting at the National Institutes
of Health (NIH), testing in patients with relapsed or refractory multiple myeloma
(clinicaltrials.gov).
14.10.2 Omacetaxine Mepesuccinate
Cephalotaxus alkaloids were initially identified from the seeds of Cephalotaxus

harringtonia and exhibited antitumor activity in vitro against leukemia L1210 and
P388 cells (Powell etal. 1972). The active alkaloids are esters of cephalotaxine and
include: harringtonine, isoharringtonine, homoharringtonine (HHT), and deoxyhar-
ringtonine. Although these alkaloids were initially classified as inhibitors of initia-
tion since they caused polysome breakdown in intact cells and cell-free systems,
this effect appears due to the very low affinity of the harringtonine alkaloids for
translating ribosomes. The available data suggests that they block translation in
the early rounds of elongation, prior to polysome formation (Pelletier and Peltz
2007). Crystallographic studies have revealed that HHT binds the A-site cleft in
the peptidyl-transferase center and prevents correct positioning of amino acid side
chains on the incoming aa-tRNAs (Gurel etal. 2009).
The preclinical toxicology and pharmacokinetic data for HHT has been exten-
sively reviewed by Kantarjian etal (Kantarjian etal. 2001). Essentially HHT is
toxic to hematopoietic, cardiac, and gastrointestinal systems when given IV as a

single bolus or as a five-day infusion (Grem etal. 1988; Newman etal. 1981; Zhang
etal. 1979). In patients given HHT by six-hour infusion, HHT shows a biphasic
plasma decay with a terminal half-life of 14.4h (Savaraj etal. 1986). HHT was also
found to penetrate the CNS (Savaraj etal. 1987).
In phase I clinical trials, cardiovascular complications, primarily hypotension
and tachycardia, were dose-related and dose-limiting when HHT was delivered as
bolus short infusions (10360min daily for 110 days, escalating from 0.2 to 8mg/
m2 daily) (Legha etal. 1984). When HHT was delivered in continuous infusion
schedules, hypotension and cardiovascular disturbances were reduced significantly,
and dose-limiting toxicity became myelosuppression (3.254mg/m2 for 510 days)
(Coonley etal. 1983; Neidhart etal. 1986).
In phase II clinical trials, using a dosing schedule of 34mg/m2 by short infusion
daily for 5 days every 21 days, HHT was ineffective against advanced solid tumors
including malignant melanoma, sarcoma, head and neck cancer, breast carcinoma,
and CRC (Ajani etal. 1986). HHT was found to also be ineffective against recurrent
or progressive malignant glioma, renal carcinoma, and ovarian cancer (summarized
in (Kantarjian etal. 2001)). The results of HHT in phase II trials against acute leu-
kemia, myelodysplastic syndrome, acute promyelocytic leukemia, and in a chronic
myeloproliferative disorder, such as CML, indicated an effective antiproliferative
activity (reviewed in (Kantarjian etal. 2001; Wetzler and Segal 2011)). On October
26, 2012, HHT (renamed to omacetaxine mepesuccinate) was approved by the FDA
for the treatment of CML showing resistance to two or more TKIs (http://www.fda.
gov/NewsEvents/Newsroom/PressAnnouncements/ucm325895.htm).
At first glance, the use of elongation inhibitors as chemotherapeutics creates
somewhat of a paradox as these would not be expected to be selective in their mode
of action and should not possess a good therapeutic window. However, mutations
or epigenetic changes that lead to elevated protein synthetic rates likely create a
dependency on the latter to support the higher metabolic requirements of a tumor
cell. Since proteins like MYC, MCL-1 and cyclin D1 have short half-lives, prevent-
ing their production (either by an initiation or elongation inhibitor) is expected to
have profound consequences on tumor cell survival and proliferation. Indeed, in
preclinical models, several inhibitors of translation elongation caused reductions in
levels of the prosurvival protein, MCL-1, and sensitized the target cells to apoptotic
triggers (Robert etal. 2009). Consistent with this, Omacetaxine causes a rapid loss
(<4h) of MCL-1, MYC, cyclin D1, and XIAP in CML, AML, and multiple my-
eloma cells (Kuroda etal. 2008a, b; Tang etal. 2006). Given the central role played
by these proteins in tumor maintenance, their dysregulation is expected to affect
proliferation and survival. That elongation inhibitors can display significant antitu-
mor activity is encouraging for the development of initiation inhibitors as these are
expected to be more selective in the spectrum of mRNAs they inhibit (and hence
maybe also less toxic).
14.11Targeting Translation to Sensitize Tumor Cells

to Standard-of-Care Therapeutics
The E-MYC mouse provides a powerful model to link tumor cell genotype with
drug response (Wendel etal. 2004). In this model, lesions in the PI3K/AKT/mTOR
signaling pathway promote drug resistance in vivo (Wendel etal. 2004). Hence, E-
MYC/AKT, PTEN//E-MYC, and TSC2/+/E-MYC tumors are generally drug
resistant and enable screening for sensitizers to chemotherapy. There are several
points that encourage combining anti-eIF4F drugs with other therapeutic strategies.
Firstly, although xenograft models are an important component of drug develop-
ment programs, they do not report on drug resistance in the clinical setting. Indeed,
the eIF4E ASO described above, LY2275796, showed very encouraging results as
a single agent in preclinical models yet no tumor response was noted in phase I
trials (Hong etal. 2011). Secondly, molecular targeted therapies to BCR-ABL and
BRAF ultimately fail due to acquired resistance in the clinic and there is no reason
to believe that the situation will be any different for eIF4F inhibitors. Indeed, eIF4E
amplifications have been found associated with PI3K targeted therapies (Ilic etal.
2011) and may well arise to therapies targeting eIF4E. Thirdly, the role of eIF4F
in E-MYC lymphoma cell survival can be directly linked to the eIF4E-responsive
target: MCL-1, one of five BCL-2 family members. Hence depending on the situa-
tion, inhibition of eIF4F may not directly lead to cell death but rather to sensitization
to an apoptotic trigger. Indeed, this seems to be the case with the eIF4A inhibitors,
silvestrol, rocaglamide hydroxamates, and hippuristanol, which are ineffective in
the E-MYC model as single agents, but quite effective as chemosensitizing agents
(Bordeleau etal. 2008; Cencic etal. 2013; Rodrigo etal. 2012). Consistent with this
model, overexpression of MCL-1 blunts the chemosensitization effect obtained by
inhibiting eIF4A (Cencic etal. 2013).
14.12Perspectives
There are several issues to consider as clinical development of translation initiation

inhibitors proceeds. Firstly, a basis for patient stratification needs to be defined.
Which cancer type(s) are most likely to respond to the type of inhibitors described
in this review? Will this relate to the amount of aberrant signaling flux through the
PI3K/mTOR pathway or will it relate to a specific eIF4F gene expression signature?
What might prove useful here is a better definition of what constitutes an eIF4F-re-
sponsive mRNA and in what context. Defining mRNAs whose translation are most
responsive to altered eIF4F levels is still a significant challenge, open to analytical
and interpretational pitfalls, and likely to differ between cell types due to transcrip-
tome differences affecting the competitive ability of the mRNA populations. As
well, it will be important to identify a reliable biomarker that can report on transla-
tion initiation inhibition in vivo. This might be a short-lived plasma protein whose
expression is eIF4F-responsive and which could be used as a surrogate reporter for

in vivo dosing. Lastly, since in some instances, translation inhibitors synergize with
standard-of-care drugs it may be important to rationalize what compounds currently
in clinical use might synergize best with translation inhibitors. The next few years
will be an exciting time as we watch how these compounds fare in a different trans-
lation arenathat of clinical trials.
Acknowledgments Gabriela Galicia-Vazquez was supported by a Cole Foundation and a CIHR

Strategic Training Initiative in Chemical Biology Fellowships. We sincerely apologize to those
authors whose work is not cited herein due to space constraints. Work in the authors laboratory on
targeting dysregulated translational control is supported by grants from the Canadian Institutes of
Health Research and Canadian Cancer Society Research Institute.
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Part II
Regulation of Translation by Signaling
Pathways in Cancer
Chapter 15
mTOR and Regulation of Translation
Yoshinori Tsukumo, Mathieu Laplante, Armen Parsyan, Davide Ruggero and

Bruno Fonseca
Contents
15.2Mechanistic and Functional Aspects of mTOR and Its Signaling 308
15.3mTORC1 Pathway and Translational Control 312
15.3.14E-BPs 312
15.3.2S6 Kinase 314
15.3.3eIF4G 315
15.3.4eEF2K 316
15.4Translation and mTORC1 in Cell Proliferation and Growth 316
15.5Translation and mTORC1 in Invasion and Metastasis 317
15.6Translation and mTORC1 in Apoptosis and Tumor Survival 318
15.7mTOR Pathway and Translational Regulation in Etiology and
Pathogenesis of Cancer 319
15.8Targeting the mTOR Pathway in Cancer 320
15.8.1Renal Cell Carcinoma 321
15.8.2Pancreatic Neuroendocrine Tumors 321
15.8.3Tuberous Sclerosis Complex and Subependymal Giant-Cell Astrocytomas 322
15.8.4Mantle Cell Lymphoma 322
15.8.5Sarcoma 323
15.8.6Diffuse Large B-Cell Lymphoma 323
15.8.7Breast Cancer 323
Y.Tsukumo() B.Fonseca
Department of Biochemistry, Rosalind and Morris Goodman Cancer Research Centre, McGill
University, Montreal, QC, Canada
e-mail: yoshinori.tsukumo@mcgill.ca
M.Laplante
Centre de Recherche de lInstitut Universitaire de Cardiologie et de Pneumologie de Qubec
(CRIUCPQ), Facult de Mdecine, Universit Laval, Quebec City, QC, Canada
A.Parsyan
D.Ruggero
Helen Diller Family Comprehensive Cancer Center, Department of Urology, School of Medicine,
University of California San Francisco, San Francisco, CA, USA
308 Y. Tsukumo et al.
15.9New Compounds Targeting the mTOR Pathway 324

15.9.1Reasons for Low Therapeutic Efficacy of Rapalogs 324
15.9.2ATP-Competitive mTOR Inhibitors 325
15.9.3Dual mTOR/PI3K Inhibitors 326
15.9.4Other Compounds 327
15.10mTOR Inhibition and Therapeutic Resistance and Radioresistance 327
References 330
Abstract The PI3K/AKT/mTOR pathway is implicated in various cellular events

including translation, cell proliferation, growth and metabolism, and is frequently
dysregulated in cancer. In this chapter, we unveil how the mTOR pathway regu-
lates the translation machinery and how dysregulation of translation via this path-
way participates in cancer biology. We also discuss the clinical and therapeutic
importance of the control of translation by the mTOR signaling in various types of
cancers.
15.1Introduction
Mammalian target of rapamycin (mTOR), also known as mechanistic target of ra-

pamycin, is a well-conserved serine/threonine kinase that, as a key component of
the phosphoinositide 3-kinase (PI3K)/v-Akt murine thymoma viral oncogene ho-
molog (AKT, also known as PKB or protein kinase B)/mTOR signaling pathway
(hereinafter mTOR pathway), regulates a variety of cellular processes, including
protein and lipid synthesis, transcription and ribosome biogenesis, impacting on
cell proliferation, growth, motility, apoptosis and autophagy in normal physiology
and a variety of diseases, such as cancer, obesity, type 2 diabetes and neurological
disorders (see Laplante and Sabatini 2009, 2012). Notably, the mTOR pathway is
frequently dysregulated in human cancers (Guertin and Sabatini 2005). A descrip-
tion of the mTOR pathway that contains dozens of proteins and hubs deserves a
separate publication. Importantly, however, this pathway converges on the control
of translation. This chapter will introduce the role of mTOR in regulation of protein
synthesis in physiology and cancer.
15.2Mechanistic and Functional Aspects of mTOR and

Its Signaling
As reflected by its abbreviated name, TOR is a target of a compound named

rapamycin, a macrolide produced by the bacterial species Streptomyces hygro-
scopicus, which was isolated from a soil sample collected on the Easter Island
(Rapa Nui) in the 1970s (Vezina etal. 1975). mTOR is a large (290kDa) protein
and a member of the PI3K-related kinase (PIKK) family. Despite its homology to
15 mTOR and Regulation of Translation 309
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Fig. 15.1 TOR structure. The N-terminus of target of rapamycin (TOR) contains tandem HEAT
repeats that are important for proteinprotein interactions. The FAT and FATC domains modulate
the activity of the kinase domain. The FRB domain is the docking site of the FKBP12rapamycin
complex.
PI3Ks and PI4Ks, mTOR has not been demonstrated to have lipid kinase activ-
ity, and it essentially functions as a Ser/Thr protein kinase (Benjamin etal. 2011).
mTOR contains multiple domains, which are required for a large functional com-
plex formation (Fig.15.1). The N-terminus contains up to 20 tandem HEAT re-
peats (a proteinprotein interaction domain). These are followed by a FAT (FRAP,
ATM, TRRAP) domain. The Cterminal portion of mTOR contains the kinase do-
main and the FKBP12rapamycin-binding (FRB) domain. The C-terminus also
contains a FATC (FRAP, ATM, TRRAP C-terminal) domain that is paired with
the upstream FAT domain in all PIKKs to modulate kinase activity in an unknown
manner (Fig.15.1).
mTOR interacts with many proteins to form two distinct multiprotein complex-
es: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Laplante and
Sabatini 2012). Both mTOR complexes share a catalytic mTOR subunit, as well as
mammalian lethal with sec-13 protein 8 (mLST8), DEP domain-containing mTOR-
interacting protein (DEPTOR) and the TTI1/TEL2 complex (Laplante and Saba-
tini 2012). In contrast, the regulatory-associated protein of mTOR (RAPTOR) and
proline-rich AKT substrate 40kDa (PRAS40) are specific to mTORC1, whereas
rapamycin-insensitive companion of mTOR (RICTOR), mammalian stress-activat-
ed MAPK-interacting protein 1 (mSIN1), and protein observed with RICTOR 1 and
2 (PROTOR1/2) are only part of mTORC2 (Laplante and Sabatini 2012). Impor-
tantly, each mTOR complex has different sensitivities to rapamycin and regulates
distinct biological processes (Fig.15.2) (Laplante and Sabatini 2012).
The activity of the mTORC1 complex is governed by five major signals: (1)
growth factors, (2) stress, (3) energy level, (4) oxygen and (5) amino acids. These
signals aim at regulating many processes involved in cell growth and proliferation
(Fig.15.2). With an exception of amino acids, these signals regulate mTORC1 by
modulating the activity of TSC1/2. TSC1/2 negatively regulates mTORC1 activ-
ity by converting a GTPase, RAS homolog enriched in brain (RHEB) (Inoki etal.
2003a), into its inactive GDP-bound form. Growth factor stimuli inhibit TSC1/2
function by phosphorylation through the upstream signaling pathways PI3K/AKT or
RAS/MAPK, resulting in the activation of mTORC1 (Fig.15.2). Like growth factor
inputs to mTORC1, many stress stimuli, including low oxygen or energy levels or
presence of DNA damage, signal to mTORC1 through TSC1/2. In response to hy-
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Fig. 15.2 mTORC1 and mTORC2 complexes. The mTOR kinase forms two distinct protein
complexes called mTORC1 and mTORC2. mTORC1 responds to growth factor stimuli, oxygen,
energy, stress and amino acids. mTORC1 regulates translation via the best characterized substrates
4E-BPs and S6Ks; lipid biogenesis by controlling function or expression of PPAR and SREBP1/2
at the multiple levels (Duvel etal. 2010; Kim and Chen 2004; Li etal. 2010; Porstmann etal. 2008;
Wang etal. 2011a; Zhang etal. 2009); lysosome biogenesis via nuclear translocation of a transcrip-
tion factor TFEB, that controls many genes with key roles in lysosomal function (Settembre etal.
2013); energy metabolism via glucose metabolism by activating transcription and translation of
HIF-1, which is a positive regulator of many glycolytic genes (Brugarolas etal. 2003; Duvel etal.
2010; Hudson etal. 2002; Laughner etal. 2001), or via mitochondrial oxidative metabolism, by
mediating the nuclear association between PPAR coactivator 1 (PGC1) and the transcription
factor Ying-Yang 1 (YY1), which positively regulate mitochondrial biogenesis and oxidative func-
tion (Cunningham etal. 2007); and autophagy via phosphorylation of its substrates, the ULK1/
ATG13/FIP200 complex and DAP1 (Ganley etal. 2009; Hosokawa etal. 2009; Jung etal. 2009;
Koren etal. 2010). mTORC2 responds to growth factors and regulates cell survival by phosphory-
lating and activating AKT and SGK1 (Garcia-Martinez and Alessi 2008; Sarbassov etal. 2005),
and cytoskeletal organization by affecting the actin cytoskeleton via activation of PKC, as well as
other effectors, such as paxilin and RHO GTPases in cell type-specific fashion (Jacinto etal. 2004;
poxia or a low energy state, AMP-activated protein kinase (AMPK) phosphorylates

TSC2 and increases its GAP activity toward RHEB (Inoki etal. 2003b). Like AKT,
AMPK also communicates directly with mTORC1 through RAPTOR phosphoryla-
tion, leading to 14-3-3 binding and the allosteric inhibition of mTORC1 (Gwinn
etal. 2008). Hypoxia also induces the expression of REDD1 (regulated in develop-
ment and DNA damage responses 1), which activates TSC2 function in a still poorly
understood manner (Brugarolas etal. 2004; DeYoung etal. 2008; Reiling and Hafen
2004). DNA damage also signals to mTORC1 through p53-dependent induction of
TSC2 and PTEN, causing downregulation of the entire PI3K/mTORC1 axis (Feng
etal. 2005; Stambolic etal. 2001), and activates AMPK through a mechanism that
depends on the induction of sestrin 1/2 (Budanov and Karin 2008). Unlike other
stimuli, amino acids regulate mTORC1 independently of TSC1/2, via a mechanism
that depends on the RAS-related GTPase (RAG), a group of proteins composed
of RAG A, B, C and D (Kim etal. 2008; Sancak etal. 2008). Under amino acid
starvation conditions, inactive mTORC1 is diffusely dispersed throughout the cyto-
plasm. Amino acid stimulation signals to vacuolar H+-ATPase (VATPase), which
is required to induce the GEF activity of RAGULATOR, a multisubunit complex
that activates the RAGs by promoting the GTP loading of RAG A/B (Sancak etal.
2010). When active, the RAG complex interacts with mTORC1, which promotes its
translocation from the cytoplasm to lysosomal membranes, where RHEB is thought
to activate mTORC1 (Laplante and Sabatini 2012; Zoncu etal. 2011).
mTORC1 regulates a variety of cellular events (such as translation, lipid synthe-
sis, lysosome biogenesis, autophagy and energy metabolism) via multiple down-
stream targets (Fig.15.2). Activated mTORC1 promotes the translation initiation
process by phosphorylating two major targets: ribosomal protein S6 kinase (S6K)
and the translation regulator eIF4E-binding proteins (4E-BPs) (Fig.15.2) (Gingras
etal. 2001; Laplante and Sabatini 2012; Ma and Blenis 2009; Silvera etal. 2010;
Sonenberg and Hinnebusch 2009). mTORC1 also induces lipid biogenesis by ac-
tivating sterol regulatory element-binding protein 1 (SREBP1) and PPAR tran-
scription factors (Fig.15.2) (Laplante and Sabatini 2009). In addition to promoting
anabolism, mTORC1 also inhibits catabolism by blocking autophagy through the
phosphorylation of the ULK1/ATG13/FIP200 complex (UNC-51-like kinase 1/au-
tophagy-related 13/FAK-interacting protein of 200kDa) (Fig.15.2) (Laplante and
Sabatini 2012; Settembre etal. 2013). Importantly, sustained mTORC1 activation
blocks growth factor signaling by activating various negative feedback loops that
inhibit PI3K signaling (Laplante and Sabatini 2009, 2012).
Compared to mTORC1, much less is known about mTORC2. mTORC2 is activated
by growth factors through a mechanism that is not well understood. mTORC2 has been
shown to regulate cell survival, metabolism, motility and cytoskeletal organization by
Sarbassov etal. 2004). Rapalogs inhibit mTORC1 activity, but not mTORC2. However, chronic
exposure to the drug can reduce mTORC2 activity. Active-site mTOR inhibitors (asTORi) inhibit
both mTORC1 and mTORC2 complexes more strongly than rapalogs. Specific components or
substrates of mTORC1 or mTORC2 are shown in red or green. Common components are shown
in purple. See List of Abbreviations.
phosphorylating many AGC kinases, including AKT, serum/glucocorticoid regulated

kinase 1 (SGK1), and PKC (Laplante and Sabatini 2012; Zoncu etal. 2011). In con-
trast to mTORC1, mTORC2 is insensitive to acute treatment with rapamycin. Never-
theless, reports indicate that prolonged exposure to rapamycin blocks mTORC2 func-
tion in certain cell lines (Sarbassov etal. 2006). Hence in this chapter and throughout
the book, unless otherwise specified, we refer to mTORC1 by referencing to mTOR.
15.3mTORC1 Pathway and Translational Control
Components of the translation machinery are the well-studied targets of mTORC1.

Translation initiation is a rate-limiting step commencing with ribosome recruitment
to the 5 end of an mRNA and ending with an elongation competent 80S ribosom-
al complex positioned at an initiation codon (Gingras etal. 1999b) (see Chap.2).
Ribosome binding is facilitated by a number of translation initiation factors that
recruit the ribosome to the mRNA 5 end. The 5 end of mRNAs possesses a cap
structure (m7GpppN, in which m represents a methyl group and N represents,
any nucleotide) that is specifically recognized by eIF4E. eIF4E is a part of a tri-
meric complex referred to as eIF4F that also includes scaffolding protein eIF4G and
RNA helicase eIF4A (Gingras etal. 1999b; Parsyan etal. 2011; Sonenberg and Hin-
nebusch 2009). Following its binding to the 5 cap, eIF4A activity within eIF4F is
thought to unwind the mRNA 5 proximal secondary structure to facilitate the bind-
ing of the 40S ribosomal subunit and its subsequent 5 to 3 scanning towards the
initiation codon. The scanning process is thought to be enhanced by other proteins,
such as the eIF4A interacting partner eIF4B and a RNA helicase, DHX29 (Gingras
etal. 1999b; Parsyan etal. 2011; Sonenberg and Hinnebusch 2009) (see Chap.5).
Importantly, mTORC1 directly and indirectly regulates several factors involved in
translation initiation, such as 4E-BPs, eIF4G, S6K, rpS6, eIF4E, eIF4B, and elonga-
tion factor eEF2 (see Fig.15.3 and Sonenberg and Hinnebusch 2009).
15.3.1 4E-BPs
The interaction between eIF4E and eIF4G is regulated by the 4E-BPs. These pro-
teins are translational repressors. In mammals, there are three 4E-BP variants: 4E-
BP1, 4E-BP2 and 4E-BP3, each encoded by a distinct gene (Pause etal. 1994; Pou-
lin etal. 1998). 4E-BPs are negatively regulated through phosphorylation at mul-
tiple sites by mTORC1. Hypophosphorylated 4E-BPs compete with eIF4G proteins
for an overlapping binding site on eIF4E and block the eIF4F initiation complex
formation, while the hyperphosphorylated 4E-BPs dissociate from eIF4E, resulting
in the eIF4F formation and promoting translation (Haghighat etal. 1995; Mader
etal. 1995; Marcotrigiano etal. 1999). Most of the studies on the roles of these
proteins in translation and their control were conducted with 4E-BP1, although this
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Fig. 15.3 Oncogenic signals promote translation of specific mRNAs and global changes in protein
synthesis. Hyperactivation of PI3K, AKT, c-MYC or loss of PTEN and TSC1/2 tumor suppresser
genes promote protein synthesis by coordinating the regulation of translation initiation, translation
elongation and ribosome biogenesis. Activated mTORC1 phosphorylates and inactivates 4E-BP1,
resulting in stimulating translation initiation by eIF4E. Furthermore, mTORC1 also phosphory-
lates S6K and activates translation initiation and elongation. c-MYC promotes protein synthe-
sis by increasing the transcription of multiple translational components including eIF4E mRNA
and by promoting rRNA synthesis (Barna etal. 2008; Hannan etal. 2003; Martin etal. 2004;
Mayer etal. 2004; Ruggero 2009). Together, oncogenic activation of the mTOR pathway as well
as MYC leads to promoting both translation of specific mRNAs and global changes in protein
synthesis. mTORC1 controls also many negative feedback loops that regulate RTK/PI3K signal-
ing. mTORC1 directly, or indirectly via S6K1, phosphorylates IRS-1 to promote its degradation,
resulting in reducing the ability of growth factors to signal downstream of RTK (Harrington etal.
2004; Tzatsos and Kandror 2006; Um etal. 2004). Additionally, mTORC1 directly phosphorylates
growth factor receptor-bound protein 10 (GRB10), which is a suppressor of RTK signaling (Hsu
etal. 2011; Yu etal. 2011). Owing to the existence of these negative feedback loops, mTORC1
inhibition can activate the RTK/PI3K signaling pathway and promote cancer cell survival. Nega-
tive regulators are shown in blue.
isoform is not the most abundant one uniformly in all tissues. For example, 4E-BP2
is the major form in the mammalian brain and has an important role in long-lasting
synaptic plasticity, learning and memory (Banko etal. 2005; Gkogkas etal. 2013;
Tsukiyama-Kohara etal. 2001). 4E-BP3 appears to be expressed broadly but more
abundantly in the human skeletal muscle, kidney and pancreas (Poulin etal. 1998).
Differences in the kinetics and phosphorylation sites have been observed among the
three 4E-BPs (Tee and Proud 2002; Wang etal. 2005).
mTORC1 directly phosphorylates two priming sites on 4E-BP1, Thr37 and
Thr46 (Brunn etal. 1997; Gingras etal. 2001). This phosphorylation event is
required for subsequent phosphorylation of Thr70, followed by Ser65, ultimately
resulting in the release of 4E-BP1 from eIF4E (Gingras etal. 1999a; Heesom and
Denton 1999; Mothe-Satney etal. 2000). Biological and clinical importance of
mTORC1-mediated 4E-BPs phosphorylation has been supported by a variety of
studies (Alain etal. 2012; Dowling etal. 2010; Hsieh etal. 2010). 4E-BP1/2 DKO
MEFs continue to proliferate even under serum-starved conditions, which in wild-
type MEFs would result in growth arrest due to the severe inhibition of the mTOR
cascade (Dowling etal. 2010). Similarly, RAS-transformed 4E-BP DKO MEFs
become resistant to mTOR inhibitors in vivo and, in contrast, the overexpression
of mTOR-mediated phosphorylation-resistant 4E-BP1 strongly reduces tumorigen-
esis (Alain etal. 2012; Hsieh etal. 2010). These studies, as well as another study
(Thoreen etal. 2012), indicate that 4E-BPs are critical mediators of mTORC1 ac-
tion in vitro and in vivo.
15.3.2 S6 Kinase
Mammalian cells contain two similar rpS6 kinase proteins (S6K1 and S6K2), which
are direct targets of mTORC1 and regulate cell growth (Grove etal. 1991; Shima
etal. 1998) (see Chap.16). S6K1 has been well-studied and its impact on cell
growth also has been better characterized than that of S6K2, partly because S6K2
was discovered much later than S6K1 (Shima etal. 1998). The activation of S6K1
promotes protein synthesis and cell growth through the phosphorylation of several
effectors (Ben-Sahra etal. 2013; Magnuson etal. 2012; Pende etal. 2004; Robitaille
etal. 2013; Ruvinsky etal. 2005). S6K1 was originally thought to control transla-
tion of an abundant subclass of mRNAs characterized by an oligopyrimidine tract
at the 5 end (5 TOP mRNAs), through the phosphorylation of rpS6 (Ruvinsky and
Meyuhas 2006). It appears that many proteins belonging to the translation machin-
ery are encoded by these 5 TOP mRNAs (Ruvinsky and Meyuhas 2006). However,
inhibition of mTORC1 with rapamycin only mediates a partial inhibitory effect on
5 TOP mRNA translation while causing complete dephosphorylation of S6K1 and
rpS6 (Stolovich etal. 2002; Tang etal. 2001). In addition, MEFs lacking S6K1/2
or knocked-in with an unphosphorylatable form of S6 still translate 5 TOP mRNA
(Pende etal. 2004; Ruvinsky etal. 2005). Thus, it is likely that the S6K/rpS6 axis is
not the major signaling node regulating 5 TOP mRNA translation. Interestingly, a
recent study has proposed that 4E-BPs may play a role in the control of translation
of 5 TOP mRNAs (Thoreen etal. 2012).
PDCD4 is one of the targets of S6K1. PDCD4 was originally identified as a sup-
pressor of neoplastic transformation (Cmarik etal. 1999) (see Chap.6). Dorrello
etal. found that PDCD4 inhibits eIF4A helicase (Dorrello etal. 2006). In response
to mitogens, PDCD4 is rapidly phosphorylated on Ser67 by S6K1 and subsequent-
ly degraded via -TRCP (see Chap.6). In cultured cells, expression of a stable
PDCD4 mutant that is unable to bind -TRCP inhibits translation of mRNA with
a structured 5 UTR, leading to a reduction in cell size and a decrease in cellular
proliferation (Dorrello etal. 2006; Schmid etal. 2008). Thus, S6K1-regulated
degradation of PDCD4 in response to mitogens can control protein synthesis and
cell growth downstream of mTORC1.
eIF4B is a physiologically relevant target of S6K1 that could explain its effect
on translation and cell growth. eIF4B is an RNA-binding protein that mediates effi-
cient recruitment of ribosomes to mRNA and stimulates the ATPase and RNA heli-
case activities of eIF4A (Rogers etal. 2002) (see Chap.5). eIF4B is phosphorylated
in response to a variety of extracellular stimuli that promote cell growth and pro-
liferation, such as serum, insulin, and phorbol esters (Duncan and Hershey 1985).
Ser422, one of the phosphorylation sites in eIF4B, is specifically phosphorylated by
S6Ks (Raught etal. 2004). In addition, a recent report showed that eIF4B regulates
translation of proliferative and prosurvival mRNAs, which have complex structures
in their 5 UTR (Shahbazian etal. 2010). Thus, eIF4B is an important mediator of
some of the effects of S6Ks on translation and cell growth.
15.3.3eIF4G
eIF4G is a scaffolding protein that plays a key role in the assembly of the initiation
complex (see Chap.7). All eukaryotes have two related eIF4G proteins, namely
eIF4G1 and eIF4G2. Both eIF4Gs are phosphoproteins (Raught etal. 2000; Tuazon
etal. 1989). Phosphorylation of eIF4G1, but not eIF4G2, is induced in an mTOR-
dependent manner in response to extracellular stimuli, such as serum, insulin, and
other factors that promote cell growth (Raught etal. 2000; Tuazon etal. 1989).
eIF4G1 has a cluster of serum-stimulated phosphorylation sites (Ser1148, Ser1188,
and Ser1232), which are sensitive to PI3K and mTOR inhibitors (Raught etal.
2000). The physiological significance of eIF4G1 phosphorylation has not been
determined because no evidence for changes in activity or association with other
initiation factors has been reported so far following its phosphorylation. On the
other hand, a recent study showed that the depletion of initiation factor eIF4G1
blocks cell proliferation and diminishes cell size, similarly to rapamycin treatment
or silencing of the mTORC1 component, RAPTOR (Ramirez-Valle etal. 2008).
Therefore, eIF4G1 may mediate some of the effects of mTORC1 on the promotion
of cell growth and proliferation.
15.3.4 eEF2K
eEF2K phosphorylates and inhibits translation elongation factor eEF2, which

mediates the translocation step of peptide-chain elongation on the ribosome (see
Chap.12). mTORC1 indirectly promotes the phosphorylation of at least three eEF2K
sites (Ser366, Ser78 and Ser359). The phosphorylation of these sites downstream
of mTORC1 reduces the activity of eEF2K, which decreases the phosphorylation of
eEF2 and promotes translation elongation (Proud 2009). Ser366 is phosphorylated
directly by S6Ks. Phosphorylation of Ser78 more strongly inhibits eEF2K activity,
but the direct upstream kinase has not been identified. Recent work has shown that
the CDK1/cyclin B complex phosphorylates Ser359 in eEF2K, and that its activity
appears to be promoted by mTORC1 (Proud 2009). Thus, mTORC1 decreases the
eEF2K activity in different ways, resulting in regulation of the elongation step of
protein synthesis. eEF2K activity is increased in various cancers, including glioma
(Arora etal. 2003). In addition, the latest study revealed that AMPK-mediated
eEF2K activation is important for tumor cell adaptation to nutrient deprivation
(Leprivier etal. 2013). eEF2K may be a valid target for treatment of cancer cells
that adapt to poor nutrient conditions (Manning 2013).
15.4Translation and mTORC1 in Cell Proliferation and

Growth
mTORC1 controls growth (increase in cell mass) and proliferation (increase in cell
number) by modulating mRNA translation through phosphorylation of 4E-BPs
(4E-BP1, 2 and 3) and S6K1 and 2 (Dowling etal. 2010; Ohanna etal. 2005).
Pharmacological or genetic inhibition of mTORC1 leads to decreased cell prolif-
eration and cell size (Dowling etal. 2010). The importance of the mTORC1/4E-BP
axis on cell proliferation has been demonstrated with the use of 4E-BPs KO MEFs
(Dowling etal. 2010). The cells lacking 4E-BPs sustained proliferation, but not cell
growth, under conditions of mTOR inhibition (Dowling etal. 2010). Consistently,
4E-BPs-deficient cells sustained translation of mRNAs that encode proprolifera-
tive and cell cycle progression proteins (Dowling etal. 2010). On the other hand,
S6Ks-deficient cells, but not 4E-BPs-deficient cells, exhibited smaller cell size than
wild-type counterparts and were resistant to reduction in cell size induced by mTOR
inhibition (Dowling etal. 2010). These phenotypes of 4E-BPs-deficient or S6Ks-
deficient cells were clearly rescued by re-expression of each protein via a recombi-
nant construct (Dowling etal. 2010). Together with other key reports (Alain etal.
2012; Deng etal. 2010; Larsson etal. 2012; Ohanna etal. 2005; Richardson etal.
2004), these studies provide evidence that mTORC1 regulates cell proliferation and
growth via translational control using distinct translational hubs. Owing to its role
in promoting cell growth and proliferation, it is not hard to imagine that dysregula-
tion of mTORC1 could play a fundamental role in cancer (Fig.15.4).
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Fig. 15.4 Hyperactivated mTOR pathway leads to dysregulation of translational control and can-
cer development and progression. Upon receiving genetic insults, such as an activating mutation
of proto-oncogenes (RTK and PI3K) or inactivation of tumor suppressor genes (PTEN, TSC1/2 and
LKB1), the mTOR pathway becomes hyperactivated, leading to increased translation of specific
mRNAs coding prometastatic, proangiogenic, proproliferative and antiapoptotic proteins (such as
VEGF, MMP-9, YB-1, cyclin D, ODC, MCL-1, BCL-2 and survivin).
15.5Translation and mTORC1 in Invasion and

Metastasis
Translational regulation by mTORC1 is implicated in many aspects of cancer biol-

ogy including invasion, metastasis and apoptosis (Del Bufalo etal. 2006; Hsieh etal.
2012; Zhou etal. 2004). Using ribosome profiling in a prostate cancer model, Hsieh
etal. showed that mTORC1 regulates translation of a large number of invasion- and
metastasis-related genes (Hsieh etal. 2012). Treatment of a human prostate cancer
cell line with an ATP-competitive mTOR inhibitor (INK128) reduced the expression
of several invasion-related proteins, such as CD44, vimentin and YB-1. Importantly,
4E-BPs depletion sustained the expression of these proteins (Hsieh etal. 2012). In
association with these observations, a previous clinical study of 165 invasive breast
cancers has shown that the phosphorylation of 4E-BP1, which activates eIF4E and
thus promotes translation, increased progressively from normal breast epithelium
to hyperplasia and from hyperplasia to invasive neoplasia (Zhou etal. 2004). Using
transgenic and allograft breast cancer models, Nasr etal. demonstrated that sup-
pressing mTOR activity or repressing its downstream translation regulators (eIF4E
and eIF4A) delayed breast cancer progression, onset of associated pulmonary metas-
tasis in vivo and breast cancer cell invasion and migration in vitro (Nasr etal. 2013).
Consistently, translation of VEGF, MMP-9 and cyclin D1, which encode products
associated with invasive and metastatic phenotypes, is inhibited upon eIF4E sup-
pression (Nasr etal. 2013). Another study has also shown that the mTORC1/eIF4E
axis appears to control translation of VEGF that is important for tumor angiogenesis.
Indeed, Del Bufalo etal. (2006) showed that treatment of human breast cancer cell
line BT474 with mTOR inhibitor temsirolimus inhibited VEGF production in vitro
under both normoxic and hypoxic conditions. The specificity of this effect via the
mTORC1/eIF4E axis was further demonstrated by the decrease in VEGF production
upon mTOR or eIF4E knockdown. Consistent with reduced VEGF production, tem-
sirolimus treatment inhibited vessel formation in vitro and in vivo (Del Bufalo etal.
2006). These observations can partly explain the antiangiogenic effects of rapamycin
reported previously in preclinical studies (Guba etal. 2002). Overall, these studies
indicate that the mTORC1/4E-BPs pathway is an important contributor to tumor
invasion, angiogenesis and metastasis (Fig.15.4). We refer the reader to chapters on
translational control and specific cancers presented in this book for more detailed
information on aberrations within the mTORC1/4E-BP axis and their clinical rel-
evance in various malignancies.
15.6Translation and mTORC1 in Apoptosis and Tumor

Survival
The mTORC1/4E-BP axis plays an important role in tumor survival and apop-
tosis (Fig.15.4) (Graff etal. 2007, 2009; Mamane etal. 2007; Mills etal. 2008).
mTORC1 inhibition by rapamycin leads to specific reduction in antiapoptotic
protein MCL-1 in MYC-driven mouse lymphoma and sensitizes the cells to che-
motherapy (Mills etal. 2008). eIF4E knockdown in a mouse cell line resulted in
reduced translation of specific mRNAs, including antiapoptotic protein survivin, and
induced apoptotic cell death (Mamane etal. 2007). In addition to these studies in
mouse cells, Graff etal. (2007, 2009) showed that the suppression of eIF4E by ASO
significantly induced apoptosis of human cancer cells and suppressed tumorigenesis
in vivo in a xenograft model. These ASOs repressed translation of eIF4E-sensitive
mRNAs, such as VEGF, cyclin D1, survivin, c-MYC and BCL-2 (Graff etal. 2007).
The effects of eIF4E knockdown also prevented endothelial cells from forming
vessel-like tube structures (Graff etal. 2007). Importantly, intravenous ASO ad-
ministration selectively and significantly reduced eIF4E expression and suppressed
tumorigenesis in the xenografts without major adverse effects (Graff etal. 2007). In
another study, Graff etal. (2009) also showed that eIF4E function is elevated in hu-
man prostate cancer, and that eIF4E ASO reduced expression of the eIF4E-regulated
proteins, including BCL-2 and c-MYC, and induced apoptosis. Collectively, these
data suggest that the 4E-BP/eIF4E axis is as an attractive target for cancer therapy.
15.7mTOR Pathway and Translational Regulation in

Etiology and Pathogenesis of Cancer
The mTOR pathway is frequently dysregulated in human cancers (Figs.15.3 and

15.4). For example, overexpression or constitutive active mutations of receptor
tyrosine kinases (RTKs), such as EGFR and HER2/neu, are frequently observed
in many cancers including lung and breast (Guertin and Sabatini 2005). In addi-
tion, amplification of the p110 catalytic subunit of PI3K, loss of PTEN (which is
a phosphatase that opposes activity of PI3K), amplification of AKT2, as well as
mutations of TSC1, TSC2 and TP53, are frequently observed in many cancers and
result in activation of mTORC1 (Cheng etal. 1996; Feng etal. 2005; Guertin and
Sabatini 2005; Inoki etal. 2003b; Laplante and Sabatini 2012). Furthermore, inacti-
vating mutations in serine/threonine kinase 11 (STK11) (also called liver kinase B1
(LKB1)) and TSC1/2, which are tumor suppressor proteins upstream of mTORC1,
occur in PeutzJeghers and tuberous sclerosis syndromes that engender predisposi-
tion for cancer development (Guertin and Sabatini 2005).
There is much evidence that dysregulation of translational control serves as a
common mechanism by which the PI3K/AKT/mTORC1 pathway promotes cellular
transformation and tumor development (Silvera etal. 2010). In a normal cell, this
pathway acts as a sensor for energy, stress, nutrient availability and growth factor
signaling. The pathway integrates these inputs to regulate ribosome production and
gene expression at the translational level. Importantly, this pathway becomes onco-
genic when hyperactivated or dysregulated. Although it remains unknown how the
PI3K/AKT/mTORC1 pathway alters translational control in cancer, recent studies
have shown that this signaling cascade directly modulates activity and expression of
specific components of the translation machinery (Alain etal. 2012; Dowling etal.
2010; Hsieh etal. 2010; Thoreen etal. 2012).
The activated PI3K/AKT/mTORC1 pathway leads to activation of translation
initiation and protein synthesis. mTOR phosphorylates 4E-BPs and leads to the
activation of eIF4E (Sonenberg and Hinnebusch 2009). The 4E-BPs/eIF4E axis
plays a primary role in translational control and cancer. As follows later in this
book, eIF4E overexpression or pathologic activation have been reported in a va-
riety of tumors including human breast, head and neck and other cancers (Berkel
etal. 2001; Rosenwald etal. 2001; Salehi and Mashayekhi 2006; Sorrells etal.
1998, 1999; Wang etal. 2009; Yoshizawa etal. 2010). The oncogenic potential
of eIF4E has been faithfully recapitulated both in vitro and in vivo (Avdulov etal.
2004; Furic etal. 2010; Lazaris-Karatzas etal. 1990; Ruggero etal. 2004). Over-
expression of eIF4E is sufficient to induce transformation of immortalized murine
fibroblasts and human epithelial cells (Avdulov etal. 2004; Lazaris-Karatzas etal.
1990). Transgenic mice, in which eIF4E expression is driven by the ubiquitous
-actin promoter, have increased susceptibility to cancer (Ruggero et al. 2004).
These mice develop lymphomas, angiosarcomas, lung carcinomas, and hepatomas
(Ruggero etal. 2004).
Activation of eIF4E either via inactivation of 4E-BPs or phosphorylation by the
MAPK pathway also plays an important role in cancer biology ((Alain etal. 2012;
Dowling etal. 2010; Furic etal. 2010; Hsieh etal. 2010; Ueda etal. 2010) and
see Chaps.4 and 17). Indeed, combined deficiency of the MAPK-interacting ki-
nases MNK1 and MNK2, which are upstream kinases that phosphorylate eIF4E at
Ser209, delays tumorigenesis in T-cell-specific PTEN-deficient mice (Ueda etal.
2010). In addition, eIF4ES209A knock-in mice, in which eIF4E cannot be phosphory-
lated on the 209th residue, are resistant to prostate cancer induced by the PTEN loss.
These results support a recent study showing that eIF4E phosphorylation is required
for translational upregulation of several proteins implicated in tumorigenesis (Furic
etal. 2010).
The most studied and, possibly the most fundamental mechanism supporting
cell growth and proliferation in cancer is the control of eIF4E activity via the
mTORC1/4E-BP axis. It has recently been shown (both in vitro and in vivo) that the
4E-BPs/eIF4E axis exerts significant control over cap-dependent translation, cell
proliferation, cancer initiation and progression downstream of mTORC1 hyperacti-
vation (Alain etal. 2012; Dowling etal. 2010; Hsieh etal. 2010). Most importantly,
restoring eIF4E oncogenic activity to normal levels downstream of AKT/mTORC1
hyperactivation results in blockage of tumor progression in a mouse model for
lymphomagenesis and associates with a drastic increase in overall survival (OS)
(Hsieh etal. 2010). Furthermore, using ribosome profiling, Hsieh etal. uncovered
specialized translation of the prostate cancer genome by oncogenic mTOR signal-
ing, revealing a remarkably specific repertoire of genes involved in cell prolifera-
tion, metabolism, and invasion (Hsieh etal. 2012). This signifies that translation,
downstream of the 4E-BPs/eIF4E axis, represents a key cellular process linking ab-
errant mTORC1 signaling to cancer etiology and pathogenesis. Specific examples
into the role of the activated mTORC1/4E-BPs/eIF4E axis in the promotion of
translation in different types of cancers can be found in other chapters of this book
(see Part IV).
Another mTORC1 substrate S6K1 is frequently amplified in a variety of tu-
mors including breast and ovarian cancer (Andersen etal. 2002; Barlund etal.
2000; Ip and Wong 2012). In breast cancer, both S6K1 and S6K2 are situated in
the commonly amplified chromosome regions 17q21-23 and 11q13 (Perez-Teno-
rio etal. 2011). Importantly, S6Ks amplification and protein overexpression have
been reported to be associated with a worse outcome in breast cancer (Perez-Teno-
rio etal. 2011).
Taken together, these reports indicate that the mTORC1-mediated translational
control has an essential role in maintaining the transformed phenotype and suggest
that inhibition of the mTOR pathway could be efficient for the treatment of a variety
of cancers.
15.8Targeting the mTOR Pathway in Cancer
Several analogs of rapamycin called rapalogs have been designed in the past 10
years and have been approved for treatment of metastatic RCC, subependimal giant
cell astrocytoma and pancreatic neuroendocrine tumors (PaNETs) (Baselga etal.
2012; Hudes etal. 2007; Krueger etal. 2010; Motzer etal. 2010, 2008; Yao etal.
2011). These rapalogs include temsirolimus (CCI-779), everolimus (RAD001), and
ridaforolimus (AP-23573) and have therapeutic effects similar to rapamycin but
possess better pharmacokinetics. (Gabardi and Baroletti 2010; Mita etal. 2008;
Rini 2008). Currently, the National Institute of Health (NIH) website (clinicaltrials.
gov) lists a significant number of clinical trials testing rapalogs as single agent or in
combination therapy for the treatment of many cancer types.
15.8.1Renal Cell Carcinoma
Renal cell carcinoma (RCC) is a cancer type in which activation of the mTOR path-
way correlates with tumor aggressiveness and poor prognosis (Pal and Quinn 2013)
(see Chap.34). It has been shown that temsirolimus (Torisel) increases survival of
patients with previously untreated metastatic RCC with poor prognostic features
(Hudes etal. 2007). In a randomized phase II trial with 111 patients with treatment-
refractory metastatic RCC, the temsirolimus-treated group showed 7% of overall
response rate and 6 months progression-free survival (PFS) (Atkins etal. 2004). In
the phase III trial, treatments with temsirolimus or IFN, or a combination of these
two drugs, were compared (Hudes etal. 2007). Of the three treatment options, the
group treated with temsirolimus alone showed the longest OS and PFS with mild-
est adverse effects. Based on these findings, temsirolimus was approved for first-
line treatment of metastatic RCC patients with poor prognosis (Hudes etal. 2007).
Everolimus has also been approved for treatment of RCC. In a phase III study with
410 RCC patients progressing after chemotherapy, median PFS (but not OS) of pa-
tients in the everolimus group was significantly longer than placebo (4.9 versus 1.9
months) (Motzer etal. 2010). Accordingly, everolimus was approved by the Food
and Drug Administration (FDA) for treatment of advanced RCC progressing after
chemotherapy (Gomez-Pinillos and Ferrari 2012).
15.8.2Pancreatic Neuroendocrine Tumors
Pancreatic neuroendocrine tumors (PaNETs) represents about 1% of all pancreatic

malignant neoplasms. Although these are rare, their incidence has doubled over the
past 20 years (Yao etal. 2008, 2011). mTOR pathway abnormalities in PaNET are
reported to be associated with increased aggressiveness and shortened PFS and OS
among patients with PaNET (Jiao etal. 2011; Yao etal. 2011) (see Chap.31). These
clues from somatic or germline mutations and protein expression of the mTOR
pathway components supported the conduct of clinical studies of mTOR inhibitor
everolimus in patients with PaNET. A phase III study of everolimus named RADI-
ANT III was conducted in 410 patients with metastatic unresectable PaNETs (Yao
etal. 2011). Treatment with everolimus significantly prolonged the median PFS (11
versus 4.6 months), but not OS compared to placebo (Yao etal. 2011). Everolimus
has been recently approved by the FDA for the treatment of metastatic or unresect-
able PaNET (Yao etal. 2011).
In addition to PaNETs, everolimus exhibited clinical benefit for patients with

advanced neuroendocrine tumors (NETs), which are neoplasms that arise from
cells of the endocrine and nervous systems. In the phase III RADIANT-2 trial
with 429 patients, the addition of everolimus to long-acting octreotide, which is
a somatostatin analog that blocks the actions of growth hormone, glucagon and
insulin, increased the median PFS to 16.4 compared to 11.3 months for place-
bo. Currently, everolimus is underway in the phase III RADIANT-4 trial among
patients with advanced nonfunctional neuroendocrine tumors of gastrointestinal
or lung origin (see clinicaltrials.gov identifier: NCT01524783, Novartis Protocol
CRAD001T2302).
15.8.3 T
uberous Sclerosis Complex and Subependymal Giant-
Cell Astrocytomas
Tuberous sclerosis is a genetic disorder characterized by the growth of numerous

benign tumors in many organs including the skin, brain, lungs and kidneys (Krueger
and Franz 2008). Tuberous sclerosis is caused by a mutation of either of two genes,
TSC1 and TSC2, which are negative regulators of mTORC1, as mentioned pre-
viously. Subependymal giant-cell astrocytomas (SEGAs) occur in up to 20% of
people with tuberous sclerosis and are more likely to develop during childhood
and adolescence (Krueger and Franz 2008). The phase III clinical trial in tuberous
sclerosis patients showed that 35% of subjects treated with everolimus had a 50%
or greater reduction in the size of SEGAs compared to no response in the placebo
group (Krueger etal. 2010). Recently the FDA approved everolimus for the treat-
ment of SEGA in patients with tuberous sclerosis who were not candidates for cura-
tive surgical resection.
15.8.4 Mantle Cell Lymphoma
The mTOR pathway is an important therapeutic target in mantle cell lymphoma be-
cause the commonly observed t(11;14) translocation induces overexpression of cy-
clin D1 mRNA, whose translation is highly regulated by the mTORC1/4E-BP/eIF4E
axis (Bertoni etal. 2004; Ohanna etal. 2005). In a phase III trial with 162 patients
with relapsed mantle cell lymphoma, two doses of temsirolimus monotherapy
(175mg weekly for three weeks followed by either 75 or 25mg weekly) had
significantly better effects on median PFS than the investigators choice protocol
(4.8 versus 1.9 months). However, there was no significant difference in the median
OS between the temsirolimus treatment group and the investigators choice proto-
col treatment group (Hess etal. 2009). These results established temsirolimus as a
new therapeutic option for relapsed/refractory mantle cell lymphoma and led to its
inclusion in the National Comprehensive Cancer Network (NCCN) Clinical Prac-
tice Guidelines in Oncology for non-Hodgkins lymphomas (Zelenetz etal. 2010).
15.8.5Sarcoma
The mTOR pathway is dysregulated in most sarcoma subtypes, including

leiomyosarcoma, rhabdomyosarcoma and perivascular epithelioid cell tumors
(Dobashi etal. 2009; Hernando etal. 2007; Pan etal. 2008; Rivera etal. 2011)
(see Chap.22). Ridaforolimus, one of the rapalogs, prevents cell growth and pro-
liferation in various sarcoma cell lines and exhibits antitumor activity in sarcoma
xenograft models (Rivera etal. 2011; Squillace etal. 2011). Ridaforolimus showed
promising activity in advanced sarcoma patients in a phase I trial. In a subsequent
phase II trial involving 212 patients with advanced soft-tissue or bone sarcomas
with no restrictions on prior chemotherapy, ridaforolimus produced clinical benefit
in 29% of patients with mild adverse effects (Chawla etal. 2012). Based on these
results, a double-blind phase III trial randomized 711 patients with metastatic sar-
coma was initiated. In this trial, ridaforolimus reduced the risk of progression or
mortality by 28% in patients with advanced soft tissue and bone sarcomas (Keedy
2012). However, it has not yet been approved by the FDA due to the small improve-
ment in disease control.
15.8.6 Diffuse Large B-Cell Lymphoma
Recent studies have highlighted dysregulation of the mTOR pathway and its role
in controlling tumor cell proliferation and survival in DLBCL (Uddin etal. 2006;
Wanner etal. 2006). In a phase II trial with everolimus including 77 patients with
aggressive non-Hodgkins lymphoma (of which, eight follicular lymphoma, 47
DLBCL and 19 mantle cell lymphoma patients) who were heavily pretreated, the
overall response rate was 30% (14/47) in DLBCL (Witzig etal. 2011). Current-
ly, everolimus is being investigated in a phase III trial as maintenance therapy for
DLBCL in patients with a complete response after R-CHOP chemotherapy (see
clinical trial, NCT00790036).
15.8.7 Breast Cancer
As indicated in preclinical breast cancer models, aberrant activation of the mTOR

pathway through growth factor signaling confers resistance to hormonal therapy,
while mTOR inhibition restores this response (deGraffenried etal. 2004; Kurokawa
and Arteaga 2003; Kurokawa etal. 2000) (see Chap.26). In a phase III trial with
rapalogs combined with hormone therapy in metastatic breast cancer, temsiroli-
mus showed no benefit in median PFS, while everolimus showed a significant im-
provement in the median PFS (Ellard etal. 2009). Everolimus, in combination with
hormonal therapy (exemestane), is approved by the FDA for advanced hormone
receptor-positive, HER2-negative, breast cancer (Baselga etal. 2012).
In addition to the cancers mentioned above, several clinical trials are presently
testing the effect of rapalogs in other malignancies including lung, endometrial and
gastric cancers (Doi etal. 2010; Gomez-Pinillos and Ferrari 2012; Oza etal. 2011;
Pandya etal. 2007; Slomovitz etal. 2010; Soria etal. 2009; Tarhini etal. 2010;
Yoon etal. 2012). However, while monotherapy with these mTOR inhibitors shows
some benefits, feedback activation of other pathways that lead to upregulation of
translational control (see below) represents a limitation of rapalogs in achieving
high efficacy of the therapy. We also refer the reader to the Part IV of this book
for more information on the role of the mTOR pathway and its targeting in various
cancers.
15.9New Compounds Targeting the mTOR Pathway
15.9.1 Reasons for Low Therapeutic Efficacy of Rapalogs
As described in the previous section, rapalogs have exhibited clinical efficacy

against a variety of malignancies and have been approved for treatment of several
cancers (Benjamin etal. 2011; Dancey 2010). However, despite observed antitu-
mor responses, rapalogs showed modest clinical responses as a single-agent therapy
(Benjamin etal. 2011; Dancey 2010). There could be several reasons for the de-
creased performance of rapalogs in certain situations. First, it is well known that
mTORC1 activation downregulates IRS-1 via several signaling routes and induces
the retro-inhibition of PI3K/AKT signaling (Fig.15.3) (Laplante and Sabatini 2012).
Previous studies have shown that inhibition of mTORC1 with rapamycin relieves
the negative feedback loops and massively promotes PI3K/AKT signaling (Har-
rington etal. 2004; Hsu etal. 2011; Tzatsos and Kandror 2006; Um etal. 2004; Yu
etal. 2011). In addition, several preclinical and clinical studies reported that rapalog
treatment resulted in higher levels of activated AKT (Floch etal. 2012; OReilly
etal. 2006; Tabernero etal. 2008; Wan etal. 2007; Wang etal. 2008a). Considering
that PI3K/AKT signaling engenders prosurvival and antiapoptotic functions, it is
believed that the aforementioned could contribute to reduced efficacy of rapalogs.
Another possible reason for the limited efficacy of rapalogs in cancer treatment
would be that these molecules only partially inhibit the phosphorylation of 4E-
BP1. Indeed, studies indicate that although rapalogs are very potent in inhibiting
S6K phosphorylation, these molecules incompletely inhibit the phosphorylation of
4E-BP1 (Choo etal. 2008; Feldman etal. 2009; Thoreen etal. 2009). Inability to
sustain 4E-BP1 dephosphorylation can lead to the persistence of protein synthe-
sis and cancer cell proliferation even in the presence of rapamycin (Feldman etal.
2009; Thoreen etal. 2009). Finally, rapamycin is largely ineffective in inhibiting
mTORC2, which is involved in cell motility and invasion (Jacinto etal. 2004; Masri
etal. 2007; Sarbassov etal. 2004).
Rapamycin is an allosteric inhibitor of mTORC1. New generation mTOR inhibi-

tors, which inhibit mTOR kinase activity by competing with ATP for binding to the
kinase domain of mTOR, have been designed and reported to have stronger anti-
tumor effects compared to rapalogs (Benjamin etal. 2011; Dancey 2010; Feldman
etal. 2009; Thoreen etal. 2009). Whereas rapamycin inhibits only mTORC1, the
active-site mTOR inhibitors (asTORi) inhibit both mTORC1 and mTORC2 (Feld-
man etal. 2009; Thoreen etal. 2009). mTOR and PI3Ks have similarities between
each kinase domains and, therefore, some of these compounds inhibit both kinases,
which are known as dual mTOR/PI3K inhibitors (Baumann etal. 2009; Manara
etal. 2010; Molckovsky and Siu 2008). Recent efforts lead to the development
of compounds that selectively inhibit both mTORC1 and mTORC2. Overall, the
anticancer efficacy of the ATP-competitive inhibitors in preclinical settings has
generally been superior to that of the rapalogs, as shown by effective inhibition of
mTORC2 activity and complete inhibition of 4E-BP1 phosphorylation (Feldman
etal. 2009; Janes etal. 2010).
15.9.2 ATP-Competitive mTOR Inhibitors
Compared to rapalogs, PP242 is a stronger mTOR inhibitor of both mTORC1

(including rapamycin-resistant 4E-BP1 phosphorylation) and mTORC2 (Feld-
man etal. 2009; Thoreen etal. 2009). In a preclinical trial, PP242 sustained 4E-
BP1 dephosphorylation and strongly suppressed tumor growth in a mouse model
of AKT-driven lymphangiogenesis, whereas rapamycin was generally ineffective
(Hsieh etal. 2010). In another study, PP242 synergized with imatinib or dasatinib
to induce apoptosis in BCR-ABL-positive cells (Janes etal. 2010). A derivative
of PP242, INK128 (MLN0128), inhibited the three primary downstream mTOR
effectors 4E-BP1, S6K1/2 and AKT in human prostate cancer PC-3 cells (Hsieh
etal. 2012). INK128 also inhibited prostate cancer cell invasion in vitro and in an
in vivo mouse model (Hsieh etal. 2012). Furthermore, recent reports from preclini-
cal model studies show that INK128 was also effective against BCR-ABL-positive
B-cell acute lymphoblastic leukemia (B-ALL) and HER2-therapy-resistant breast
cancer (Garcia-Garcia etal. 2012; Janes etal. 2013). Currently, INK128 is under
study in an early clinical trial for advanced malignancy.
AZD8055 completely dephosphorylated phospho-4E-BP1 Thr37/46 compared
with rapamycin and has a greater effect on inhibition of cap-dependent translation
in cell-line experiments (Chresta etal. 2010). In addition, AZD8055 inhibited tumor
development more than rapamycin in the mouse xenograft model (Chresta etal.
2010). AZD8055 treatment induced HER3 phosphorylation in several HER2am-
plified breast cancer cells while combination of AZD8055 with the HER2 inhibitor
lapatinib induced synergistic cell death consistent with inhibition of feedback ac-
tivation of phospho-HER3 (Marshall etal. 2011). Similarly, AZD8055 synergized
with the MEK inhibitor AZD6244 in a NSCLC xenograft model, as evidenced by
AZD8055-driven increase in ERK phosphorylation (Holt etal. 2012; Marshall etal.
2011). Thus, combining mTOR and MAPK pathway inhibition is a potentially ef-
fective strategy for cancer treatment.
Compared to rapamycin, OSI-027 (OSI Pharmaceuticals) exhibited stronger
translational inhibition activity and apoptotic effects against leukemic cell lines
expressing the T315I BCR-ABL mutation, which is refractory to all BCR-ABL
inhibitors currently in use (Carayol etal. 2010). OSI-027 had also similar and
suppressive effects on primitive leukemic progenitors from AML patients (Altman
etal. 2011).
Torin 1 is a highly potent and selective mTOR kinase inhibitor with strong bio-
logical activity in various preclinical settings (Thoreen etal. 2009). It potently in-
hibits mTORC1 (including sustained inhibition of 4E-BP1 phosphorylation) and
mTORC2. A medicinal chemistry program was initiated based on the original Torin
1 backbone to address some of its unfavorable properties, such as a low-yielding
synthetic route, poor water solubility and oral bioavailability and low stability (Liu
etal. 2011). This led to the production of Torin 2, which has better pharmacokinetic
properties and an improved synthetic route, allowing further in vivo evaluation of its
therapeutic potential (Liu etal. 2011). In a preclinical model, combination of Torin
2 with the MEK inhibitor AZD6244 yielded significant growth inhibition against
K-RAS-driven lung tumors (Liu etal. 2013).
15.9.3 Dual mTOR/PI3K Inhibitors
In a phase I clinical trial, dual mTOR/PI3K inhibitor BEZ235 was effective for mul-
tiple myeloma and sarcoma as a single agent and, even more so, in combination with
other drugs (bortezomib, vincristine, doxorubicin or melphalan) (Baumann etal.
2009; Manara etal. 2010). It is currently in several phase I and II trials of advanced
solid tumors and metastatic breast cancer, either as a monotherapy or a combination
therapy with paclitaxel, trastuzumab, BKM120 or BKM162. On the other hand, in a
mouse lung cancer model driven by mutant KRAS (G12D), BEZ235 was ineffective
as a single agent; however the combination with MEK inhibitor AZD6244 led to a
more significant tumor regression (Engelman etal. 2008). Similarly, a combination
of BEZ235 and AZD6244 has been reported to more effectively suppress growth
of CRC and melanoma (Aziz etal. 2010; Migliardi etal. 2012; Roberts etal. 2012;
Simmons etal. 2012). A clinical trial of BEZ235 combined with a MEK inhibitor
MEK162 has started for selected advanced solid tumors. Another dual mTOR/PI3K
inhibitor and the first orally available pharmaceutical of its class, XL765, is also
currently being investigated in clinical trials for several solid tumors (Molckovsky
and Siu 2008).
15.9.4Other Compounds
SF1126 is a novel prodrug that consists of a well-characterized PI3K inhibitor

LY294002 conjugated to an RGDS (Arg-Gly-Asp-Ser) peptide fragment (Garlich
etal. 2008). The RGDS peptide binds integrin within the tumor compartment, re-
sulting in enhanced delivery of the active compound LY294002 to the tumor vascu-
lature and the tumor cells themselves, and inhibiting PI3Kdependent angiogenesis
specifically in the tumor (Garlich etal. 2008). SF1126 was tested in a dose es-
calation study in patients with advanced solid tumors. This compound was well
tolerated with low-grade adverse events and 46% of patients showed stabilization
of their disease (Mahadevan etal. 2012; Zhang etal. 2011).
Metformin is used for the treatment of type 2 diabetes. Epidemiological studies
have consistently shown a lower incidence of various cancer types among diabetic
patients taking metformin (Evans etal. 2005; Libby etal. 2009). Metformin in-
hibits mTORC1 through activation of AMPK, which activates mTORC1 negative
regulator TSC2, and subsequently decreases cap-dependent translation (Dowling
etal. 2007; Zakikhani etal. 2006). This compound consistently displays a strong
antiproliferative effect in a wide range of cancer cell lines in vitro and in xenograft
models (Zhou etal. 2004). A recent study revealed that metformin, as well as ca-
nonical mTOR inhibitors, affect translation of genes encoding cell-cycle regulators
via the mTORC1/4E-BP axis (Larsson etal. 2012). Since metformin is a safe and
remarkably well-tolerated drug, numerous clinical trials on its anticancer potential
are underway (Pollak 2012).
Farnesyltransferase inhibitors (FTI) block the activity of RHEB, which is a
GTPase that directly activates mTORC1 (McMahon etal. 2005). Like RAS, RHEB
activity requires farnesylation. RHEB activity is blocked by FTI. In PTEN-deficient
tumor cells, inhibition of RHEB by FTI is responsible for the specific antineoplastic
effects that are dampened in the farnesylation-independent mutant of RHEB (Ma-
vrakis etal. 2008). RHEB overexpression, observed in some human lymphomas,
results in mTORC1 activation and increased sensitivity to rapamycin and FTI treat-
ment (Mavrakis etal. 2008). The FTI tipifarnib is currently in phase I and II trials
for the treatment of AML in adults.
15.10mTOR Inhibition and Therapeutic Resistance and

Radioresistance
Dysregulation of the PI3K/AKT/mTOR pathway in cancer not only contributes to

a tumorigenic phenotype, such as increased proliferation, motility and invasion, but
also leads to drug resistance. In this section, we review the role of the PI3K/AKT/
mTOR pathway in resistance to chemotherapy including conventional anticancer
medications, molecular targeted agents, hormonal therapy and radiation.
In HER2-positive breast cancer, loss of PTEN protein expression (3040% of

tumors) and activating mutations in PIK3CA (E545K or H1047R; 2025% of tu-
mors) are amongst the genetic alterations that lead to the activation of the mTOR
pathway (Hernandez-Aya and Gonzalez-Angulo 2011). Loss of PTEN is associated
with resistance to trastuzumab, which is a monoclonal antibody that interferes with
the HER2 receptor, since trastuzumabs antitumor activity requires PTEN activa-
tion (Nagata etal. 2004). As previously described, PTEN is a tumor suppressor that
inactivates PI3K signaling and mTOR activation. Recent clinical studies showed
that activation of the PI3K/AKT/mTOR pathway through genetic alterations was
associated with significantly shorter PFS (Berns etal. 2007; Wang etal. 2011b)
and poor OS (Gallardo etal. 2012) in patients receiving trastuzumab therapy.
The trastuzumab-resistance caused by activation of the PI3K/AKT/mTOR path-
way indicates that inhibition of this pathway can suppress proliferation in HER2-
positive resistant breast cancer cells. Indeed, the pan-PI3K inhibitors GDC-0941
and LY294002 and dual PI3K/mTOR inhibitor BEZ235 were able to significantly
inhibit PI3K/AKT/mTOR signaling and cell proliferation in trastuzumab-resistant
breast cancer cell lines in a dose-dependent manner (Eichhorn etal. 2008; Junttila
etal. 2009; Kataoka etal. 2010; Nagata etal. 2004). These findings indicate that
inhibition of the PI3K/AKT/mTOR pathway may be an effective approach to treat
tumors that are resistant to trastuzumab.
In the case of a lapatinib, which is a small compound that inhibits both EGFR
and HER2 activity, it has been reported that activation of PIK3CA through hotspot
E545K or H1047R mutations conferred resistance to lapatinib in HER2-overex-
pressing breast cancer cells (Eichhorn etal. 2008; OBrien etal. 2010). Correspond-
ingly, BEZ235 reversed lapatinib resistance in PTEN-deficient or PIK3CA-acti-
vated breast cancer cell lines (Eichhorn etal. 2008). Furthermore, mTOR inhibi-
tion with ridaforolimus (mTORC1 inhibitor) or INK-128 (mTORC1/2 inhibitor) in
combination with lapatinib resulted in significantly higher rates of tumor response
than either agent given alone in cell-based trastuzumab/lapatinib-resistant xenograft
models (Garcia-Garcia etal. 2012; Gayle etal. 2012). These studies support clinical
evaluation of mTOR inhibition in patients with HER2-overexpressing breast can-
cers, which are resistant to both trastuzumab and single-agent lapatinib. In phase I
and I/II study, everolimus combined with weekly paclitaxel and trastuzumab was
generally well tolerated and had encouraging antitumor activity in patients with
trastuzumab-pretreated and trastuzumab-resistant metastatic HER2-overexpressing
breast cancer (Andre etal. 2010; Morrow etal. 2011). The recently completed phase
III study (BOLERO-3) showed that everolimus in combination with trastuzumab
plus vinorelbine, which is an antimitotic drug, improved PFS compared to trastu-
zumab plus vinorelbine alone in trastuzumab-resistant metastatic breast cancer pa-
tients (Sendur etal. 2013).
Preclinical studies indicate that inhibition of the PI3K/AKT/mTOR pathway
can also overcome or prevent resistance to hormonal therapy (Cavazzoni etal.
2012; Ghayad etal. 2008, 2010; Miller etal. 2010). Treatment of breast cancer
with the dual PI3K/mTOR inhibitor BEZ235 has been shown to prevent cells from
developing resistance to hormone therapy by inducing apoptosis in long-term
estrogen-deprived cells (Miller etal. 2010). Furthermore, BEZ235 increased hor-

mone sensitivity, consistent with restoring levels of estrogen receptor (ER) and ER-
inducible target genes, and resulting in enhancement of sensitivity to tamoxifen.
Similarly, both everolimus and BEZ235 were able to restore sensitivity to hormone
therapy in resistant cell lines (Cavazzoni etal. 2012; Ghayad etal. 2008). Inclu-
sion of a MEK1 inhibitor in this combination further increased this effect (Ghayad
etal. 2010). A randomized phase III study (BOLERO-2) evaluated the efficacy
of an aromatase inhibitor, exemestane, with or without everolimus in postmeno-
pausal women with ER-positive/HER2-negative locally-advanced or metastatic
breast cancer who were refractory to nonsteroidal aromatase inhibitors and had
documented disease recurrence or progression (Baselga etal. 2012). This study
revealed that everolimus, combined with an aromatase inhibitor, improved PFS in
patients with hormone receptor-positive advanced breast cancer previously treated
with nonsteroidal aromatase inhibitors. Everolimus in combination with exemes-
tane for the treatment of postmenopausal women with advanced hormone receptor-
positive/HER2-negative breast cancer was approved by the FDA and the European
Medicines Agency (EMA) in 2012. Other trials are also ongoing with the PI3K/
AKT/mTOR pathway inhibitors in combination with hormonal therapy in resistant
patients (Burris 2013) (see also clinicaltrials.gov).
Loss of PTEN and activation of the PI3K/AKT/mTOR pathway are also fre-
quent and well-established mechanisms of prostate cancer progression (Bertram
etal. 2006; Mulholland etal. 2006; Sircar etal. 2009), that, by crosstalk induction
of ligand-independent activation of androgen receptors, support the development
of resistance to androgen deprivation therapy (Lin etal. 2004; Pienta and Bradley
2006). In turn, although androgen receptors upregulate mTOR activity by increas-
ing the androgen receptordependent transcription of nutrient transporters (Xu etal.
2006), the inhibition of mTOR with rapamycin increases androgen receptor mRNA
and transcriptional activity (Cinar etal. 2005; Wang etal. 2008b). Therefore, in-
hibiting both pathways would be essential to achieve complete responses. A phase
II trial is currently studying bicalutamide, which is an oral non-steroidal antian-
drogen, and everolimus to see how well they work in combination compared with
bicalutamide alone in treating patients with recurrent or metastatic prostate cancer
(Gomez-Pinillos and Ferrari 2012).
In glioblastoma multiforme, radiosensitization is an important therapeutic op-
tion because standard therapy for glioblastoma multiforme comprises adjuvant ra-
diotherapy combined with the alkylating agent temozolomide (TMZ) (Stupp etal.
2007). Ionizing radiation damages DNA and leads to apoptosis, resulting in tumor
regression. However, activation of some signaling pathways can promote cell sur-
vival even after radiation-induced DNA damage, thus limiting efficacy. One such
pathway is the PI3K/AKT/mTOR cascade, which is widely hyperactivated in glio-
blastoma multiforme through multiple genetic aberrations, such as loss of PTEN
expression; activating mutations in the genes encoding PI3K (such as PIK3CA or
PIK3R1); and overexpression of RTKs (such as EGFR (HER1), HER2 (also known
as ERBB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2)),
platelet-derived growth factor receptor (PDGFR) and MET) (The Cancer Genome
Atlas Research Network 2008). The frequency of genetic alterations in RTK/RAS/

PI3K in glioblastoma multiforme has been estimated to be about 90% (The Cancer
Genome Atlas Research Network 2008). Chakravarti etal. found that glioblastoma
multiforme patients with an activated PI3K/AKT/mTOR pathway showed signifi-
cantly worse survival than patients without oncogenic activation of the pathway
(Chakravarti etal. 2004). Several clinical studies have evaluated mTORC1 inhibi-
tors in combination with TMZ and radiation in glioblastoma multiforme (Sarkaria
etal. 2010, 2011). These trials showed different results. In a phase I trial, a com-
bination of temsirolimus, TMZ and radiation was associated with high infection
rates (Sarkaria etal. 2010). In contrast, a similar trial of everolimus in combination
with TMZ and radiation, followed by everolimus plus adjuvant TMZ, demonstrated
partial metabolic responses in 22% of patients, and stable metabolic disease in 78%
(Sarkaria etal. 2011). A phase I clinical trial is ongoing to examine whether the ad-
dition of the dual PI3K/mTOR inhibitor can sensitize tumors to TMZ (Burris 2013).
mTOR regulates translation of specific mRNAs responsible for cell proliferation,

survival and invasion through the phosphorylation of several downstream sub-
strates, such as 4E-BPs and S6K. Therefore, the dysregulation of the mTOR path-
way leads to transformation accompanied by aberrant cell growth and proliferation
and, moreover, contributes to tumor angiogenesis, invasion and metastasis. In fact,
the overactivation of the mTOR pathway is observed in a variety of human cancers.
Consequently, inhibition of mTOR activity in cancer cells has great therapeutic po-
tential. Rapalogs are FDA-approved for the treatment of several types of cancers.
Despite these promising data, single treatment with rapalogs is ineffective for the
treatment of most tumor types. Given that rapalogs have been shown to restore
sensitivity to many standard chemotherapeutic agents, they may be best utilized
in combination chemotherapy. Second-generation mTOR inhibitors, which target
both mTORC1 and mTORC2, as well as dual PI3K/mTOR inhibitors, have already
been developed. Importantly, compared to rapalogs, these inhibitors have shown
improved efficacy in preclinical studies and appear to have a potential to circum-
vent rapalog resistance in some cancer types. Some of these inhibitors are currently
in phase I trials.
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Chapter 16
Ribosomal Protein S6 and S6 Kinases
Mario Pende and Caroline Treins
Contents
16.2Molecular Mechanisms of Growth and Metabolic Control 348
16.2.1 Substrates Involved in Protein and Nucleic Acid Synthesis 350
16.2.2 Substrates Involved in Feedback Regulation 353
16.2.3 Substrates Involved in Cellular Metabolism 354
16.2.4 Substrates Acting as Tumor Suppressors or Oncogenes 355
16.3 S6 Kinases and Cancer 356
16.4 Potential Mechanisms of Resistance 357
References 358
Abstract Ribosomal protein S6 (rpS6) kinases 1 and 2 (S6K1 and 2) are substrates
of mTOR complex 1 (mTORC1), participating in specific growth and metabolic
programs. Although initially discovered as the kinases phosphorylating the 40S
ribosomal subunits on the C-terminal tail of rpS6, it is becoming increasingly appre-
ciated that S6Ks also control additional outputs beyond protein translation. Here
we review the molecular mechanisms of S6Ks and their targets, as well as discuss
the role of S6Ks in cancer biology and the ways of using them as targets for future
therapies.
16.1Introduction
Forty years ago, the phosphorylation on the C-terminal tail of rpS6 was the first
posttranslational modification described for ribosomal proteins in eukaryotic cells
(Gressner and Wool 1974; Thomas etal. 1980). It takes place on the 40S subunit
M.Pende() C.Treins
Institut National de la Sant et de la Recherche Mdicale (Inserm) and Facult de Mdecine,
Universit Paris Descartes, Paris, France
e-mail: mario.pende@inserm.fr
346 M. Pende and C. Treins
of the ribosomes in the nucleolus and the cytosol, typically 15 to 60min after the
cells are exposed to growth factors, mitogens, nutrients, or agents that raise the
intracellular amino acid concentration, such as the protein synthesis inhibitor cyclo-
heximide. Hence, it became one of the paradigms in the growth signal transduction
mechanisms to adapt to environmental cues and sustain long-term responses, pos-
sibly by acting on protein synthesis.
The kinase phosphorylating rpS6 in mammalian cells was identified as a pro-
tein of 70kDa, named p70S6 kinase (p70S6K) and encoded by the RPS6KB1 gene
(Kozma etal. 1989; Price etal. 1989). Later on, a homolog mammalian RPS6KB2
gene was identified and p70S6K was renamed to S6K1, and the homolog protein
was named S6K2 (Gout etal. 1998; Shima etal. 1998; Lee-Fruman etal. 1999).
S6K1 and S6K2 share a high level of sequence identity and belong to the AGC fam-
ily of protein kinases, phosphorylating serine (Ser, S) or threonine (Thr, T) residues
preceded by basic arginine (Arg, R) or lysine (Lys, K) residues at the 3, 5 posi-
tions (R/KXR/KXXS/T, where X is any amino acid) (Pearce etal. 2010).
Both S6K1 and S6K2 are ubiquitously expressed, with the abundance of S6K2
being reportedly lower than S6K1 (Shima etal. 1998). Three S6K1 isoforms have
been described: (1) p70S6K1; (2) p85S6K1, which is generated by the usage of an
alternative translational start codon and contains an additional 21 amino acid stretch
at the N-terminus (Grove etal. 1991; Reinhard etal. 1994); and (3) p31S6K1, which
is generated by alternative splicing and lacks a catalytic domain (Karni etal. 2007).
Although p70S6K1 was initially proposed to be cytosolic and p85S6K1 to be nu-
clear, due to the presence of a putative nuclear localization signal in the 21 amino
acid N-terminal stretch, rigorous biochemical fractionation experiments have now
demonstrated that p70S6K1 and p31S6K1 can also be detected in the nucleus (Ros-
ner and Hengstschlager 2011). In addition, immunofluorescent detection of rpS6
phosphorylation in cells lacking S6K1, S6K2 or both, have provided evidence that,
similar to S6K1, S6K2 can also act in the nucleolus and cytosol (Pende etal. 2004).
Thus, with the exception of a report suggesting a centrosomal localization specific
to S6K2 (Rossi etal. 2007), the tissue and cellular distribution of S6Ks largely
overlap.
S6Ks also share a similar mode of regulation that has been better characterized
for p70S6K1 but largely applies to the other isoforms. They are substrates of the
mTOR complex 1 (mTORC1) that phosphorylates Thr389 of p70S6K1 in the hy-
drophobic motif (Hara etal. 2002; Kim etal. 2002). This modification is a key event
for S6K activation, as it renders Thr229 in the T loop of the catalytic domain ac-
cessible for phosphorylation by phosphoinositide-dependent kinase 1 (PDK1), thus
facilitating the interaction with the substrates (Alessi etal. 1998; Pullen etal. 1998).
mTORC1 integrates a large variety of environmental signals to decipher condi-
tions favorable for growth (Laplante and Sabatini 2012). It is activated by growth
factors, peptides, mitogens, inflammatory cytokines, nutrients, oxygen and energy
status. The specific kinase activity of mTORC1 towards S6K1 is relatively low, as
compared to other mTORC1 substrates (Kang etal. 2013). This makes S6K1 phos-
phorylation and activation exquisitely sensitive to environmental conditions affect-
ing mTORC1. For instance, low extracellular amino acid levels or treatment with
the mTORC1 allosteric inhibitor rapamycin affect S6K1 phosphorylation more than
16 Ribosomal Protein S6 and S6 Kinases 347
other mTORC1 substrates, such as 4E-BPs (Kang etal. 2013). Mutation of S6K1
Thr389 to Ser is sufficient to improve the kinetics of phosphorylation by mTORC1,
and provides a proliferative advantage to the mutant-over-wild-type S6K1-express-
ing cells in nutrient poor conditions (Kang etal. 2013). In conclusion, the tight
regulation of wild-type S6Ks by mTORC1 is ideal to fine-tune growth responses
depending on the availability of anabolic signals.
The first demonstration of the growth promoting action of S6Ks came from ge-
netic studies in Drosophila, in which loss of function mutations of the single S6K
gene affect cell size but not cell number in adult tissues of the flies (Montagne etal.
1999). However during larval development, the proliferation rate of S6K-deficient
cells is lower as compared to wild type, thus causing a developmental delay. These
functions are largely conserved during evolution, as indicated by gene deletion ex-
periments in mice. S6K1 KO mice display reduced cell size, while the combined
deletion of S6K1 and S6K2 leads to perinatal lethality (Pende etal. 2000, 2004).
Growth (increase in cell size), but not proliferation (increase in cell number), of
S6K deficient cells in culture is reduced and resistant to the inhibitory effects of
rapamycin (Ohanna etal. 2005; Dowling etal. 2010). Conversely, the deletion of
another class of mTORC1 substrates, 4E-BP1 and 4E-BP2, renders proliferation of
MEFs, but not growth, resistant to ATP-competitive inhibitors of mTOR (Dowling
etal. 2010). The phosphorylation of 4E-BPs by mTORC1 releases the cap-binding
protein eIF4E, thus promoting eIF4E interaction with eIF4G and the formation of
the eIF4F complex that associates with the mRNA. Taken together, this evidence
led to the hypothesis that the S6K branch of mTORC1 signaling mainly controls
cell size, while the 4E-BP branch controls cell number. Although this model may
hold true in a number of experimental settings, care should be taken not to general-
ize these findings, as S6Ks can also participate in the regulation of cell proliferation
and survival in specific pathophysiological conditions (Gonzalez-Rodriguez etal.
2009; Espeillac etal. 2011).
Consistent with the high sensitivity of S6K activity to nutrient availability, the
phenotype of S6K loss-of-function mutants mimics metabolic adaptations to a ca-
loric restriction state. S6K1 deficient animals burn fat and spare glucose as source
of energy (Um etal. 2004; Aguilar etal. 2007), have low plasma insulin levels
(Pende etal. 2000), are hypersensitive to the hypoglycaemic action of insulin (Um
etal. 2004), and display a mild energy stress leading to the activation of the AMPK
(Aguilar etal. 2007). Similar to rapamycin treatment and caloric restriction (Har-
rison etal. 2009; Kenyon 2005), S6K1 deletion prolongs lifespan (Selman etal.
2009). These metabolic effects of S6K inhibition are noteworthy in the context of
tumorigenesis and may be therapeutically relevant. As we will see below, they may
affect tumor growth and resistance to stress conditions.
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Fig. 16.1. Schematic representation of S6K actions. S6Ks are mTORC1 substrates integrating
the anabolic signals from nutrients and growth factors. A key event for mTORC1 activation is the
GTP loading on RHEB and its recruitment at the lysosomal membrane. In turn, S6Ks phosphory-
late protein substrates, which belong to the indicated main functional categories: nucleic acid and
protein synthesis, feedback control of signal transduction, metabolism and oncogenic factors.
16.2Molecular Mechanisms of Growth and Metabolic

Control
A model, which is widespread in the scientific literature for historical reasons, pro-
poses that S6Ks control growth via the upregulation of protein synthesis. Although
it is widely accepted that S6Ks participate in the growth program downstream of
mTORC1, it is less clear as to how S6Ks precisely affect protein synthesis and
whether this regulation plays an essential role in growth and tumorigenesis. It is
becoming increasingly appreciated that S6Ks have additional cellular targets that
are not directly implicated in translation. In the following sections, we will review
the direct substrates and interacting partners of S6Ks and discuss their functional
relevance. The growing list of S6K substrates can be classified into the following
main categories: substrates involved in protein and nucleic acid synthesis; those
Table 16.1 List of validated S6K substrates.

Name Gene Phospho Site Redundancy Function Function- References
Symbol phosphorylation
Nucleic acid rpS6 RPS6 230- S6K1, S6K2, Essential component of ? (Gressner and Wool,
and Protein krrrlsslrastsksessqk RSK small ribosomal subunit 1974);
(Thomas et al., 1980)
synthesis
eIF4B EIF4B 419-rersrtgse S6K1, S6K2, Cofactor of eIF4A Promoting association with (Raught et al., 2004;
RSK helicase eIF4A Shahbazian et al.,
2006)
eEF2K EEF 2K 361-rvrtlsgs S6K1, S6K2, Kinase negatively Inhibitory on eEF2K activity (Wang et al., 2001)
RSK controlling eEF2
PDCD4 PDCD4 58-kakrrlrknssr S6K1, AKT Negative regulator of promoting PDCD4 degradation (Dorrello et al., 2006)
eIF4A by E-TRCP dependent
ubiquitination
CBP80 NCBP1 3-rrrhsd; S6K1 Cap-binding complex ? (Wilson et al., 2000; Ma

17-krrktsd; component binding the et al., 2008)
5'end of pre mRNA
SKAR POLDIP3 121-islkrssp; S6K1 Component of Recruitment of S6K1 to (Ma et al., 2008)
379-rrvnsas spliceosome, DNA spliceosome
polymerase interaction
protein
CAD CAD 1852-pprihrasdp S6K1 Rate-limiting enzyme for Promoting de novo pyrimidine (Robitaille et al., 2013;
de novo pyrimidine biosynthesis Ben Sahra et al., 2013)
biosynthesis
CCTE; TCPB 255-rvrvdstak S6K1, S6K2, Component of Promoting folding? (Abe et al., 2009;
RSK chaperonin complex for Jastrzebski et al., 2011)
TCP1E protein folding
URI; RMP 366-krkrknstg S6K1 Chaperone and binding Releasing the phosphatase (Gstaiger et al., 2003;
RMP partner of PP1g that becomes active Djouder et al., 2007;
phosphatase Theurillat et al., 2011)
GPR75 GPR75 ? ? Mitochondrial chaperone ? (Jastrzebski et al.,

2011)
Retrograde RICTOR RICTOR 1128-nrrirtltep S6K1 Component of mTORC2 Mild inhibition of mTORC2 (Dibble et al., 2009;
control Treins et al., 2010;
Julien et al., 2010)
mTOR MTOR 2440-krsrtrtdsy S6K1, S6K2, Upstream kinase ? (Holz and Blenis, 2005)
AKT activating S6Ks, master
regulator of cell growth
SIN1 MAPKAP1 79-girrrsnta; S6K1, AKT Component of mTORC2 Inhibition of mTORC2 (Liu et al., 2013)
391-ihrlrfttd
IRS-1 IRS1 301-rrsrtesl; S6K1 Insulin receptor substrate Inhibitory (Harrington et al., 2004;
1096-rrrhsse Tremblay et al., 2007)
Metabolic AMPK PRKAA2 486-pqrscsa S6K1 Kinase sensing energy Inhibitory (Dagon et al., 2012)
program stress and
D2
reprogramming cell
metabolism
PGC1D PPARGC1A 564-rmrsrsr; S6K1, AKT Transcriptional co factor Inhibitory on gluconeogenesis (Lustig et al., 2011)
568-rsrsrsfsr induced by starvation, program
programming metabolism
GSK3D GSK3A 16-rartssf S6K1, S6K2, Inhibition of glycogen Inhibitory (Zhang et al., 2006)
AKT synthase activity, cell
cycle progression
GSK3E GSK3B 4-rprttsf S6K1, S6K2, Inhibition of glycogen Inhibitory (Zhang et al., 2006)
AKT synthase activity, cell
cycle progression
Control of MDM2 MDM2 159-ssrrraise S6K1, S6K2, E3 ubiquitin ligase for Inhibitory, promoting cytosolic (Lai et al., 2010)
Oncogenic AKT p53 retention
factors
GLI1 GLI1 79-kkralsi S6K1 Transcription factor Increasing transcriptional (Wang et al., 2012)
activity and reducing inhibition
by SuFu
BAD BAD 94-rgrsrsa S6K1, AKT Proapoptotic proteins Promoting 14-3-3 binding and (Harada et al., 2001)
binding BCL-2 and BCL- releasing survival factors BCL-
XL 2 and BCL-XL
Nomenclature and sequence are for human genes. Phosphorylation sites are indicated in red, the
surrounding basic residues are in bold.
involved in feedback regulation of signal transduction and cellular metabolism; and

substrates acting as tumor suppressors or oncogenes (Fig.16.1 and Table16.1).
In many cases, the mutation of the S6K phosphorylation site in one single sub-
strate has deceivingly modest effects on functional readouts. However, as we will
see below, one should keep in mind that S6Ks phosphorylate multiple proteins in
each functional class. Possibly, it is the combination of the phosphorylation events
that is required to turn on the whole growth or metabolic program.
16.2.1 Substrates Involved in Protein and Nucleic Acid Synthesis
In starving cells, S6Ks stably associate with the translation initiation factor eIF3
(Holz etal. 2005). Upon growth factor stimulation, mTORC1 is recruited to the 43S
PIC, which is composed of the eIF2/GTP/Met-tRNAiMet ternary complex, the 40S
ribosomal subunit and eIF3, eIF1, eIF1A, and eIF5 translation initiation factors (see
Chap.2). mTOR can then phosphorylate its substrates, S6Ks and 4E-BPs. Active
S6K1 dissociates from eIF3 and in turn phosphorylates components of the transla-
tion machinery, namely the 40S subunits on rpS6 and the initiation factor eIF4B
(Holz etal. 2005).
eIF4B is a cofactor that increases the ATPase activity of the eIF4A helicase that
unwinds secondary structures in the 5 untranslated region (UTR) and stabilizes the
linear mRNA (see Chap.5). eIF4B phosphorylation strongly promotes in vitro as-
sociation with eIF4A (Raught etal. 2004; Shahbazian etal. 2006).
In addition, S6K1 also phosphorylates PDCD4, a tumor suppressor that binds
eIF4A and eIF4G (Dorrello etal. 2006). PDCD4 may function as a negative regula-
tor of eIF4A, either by directly inhibiting helicase activity or by competing for bind-
ing to eIF4G (see Chaps.5, 6 and 16). Growth factor stimulation, through S6K1-
dependent phosphorylation of PDCD4, promotes PDCD4 ubiquitination by the E3
ubiquitin ligase TRCP, thus decreasing PDCD4 protein stability (see Chap.6).
Thus, S6Ks may impact on eIF4A helicase activity by two distinct phosphorylation
events targeting eIF4B and PDCD4. Overexpression of the phosphorylation mutant
PDCD4 slows down cell growth and proliferation, suggesting that this substrate
may participate in the action of S6Ks on growth (Dorrello etal. 2006). However
caution should be used in the interpretation of overexpression studies that may lead
to the titration of other signal transduction elements. In particular, these artefactual
elements may explain the controversial effects observed in the studies of eIF4B
overexpression (Raught etal. 2004; Shahbazian etal. 2006).
Although these modifications may be consistent with the role of S6Ks in trans-
lation initiation, the effects of S6K inhibition on global translation initiation are
mild. S6K-deficient cells have polysomal profiles and methionine incorporation
rates (that reflect the translational status of the cell) similar to control cells (Mieu-
let etal. 2007; Espeillac etal. 2011; Chauvin etal. 2013). Consistently, the po-
tency of rapamycin and the novel ATP-competitive mTOR inhibitors on transla-
tion initiation correlates with their ability to inhibit 4E-BP phosphorylation, rather
than S6K (Choo etal. 2008; Feldman etal. 2009; Thoreen etal. 2009). Rapamycin
treatment, which fully blocks S6K phosphorylation, but has partial and reversible
effects on 4E-BP phosphorylation, is not a potent inhibitor of protein synthesis,
typically reducing translation of a cap-dependent reporter by 1025% (Choo etal.
2008).
One possibility is that S6K activity favors the translation of a subset of mRNAs.
However, it is now clear that the translation of 5 TOP mRNAs, which is controlled
by mTORC1 activity and is inhibited by rapamycin, does not require S6K activity
and rpS6 phosphorylation (Pende etal. 2004; Ruvinsky etal. 2005). Furthermore,
microarray analysis on polysome-associated translating mRNAs in S6K1/S6K2 /
cells failed to identify specific mRNAs under translation control by this pathway
(Chauvin etal. 2013). Future studies, using ribosome profiling experiments, should
provide the accuracy to detect putative modifications in translation initiation rates
of specific mRNAs.
Intriguingly, S6Ks may have a key role in the pioneer round of translation. During
splicing in the nucleus, the cap-binding complex, the transcription-export complex
and the exon junction complex are recruited to the precursor mRNAs (pre-mRNAs).
After export to the cytosol, these complexes remain associated to the mRNA at the
beginning of the pioneer round of translation and improve translation efficiency.
During or at the end of the pioneer round of translation, these complexes dissoci-
ate from the mature mRNA and the cap-binding complex at the 5 end is replaced
by the cap-binding protein eIF4E (Ma etal. 2008). While active S6K1 dissociates
from eIF4E-bound mRNAs, it remains stably associated with cap-binding complex-
bound mRNAs and is required for the phosphorylation of multiple proteins associ-
ated with newly synthesized transcripts in mRNP (Ma etal. 2008). Among the iden-
tified S6K1-dependent phosphoproteins are the cap-binding protein CBP80 and the
exon junction complex constituent, the S6K1 Aly/REF-like target (SKAR). Of note,
knockdown of S6K1 decreases translation efficiency of intron-containing reporter
mRNAs (Wilson et al. 2000; Ma etal. 2008). Thus, the prominent role of S6K1
in the pioneer round of translation versus the translation of eIF4E bound mRNAs,
might explain the scarce impact of S6K inhibition on global translation initiation.
S6Ks may affect other steps in the translation process, such as translation elon-
gation, termination and fidelity that are not detected by classical translation initia-
tion assays. A major regulatory node in the control of translation elongation is the
phosphorylation of the elongation factor eEF2 on Thr56 by eEF2K, which has an
inhibitory role on eEF2 activity (Wang etal. 2001) (see Chap.12). eEF2K, in turn,
contains multiple sites of phosphorylation targeted by upstream kinases. S6K1 and
S6K2 phosphorylate Ser366 of eEF2K, thus decreasing eEF2K catalytic activity
and promoting eEF2 action on the translocation of ribosomes one codon forward on
the mRNA (Wang etal. 2001).
The role of rpS6 phosphorylation remains unclear. In a recent atomic structure
of the yeast ribosomes by crystallography, rpS6 is found close to the binding site
for eIF4G and interacts with large ribosomal subunit protein rpL24 of the large 60S
subunit (Jenner etal. 2012). Interestingly, rpL24 is involved in translation reini-
tiation of polycystronic mRNAs by mediating the retention of eIF3 to ribosomes.
However, it is presently unknown whether rpS6 phosphorylation modifies these
interactions, as the recombinant rpS6 used in these studies lacked the C-terminal tail
containing the phosphorylation sites.
Both S6K1 and S6K2 phosphorylate five serine residues in mammalian rpS6,
Ser 235, 236, 240, 244 and 247 (Krieg etal. 1988). Strikingly, knock-in muta-
tions of these Ser residues to Ala (rpS6P/), which impair rpS6 phosphorylation,
have the tendency to increase, rather than decrease, protein synthesis (Ruvin-
sky etal. 2005). This surprising evidence has been accumulated in a num-
ber of experimental systems, including cultured MEFs, liver tissue after feed-
ing and lymphomas expressing AKT (Ruvinsky etal. 2005; Hsieh et al. 2010;
Chauvin et al. 2013). The simplest explanation would be that rpS6 phosphoryla-
tion inhibits, rather than stimulates, protein synthesis. It is also possible that the
rpS6 phosphorylation mutants may trigger compensatory mechanisms stimulating
translation initiation, for instance through an increase in 4E-BP phosphorylation.
Finally, rpS6 phosphorylation may not play a direct role in translation, but rather
act by coordinating cellular responses with the protein synthetic demand. Along
this line, it is interesting that both S6K activity and rpS6 phosphorylation are in-
volved in the synthesis of ribosome biogenesis factors that are small nucleolar
ribonucleoproteins (snoRNPs) and proteins required for the rRNA processing
and its assembly with ribosomal proteins. Livers from S6K1/S6K2/ and rpS6P/
mice downregulate the RNA of many ribosome biogenesis factors, suggesting a
direct or indirect effect of rpS6 phosphorylation on their transcription in the nu-
cleus (Chauvin etal. 2013).
The regulation of chaperon-like proteins may be an emerging role of S6Ks. S6Ks
phosphorylate the chaperonin containing TCP1, subunit 2 (CCT) of the chapero-
nin complex (Abe etal. 2009; Jastrzebski etal. 2011). Chaperons interact with the
hydrophobic regions of unfolded proteins, preventing aggregation and promoting
refolding. Although CCT phosphorylation by the mTORC1/S6K axis correlates
with increased folding efficiency, as assessed by globular G actin folding assays,
both phospho-mimetic and phospho-ablating CCT mutants display impaired fold-
ing as compared to wild-type counterparts (Jastrzebski etal. 2011). It is also puz-
zling that overactivation of the mTORC1 pathway, following high fat diet or genetic
inactivation of the tumor suppressors TSC1/2, causes endoplasmic reticulum stress
and protein folding defects (Ozcan etal. 2008). It would be interesting to assay the
functional output of chaperonin complex regulation by S6Ks in conditions of endo-
plasmic reticulum stress triggered by mTORC1 overactivation.
In addition to CCT another putative substrate of S6Ks is the mitochondrial
chaperone GRP75 (also known as heat shock 70kDa protein 9 (HSPA9)), though
this requires further validation (Jastrzebski etal. 2011). The unconventional pre-
foldin RPB5 interactor 1 (URI) protein, a member of the prefoldin family of chap-
erones and a binding partner of the PP1 phosphatase, is also phosphorylated by
S6K1 (Gstaiger etal. 2003; Djouder etal. 2007; Theurillat etal. 2011). This event
frees the PP1 phosphatase that can act on S6K1 itself and on specific S6K1 sub-
strates, thus terminating S6K1 signaling by a negative feedback mechanism.
A new aspect of the biological actions of S6Ks has recently come from two
studies using phosphoproteomics or metabolomics approaches in cells with per-
turbed mTORC1 signaling. The first demonstrates that CAD (carbamoyl-phos-

phate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a rate-limit-
ing enzyme in de novo pyrimidine biosynthesis, is differentially phosphorylated in
mTORC1-deficient cells versus mTORC1 positive control (Robitaille etal. 2013).
The latter reveals that N-carbamoyl aspartate, the metabolite produced by CAD, is
accumulated in cells with hyperactive mTORC1 (Ben Sahra etal. 2013). De novo
pyrimidine biosynthesis leads to the production of UTP, CTP, dTTP and dCTP
from glutamine, HCO3, aspartate and ribose. S6K1 plays a key role in the trans-
duction of mTORC1 signals to the de novo synthesis of pyrimidines, by directly
phosphorylating CAD on Ser1859 and promoting its activity. Consistently, modu-
lating S6K1 activity affects incorporation of pyrimidines into RNA and DNA in
situations that rely on their de novo production from aspartate. In conclusion, these
elegant studies reveal metabolic conditions in which proliferation, rRNA synthe-
sis and ribosome biogenesis, and ultimately protein synthesis, may significantly
depend on S6K1 due to its function in replenishing the intracellular pyrimidine
pools.
16.2.2 Substrates Involved in Feedback Regulation
By looking at the list of S6K substrates, it is striking that many of these are actually
upstream elements in the mTOR signal transduction pathway (see second section
of Table16.1). Therefore it is clear that an important function of S6Ks is the ret-
rograde feedback control of the mTOR pathway and growth factor and nutrient-
dependent signaling. S6Ks phosphorylate mTOR itself on Thr2446 and Ser2448,
however the functional relevance of these phosphorylation sites is unclear (Holz
and Blenis 2005). S6Ks also phosphorylate two components of the mTOR complex
2 (mTORC2), namely RICTOR and stress-activated MAPK-interacting protein 1
(SIN1) (Dibble etal. 2009; Julien etal. 2010; Treins etal. 2010). Although there are
some controversies on the function of each phosphorylation event, there is a general
consensus that S6Ks act by inhibiting the mTORC2 substrate AKT. This molecular
mechanism likely plays an important role in the well-known insulin-resistant state
triggered by an excess of nutrients (Ozcan etal. 2008). Since one major function
of insulin and other growth factor peptides is to stimulate nutrient uptake and us-
age, cells have developed negative feedback mechanisms in which nutritional cues
inhibit action of the growth factors. The inhibitory effects of the nutrient-stimulated
mTORC1/S6K axis on the insulin/growth factor-stimulated mTORC2/AKT axis is
certainly involved in this important nutritional checkpoint. Phosphomimetic SIN1
mutants are unable to interact with RICTOR and mTOR, while phospho-ablative
mutants increase AKT phosphorylation (Liu etal. 2013). Similarly, two studies
have shown a mild inhibitory effect of RICTOR phosphorylation on AKT activity
(Dibble etal. 2009; Julien etal. 2010).
Another major node of feedback regulation is the insulin receptor substrates 1
and 2 (IRS-1 and IRS-2). The IRS phosphorylation on tyrosine (Tyr, Y) residues
by the insulin receptor and IGF-1 receptor (IGF-1R) initiates the growth factor ac-
tion. Subsequently, the IRS phosphorylation on Ser and Thr residues contributes to
shutting down the signal by promoting protein degradation or decreasing the inter-
action with the receptors (Harrington etal. 2004; Tremblay etal. 2007). S6Ks are
among the kinases regulating Ser/Thr phosphorylation of IRS. The increase in in-
sulin sensitivity of S6K1-deficient mice correlates with a decrease in IRS-1 Ser/Thr
phosphorylation (Um etal. 2004). Because of the multiple phosphorylation sites on
IRS-1 and the general amelioration of insulin sensitivity upon S6K1 inactivation,
it is difficult to demonstrate which IRS-1 sites are directly targeted by S6Ks. How-
ever, considering the phosphorylation motif and the results of in vitro kinase assays,
Ser307 and Ser1101 of IRS-1 are likely to be direct substrates of S6K1 (Harrington
etal. 2004; Tremblay etal. 2007).
16.2.3 Substrates Involved in Cellular Metabolism
It is becoming increasingly appreciated that metabolic adaptations play a crucial

role in tissue growth and tumorigenesis, as they may provide cellular components
to increase cell mass during proliferation, as well as the energy to face low oxygen
and low nutrient environment (Vander Heiden etal. 2009). S6Ks can be considered
as an anabolic switch for metabolic programs, while AMP-activated protein kinases
(AMPKs) function as catabolic switches (Hardie 2011). The former are activated
in conditions of food plenty, while the latter during starvation and increased AMP/
ATP ratio. S6Ks promote storage of nutrient excess as fat by multiple mechanisms,
possibly including commitment of embryonic stem cells to adipogenic precursors
and sterol regulatory element-binding protein (SREBP) processing, although some
of these effects are controversial (Carnevalli etal. 2010; Lewis etal. 2011; Yecies
etal. 2011). Conversely, AMPKs stimulate fatty acid oxidation, mainly by con-
trolling acetyl-CoA carboxylase activity (Fullerton etal. 2013). The crosstalk be-
tween S6K and AMPK pathways is reinforced by their mutual regulation. AMPKs
inhibit mTORC1 signaling through the phosphorylation of RAPTOR and TSC2
(Inoki etal. 2003; Gwinn etal. 2008). S6K1 inhibits AMPK activity by directly
phosphorylating the catalytic subunit of AMPK and by modulating the AMP/ATP
ratio (Aguilar etal. 2007; Dagon etal. 2012).
Besides AMPK, another master regulator of the metabolic program in response
to starvation is the PPAR coactivator 1 (PGC1 that is encoded in humans by the
PPARGC1A gene), which associates with different transcription factors to upregu-
late the expression of genes involved in mitochondrial biogenesis, fatty acid oxida-
tion and gluconeogenesis (Lin etal. 2005). PGC1 contains a basic domain with
multiple putative phosphorylation sites for AGC protein kinases. Recently, it has
been proposed that Ser569 and Ser573 PGC1 are phosphorylated by S6Ks, based
on their sensitivity to rapamycin and the ability of S6K1 to phosphorylate them in
vitro (Lustig etal. 2011). However, S6K1 loss-of-function approaches were not
tested in vivo to validate these results. Of note, adenoviral mediated overexpression
of the phospho-ablating PGC1 mutant potentiates the gluconeogenic gene pro-
gram in cultured hepatocytes and in liver in vivo, without altering the mitochondrial
biogenesis and fatty acid oxidation programs. Thus the phosphorylation of PGC1
on these residues has the potential to suppress gluconeogenesis but not fatty acid
oxidation, reinforcing a metabolic program that may be suitable in high caloric diet
conditions. It is confusing that S6K1-deficient mice display increased fatty acid
oxidation without apparent alterations in gluconeogenesis (Um etal. 2004). One
possibility is that the additional S6K-dependent programs on fatty acid oxidation,
including the AMPK regulation, may be more prominent in standard chow condi-
tions. Clearly, further in vivo studies under different nutritional regimens are needed
to dissect the role of S6Ks on PGC1 function.
16.2.4 Substrates Acting as Tumor Suppressors or Oncogenes
A number of oncogenes and tumor suppressors are directly controlled by S6Ks (see
fourth section of Table16.1). Although control of these master regulators of growth
and development is complex, integrating a variety of upstream signals, the input
from S6Ks might have a specific role in the transduction of signals from nutrition.
MDM2 is the E3 ubiquitin ligase controlling the degradation of p53, the tumor
suppressor most commonly mutated in human cancer. In cultured MEFs, in the
presence of serum and the DNA-damaging agent doxorubicin, S6K1 contributes to
the phosphorylation of MDM2 on Ser166 and its retention in the cytosol (Lai etal.
2010). This potentiates the p53-dependent transcriptional response to DNA-damag-
ing agents, and in particular may explain how nutrients and growth factors can favor
the proapoptotic and prosenescent function of p53. However it should be noted that
the effects of S6K1 inhibition on MDM2 and p53 are partial, suggesting parallel
modes of regulation. In addition, they could be observed in primary cells, but not in
cancer cell lines, indicating that some tumors have escaped this checkpoint.
While mTORC1/S6Ks may promote apoptosis during DNA damage, they may
promote survival in growth permissive conditions. BCL-2 antagonist of cell death
(BAD) is a proapoptotic BH3-only protein acting on BCL-2 and B-cell lymphoma
extra large (BCL-XL) proteins. Upon growth factor stimulation, BAD phosphoryla-
tion by AGC kinases, including S6Ks, promotes association with 14-3-3 proteins
and sequesters BAD in the cytosol, thus freeing mitochondrial BCL-2 and BCL-XL
to sustain survival (Harada etal. 2001).
Some developmental programs regulating cell fate determination and pattern
formation can also be regulated by S6Ks. Glioma-associated oncogene homolog
proteins (GLI) are transcription factors regulating survival genes downstream the
Hedgehog pathway. They may have a tumorigenic role, especially in specific can-
cers, such as those of the esophagus (Wang etal. 2012). TNF can also activate GLI
activity in a Hedgehog pathway-independent and mTOR/S6K-dependent manner
(Wang etal. 2012). S6K1 can phosphorylate GLI1 on Ser84 after TNF treatment,
enhancing its oncogenic function and reducing inhibition by suppressor of fused
(SUFU) factors (Wang etal. 2012).
16.3 S6 Kinases and Cancer
As described in various chapters of this book, the mTORC1 pathway is upregu-

lated in the majority of human cancers. A variety of somatic mutations leads to
unrestrained mTORC1 activation. These include gain of function mutations in
PI3K, RAS, AKT, and loss of function mutations in TSC1, TSC2, PTEN, LKB1
and AMPK. Thus, one may predict that S6K activity favors progression in a subset
of cancers relying on mTORC1 activity. This hypothesis has been proven in mice
carrying inactivation of one PTEN allele (Nardella etal. 2011). These mice develop
a wide spectrum of neoplasia in different tissues, and in particular a deadly lymph-
adenopathy. Strikingly, while S6K1 inactivation does not improve the lymphade-
nopathy and survival curve of PTEN+/ mice, it is sufficient to strongly reduce the
penetrance of pheochromocytomas, due to an antiproliferative effect on the chro-
maffin cells in the medulla of adrenal glands. S6K1 is also required for insulinoma
formation in pancreatic -cells expressing oncogenic AKT1 (Alliouachene et al.
2008). Taken together, these initial screenings for S6K1-dependent tumorigenesis
suggest that endocrine tumors particularly rely on this pathway.
The future challenge is to predict the tumors that respond to therapies against
S6K activity. For this purpose, further studies in the mouse model of cancer will
prove to be valuable. In addition, it is conceivable that the S6K-dependent tumors
will be a subset of the human cancers that respond to therapies with rapamycin de-
rivatives (rapalogs), as rapamycin is a potent inhibitor of the S6K branch of mTOR
signaling. As presented in other chapters of this book, rapalogs are approved for
treatment of RCC, mantle cell lymphomas, and the clinical manifestations of tuber-
ous sclerosis complex, such as angiomyolipomas, lymphangioleiomyomatosis and
SEGAs (see PartIV). It is likely that ongoing clinical trials will increase the spectra
of neoplasia that can be treated with rapalogs. Moreover, the response rate to the
rapalog treatment should be improved if patients with mutations and biomarkers
corresponding to mTORC1/S6K hyperactivity were selectively recruited.
Is there an interest in developing S6K specific inhibitors for cancer? Although
rapalogs are relatively well tolerated, long-term administration may have undesir-
able side effects, including dyslipidemia, hyperglycaemia, stomatitis, skin rash,
lung fibrosis, leucopoenia and anemia. It is likely that some of the adverse meta-
bolic effects are due to mTORC2 inhibition by long-term rapalog treatment. In this
case, it can be envisaged that S6K specific inhibitors would present the advantage to
limit insulin resistance, and potentially improve insulin sensitivity. Conversely, the
therapeutic response rate is not expected to be higher than with rapalogs. Combina-
torial therapies could be required, also considering the development of resistance.
16.4 Potential Mechanisms of Resistance
Tumor cells, initially relying on mTORC1/S6K signaling for proliferation, growth

and survival, may develop resistance to inhibitors of the pathway. A major mecha-
nism of resistance is the functional redundancy between S6K, AKT and RSK (Ta-
ble16.1). Many S6K substrates can also be phosphorylated by RSK in vivo. This
has been demonstrated for rpS6, eEF2K, eIF4B, CCT but probably also applies
to additional targets (Wang etal. 2001; Pende etal. 2004; Shahbazian etal. 2006;
Abe etal. 2009). Similarly, BAD, PGC1, MDM2, GSK3 phosphorylation may be
regulated by both S6K and AKT (Harada etal. 2001; Zhang etal. 2006; Li etal.
2007). Thus, tumor cells can, in a compensatory way, upregulate AKT and RSK to
circumvent a block of S6K activity.
The regulation of AKT activity is actually an important biological action of S6Ks
(see above). Depending on the tumor type, transformed cells might directly have
a growth advantage in suppressing S6K action, as this may result in unrestrained
AKT activity. Along this line, SIN1 mutations in cancer tend to disrupt the S6K
phosphorylation motif. This leads to the inability of S6K in phosphorylating SIN1
and in suppressing AKT activity, which is responsible for augmented cancer cell
survival and resistance against chemotherapeutic agents, such as etoposide and cis-
platin (Liu etal. 2013).
Albeit rarer than the previously described cases, another possible mechanism of
resistance are adaptive changes that bypass the need of high mTORC1 signaling and
maintain S6K activity even in presence of mTOR inhibitors, such as rapalogs. URI
overexpression, which is detected in some ovarian cancer cell lines due to gene am-
plification, can act in this way by extinguishing the PP1 -mediated dephosphoryla-
tion of S6K1 and BAD (Theurillat etal. 2011).
Even when the upregulation of the S6K pathway confers an advantage to cancer
cells for tumor initiation, the use of S6K inhibitors should be considered with cau-
tion as it could favor survival mechanisms of cancer cells in stress conditions. For
instance, the AMPK activation following S6K inhibition might slow down cancer
proliferation, but could also allow cancer cells to cope with the energy stress in
nutrient poor conditions. Similarly, transformed cells display an increase in transla-
tion elongation rates but also an increased susceptibility to cell death triggered by
nutrient deprivation. In this context, it is interesting to consider recent studies on
eEF2K, whose inhibitory activity on eEF2 is partially relieved by S6Ks (Leprivier
etal. 2013). Of note, eEF2K overexpression, as it has been observed in glioblas-
tomas, can provide a survival advantage (Leprivier etal. 2013). eEF2K deletion,
while upregulating translation elongation, increases sensitivity of tumors to caloric
restriction.
In conclusion, S6Ks appear as key mediators of organismal growth, development

and metabolism, sensing nutrient availability and coordinating specific anabolic
programs in the cell. From the available list of S6K substrates, four distinct pro-
grams have insofar clearly emerged (also see Table16.1 and Fig.16.1). Among
them, the control of nucleic acid and protein synthesis plays an important role, if
we consider the amount of targets. However, we still need to define the function
of some regulatory mechanisms. The first one, but not the sole, is the function of
rpS6 phosphorylation. New technical developments (e.g. ribosome profiling) and
simple organism (e.g. yeast) models should help address these outstanding ques-
tions. We should also consider some underappreciated aspects of translational con-
trol by mTORC1/S6K, such as protein folding and fidelity, which may be particu-
larly important in the pathophysiological conditions of cancer and ageing. Whether
the translational control or other S6K-dependent anabolic programs are relevant for
tumorigenesis also remains to be clarified. It is likely that mechanistic studies on
endocrine tumors that heavily rely on S6K activity, such as pheochromocytomas
and insulinomas, would provide valuable insights. These perspectives will be de-
veloped in an exciting combination of basic science on translation and metabolism,
nutrition and ageing, animal models, clinical trials, pharmacology, human genetics.
Therefore, S6Ks should be considered as therapeutic targets against cancer with
the following rationale: (1) for tumors that heavily rely on this pathway, if the prom-
ising results of preclinical trials on endocrine tumors can be transposed from mice
to humans; (2) in a context of personalized medicine, when the genetic and bio-
marker signatures point to the hyperactivation of S6Ks as a putative initiating event
in tumorigenesis; and (3) in combinatorial therapies with other pharmacological
agents, to limit resistance and improve response rates. Although the adaptive chang-
es in cancer cell signal transduction add a high degree of complexity in the design
of therapeutic strategies against tumors, they also offer the opportunity of synthetic
lethal approaches that specifically affect mechanisms of resistance in cancer cells
and that are better tolerated by the healthy cells.
Acknowledgements We are grateful to the members of INSERM-U845 for support, and to Ste-
fano Fumagalli, Ganna Panasyuk and Yves de Keyzer for critically reading the manuscript. We
thank Jean Pierre Laigneau for his expert help on the preparation of figures. M.P. is a recipient of
grants from European Research Council, Institut National du Cancer, Association Sclerose Tuber-
euse de Bourneville, the Association pour la Recherche sur le Cancer, Contrat dinterface avec
lHopital. C.T. is supported by a grant from Institut National du Cancer.
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Chapter 17
eIF4E Phosphorylation Downstream of MAPK
Pathway
Luc Furic, Emma Beardsley and Ivan Topisirovic
Contents
17.2eIF4E Phosphorylation Downstream of the MEK/ERK/MNK and
p38/MNK Pathways in Cancer 365
17.3Pharmacological Inhibitors of eIF4E Phosphorylation 367
17.4Therapeutic Resistance and Radioresistance 367
17.5Novel Therapies Impacting eIF4E Activity 368
References 370
Abstract Tumor cells are dependent on high protein synthesis rates to fuel their
neoplastic growth. eIF4E is a 5 mRNA cap-binding protein that recruits mRNA
to the ribosome and exhibits oncogenic properties. Mitogens, nutrients and growth
factors stimulate the MAPK and PI3K pathways that converge on eIF4E. eIF4E is
phosphorylated by the MNKs at a single site (Ser209 in mammals), which stim-
ulates translation of a subset of tumor-promoting mRNAs, thereby bolstering its
oncogenic properties. In this chapter, we will discuss the molecular mechanisms
underlying the role of eIF4E phosphorylation in neoplasia and its potential clinical
applications.
L.Furic() E.Beardsley
Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC,
Australia
e-mail: luc.furic@monash.edu
I.Topisirovic
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital,
364 L. Furic et al.
17.1Introduction
Increased level of activity of the mitogen-activated protein kinase (MAPK) path-

way and the PI3K/AKT/mTOR pathway are responsible for survival and prolifera-
tion signaling in a wide spectrum of tumors (Castellano and Downward 2010; Furic
etal. 2009; McKay and Morrison 2007). These two pathways impinge on one of
the most energy-consuming process in the cell, protein synthesis (Buttgereit and
Brand 1995; Rolfe and Brown 1997; Roux and Topisirovic 2012). Cancer cells ex-
hibit elevated rates of protein synthesis (Silvera etal. 2010; Topisirovic and Sonen-
berg 2011; Topisirovic etal. 2011; van Riggelen etal. 2010). eIF4E is a 5 mRNA
cap-binding protein that recruits mRNA to the ribosome and exhibits oncogenic
properties (Avdulov etal. 2004; Lazaris-Karatzas etal. 1990; Ruggero etal. 2004)
(see Chap.4). eIF4E associates with eIF4G, a large scaffolding protein, and the
helicase eIF4A to form the heterotrimeric protein complex termed eIF4F (Jackson
etal. 2010). Availability of eIF4E to bind the 5 cap and to associate with eIF4G is
regulated by the phosphorylation status of 4E-BPs (Gingras etal. 1999, 2001; Pause
etal. 1994) via mTOR (see Chap.4).
eIF4E was first identified as a phosphoprotein by isoelectric focusing (Rychlik
etal. 1986). Subsequent determination of phospho-acceptor sites was performed by
phosphopeptide mapping and phospho-amino acid analysis using thin layer chro-
matography and thin layer electrophoresis (Rychlik etal. 1987). The first reported
site of phosphorylation was mistakenly located at serine 53 near the cap-binding
pocket. The addition of a negatively charged phosphate group on eIF4E lead to
the hypothesis that phosphorylation would essentially increase or decrease the af-
finity for the 5 cap structure. The actual phosphorylation site was later reassigned
to Ser209, a serine residue located in the C-terminal region of eIF4E (Flynn and
Proud 1995; Joshi etal. 1995; Makkinje etal. 1995). Based on the crystal structure
obtained of eIF4E bound to a cap analog (Marcotrigiano etal. 1997a, b), it was
hypothesized that the phosphate group on Ser209 could form a salt bridge with
K159 and potentially increase or stabilize the association of eIF4E with the 5 cap.
Subsequent studies have reported conflicting results on the direct impact of eIF4E
phosphorylation on cap-binding, whereby it was suggested that phosphorylation in-
creases (Shibata etal. 1998) or decreases (Scheper etal. 2002; Zuberek etal. 2003)
the affinity of eIF4E for the cap. Notwithstanding that it remains unclear to which
extent phosphorylation affects the in vivo affinity of eIF4E for the cap structure,
eIF4E phosphorylation is not required for global protein synthesis, as shown by the
ability of eIF4ES209A mutant to rescue translation in vitro and in vivo (McKendrick
etal. 2001). However, a subset of mRNAs display increased level of translation in
response to eIF4E phosphorylation (Furic etal. 2010; Wendel etal. 2007).
Two kinases were identified with the capacity to phosphorylate eIF4E on a sin-
gle residue (Serine 209), namely the MAPK-interacting serine/threonine-protein
kinases 1 and 2 (MNK1 and MNK2) (Fukunaga and Hunter 1997; Waskiewicz
etal. 1997). Although, initial reports suggested that PKC could also directly phos-
phorylate eIF4E (Whalen etal. 1996), no strong evidence supports the existence of
such an event in vivo (Frederickson etal. 1991, 1992; Frederickson and Sonenberg
17 eIF4E Phosphorylation Downstream of MAPK Pathway 365
1992). The phosphorylation of eIF4E occurs via a two-step process requiring the in-
teraction of both eIF4E and MNK with eIF4G, which acts as a scaffold, to position
the two proteins for the catalytic event. This substrate presentation event potentially
allows for increased specificity. MNK1 and MNK2 are alternatively spliced and
yield two isoforms each termed MNK1a/1b and MNK2a/2b (OLoghlen etal. 2004;
Slentz-Kesler etal. 2000). Only the longer versions of both kinases contain the
ERK-interacting site in its C-terminus (Fukunaga and Hunter 1997; Scheper etal.
2001; Waskiewicz etal. 1997). All MNK isoforms contain an eIF4G-interacting
domain in their N-terminus. The splicing factor SF2/ASF controls the splicing of
MNK2 pre-mRNA stimulating the expression of the MNK2b isoform (Karni etal.
2007). Even though MNK2b lacks the MAPK-interacting site and has low basal ac-
tivity relative to other MNK isoforms, it is considered to be a constitutively-active
isoform of MNK2 that can lead to an increase in eIF4E phosphorylation when
overexpressed (Scheper etal. 2003). It has also been proposed that the dephos-
phorylation of eIF4E could be regulated via the interaction of PP2A with eIF4E and
MNK (Li etal. 2010).
The mechanistic implications of eIF4E phosphorylation remain unanswered.
Potential mechanisms include: altering the affinity of eIF4E for the 5cap; affect-
ing the stability of the eIF4F complex; modulating the affinity of the 4E-binding
proteins (4E-BPs) for eIF4E; and modulating the rate of eIF4F formation (Morley
etal. 1993a, b).
The role of Ser209 phosphorylation in homeostasis remains largely speculative.
Some studies have suggested that Ser209 could be involved in stimulating the local
translation of silenced mRNAs at the synapse (Dagestad etal. 2006; Kanhema
etal. 2006). High levels of eIF4E phosphorylation have also been reported in non-
cancerous tissues associated with neurodegenerative diseases (e.g. Alzheimers le-
sions) (Li etal. 2004). Angiotensin II was reported to stimulate eIF4E phosphoryla-
tion in vascular smooth muscle cells (Rao etal. 1994). However, numerous reports
of increased levels of phosphorylated eIF4E in cancerous compared to normal tis-
sues exist and many of these studies have linked increased levels of phosphorylated
eIF4E with pathophysiological parameters and clinical outcome (see below).
17.2eIF4E Phosphorylation Downstream of the MEK/

ERK/MNK and p38/MNK Pathways in Cancer
eIF4E was first reported to be an oncogene in 1990 (Lazaris-Karatzas etal. 1990)

. Since then, high levels of Ser209 phosphorylation have been reported in multiple
human cancerous tissues (Fan etal. 2009) including brain (Tejada etal. 2009) (high-
er in meningioma than in astrocytic and oligodendroglial tumors) , breast (Wheater
etal. 2010), prostate (Furic etal. 2010; Graff etal. 2009), lung (Yoshizawa etal.
2010), head and neck (Frederick etal. 2011), leukemic cells (Lim etal. 2013), ovary
(Noske etal. 2008), penile (Ferrandiz-Pulido etal. 2013) and gastric (Liang etal.
2013) cancers, among others.
366 L. Furic et al.
Fig. 17.1 Signaling pathways

regulating phosphorylation
of eIF4E. eIF4E activity is
regulated by the PI3K (blue)
and MAPK pathways. Avail-
ability of eIF4E to assemble
into the eIF4F complex by
binding eIF4G is regulated
by the phosphorylation status
of the 4E-BPs. When bound
to eIF4G, eIF4E can be phos-
phorylated by the MNKs.
MNKs activity is regulated
downstream of the p38/
MAPK stress signaling path-
way (orange) and the RAS/
RAF/MERK/ERK mitogenic
signaling pathway (green).
An increase in phosphoryla-
tion of eIF4E on Ser209
stimulates the translation of
a subset of tumor-promoting
mRNAs.
MNK1 and MNK2 are activated downstream of stress (p38) and mitogenic (MEK/
ERK) signaling (Fig.17.1) (Buxade etal. 2008). Overexpression of MNK1 has been
observed in glioblastoma (Grzmil etal. 2011). Although MNK2 is constitutively ac-
tive, little data supports the overexpression of this protein in cancer. Development
of more reliable antibodies will help in determining if MNK2 is overexpressed in
cancer. Results have emerged adding a layer of complexity to the oncogenic activity
of MNK2 through the selective expression of the constitutively active MNK2b iso-
form in cancer cells (Adesso etal. 2013). A recent report (Stead and Proud 2013) un-
covered a mechanism by which partial inhibition of mTOR activity with rapamycin
decreases the phosphorylation of Ser437 in MNK2, leading to increased phosphory-
lation of eIF4E. The authors hypothesized that a change in conformation, possibly
relieving steric hindrance of the C-terminus on the N-terminus (Parra etal. 2005), is
responsible for the increase in MNK2 activity towards eIF4E. Further characteriza-
tion will be required especially considering that conflicting results were obtained
with MNK2 cells treated with rapamycin (Wang etal. 2007) .
The deletion of both MNK1/2 genes or the expression of a phosphorylation de-
ficient mutant (Ser209Ala) of eIF4E in primary and immortalized mice cells and
xenografts demonstrated that phosphorylation of eIF4E bolsters its oncogenic po-
tential (Furic etal. 2010; Topisirovic etal. 2004; Ueda etal. 2010; Wendel etal.
2007). One of the key studies to first demonstrate the in vivo importance of eIF4E
phosphorylation in tumor progression used a model of lymphocytes transfer to re-

constitute the immune compartment following lethal dose irradiation. This model
allowed demonstrating that eIF4E phosphorylation was required for eIF4E tumori-
genic potential in vivo (Wendel etal. 2007). This study was based on forcing the ex-
pression of eIF4E, but did not answer the question regarding the dependence of tu-
mors on eIF4E. Two papers later confirmed this. First, Ueda and colleagues used the
MNK1/2 DKO mice to demonstrate that lymphoma formation was inhibited in the
absence of eIF4E phosphorylation (Ueda etal. 2010). These findings showed the re-
quirement of MNK1/2 for tumorigenesis but did not underpin the substrate through
which MNKs are involved in cancer biology, leaving the possibility of substrates
other than eIF4E that might be responsible for the reduced tumor burden. Second,
prostate tumorigenesis initiated by the loss of the tumor suppressor PTEN requires
eIF4E phosphorylation to form invasive adenocarcinomas (Furic etal. 2010). When
considered collectively, these studies demonstrate that eIF4E phosphorylation plays
a major role in tumorigenesis and tumor progression.
17.3Pharmacological Inhibitors of eIF4E

Phosphorylation
The majority of the literature interested in small molecule inhibitors of MNK1/2

reports results obtained using CGP57380 (Chrestensen etal. 2007; Morley and
Naegele 2002; Pons etal. 2011; Shi etal. 2013). However, CGP57380 turned out to
possess MNK-independent effects (Bain etal. 2007). Therefore, many laboratories
have performed screens to identify and develop more specific MNK1/2 inhibitors
(Hou etal. 2012; Konicek etal. 2011; Oyarzabal etal. 2010). One of these efforts
led to the identification of cercosporamide as a low nanomolar range inhibitor of
MNK1/2. Cercosporamide was initially identified as an antifungal agent targeting
a yeast-specific PKC isoform (Sussman etal. 2004). It has shown promising results
in inhibiting myeloid leukemia precursor cells (Altman etal. 2013) and lung metas-
tasis after injection of B16 melanoma cells in mouse (Konicek etal. 2011). This in-
hibitor was screened for selectivity against a panel of 76 kinases and demonstrated
high selectivity and affinity to MNK, with an IC50 of 116nM for MNK1 and 11nM
for MNK2. Additional analogs of cercosporamide are currently being developed
(Bazin etal. 2013).
17.4Therapeutic Resistance and Radioresistance
Initial reports showed that treating cells with rapamycin would induce and increase
the amount of eIF4E phosphorylated at Ser209 (Sun etal. 2005). These counterin-
tuitive results were pivotal to the elucidation of the feedback loop downstream of
S6K, which attenuates insulin receptor substrate (IRS-1/2) driven signaling (Har-
rington etal. 2005).
368 L. Furic et al.
Gemcitabine, a chemotherapeutic drug, was also shown to increase eIF4E phos-

phorylation through an MNK2-dependent mechanism (Adesso etal. 2013). Previ-
ous reports of rapamycin induced eIF4E phosphorylation were explained by the
partial inhibition of mTORC1 resulting in inhibition of phospho-S6K, but not
phospho-4E-BP (Choo etal. 2008). This asymmetry in mTORC1 substrates inhi-
bition with rapamycin supports this model, considering that active site inhibitors
of mTOR (asTORi) do not stimulate eIF4E phosphorylation (Furic etal. 2010).
asTORi-induced inhibition of eIF4E phosphorylation is caused by the increase in 4E-
BP/eIF4E complex formation, which precludes eIF4E from interacting with eIF4G
and being phosphorylated by MNK (Mller etal. 2013). In addition, it remains
possible that asTORi inhibitory activity towards PI3K also inhibits upstream signal-
ing responsible for eIF4E phosphorylation.
17.5Novel Therapies Impacting eIF4E Activity
If we consider that the relative ratio of 4E-BPs versus eIF4E could be an indicator
of eIF4E targeting efficiency it may prove important to assess the ratio in tumor
biopsies to specifically target tumors which have very high levels of eIF4E expres-
sion (Alain etal. 2012), considering that targeting eIF4E phosphorylation could
be of higher therapeutic efficacy in the context of high eIF4E/4E-BP ratio. This is
explained by the fact that in that context, there is not enough 4E-BP available to se-
quester eIF4E and preclude eIF4F complex formation. The current literature points
to the 4E-BPs as master integrators of MAPK and PI3K signaling and therefore
key target in limiting the proliferative potential of tumor cells (She etal. 2010). In
this context, the inhibition of MNK1/2 could serve as a second line of attack to add
potency to the targeting of the eIF4E/4E-BP axis when mTOR-directed inhibition
fails as a single agent or as a combination therapy.
ISIS Pharmaceuticals have been testing the efficacy of ASO against eIF4E de-
veloped in collaboration with Eli Lilly and Co. (ISIS183750) (Graff etal. 2007).
A dose escalation clinical trial has been conducted in patients with advanced stage
4 cancers (Hong etal. 2011). The eIF4E ASO is currently used in clinical trials
to determine efficacy in treating castrate-resistant prostate cancer (http://clinical-
trials.gov/ct2/show/NCT01234025) and CRC (http://clinicaltrials.gov/ct2/show/
NCT01675128). Other ongoing trials are testing ISIS183750 efficacy in NSCLC
(http://clinicaltrials.gov/show/NCT01234038). The eIF4E ASO also showed effi-
cacy in vitro against mesothelioma cells (Jacobson etal. 2013).
In this last part of the chapter, we will discuss current therapeutic approaches and
clinical trials that are underway where we believe inhibiting eIF4E phosphorylation
could be explored further. In a clinical setting, inhibition of eIF4E phosphorylation
could be achieved by: (1) Direct inhibition of the MNKs or (2) Inhibition of 4E-BP
phosphorylation and sequestration of eIF4E by unphosphorylated 4E-BPs (as stated
previously, this option is dependent on the 4E-BPs/eIF4E ratio).
Currently, trials in solid tumors are exploring the efficacy of inhibiting PI3K/
mTORC1/2 axis alone (e.g. BKM120, BEZ235, PF05212384, GSK1059615) or in
combination with standard of care. It has been demonstrated that inhibiting MEK
in combination with mTORC1 could inhibit tumors in preclinical models (Kinkade
etal. 2008). Therefore, inhibiting phosphorylation of eIF4E could be a promis-
ing target downstream of MAPK in combination with PI3K/mTORC1/2 inhibi-
tors. Trials have tested the efficacy of inhibiting MEK alone using PD-0325901 in
advanced cancers (NSCLC, melanoma, colon cancer and breast cancer) (LoRusso
etal. 2010). Although initial results were promising, the trial was terminated due
to neurotoxicity. The combined inhibition of PI3K and MEK using a lower dose of
PD-0325901 in combination with the PI3K/mTOR inhibitor (PF-05212384) is cur-
rently in clinical trial (http://clinicaltrials.gov/ct2/show/NCT01347866).
Recent results have shown the efficacy of targeting VEGF and EGFR using
bevacizumab and erlotinib in glioblastoma (Sathornsumetee etal. 2010). These
promising results also demonstrate the potential of targeting MAPK and PI3K sig-
naling in glioblastoma. Moreover, inhibiting eIF4E activity in glioblastoma has
already shown encouraging results in preclinical models (Grzmil etal. 2011; Ueda
etal. 2010).
Overall, the idea of inhibiting eIF4E phosphorylation via MNK inhibition to
sensitize tumors to standard of care therapy is an appealing therapeutic avenue.
eIF4E has been an attractive therapeutic target for years and clinical trials have
recently begun to test the efficacy of an eIF4E ASO. Moreover, recent work has
demonstrated that inhibiting or abolishing the phosphorylation of eIF4E might
achieve similar results as decreasing the total amount of the protein. Future work
will be required to develop better MNK1/2 inhibitors and validate their efficacy
as single agents and in combination therapy in preclinical cancer models. If such
inhibitors are successfully developed and show efficacy in inhibiting tumor growth,
the remaining challenge will be to investigate at which stage of the disease MNK
inhibition should be tested.
Acknowledgments LF is supported by Prostate Cancer Foundation of Australia grants (#YI-

0310, #CG1511) and Australian Research Council grant (#DE120100434). EB is supported by
a philanthropic grant from the EJ Whitten Foundation. IT is supported by grants from the Cana-
dian Institutes of Health Research (MOP-115195), Terry Fox Research Institute team grant (TFF-
116128), The Canada foundation for innovation Leaders Opportunity Fund (CFI-LOF), Fonds de
la Recherche en Sant du Qubec (FRSQ) and is a recipient of a CIHR New Investigator Salary
Award.
370 L. Furic et al.
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Part III
Cell Fate and Translation in Cancer
Chapter 18
Translational Control of Cell Proliferation
and Viability in Normal and Neoplastic Cells
Svetlana Avdulov, Jos R. Gmez-Garca, Peter B. Bitterman

and Vitaly A. Polunovsky
Contents
18.2Upstream Regulation of eIF4F-Mediated Translation 379
18.3Downstream Effectors of eIF4F-Mediated Signaling 380
18.4Translational Control of the Cell Cycle 381
18.4.1Translational Control of G0 Exit and Prereplicative Events 381
18.4.2Translational Control of G1 Progression, DNA Synthesis and
Postreplicative Events 382
18.5Translational Control of the Cellular Tumor Suppression Programs 383
18.5.1Cellular Senescence 383
18.5.2Apoptosis 384
18.5.3Autophagy 385
18.6Translational Control of Cancer Genesis 386
18.7Concluding Remarks: Implication for Cancer Prevention and Therapy 388
References 389
AbstractAvailable evidence indicates that the initiation step in translation of

mRNA into proteina step mediated by the translational complex eIF4Fis an
obligatory regulatory hub in the circuitry of the normal cell physiology and can-
cer. Control of cell cycle progression, apoptosis, premature cell senescence and
authophagy by eIF4F is required in the developmental and physiological setting for
maintenance of cell homeostasis, whereas usurping eIF4F-mediated translational
control is oncogenic. The members of eIF4F are ubiquitously dysregulated or over-
expressed in human malignancies. When hyperactivated by upstream oncogenic
signaling, eIF4F selectively stimulates translation of a group of mRNAs required
for the acquisition and maintenance of the basic capabilities of cancer, including
cell autonomy for growth signals, tissue invasion and evasion of cancer defense
V.A.Polunovsky() S.Avdulov J.R.Gmez-Garca P.B.Bitterman

Department of Medicine and Masonic Cancer Center, University of Minnesota,
Minneapolis, MN, USA
e-mail: polun001@umn.edu
378 S. Avdulov et al.
systems, such as apoptosis and premature senescence. These findings place aberrant
eIF4F in a list of primary candidates for targeting by cancer therapy.
18.1Introduction
Activation of protein synthesis is an essential event during transition of a cell to

malignancy. Translation initiation is the primary step determining the rate of protein
synthesis. The cap-dependent initiation apparatus is a regulatory hub where canoni-
cal oncogenic pathways converge (Polunovsky and Bitterman 2006; Sonenberg and
Hinnebusch 2009). Tyrosine kinases and oncoproteins such as MYC and RAS lie
at the apices of the branched signaling networks that converge to activate eIF4F
during the physiological cell cycle entry and oncogenic propagation of transformed
cells. Recent studies demonstrated that sustained activation of eIF4F leads to a se-
lective increase in the recruitment of ribosomes to cancer-related mRNAs encod-
ing growth factors, growth factor receptors and components of receptor signaling
cascades including MYC and RAS that regulate protein synthesis, cell proliferation,
viability and invasion (Bitterman and Polunovsky 2010; Larsson etal. 2006; Ma-
mane etal. 2007; Rajasekhar and Holland 2004; Silvera etal. 2010). These results
suggest that oncogenic mutations upregulate eIF4F to boost their own proneoplastic
activity thereby creating a self-amplifying cancer-promoting loop. However, the
signals that drive uncontrolled cell cycle progression also trigger a variety of in-
trinsic tumor suppressor programs. These programs counterbalance oncogenic sig-
naling via triggering of cell cycle arrest by premature senescence or cell death by
apoptosis or destructive autophagy. In the contemporary version of the classical
paradigm of oncogenic cooperation, cancer arises only when somatic cells accu-
mulate a threshold number of oncogenic alterations to corrupt these innate cellular
anticancer programs and promote progressive neoplastic transformation (Ashworth
etal. 2011; Lowe etal. 2004). Since the activity of oncogenes also mediates prolif-
eration of normal cells, it is clear that the cancer-defense systems must discriminate
between neoplastic and normal states of a cell to restrict activation of antiprolifera-
tive and cell death-inducing pathways to situations when signaling is oncogenic.
Mounting evidence indicates that the redirection of eIF4F-mediated translational
program is required for the genesis and maintenance of neoplastic growth. As a con-
sequence, cancers become addicted to a proneoplastic pattern of hyperactivated
eIF4F -mediated translation (Mader etal. 1995). We conceptualized that resending
the translational landscape at the earliest stages of oncogenesis is directed to driving
ectopic cell proliferation in combination with the suppression of the tumor suppres-
sor pathways that restrict the cell neoplastic conversion (Bitterman and Polunovsky
2010; Bitterman and Polunovsky 2012b).
18 Translational Control of Cell Proliferation and Viability in Normal 379
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Fig. 18.1 Mitogen-induced activation of eIF4F-mediated translation. a Translational repression

of eIF4F by the eIF4E- binding proteins (4E-BPs). b Mitogen-induced phosphorylation of the
translation initiation factors and dissociation of the eIF4E/4E-BP complex. c Assembly of eIF4F
and formation of the RNA-binding initiation complex.
18.2Upstream Regulation of eIF4F-Mediated Translation
As discussed in previous chapters, eIF4F is a trimeric complex, consisting of

eIF4E, eIF4G and eIF4A. The least abundant and the rate-limiting member of the
eIF4F complex is the cap-binding protein eIF4E, which is also a major target for
regulation of translation initiation by the cellular circuitry. Translational repres-
sion mainly takes place via the eIF4E-binding proteins, 4E-BPs, sequestering of
eIF4E (Fig.18.1a). A wide range of extracellular cues, such as growth factors and
hormones, stimulate eIF4F assembly and cap-dependent translation. These stimuli
trigger 4E-BP phosphorylation, their release from eIF4E and subsequent associa-
tion of the latter with eIF4G to form the eIF4F complex and to initiate translation.
The primary regulation of eIF4E activity is exerted through the PI3K/AKT/mTOR
kinase cascade that leads to phosphorylation of the 4E-BPs on serine/threonine sites
(Fig. 18.1b and Chap.15). The MAPK pathway also regulates eIF4E via phos-
phorylation at Ser209 resulting in modulation of the translational activity of eIF4F
(see Chap.17) (Hay and Sonenberg 2004; Ma and Blenis 2009). Phosphorylation
of eIF4E is mediated by the MAPK kinase-interacting kinases, MNKs, which is
activated through the RAS-regulated MAPK/ERK and p38/JNK kinase cascades

(Waskiewicz etal. 1999). Transcriptional control of eIF4E occurs through c-MYC,
which transcriptionally activates the eIF4E gene through two functional c-MYC-
binding sites (E-boxes) in the eIF4E promoter (Cole and Cowling 2008; Eilers and
Eisenman 2008). Another mTORC1 target, S6K, also plays an important role in
translation initiation via phosphorylation of a diverse range of targets including
ribosomal proteins, the eIF4A helicase cofactors and regulators, such as tumor
suppressor PDCD4 and eIF4B (see Chaps.5, 6 and 16). Stimulation of cells with
growth factors leads to association of activated mTORC1 with eIF3, recruitment of
the 40S ribosomal subunit to eIF4F and subsequent scanning of the complex toward
the initiation codon (Fig.18.1c).
Since eIF4E represents the most downstream target of the multiple oncogenic
pathways, it is not surprising that eIF4F-mediated cap-dependent translation is
commonly upregulated in most, if not all, human malignancies (De Benedetti and
Graff 2004). Mutations of genes encoding eIF4F are relatively rare in human tu-
mors. More frequently, normal eIF4F function is compromised by mutations in one
or more of its upstream regulators, such as MYC, RAS and PI3K, which down-
stream pro-oncogenic pathways converge on eIF4F (Mader etal. 1995; Richter and
Sonenberg 2005).
18.3Downstream Effectors of eIF4F-Mediated Signaling
Genome-wide studies document the pleiotropic nature of the eIF4F effector path-
ways. Although the net effect of hyperactivated eIF4F on overall translational rates is
modest ranging from 2025%, its stimulating effects on translation of specific sub-
sets of mRNAs can reach several orders of magnitude (De Benedetti and Graff 2004;
Silvera etal. 2010). The mRNAs that are the most affected by activated eIF4F are
those encoding proteins regulating hallmark capabilities of cancer cells. Established
examples of translationally regulated mRNAs include those for growth factors and
cytokines (FGF2, IGF-2, PDGF, TGF, VEGF, IL-15, WNT/-catenin), protein kinases
(MOS, PIM1), transcription factors (FOS, MYC), enzymes of polyamine biosynthe-
sis (ornithine decarboxylase and ornithine aminotransferase), inhibitors of apoptosis
(survivin, BCL-2 and BCL-XL) and promoters of cell cycle transit (RAS, CDK4 and
cyclin D1) (Bitterman and Polunovsky 2010; De Benedetti and Graff 2004; Larsson
etal. 2007; Pond etal. 2010; She etal. 2010). Although some genes that are positively
regulated by oncogenic eIF4F activity encode inhibitors of cell cycle progression and
inducers of apoptosis (e.g. p27kip, p53), the net effect of upregulated cap-dependent
translation is an increase in cell proliferation and viability (Larsson etal. 2006; Ma-
mane etal. 2007). A subset of eIF4F-regulated proteins is also represented by the
upstream eIF4E activators. These include growth factors, such as the platelet-derived
growth factor (PDGF) and epidermal growth factor (EGF) (Clemens and Bommer
1999), AKT (Culjkovic etal. 2008), RAS (Larsson etal. 2007; Lazaris-Karatzas and
Sonenberg 1992) and c-MYC (Lin etal. 2008) creating self-amplifying feed-forward
signaling loops. Thus, the translation initiation machinery couples the cellular growth
and survival circuits to a variety of trophic extracellular signalsmaking it a strategic

downstream target of nearly any oncogenic mutation.
18.4Translational Control of the Cell Cycle
18.4.1Translational Control of G0 Exit

and Prereplicative Events
Physiological control of the size of cell populations in multicellular organisms rep-

resents a balance between cell proliferation, non-proliferative states and cell death
(Prescott 1968). Metazoan somatic cells require continuous signals from the extra-
cellular milieu to maintain homeostasis, survive and proliferate (Raff 1992; Sherr
and Roberts 1995). In the absence of these signals, cells undergo apoptosis (Kerr
etal. 1972) or exit the cell cycle after completing mitosis and accumulate in the
reversible phase of proliferative quiescence known as G0 (Epifanova and Polu-
novsky 1986; Pardee etal. 1978; Smith and Martin 1973). The state of prolifera-
tive quiescence functionally and metabolically differs from other more durable and
frequently irreversible non-proliferative states, such as terminal cell differentiation
and senescence. G0 is an active metabolic state in which cells express a set of genes
to counteract intrinsic proliferative signals by synthesis of endogenous inhibitors of
the G1 onset and progression to S phase (Coller 2007; Epifanova etal. 1978; Smith
and Martin 1973). Growth factors signal G0 exit and transit of cells through G1
by reprogramming the cellular metabolic machinery towards production of moieties
neutralizing the G1 inhibitors and directing synthesis of CDKs, which propel the cell
transit to the S phase (Polunovsky etal. 1983). Cell cycle reentry and progression
are driven by periodic expression of genes responsible for G0 exit, G1 transit and
transition through the checkpoints prior to and after the S phase (Sherr and Roberts
1999; Whitfield etal. 2006). In eukaryotic cells, these transcripts are subject to ex-
tensive posttranscriptional regulation at the level of mRNA stability and ribosome
recruitment, which profoundly affect the gene expression landscape as cells switch
from proliferation to quiescence and vice versa (Keene 2007). Dysregulation of these
mechanisms leads to abnormal organogenesis and is involved in the pathogenesis of
cancer and other disease states that are associated with abnormal cell proliferation.
Regulation of translation integrates the succession of events governing transi-
tion from the proliferative quiescence to the cell cycle progression. The classical
observations established that global rates of protein synthesis and expression levels
of translation initiation factors in quiescent cells are depressed congruently (Duncan
and Hershey 1985). On the other hand, mitogenic stimulation leads to an immedi-
ate increase in recruitment of mRNA to polyribosomes. Subsequent studies defined
the steps governing translational control of maintenance of the quiescence state
and growth factor-mediated cell cycle reentry (Averous and Proud 2006; Guertin
and Sabatini 2007; van der Heijden and Bernards 2010). They established that the
translationally inert eIF4E/4E-BP complex and the activator of translation initiation
mTORC1 are spatially separated in a quiescent cell: while eIF4E and 4E-BPs are
localized in the G0 nucleus, mTORC1 localizes predominantly in the cytoplasmic

compartment and migrates to the nucleus in response to growth factor signaling (Kim
and Chen 2000; Zhang etal. 2002). In the close proximity to its substrate, mTORC1
phosphorylates 4E-BP1 resulting in dissociation of the eIF4E/4E-BP complex and
translocation of eIF4E to the cytoplasm (Fig.18.1b and c). This process is accom-
panied by activation of the eIF4E-dependent nuclear export of a set of transcripts
encoding promoters of cell cycle transit (Culjkovic etal. 2007). Phosphorylation of
the translational regulators S6K1 and 4E-BP1 is mandatory for the growth factor-
induced transition of non-proliferating cells into the S phase (Dowling etal. 2010;
Fingar etal. 2004). When proliferative signals abate, this configuration rapidly re-
verses. Oncogenic insults switch off the nuclear-cytoplasmic shuttling of eIF4E to
a state of constitutive cytoplasmic localization, a mode characteristic of constantly
proliferating cells (Pyronnet and Sonenberg 2001).
18.4.2 T
ranslational Control of G1 Progression, DNA Synthesis
and Postreplicative Events
While cell cycle entry and progression of cells through the G1/S checkpoint criti-
cally depend upon extracellular proliferative signals, transition of cells through
the S phase, G2 and mitosis does not require exogenous stimuli. This switch from
signaling-dependent to autonomous cell cycle phases is associated with two pro-
found changes in translational control. First, whereas cell cycle entry and progres-
sion through the G1/S checkpoint are controlled by the activity of the cap-dependent
translation apparatus, progression of cells through S, G2 and M relies on translation
of mRNA in a cap-independent manner using IRES (Pyronnet and Sonenberg 2001;
Qin and Sarnow 2004; Sivan and Elroy-Stein 2008). Second, regulation could be
exerted by the cytoplasmic polyadenylation-element binding proteins (CPEBs) that
modify mRNA poly(A) tail length for a subset of transcripts regulating G2/M and
anaphase checkpoints (Novoa etal. 2010).
The translational landscape of cells traversing the eukaryotic cell cycle has been
recently discovered by using the advanced methodology of ribosome profiling
(Stumpf etal. 2013). This approach revealed several hundreds of mRNAs, whose
coordinated translational efficiency is embedded in the mechanisms driving pro-
gression of cell through the cell cycle. During physiological cell cycle transit, the
switch from cap-dependent to cap-independent translation is tightly regulated by a
still unclear mechanism, which is circumvented by oncogenic insults such as the
constitutive expression of c-MYC. When dysregulated, c-MYC promotes aberrant
hyperactivation of eIF4F and increases cap-dependent translation in both the pre-
replicative and postreplicative phases of the cell cycle (Barna etal. 2008). This
leads to autonomous cell proliferation and genome instability, events that are char-
acteristic of early stages of neoplastic transformation. Thus, eIF4F mediated trans-
lational control is an integral part of the oncogene-induced dysregulation of cell
proliferation, resulting in the self-sufficiency of cell signals driving entry into and
progression through the cell cycle, and of the relaxation of the oncogene-induced
dysregulation of cell proliferation resulting in intrinsic control for genome integrity.
18.5Translational Control of the Cellular Tumor

Suppression Programs
To mitigate the risk of neoplasia, evolution conferred metazoan organisms with a

constellation of anticancer defense systems. One well-known barrier for tumorigen-
esis is replicative cellular senescence which occurs during consecutive replication
cycles and which is associated with the progressive attrition of telomeres. Prototypi-
cal oncogenes, in addition to promoting the dysregulated cell cycle, trigger a variety
of tumor suppressor programs that restrain oncogenesis (Lowe etal. 2004). Estab-
lished examples include the ability of c-MYC (Askew etal. 1991; Evan etal. 1992)
and transcription factor E2F1 (Nahle etal. 2002) to stimulate apoptosis; the capabil-
ity of RAS to induce destructive autophagy (Elgendy etal. 2011) and the paradoxi-
cal effects of various oncogenic insults to promote a senescence-like cell cycle arrest
(Braig and Schmitt 2006; Collado and Serrano 2010; Minamino etal. 2004; Serrano
etal. 1997). Escape from these barriers at the earliest stages of tumorigenesis is a
prerequisite for transformation. Growing number of evidence points at the aberrant
activation of eIF4F as a central component of such early proneoplastic events.
18.5.1Cellular Senescence
Replicative senescence was first described as a permanent cell cycle arrest after a
finite numbers of cell divisions of the primary cells (Hayflick and Moorhead 1961;
Shay and Wright 2005). This growth factor-refractory state is characterized by
telomere shortening, specific morphological changes, lock down of the RB- and
p53-regulated checkpoints and long-term viability. The other markers commonly
used for identification of senescent cells include increased SA--GAL activity, the
production of senescence-associated heterochromatic foci (SAHF) and activation of
the DNA damage response. Replicative senescence has been originally described as
a factor that limits tumorigenesis (Hayflick and Moorhead 1961). Indeed, conver-
sion of a cell from a mortal to an immortal state is an essential and a rate-limiting
step in cancer (Shay and Wright 2005). Another type of permanent cell cycle block-
ade that is phenotypically close to the replicative senescence state is premature or
telomere-independent senescence. Primary (Serrano etal. 1997) or immortalized
(Wei etal. 1999) cells can be arrested due to excessive mitogenic stimulation by
oncoproteins such as RAS, BRAF and AKT (designated proto-oncogene-induced
senescence) (Braig and Schmitt 2006; Collado and Serrano 2010; Hemann and
Narita 2007; Serrano etal. 1997) or by inactivation of some tumor suppressors like
PTEN or RB or RB (Alimonti etal. 2010; Chicas etal. 2010). Cultured HMECs
can also undergo premature senescence due to accumulation of p16INK4A and other
inhibitors of the G0-to-S progression, which is believed to result from in vitro culti-
vation stress (Ramirez etal. 2001; Romanov etal. 2001; Sang etal. 2008). Despite
the similarity in the pattern of morphological transformation and in the involvement
of the p53 and RB pathways, replicative and premature senescence differ in sev-
eral important aspects indicating that these two types of cell cycle arrest represent
two distinct pathophysiological phenomena (Braig and Schmitt 2006; Hemann and
Narita 2007).
Although eIF4E displays attributes of an oncogene, the effects of constitutive
eIF4E hyperactivation on primary mammalian cell proliferation differ profoundly
from those induced by conventional oncoproteins, such as RAS or AKT. Sustained
overexpression of eIF4E accelerates proliferative senescence of mouse fibroblasts
(Ruggero etal. 2004). Similarly, upregulation of eIF4F due to loss of 4E-BPs re-
duces the life span of mouse and human fibroblasts and antagonizes tumorigenesis
by accelerating senescence in a p53-dependent manner (Petroulakis etal. 2009).
In agreement with these data, primary HMECs harboring constitutively activated
eIF4E manifest accelerated progress to the terminal stage of replicative senescence
(Larsson etal. 2007) confirming that sustained activation of eIF4E-mediated trans-
lation accelerates the onset of proliferative senescence.
Can activated eIF4F promote oncogene-induced cellular senescence? Oncogenic
K-RAS induces premature senescence operating, in part, through the eIF4E-depen-
dent translational upregulation of promyelocytic tumor suppressor protein (PML)
(Scaglioni etal. 2012). This indicates that some mediators of oncogene-induced
cellular senescence can be subjects to positive translational control. However,
overexpression of eIF4E on its own does not activate premature senescence. In-
stead, sustained expression of ectopic eIF4E enables primary HMECs to bypass the
senescence-like growth arrest induced by environmental stresses (Avdulov etal.
2004). Moreover, acting in collaboration with the catalytic subunit of hTERT, over-
expressed eIF4E promotes a spectrum of pre-neoplastic properties in immortalized
HMECs including autonomy for cell cycle progression, viability and clonogenic
expansion (Larsson etal. 2007). In this context, therefore, ectopic activation of
eIF4E-mediated translation rather counteracts premature senescence. These find-
ings support the notion that sustained activation of the PI3K/AKT/mTOR/eIF4E
axis downstream of RAS may overcome RAS-induced senescence (Kennedy
etal. 2011). Indeed, our preliminary experiments show that concurrent activation
of HRASV12 and eIF4E impedes the RAS-induced senescence-like phenotype in
hTERT immortalized HMECs (Gomez-Garcia, unpublished).
18.5.2Apoptosis
Many cellular phenomena related to alterations in activities of cap-dependent and

IRES-driven translation during programmed cell death are described in details in
Chap.19. Here, we focus on the role of eIF4F-mediated translation in oncogene-
induced apoptosis which functions as a part of tumor surveillance programs. When
activated, eIF4F collaborates with c-MYC (Lin etal. 2009; Rinker-Schaeffer etal.
1993; Ruggero 2009) to promote malignant transformation. This effect is mediated
by the ability of eIF4F to block MYC-induced apoptosis (Polunovsky etal. 1996;
Wendel etal. 2004) through translational activation of negative regulators of the
apoptotic machinery such as BCL-XL (Li etal. 2003; Soni etal. 2008; Tan etal.
2000). Along with this, enforced expression of 4E-BP1 sensitizes RAS-transformed
cells to drug-induced apoptosis in a manner dependent on the ability of 4E-BP1

to sequester eIF4E (Li etal. 2003; Polunovsky etal. 2000). Survival signals from
serum growth factors (Polunovsky etal. 1996) and IL-6 (Yamagiwa etal. 2004)
operate through the PI3K/AKT/mTORC1/eIF4E cascade to suppress the innate
apoptotic machinery. When upregulated, eIF4E suppresses apoptosis through both
nuclear (Culjkovic etal. 2008) and cytoplasmic (Larsson etal. 2006) mechanisms
of posttranscriptional gene expression control.
18.5.3Autophagy
The catabolic self-degradation process called authophagy serves as a mechanism

to maintain intracellular homeostasis in both normal and cancer cells (Amaravadi
etal. 2011; Maiuri etal. 2007). Early autophagic events include formation of dou-
ble-membrane autophagosome vesicles that enclose a portion of the cytoplasm with
available organelles. This process is triggered by the nutrient deprivation or exten-
sive stress signaling and driven by the evolutionary conserved autophagy-related
proteins. During the later stages of autophagy autophagosomes fuse with lysosomes
to degrade the enclosed material by lysosomal enzymes and to subsequently reuse
the degradation products for anabolic processes, such as de novo protein synthesis.
Although autophagy generally functions as a cytoprotective process, extensive and
dysregulated autophagy may induce cell death (Kroemer and Levine 2008). This
type of autophagy could be classified as destructive as opposed to cytoprotective
autophagy. Mitogenic activation of some oncoproteins, such as RAS, is accom-
panied by extensive destructive autophagy (Elgendy etal. 2011), which functions
as one of the effector mechanisms of oncogene-induced senescence (Young etal.
2009). During neoplastic cell transformation, cooperative oncogenic signals switch
the function of upregulated autophagy from its suicidal activity to its cytoprotec-
tive mission, thus enabling neoplastic growth (Cheng etal. 2011; Guo etal. 2011).
Thus, depending upon genetic background and cellular context, oncogene-induced
autophagy can function to either promote or prevent cancerous growth. Translation
and autophagy are regulated in an opposite manner through the PI3K/AKT/mTOR
pathway: trophic signals stimulate eIF4E-mediated translation through activation of
mTORC1 and suppress autophagy by mTORC1-mediated phosphorylation of the
autophagy initiator ULK1 (Kim etal. 2011). Accordingly, downregulation of trans-
lation in cancer cells stimulates autophagy (Cheng etal. 2011; Narita etal. 2011).
Hence, cells use high levels of translational activity for curbing the autophagic flux
of metabolites under conditions of trophic well-being. On the contrary, starvation
decreases the translational activity resulting in a compensatory cytoprotective au-
tophagic response.
The data summarized here suggest that activated cap-dependent translation gen-
erally serves to increase cell viability and defeat tumor surveillance pathways. Since
cancers can only arise once anticancer defenses are alleviated, dysregulation of cap-
dependent translation by upstream oncogenic signals can be one of the earliest steps
in tumorigenesis, which is used by oncogenic lesions to quell the intrinsic tumor
suppressive mechanisms embedded into oncogenic programs.
18.6Translational Control of Cancer Genesis
The data summarized here allow formulating the basic principle of translational
control of cell proliferation and viability in normal and neoplastic cells:
1. Cell cycle entry and progression of nontransformed cells require activated eIF4F;
2. Oncogenic signals redirect translational control from regulation of physiological
cell proliferation to driving uncontrolled neoplastic progression;
3. Activated oncogenes trigger a tumor suppressor program that can be abrogated
by activated eIF4F.
Hence, oncogenic signals emanate the contradictory intracellular pathways modulat-
ing cell viability: the tumor suppressor pathway leading to cell death and the eIF4F-
mediated prosurvival program. Therefore, the net outcome of oncogene activation
can be either normal cell proliferation, or neoplastic progression, or growth arrest
and cell death. Each outcome is presumably dictated by a cellular context and
downstream interactions that independently potentiate or mitigate each pathway.
Tumorigenic activity of eIF4F is a derivative of a prosurvival eIF4F function, which
is embedded in the normal cell cycle to protect cells from self-destruction when
physiological proliferation is needed. However, this protection is not absolute. One
reasonable view on the cancer genesis is a synthetic viability model. Accord-
ing to this formulation, two or more proneoplastic genetic or epigenetic changes
cooperate, so that each counteracts the anticancer activity of the other (Lowe etal.
2004; Nowell 1976; Renan 1993). For example, activated c-MYC suppresses RAS-
induced senescence (Hydbring etal. 2010); whereas oncogenic RAS rescues cells
from c-MYC-induced apoptosis (Lowe etal. 2004). Notably, the capacity of onco-
genes to induce dysregulated cell proliferation or promote self-destruction depends
upon oncogene expression levels. Whereas high-level expression of c-MYC and
RAS triggers tumor suppressor programs, low levels of expression of dysregulated
oncogenes drive cell cycle progression and hyperplasia (Lee and Bar-Sagi 2010;
Murphy etal. 2008; Sarkisian etal. 2007; Tuveson etal. 2004). It is generally ac-
cepted that the intensity and duration of mitogenic signals are sensed by the cellular
anticancer surveillance systems, allowing it to discriminate physiological signals
for orderly proliferation from aberrant signals that promote uncontrolled prolifera-
tion and tumorigenesis (Lowe etal. 2004). This notion suggests that transition of
a cell from a normal to malignant state requires at least the following changes: (a)
mutation/s that serve a foundation for an uncontrolled cell proliferation; (b) inten-
sification of these proliferative signals to a threshold level necessary for tumori-
genesis and (c) disabling of anticancer surveillance and/or defense programs (Ra-
jasekhar and Holland 2004). Here we propose a model that extends the synthetic
viability view by including the translational control of cell viability. According to
this model, the net outputs of oncogenic insultsneoplastic proliferation or cell
deathare dictated by interactions between the cooperative intensity of the signals
triggering anticancer defenses and the eIF4F-mediated rescue of transformed cells
from the tumor suppressor programs (Fig.18.2).
a W
/&&
K E
b /&&
K

d
c
K
, E
/&&
K

d
Fig. 18.2 Simplified scheme of eIF4F-mediated translational control for oncogene interdepen-
dence. a Physiological cell proliferation requires activation of the eIF4F-driven translational appa-
ratus. b Oncogenic signaling reprograms the translation machinery. This results in differentiated
recruitment into or out of polyribosomes of a substantial subset of mRNAs in a manner regulated
by these signals. The mRNAs most affected are those encoding proteins involved in multiple
signaling pathways, cell cycle progression, cell motility and other cancer-related functions. Onco-
gene-induced alterations of the transcriptome and translatome lead to proneoplastic cell prolif-
eration, which is largely suppressed by the intrinsic tumor suppressor programs. c Prosurvival
interdependence of two oncogenic insults (synthetic viability) arises due to hyperactivation of
eIF4E-mediated translation by oncogenic signals that converge on and hyperactivate eIF4F. In
this example, oncogenic signal A may phosphorylate eIF4E whereas signal B may result in dis-
sociation of 4E-BPs. Hyperactivation of eIF4E produces a radical shift in the composition of the
translatome, leading to the abrogation of the tumor suppressor program.
What is the role of translational control in these cancer-initiating events? In most

tumors, the genes encoding the eIF4F constituents are not mutated. Instead, persis-
tently upregulated eIF4F-mediated translation in cancers is due to sustained signals
emanating from constitutively activated cell cycle promoters such as MYC, RAS
and AKT .
How does increased intensity eIF4F-mediated translation promote oncogenesis?
A genome-wide analysis of the transcriptome and translatome showed that acquisi-
tion of eIF4E-induced neoplastic lesions was associated with altered translational
control of genes assigned to regulation of cell cycle progression, translation, viabil-
ity and invasion (Larsson etal. 2007; Mamane etal. 2007). The data summarized
here show that hyperactivated eIF4E elicits the program that robustly inhibits on-
cogene-induced anticancer defenses. Recent findings also indicated that increased

expression levels of the other component of eIF4F, the docking protein eIF4G1,
contribute to breast cancer maintenance by reprogramming eIF4F-mediated and
IRES-driven translation for promoting increased cell proliferation and cell viability
(Badura etal. 2012). This salvage function of hyperactivated eIF4F contributes to
cancer genesis by abrogating tumor suppressor pathways and makes cancer cells
dependent upon upregulated eIF4F-dependent translation.
What serves to restrain the oncogenic potential of eIF4F-mediated translation?
In physiological conditions, the activity of eIF4F is under strict regulation by ho-
meostatic mechanisms mediated by a set of translational repressors such as 4E-BPs,
PML and maskin (Richter and Sonenberg 2005). Hyperactivation of eIF4F leads to
a compensatory 4E-BP dephosphorylation enabling feedback inhibition of the eI-
F4F complex (Khaleghpour etal. 1999). Strategies to tackle this negative regulation
have been revealed in studies of the transition from early stages of neoplasia to
advanced breast cancer. In this situation, overproduction of 4E-BP1 is met with
increased levels of eIF4G1 and a switch from predominantly cap-dependent to cap-
independent translation (Braunstein etal. 2007). The importance of the 4E-BPs in
the anticancer program has been highlighted in the studies of mice with genetic
deletion of 4E-BP1 and 4E-BP2. Such mice display changes in molecular profiles
suggestive of translational activation of genes governing angiogenesis, growth and
proliferation (Kim etal. 2009). These animals are also sensitized to carcinogen-
induced lung tumorigenesis. These findings show that while knocking down of 4E-
BPs themselves does not induce cancer, it efficiently collaborates with tumorigenic
mutational events by preventing oncogene-induced tumor suppression pathways.
These data support the idea that the 4E-BPs function as guardians of a translational
checkpoint in tumor defense and that disabling the 4E-BPs enables cancer develop-
ment and progression programs.
18.7Concluding Remarks: Implication for Cancer

Prevention and Therapy
Aberrant activation of eIF4F is a significant predictor of reduced OS and decreased

time to relapse in many solid malignancies (Bitterman and Polunovsky 2010; Sil-
vera etal. 2010). Direct mutational activation of the genes encoding the eIF4F
members is relatively uncommon in tumors; rather, the dysregulated and high-level
expression of the eIF4F constituents is frequently a consequence of their relent-
less induction by upstream oncogenic signals. The summary of data presented here
shows that activation of eIF4F outside of the context of normal regulation at the
wrong time or the wrong place promotes specialized translation of mRNAs. These
mRNAs encode proteins involved in abrogating tumor suppressor pathways, cell
cycle progression, survival and other function known to be involved in cancer biol-
ogy. Hence, differential expression of genes governing the range of eIF4F activity
could be an important factor underlying genetic variations in cancer susceptibility
in populations. In addition to genetic factors, susceptibility to cancer could be influ-

enced by environmental or physiological stimuli leading to persistent hyperactiva-
tion of eIF4F. Therefore, compounds that may counter these signals could be used
as therapeutic agents.
The hyperactivation of eIF4F in advanced cancers and the dependence of malig-
nant growth upon the eIF4F activation status have made it an attractive target for
cancer therapy (Bitterman and Polunovsky 2012a; Fischer 2009; Hsieh and Rug-
gero 2010; Malina etal. 2011; Mavrakis and Wendel 2008). Most aspects of novel
approaches to cancer therapy by antagonizing aberrant translation are presented in
Chap.14 and Part IV. The translation machinery is an extremely attractive thera-
peutic target since the goal is to normalize not extinguish the function of the former.
In this context, therapies targeting translation are promising to be potentially well-
tolerated ones and to be a widely applicable adjunct to currently available cytotoxic
and targeted therapeutics. Based on our current knowledge, targeting eIF4F is much
more likely to chemosensitize tumors by restoring cancer defense programs, rather
than directly leading to cancer cell death. Thus, we predict that such therapy, tar-
geting translation, is likely to be effective in combination with more conventional
approaches, whose mechanisms are based on induction of premature senescence,
autophagy and apoptosis.
Acknowledgements We thank members of the Bittermans and Polunovskys research groups for
the productive discussions.
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Chapter 19
Translation and Apoptosis in Cancer
Martin Holcik
Contents
19.2Regulation of Global Translation During Apoptosis 398
19.3Processing of Translation Factors by Caspases 399
19.3.1eIF4G 399
19.3.2eIF3 401
19.3.3eIF4B 402
19.4Phosphorylation of Initiation Factors 402
19.4.14E-BPs 402
19.4.2eIF4E 403
19.4.3eIF2 403
19.5IRES-Dependent Translation of Cellular mRNAs Involved in Stress Response 405
19.6Antiapoptotic Proteins 406
19.6.1IAP Family 406
19.6.2BCL-2 Family 408
19.7Proapoptotic Proteins 409
19.7.1APAF-1 410
19.7.2DAP5 410
19.7.3c-MYC 411
19.8IRES-Dependent Translational Control of EMT 411
19.9eIF4E-Independent and IRES-Independent Translation 412
19.10Concluding Remarks 412
References 413
Abstract Regulation of protein synthesis (translation) is a key cellular process that

is intimately linked to cellular survival. Given that translation consumes more than
50% of cells energy, it is not surprising that translation is tightly regulated and
that perturbations in translation are not well-tolerated. Conversely, many aberrant
cellular processes require that the translation machinery and translation output (i.e.
proteome) to be modified. This is often the case in various stress response and
M.Holcik()
Apoptosis Research Centre, Childrens Hospital of Eastern Ontario Research Institute,
Department of Pediatrics, University of Ottawa, Ottawa, Canada
e-mail: martin@arc.cheo.ca
396 M. Holcik
disease states, and is probably best exemplified in cancer. Indeed, the link between
dysregulated translation and cancer has long been suspected, and in recent years
strong experimental evidence has been obtained by many laboratories. One specific
aspect of this link is the ability of cancer cell to evade death (apoptosis) by changing
and reprograming of translation. A key advantage of translational control is the abil-
ity of cells to rapidly reprogram the protein output in response to triggers that may
lead to apoptosis, thereby fine-tuning the mechanism that tips the balance between
cell survival and apoptosis. While several of the key apoptotic factors are regulated
in response to increased levels of canonical factors such as eIF4E and eIF4G, and
are discussed elsewhere in this book, an alternative translation initiation, best exem-
plified by an internal initiation via IRES, has been shown to play a critical role in the
regulation of apoptotic response and tumorigenesis, and is the focus of this chapter.
19.1Introduction
Although translation is a continuous process starting with the recognition of mRNA

by the cellular translation machinery and ending with the production of a protein, it
is often, primarily for didactic purposes, divided into four stepsinitiation, elonga-
tion, termination and ribosome recycling (see Chap.2). While all these steps are
regulated, the majority of the regulatory processes center on translation initiation.
The reason for this is that it is more effective to regulate (e.g. stop) the beginning of
the given process, rather than to deal with unfinished, partly finished or unwanted
proteins that might have arisen spuriously or at the wrong time, and thus be detri-
mental to cellular homeostasis and/or cell survival.
Following synthesis in the nucleus, virtually all eukaryotic mRNAs are modified
at both the 5 and 3 ends by addition of m7G cap and poly(A) tail, respectively. The
purpose of these modifications is two-fold; they protect the mRNA against degrada-
tion, as well as promote engagement of the mRNA by the translation machinery, and
therefore play a key role in translation initiation. The recognition of mRNA by the
translation machinery occurs through the m7G cap and a specialized cap-binding
protein, eIF4E (but see exceptions below). In turn, eIF4E is bound by eIF4G (a scaf-
folding protein) and an RNA helicase eIF4A (the three-protein complex is termed
eIF4F), and subsequently a cohort of additional initiation factors, including eIF3E,
which recruit the small ribosomal submit to the 5 end of the mRNA (Fig.19.1). It
is believed that the mRNA-bound 43S ribosomal complex then relocates (scans)
along the mRNA until the correct initiation codon (most often AUG) in the proper
context is recognized and the polypeptide chain synthesis commences. It should be
noted that the poly(A) tail and its associated poly(A)-binding protein PABP play a
role of a translational enhancer, since the recruitment of 43S to the mRNA is greatly
enhanced by eIF4G-PABP interaction (Hentze etal. 2007). This type of translation
is referred to as cap-dependent, implying that the ability of the translation machin-
ery to recognize mRNA is absolutely dependent on the presence of m7G cap at the
5 end of the mRNA.
19 Translation and Apoptosis in Cancer 397
Fig. 19.1 Schematic diagram outlining the key points of regulation during translation initiation.
For simplicity, not all initiation factors are shown. Initiation factors that are described in this
review with greater detail are indicated with asterisks. Adapted and modified from Holcik etal.
(2000a).
398 M. Holcik
Fig. 19.2 A model of the interconnectedness of translation and apoptosis. The left side of the
model depicts regulation of general (cap-dependent) translation; the right side depicts selective
(IRES-mediated) translation. Green lines indicate positive, while red line negative interactions.
Dotted line depicts indirect effect. For simplicity, not all factors are shown. Adapted and modified
from Liwak etal. (2012a).
Existence of an alternative translation initiation was also suspected, since it was

observed that under conditions when cap-dependent translation is severely compro-
mised (e.g. nutrient deprivation, hypoxia and viral infection), a subset of mRNAs
is still efficiently translated (Bushell etal. 2006; Johannes etal. 1999; Morley etal.
2005). Furthermore, the reprograming of the cellular proteome, which is allowed
by the alternative initiation, was found to be required for stress response (Spriggs
etal. 2010; Spriggs etal. 2008). It was therefore proposed that selective trans-
lation, which occurs by the alternative mode of translation initiation (most com-
monly by the internal ribosome entry site (IRES) mechanism), is a key mechanism
which is required for cellular survival under stress and is used by cells to fine-tune
their stress response (Holcik and Sonenberg 2005; Holcik etal. 2000a; Liwak etal.
2012a; Ruggero 2013; Silvera etal. 2010) (Fig.19.2).
19.2Regulation of Global Translation During Apoptosis
The ability of the cell to regulate translation is frequently focused on two key con-
trol points which are shared by virtually all mRNAscomponents of the eIF4F
complex and eIF2. These regulatory points are utilized under normal growth con-
ditions to match the protein output to the cells growth, but also under physiological
and pathophysiological conditions, which require adaptive changes in gene expres-

sion. Induction of apoptosis by physical (e.g. radiation), chemical (e.g. chemotox-
ic drugs), or pathophysiological triggers is accompanied by substantial and rapid
downregulation of protein synthesis. The decrease in global translation rate is due
to changes in the activity or abundance of canonical translation initiation factors, al-
though some evidence suggests that degradation of mRNAs also plays an important
role in this process (Bushell etal. 2004).
The proteolytic processing of initiation factors during apoptosis is accomplished
by caspases, whereas alteration in their activity is accomplished by changes in their
phosphorylation status (Table19.1). Interestingly, both of these changes affect the
assembly of normal initiation complexes and lead to global inhibition of transla-
tion (Clemens 2001; Clemens etal. 2000; Morley 2001). At the same time, how-
ever, they promote assembly of modified or alternative initiation complexes that are
believed to promote selective translation of those mRNAs that are required either
for completion of apoptosis or cell survival (see below). In addition, unlike caspase-
mediated cleavage, the phosphorylation changes occur very early on following ex-
posure of cells to stress, which eventually may lead to apoptosis. Thus, the initial
cellular response to stress could be divided into two phases; (1) early signaling
events, whose purpose is to temporarily attenuate general translation, and set stage
for selective reprograming of the translation machinery, and (2) assembly of the
alternative initiation complexes comprised of fragments of canonical factors that
are resistant to normal regulatory signals and support selective translation during
the progression of apoptosis.
19.3Processing of Translation Factors by Caspases
It was observed that initiation of apoptosis by various triggers (such as cisplatin,

etoposide, TNF or TRAIL) results in the caspase-mediated cleavage of a number
of initiation factors including eIF2, 4E-BP1, eIF4B, eIF3j, DAP5, PABP, eIF4G1
and eIF4G2 (Bushell etal. 2000; Clemens etal. 2000; Clemens etal. 1998; Henis-
Korenblit etal. 2000; Marissen etal. 2000a; Marissen etal. 2000b; Marissen etal.
2004; Tee and Proud 2001, 2002). This primarily targets the mRNA-binding step of
translation initiation.
19.3.1eIF4G
Cleavage of eIF4G1 generates three fragments termed fragments of apoptotic cleav-

age of eIF4G (N-FAG, M-FAG, and C-FAG) which appear to play distinct functions
(Morley etal. 2005). The M-FAG retains its ability to interact with eIF4A, eIF4E
and eIF3 and assembles into a modified form of the eIF4F complex, which supports,
at least temporary, cap-dependent and perhaps even cap-independent translation
Table 19.1 Modification of translation initiation factors during apoptosis. The table provides a list of selected eukaryotic translation initiation factors that are
400
modified during apoptosis induced by different triggers.

Translation Modifications Effects Apoptotic triggers References
initiation
factors
eIF2 Phosphorylation of eIF2 Inhibition of GDP to GTP exchange on eIF2. Iron deficiency, heavy metals, Gebauer and Hentze (2004) and Row-
subunit at Ser51 Inhibition of global translation and apoptosis osmotic or oxidative stress, lands etal. (1988)
and translation of specific transcripts and cell heat shock, double-stranded
survival RNA, amino acid starvation,
unfolded protein response
(UPR)
eIF4E Dephosphorylation Global translation inhibition Stimuli that activate protein Mathews etal. (2007)
phosphatase 2A (PP2A)
4E-BPs Dephosphorylation Competition with eIF4G on eIF4E. Global DNA damage, TRAIL, stauro- Bushell etal. (2000); Jeffrey etal.
translation inhibition and apoptosis sporine, rapamycin (2002); Tee and Proud (2000, 2001)
4E-BPs Cleavage by caspases at Cleaved form strongly binding eIF4E and inhi- Staurosporine, etoposide, p53 Tee and Proud (2000, 2001)
Asp24 bition of cap-dependent translation activation
eIF4G1 and Cleavage by caspase 3 at Cleaved forms (except for M-FAG from TNF, TRAIL, cisplatin, Bushell etal. (2000); Holcik and
eIF4G2 Asp532 and Asp1176 of eIF4G1) cannot bridge eIF4E, 4A and eIF3 etoposide, cycloheximide, Sonenberg (2005); and Morley
eIF4G1Cleavage by cas- together. Global translation and attenuation MG132, serum deprivation, etal. (1998)
pase 3 at Asp 560, 851, 978, of antiapoptotic response FAS receptor activation
1162 and 1407 of eIF4G2
DAP5/p97/ Cleavage by caspase 3at p86 fragment with eIF4A and eIF3 binding sites UPR, FAS receptor activation Henis-Korenblit etal. (2002); Hunds-
NAT1 Asp790 but no eIF4E site. Inhibition of cap-depen- doerfer etal. (2005); Nevins etal.
dent translation but stimulation of specific (2003); Warnakulasuriyarachchi
IRES-dependent translation etal. (2004)
eIF4B Cleavage by caspase 3 at Fragment is still able to interact with eIF4F and Cycloheximide, FAS receptor Bushell etal. (2001); Bushell etal.
Asp45 eIF3 but lacks the region mediating PABP activation (2000); Shahbazian etal. (2010a);
binding Shahbazian etal. (2010b)
eIF3j (p35) Cleavage by caspase 3 at Reduced affinity of the eIF3 complex for the Cycloheximide, FAS receptor Bushell etal. (2000) and Fraser etal.
Asp242 40S ribosomal subunit. Inhibition of global activation (2004)
protein translation
eIF3f (p47) Phosphorylation Enhanced association with the core subunits of Staurosporine Shi etal. (2009)
M. Holcik
eIF3. Inhibition of global protein translation

during early phases of apoptosis. M-FAG is eventually degraded with prolonged

exposure to stress, coinciding with the inhibition of cap-dependent translation and
attenuated stress response. In contrast, neither N-FAG nor C-FAG appears to have
any influence on translation, whether cap-dependent or IRES-dependent, despite
the fact that both fragments persist in apoptotic cells. N-FAG retains its ability to
interact with PABP and could therefore potentially disrupt PABP interaction with
eIF4F; however, it is sequestered in the nuclei. It is possible that the loss of integ-
rity of nuclear envelope will eventually lead to cytoplasmic localization of N-FAG.
However, this occurs at very late stages of apoptotic death and likely will have no
meaningful impact on the progression of apoptosis or the ability of cells to recover.
In addition, PABP has also been shown to be cleaved in apoptotic cells, although
significantly later than eIF4G1 (Marissen etal. 2004). eIF4G2 is cleaved in mul-
tiple fragments, but none of them have been shown to participate in either global
or selective translation in apoptotic cells, and are more likely degraded primar-
ily for the purpose of inhibiting global translation (Marissen etal. 2000a; Morley
2001; Morley and Pain 2001). The final member of the eIF4G family, DAP5/p97,
is the caspase-activated translation initiation factor (Imataka etal. 1997; Imataka
and Sonenberg 1997; Levy-Strumpf etal. 1997; Yamanaka etal. 1997). In normal
cells, DAP5/p97 functions as an inhibitor of cap-dependent translation. However,
removal of the 11kDa C-terminus generates a DAP5/p86 variant that drives se-
lective translation (see below). The cleavage of eIF4G and DAP5/p97 therefore
functions as an apoptotic translation switch that regulates cell fate by tipping the
balance between the translation of proapoptotic and antiapoptotic proteins (Holcik
and Sonenberg 2005; Holcik etal. 2000a). In addition, DAP5/p97 activates its own
translation both in caspase-dependent and caspase-independent manner suggesting
the existence of a positive feedback loop that ensures elevated levels of DAP5/
p97 to support IRES-dependent translation of select mRNAs during conditions of
reduced global protein synthesis (Lewis etal. 2008).
19.3.2eIF3
eIF3 is an essential factor that brings together the 43S ribosomal PIC and eIF4F-
bound mRNA and is composed of at least 13 subunits (see Chap.8). The p35 sub-
unit (eIF3j) mediates stable association of eIF3 with the ribosome (Fraser etal.
2004) and was shown to be cleaved following induction of apoptosis by cyclo-
heximide in BJAB cells in a caspase-dependent manner, although at a slower rate
than eIF4G1 (Bushell etal. 2000). Consequently, eIF3 is poorly recruited to the
ribosome resulting in an inhibition of general translation. Similar to the cleavage
of eIF4G2, the most likely purpose for this cleavage is to block translation at later
stages of apoptosis. Recently, a link was proposed between expression of the trun-
cated form of eIF3e and cap-independent translation. Integration of MMTV oc-
curs commonly in the locus encoding eIF3e resulting in the production of truncated
eIF3e. Ectopic expression of this truncated form caused a shift from cap-dependent
402 M. Holcik
to IRES-dependent translation, and specifically enhanced IRES-dependent transla-

tion of c-MYC, XIAP, PIM1 and CYR61 (cysteine-rich angiogenic inducer 61), all
proteins previously shown to be involved in tumorigenesis (Chiluiza etal. 2011).
Similarly, eIF3 appears to play a role in regulating IRES-mediated translation dur-
ing EMT (see below).
19.3.3eIF4B
eIF4B stimulates eIF4A helicase activity and consequently ribosome binding to the
mRNA (Hinnebusch and Lorsch 2012). eIF4B was shown to be cleaved in a caspase
3 dependent (Bushell etal. 2000) and independent (Jeffrey etal. 2002) manner in
BJAB and MCF-7 cells, respectively. Because the cleaved fragment of eIF4B still
forms a complex with eIF4F, it is not clear what the purpose of the cleavage is. The
kinetic analysis of the caspase 3 mediated cleavage indicates that it occurs in later
stages of apoptosis, suggesting that it could be a non-specific consequence of cas-
pase activation without any regulatory function. However, recent data showed that
eIF4B controls cell survival by promoting selective translation of genes involved in
cell survival (e.g. BCL-2, XIAP) and cell proliferation (e.g. c-MYC, CDC25C and
ODC) (Shahbazian etal. 2010b). It is therefore possible that the cleavage of eIF4B
might specifically affect translation of these genes.
19.4Phosphorylation of Initiation Factors
In addition to non-reversible modifications of translation initiation factors by pro-

teolytic cleavage, some factors are modified by phosphorylation that affects either
their activity or their ability to interact with other components of the translation
machinery, or both.
19.4.1 4E-BPs
The primary mechanism which regulates binding of eIF4E to mRNAs is the com-
petitive binding of eIF4E by eIF4E-binding proteins 1, 2 and 3 (4E-BPs) (reviewed
in Roux and Topisirovic 2012). Binding of 4E-BPs to eIF4E is mutually exclusive
with that of eIF4G ; thus, eIF4E, which is sequestered in a complex with 4E-BPs,
cannot interact with eIF4G and recruit mRNA to eIF4F, resulting in an inhibition of
general translation. The affinity of 4E-BPs to eIF4E is regulated by phosphorylation
by mTOR such that hyperphosphorylated 4E-BPs cannot bind eIF4E, favoring ac-
tive translation. Conversely, dephosphorylation of 4E-BPs which occurs during cell
stress leading to apoptosis (such as DNA damage, TRAIL, FAS ligand, staurospo-
rine, etoposide, or cisplatin treatment) leads to sequestration of eIF4E in a complex
with 4E-BPs and inhibition of translation (Morley and Coldwell 2007). In addition,
several DNA damage-inducing treatments such as etoposide, cisplatin and activa-
tion of p53 result in the cleavage of 4E-BP1 with the fragments exhibiting higher
affinity for eIF4E than the full-length protein (Constantinou and Clemens 2005).
19.4.2eIF4E
The affinity of eIF4E for the cap is also regulated by phosphorylation at Ser209 by
the MAPK-integrating kinases MNK1 and MNK2, which lie downstream of ERK
and p38 MAPK pathways and respond to stimulation of cells with growth factors,
anisomycin or UV irradiation (Waskiewicz etal. 1997). Phosphorylation of eIF4E
enhances general translation by reducing its affinity for cap; conversely, dephos-
phorylation of eIF4E (and MNK) by PP2A leads to inhibition of translation (Li etal.
2010). It has been proposed that while the changes in eIF4E affinity or availability
alter general translation in the apoptotic cell, selective translation of mRNAs that do
not require eIF4E for their translation should be able to continue unhindered, even
enhanced. Indeed, translation of IRES-containing mRNAs such as BCL-2, cIAP1
(cellular inhibitor or apoptosis 1), NOTCH2, or c-MYC continued in etoposide-
treated (Sherrill etal. 2004; Van Eden etal. 2004) or TRAIL-exposed (Bushell etal.
2006) cells, despite compromised translation machinery.
19.4.3eIF2
The second critical regulatory node of translation regulation is eIF2. eIF2 is re-
quired for bringing the initiator Met-tRNAi (in the form of ternary complex con-
sisting of eIF2, Met-tRNAi and GTP) to the 40S ribosomal subunit and formation
of the 43S PIC (reviewed in Hinnebusch and Lorsch 2012). eIF2 exists in GDP or
GTP bound forms, and the GDP to GTP exchange, catalyzed by eIF2B, is necessary
for regenerating active eIF2, which can subsequently bind Met-tRNAi. In response
to a variety of stresses, the subunit of eIF2 is phosphorylated at serine 51, which
greatly increases its avidity for eIF2B and locks both proteins in an inactive eIF2/
eIF2B complex. As a result, availability of the ternary complex significantly de-
clines, leading to inhibition of 43S formation and translation initiation. Virtually
every cellular stress regulates translation through phosphorylation of eIF2 via one
of four eIF2 kinases, which show preferential activation by distinct stresses. These
kinases include HRI, PKR, GCN2 and PERK (see Chap.9). HRI is activated by
iron deficiency, heavy metals, osmotic or oxidative stress and heat shock. PKR
is activated by dsRNA during viral infection or IFN-induced apoptosis. GCN2 is
activated by amino acid starvation and UV irradiation. PERK is activated in re-
sponse to endoplasmic reticulum stress and UV irradiation (see Chap.9). Phos-
phorylation of eIF2 is increased in cells exposed to a variety of apoptotic stimuli
such as TRAIL, TNF, FAS ligand, serum deprivation, hypoxia, osmotic shock, and
404 M. Holcik
various drug treatments. In addition, eIF2 can also be cleaved by caspases. The
link between eIF2 modifications and apoptosis is not straightforward, however,
as phosphorylation of eIF2 can be both the cause and a consequence of the cells
commitment to apoptosis. For example, phosphorylation of eIF2 in MCF-7 cells
treated with TRAIL is a caspase-8-dependent process (Jeffrey etal. 2002), whereas
caspase 3 activation in MEFs treated with TNF requires phosphorylation of eIF2
first (Scheuner etal. 2006). In addition, eIF2 itself can be a target of caspases 3,
6, 8 and 10 (Bushell etal. 2000; Marissen etal. 2000b). The truncated eIF2 is no
longer dependent on eIF2B for the GDP to GTP exchange and the expression of this
fragment can restore PKR-induced repression of translation (Marissen etal. 2000b;
Satoh etal. 1999).
The ternary complex is absolutely required for the translation of most mRNAs,
therefore phosphorylation of eIF2 would be expected to serve only one purpose: to
inhibit translation. Paradoxically, however, it is not the case and phosphorylation of
eIF2 enhances the translation of select mRNAs which encode for proteins required
for stress adaptation and recovery (reveiwed in Holcik and Sonenberg 2005). Some
of these mRNAs, best exemplified by the transcription factor ATF4, harbour short
uORFs in their 5 UTRs that engage the ribosome and therefore render the main
coding ORF of these mRNAs poorly translated when ternary complex is abundant.
In contrast, during stress conditions when the ternary complex is scarce ribosomes
pass through the uORFs without initiating leading to productive translation of the
main ORF. However, it is expected that under conditions of severe stress (when no
ternary complex is available) even translation of these mRNAs would be inhibited.
A completely different mode of eIF2-independent translation was described for
some cellular mRNAs that utilize IRES for initiation. For example, translation of
XIAP is resistant to the global attenuation of translation due to eIF2 phosphoryla-
tion during hypoxia (Aird etal. 2008; Aird etal. 2010), glucose deficiency (Muaddi
etal. 2010), or serum deprivation (Holcik etal. 1999; Riley etal. 2010; Thakor and
Holcik 2012), while enhanced translation of VEGF and p120 catenin was observed
in hypoxic inflammatory breast tumors that also show high levels of phosphory-
lated eIF2 (Silvera etal. 2009; Silvera and Schneider 2009). We have investigated
the molecular mechanism of eIF2-independent translation of XIAP and showed
that during normal proliferative conditions, when the ternary complex is abundant-
ly available the XIAP translation proceeds in eIF2-dependent mode, similar to
other cellular mRNAs. However, upon pathophysiological stress the XIAP IRES-
dependent translation switches to an alternative eIF5B-dependent mode to avoid
attenuation due to eIF2 phosphorylation. This mode of translation resembles that
of bacterial translation which uses eIF5B analog IF2 to interact with the initiator
tRNA and the ribosome. In fact, this bacterial-like mode of translation mediated by
eIF5B was also described for the translation initiation of viral IRESs of HCV and
CSFV (classical swine fever virus) (Pestova etal. 2008; Terenin etal. 2008). In
addition to eIF5B, ligatin, MCT-1/DENR (Skabkin etal. 2010) and eIF2D (Dmit-
riev etal. 2010) were all reported to be able to deliver tRNA into the P-site of the
ribosome during HCV IRES-mediated translation, although their role in supporting
cellular mRNA translation during stress has not been determined. The ability of cell
to bypass requirement for typical ternary complex suggests that cells have evolved
an alternative, eIF2-independent mechanism of tRNA delivery to support a res-
cue translation of critical survival proteins under circumstances when the normal
mechanism is unavailable.
19.5IRES-Dependent Translation of Cellular mRNAs

Involved in Stress Response
Some cellular mRNAs appear to be translated by an alternative mechanism that

does not rely on m7G cap and its associated factors. This phenomenon, termed in-
ternal initiation, was first observed with RNA viruses (in particular picornaviridae)
whose RNA is naturally uncapped and yet efficiently translated by the host transla-
tion machinery (Pelletier and Sonenberg 1988). Instead of cap, distinct functional
RNA elements, termed IRES, precede the protein-coding portion of viral RNA and
directly recruit the 40S ribosome to the vicinity of initiation codon (Fig.19.3). This
recruitment can occur in the absence of any other protein factors (as with dicistro-
virus intergenic IRES) or with the aid of various combinations of canonical initia-
tion factors (such as eIF3E, eIF5, and eIF5B) and auxiliary proteins in case of the
remaining viral IRES (reviewed in Jackson 2013). In addition, many of these vi-
ruses actively alter the host translation machinery by cleaving key initiation factors,
such as eIF4G, thereby disabling host cap-dependent translation and usurping it for
translation of viral proteins. Similar to the viral internal initiation, several cellular
mRNAs were suggested to be translated by this alternative mode of translation. This
notion was brought forward since it was observed that a subset of cellular mRNAs
(up to 10%) was efficiently translated in cells infected with poliovirus, which is
known to cleave eIF4G and hence inhibit cap-dependent translation (Johannes etal.
1999). Indeed, subsequent studies described functional IRES elements in a variety
of cellular mRNAs, although it needs to be emphasized that many of these IRES
elements were subsequently shown to be artefacts, and the validity of cellular IRES
is still hotly debated (reviewed in Jackson 2013; Komar etal. 2012). Nevertheless,
several cellular IRES were shown to be bona fide regulatory elements that drive
translation of their respective proteins in a cap- and/or eIF4E-independent manner
during cellular stress. Interestingly, many of the IRES-harbouring mRNAs encode
proteins that are key regulators of cell proliferation and survival/apoptosis. This is
not surprising given that the aforementioned cellular processes require strict control
of gene expression. IRES-mediated translation provides a means for escaping the
global suppression of the protein synthesis and allows the selective translation of
specific mRNAs. Based on this notion, we and others have proposed that the selec-
tive regulation of IRES-mediated translation is important for the regulation of cell
death and survival (Holcik and Sonenberg 2005; Holcik etal. 2000a; Lewis and
Holcik 2005; Sachs etal. 1997; Silvera etal. 2010). Indeed, the experimental data
from many laboratories have now validated this hypothesis in a number of models
(reviewed in Elroy-Stein and Merrick 2007; Silvera etal. 2010). The in vivo evi-
406 M. Holcik
Fig. 19.3 Mechanisms of cap-dependent (left) and IRES-dependent (right) translation initiation.
Initiation factors thought to favor one mode of translation initiation over the other are indicated.
Only factors pertinent to this chapter, and shown to be modified during apoptosis are indicated.
ITAFs represent IRES-binding proteins that has been demonstrated or suggested by several labora-
tories to be involved in translation of distinct IRESs. Question mark denotes suspected interaction.
Adapted and modified from Holcik etal. (2000a).
dence supporting this concept, and particularly in the context of cancer formation
is particularly striking. For example, the selective impairment of IRES-mediated
translation but not cap-dependent translation results in enhanced apoptosis of hema-
topoietic progenitors and stem cells, leading to the fatal progressive bone marrow
failure syndrome known as dyskeratosis congenita and its associated tumorigenesis
(Bellodi etal. 2010a; Bellodi etal. 2010b; Yoon etal. 2006). A switch to IRES-
dependent translation has been recently implicated in breast cancer growth and an-
giogenic potential in vivo (Braunstein etal. 2007), and in the acquired resistance
of cancer cells to radiation-induced apoptosis (Gu etal. 2009). Similarly, HPV-
induced transformation of human keratinocytes is accompanied by a switch from
cap-dependent to IRES-dependent translation (Mizrachy-Schwartz etal. 2007).
19.6Antiapoptotic Proteins
19.6.1IAP Family
The inhibitor of apoptosis (IAP) proteins are a family of potent inhibitors of apop-
tosis that bind to and inhibit key caspases involved both in the initiation and the
execution steps (Holcik and Korneluk 2001; Salvesen and Duckett 2002). Since the
aberrant regulation of apoptosis has been implicated in various disorders ranging
from cancer to autoimmunity to neurodegeneration, the control of apoptosis has
emerged as a key pharmacological target (Reed 1999). There is now overwhelming
evidence for the role of the IAP genes in cancer (Carter etal. 2011; Dean etal. 2009;
LaCasse etal. 2006; Schimmer etal. 2009; Tamm etal. 2000). Targeting of IAP
expression is a promising therapeutic opportunity in the treatment of cancer and
is now in clinical trials worldwide (Flygare and Fairbrother 2010). IAPs regulate
many key cellular processes including signaling, cell division, and apoptosis (de
Almagro and Vucic 2012).
Two of the eight mammalian IAPs, cIAP1 and XIAP, were shown to be regu-
lated primarily at the level of protein synthesis. Both cIAP1 and XIAP have long 5
UTRs with a high degree of predicted secondary structure and contain larger than
expected number of initiation AUG codons, features suggestive of poor translation
(Baird etal. 2006; Holcik etal. 1999; Riley etal. 2010; Warnakulasuriyarachchi
etal. 2004; Warnakulasuriyarachchi etal. 2003). Several studies have now estab-
lished that cIAP1 and XIAP expression is selectively upregulated in response to
various apoptotic stimuli and is driven by their respective IRES elements. For ex-
ample, cIAP1 translation is activated following treatment of cells with tunicamycin
or thapsigargin (Graber etal. 2010; Warnakulasuriyarachchi etal. 2004), etopo-
side or sodium arsenite (Van Eden etal. 2004), or viral infection (Yoshimura etal.
2008). Similarly, XIAP translation is activated in response to low dose irradiation
(Gu etal. 2009; Holcik etal. 2000b), serum deprivation (Holcik etal. 1999; Riley
etal. 2010; Thakor and Holcik 2012), or treatment of cells with FGF2 (Liwak etal.
2012b). In all cases, IRES-mediated translation of either cIAP1 or XIAP is critical
for survival of cells under stress (Gu etal. 2009; Holcik etal. 2000b; Riley etal.
2010; Van Eden etal. 2004; Warnakulasuriyarachchi etal. 2004).
The sequence and structure determinants of cIAP1 and XIAP IRESs, as well
as the proteins that specifically interact with each IRES, were determined. As re-
ported previously for other cellular IRESs, cIAP1 and XIAP IRES do not share
much sequence or structural similarity (Baird etal. 2007; Faye etal. 2013). The
initial RNA chromatography approaches to identify IRES binding proteins revealed
completely different sets of ITAFs for these two IRES (Graber etal. 2010; Lewis
etal. 2007). Not surprisingly therefore, the promotion of IRES-mediated transla-
tion of cIAP1 differs from that of XIAP. Activation of cIAP1, but not XIAP IRES
in the context of endoplasmic reticulum stress is dependent on caspase activation
and requires proteolytic processing of DAP5/p97 into DAP5/p86 isoform (Lewis
etal. 2008; Warnakulasuriyarachchi etal. 2004). Under conditions of reduced cap-
dependent translation, cIAP1 mRNA is selectively recruited to the polysomes via
IRES, and this process is dependent on DAP5/p97, as reducing the levels of en-
dogenous DAP5/p97 by siRNA abrogated both the translation and IRES activity of
cIAP1 during endoplasmic reticulum stress. Consistent with the role of DAP5/p97
as an activator of translation of select mRNAs, in particular as part of the cellular
stress response, DAP5/p97 knockdown is not compatible with cell survival (Lee
and McCormick 2006; Nousch etal. 2007; Yamanaka etal. 2000). Although the
precise mechanism of how DAP5/p86 enhances cIAP1 IRES activity is not known,
it is likely that it involves association with other accessory proteins. For example,
another cIAP1 ITAF, NF45 (nuclear factor 45), was shown to be required for activ-
ity of cIAP1 IRES in response to endoplasmic reticulum stress (Graber etal. 2010).
Interestingly, reducing the levels of NF45 by RNA interference resulted in reduced
translation of both XIAP and cIAP1, and it was subsequently shown that NF45
preferentially binds to and regulates a cohort of AU-rich IRES-containing mRNAs,
such as cIAP1 and XIAP (Faye etal. 2013). Whether NF45 and DAP5/p97 interact
with each other, however, remains to be determined.
408 M. Holcik
XIAP protein is encoded by two mRNA splice variants that differ only in their 5
UTR regions (Riley etal. 2010). Under normal growth conditions the more abun-
dant shorter transcript produces the majority of XIAP protein by cap-dependent
translation. However, during cellular stress, the longer, IRES-containing, transcript
supports efficient translation even though global cap-dependent translation is atten-
uated (Riley etal. 2010; Thakor and Holcik 2012). Translation of XIAP IRES can
proceed efficiently even in the absence of the ternary complex since the XIAP IRES
uses an alternative mode of Met-tRNA delivery facilitated by eIF5B (see above)
(Thakor and Holcik 2012). In addition, XIAP IRES has been shown to be activated
by growth signals, such as FGF2. Treatment of SCLC with FGF2 protects them from
etoposide-induced cell death by upregulating XIAP, and another antiapoptotic pro-
tein, BCL-XL (Pardo etal. 2003; Pardo etal. 2006). Stimulation of cells with FGF2
leads to activation of S6K2 which subsequently phosphorylates a known tumor
suppressor PDCD4, leading to its proteasomal degradation (Liwak etal. 2012b).
PDCD4 is a translational regulator of XIAP and BCL-XL IRES, which normally
binds to and represses XIAP and BCL-XL IRES; thus, removal of PDCD4 results
in derepression of XIAP and BCL-XL translation (Fig.19.4). Interestingly, loss of
PDCD4 has been correlated with more aggressive and invasive tumors (Qian etal.
2009), and this correlation is further extended to increased expression of BCL-XL
and enhanced chemoresistance, in particular in glioblastomas (Liwak etal. 2013).
19.6.2 BCL-2 Family
At least two members of the BCL-2 family of apoptotic proteins, BCL-2 and BCL-
XL, are also translated by the IRES-dependent mechanism during stress. Both BCL-
2 and BCL-XL are prosurvival factors of the BCL-2 family of proteins that regulate
mitochondrial membrane permeability and the release of proapoptotic factors such
as cytochrome c and SMAC /DIABLO, thus triggering cell death (Youle and Stras-
ser 2008). It was shown that in cells treated with etoposide or sodium arsenite the
BCL-2 translation was induced through its IRES element to maintain the balance
between proapoptotic and antiapoptotic proteins and to delay unwanted induction
of apoptosis (Sherrill etal. 2004). Interestingly, the IRES activity of BCL-2 requires
DAP5/p97 since cells depleted of DAP5/p97 show reduced expression of BCL-2,
reduced BCL-2 IRES activity, and increased cell death (Marash etal. 2008). Simi-
larly, preliminary data suggested that BCL-XL protein levels were also reduced in
DAP5/p97-depleted cells without concomitant changes in BCL-XL mRNA levels,
although it was not directly tested if the activity of BCL-XL IRES was abrogated
in these conditions (Liberman etal. 2009). BCL-XL IRES was initially identified
in an unbiased screen. Yoon and colleagues have shown that translation of BCL-
XL mRNA is specifically impaired in cells harbouring mutation of dyskerin, the
gene mutated in patients suffering from dyskeratosis congenita (Yoon etal. 2006).
This group further demonstrated that the 5 UTR of BCL-XL contains an IRES el-
ement, which drives BCL-XL translation under stress. Additional recent reports
confirmed the 5 UTR of BCL-XL as a key regulatory cis-element controlling ex-
Fig. 19.4 FGF2 modulates survival through S6K2 and PDCD4. Normally, IRES-bound PDCD4
blocks access of the ribosome to the mRNA, thus inhibiting efficient translation (top). FGF2-medi-
ated phosphorylation, and subsequent degradation of PDCD4 through the proteosome, leads to
derepression of the IRES-dependent translation of cellular mRNAs (such as XIAP and BCL-XL)
that mediate FGF2/S6K2 prosurvival signaling, which results in enhanced survival and apoptotic
resistance of cancer cells (bottom).
pression of BCL-XL. Cytoplasmic accumulation of hnRNP A1 in cells undergoing

osmotic shock was shown to tip the survival balance in favor of apoptosis by inhib-
iting translation of the BCL-XL mRNA through binding to its 5 UTR (Bevilacqua
etal. 2010). Similarly, binding of PDCD4 to the 5 UTR of BCL-XL specifically
repressed BCL-XL translation (Liwak etal. 2012b). This repression was relieved in
cells treated by FGF2, resulting in the degradation of PDCD4 and de-repression of
BCL-XL translation. Finally, RNA-binding protein HuR was shown to specifically
and directly bind to the 5 UTR of BCL-XL and function as a repressor of BCL-XL
translation (Durie etal. 2013). Although the HuR levels and subsequent changes in
BCL-XL expression were not linked directly to cell death, reduction in HuR expres-
sion by siRNA resulted in a marked fragmentation of the mitochondrial network
suggesting that HuR contributes to the regulation of the mitochondrial dynamics by
controlling IRES-dependent expression of BCL-XL.
19.7Proapoptotic Proteins
Although a majority of the cellular proteins encoded by mRNAs with IRES fall into
the category of proteins that oppose cell death, the following examples represent
proteins whose expression drives apoptosis.
410 M. Holcik
19.7.1 APAF-1
APAF-1 (apoptotic protease activating factor 1) is an essential component of the

apoptosome, a large multiprotein structure that is required for the activation of cas-
pase 9 (reviewed in Yuan and Akey 2013). The IRES of APAF-1 mRNA supports a
low level of translation and requires binding of at least two RNA-binding proteins,
PTB (polypyrimidine tract-binding protein) and UNR, to attain proper conforma-
tion which renders the IRES amenable to ribosome loading (Coldwell etal. 2000;
Mitchell etal. 2001; Mitchell etal. 2003). It should be noted, however, that an al-
ternative interpretation also exists for APAF-1 translation. The APAF-1 5 UTR has
been postulated to support translation which is m7G cap- and IRES-independent,
but instead relies on 5-end dependent scanning which utilizes specific features of
its 5 UTR (Andreev etal. 2012, 2013). Irrespective of the precise molecular mecha-
nism of how APAF-1 5 UTR recruits the ribosome, the data shows congruently that
translation of APAF-1 mRNA continues selectively when the global translation is
attenuated. Given the essential role for APAF-1 in the progression of intrinsic apop-
tosis, the IRES-mediated translation of APAF-1 has been suggested to be required
for the execution of apoptosis (Coldwell etal. 2000). This is indeed the case during
apoptosis induced by etoposide (Andreev etal. 2012; Nevins etal. 2003) and UV
irradiation (Ungureanu etal. 2006) in mammalian cells, or by siRNA targeting of
specific eIF4G isoforms during oogenesis in C. elegans (Contreras etal. 2011).
However, the APAF-1 IRES does not function well during TRAIL- or staurospo-
rine-induced apoptosis (Bushell etal. 2006; Stoneley and Willis 2004) suggesting
that it might be subjected to differential regulation depending on the apoptotic trig-
ger.
19.7.2 DAP5
As mentioned earlier, DAP5/p97 is a member of the eIF4G family of proteins. In

addition to being the critical switch for enabling IRES-dependent translation dur-
ing cell stress, DAP5/p97 mRNA is itself translated by an IRES-dependent mecha-
nism, and is activated in cells undergoing apoptosis (Henis-Korenblit etal. 2002;
Henis-Korenblit etal. 2000; Nevins etal. 2003). This mechanism is similar to that
of APAF-1, where the caspase-cleaved fragments of eIF4G1 and DAP5/p97 itself
drives DAP5/p97 IRES-dependent translation, thus providing a positive feedback-
loop. In addition, during endoplasmic reticulum stress , the expression of DAP5/
p97 is enhanced by selective recruitment of DAP5/p97 mRNA into polysomes via
the DAP5/p97 IRES (Lewis etal. 2008). However, unlike in etoposide-treated cells,
the induction of DAP5/p97 during endoplasmic reticulum stress is caspase-inde-
pendent, such that it is the full length unprocessed DAP5/p97 which functions as a
translational activator of its own mRNA.
19.7.3 c-MYC
A transcription factor and potent oncogene c-MYC is translated through an IRES-

dependent as well as cap-dependent mechanism (Nanbru etal. 1997; Stoneley etal.
1998). c-MYC IRES is activated in response to genotoxic stress (Subkhankulova
etal. 2001), viral infection (Johannes etal. 1999) and following induction of apop-
tosis by FAS ligand (Stoneley etal. 2000) or taxol (Pineiro etal. 2010). A mutated
form of the c-MYC IRES, which enhances IRES activity and results in increased
expression of c-MYC was identified in a cohort of multiple myeloma patients
(Chappell etal. 2000). It was later determined that two c-MYC IRES-binding pro-
teins, YB-1 and PTB, and PTB, bind more strongly and synergistically to the mu-
tated c-MYC IRES (Cobbold etal. 2010). Interestingly, the expression of PTB and
YB-1 was higher in multiple myeloma-derived cell lines, suggesting that both the
increased affinity of the mutated c-MYC IRES, as well as enhanced expression of
YB-1 and PTB, contribute to high levels of c-MYC in these cells. Similarly, stimu-
lation of multiple myeloma cells by IL-6 resulted in an increase in IRES-dependent
translation of c-MYC, in this case by the phosphorylation-dependent increase in the
affinity of hnRNP A1 to c-MYC IRES (Shi etal. 2008). Recently, cardiac glycoside
compounds were identified as inhibitors of c-MYC IRES-dependent translation,
suggesting that specific inhibition of IRES-mediated translation may be a success-
ful strategy to block tumorigenesis (Didiot etal. 2013).
19.8IRES-Dependent Translational Control of EMT
Epithelial-mesenchymal transition (EMT) plays a key role in normal embryonic

development as well as in cancer progression and metastasis. EMT is associated
with higher cell motility, increased resistance to apoptosis and enhanced chemo-
resistance (reviewed in Thiery etal. 2009). Although EMT is primarily controlled
at the transcriptional level (Groger etal. 2012; Zeisberg and Neilson 2009), recent
data suggests that translational control plays an important role as well, particularly
in controlling the expression of EMT master regulators, such as SNAIL1 and ZEB2
(zinc finger E-box-binding homeobox 2) (Zheng and Kang 2013). Enforced expres-
sion of a transcription/translation regulatory protein YB-1 in noninvasive breast
epithelial cells induced EMT and directly activated IRES-dependent translation of
several transcription factors, including SNAIL1 (Evdokimova etal. 2009). Simi-
larly, decreased expression of eIF3e observed during EMT in breast epithelial cells
resulted in preferential translation of SNAIL1 and ZEB2 while concurrently re-
ducing global translation (Gillis and Lewis 2012). Like SNAIL1, ZEB2 has been
shown to contain an IRES in its 5 UTR (Beltran etal. 2008). Expression of a critical
extracellular matrix protein, laminin B1 (LAMB1), is also enhanced during EMT
of malignant hepatocytes. The 5 UTR of LAMB1 harbours an IRES element which
is activated during EMT, resulting in increased translation of LAMB1 (Petz etal.
412 M. Holcik
2007). This EMT-associated increase in LAMB1 IRES activity and LAMB1 protein
levels is dependent on the cytoplasmic accumulation of RNA-binding protein La
(Petz etal. 2012a), which requires PDGF (Petz etal. 2012b).
19.9eIF4E-Independent and IRES-Independent

Translation
During low oxygen conditions (hypoxia) the general protein synthesis is severely
reduced due to sequestration of eIF4E by 4E-BPs, a consequence of reduced ac-
tivity of mTOR (Connolly etal. 2006). Although several mRNAs continue to be
translated via the IRES mechanism (for example, VEGF (Braunstein etal. 2007),
HIF-1 (Lang etal. 2002) or BCL-2 (Sherrill etal. 2004)) that relies on eIF4G and
4E-BPs to reprogram cellular translational output, a completely different transla-
tional oxygen-regulated switch was recently described that does not utilize an IRES.
It was observed that translation of EGFR mRNA is activated during hypoxia, and
this activation requires activity of an oxygen-regulated hypoxia-inducible factor
HIF-2, a transcription factor involved in the maintenance of cellular oxygen ho-
meostasis (Franovic etal. 2007). HIF-2 nucleates an oxygen-regulated translation
initiation complex that promotes selective translation of a cohort of mRNAs in a
cap-dependent, but eIF4E-independent manner (Uniacke etal. 2012). This com-
plex consists of HIF-2, the RNA-binding protein RBM4 and a homolog of eIF4E,
the cap-binding protein eIF4E2. Interestingly, this complex is recruited to its target
mRNAs via an RNA hypoxia response element (rHRE), which is located in the 3
UTR of a variety of hypoxia inducible mRNAs (for example, EGFR, PDGFRA
and IGF-1R), and subsequently promotes loading of these mRNAs to polysomes
for active translation. Importantly, since 4E-BPs have a significantly lower affinity
for eIF4E2 than for eIF4E, and since eIF4E cannot replace eIF4E2 in the HIF-2/
RBM4/eIF4E2 complex, this complex supports cap-dependent yet eIF4E-indepen-
dent protein synthesis that is resistant to hypoxia-induced repression of translation
(Uniacke etal. 2012).
19.10Concluding Remarks
Fine-tuning of cellular stress response by means of modulating translation is now

a widely accepted notion. Yet, the precise mechanisms and the scope of variations
on these mechanisms are still not completely understood and their interrogations
keep providing us with new and surprising findings. This is true particularly in the
context of cancer cells, which usurp processes that non-cancerous cells use only
in extreme situations, such as response to death stimuli. The examples shown here
illustrate how dysregulated translation plays a key role in apoptotic response, and
thus tumorigenesis and chemoresistance of cancer cells. In addition to providing us
with an in-depth understanding of the translational control mechanisms, this knowl-

edge also offers an opportunity for designing strategies for selective targeting of
these processes with the hope of therapeutic utility in the future.
Acknowledgments I am indebted to my colleagues and students at the Apoptosis Research Centre

for fruitful and frequently spirited discussions of the subject covered in this chapter. I apologize to
those colleagues whose work was not cited due to space considerations. The work in my laboratory
is supported by operating grants from the Canadian Institutes of Health Research, Cancer Research
Society, and Natural Sciences and Engineering Research Council of Canada.
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Chapter 20
Translation in Cancer at Hypoxia
Tingfang Yi and Gerhard Wagner
Contents
20.1Hypoxia in Cancer 421

20.2Translation in Cancer Cells Under Hypoxic Conditions 422
20.2.1Uncoupling of mTOR Signaling Inhibition 423
20.2.2HIF-1/eIF4E1 Cap-Dependent Translation Pathway 424
20.2.3HIF-2/RBM4/eIF4E2-Mediated Translation 425
20.2.4IRES-Dependent Translation 425
20.3Translation Regulation in Hypoxia-Promoted Tumor Angiogenesis 426
20.4Translation Regulation in Cancer Stem Cells in the Hypoxic Niche 427
20.5Translational Regulation in Hypoxia-Enhanced Tumor Metastasis 427
20.6Development of Therapies Targeting Translation in Hypoxic Cancer Cells 428
References 429
Abstract Low oxygen concentration (hypoxia) promotes cancer evolution, tumor

angiogenesis and tumor metastasis. Under hypoxic conditions cancer cells undergo
genetic and adaptive changes in regulation of translation that facilitate production
of proteins involved in tumor angiogenesis, cancer cell survival, proliferation and
invasion. Hence, the translation machinery in cancer cells exposed to low oxygen
levels is a candidate therapeutic target. This chapter focuses at the role of hypoxia
in cancer biology vis--vis translation and its regulation.
20.1Hypoxia in Cancer
About 60 years ago, Thomlinson and Gary postulated the presence of hypoxia in
human tumors based on their observations of necrosis far from vessels within tu-
mors. They also found that hypoxic cancer cells are resistant to radiotherapy, which
G.Wagner() T.Yi
Department of Biological Chemistry and Molecular Pharmacology,
e-mail: Gerhard_Wagner@hms.harvard.edu
422 T. Yi and G. Wagner
kills cells by introducing oxidation-dependent DNA damage (Gray etal. 1953;

homlinson and Gray 1955). Solid tumors associate with chronic hypoxia (when
T
tumors outgrow their blood supply due to uncontrolled proliferation) and acute
hypoxia (transient periods of low oxygen caused by aberrant blood flow). Oxygen
concentrations in human tumors are highly heterogeneous with many regions of
the tumor having very low levels of partial pressure of oxygen, such as 2.4mmHg
in prostate (Movsas etal. 2001) and 2.7mmHg in pancreatic cancer (Koong etal.
2000). These levels of partial pressure of oxygen are substantially low in compari-
son to those of 1465mmHg observed in normal organs and tissues (Brahimi-Horn
and Pouyssegur 2007). Intratumor hypoxia contributes to genome instability by re-
pressing DNA-repair pathways, enhanced angiogenesis and vasculogenesis, cancer
stem cell maintenance, EMT, induction of autophagy, anaerobic metabolic switch,
suppression of immune surveillance, selection of clonal populations resistant to
apoptosis, and more metastatic phenotypes (Brown and Wilson 2004).
Cancer cells at hypoxia within solid tumors generally present resistance to most
anticancer agents due to several reasons. First, resistance may be due to inadequate
exposure of some regions of cancer to anticancer drugs as a consequence of the
distance of hypoxic cancer cells from blood vessels and as a result of aberrant tu-
mor vessel structure-associated leakiness (Stylianopoulos and Jain 2013; Tannock
1998). Second, hypoxia selects for cells with lost sensitivity to p53-mediated apop-
tosis, which subsequently might decrease sensitivity to some anticancer drugs (Kim
etal. 1997). Third, hypoxia promotes the expression of genes involved in drug
resistance (Comerford etal. 2002; Wartenberg etal. 2003). Fourth, hypoxia pro-
motes the enrichment of cancer stem cells, which have enhanced drug resistance
(Kim etal. 2009; Li etal. 2009). Fifth, hypoxia enhances tumor angiogenesis and
therefore facilitates tumor metastasis (Folkman 2007). Lastly, hypoxic cancer cells
utilize alternative translation pathways to bypass translation suppression induced by
hypoxia (Silvera etal. 2010).
Hypoxia promotes cancer evolution through upregulating transcription of at least
60 putative genes in cancer cells, which has been elegantly reviewed (Harris 2002).
Here we focus on the effects of hypoxia on translation regulation and therapeutic
strategies targeting elements of the translation machinery in hypoxic tumors.
20.2Translation in Cancer Cells Under Hypoxic Conditions
Although translation is considered to be globally attenuated in hypoxic cells, which

contributes to conserving energy and sustaining survival during periods of ineffi-
cient ATP production, a subset of mRNAs believed to be involved in the adaptive
responses to hypoxia are preferentially translated in cancer cells (Connolly etal.
2006; Koritzinsky etal. 2006). For instance, high levels of the main isoform of
translation initiation factor eIF4E, eIF4E1; transcription factors such as hypoxia
inducible factor 1 (HIF-1), CREB, and NF-B; oncoprotein c-MYC and cell cy-
cle regulator cyclin D1; proangiogenic factor VEGF; VEGF receptor 2 (VEGFR2)
and Tie2 have been observed in many types of solid tumors (Mamane etal. 2004;
20 Translation in Cancer at Hypoxia 423
Ruggero etal. 2004). Even at anoxia (0% O2), eIF4G and activating transcription
factors 4 and 6 (ATF4 and ATF6) have been shown to remain associated with heavy
polysomal fractions, representing an active translation state (Bauer etal. 2002;
Blais etal. 2004).
In normal cells, suppressed translation at acute anoxia (0% O2 for 14h) is
regulated by phosphorylation of eIF2, whereas in prolonged anoxia (0% O2 for
16h) repression of translation is maintained by inhibition of eIF4E via its binding
proteins 4E-BPs and the eIF4E transporter, 4E-T. Hypoxia (0.51.5% O2) inhibits
protein synthesis through suppression of multiple key regulators, including eIF2,
eEF2, S6K, mTOR and rpS6 (Knaup etal. 2009; Romero-Ruiz etal. 2012; Zhu
etal. 2009). Moderate hypoxia (1.5% O2) in combination with serum deprivation
effectively inhibits mTOR activity and causes hypophosphorylation of the mTOR
substrates 4E-BP1 and S6K in normal cells. Translational repression in cancer cells
under chronic hypoxia and glucose depletion is independent of the eIF2 kinase
PERK (Liu etal. 2006).
However, cancer cells are believed to employ several alternative translation path-
ways to bypass hypoxia-induced translational repression in response to adaption to
low oxygen circumstances that include: uncoupling of mTOR signaling inhibition
of cap-dependent translation; upregulating translation of a subset of mRNAs via
HIF-1/eIF4E1-dependent mechanism; upregulating translation via HIF-2/RBM4/
eIF4E2 mediated mechanism; and using internal ribosome entry site or IRES-depen-
dent translation (Fig.20.1). These mechanisms are further discussed below.
20.2.1 Uncoupling of mTOR Signaling Inhibition
eIF4E-binding proteins, 4E-BPs, are potent endogenous repressors of translation

(see Chaps.3 and 4). The main isoform of 4E-BPs is 4E-BP1, on which a majority
of studies in translational regulation are focused. Hypophosphorylated 4E-BPs bind
to eIF4E and prevent the recruitment of eIF4G, thereby disrupting the assembly of
the eIF4F complex (see Chap.4). The mTOR complex hyperphosphorylates 4E-
BPs, leading to their dissociation from eIF4E and its binding to eIF4G. The eIF4F
complex is formed to initiate cap-dependent translation.
Hypoxia-stimulated AMPK activates TSC2, which together with TSC1 inhibits
mTOR and subsequently represses translation initiation in normal cells (Bjornsti
and Houghton 2004) (see Fig.20.1). However, in hypoxia, some cancer cells, such
as breast cancer cells, elicit ability to uncouple TSC2 to block mTOR (Connolly
etal. 2006). In addition, control of mTOR activity by its negative regulator REDD1
is also uncoupled in hypoxic cancer cells (Connolly etal. 2006). However, the
mechanisms of how cancer cells uncouple regulation of mTOR in cancer are still
unclear. The uncoupling of mTOR leads to uninhibited signaling downstream to the
4E-BPs/eIF4E axis. As described in previous chapters eIF4E appears to selectively
facilitate translation of mRNAs that encode for prosurvival, pro-proliferative and
proangiogenic proteins (see Chap. 4). Hence, uninhibited eIF4E, as in case of un-
coupling described above, would in theory create a protumorigenic state.
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pression in tumors. 1 Uncoupling of mTOR signaling inhibition. 2 HIF-1/eIF4E1 cap-dependent
translation pathway. 3 HIF-2/RBM4/eIF4E2 mediated translation. 4 IRES-Dependent transla-
tion. See text for more details.
20.2.2 HIF-1/eIF4E1 Cap-Dependent Translation Pathway
Most eukaryotic mRNAs have the m7GpppN cap structure at the most 5 end of the
mRNA (see Chap.2). eIF4E is the cap-binding component of the eIF4F complex
that is essential for cap-dependent translation and ribosome recruitment to mRNAs
(Marintchev and Wagner 2004).
The human eIF4E family consists of three members: eIF4E1, eIF4E2 (4EHP),
and eIF4E3. The canonical isoform is eIF4E1 that is mostly studied and is broadly
referred in this book as eIF4E. Only eIF4E1 and eIF4E3 bind to eIF4G (Joshi etal.
2004). eIF4E3 competes with eIF4E1 for cap-binding and fails to bind the 4E-BPs
(Osborne etal. 2013). eIF4E1 is the dominant eIF4E family member, which pri-
marily suppresses eIF4E3 in breast cancer cells at normoxia and hypoxia (Yi etal.
2013). At low oxygen levels, hypoxia-stabilized HIF-1 binds to the hypoxia re-
sponse elements (HREs) at the proximal promoter region of human eIF4E1 and
facilitates eIF4E1 expression (see Fig.20.1). The enhanced eIF4E1 in hypoxic
cancer cells upregulates cap-dependent translation of a subset of mRNAs that code
for proteins implicated in cell proliferation and tumor growth, such as cyclin D1,
c-MYC, VEGF and eIF4G1 (Yi etal. 2013) (see also Chap.4). Thus, the increased
sensitivity of breast cancer cells to the eIF4E1/eIF4G1 interaction inhibitor 4EGI-1
could represent an effective strategy towards inhibiting eIF4E prosurvival and pro-
proliferative function in hypoxia (Yi etal. 2013).
20.2.3 HIF-2/RBM4/eIF4E2-Mediated Translation
In glioblastoma cells, hypoxia stimulates the formation of HIF-2/RBM4/eIF4E2

complex at the RNA hypoxia response element (rHRE). HIF-2/RBM4 recruits
the mRNA cap to eIF4E2 and targets mRNAs to polysomes for activation, which
evades hypoxia induced translation repression (Uniacke etal. 2012) (see Fig.20.1).
The HIF-2/RBM4/eIF4E2 complex preferentially promotes cap-dependent trans-
lation of selective mRNAs coding for proteins implicated in the adaptive response
to hypoxia, as well as a wide variety of biological processes including cancer, such
as EGFR, PDGFR and IGF-1R (Uniacke etal. 2012). As eIF4E2 does not bind to
eIF4G, which is a scaffold platform for several translation initiation related factors,
such as eIF4AI, eIF3 and poly(A)-binding protein 1 (PABP1), the mechanisms as
to how the HIF-2/RBM4/eIF4E2 complex recruits the ribosome and effectively
induces this alternative translation initiation in hypoxic cancer cells are unclear and
require further studies.
20.2.4 IRES-Dependent Translation
The 5 UTR of some mRNAs harbor IRES cis-elements, which facilitate direct ri-
bosome binding independent of formation of the eIF4F complex and its binding
to the cap (Pelletier and Sonenberg 1988) (see Chaps.3 and 19). IRES-dependent
translation was first discovered to be used by some viruses to translate their RNA
and switch the translation machinery from cap-dependent translation of cellular
mRNAs. However, further studies suggested that this mechanism could be used
to translate endogenous mRNAs as well. It is now estimated that about 10% of
mRNAs in human cells have the capacity to initiate translation via IRES (Stoneley
and Willis 2004). Cancer cells at hypoxia employ IRES-dependent translation to
express proteins involved in cell growth, proliferation, survival and angiogenesis,
such as transcription factors c-MYC, c-JUN and HIF-1; growth factors and recep-
tors FGF2, PDGF2, VEGF-A, IGF-2, IGF-1 and IGF-1R; translation and RNA pro-
cessing factors eIF4G1 and eIF4G2 (DAP5); and PKC and calcium/calmodulin-
dependent protein kinase II (CaMKII) (Henis-Korenblit etal. 2000; Morrish and
Rumsby 2002; Stoneley and Willis 2004) (see Fig.20.1). Although the mechanisms
of viral IRES translation have been well characterized, the translation mechanisms
of cellular IRESs are yet to be determined.
It is important to note, that there is controversy regarding the existence of the
cellular IRES-dependent translation initiation mechanism. For, example, there are
seemingly controversial observations that VEGF-A mRNA can be translated by cap-
dependent, but also IRES-dependent mechanisms in hypoxic cancer cells (Young
etal. 2008). Speculatively, the difference between viral and cellular IRES containing
RNA is that cellular signals are usually capped, so in a setting where more efficient
cap-dependent translation is suppressed IRES-dependent translation surfaces as
a mechanism of translation of specific mRNAs. This could be a mechanism that is,
at least in part, employed during hypoxia. However, understanding of the possible
dynamic switches between IRES- and cap-dependent translation of some mRNAs
in cancer cells at low oxygen levels is still limited.
20.3Translation Regulation in Hypoxia-Promoted Tumor

Angiogenesis
Tumor angiogenesis, new vessel generation and growth within tumors, is consid-
ered as a hallmark of cancer development and metastasis. Intratumor hypoxia po-
tently promotes tumor angiogenesis and vasculogenesis within tumors (Folkman
1995). Interestingly, cancer stem cells, in brain and breast cancer models, have been
shown to differentiate into vessel endothelial cells and pericytes, that are contractile
cells that wrap around the endothelial cells, and therefore promote tumor angiogen-
esis (Cheng etal. 2013; Ricci-Vitiani etal. 2010).
While the mechanisms of hypoxia-induced angiogenesis vis--vis translation-
al regulation still remains to be better elucidated, there are strong indications that
translational regulation is involved in tumor angiogenesis in hypoxia. Hypoxia-
elevated HIFs (HIF-1, HIF-2 and HIF-1/ARNT (aryl hydrocarbon recep-
tor nuclear translocator)) upregulate transcription of multiple genes encoding
proangiogenic and pro-proliferative factors, such as VEGFs, VEGFRs, FGF2,
PDGF2, IGF-2, IGF-1R TGF- and TGF- receptors (Caniggia etal. 2000; Huss
etal. 2001; Zhang etal. 2003). Most of these transcripts contain IRES struc-
tures, that likely are used to evade hypoxia-induced cap-dependent translational
repression of their mRNAs (Kerbel and Folkman 2002). The 5 UTR of VEGF
mRNA harbors both canonical AUG, but also CUG start codons as well as two
IRES structures, which may endow the redundant IRES-dependent and IRES-
independent translation of VEGF mRNAs within cancer cells under hypoxia
conditions (Akiri etal. 1998).
Hypoxia upregulates 4E-BP1 and eIF4G levels and promotes IRES-dependent
translation of VEGF and HIF-1, and subsequently enhances tumor angiogenesis
and vascularization in breast tumors (Azar etal. 2013; Braunstein etal. 2007) In-
creased levels of 4E-BP1 trigger hypoxia-induced inhibition of cap-dependent trans-
lation of VEGF, while elevated eIF4G promotes VEGF IRES-dependent translation
and selective translation of mRNAs containing IRES that encode proteins important
for angiogenesis, hypoxia adaptation and survival. The switch from cap-dependent
to cap-independent translation of selective mRNAs facilitates angiogenesis within
tumors (Braunstein etal. 2007).
Although intensive efforts have been made in the therapy of cancer by targeting
hypoxic tumor angiogenesis, the effects of various pharmaceutical approaches are
transient, modest and frequently accompanied by resistance and further worsening

of malignant characteristics (Rapisarda and Melillo 2012). These clinical observa-
tions suggest that the mechanisms of tumor angiogenesis and proangiogenic protein
synthesis within hypoxic tumors require intensive research efforts.
20.4Translation Regulation in Cancer Stem Cells

in the Hypoxic Niche
Cancer stem cells enrich in the hypoxic niche that is a region relatively far from the
vasculature and thus is oxygen depleted (Smalley and Ashworth 2003). The hypoxic
microenvironment is required for cancer stem cell maintenance and self-renewal
(Keith and Simon 2007). HIF-2 is critical for brain cancer stem cell self-renewal
and multipotency through upregulating expression of OCT4 and NOTCH (Covello
etal. 2006). In addition, VEGF, proto-oncogene c-MYC, transcription factor SOX2
(sex determining region Y-box 2) and zinc-finger transcriptional repressor SNAIL
are found to be elevated in multiple cancer stem cells under hypoxic conditions
(Keith and Simon 2007). These mRNA have been shown to be regulated by transla-
tion (Mohyeldin etal. 2010). Direct studies to the mechanism of translational regu-
lation of proteins involved in cancer stem cell differentiation in the hypoxic niche
might prove to be important for our understanding of the cancer biology.
20.5Translational Regulation in Hypoxia-Enhanced

Tumor Metastasis
Tumor metastasis is a multistep process that involves tumor cell invasion, intrava-
sation, survival in the peripheral blood, organ-specific extravasation, colonization
and, ultimately, proliferation of dormant micro-metastases to life-threatening le-
sions. Hypoxia associates with most of these steps. HIF-1 translation is increased
in metastatic lesions in comparison to primary tumors and HIF-1 promoted gene
expression is broadly involved in tumor metastasis (Keith and Simon 2007). EMT
and its reverse program of mesenchymal-epithelial transition are considered to be
involved in hypoxia-enhanced tumor metastasis (Savagner etal. 1994; Thiery 2002).
During EMT, the expression levels of non-epithelial cadherins (such as N-cadherin)
and cell surface proteins (such as CD44 and integrin 6) are increased, while the
expression levels of epithelial junction proteins (such as E-cadherin, -catenin and
-catenin) are decreased (Tsai and Yang 2013). In addition, expression of SNAIL1,
SNAIL2, twist-related proteins (TWIST) 1 and 2, and TGF-, WNT and NOTCH
is elevated in metastatic tumors (Kim etal. 2011; Lo etal. 2007; Yuen etal. 2007).
The fact that the orchestration of protein synthesis is important for tumor invasive-
ness and relocation suggests that appropriate translational regulation is essential for
hypoxia-promoted tumor metastasis.
20.6Development of Therapies Targeting Translation

in Hypoxic Cancer Cells
Translation initiation is a highly regulated multifaceted biological process and it

is often abnormally usurped in cancer cells, and therefore is considered a poten-
tial antineoplastic target. Dysregulation of the eIF4F complex-controlled trans-
lation initiation is proposed to play an important role in cancer cells at hypoxia.
This hub of translation is becoming a focus of attention in terms of therapeutic
targeting. In recent years, several inhibitors have been developed to target eIF4F.
These include pateamine (Bordeleau etal. 2005), 4EGI-1 (Moerke etal. 2007), hip-
puristanol, silvestrol, 4E1RCat (Cencic etal. 2011), 4Ei-1 (Ghosh etal. 2009) and
4EASO4 (Graff etal. 2007). In relation to hypoxia, 4EGI-1, that is a small molecule
binding to eIF4E and inhibiting eIF4F assembly, has been found to preferentially
inhibit breast tumorsphere growth in hypoxic (compared to normoxic) conditions
and suppress breast and melanoma tumor growth (Chen etal. 2012; Yi etal. 2013).
Therefore, agents targeting selective translation on which hypoxic cancer cells are
dependent may have the benefit of opening novel avenues of therapeutic success.
While still in its late infancy, the research into the role of translation in cancer biol-
ogy under hypoxic condition indicates at the intricate involvement of the translation
initiation step of the protein biosynthesis pathway. This review elucidates the com-
plexities and multiplicity of mechanisms employed by the hypoxic cancer cell and
its environment to further build on the progression of the malignant disease. These
mechanisms appear to range from specific upregulation of translation of a subset
of mRNAs by canonical translation pathways, such as cap-dependent translation,
to switching to more rare forms of translation initiation, such as the IRES mecha-
nism. A great deal of these changes in translation occur via its aberrant regulation,
such as uncoupling of canonical signaling pathways and making them insensitive
to hypoxia-induced repression of protein synthesis observed in normal physiology.
These changes in translational regulation and, subsequently, changes in the patterns
of translation at low oxygen levels are essential for the orchestration of synthe-
sis of proteins important for tumorigenesis, cancer stem cell self-renewal, tumor
angiogenesis, tumor development and metastasis, drug resistance, and evasion of
apoptosis .
Recent developments in cancer therapeutics already provide an interesting arse-
nal that can be employed to target translation and its regulation in cancer at hypoxia.
Pharmacological therapy targeting translation in hypoxia-affected cancer may in-
hibit tumor evolution and subsequently lead to clinical benefits. However, it also
appears that we have to go a long way to understand the elegant pathological mech-
anisms that dysregulate translation in cancers at hypoxia. These efforts, however,
have a potential to enrich our yet limited spectrum of pharmaceutical compounds

that are able to effectively fight and cure cancer.
Acknowledgments Research described here was supported by NIH grants CA68262 and
GM047467.
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Part IV
Translation and Its Regulation
by Cancer Types
Chapter 21
Melanoma and Non-Melanoma Skin Cancers
Armen Parsyan, Ryan J. Sullivan, Ari-Nareg Meguerditchian

and Sarkis Meterissian
Contents
21.2Translation and Its Regulation in Melanoma 437
21.2.1eIF4E Protein and Its Regulation in Melanoma 437
21.2.2Increased Expression of eIF4E in Melanoma 438
21.2.3Activation of eIF4E in Melanoma 439
21.2.4Other Translation Factors in Melanoma 440
21.2.5eIF2 in Melanoma 441
21.2.6Role of eIF2 in Melanoma Treatment and Resistance to Chemotherapy 442
21.3Novel Therapeutics Based on Translation Targeting 443
21.4Targeted Therapies Directed at the Downstream Targets of eIF4E 443
21.4.1BCL-2 and Survivin 444
21.4.2Cyclin D 444
21.4.3VEGF/VEGFR 444
21.5Direct Targeting of Translation Factors in Melanoma 445
21.6Non-Melanoma Skin Cancers and Translation 446
References 448
AbstractCutaneous malignancies affect millions of people worldwide. These

cancers cover a wide clinical spectrum, from relatively indolent types, such as the
most common form of cancer basal cell carcinoma, to more lethal ones, such as
melanoma. In this chapter we uncover the role of the translation machinery and
its regulatory mechanisms in the biology of skin cancers with a special emphasis
on melanoma. We show that, while a plethora of translation factors appears to be
A.Parsyan() A.-N.Meguerditchian S.Meterissian

R.J.Sullivan
Center for Melanoma, Massachusetts General Hospital Cancer Center, Boston, MA, USA
A.-N. Meguerditchian S. Meterissian
Department of Oncology, Faculty of Medicine, McGill University, Montreal, QC, Canada
436 A. Parsyan et al.
implicated in the development and progression of melanoma and other skin cancers,
translation initiation factor eIF4E represents a fundamental component of their eti-
ology and pathogenesis. This paradigm is emphasized by the recent groundbreaking
discoveries of mutations and alterations in the key signaling pathways that serve as
the foundation of melanoma biology and that converge on the control of translation
via eIF4E. Different aspects of melanoma biology, treatment and drug-resistance
will be also highlighted in relation to translation and its control.
21.1Introduction
Skin cancers can be divided into melanoma (tumors of melanocytes) and non-
melanoma cancers. The most common types of non-melanoma skin cancers are
represented by cutaneous squamous cell carcinomas that are malignant tumors of
keratinizing epidermal cells and basal cell carcinoma that arises from the basal layer
of the epidermis and its appendages. Basal cell carcinoma is the most common can-
cer worldwide, with an exceptionally low metastatic potential and mortality rates
(Lewis and Weinstock 2007; Lomas etal. 2012). On the other hand, melanoma is
the deadliest form of skin cancers that has a high incidence that is steeply rising
(CDC/NCI 2013; Ferlay etal. 2010; Forsea etal. 2012; Howlader etal. 2012).
Surgical resection remains the mainstay of treatment and a few non-surgical
therapeutic options exist (Coit etal. 2013; Pflugfelder etal. 2013). The role of ef-
fective therapeutics is especially important for metastatic melanoma that has a very
poor prognosis (Balch etal. 2009). Current therapeutic options include radiothera-
py, conventional chemotherapeutic agents (such as dacarbazine, TMZ, paclitaxel,
carboplatin and others), biochemotherapy/immunotherapy (such as IFN-, IL-2 and
anti-cytotoxic T-lymphocyte antigen 4 antibody, ipilimumab) that generally confer
minimal to moderate survival benefits (Coit etal. 2013; Ives etal. 2007; Jang and
Atkins 2013). Recently-developed targeted therapies have been proven effective,
albeit in a select group of patients with metastatic disease and specific genetic find-
ings. For example, the BRAF inhibitors vemurafenib and dabrafenib, as well as the
MEK inhibitor trametinib, are approved by the FDA for the treatment of patients
with Val600 (V600) mutations of the BRAF gene (Coit etal. 2013). Additionally,
imatinib has shown promising activity in phase II studies of c-KIT mutated and
amplified tumors (Carvajal etal. 2011; Coit etal. 2013; Guo etal. 2011; Hodi
etal. 2013). However, even these targeted therapies are only rarely associated with
complete regression, and the therapeutic benefits are typically temporary due to the
development of cellular resistance (Lito etal. 2013).
Due to high mortality, the increasing incidence and the need for improved treat-
ment modalities major research efforts have been directed towards understanding
the molecular basis of melanoma in order to devise novel therapeutic strategies
and to improve the existing ones. Recent years have been very productive in terms
of identifying molecular targets, but a comprehensive understanding of melanoma
biology remains elusive.
21 Melanoma and Non-Melanoma Skin Cancers 437
Evidence suggests that melanoma is a disease of melanocytes with a wide spec-

trum of histopathologic subgroups hallmarked by a distinct pattern of oncogenic
mutations. Recent developments in melanoma research have identified that muta-
tions and aberrations of several molecular targets lead to dysregulated cell signaling
that is important in the etiology and pathogenesis of this disease (for a review see
Bello etal. 2013; Lito etal. 2013; Yajima etal. 2012). As mentioned on numerous
occasions in this book, major signaling pathways that lead to tumorigenesis con-
verge on the translation machinery. This is not an exception for melanoma, as we
will learn in this chapter. Before embarking on the analysis of the role of translation
and its control in melanoma, let us briefly review the molecular targets known to
date to be involved in melanoma biology and that are relevant to this discussion.
Two major signaling pathways appear to be involved in the pathobiology of mel-
anoma (Bello etal. 2013; Yajima etal. 2012): the PI3K/AKT/mTOR pathway and
the MAPK pathway that involves RAS/RAF/MEK/ERK cascade (see Chaps.15
and 17). Various genes (the majority of which are involved in these pathways)
are mutated in melanoma, such as BRAF, CDKN2A, NRAS, TP53, PTEN, MEK
(MAP2K1), KIT and others (Bello etal. 2013; Yajima etal. 2012). While an in-
depth discussion of these mutations is beyond the scope of this chapter, it is worth
mentioning that the majority of melanomas appear to have activating mutations in
the MAPK pathway (reviewed in Bello etal. 2013; Roring and Brummer 2012; Ya-
jima etal. 2012). BRAF is the most commonly mutated gene in melanoma involv-
ing up to 4050% of patients (Brose etal. 2002; Chapman etal. 2011; Davies etal.
2002; Pollock etal. 2003). By far the most common mutation within BRAF is the
substitution of Val by Glu at the 600 position (BRAFV600E) that makes the protein
constitutively active and constantly firing downstream of the MAPK signaling path-
way, culminating in phosphorylation of eIF4E at Ser209. Hyperactivation of MEK
by oncogenic BRAFV600E causes activation of ERK, which in turn is associated with
induction of activating phosphorylation of eIF4E (Croft etal. 2013). As discussed
in previous chapters, eIF4E is a key translation initiation factor and regulator of
translation, which is often involved in cancer biology.
Another signaling pathway involved is the PI3K/AKT/mTOR cascade that has
been associated with melanoma biology via mutations or activation of its compo-
nents or controllers, such as PTEN and AKT (reviewed in Bello etal. 2013; Davies
etal. 2009; Roring and Brummer 2012; Yajima etal. 2012). This pathway also
converges on eIF4E via control of the eIF4E-binding proteins, 4E-BPs, by mTOR.
21.2Translation and Its Regulation in Melanoma
21.2.1 eIF4E Protein and Its Regulation in Melanoma
The role of the translation machinery in skin cancers in general and melanoma in
particular was first investigated only a few years ago. Initial attempts to look into
the role of translation in melanoma mainly focused on eIF4E and failed to provide
evidence of involvement of this protein in this disease. While details regarding

eIF4E function in physiology and disease are described in Chaps.2 and 4, it is worth
mentioning that this is a proto-oncogene and a part of the trimeric eIF4F transla-
tion initiation complex that binds to a so-called cap structure on the mRNA. The
current paradigm of the eIF4E involvement in cancer biology is based on the fact
that this protein, while being a general translation factor, preferentially enhances
translation of mRNAs that possess moderate to strong secondary structures at their
5 UTR. These mRNAs often encode proteins that play essential roles in cell prolif-
eration, cell cycle, survival and angiogenesis and are represented by such molecules
as VEGF and cyclin D1 (see Chap.4). Increased levels of expression of eIF4E, its
activating phosphorylation or activation via 4E-BPs lead to increased translation of
those messages and can create a tumorigenic state. It is important to mention that
4E-BPs bind and negatively regulate eIF4E, however, upon activating signaling
from mTOR, 4E-BPs are phosphorylated and dissociate from eIF4E, liberating it to
participate in translation.
21.2.2 Increased Expression of eIF4E in Melanoma
Data from small case series indicated that eIF4E, albeit minimally expressed in
normal melanocytes, showed minimal or no increase of its expression in benign
nevi and melanomas (Rosenwald etal. 2003). This study also reported very low
levels of expression of an eIF4E target cyclin D1 in normal and malignant melano-
cytes. However, later studies, using likely more sensitive assays and larger patient
cohorts, strongly suggested that eIF4E is overexpressed in a majority of melanoma
specimens (Wen etal. 2010; Yang etal. 2007). Using tissue microarray, Yang etal.
showed that eIF4E and its translational targets (such as VEGF and cyclin D1) had
elevated expression levels in almost 6070% of melanoma specimens (Yang etal.
2007). Moreover, levels of eIF4E strongly and significantly correlated with VEGF
and cyclin D1 in melanoma (Spearmans r=0.97 and 0.77; all p<0.0001), provid-
ing indirect evidence in support of the hypothesis that there is eIF4E-dependent
control of translation of these proteins in general and in melanoma in particular.
The mechanism of eIF4E overexpression likely includes activated transcription
of its gene by proto-oncogene c-MYC (Jones etal. 1996) or other factors, such as
heterogeneous nuclear ribonucleoprotein K (hnRNP K) (Lynch etal. 2005). Inter-
estingly, a higher hnRNP K protein level in both melanoma cell lines and melanoma
tissue specimens, compared to normal or benign tissues, has been reported, cor-
relating with a higher c-MYC and eIF4E expression (Wen etal. 2010). Aberrant
or increased expression of c-MYC was reported for melanomas and linked to its
pathogenesis (Greulich etal. 2000; Shin etal. 1987; Zhuang etal. 2008). Thus,
overexpression of eIF4E in melanomas could be, at least in part, related to activa-
tion of its transcription.
A recent publication provided interesting data regarding regulation of the lev-
el of eIF4E expression by its targeting miRNA, miR-768-3p (Jiang etal. 2013).
Confirming previous reports, an increased level of expression of eIF4E was ob-

served in melanoma samples in this study. This increase in eIF4E levels was spe-
cifically associated with the downregulation of miR-768-3p. Overexpression of this
miRNA led to the downregulation of the endogenous eIF4E protein and inhibition
of cell survival and proliferation. Interestingly, downregulation of miR-768-3p ap-
peared to be mediated by activation of the MAPK pathway. In addition, BRAF in-
hibitor PLX4720 or MEK inhibitor U0126 resulted in the upregulation of miR-768-
3p, showing a potential mechanism of action of these pharmaceutical compounds
via eIF4E (Jiang etal. 2013).
21.2.3 Activation of eIF4E in Melanoma
As discussed throughout this book, the role of eIF4E in cancer biology is not lim-
ited to its overexpression but also its activation. Activation of eIF4E takes place
through various signaling cascades. eIF4E can be activated via phosphorylation by
the MAPK/ERK pathway kinase MNK (Hou etal. 2012; Waskiewicz etal. 1997).
MNKs, through the phosphorylation of eIF4E, promote cellular proliferation and
survival in cancer (see Chaps.4 and 17). The basal levels of phosphorylated eIF4E
are generally higher in melanoma cells than in melanocytes (Croft etal. 2013).
This increase in phospho-eIF4E is likely mediated by the activated MAPK pathway,
since its phosphorylation can be rapidly reduced by a V600 mutant BRAF inhibitor
PLX4720 in BRAFV600E, and by a selective MEK1/2 inhibitor U0126 in both the
BRAFV600E and wild-type BRAF melanoma cells (Croft etal. 2013). It indeed ap-
pears that the MNK/eIF4E axis is important in the pathophysiology of melanoma.
Signifying the clinical importance of this axis, Konicek and colleagues, using se-
lective MNK inhibitors that block eIF4E phosphorylation, showed suppression of
metastatic growth in the experimental B16 melanoma pulmonary metastases mouse
model (Konicek etal. 2011).
Another signal transduction cascade that can activate eIF4E is the PI3K/AKT/
mTOR pathway, that by phosphorylating and thus inactivating the inhibitory 4E-
BPs, activates eIF4E by liberating it for binding to eIF4G and forming the eIF4F
complex. A major regulator of eIF4E activity, the 4E-BP1 protein, is hyperphos-
phorylated in the majority of melanoma cell lines harboring BRAF and PTEN mu-
tations compared with untransformed melanocytes or RAS/RAF/PTEN wild-type
melanoma cells (OReilly etal. 2009). Both RAF and PTEN are part of signaling
modules involving the MAPK and PI3K/AKT/mTOR pathways that converge in
regulating the activity of eIF4E directly or via 4E-BPs (see Chaps.4, 15 and 17).
High levels of phosphorylated 4E-BP1 are found in metastatic melanoma and are
associated with significantly worse overall and postrecurrence survival (OReilly
etal. 2009). The role of AKT signaling in melanoma is further emphasized by find-
ings that AKT, namely AKT3 protein, is selectively activated in up to 60% of non-
familial melanomas, and its levels positively correlate with advanced stage disease
(Stahl etal. 2004). A tumor suppressor candidate gene RAS association domain
family 1, isoform A (RASSF1A), that appears to suppress melanoma development

by modulating apoptosis and cell cycle progression, does so, at least in part, via
reduction of phosphorylation of AKT and its downstream targets p70S6K and sub-
sequently eIF4E (Yi etal. 2011).
Thus, while various aberrant signaling events could be described and theorized
in melanoma, it appears that they eventually converge on the translation initiation
machinery, making the latter a perfect target for therapeutics.
21.2.4 Other Translation Factors in Melanoma
Various other translation factors have been studied in melanoma. However, our un-
derstanding of their role in this disease is much less clear than that of eIF4E. Early
reports described that cultures of congenital melanocytic nevi exhibited intermedi-
ate expression of eIF4AI (Eberle etal. 1997). This increase was associated with a
concomitant increase of expression of eIF4AI mRNA in a panel of 14 melanoma
cell lines (Eberle etal. 1997). Notably, in this report, the mRNAs of eIF4E, eIF4AII
or eIF4B were not found to be significantly elevated. There is indirect evidence sug-
gesting that eIF4B might be important for melanogenesis via regulation by agouti
signaling protein (ASIP) involved in pigment production in melanocytes (Voisey
etal. 2003). Another recently-characterized translation initiation helicase DHX29
was found overexpressed by ONCOMINE database predictions in metastatic mela-
noma (Parsyan etal. 2009).
The subunits of the eIF3 complex have been linked to melanoma biology. Pro-
tein and mRNA levels of the eIF3f (p47) subunit of eIF3 have been shown to be
decreased in melanoma and pancreatic cancers (Doldan etal. 2008; Shi etal. 2006).
Overexpression of eIF3f inhibits translation and induces apoptosis in melanoma
cells, while silencing of this protein protects melanoma cells from apoptosis. The
eIF3f gene is located at chromosome region 11p15.4, which is often lost in melano-
mas and other tumors. Doldan etal. showed that the loss of heterozygosity of eIF3f
in melanoma ranged from 7592% (Doldan etal. 2008), further suggesting a pos-
sible association between this protein and melanoma pathobiology.
In a recent study, using five different cancer cell lines NCI-ADR/RES (NAR
adriamycin resistant ovarian carcinoma), HeLa (cervical carcinoma), MCF-7
(breast carcinoma), HCT116 (colon carcinoma) and B16F10 (mouse melanoma),
it was demonstrated that another subunit of eIF3, eIF3c, is essential for translation
initiation in vivo (Emmanuel etal. 2013). Downregulation of eIF3c decreases global
protein synthesis, mediates G0/G1 or G2/M arrest in a tissue-dependent manner,
which leads to a reduction in cell proliferation and eventually to cell death. Further-
more, this study reported an efficient delivery of eIF3c-siRNAs using hyaluronan-
coated lipid-based nanoparticles to mouse melanoma cells, which could be further
studied in preclinical and clinical models.
A recent study demonstrated decreased levels of tumor suppressor PDCD4
mRNA and protein in malignant melanoma, compared with normal tissues (Wang
etal. 2013). Additionally, using antagomiR technology (that targets microRNAs) or

a curcumin analog, EF24 (that appears to downregulate miR-21) leads to upregu-
lation of some of its targets, such as tumor suppressors PTEN and PDCD4 (Yang
etal. 2011, 2013). Targeting miR-21 by these compounds in the B16 melanoma
cells mice xenograft model led to the formation of smaller lung metastatic lesions
and prolonged survival compared with control mice, correlating with decrease in
miR-21 and increase in PDCD4 levels (Yang etal. 2011, 2013). Indeed, the miR-21/
PDCD4 axis appears to be a promising therapeutic target.
While the role of elongation, termination and ribosome recycling in melanoma
is generally unknown, a single study of the serum samples from melanoma patients
reported an increase in eukaryotic elongation factor eEF2 compared to healthy sub-
jects (Suzuki etal. 2010). While the physiological role of this observation is not
clear, it was proposed as a possible biomarker for melanoma early detection.
21.2.5 eIF2 in Melanoma
eIF2 is a translation initiation factor and a part of the eIF2 ternary complex that
is significant by the fact of its phosphorylating regulation by various kinases re-
sponsive to numerous stress stimuli (see Chap.9). These stress stimuli specifically
activate kinases (such as PKR or PERK) that phosphorylate eIF2 and hence down-
regulate translation (see Chap.9). The mechanism of eIF2 involvement in cancer
is still unclear (we refer the reader to Chap.9 for more details), however its overex-
pression or activation is generally expected to increase translation and hence induce
a proproliferative state.
Early studies of the role of eIF2 in melanoma identified that its expression is
increased in melanomas and melanocytic nevi compared to normal tissues (Rosen-
wald etal. 2003). This finding suggested that increased expression of eIF2 alone
is not sufficient for malignant transformation, since benign nevi had levels of ex-
pression of this protein comparable to melanoma cells. However, overexpression
of eIF2 may be a predisposing factor, provided that additional changes take place.
Indirect evidence from pharmacological targeting of eIF2 by small molecule in-
hibitors supports the notion that this protein is an important player in melanoma
(Chen etal. 2012).
eIF2 is subject of control by phosphorylation (see Chap.9). Hence, its role in
melanoma is likely to be not only limited to the expression levels but also char-
acterized by inactivating phosphorylation at Ser51 by its regulatory kinases, such
as the aforementioned PKR and PERK. In some cancer models phosphorylation
of eIF2 leads to proapoptotic and tumor suppression phenotype (Tuval-Kochen
etal. 2013). However, in melanoma cells, as opposed to nontransformed cultured
melanocytes, elevated levels of phospho-eIF2 are commonly found (Kim et al.
2002). Additionally, ectopic expression of BRAFV600E in melanocytes increases
levels of phospho-eIF2 (Croft et al. 2013). The increase in phospho-eIF2 cor-
relates with elevated levels of PKR and PKR autophosphorylation activity (Kim
etal. 2002). Notably, inactivation of tumor suppressor PTEN that is often found in
various cancers and melanoma (Bello etal. 2013) has been demonstrated to con-
trol the eIF2 phosphorylation pathway (Mounir etal. 2009). This inactivation of
PTEN in human melanoma cells reduces eIF2 phosphorylation. The antiprolifera-
tive and proapoptotic effects of PTEN were compromised in MEFs that lacked PKR
or contained a phosphorylation-defective eIF2 (Mounir etal. 2009). It could be
hence hypothesized that eIF2 plays different roles in cancer biology depending on
a specific molecular profile of the tumor. More specifically, eIF2 phosphorylation
in a milieu of oncogenic programs that do not directly involve eIF2 can represent a
reactive defense mechanism to oppose activated tumorigenic signaling and repress
translation. While on the other hand, when eIF2 lies within an oncogenic program,
similarly to the case of PTEN mutations described above, it would be expected to
be activated to provide upregulation of translation. However, evidence towards this
hypothesis needs experimental corroboration. Due to the complexities in our under-
standing of the involvement of eIF2 in cancer, we direct the reader to a specific
chapter (see Chap.9).
21.2.6 R
ole of eIF2 in Melanoma Treatment
and Resistance to Chemotherapy
The role of the antitumor defense mechanism of eIF2 is also indirectly evident
from studies of its activation in melanoma treatment. Phosphorylation of eIF2 is
regulated by PKR that is inducible by IFN-2b (Kim etal. 2002) and PERK that
is activated by endoplasmic reticulum stress induced by docetaxel (Mhaidat etal.
2008) or cisplatin (Yacoub etal. 2010). Similarly, treatment of neuroblastoma and
melanoma cell lines with endoplasmic reticulum stress inducers like fenretinide,
bortezomib or thapsigargin results in an increase of eIF2 signaling, characterized
by elevated levels of phosphorylated eIF2 (Armstrong etal. 2010). The effects of
the Pseudomonas exotoxin A-based immunotoxin, 9.2.27PE, and BH-3 mimetic
compound ABT-737 in a panel of melanoma cell lines caused anticancer effects,
likely via induction of endoplasmic reticulum stress and phosphorylation of eIF2
protein levels (Risberg etal. 2011). Another agent, thiostrepton, displayed selec-
tive anti-melanoma activity causing various anticancer effects but also endoplas-
mic reticulum stress response with increase in phospho-eIF2 levels (Qiao et al.
2012b). D-Penicillamine, that appeared to rapidly activate cytotoxic unfolded pro-
tein response, including phospho-PERK and phospho-eIF2, was found inducing
caspase-dependent cell death in cultured human metastatic melanoma cells (A375,
G361) without compromising viability of primary epidermal melanocytes (Qiao
etal. 2012a). 11-dehydrosinulariolide from coral Sinularia leptoclados possessed
antiproliferative, antimigratory and apoptosis-inducing activities against A2058
melanoma cells via endoplasmic reticulum stress, at least in part by the upregula-
tion of PERK/eIF2/ATF4/CHOP and ATF6/CHOP (Su etal. 2012).
eIF2 also likely plays a role in the resistance to some of the current melano-
ma treatment modalities, such as INF and vemurafenib. It has been proposed that
in various cancer types, including melanoma, activation of diverse eIF2 kinases
(such as PERK, PKR and GCN2), followed by IFN-/ receptor chain IFNAR1
downregulation enables multiple cellular components of tumor tissue to evade the
direct and indirect antitumorigenic effects of type 1 IFN (Bhattacharya etal. 2013).
In patients with BRAFV600E -mutated melanoma BRAFV600E kinase inhibitor vemu-
rafenib has remarkable antitumor activity that is, however, limited by the onset
of drug resistance (Beck etal. 2013). Vemurafenib induces endoplasmic reticulum
stress response with associated increase in phospho-eIF2. In melanoma cells with
low sensitivity or resistance to vemurafenib, combination treatment with thapsigar-
gin or other inducers of endoplasmic reticulum stress may be useful to overcome
vemurafenib resistance (Beck etal. 2013).
21.3Novel Therapeutics Based on Translation Targeting
Based on the growing knowledge of melanoma pathobiology, novel targeted thera-

pies are being developed and tested in preclinical and clinical settings (for review
see Britten 2013; Neuzillet etal. 2013; Populo etal. 2012) (also see clinicaltrials.
gov for details on clinical trials in melanoma). Numerous types of targeted therapies
are being tested, approved or already used in clinical settings including inhibitors
of BRAF, mTOR (such as everolimus and temsirolimus), PI3K or combined PI3K/
mTOR inhibitors (such as BMK120 and BEZ235), MEK (such as trametinib, cobi-
metinib, selumetinib and pimasertib) and others (Britten 2013; Neuzillet etal. 2013;
Populo etal. 2012). Notably, most of these targeted approaches affect MAPK and
PI3K/AKT/mTOR pathways that converge on translational control and notably on
regulation of eIF4E activation. In addition, targeting these pathways, or co-targeting
them, appears to provide increased protection against chemoresistance (Atefi etal.
2011; Sullivan and Flaherty 2013; Tentori etal. 2013). However, since focusing on
the compounds targeting these pathways is outside of the scope of this work, we
would direct the interested reader to the aforementioned excellent reviews to obtain
more detailed information (see also Chaps.14, 15 and 17). For the sake of our dis-
cussion we will concentrate on the regulatory targets just upstream of the translation
machinery or those directly affecting translation factors.
21.4Targeted Therapies Directed at the Downstream

Targets of eIF4E
A number of clinical trials have been performed with agents that affect targets of
eIF4E, including BCL-2, survivin, cyclin D1 and VEGF/VEGFR.
21.4.1 BCL-2 and Survivin
Targeting apoptosis via BCL-2 and survivin antagonism has been and remains an
important strategy for the treatment of patients with metastatic melanoma. In par-
ticular, the BCL-2 antisense oligodeoxynucleotide, oblimersen, showed promising
activity in a randomized phase II study of dacarbazine with or without oblimersen,
with improvement of response rate, PFS and OS seen in the subgroup of patients
with a normal lactate dehydrogenase (LDH) that is used as a melanoma marker
(Bedikian etal. 2006). Unfortunately, there was no benefit to the addition of oblim-
ersen to chemotherapy in a randomized phase III trial of patients with metastatic
melanoma and a normal LDH (NCT00518895; Press Release October 29, 2009).
Additionally, the survivin inhibitor YM155 was tested as a single agent in patients
with metastatic melanoma. In this phase II open-labeled study there was only one
responder among 34 enrolled patients and, as a result, has not been developed fur-
ther as a monotherapy in this disease (Lewis etal. 2011).
21.4.2 Cyclin D
Cell cycle regulation is another emerging target in melanoma. Nearly 8090%

of melanomas have dysregulated cell cycle signaling (either through CDKN2A
(p16INK4A) loss, cyclin D amplification or CDK4 mutation), prompting the active
clinical development of CDK4/6 inhibitors. Based on this knowledge and promis-
ing preclinical data, suggesting that the combination of MEK and CDK4/6 inhi-
bition is a uniquely effective strategy in melanoma, a phase I/II clinical trial of
MEK162 and LEE011 has recently opened (NCT01777776) (Kwong etal. 2012;
Sheppard and McArthur 2013).
21.4.3 VEGF/VEGFR
Tumor angiogenesis in melanoma has been well documented and well targeted
through a number of clinical trials. In particular, several studies have investigat-
ed the role of bevacizumab, a monoclonal antibody targeting VEGF, in patients
with melanoma, and the most promising results were seen with the combination
of carboplatin/paclitaxel and bevacizumab. In an initial phase II trial 53 patients
were treated, with 17% achieving a partial remission and an additional 57% having
stable disease for at least eight weeks (Perez etal. 2009). Based on these results, a
multicenter, randomized phase II trial (BEAM trial) was performed to evaluate the
combination of carboplatin/paclitaxel with (143 patients) or without (71 patients)
bevacizumab. The response rate, median PFS, median OS, and one year OS were
all better but not statistically significant in patients receiving carboplatin/paclitax-
el plus bevacizumab (Kim etal. 2012). The addition of bevacizumab appeared to
provide statistically significant benefit with regard to improved OS in patients with

M1c disease, especially among those patients who had an elevated LDH. However,
a follow up study to the BEAM trial has not been pursued. A second angiogenesis
inhibitor, axitinib, which has potent antagonism against VEGFR 1, 2, and 3, has
also been shown to have activity in melanoma. Specifically, in a phase II trial of 32
patients, the response rate was 18% (Fruehauf etal. 2011). Another phase II trial of
axitinib is actively accruing (NCT01533948).
21.5Direct Targeting of Translation Factors

in Melanoma
Novel therapeutic approaches to target translation factors are also being developed
and studied in melanoma (Chen etal. 2012; Hong etal. 2011; Konicek etal. 2011).
Since the signaling pathways described above converge on eIF4E, it might be pru-
dent to directly target that proto-oncogenic molecule. Strategies targeting eIF4E in
general are discussed in Chaps.4 and 14, while the data looking into depressing
the activity of eIF4E in melanoma are already presented throughout this chapter,
especially in the context of targeting the MAPK and PI3K/AKT/mTOR pathways
(see section on eIF4E in melanoma). On the other hand, there are few options and
studies looking at the downregulation of eIF4E expression. Some of these, such as
using ASOs, have been applied in melanoma, although no evidence to the effective-
ness of this approach has been obtained (Hong etal. 2011). A phase I trial evaluated
such an oligonucleotide (LY2275796) in patients with advanced cancer, including
melanoma (Hong etal. 2011). While LY2275796 was well tolerated and eIF4E tu-
mor tissue expression was decreased, there was no tumor response observed. This
result could be attributed to the diversity of the studied population and their diverse
genetic profile.
Another approach is to target eIF4E interaction with its partners. In a recent
study, two small molecule inhibitors of translation initiation, one disrupting eIF4E
interaction with eIF4G and the other (#1181) phosphorylating eIF2, were inves-
tigated in melanoma and breast cancer models (Chen etal. 2012). Both molecules
inhibited proliferation of human cancer cells likely via observed preferential down-
regulation of translation initiation of oncoproteins. Both #1181 and 4EGI-1 strongly
inhibited growth of human breast and melanoma cancer xenografts with a good
toxicity profile (Chen etal. 2012).
PDCD4 is another hub of translational control that might be worthwhile tackling.
PDCD4 targeting appears to be possible via downregulation of its negative regula-
tor miR-21. AntagomiR technology involves oligonucleotides that target and inhibit
miRNAs. As discussed earlier in this chapter, agents such as antagomiRs, as well as
a curcumin analog EF24, showed promising anticancer effects related to the down-
regulation of miR-21, leading to the upregulation of some of its targets, including
PDCD4 (Yang etal. 2011, 2013).
Therapeutic strategies that are based on mRNA or miRNA silencing could pres-
ent an interesting opportunity for a local treatment of melanoma or treatments of
advanced stage disease, including limb perfusion or infusion, where they can be
locally applied. Xenograft experiments with these molecules have provided prom-
ising results. However, further testing in preclinical settings is required. Overall, it
becomes increasingly obvious that targeting translation directly or via its regulatory
systems might represent a novel effective approach in melanoma therapy.
21.6Non-Melanoma Skin Cancers and Translation
The role of the translation machinery in non-melanoma skin cancers is less well
studied, due to the fact that these tumors rarely metastasize, and hence there is less
of a sense of urgency to perform extensive preclinical evaluation of these diseases in
comparison to melanoma. Involvement of eIF4E in non-melanoma skin cancer was
suggested by Salehi etal. who demonstrated eIF4E overexpression in squamous
cell carcinoma samples (Salehi etal. 2007). This study also reported a positive as-
sociation between increasing levels of expression of eIF4E and advancing stage of
squamous cell carcinoma. It appears that in these forms of skin cancer, control of
eIF4E activity via the mTOR pathway carries an important function. Constitutive
activation of the AKT/mTOR pathway with activation of its downstream effectors
4E-BP1 and S6K was frequently found in epidermal tumors, especially in malig-
nant ones (Chen etal. 2009). AKT-mediated signaling, that has been shown to be
functionally involved in keratinocyte transformation and upregulated angiogenic
profiles, has been found to be associated with increased levels of VEGF protein
but not its mRNA (Segrelles etal. 2004). The induction of VEGF protein by AKT
is associated with increased phosphorylation and thus activation of p70S6K and
4E-BP1, leading to increased VEGF translation. The antidiabetic drug metformin,
which is currently explored as a chemotherapeutic and chemoprevention agent, ap-
pears to block squamous cell carcinoma growth by inhibiting NF-B and mTOR
signaling pathways via regulation of translation by the phosphorylation of p70S6K
and 4E-BP1 (Chaudhary etal. 2012).
An aggressive form of cutaneous malignancy, Merkel cell carcinoma, is caused
by a Merkel cell polyomavirus that expresses a small T (sT) antigen which acts
as an oncogene. sT appears to act downstream of mTOR to preserve eukaryotic
4E-BP1 hyperphosphorylation, resulting in dysregulated translation (Shuda etal.
2011). Expression of a constitutively active 4E-BP1 that could not be phosphory-
lated antagonizes the cell transformation activity of Merkel cell polyomavirus sT.
This is one of the examples where viruses modulate the translation machinery to
assist tumorigenesis (see also Chap.4).
A recent report implicates MAPK pathway-driven translational activation result-
ing in an upregulation of translation of structured 2 mRNA, which is the subunit of
laminin-332 frequently abnormally expressed in oral dysplasia and epidermal squa-
mous cell carcinoma (Degen etal. 2012). Single studies report involvement of other
translation factors in non-melanoma skin cancers. For example, in a mouse skin

epidermal JB6 Cl41 model, ultraviolet A (UVA) radiation, which is an important
etiological factor in skin tumorigenesis, induced phosphorylation of PKR and eIF2
(Zykova etal. 2007), implicating a role of the latter in UV-mediated skin cancers.
One of the first reports of the role of PDCD4 as a tumor suppressor (see Chap.6)
came from studies in skin cancer models where it was shown to inhibit neoplastic
transformation and carcinogenesis (Cmarik etal. 1999; Jansen etal. 2005). On the
other hand, it appears that tumor promoters, such as 12-O-tetradecanoylphorbol-
13-acetate, likely via increased proteasomal degradation, decrease levels of PDCD4
in mouse skin papillomas and in keratinocytes (Schmid etal. 2008). This effect is
likely mediated by an activation of the PI3K/AKT/mTOR and MAPK pathways
with subsequent phosphorylation of PDCD4 and its targeted degradation. Given the
known function of PDCD4 as a tumor suppressor in general and in skin cancers in
particular, its targeting represents a viable alternative. One of the lipoxygenase me-
tabolites of linoleic acid from corn germ, (+/)-13-Hydroxy-10-oxo-trans-11-oc-
tadecenoic acid (13-HOA), exhibits properties of an antitumor chemopreventive
agent in a carcinogen-induced mouse skin tumor model and has a unique mode
of action that might involve induction of PDCD4 (Yasuda etal. 2009). Another
potential mechanism to upregulate PDCD4 expression for cancer therapy is to
downregulate miR-21 that controls its expression. Indeed, knocking out miR-21 in
mice significantly reduces papilloma formation in response to skin carcinogenesis
via targeting of PDCD4, but also via Spry1 and PTEN (Ma etal. 2011). On the
other hand, induction of miR-21 in epidermal cell lines is caused by ultraviolet B
(UVB) radiation with subsequent inhibition of PDCD4 (Hou etal. 2013). UVB
induces PDCD4 inhibition, which might be mediated through reactive oxygen spe-
cies (ROS).
An increasing body of evidence points to the role of dysregulation in translation and

its control in the biology of melanoma and non-melanoma skin cancers. Decades of
discoveries have shed light on the importance of alterations in signaling pathways
in these conditions. It appears that melanoma favors mutations and perturbations
in the MAPK pathway, specifically in its RAS/RAF/MEK/ERK axis (Bello etal.
2013; Roring and Brummer 2012; Yajima etal. 2012), while non-melanoma can-
cers often present with aberrations in the PI3K/AKT/mTOR cascade (Chaudhary
etal. 2012; Chen etal. 2009; Salehi etal. 2007; Segrelles etal. 2004). Both of these
pathways converge on the translation machinery, notably on the proto-oncogene
eIF4E, emphasizing a fundamental role of dysregulated translation in the etiology
and pathogenesis of skin cancers.
Although less well studied, various other factors are implicated in skin cancer
biology. One of these in melanoma includes another fundamental hub of transla-
tional control, eIF2, that is activated by signal transduction via kinases responding
to stresses, such as amino acid deprivation (GCN2), endoplasmic reticulum stress

(PERK), the presence of dsRNA (PKR) or heme deficiency (HRI) (see Chap.9).
Our understanding of the role of this factor is incomplete and requires further stud-
ies. However, regulation of eIF2 is likely important for the activity of and resis-
tance to chemotherapeutic agents. Additionally, the role of the bona fide tumor sup-
pressor PDCD4, a regulator of eIF4A helicase, as a contributing pathogenetic factor
in melanoma and non-melanoma skin cancers is being unveiled.
Thus, in skin cancers, a pattern is emerging pointing to three axes of translation
and its control: 4E-BP/eIF4E and PDCD4/eIF4A, which represent the initial stage
of translational control at the eIF4F complex, and eIF2, that represents the later
stage of control of translation initiation. These pieces of evidence provide fruitful
ground for the potential development of prognostic and diagnostic modalities, as
well as therapies for both melanoma and non-melanoma skin cancers.
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Chapter 22
Sarcomas
Armen Parsyan, James L. Chen, Raphael Pollock and Sarkis Meterissian
Contents
22.2eIF4E and Its Regulation by mTOR/4E-BPs in Sarcomas 455
22.2.1Tumors of Muscle Origin 455
22.2.2Bone, Cartilage and Joint Tissue Tumors 457
22.2.3Adipose Tissue Tumors 457
22.2.4Tumors of Vascular Origin 458
22.2.5Other Types of Sarcoma 458
22.2.6Gastrointestinal Stromal Tumors 459
22.3eIF2 in Sarcomas 459
22.4mTOR Inhibitors as Single Agents 460
22.5mTOR Inhibition as Combination Therapy 461
References 462
Abstract Sarcomas, as opposed to carcinoma, are rare forms of human cancer com-
prising less than 1% of adult cancers. While represented by a broad range of entities,
sarcomas share a common ground that is a mesenchymal origin of the tumors. With
the understanding of these various types of sarcomas at the molecular level recently
gaining its momentum, the role of translation in the pathobiology of this group
A.Parsyan() S.Meterissian
Division of General Surgery, Department of Surgery,
J.L.Chen
Department of Biomedical Informatics and Division of Medical Oncology,
Department of Internal Medicine, The Ohio State Wexner Medical Center, Columbus, OH, USA
R.Pollock
Division of Surgical Oncology, Department of Surgery,
The Ohio State Wexner Medical Center, Columbus, OH, USA
S.Meterissian
of diseases started to uncover, pointing at a common theme: the activation of the

mTOR pathway with subsequent derepression of the function of eIF4E via 4E-BPs
and the activation of the proproliferative translation program. In this chapter, we, for
the first time, summarize the current knowledge regarding translation, its regulation,
as well as diagnostic and therapeutic potential in various types of sarcomas.
22.1Introduction
In contrast to carcinomas that arise from epithelial cells, sarcoma is a relatively rare
form of cancer that originates from cells of mesenchymal origin and comprises less
than 1% of adult cancers. This would include a broad range of tissues, such as bone,
cartilage, adipose, muscular, vascular, or hematopoietic tissues. According to the
World Health Organizations (WHO) International Classification of Diseases for
Oncology, 3rd Edition (ICD-O-3) (Fritz etal. 2000) sarcomas are classified as soft
tissue sarcomas (with numerous entities, ranging from angio-, fibro-, neuro-, heman-
gio-, leiomyo- and other sarcomas) and non-soft tissue types, such as chondro- and
osteosarcomas, Ewings tumor etc. While this classification basically reflects the
tissue origin of sarcomas, a novel WHO classification has made substantial steps
forward regarding the molecular, genetic and cytogenetic characterization of soft
tissue sarcomas, providing more reproducible pathogenetic and diagnostic informa-
tion (Fletcher 2014; Fletcher etal. 2013). This new genetic information on sarcomas
further indicates the etiological and pathogenetic diversity of this entity of diseases.
Despite the aforementioned heterogeneity of sarcomas, our understanding of
the disease and its treatment options has progressed slowly. Like most cancers, the
standard of care therapies include surgery, radiotherapy, and chemotherapy. In the
locally-advanced and metastatic setting, chemotherapy remains the most popular
treatment. With a few notable exceptions, the anthracycline-based cytotoxic chemo-
therapy has remained the mainstay of most clinical treatment regimens with modest
responses of up to 25% for single-agent therapy and 3040% when used in combi-
nation (Spira and Ettinger 2002). Recent drug development efforts have focused on
novel combinations of existing cytotoxic chemotherapies in the absence of effective
targeted therapies.
Gastric leiomyosarcoma, also known as gastrointestinal stromal tumor (GIST),
reflects a treatment paradigm in the management of sarcoma (also discussed be-
low). GIST sarcomas are driven by c-KIT and PDGFR-, which are targetable by
imatinib (Heinrich etal. 2003). This finding demonstrated clearly that understand-
ing the molecular underpinnings of the tumor biology could permit targeted therapy
that directly impacts patient survival and morbidity. Other sarcomas remained with-
out a targeted agent until pazopanib (a tyrosine kinase inhibitor (TKI) of VEGFR,
PDGFR and c-KIT) was FDA-approved for the use in soft tissue sarcoma after
having demonstrated a modest benefit (van der Graaf etal. 2012). The momentum
appears to be building up. For example, the recent work in various liposarcomas
has demonstrated that increased MDM2 and CDK4 expression might represent a
potential therapeutic target (Dickson etal. 2013; Ray-Coquard etal. 2012).
22Sarcomas 455
The fusion protein NAB2-STAT6 has been implicated in solitary fibrous sarcoma
with downstream activation of RTK of the early growth response protein (EGR1).
Thus it remains to be seen if TKIs that have been developed are effective in this tu-
mor type (Robinson etal. 2013). In synovial sarcoma, the fusion protein SS18-SSX2
(synovial sarcoma translocation, chromosome 18synovial sarcoma, X breakpoint
2) appears to augment the expression of the antiapoptotic gene BCL-2, thus opening
the door to anti-BCL-2-directed therapy (Jones etal. 2013). Similarly, PAX3-FOXO1
appears to drive growth of a subset of alveolar rhabdomyosarcomas, since inhibition
of this fusion protein abrogates tumor growth in vitro and in vivo (Jothi etal. 2013).
Excitingly, there are many other druggable molecular pathways that have been
identified in different sarcoma subtypes. A promising targeting pathway remains the
mTOR cascade whose activation leads to the upregulation of translation initiation
via eIF4E. There has been much progress made in both the clinical and basic sci-
ence level understandings of this pathway in sarcoma. While facing this diversity
of entities, we will nevertheless attempt to summarize the role of translation and its
control in these neoplasms.
22.2eIF4E and Its Regulation by mTOR/4E-BPs

in Sarcomas
Early on, reports suggested the importance of the PI3K/AKT/mTOR pathway via
4E-BPs/eIF4E- and p70S6K- mediated translational control in various types of sar-
comas, as well as the effectiveness of mTOR inhibitors, such as rapamycin and rapa-
logs, in targeting this pathway (Dudkin etal. 2001; Huang etal. 2004; Lekmine etal.
2004; Tuhackova etal. 1999; Wan etal. 2005). IHC analysis of 140 cases of bone
and soft tissue tumors showed activation of AKT in 55% (61% in malignant and
27% in benign tumors) and mTOR expression in 61% (66% in malignant and 39%
in benign) of various sarcomas, such as rhabdomyosarcomas, chordomas, schwan-
nomas and synovial sarcomas (Dobashi etal. 2009). As evidenced by the f ollowing
discussion, various types of sarcomas exhibit activation of the AKT/mTOR/4E-BP1
axis that, via translational control, regulates proliferation, differentiation and main-
tenance of various morphological phenotypes of sarcoma cells (Dobashi etal. 2009).
Elevation of phosphorylated 4E-BP1 in sarcomas, as an indicator of eIF4E-driven
cap-dependent translation, is associated with a higher likelihood of metastases, as
well as adverse prognosis, clinicopathologic findings and poor survival (Conti etal.
2014; Petricoin etal. 2007; Setsu etal. 2013; Setsu etal. 2012).
22.2.1 Tumors of Muscle Origin
Rhabdomyosarcoma originates from skeletal muscle progenitors and probably

represents the most studied type of sarcoma vis--vis translation. In various types
of rhabdomyosarcomas, activation of the AKT/mTOR pathway and its down-
stream effectors p70S6K, rpS6 and 4E-BP1 is commonly observed (Cen etal.
2007; Dudkin etal. 2001; Petricoin etal. 2007). In both alveolar and embryonal
r habdomyosarcomas, phosphorylation levels of mTOR and its downstream targets,

p70S6K, rpS6, and 4E-BP1, have been reported increased between 50 and 72%
(Cen etal. 2007). Activation of the AKT/mTOR pathway was found to be negative-
ly associated with childhood rhabdomyosarcoma survival (Petricoin etal. 2007). As
an indication of this activation, high phosphorylation levels of translation factors,
such as 4E-BP1 and eIF4G (on Ser1108), were observed and associated with poor
overall and poor disease-free survival (DFS).
Studies from rhabdomyosarcoma xenograft models suggest that rapamycin and a
rapalog temsirolimus cause rapid inactivation of eIF4E by 4E-BP1, as well as subse-
quent inhibition of cell growth and activation of antiangiogenesis via translational con-
trol of HIF-1 and VEGF (Dudkin etal. 2001; Petricoin etal. 2007; Wan etal. 2006).
In a p53-mutant Rh30 rhabdomyosarcoma cells, inhibition of mTOR by rapamycin
was also shown to activate apoptosis signaling regulating kinase 1 (ASK1) signaling
by suppressing protein phosphatase 5 activity (Huang etal. 2004). ASK1, also known
as MAP kinase kinase kinase 5 (MAP3K5), is a member of the MAPK family and, as
such, a part of the mitogen-activated protein kinase signaling pathway. The aforemen-
tioned effect was found to be dependent on expression of 4E-BP1 (Huang etal. 2003;
Huang etal. 2004). High correlation was observed between 4E-BP1 dephosphoryla-
tion and growth inhibition of rhabdomyosarcoma cells upon treatment with temsiroli-
mus (CCI-779), another rapalog (Dudkin etal. 2001).
In various sarcoma and carcinoma models, rapamycin was also reported to in-
hibit cell motility by preventing stimulated F-actin reorganization and phosphoryla-
tion of focal adhesion proteins, including focal adhesion kinase (FAK), paxillin and
p130(Cas) (Liu etal. 2008; Liu etal. 2010). Both the S6K1 and the 4E-BP1 axes of
the mTOR pathway were found to be involved in the regulation of IGF-1-stimulated
F-actin reorganization, while S6K1 alone controlled IGF-1-stimulated phosphory-
lation of the focal adhesion proteins (Liu etal. 2008). In addition, treatment of
human Ewing sarcoma (Rh1) and rhabdomyosarcoma (Rh30) cells with rapamycin
inhibited cell motility via activation of the PP2A phosphatase and concurrent inhibi-
tion of IGF-1-stimulated phosphorylation of ERK1/2 (Liu etal. 2010).
Another pharmaceutical compound, curcumin, a component of a popular South
Asian spice turmeric and novel anticancer agent, was shown to reduce growth of
Rh1 and Rh30 rhabdomyosarcoma cells via inhibition of mTOR and its down-
stream effector molecules p70S6K and eIF4E (Beevers etal. 2009; Beevers etal.
2006). Curcumin was suggested to act by disrupting the mTOR/RAPTOR complex
(Beevers etal. 2009; Beevers etal. 2013).
These findings further emphasize importance of the mTOR activation in sarco-
mas and the dependence of its effects on translational regulation via 4E-BP1 and
eIF4E. Indeed, direct reduction of 4E-BP1 expression levels (and thus potential
activation of eIF4E) by shRNA has significant effects on tumor development as-
sociated with enhanced proliferation and survival of Rh30 rhabdomyosarcoma cells
(Barnhart etal. 2008). 4E-BP1 knock-down in these cells led to a failure to develop
the extensive necrotic zones and edema observed in control tumors in a xenograft
model, as well as relieved a hypoxia-mediated inhibition of cycle progression,
correlating with increased expression of cyclin D1, c-MYC and antiapoptotic fac-
tors, BCL-XL and XIAP (Barnhart etal. 2008).
22Sarcomas 457
In other types of sarcomas of muscle origin, an anecdotal report suggests that the
AKT/mTOR pathway is activated in soft tissue leiomyosarcomas (a smooth muscle
sarcoma) (Setsu etal. 2012). By IHC, phosphorylated forms of AKT, mTOR, rpS6,
and 4E-BP1 were positive in approximately 7080% of the soft tissue leiomyosar-
coma samples and correlated with adverse prognosis and aggressive pathological
findings (Setsu etal. 2012).
22.2.2Bone, Cartilage and Joint Tissue Tumors
Osteosarcoma, a bone cancer, is the most prevalent primary malignant tumor of

the bone and has a high propensity for metastasis that depends on the expression of
ezrin, a protein that plays a key role in cell surface adhesion, migration, and organi-
zation. Ezrin-related metastatic behavior has been linked to translation via mTOR
to S6K1 and 4E-BP1 signaling that is sensitive to rapalogs treatment (Wan etal.
2005). Indeed, findings from these sarcoma types also highlight the activation of
the mTOR pathway and dependent involvement of the translation machinery in the
biology of these tumors. A study of osteosarcomas using U2OS line demonstrated
that, via PI3K/mTOR signaling, p70S6K is rapidly phosphorylated and activated
during engagement of the IFN- receptor in sensitive cell lines (Lekmine et al.
2004). The IFN--activated p70S6K subsequently phosphorylates the rpS6 and also
induces the phosphorylation of 4E-BP1, resulting in activation of translation. While
eIF4E was found to be uniformly expressed in osteosarcoma patient samples, no
association was reported between eIF4E and the clinical outcome (Osborne etal.
2011). Studies of the translation machinery in other components of the bone, such
as cartilage (chondrosarcoma) or synovium of the joint (synovial sarcoma) are an-
ecdotal. A single report indicates that everolimus, an analog of rapamycin, inhibited
chondrosarcoma proliferation via its mTOR effects and 4E-BP1 in the rat orthotopic
Schwarm chondrosarcoma model (Perez etal. 2012). The AKT/mTOR pathway
was found activated and associated with worse clinical and pathologic behavior in
patients with synovial sarcoma (Setsu etal. 2013). AKT, mTOR, rpS6 and 4E-BP1
were activated in 76.5, 67.6, 59.6, and 42.6% of samples, respectively. Activation
of 4E-BP1 correlated with higher mitotic activity, greater necrosis and shorter over-
all and event-free survival (Setsu etal. 2013).
22.2.3Adipose Tissue Tumors
Overexpression of eIF4E may be a primary event in the initiation of liposarcomas, a

tumor that originates from adipose tissues, since it was reported to be strongly upreg-
ulated in normal adipose tissue of FUS-DDIT3 (DNA-damage-inducible transcript
3) transgenic mice (Perez-Mancera etal. 2008). FUS-DDIT3, a chimeric protein
generated by the chromosomal translocation t(12;16)(q13;p11), is a protein that dis-
rupts normal adipocyte differentiation (Engstrom etal. 2006; PerezMancera etal.
2007). It has been shown that FUS-DDIT3 interferes with the control of translation
initiation by the upregulation of eIF2 and eIF4E in FUS-DDIT3 mice and human
liposarcoma cell lines (PerezMancera etal. 2008).
Aberrations in the PI3K/AKT/mTOR pathway in myxoid and round cell lipo-
sarcomas were shown in a tissue microarray composed of 165 tumors from 111
patients, revealing activating PIK3CA mutations in 6/44 cases, complete loss of
PTEN in 13/111 and strong IGF-1R expression in 25/97 cases (Demicco etal.
2012). Activation of the PI3K pathway and increased phosphorylation of 4E-BP1 in
round cell tumors has also been reported recently (Demicco etal. 2012).
22.2.4Tumors of Vascular Origin
Sarcomas of vascular origin, such as angiosarcoma and hemangiosarcoma, are

also poorly studied in terms of translational control. In concordance with the
overall theme found in sarcomas, activated AKT/mTOR pathway is also found in
angiosarcomas with a concomitant increase in phosphorylated S6K and 4E-BP1
(Italiano etal. 2012). In a study of patients with 43 metastatic and 179 local-
ized angiosarcomas, IHC analysis identified significant overexpression of eIF4E,
VEGF-A and C, as well as phosphorylated AKT and 4E-BP1 (Lahat etal. 2010).
Expression intensity of phosphorylated 4E-BP1 was found to be significantly high-
er in metastatic angiosarcoma samples compared with localized lesions, suggesting
correlation between activation of the translation machinery and metastatic disease.
While data in humans are lacking, studies of canine hemangiosarcoma (that is a ma-
lignant tumor with poor long-term prognosis due to the development of metastasis
despite aggressive treatment) describe findings similar to those in angiosarcoma
and are suggestive of activation of eIF4E via activation of mTOR/4E-BPs (Murai
etal. 2012a; Murai etal. 2012b).
22.2.5Other Types of Sarcoma
The altered signaling to the phosphorylation of 4E-BP1 and subsequent disin-

hibition of eIF4E were also found in Rous sarcoma oncovirus-transformed cells
(Tuhackova etal. 1999). In Rous sarcoma virus-transformed hamster fibroblasts,
the disruption of the SRC kinase activity, which has been observed in a large num-
ber of human malignancies results in substantial reduction of the phosphorylation of
mTOR, S6K1, rpS6, and 4E-BP1 (Vojtechova etal. 2008). Another virus-associated
sarcoma model, Kaposis sarcoma, is caused by the Kaposis sarcoma-associated
herpesvirus (KSHV), which encodes an oncogene, viral G protein-coupled recep-
tor, that promotes potent activation of the PI3K/AKT/mTOR pathway (Bhatt and
Damania 2012; Sodhi etal. 2006). The effects of the mTOR pathways on Kaposis
sarcoma development by KSHV were shown to be dependent on 4E-BP/eIF4E
activity, further emphasizing the importance of the translation machinery in sar-
comas (Martin etal. 2013). Of note, treatment of Kaposis sarcoma patients with
22Sarcomas 459
r apamycin, also known as sirolimus, provided one of the first pieces of evidence of
the antineoplastic activity of mTOR inhibitors in humans, becoming a foundation of
care for Kaposis sarcoma arising in renal transplant patients (Stallone etal. 2005).
22.2.6Gastrointestinal Stromal Tumors
Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tu-
mors of the gastrointestinal tract, arising from interstitial cells of Cajal. GISTs
are often genetically characterized by the mutations of the proto-oncogene KIT or
PDGFRA (-type PDGF receptor A) genes, leading to the constitutional activation
of the RTKs (Heinrich etal. 2003; Hirota etal. 1998). These RTKs represent the
core mechanism of PI3K/AKT/mTOR pathway activation with further downstream
signaling to 4E-BPs and p70S6K (Rios-Moreno etal. 2011; Sapi etal. 2011). In-
creased KIT signaling, which correlates with accelerated proliferation and shorter
DFS, leads to the activation of RAS/RAF/ERK, PI3K/AKT/mTOR/EIF4E and
Janus kinase (JAK)/STATs cascades and is associated with a high expression of
eIF4E and the upregulation of cyclin D (Haller etal. 2008). As evidenced in other
chapters, cyclin D1 is regulated by eIF4E (Rosenwald etal. 1995; Rosenwald etal.
1993) (see Chap.4 and Part IV). While more research is required to understand the
role of the translation machinery in GIST, it appears that the therapeutic targeting
of RTKs leads to promising antitumor effects via 4E-BPs and p70S6K (Yang etal.
2006). While the role of other translation factors in GIST etiology and pathogenesis
is generally unknown, there is a report that a tumor suppressor and translational
regulator PDCD4 plays an important role in the progression and malignant prolif-
eration of GIST (Ding etal. 2012). PDCD4 mRNA and protein expression in a total
of 63 GIST samples was diminished in almost 70% of tumor specimens compared
to adjacent normal gastrointestinal tissues and was inversely associated with tumor
size, mitotic rate and the Ki-67 labeling index.
22.3 eIF2 in Sarcomas
The role of eIF2 in cancer is entertained (see Chap.9). There are a handful of stud-
ies of eIF2 in sarcomas. Western blot analysis of osteosarcoma tissues showed that
the levels of the phosphorylated form of eIF2 are decreased in tumors, compared
to normal controls, which would be expected to lead to the upregulation of transla-
tion and thus protumorigenic effects (Wimbauer etal. 2012). Increased phosphory-
lation of eIF2 at the G2/M cell cycle transition boundary in human osteosarcoma
cells has also been observed to correlate with a decreased rate of protein synthesis,
but the significance of this finding in sarcoma biology needs further investigation
(Datta etal. 1999). In the aforementioned FUS-DDIT3 liposarcoma model and
liposarcoma cell lines, FUS-DDIT3 appears to upregulate eIF2 and eIF4E, which
would be expected to enhance translation and to lead to protumorigenic effects

(PerezMancera etal. 2008). These studies point at the contribution of eIF2 in the
pathogenesis of sarcoma, however the role of this protein in this disease is still not
understood and requires further studies.
22.4mTOR Inhibitors as Single Agents
The most studied class of drugs affecting the translation machinery in sarcoma
is the mTOR pathway inhibitors, such as rapalogs: everolimus, ridaforolimus,
sirolimus (rapamycin) and temsirolimus. Despite the fact that everolimus is a stan-
dard of care in various tumor types its efficacy in sarcoma is not well understood.
The largest study is a multicenter phase II clinical trial of 38 patients who failed
standard anthracycline-based therapy (Yoo etal. 2013). In this study, one patient
with an angiosarcoma had partial response and ten had stable disease at 16 weeks.
Modest median PFS benefit of 1.9 months had been observed. Similarly, there is
limited clinical data regarding sirolimus as a single agent. Three patients with pe-
ripheral vascular epithelioid cell tumors (PEComas) were treated with sirolimus
and all demonstrated radiographic responses (Wagner etal. 2010). Thus, at least for
PEComas, sirolimus may be considered as a therapy. Another rapalog, temsirolimus
has been studied in a phase II trial of 40 patients who could not have received che-
motherapy for their metastatic disease. There were two partial responders, one with
undifferentiated fibrosarcoma and another with uterine leiomyosarcoma. Similarly
to everolimus, median PFS was 2.0 months (Okuno etal. 2011).
The largest studies of mTOR inhibition in sarcoma have been conducted with
ridaforolimus (previously known as deforolimus). Ridaforolimus demonstrated
promising results in a large phase II study of 162 patients with soft tissue sarco-
mas and 54 patients with bone sarcomas. The overall clinical benefit rate (clinical
or partial response or stable disease at 16 or more weeks) was 28.8%, and PFS
at 6 months was 23.4% (Chawla etal. 2012). Four patients had a confirmed par-
tial response (two with osteosarcoma, one with spindle cell sarcoma and one with
malignant fibrous histiocytoma). Based on this promising data, a multinational
randomized double-blinded placebo-controlled phase III trial of ridaforolimus,
called SUCCEED, was initiated. The trial tested oral ridaforolimus against placebo
in 711 patients with advanced sarcoma who achieved clinical benefit with prior
cytotoxic therapy. Clinical benefit at four months was achieved more often with
ridaforolimus than placebo (40.6% versus 28.6%; p<0.001). The ridaforolimus
group had a 28% reduction in the risk of progression or death (p<0.001), but me-
dian PFS benefit was small (17.7 weeks for ridaforolimus versus 14.6 weeks for
placebo), while the OS at 24 months was not significant (Demetri etal. 2013).
These results were not sufficient for the FDA approval of ridaforolimus.
Taken together, mTOR inhibitors as a monotherapy provide small benefits in a
modest number of patients. However, it is worth noting that all four aforementioned
compounds are first-generation mTOR inhibitors, which target mTORC1, but not
mTORC2 (see Chap.15). These mTOR inhibitors can cause feedback activation of
22Sarcomas 461
the PI3K/AKT pathway and diminution of anticancer effects (see Chap.15). Hence
the use of combined mTORC1/C2 inhibitors in sarcomas warrants further studies
(see Chap.15).
22.5mTOR Inhibition as Combination Therapy
Due to a lack of promising results with rapalogs as a monotherapy in sarcoma,

their use in combination therapy is being explored. Combinations with liposomal
doxorubicin (Thornton etal. 2013), cyclophosphamide (Schuetze etal. 2012), and
irinotecan (Verschraegen etal. 2013) have all been attempted, however results
remain to be observed. There is an ongoing clinical study of rapamycin with gem-
citabine versus gemcitabine alone in advanced soft tissue sarcomas (NCT01684449,
see clinicaltrials.gov). Combinations with different molecular pathway inhibition
are also underway. There are phase II studies of everolimus with bevacizumab
(anti-VEGF monoclonal antibody) in refractory malignant peripheral nerve sheath
tumors (NCT01661283) and everolimus with sorafenib (anti-VEGF TKI) in osteo-
sarcoma (NCT01804374).
Another strategy is a combined RAS/MEK and mTOR pathway inhibition. In-
deed, dual inhibition of RAS/MEK and mTOR appears to be a promising modality
in vitro (Renshaw etal. 2013). A phase II clinical trial of temsirolimus and MEK
inhibitor selumetinib in metastatic and advanced soft tissue sarcomas is underway
(NCT01206140).
Compensatory activation of the PI3K/AKT pathway due to mTORC1 inhibition
by rapalogs can occur through an insulin-like growth factor 1 receptor (IGF-1R)
dependent mechanism (OReilly etal. 2006; Wan etal. 2007). This observation
has spurred a phase II trial combining temsirolimus with anti-IGF-1R antibody
cixutumumab that showed promising results in patients with IGF-1R positive bone
sarcoma (Schwartz etal. 2013).
While our understanding of the role of the translation machinery in sarcoma still
in its infancy and mainly comes from studies dissecting the signaling cascades
that control this process, the emerging model in sarcoma could be summarized by
the activation of the mTOR pathway, leading to the phosphorylation of 4E-BP1
and activation of eIF4E proto-oncogene. It is notable that studies from various
sarcoma types indicate on the validity of this model. Very scarce data exist on the
role of other translation factors in the development and progression of sarcomas,
with anecdotal findings associated with increased eIF4B (Kuang etal. 2011; Le
etal. 2013) and eIF4G phosphorylation (Petricoin etal. 2007), eIF4H upregulation
(Pawlowski etal. 2011) and decrease in PDCD4 protein, associated with increased
levels of miR-21 (Pan etal. 2010) in this type of cancer. Overall, these reports
provide very indirect associative evidence to make any meaningful conclusions on

the role of other translation factors in sarcoma biology. However, all these findings
indicate the ongoing theme of the importance of the eIF4F complex and its con-
trol by the signaling cascades, such as mTOR, in sarcomas. For examples, eIF4B,
eIF4H and PDCD4 control the activity of eIF4A, a component of the trimeric eI-
F4F complex, which also contains above-examined eIF4E and eIF4G. eIF4B activ-
ity, via phosphorylation, is controlled by the PI3K/mTOR/S6K axis and the MAPK
pathway (Holz etal. 2005; Shahbazian etal. 2006). The role of S6K in sarcomas
has been discussed above. eIF4H is a factor similar to eIF4B in that it controls
the activity of eIF4A helicase, which is essential for translation of mRNAs with
structured 5 UTRs important for cancer development and progression. We refer
the reader to specific chapters in this book for more detailed information regard-
ing these proteins and their emerging role in cancer. We also would like to point
out that a large body of molecular biology and biochemical research into transla-
tion and its regulation in cancer was performed using various forms of fibroblasts,
including MEF that represent cells of mesenchymal origin. Various findings to
the protumorigenic roles of translation factors from these systems are likely more
directly interpretable in the context of sarcomas rather than carcinomas, the malig-
nancies of epithelial origin. In summary, it appears that studies of these proteins in
sarcoma would provide interesting and important results regarding the role of the
translation machinery, and could provide us with novel tools to diagnose and treat
this group of diseases.
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Chapter 23
Hematological Malignancies and Premalignant
Conditions
Markus Reschke, Nina Seitzer, John G. Clohessy and Pier Paolo Pandolfi
Contents
23.2 Inherited Bone Marrow Failure Syndromes 468
23.2.1 X-linked Dyskeratosis Congenita 469
23.2.2 Diamond-Blackfan Anemia 469
23.2.3 ShwachmanBodianDiamond Syndrome 470
23.2.4 Myelodysplastic Syndrome and the 5q- Syndrome 470
23.3Ribosomal Alterations in Hematological Malignancies 472
23.4 Translation Factors in Hematological Malignancies 473
23.4.1eIF4E 474
23.4.2eIF2 475
23.4.3eIF6 476
23.4.4 Other Translation Initiation Factors 476
23.5Treatment Strategies Targeting the Translation Machinery in
Hematological Malignancies 477
References 480
AbstractHematological malignancies and premalignant conditions represent a

wide spectrum of diseases that are characterized by various molecular alterations.
One important etiologic and pathogenetic role of these conditions is defined by dys-
regulation and malfunction of the translation machinery, including aberrations in
ribosome biogenesis and function and involvement of translation factors. While the
P.P.Pandolfi() M.Reschke N.Seitzer

Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine
e-mail: ppandolf@bidmc.harvard.edu
J.G.Clohessy
Preclinical Murine Pharmacogenetics Facility, Cancer Research Institute,
Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology,
Beth Israel Deaconess Medical Center, Harvard Medical School,
468 M. Reschke et al.
role of ribosomal aberrations in other cancers is still unclear, enough evidence has
been obtained in support of the notion that it may serve a causative foundation in a
number of premalignant hereditary syndromes called ribosomopathies. This chapter
will summarize a vast field of research concerning the involvement of the translation
machinery in malignant and premalignant conditions of the hematopoietic system
and will discuss ways to target this machinery in these neoplastic processes.
23.1Introduction
Hematological malignancies account for approximately 10% of newly diagnosed can-

cer cases in the United States (Mehta 2011). They are typically classified as either
leukemia or lymphoma because of their predominant organ involvement and clinical
presentation. Leukemias are blood cancers that affect immature white blood cells of
either the myeloid or lymphoid lineage in the bone marrow, with a typical involvement
of the peripheral blood at presentation (Lowenberg etal. 1999). Lymphoma represents
a heterogeneous group of cancers that predominantly affect the lymphoid compartment
at presentation (Kppers 2005; Shankland etal. 2012). The treatment of hematological
malignancies ranges historically from chemotherapy and radiotherapy, to bone mar-
row transplantation, and targeted therapies are now becoming more widely available,
depending on the specific subtype (Bendandi etal. 2004; Burnett etal. 2011).
It is becoming increasingly clear that malfunctions of the translation machinery
contribute to the development and progression of different forms of hematological
diseases including overt malignancies (Ruggero and Pandolfi 2003). These mal-
functions range from aberrant expression and regulation of translation factors to
mutations of ribosomal proteins.
In order to understand the role of the translation machinery in hematological
malignancies it is also important to understand the biology of premalignant hemato-
logic conditions (i.e. myelodysplastic syndrome (MDS) and inherited bone marrow
failure syndromes) that confer an increased risk of malignant transformation. These
disorders are rare and typically characterized by defects in multiple hematopoietic
cell types (Narla and Ebert 2010). It is now clear that these conditions are often
based on mutations or alterations to ribosome biogenesis proteins, ribosome-asso-
ciated proteins and ribosomal proteins themselves (Narla and Ebert 2010; Ruggero
and Pandolfi 2003; Teng etal. 2013). Thus, this chapter will dissect our knowledge
on the dysregulation of translation in premalignant and malignant hematological
disorders from the perspective of ribosomal alterations and translation factors.
23.2 Inherited Bone Marrow Failure Syndromes
In recent years, a number of inherited bone marrow failure syndromes have been
identified that are characterized by distinct genetic abnormalities affecting ribo-
some biogenesis, resulting in increased cancer susceptibility. This class of diseases
is now collectively referred to as ribosomopathies (summarized in Table23.1).
23 Hematological Malignancies and Premalignant Conditions 469
Table 23.1Summary of ribosomopathies. The table was adapted and modified from Narla and
Ebert (2010), Shenoy etal. (2012) and Teng etal. (2013).
/ ' & ^
s D D^ D>
Z^ Z^ Z^ Z> Z> Z> Z ^
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^
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I mportantly, several studies on the pathogenesis of ribosomopathies, as outlined in

the following paragraphs, demonstrate that alterations to the ribosomal machinery
can alter the translational landscape of a cell, and it is likely that such changes con-
tribute to their development and progression to tumorigenesis.
23.2.1 X-linked Dyskeratosis Congenita
X-linked dyskeratosis congenita results from specific mutations to the dyskerin (DKC1)
gene located on chromosome X. Dyskeratosis congenita is a rare bone marrow failure
syndrome characterized by skin hyperpigmentation, nail dystrophy and oral leukopla-
kia (Heiss etal. 1998; Shenoy etal. 2012) but more importantly, dyskeratosis congenita
confers a high risk of tumor development including leukemia (Narla and Ebert 2010).
DKC1 is an evolutionary conserved gene that mediates the modification of rRNA by
converting specific uridine nucleotides to pseudouridine. These modifications are criti-
cal for efficient ribosome assembly and the maintenance of translational fidelity (Rug-
gero etal. 2003; Yoon etal. 2006). Animal models with DKC1 mutations recapitulate
the phenotype of patients with dyskeratosis congenita and confer increased cancer sus-
ceptibility (Ruggero etal. 2003). Interestingly, cells of dyskeratosis congenita mice do
not exhibit changes in global translation but rather in the translation of a specific subset
of IRES-containing mRNAs, including the tumor suppressors p27 and p53 and the an-
tiapoptotic factors BCL-XL and XIAP (Bellodi etal. 2010; Yoon etal. 2006). Thus, im-
pairments in rRNA modifications and subsequent defects in IRES-mediated translation
of specific mRNAs likely contribute to disease pathogenesis of dyskeratosis congenita.
23.2.2 Diamond-Blackfan Anemia
Diamond-Blackfan anemia (DBA) is characterized by anemia, macrocytosis, re-

ticulocytopenia and a selective decrease in erythroid progenitors. DBA patients
further display an increased risk of developing MDS and AML (Narla etal. 2011).
In contrast to dyskeratosis congenita patients, up to 50% of DBA patients harbor
mutations or deletions in ribosomal proteins themselves (Farrar etal. 2011; Narla
etal. 2011). Mutation to the RPS19 gene is most frequently observed in DBA, with
approximately 25% of DBA families harboring this mutation (Boria etal. 2010).
Several studies have demonstrated that mutation to RPS19 and other ribosomal pro-
teins results in impaired ribosome biogenesis, which leads to a defect in 40S sub-
unit maturation and decreased numbers of mature ribosomes (Choesmel etal. 2007,
2008; Flygare etal. 2007). Generally, it would be expected that a decrease in mature
ribosomes, such as in the case of DBA, would have multiple effects in any given
cell type. However, DBA patients present with a remarkable tissue specific pheno-
type and a particularly dramatic erythroid phenotype. One possible explanation for
this phenomenon is the observation that ribosomal proteins can exert extra-ribo-
somal functions, leading to the activation of p53 (Teng etal. 2013). Indeed, it has
been shown that several ribosomal proteins bind to and sequester MDM2, which
in turn leads to p53 stabilization, cell-cycle arrest and apoptosis, specifically in the
erythroid lineage (Danilova etal. 2008; Dutt etal. 2011). While p53 activation may
explain certain features of DBA pathology, its tumor suppressive properties do not
provide an explanation for the increased cancer susceptibility observed in DBA
patients. Interestingly, the existence of specialized ribosomes has been recently
proposed (Xue and Barna 2012), whereby ribosomes containing mutant ribosomal
proteins could potentially control a specific subset of mRNAs that subsequently
contribute to cancer development in DBA patients in a tissue specific manner.
23.2.3 ShwachmanBodianDiamond Syndrome
ShwachmanBodianDiamond syndrome (SDS) is a rare autosomal recessive pedi-

atric disorder characterized by exocrine pancreatic insufficiency, bone marrow dys-
function, skeletal abnormalities and an increased risk of progression to MDS and
AML (Donadieu etal. 2005; Dror 2005). SDS is always associated with mutations
in the Shwachman-Bodian-Diamond syndrome (SBDS) gene where almost 90% of
patients carry a bi-allelic SBDS mutation (Boocock etal. 2003). It has been shown
that SDS patients display altered ribosome profiles and impaired association of the
40S and the 60S ribosomal subunits (Burwick etal. 2012) and in fact, several stud-
ies have linked SBDS to the control of ribosomal subunit joining. Mechanistically,
it has been demonstrated that SBDS cooperates with the GTPase elongation factor-
like 1 (EFL1) to catalyze eIF6 removal from mammalian pre-60S subunits (Finch
etal. 2011). In addition, SBDS has been also shown to directly interact with rRNA
suggesting a role for SBDS in rRNA processing (Ganapathi etal. 2007).
23.2.4 Myelodysplastic Syndrome and the 5q- Syndrome
Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal he-

matopoietic disorders characterized by anemia, erythrocyte dysfunction and bone
Fig. 23.1 Schematic presenta-

tion of the common deleted T
regions in the 5q- syndrome. T
Genes with known functions T
in ribosome biogenesis and
T
protein translation RPS14,
miR-145, NPM1) are depicted T &711$(*5
in red. The figure was adapted T
and modified from Ebert
(2011).
T US6PL5
T
T
T
T 130
T
T
marrow failure with predisposition to a higher risk of developing AML (Nimer

2008). At diagnosis, 4060% of all MDS patients show identifiable chromosomal
aberrations (Nimer 2008). Within this population, the 5q- syndrome constitutes a
distinct entity that is characterized by specific deletions of the long arm of chromo-
some 5, which share a common deleted region (CDR). In recent years, several stud-
ies have shed light on the molecular mechanisms underlying 5q- development. In
particular, the ribosomal protein rpS14 and nucleophosmin (NPM1) have emerged
as key players in 5q- pathology (Fig.23.1).
Although the mechanism is not fully understood, RPS14 has been identified as
a critical gene for the erythroid phenotype of the 5q- syndrome (Ebert etal. 2008).
Specifically, suppression of rpS14 expression causes a severe block of erythroid dif-
ferentiation, whereas its overexpression in cells with 5q- MDS rescued the erythroid
differentiation defect. Similar to DBA patients, decreased expression of rpS14 in-
duced p53 expression (Dutt etal. 2011). Indeed, this induction of p53 has been
shown to cause a cell cycle arrest in erythroid progenitor cells, which eventually
results in the anemia phenotype observed in 5q- patients (Barlow etal. 2009; Dutt
etal. 2011).
In addition to rpS14, the NPM1 gene has been proposed to play an important
role in the 5q- syndrome. Although NPM1 has a number of distinct biological func-
tions, it has been implicated in the control of ribosome biogenesis at multiple levels.
Specifically, NPM1 regulates ribosome biogenesis at the level of both ribosome
processing and assembly (Dumbar etal. 1989; Haga etal. 2013; Maggi etal. 2008;
Wang etal. 1994; Yu etal. 2006; Yun etal. 2003). The NPM1 locus lies distal to
the 5q CDR. However, it is frequently codeleted with RPS14 in the CDR, and/
or downregulated, and may therefore critically contribute to and modify the 5q
MDS phenotype (Ebert 2011). In support of this view, NPM1-deficient mice display
a phenotype that is characterized by several features of human MDS including a
block in erythroid differentiation and defects in the megakaryocytic and granulo-
cytic compartments (Grisendi etal. 2005).
Taken together, ribosomopathies represent an important class of hematological

diseases that are characterized by defects in ribosome biogenesis and function, in
turn collectively underscoring the very important role of translational dysregula-
tion in the pathogenesis of hematological disease. While there has been tremendous
progress in defining the genetic basis of these diseases, further studies are required
to determine the exact role of this altered ribosomal function in their etiology, so as
to allow for the development of novel therapeutics to target these disorders and to
prevent the development of the associated hematopoietic malignancies.
23.3Ribosomal Alterations in Hematological

Malignancies
Blood malignancies are frequently characterized by the malfunction of proto-onco-

genes such as mTOR and MYC as well as tumor suppressors including p53, PTEN
and NPM1. Strikingly, these key drivers profoundly affect the regulation of transla-
tion factors, as well as ribosome biogenesis and function. Accordingly, their tumori-
genic effects are achieved, at least in part, through this mechanism (Backman etal.
2002; Dowling etal. 2010; Grisendi etal. 2006; Ruggero 2009; Vogt 2001; Zhai
and Comai 2000).
One of the best-studied ribosome-associated genes that plays an important role in
the development and progression of B-cell lymphomas is the oncogenic transcrip-
tion factor c-MYC (hereafter referred to as MYC). MYC is frequently dysregulated
in lymphomas and directly impacts protein synthesis by regulating the transcription
of ribosomal proteins, elongation as well as initiation factors, such as eIF4E (Coller
etal. 2000; Ruggero 2009). Recurrent MYC gene rearrangements are commonly
found in aggressive B-cell lymphomas (Slack and Gascoyne 2011) and represent an
important oncogenic driver of this disease. In support of these findings, E-MYC
transgenic mice, where MYC expression is under the control of the E immuno-
globulin heavy chain intron transcription enhancer, are prone to develop B-cell lym-
phomas (Adams etal. 1985). Using the E-MYC transgenic mouse model system,
Barna and colleagues were able to demonstrate that MYC specifically increases
cap-dependent translation (Fig.23.2) (Barna etal. 2008). The resulting impairment
of IRES-mediated translation of the mitotic protein CDK11/p58 leads to cytokinesis
defects and genomic instability (Barna etal. 2008) (Fig.23.2). Indeed, E-MYC
transgenic mice displayed increased genomic instability suggesting that impaired
IRES-mediated translation of specific mRNAs downstream of MYC contribute to
cancer progression (Fig.23.2). The specific requirement for the effects of MYC in
ribosome biogenesis and protein synthesis has been demonstrated by crossing E-
MYC transgenic mice to mice heterozygous for RPL24 (Barna etal. 2008). These
mice displayed a delayed onset of lymphomas and had normal translation rates and
cell size suggesting that the oncogenic potential of MYC is dependent on its ability
to impact the translation machinery.
Similar to lymphoma, there is an increasing body of evidence in support of the
notion that alterations in the translation machinery may affect the development of
Fig. 23.2 The role of MYC in translation and tumorigenesis. Overexpression of MYC results
in an increase of cap-dependent translation, cell growth and cell division, which ultimately
promotes tumorigenesis. Increased cap-dependent translation in established tumors leads to
an imbalance of IRES-mediated translation, which affects key mitotic regulators like CDK11/
p58. Downregulation of CDK11/p58 translation in turn leads to genomic instability and tumor
progression (Barna etal. 2008).
different forms of leukemia. For example, mutations to RPL5 and RPL10 and het-
erozygous deletions of RPL22 have recently been identified in T-ALL patients
suggesting that alterations to ribosomal proteins may contribute to the develop-
ment and progression of T-ALL (De Keersmaecker etal. 2012; Rao etal. 2012).
Another compelling example for an alteration to factors that contribute to normal
ribosome biogenesis and function in leukemia is represented by NPM1. NPM1 is
one of the most frequent targets of genetic alterations in hematopoietic tumors. Two
well-characterized genomic events that involve the NPM1 gene are chromosomal
translocations (found in both lymphoid and myeloid disease) (Szebeni and Olson
2008), and mutation resulting in a cytoplasmic localization of the protein (NPMc+;
found in AML) (Grisendi etal. 2006). Indeed, NPMc+ is now established to be the
most frequent mutation identified in karyotypically normal AML (Grisendi 2005).
Given the importance of NPM1 for ribosome biogenesis it is likely that mutations
and translocations to the protein result in ribosome deficiencies contributing to dis-
ease-specific phenotypes. Besides AML and T-ALL, mutations to proteins involved
in the control of protein synthesis have also been identified in multiple myeloma
(Chapman etal. 2011).
Together, these findings clearly demonstrate that alterations to components of
the translation machinery or to upstream signaling proteins that impact translation
can contribute to the development and progression of hematological malignancies .
23.4 Translation Factors in Hematological Malignancies
While ribosomopathies represent an important class of hematological diseases

where defects in ribosome and ribosome-associated proteins contribute to disease
pathology, it is becoming increasingly clear that alterations to translation factors
also contribute to the development and progression of different hematologic malig-

nancies. Here, we will discuss the role of translation initiation factors in the devel-
opment of these diseases.
23.4.1eIF4E
As discussed in other chapters in this book, translational control mainly happens at

the step of translation initiation. Translation initiation is a tightly regulated process
and is mainly controlled by the eIF4F and eIF3 complexes as well as by eIF2,
which all have been implicated in the development of different human cancers
(Silvera etal. 2010). The mRNA-bound eIF4F complex facilitates cap-dependent
mRNA translation (Sonenberg and Hinnebusch 2009). The activity of the eIF4F
complex is regulated through upstream signaling pathways and its dysregulation is
strongly linked to cancer initiation and progression (Silvera etal. 2010). The eIF4F
complex consists of three major components: eIF4E, eIF4G and eIF4A. Amongst
these, eIF4E is the best-characterized factor in hematological malignancies. eIF4E
is considered to be a key and rate-limiting factor in controlling translation initiation
and is regulated through direct phosphorylation by upstream signaling pathways in-
cluding the MAPK pathway (Fig.23.3a) (Pyronnet 1999), as well as through inhibi-
tory interactions with 4E-BPs (Fig.23.3a). Hypophosphorylation of 4E-BP1 leads
to its activation and in turn to sequestration of eIF4E that subsequently blocks the
formation of the cap-dependent translation initiation complex by removing eIF4E
from eIF4G (Silvera etal. 2010). In contrast, hyperphosphorylation of 4E-BP by
the PI3K/AKT/mTOR pathway leads to the release of 4E-BP from eIF4E and to
the activation of translation (Gingras etal. 1998). Importantly, eIF4E is frequently
overexpressed in various hematological malignancies including non-Hodgkins
and B-cell lymphomas, AML and CML (Green etal. 2012; Topisirovic etal. 2003;
Wang etal. 1999). Interestingly, besides its role in translation initiation, eIF4E can
also promote the nucleocytoplasmic transport of specific transcripts thereby con-
tributing to the etiology of leukemia (Topisirovic etal. 2003). Moreover, eIF4E is
a direct transcriptional target of nuclear factor -light-chain-enhancer of activated
B-cells (NF-B) and eIF4E is frequently dysregulated in the M4 and M5 subtypes
of AML (Hariri etal. 2013). Consistent with its proposed oncogenic function, stud-
ies in genetically engineered mouse models identify eIF4E as a critical determinant
for the development of hematological malignancies. For example, transgenic ani-
mals overexpressing eIF4E develop B-cell lymphomas (Ruggero etal. 2004). It is
important to note that tumors in eIF4E transgenic animals arise with a relative long
latency suggesting that eIF4E may cooperate with other genetic lesions in B-cell
lymphoma development. Indeed, the overexpression of eIF4E markedly accelerated
the onset of MYC-induced B-cell lymphomas demonstrating that eIF4E can cooper-
ate with MYC to drive tumorigenesis (Ruggero etal. 2004; Wendel etal. 2004). In
addition, hyperactivation of eIF4E was shown to be required for the development
of AKT-induced T-cell lymphomas (Hsieh etal. 2010) further demonstrating that
eIF4E is a critical determinant for tumorigenesis.
Fig. 23.3Regulation of translation factors involved in hematological malignancies. a The eIF4F

complex is regulated by two distinct upstream signaling pathways: PI3K/AKT/mTOR and MAPK/
MNK. b Stress-activated eIF2 kinases (eIF2K) regulate the activity of EIF2. c eIF6 is mainly
regulated by growth factor signaling through the PKC pathway.
In addition to regulating the 4E-BP1/eIF4E axis, the mTOR pathway is known to

activate translation initiation via the phosphorylation of S6K (Ma and Blenis 2009).
Amongst others, S6K regulates the activity of PDCD4, a tumor suppressor protein
that inhibits protein synthesis by suppression of translation initiation (see Chaps.6
and 16). Specifically, phosphorylation of PDCD4 by S6K serves as a signal for the
SCF/-TRCP complex to bind and mark PDCD4 for degradation (see Chap.6).
PDCD4 degradation in turn triggers the release of the initiation factors eIF4A and
eIF4G to increase cap-dependent translation (Dorrello etal. 2006). Interestingly,
PDCD4 was recently demonstrated to contribute to retinoic acid-induced granulo-
cyte differentiation of human myeloid leukemia cells (Ozpolat etal. 2007).
23.4.2eIF2
The eukaryotic initiation factor eIF2 is an essential component of the ternary com-
plex that regulates global protein synthesis in response to various cellular and en-
vironmental stimuli (Wek etal. 2006) (see Chap.9). Within this complex, eIF2
binds to GTP and to Met-tRNAi to transfer Met-tRNAi to the 40S ribosomal sub-
unit (Kimball 1999). In response to various cellular stresses, a family of stress-
activated protein kinases phosphorylates the subunit of eIF2 (eIF2), resulting in
global inhibition of protein synthesis (Fig.23.3b) (Wek etal. 2006). These eIF2
kinases include GCN2, PERK, PKR and HRI and sense either amino acid depri-
vation, unfolded proteins, double-stranded RNA or heme deficiency, respectively.
Importantly, overexpression of a mutant form of eIF2 that cannot be inactivated
by these upstream kinases causes malignant transformation of NIH 3T3 cells, sug-
gesting that eIF2 has a critical role in regulating cell proliferation (Donz etal.
1995). PKR-mediated phosphorylation of eIF2 can regulate terminal differentia-
tion of AML cells (Ozpolat etal. 2012), while the PERK/eIF2 axis is upregulated
in BCR-ABL expressing CML cells and acts as a prosurvival signal downstream
of BCR-ABL signaling (Kusio-Kobialka etal. 2012). Moreover, induction of this
pathway in CML cells confers resistance to the small molecule inhibitor imatinib
(Kusio-Kobialka etal. 2012) suggesting an important role of eIF2 in CML devel-
opment and therapy resistance. In addition to CML, it has been shown that c-MYC
activates the PERK/eIF2 axis in lymphoma cells, thereby leading to increased cell
survival via the induction of cytoprotective autophagy (Hart etal. 2012).
23.4.3eIF6
In contrast to eIF4E and eIF2, which mainly regulate cap-dependent translation

downstream of the mTOR and MAPK signaling cascades, eIF6 is necessary for 60S
ribosome biogenesis in the nucleolus (Sanvito etal. 1999) as well as for the joining
of the large ribosomal subunit (60S) with the 48S initiation complex in the cyto-
plasm (Ceci etal. 2003). eIF6 is mainly regulated by the PKC pathway (Fig.23.3c)
(Ceci etal. 2003; Grosso etal. 2008) and has been shown to be a rate-limiting fac-
tor for translation and cell transformation (Gandin etal. 2008). Mechanistically,
PKCII phosphorylates eIF6 allowing for the release of eIF6 from the 60S subunit
and proper subunit joining (Miluzio etal. 2009). Importantly, MYC-driven lym-
phomas contain high levels of PKCII and phosphorylated eIF6 and the deletion of
one copy of eIF6 in E-MYC mice delays lymphomagenesis suggesting that eIF6 is
rate-limiting for MYC-induced tumorigenesis (Miluzio etal. 2011).
23.4.4 Other Translation Initiation Factors
Other translation factors have been implicated in hematological malignancies. For

example, eIF4B, a known activator of the RNA helicase eIF4A, has been implicated
in the pathogenesis of DLBCL and was characterized as a novel prognostic marker
for poor survival in human DLBCL (Horvilleur etal. 2013). Moreover, it has been
recently demonstrated that phosphorylation of eIF4B by PIM kinases is required for
BCR-ABL induced cellular transformation (Yang etal. 2013). eIF5A, a translation
factor that plays a significant role in the formation of the first peptide bond during
protein synthesis (Henderson and Hershey 2011), plays a critical role in BCR-ABL
positive leukemias (see Chap.10). eIF5A is the only translation initiation factor
that is activated by posttranslational hypusination (Caraglia etal. 2013), and was
found to be downregulated upon imatinib treatment of BCR-ABL-positive cell lines

and hypusination inhibitors exerted an antiproliferative effect on BCR-ABL-posi-
tive leukemia cell lines (Balabanov etal. 2007). These data suggest that eIF5A is
required for cell proliferation and that targeting eIF5A hypusination represents a
promising approach for the treatment of BCR-ABL-positive leukemias.
During translation initiation, the 43S PIC joins with the mRNA-bound eIF4F
complex to form the 48S initiation complex (Sonenberg and Hinnebusch 2009). The
crucial mediator in bridging both complexes is the eIF3 complex, which consists of
over a dozen subunits (Dong and Zhang 2006). However, the importance of the eIF3
complex in hematological malignancies has yet to be determined. Several other
translation factors including eIF1 as well as termination and elongation factors have
also not yet been investigated in hematological malignancies and future studies will
shed light on the role of these factors in the etiology of these diseases.
23.5Treatment Strategies Targeting the Translation

Machinery in Hematological Malignancies
In recent years, there has been an extensive body of research that established a
causal role for the dysregulation of the translation machinery in cancer develop-
ment and progression. Thus, targeting the translation machinery for the treatment of
hematological malignancies could represent a promising therapeutic approach. In
this part of this chapter, we will briefly summarize the strategies that aim to target
translation for cancer therapy.
Due to its central role in translation initiation, eIF4E represents an attractive
target for the design of anticancer therapeutics. Thus far, attempts to directly block
activity of eIF4E include the use of ASOs that directly bind to the eIF4E mRNA
leading to its destruction (Fig.23.4) (Graff etal. 2007); however, besides possible
delivery issues of the ASO, the potential efficacy of eIF4E ASO has yet to be tested
in hematological malignancies. Another strategy to target eIF4E is to block its in-
teraction with eIF4G. High-throughput screens for inhibitors that could prevent the
binding of eIF4E to eIF4G identified the compound 4EGI-1 (Fig.23.4) (Moerke
etal. 2007). Importantly, it has been shown that 4EGI-1 induces apoptosis of AML
blast cells without affecting the differentiation and survival of normal CD34+he-
matopoietic cells in vitro (Tamburini etal. 2009). In addition, 4EGI-1 was able to
induce apoptosis of multiple myeloma cells and synergizes with the BH3 mimetic
ABT-737 to induce apoptosis in chronic lymphocytic leukemia (Descamps etal.
2012; Willimott etal. 2013). Besides eIF4E, targeting the RNA helicase eIF4A rep-
resents another strategy to directly modulate the eIF4F initiation complex in cancer.
Hippuristanol is a selective eIF4A inhibitor and has been shown to sensitize E-
MYC induced lymphomas to DNA-damaging agents (Fig.23.4) (Cencic etal. 2013).
In addition, the small molecule inhibitor silvestrol interferes with eIF4A activity
and exerts antineoplastic activity in a mouse lymphoma model in vivo (Fig.23.4)
(Bordeleau etal. 2008). Thus, suppression of eIF4A in active disease represents a
promising therapeutic approach for the treatment of hematological malignancies.
Fig. 23.4 Targeting the translation machinery for the therapy of hematological malignancies.
Several inhibitors that either target upstream signaling pathways that impinge on translational
regulators (e.g. mTOR, MNK1/2) or translation initiation factors (e.g. eIF4E, eIF4A) are being
developed and are currently in various stages of preclinical and clinical testing.
Besides the direct targeting of the translation initiation machinery, the most
common strategy to interfere with aberrant translational control in cancer is the
targeting of aberrant upstream regulatory signaling pathways. Amongst the clas-
sical signaling nodes that impact on the translation machinery, mTOR represents
the most widely studied target for cancer therapy. As already mentioned in several
other chapters of this book, components of the PI3K/AKT/mTOR pathway are fre-
quently mutated in different human cancers and pathway hyperactivity contributes
to the malignant phenotype of cancer cells. In particular, mTOR hyperactivity has
been demonstrated in AML, Hodgkins lymphoma, non-Hodgkins lymphoma and
multiple myeloma (Kelly etal. 2011; Younes and Samad 2011). Several therapeu-
tic agents that target the PI3K/AKT/mTOR pathway are currently under active in-
vestigation. Rapamycin, an allosteric mTOR inhibitor targeting mTORC1, was the
first compound identified to interfere with this pathway (Fig.23.4) (Guertin and
Sabatini 2009). The mTOR kinase associates in two distinct complexes in the cell:
mTORC1 and mTORC2 (see Chap.15). Unlike rapamycin, mTOR ATP-site inhibi-
tors that function to inhibit both mTORC1 and mTORC2 complexes are also being
developed (Benjamin etal. 2011). Amongst these ATP-site inhibitors, PP242 has
been shown to suppress tumor growth in a mouse model of AKT-driven lymphoma-
genesis (Hsieh etal. 2010). Several other mTOR inhibitors have been successfully
used to assess the therapeutic effect of mTOR inhibition in a variety of other hema-
tological malignancies (Fig.23.4). Together these studies validate mTOR and the
translation machinery as a highly relevant target for the therapy of different classes
of hematological malignancies.
Another signaling pathway that has been suggested as an attractive target up-
stream of the translation machinery is the MNK pathway (Fig.23.4). As described
above, the serine/threonine kinases MNK1 and 2 directly phosphorylate eIF4E
thereby controlling the rate of protein synthesis (Ma and Blenis 2009; Pyronnet
1999; Waskiewicz etal. 1997). So far several MNK kinase inhibitors have been
described including CGP052088, CGP57380, and cercosporamide (Fig.23.4)
(Buxad etal. 2005; Chrestensen etal. 2007; Konicek etal. 2011; Rowlett etal.
2008; Tschopp etal. 2000; Worch etal. 2004). Importantly, it has been shown that
targeting of the MNK axis in blast crisis CML as well as AML inhibits leukemia
cell growth, suggesting that MNK may be a promising target for the treatment of
different leukemia subtypes (Altman etal. 2013; Lim etal. 2013).
Besides mTOR and MNK, AMPK, a sensor for cellular energy status, represents
an important target for the treatment of hematological malignancies. Activation of
AMPK leads to the phosphorylation of TSC2 and to the downstream inhibition of
the translational regulators S6K and 4E-BP1 (Inoki etal. 2003; Corradetti etal.
2004). Thus, pharmacological activation of AMPK and subsequent inhibition of
protein synthesis could be an effective means for the treatment of hematological
malignancies. Indeed, the AMPK activators, metformin and phenformin have been
shown to block growth and induce apoptosis in several leukemia cells types (Huai
etal. 2012; Leclerc etal. 2013; Rosilio etal. 2013; Vakana etal. 2011; Green etal.
2010) validating AMPK activation as a promising strategy for the treatment of dif-
ferent blood cancers.
As outlined above, it is becoming increasingly clear that the translation machinery

is an important determinant of tumorigenesis. However, we are just beginning to
appreciate how mutations in the various components of this machinery actually
contribute to the development and progression of human cancer. The discovery that
ribosomopathies are characterized by impaired ribosome biogenesis has brought
about the question whether the ribosome itself is altered in these diseases or if these
phenotypes are due to aberrant non-ribosomal functions of ribosomal components.
The lack of a high throughput platform that would allow for a systematic assess-
ment of the composition of the ribosome and its associated proteins has until re-
cently hampered our understanding whether such cancer ribosomes indeed exist.
However, a recent study by our group described the development of a SILAC-based
(stable isotope labeling by amino acids) mass spectrometry approach to probe mam-
malian riboproteomes (i.e. the ribosome and ribosome-associated proteins) in a
high-throughput fashion (Reschke etal. 2013). Applying this approach to the study
of human cancer cell lines allowed us to demonstrate that riboproteomes vary in
their composition in different cancer cell lines suggesting that such changes could
be causally involved in cancer development (Reschke etal. 2013). This approach
can now be readily applied to various cellular and biological systems and will shed
light on the relative contribution of aberrant riboproteomes to disease development.
In addition, there is an increasing appreciation for the contribution of regulatory
elements within mRNAs that are recognized by RNA-binding proteins, miRNAs
or the ribosome itself, to fine-tune the expression of mRNA translation (Ruggero
2012). Furthermore, it has been recently recognized that mRNA translation is regu-
lated by competing endogenous RNA networks (Karreth and Pandolfi 2013). It is
possible that these complex regulatory mechanisms are hijacked by the aberrant
cancer riboproteome to direct the expression of a specific subset of mRNAs that
ultimately drive tumorigenesis. In support of this notion, it has been recently shown
that mTOR signaling contributes to cancer development and progression through
the regulations of a subset of mRNAs that harbor distinct regulatory sequences
intheir 5 UTR (Hsieh etal. 2012; Thoreen etal. 2012). The detailed analysis of
these translational mechanisms and how they contribute to cancer development and
progression will be an exciting area of research in the years to come.
Importantly, several components of the translation machinery including trans-
lation factors that are dysregulated in human cancer have emerged as important
targets for therapeutic intervention. Several agents that either target important up-
stream signaling pathways that regulate translation (e.g. mTOR pathway; MNK
pathway; and PKC pathway) or the translation machinery itself (e.g. eIF4E ASOs)
are being developed (Engelman 2009; Graff 2005; Graff etal. 2007).
In conclusion, our increased understanding of translational dysregulation in he-
matopoietic disorders has led to a greater appreciation of the ribosome, ribosomal
proteins and ribosome-associated proteins as an underlying cause of malignant
transformation and provides an opportunity for the development of novel therapeu-
tics that will ultimately impact patient survival.
Acknowledgments We thank Pandolfi lab members for helpful discussions and critical reading of
the manuscript. M.R. received supported from the German Academy of Sciences Leopoldina (Leo-
poldina Research Fellowship grant number: LPDS 2009-27). P.P.P acknowledges support from
NIH Grants CA141496 and CA082328.
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Chapter 24
Brain Tumors
Armen Parsyan, Justin G. Meyerowitz and William A. Weiss
Contents
24.2eIF4E and Its Regulation by 4E-BPs 488
24.3Therapeutic Targeting of the 4E-BPs/eIF4E Axis 491
24.4PDCD4 and Its Targeting 491
24.5Other Translation Factors 492
References 494
Abstract Brain tumors in general and one of their most common and deadliest
types, glioblastoma multiforme, are poorly understood in terms of their molecular
etiology and pathogenesis. Treatment options for patients with advanced high-grade
brain tumors are very limited. The role of translation and its regulation in develop-
ment and progression of brain tumors reveals that the activity of the eIF4F complex
plays an important role in this disease. At least two points of control of this complex
are altered in brain tumors: through the increased expression and activity of the
proto-oncogene eIF4E or via control of the eIF4A helicase by the tumor suppressor
PDCD4. This chapter discusses the role of dysregulated eIF4F function in particular
and translation in general in the pathobiology of brain tumors. We also discuss brain
tumors therapeutic targeting based on regulation of translation.
A.Parsyan()
J.G.Meyerowitz
Department of Chemistry and Chemical Biology, University of California, San Francisco,
CA, USA
J.G.Meyerowitz W.A.Weiss
Department of Neurology, School of Medicine, University of California, San Francisco,
CA, USA
24.1Introduction
Tumors of the nervous system are represented by multiple entities. Relevant for
our discussion are brain tumors, on which the majority of studies into translation
and its regulation are focused. The most common brain tumors include those aris-
ing from various tissue components of the glia (astrocytomas, oligodendrogliomas,
ependymomas), meninges (meningiomas) and others, such as medulloblastoma.
The most malignant form of astrocytoma is glioblastoma multiforme, a high-grade
(grade IV) glioma. Treatment options for patients with glioblastoma usually include
chemotherapy, radiation and surgery, which provide some relatively small degree
of improvement to the otherwise dismal survival (Galanis etal. 2005; Johnson and
ONeill 2012). Similar lines of treatment are available for other types of brain tu-
mors. Molecular studies to understand gliomas and other types of brain tumors are
underway and give hope to advance treatment of these conditions (Kwiatkowska
etal. 2013; Van Meir etal. 2010). Not uncommonly, brain tumors present with
various genetic alterations that involve p53 tumor suppressor (present in 31% of
glioblastoma multiforme), PTEN (23%), EGFR, (23%) and PI3K (14%) (Ohgaki
and Kleihues 2011; Verhaak etal. 2010). These alterations, directly or via cross-
talk, regulate the PI3K/AKT/mTOR or MAPK signaling pathways that converge on
regulation of the translation machinery (see Chaps.15 and 17), on which we will
focus our current attention.
24.2eIF4E and Its Regulation by 4E-BPs
eIF4E is a cap-binding component of the eIF4F complex, which also includes he-
licase eIF4A and scaffolding protein eIF4G (see Chaps.2 and 4). eIF4E is a proto-
oncogene that is required for translation of mRNA encoding prosurvival, proangio-
genesis and proproliferative proteins, such as VEGF and cyclin D1 (see Chap.4).
As discussed later on in this section, eIF4E could participate in tumorigenesis via
increase in its expression or activation. Activation of eIF4E occurs via the mTOR
pathway-related phosphorylation and deactivation of eIF4E inhibitory binding pro-
teins (4E-BPs) and via the MAPK pathway that signals to MNK proteins to directly
phosphorylate and activate eIF4E.
An early study showed that serum growth factors are required for expression of
eIF4E mRNA in nontransformed cells; however this requirement is lost in C6 glio-
blastoma, A431 carcinoma and n-MYC transformed Rat-1 cells (Rosenwald 1995).
These data raised the possibility that neoplastic transformation in general and in
glioblastomas in particular are associated with increased expression of eIF4E that is
independent of growth factors (Rosenwald 1995). These findings also indicate that
the loss of eIF4E regulation by signaling pathways or activation of these pathways
plays a role in the pathogenesis of glioblastoma.
24 Brain Tumors 489
Despite these reports, the role of translation in brain tumors did not receive much
attention for almost a decade. In 2005, Gu etal. used IHC to study eIF4E protein ex-
pression in human brain tissue from patients without central nervous system diseas-
es and brain biopsy tissues from patients with anaplastic astrocytoma (high grade
III glioma) and glioblastoma multiforme (Gu etal. 2005). Malignant cells from ana-
plastic astrocytoma and glioblastoma showed diffuse uniform expression of eIF4E
compared to undetectable eIF4E levels in normal neuroglia and pyramidal neurons
(Gu etal. 2005). In glioblastoma, eIF4E was also present in proliferative endothelial
cells and vascular lining endothelial cells (Gu etal. 2005). This study provided the
first evidence that eIF4E was upregulated in high-grade astrocytic tumors.
Further studies confirmed that elevated expression of eIF4E correlated with ma-
lignancy in a spectrum of brain tumors, not only limited to astrocytic but also oli-
godendroglial tumors and meningiomas (Tejada etal. 2009; Yang etal. 2007). In
a tissue microarray, elevation of eIF4E was reported in 48% of the glioblastoma
multiforme samples (Yang etal. 2007). Meningiomas and oligodendroglial tumors
show higher levels of eIF4E expression than astrocytomas (Tejada etal. 2009).
Emphasizing its role in malignancy, levels of eIF4E were found to be significantly
higher in atypical than in benign meningiomas (Tejada etal. 2009). Thus, overex-
pression of eIF4E represents a common theme in various types of malignant brain
tumors.
While regulation of eIF4E expression could be mediated by various factors (see
Chap.4), such as the MYC proto-oncogene, this association in glioma biology has
not yet been established. However, a mechanism by which eIF4E could be overex-
pressed in human glioblastoma cells was suggested to take place via JNKs that are
members of the MAPK family implicated in tumorigenesis in general and gliomas
in particular (Cui etal. 2006).
Another mechanism to potentiate eIF4E oncogenic function is to control its
activity, such as through phosphorylation by MNK1/2 downstream of the MAPK
pathway (see Chap.17). High levels of phosphorylated eIF4E are found in me-
ningiomas and astrocytic tumors (Tejada etal. 2009). In astrocytic tumors, phos-
phorylated eIF4E levels were significantly higher in glioblastoma than in diffuse
astrocytoma (Tejada etal. 2009). Meningiomas showed elevated levels of eIF4E
phosphorylation, while oligodendroglial tumors had levels similar to normal brain
tissue samples. In this study, a significant positive correlation was found between
phosphorylated eIF4E and cyclin D1 in brain tumors (Tejada etal. 2009).
The MAPK pathway appears to play an important role in the pathogenesis of
glioblastoma (Sunayama etal. 2011). Inhibition of MNK1 activity in glioblastoma
cells by the small molecule CGP57380 suppressed phosphorylation of eIF4E, as
well as cellular proliferation and colony formation, whereas concomitant treatment
with CGP57380 and the mTOR inhibitor rapamycin accentuated growth inhibition
and cell-cycle arrest (Grzmil etal. 2011). This latter result emphasizes the roles
played by both the MAPK and mTOR pathways in oncogenic translation in brain
tumors. This study also showed that MNK1, likely via its translational target eIF4E,
regulates translation of mRNAs involved in control of TGF- pathway, such as
SMAD2 (Grzmil etal. 2011).
In human glioma-initiating cells, eIF4E has also been shown to translationally

activate SOX2, a master transcription factor essential for maintaining self-renew-
al, or pluripotency, of undifferentiated embryonic stem cells, and in sustaining the
self-renewal of neural stem cells and glioma-initiating cells (Ge etal. 2010). This
control of SOX2 by eIF4E could drive increased self-renewal of glioma-initiating
cells, providing a potential therapeutic target (Ge etal. 2010). Altogether these data
suggest that eIF4E, via its activation or overexpression, upregulates prosurvival and
proproliferative programs, via targets, such as cyclin D1, SMAD2 and SOX2. Nota-
bly, activation of tumor cell integrin (v)(3), implicated in brain tumor metastatic
growth and angiogenesis, leads to upregulation of VEGF under normoxic condi-
tions, via a translational mechanism of phosphorylation and inhibition of transla-
tional repressor 4E-BPs (Lorger etal. 2009).
Activation of eIF4E also occurs through suppression of the inhibitory effect of
4E-BPs via their phosphorylation by the PI3K/AKT/mTOR pathway. This pathway
is highly upregulated in gliomas due to various factors (such as deletion or mutation
of PTEN, as mentioned above), and its activation is associated with tumor grade
(Aeder etal. 2004; Cui etal. 2006; Ermoian etal. 2009; Hanrahan and Blenis 2006;
Korkolopoulou etal. 2012; Mueller etal. 2012; Riemenschneider etal. 2006). In
addition to its effects on 4E-BPs, mTOR plays an important role in regulating pro-
tein translation through phosphorylation of p70S6K, a protein involved in ribosome
biogenesis via control of rpS6. One study revealed that activation of the mTOR
pathway and its upstream and downstream effectors (such as phosphorylated 4E-
BP1, phosphorylated AKT, PTEN, TSC1 and TSC2) significantly correlates with
worsening grade of gliomas (Ermoian etal. 2009).
In astrocytic tumors, phosphorylation of 4E-BP1 represents one of the major
clinicopathologic markers of an aggressive and angiogenic phenotype and corre-
lates with worsening tumor grade (Korkolopoulou etal. 2012). Phosphorylation of
4E-BP1 reflected and correlated with the activated state of mTOR that adversely
affected OS and DFS in both univariate and multivariate analyses. Interestingly,
phosphorylation of another downstream target of mTOR, p70S6K, only marginally
correlated with phosphorylation of mTOR (Korkolopoulou etal. 2012). A recent
study of pediatric grade IIIV gliomas correlated phosphorylation of rpS6 and 4E-
BP1 with decreased PFS; however only the latter showed statistical significance
(Mueller etal. 2012). This suggests that the mTOR/4E-BPs axis might have more
bearing on translational regulation and clinicopathologic characteristics in brain tu-
mors than mTOR/S6K.
In addition to their deactivation through phosphorylation by activated mTOR,
decreased expression of 4E-BPs in an experimental setting was associated with
prosurvival effects in glioblastoma. Loss of 4E-BP1 expression did not signifi-
cantly alter growth in vitro but accelerated the growth of glioblastoma U87 tumor
xenografts (Dubois etal. 2009). Cells lacking 4E-BP1 were significantly more sen-
sitive to hypoxia-induced cell death in vitro and were more sensitive to radiation
(Dubois etal. 2009).
24 Brain Tumors 491
24.3Therapeutic Targeting of the 4E-BPs/eIF4E Axis
Inhibitors of the mTOR pathway are being tested in preclinical and early stage trials
and show promising results in terms of safety and efficacy. PI3K/mTOR pathway
inhibitors are broadly divided into mTOR allosteric inhibitors (rapamycin analogs,
or rapalogs), ATP-competitive mTOR inhibitors, and dual inhibitors of PI3K and
mTOR (see Chap.15). Rapalogs, the first generation of mTOR inhibitors (such as
temsirolimus, everolimus and ridaforolimus), potently inhibit mTORC1 phosphory-
lation of p70S6K but generally spare mTORC2 and mTORC1 phosphorylation of
4E-BPs. This results in both continued eIF4E activity as well as feedback activation
mechanisms that hinder their effects (see Chap.15). While rapalogs have shown
success in specific contexts, such as RCC and endometrial cancers (see Chap.32
and 34), they have largely failed in trials for glioblastoma. Single-agent rapalog tri-
als have generally shown modest radiographic improvement of disease without sig-
nificant increase in OS or PFS (Chang etal. 2005; Faivre etal. 2006; Galanis etal.
2005). Similarly, combination trials of rapamycin with the chemotherapeutic agent
TMZ (Josset etal. 2013), VEGF inhibitor bevacizumab (Hainsworth etal. 2012;
Lassen etal. 2013) or others (Puli etal. 2010) have shown little to no improvement
in PFS and OS.
In contrast, ATP-competitive mTOR inhibitors and dual PI3K/mTOR inhibitors
have shown activity in preclinical studies and are beginning to enter clinical trials.
These agents inhibit both mTORC1 and mTORC2 in an ATP-competitive manner
that blocks phosphorylation of both p70S6K and 4E-BPs. The mTOR inhibitors
AZD-8055 and WYE-125132 (WYE-132) are effective in preclinical models of
glioblastoma, and AZD-8055 is currently in a phase I trial for adults with recurrent
gliomas (Chresta etal. 2010; Yu etal. 2010). The dual PI3K/mTOR inhibitor XL765
(SAR245409) showed promise as both monotherapy and in combination with TMZ
in xenograft models of glioblastoma and is currently in phase I trials (Prasad etal.
2011). Another dual PI3K/mTOR inhibitor NVP-BEZ235 has not only shown ef-
ficacy as single agent but also as an enhancer of radiosensitivity in preclinical trials
(Cerniglia etal. 2012; Kuger etal. 2013; Sunayama etal. 2010). Other strategies,
such as MEK inhibitors (U0126) also show activity in preclinical glioma models
(Glassmann etal. 2011). Despite these developments, direct targeting of eIF4E and
4E-BPs as described elsewhere (see Chaps.4 and 14) in this volume have not been
studied in the context of brain tumors and might be an efficient strategy, especially
in terms of overcoming feedback resistance observed in the pharmacological inhibi-
tion of the signaling cascades.
24.4PDCD4 and Its Targeting
PDCD4 is a bona fide tumor suppressor that acts via inhibition of a component of the
eIF4F complex, helicase eIF4A (see Chap.6). PDCD4 is often found downregulated
in brain tumors, in part through negative regulation by miR-21 (Costa etal. 2013b;
Gaur etal. 2011; Grunder etal. 2011; Park etal. 2012). Downregulation of PDCD4
by miR-21 has been shown to facilitate glioblastoma proliferation in vivo (Gaur
etal. 2011). Inversely, downregulation of miR-21 or overexpression of PDCD4 in
glioblastoma cell lines lead to decreased proliferation, increased apoptosis, and de-
creased colony formation in soft agar and decreased tumor formation and growth in
a mouse xenograft model (Gaur etal. 2011). Decrease in PDCD4 via upregulation
of miR-21 was shown to participate in glioblastoma pathobiology and to adversely
correlate with clinicopathologic factors and metastatic potential (Chen etal. 2008;
Gaur etal. 2011; Park etal. 2012). Similar findings have been observed in medul-
loblastoma, where the miR-21/PDCD4 axis was linked with leptomeningeal dis-
semination of the disease (Grunder etal. 2011).
In glioblastoma multiforme, loss of PDCD4 also promotes chemoresistance
(Liwak etal. 2013). This effect is, at least in part, mediated via upregulation of
BCL-XL, an important antiapoptotic protein, that is thought to be regulated at the
level of translation by PDCD4. Similarly, in glioblastoma elevated levels of miR-
21 also mediate radioresistance via downregulation of PDCD4 and DNA mismatch
repair protein hMSH2 (Chao etal. 2013).
The mechanisms that lead to dysregulation of PDCD4 control and expression
in brain tumors require further understanding. miR-21 upregulation in glioblasto-
ma cells could occur via heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP
C1/2) (Park etal. 2012). Despite the clear role of miR-21 as a negative regulator of
PDCD4, other mechanisms can reduce PDCD4 expression in glioma, such as 5CpG
island methylation (Gao etal. 2009). Recent work has also provided evidence that
FAT1 may suppress the activity of PDCD4 in glioblastoma (Dikshit etal. 2013).
Targeted delivery of nucleic acids to glioblastoma using chlorotoxin (CTX, a
peptide reported to bind selectively to glioma cells) coupled to a miR-21 silencer
and packaged in lipid-based nanoparticles resulted in increased levels of the tumor
suppressors PTEN and PDCD4, activation of caspase 3/7 and decreased tumor cell
proliferation (Costa etal. 2013a). Thus this approach could be used alone or in
combination to enhance the effect of other therapeutic agents. For example, down-
regulation of miR-21 via upregulation of PDCD4 and other targets of miR-21, such
as PTEN, enhanced cytotoxicity in response to the antiangiogenic drug sunitinib
(Costa etal. 2013b). Thus PDCD4 is another regulator of translation that is closely
linked to brain tumor biology, and may represent a therapeutic target.
24.5Other Translation Factors
eIF2 is a translation initiation factor whose role in tumorigenesis is not yet fully
understood, despite evidence of its importance in cancer biology (see Chap.9).
Phosphorylation of eIF2 at Ser51 leads to inhibition of protein synthesis and
occurs in response to activation of a set of kinases responsive to specific stress
stimuli. One of these kinases is PKR (see Chap.9). Although some consequences
of eIF2 phosphorylation are cytoprotective, phosphorylation of eIF2 by PKR
24 Brain Tumors 493
could be proapoptotic and tumor suppressing (Mounir etal. 2009). As previously

mentioned, alterations in tumor suppressor PTEN play an important role in brain
tumors. PTEN requires activation of PKR/eIF2 phosphorylation for its antitumor
effects (Mounir etal. 2009). Inactivation of PTEN reduces eIF2 phosphorylation,
and reconstitution of PTEN activity in glioblastoma cells induces PKR activity and
eIF2 phosphorylation (Mounir etal. 2009). Importantly, tumor suppressive prop-
erties of PTEN were, in a series of elegant experiments, shown to be dependent on
PKR and eIF2 phosphorylation (Mounir etal. 2009). However, there were no dif-
ferences found in eIF2 levels or levels of phosphorylated eIF2 in meningiomas,
oligodendrogliomas or astrocytomas, although further analysis identified that eIF2
levels were significantly higher in atypical than in benign meningiomas (Tejada
etal. 2009).
An anecdotal report implicates eIF3, namely its eIF3b subunit in the biology of
brain tumors (Liang etal. 2012). This study showed that eIF3b was expressed in hu-
man glioblastoma and human glioblastoma cell lines (U251, U87, A172 and U373),
and that silencing eIF3b expression inhibited proliferation and induced apoptosis
of U87 cells (Liang etal. 2012). Another subunit of eIF3, eIF3c (p110), was shown
to interact with schwannomin or merlin, the neurofibromatosis 2 (NF2) tumor sup-
pressor protein that is commonly lost in benign human brain tumors (Scoles etal.
2006). Schwannomin could inhibit cellular proliferation when eIF3c was highly ex-
pressed. High eIF3c levels were observed in meningiomas that had lost schwanno-
min expression, whereas meningiomas retaining schwannomin exhibited low eIF3c
levels (Scoles etal. 2006).
Bioinformatics analysis of the DHX29 helicase, a newly described participant in
translation, suggested that this protein might be upregulated in glioblastoma multi-
forme (Parsyan etal. 2009). Results from our laboratory indicated that indeed this
protein is overexpressed in glioblastoma cell lines, and its silencing by siRNA leads
to a dramatic decrease in survival and proliferation of glioma cells (unpublished,
Parsyan and Sonenberg 2011).
A recent report implicates regulation of elongation in brain tumors. Under con-
ditions of acute nutrient depletion, eEF2K, which is activated by AMPK, confers
cell survival by blocking translation elongation (see Chap.12). In human medul-
loblastoma and glioblastoma, expression of eEF2K correlated with OS (Leprivier
etal. 2013). While the role of elongation in brain tumors is generally unknown, this
report provides the first evidence that this step of translation might influence brain
tumor biology.
The eIF4F complex plays an important role in mediating tumorigenic effects in

malignant brain tumors. Within this trimeric complex, the eIF4E proto-oncogene is
overexpressed and activated via phosphorylation, and by inhibition of 4E-BPs via
the activated mTOR. Many clinicopathologic correlates regarding these proteins
exist in relation to various types of brain cancer. However, first attempts to target
mTOR that used rapalogs have largely been unsuccessful at treating brain tumors,
likely due to feedback activation loops and the failure of these compounds to fully
inhibit the critical translational target 4E-BP. In contrast, second-generation mTOR
inhibitors and PI3K/mTOR inhibitors show greater promise in targeting translation
in brain tumors. Importantly, strategies to directly target eIF4F, eIF4E and 4E-BPs
are being developed and used in other cancers (as presented in this book). Their use
might prove more effective than indirect targeting of translation factors via complex
signaling networks that are prone to feedback activation and induction of resistance.
The PDCD4 tumor suppressor is also strongly implicated in gliomagenesis,
through downregulation by miR-21. This hub of translational control also repre-
sents a promising prognostic, diagnostic and therapeutic target. The role of other
components of the translation machinery in brain cancers is elusive due to limited
research in the area. However, preliminary reports suggest that further studies of
eIF2, subunits of eIF3, DHX29 and the elongation apparatus can provide valuable
information regarding the pathobiology of brain tumors.
Further research is required to underpin the molecular biology and biochemis-
try behind brain malignancies and their deadliest form, glioblastoma. Nevertheless,
targeting eIF4E, 4E-BPs and PDCD4 in brain tumors represent promising strategies
that, paired up with the use of these proteins as biomarkers, might improve survival
of patients with brain tumors.
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Chapter 25
Head and Neck Cancers
Cherie-Ann O. Nathan, Oleksandr Ekshyyan and Arunkumar Anandharaj
Contents
25.2eIF4E and Its Regulation 500
25.3Other Translation Factors 502
25.4Emerging Therapies Targeting Translation and Its Regulation 502
25.5Translation Machinery in Therapeutic Resistance and Radioresistance 504
25.6Components of Translation Machinery as Biomarkers 505
References 507
Abstract Recent evidence suggests that dysregulated translation and its control
significantly contribute to the etiology and pathogenesis of the head and neck can-
cers, specifically to that of squamous cell carcinoma (HNSCC). eIF4E is one of the
most studied components of the translation machinery implicated in the develop-
ment and progression of HNSCC. It appears that dysregulation of eIF4E levels and
activity, namely by the PI3K/AKT/mTOR pathway, plays an important role in the
etiology and pathogenesis of HNSCC and correlates with clinical outcomes. In this
chapter, we will discuss the role of eIF4E and some other translation factors as they
relate to the biology and treatment of HNSCC.
25.1Introduction
Head and neck malignancy represents the fifth most common cancer worldwide
(Balfour etal. 2009). However, head and neck cancer is not a single entity, but a
heterogeneous group of neoplasms encompassing a wide range of malignant tumors
that can originate in or around the organs of the upper aerodigestive tract, salivary
C.-A.O.Nathan() O.Ekshyyan A.Anandharaj

Department of Otolaryngology, Head and Neck Surgery and Feist-Weiller Cancer Center,
Louisiana State University Health Sciences CenterShreveport, Shreveport, LA, USA
e-mail: cnatha@lsuhsc.edu
500 C.-A. O. Nathan et al.
and thyroid (often classified separately) glands, skull base, paranasal sinuses, skin
and lymphatics of the head and neck. In the head and neck region, the most common
neoplastic lesions observed arise in squamous epithelium (Balfour etal. 2009).
More than 90% of head and neck cancers are squamous cell carcinomas (Marur and
Forastiere 2008), which will be the focus of this chapter.
HNSCCs account for about 34% of all cancers (Jemal etal. 2003; Balfour etal.
2009). Over 40,000 new cases of head and neck cancer are diagnosed annually in
the United States and there are over 500,000 new cases worldwide (Jemal etal.
2003; Balfour etal. 2009). HNSCC evolves through a multistep carcinogenesis pro-
cess, which is commonly attributed to the abuse of tobacco and alcohol. HPVs have
been strongly implicated as causative agents in a subset of HNSCC, predominantly
oropharyngeal cancer (Balfour etal. 2009).
HNSCC remains a challenge because of the high locoregional recurrence rates,
>50% in advanced-stage disease (Argiris etal. 2008). Numerous advances were
made in the diagnosis and treatment of this disease over the last three decades. How-
ever, despite multifaceted approaches involving chemotherapy, radiotherapy and
reconstructive surgery, only modest improvements in patient outcomes have been
achieved. Treatment failures often result from locoregional recurrence, metastasis
and second primaries (Licitra etal. 2008). Detailed elucidation of the molecular
aberrations in tobacco-induced and HPV-associated HNSCC, coupled with the use
of novel targeted therapies are expected to lead to major improvements in treatment
of this devastating disease (Balfour etal. 2009).
25.2eIF4E and Its Regulation
Research over the past decade has identified the critical role that the PI3K/AKT/
mTOR signaling pathway (mTOR pathway) plays in the pathogenesis of HNSCC
(Le Tourneau and Siu 2008; Molinolo etal. 2009; Liao etal. 2011). This pathway
has been reported to be dysregulated in as many as 99% of HNSCCs (Molinolo
etal. 2007, 2009; Clark etal. 2010). Activity of the mTOR pathway is regulat-
ed by nutrients and growth factors, but, in malignancies such as head and neck
carcinomas, can also be altered by mutations of various genes, that either control
the pathway or serve as its integral hub (such as MET, EGFR, PIK3CA, PTEN and
AKT2) (Rothenberg and Ellisen 2012; Loyo etal. 2013; Psyrri etal. 2013). Impor-
tantly for our discussion, the mTOR pathway converges on the translation machin-
ery, which in turn likely serves a direct driver of tumorigenesis by this signaling
cascade (see Chaps.15 and 16).
One of the downstream effectors of the mTOR pathway is the translation ini-
tiation factor eIF4E that facilitates translation via binding to the cap structure of
mRNA (see Chap.2). As described elsewhere in this book, various studies pro-
vide compelling evidence regarding the importance of eIF4E in cancer biology.
Overexpression of eIF4E leads to malignant transformation as evidenced by forma-
tion of transformed foci on a monolayer of untransformed cells, anchorage-inde-
pendent growth in soft agar and tumor formation in nude mice (De Benedetti and
25 Head and Neck Cancers 501
Rhoads 1990; Lazaris-Karatzas etal. 1990; Goldson etal. 2007). eIF4E appears to
selectively facilitate translation of mRNAs encoding proteins, such as VEGF, FGF2,
oncoprotein c-MYC, cyclin D1 and many others, which are involved in malignant
transformation, angiogenesis, invasion and metastasis (Zimmer etal. 2000). In ad-
dition, eIF4E might promote export of mRNAs that contain the eIF4E sensitivity
element within their 3 UTRs, such as cyclin D1, c-MYC, ODC and an oncogenic
serine/threonine kinase PIM1 (Culjkovic etal. 2007). Increased expression of these
proteins is reported in head and neck cancers and has a clinical significance. For
example, overexpression of cyclin D1 is associated with poor prognosis in head and
neck cancers (Yu etal. 2005; Hong etal. 2011). ODC activity was found to be sig-
nificantly elevated in tumor tissue samples from HNSCC patients, as compared to
adjacent normal mucosa (Weiss etal. 1992). Increased translation of antiapoptotic
PIM1 was demonstrated in HNSCC (Beier etal. 2007; Chiang etal. 2006). In other
cancer models, overexpression of these proteins has been linked to the increased
levels of expression or activity of eIF4E (Kleiner etal. 2009; Topisirovic etal. 2005;
Hoover etal. 1997).
It has been reported that the eIF4E protein is indeed overexpressed (as detected
by IHC and Western blot analysis) in tumors that originate in various head and neck
siteslarynx, esophagus, hypopharynx, pharynx, nasopharynx, tongue, oral floor
and tonsils (Franklin etal. 1999; Nathan etal. 2000; Salehi and Mashayekhi 2006;
Sorrells etal. 1999b; Wu etal. 2013). However, both head and neck tumor tissues
and adjacent normal epithelium at the tumor margin were shown to overexpress
eIF4E (Sorrells etal. 1999a). Upregulation of the eIF4E protein via eIF4E gene am-
plification is one of the early events in the tumorigenesis of HNSCC. Interestingly,
the degree of gene amplification and eIF4E protein expression increases from the
tumor-free margins to the core of the tumor (Sorrells etal. 1999b). eIF4E gene am-
plification and protein expression appear to be significantly higher in invasive carci-
nomas of head and neck when compared to normal tissues or benign tumors. In some
studies, malignant HNSCCs demonstrate a four-fold eIF4E gene amplification and
15-fold increase in protein expression compared to the normal tissue (Haydon etal.
2000). Hence, the degree of eIF4E gene amplification and resulting protein overex-
pression might correlate with progression of the disease to a malignant phenotype
(Haydon etal. 2000; Sorrells etal. 1999b). eIF4E was found to be overexpressed in
over 98% of HNSCC tumors (Franklin etal. 1999; Nathan etal. 1999, 2002).
Of a substantial clinical importance, increasing levels of eIF4E expression in
tumor-free surgical margins appear to negatively correlate with the disease-free in-
terval in HNSCC (Franklin etal. 1999; Nathan etal. 1999, 2002). About half of the
HNSCC cases that showed overexpression of eIF4E in histologically tumor-free
margins had significantly increased risk of local-regional recurrence when com-
pared to patients with eIF4E-negative tumor margins. eIF4E-positive but surgically
negative margins (those in which the basal cell layer expresses eIF4E as determined
by IHC) had significantly higher levels of AKT kinase activity, higher expression
of phospho-mTOR (Ser2448) and phospho-4E-BP1 (Thr70) than eIF4E- and surgi-
cally negative margins (Nathan etal. 2004a, b). Thus, levels of eIF4E expression
and activity at the surgical margins might serve as an important prognosticator of
disease recurrence in HNSCC.
As described in other chapters, eIF4E is controlled by various signaling cascades.

eIF4E activity is controlled by the PI3K/AKT/mTOR and MAPK pathways (De
Luca etal. 2012). The latter pathway alters eIF4E activity via its phosphorylation
on a conserved serine 209 by two MAPK-interacting kinases, MNK1 and MNK2
(see Chap.17). Interestingly, MNK1/2 do not seem to be necessary for the activity
of eIF4E in normal cells but are important for the tumor growth (Ueda etal. 2004,
2010). Constitutive activation of MNK promotes tumorigenesis in a manner similar
to eIF4E overexpression suggesting that the phosphorylation of eIF4E by MNK is
critical for the oncogenic activity of eIF4E (Topisirovic etal. 2004; Wendel etal.
2007). Indirect evidence suggests that the MAPK pathway is involved in the biology
of HNSCC via eIF4E. HNSCC cell lines have higher levels of phospho-MNK1 com-
pared to normal oral keratinocytes (Chakravarti etal. 2010). Nutraceutical curcumin,
that demonstrates anticancer activity, suppresses phosphorylation of MNK1, eIF4E
and 4E-BP1 causing an inhibition of the cap-dependent translation and expression of
cell growth-promoting proteins in these cells (Chakravarti etal. 2010).
Recently, HPV infection has been shown to be associated with head and neck
cancers (Syrjnen 2010; Psyrri etal. 2011, 2012; Rautava and Syrjnen 2012).
HPV has been detected in 25% of head and neck cancers overall and 5070% of
oropharyngeal carcinomas (Sturgis and Cinciripini 2007). Interestingly expression
of p16, a marker for HPV infection, showed a significant correlation with eIF4E
phosphorylation on Ser209 in tonsillar squamous cell carcinoma (Fury etal. 2011),
suggesting a potential link between MNK-mediated eIF4E phosphorylation and
HPV infection in head and neck cancers.
25.3Other Translation Factors
It appears that other components of the translation initiation machinery participate

in the development and progression of head and neck cancers. For example, the ma-
jority of HNSCCs demonstrate reduced mRNA and protein expression of PDCD4, a
tumor suppressor and a binding partner of eIF4A (Reis etal. 2010; Wang and Zhang
2012). This could be a result of PDCD4 gene deletion (Scapoli etal. 2011) or down-
regulation of PDCD4 expression by miR-21 (Reis etal. 2010). A matrigel invasion
assay demonstrated that PDCD4 expression suppressed invasion of oral carcinoma
cells, while siRNA-mediated suppression of PDCD4 correlated with increased cell
invasiveness (Reis etal. 2010).
25.4Emerging Therapies Targeting Translation

and Its Regulation
mTOR-targeted therapy using various rapalogs underwent extensive preclinical

testing in HNSCC and demonstrated promising results characterized by significant
effects on mTOR signaling (as evidenced by decreased phosphorylation of rpS6 and
4E-BP1 and reduced protein expression of angiogenic markers, such as VEGF-A

and FGF2) (Amornphimoltham etal. 2005; Nathan etal. 2007; Czerninski etal.
2009; Ekshyyan etal. 2009). The effects of treatment with the rapalog temsirolimus
on the key components of the PI3K/AKT/mTOR pathway (phospho-AKT, rpS6,
phospho-4E-BP1) were evaluated in an exploratory clinical trial of temsirolimus
in patients with advanced stage HNSCC (Ekshyyan etal. 2010). The study showed
that administration of three 25mg doses of temsirolimus significantly decreased
phosphorylation of two key regulators of translation, rpS6 and 4E-BP1, in the post-
treatment versus pretreatment tumor samples (Ekshyyan etal. 2010). Treatment
with mTOR inhibitor everolimus in a phase I clinical trial induced a dose- and
schedule-dependent inhibition of the mTOR pathway with a near complete inhi-
bition of phospho-rpS6 and phospho-eIF4G at 10 mg/day and 50 mg/week in
advanced solid tumors of multiple sites (Tabernero etal. 2008). Thus, these studies
provide support to the concept that inhibiting the mTOR pathway might prove to be
useful in cancer therapy in general and in HNSCC in particular.
Rapamycin (sirolimus) and its analogs, everolimus (also known as RAD001 or
Afinitor), temsirolimus (also known as CCI-779 and Torisel) and ridaforolimus
(also known as AP23573 or MK-8669, formerly deferolimus) are allosteric inhibi-
tors of mTORC1, but not of mTORC2. Since mTORC2 is resistant to rapalogs,
feedback activation of AKT signaling by mTORC2 is often observed with rapalogs
(Sun etal. 2005; OReilly etal. 2006; Wan etal. 2007). Second-generation mTOR
inhibitors target the catalytic activity of mTOR. These inhibitors are ATP competi-
tors and have high specificity for the mTOR serine/threonine kinase activity in-
hibiting both mTORC1 and mTORC2. Some of these inhibitors also target PI3K
catalytic activity. The added benefits of the second-generation mTOR inhibitors are
an improved inhibition of mTORC1 and prevention of the feedback activation of
PI3K/AKT signaling. Second-generation mTOR inhibitors displayed encouraging
antitumor activity in preclinical models of HNSCC (Erlich etal. 2012). In head
and neck cancer preclinical models, a combination of second-generation mTOR
inhibitors with anti-EGFR treatment resulted in synergistic antiproliferative effects
in vitro and significantly enhanced antitumor effects in vivo (Cassell etal. 2012).
TORC1/TORC2 inhibitor OSI-027/ASP4876 reduced phosphorylation of mTOR
downstream targets 4E-BP1 and S6K in a dose-dependent manner. Furthermore,
combined inhibition of EGFR and mTORC1/2 in HNSCC preclinical models result-
ed in enhanced inhibition of phosphorylation of the downstream targets of the path-
way, such as phospho-4E-BP1, phospho-p70S6K and phospho-PRAS40 (Cassell
etal. 2012).
mTORC1 inhibitors rapamycin, temsirolimus and everolimus are being evalu-
ated in several phase II clinical trials as adjuvant therapy for patients with head
and neck cancer (clinicaltrials.gov: NCT01111058, NCT01172769, NCT01195922;
accessed 2/11/2013). Various combinations of mTORC1 inhibitors with chemo-
therapeutic agents (cisplatin, carboplatin, paclitaxel, docetaxel), other molecular
targeted agents (cetuximab, erlotinib) and radiotherapy are being explored. The
results of a phase 1 study combining everolimus with weekly cisplatin and intensi-
ty-modulated radiotherapy in cancer patients (of which 7/13 were HNSCC patients)
showed that everolimus was well tolerated at a dose of 5mg/day (Fury etal. 2013).
Interestingly, increased expression of eIF4E was detected by IHC in histologically

negative margins in all four available resected HNSCC specimens. Median follow-
up was 19.4 months among the entire study population (N=13) and two patients
had recurred. Importantly, both patients who experienced disease recurrence had
elevated eIF4E expression in histologically cancer-free surgical margin (Fury etal.
2013).
Head and neck cancer cells have elevated eIF4E levels, which enhance transla-
tion of a subset of mRNAs (Nathan etal. 1999; De Benedetti and Graff 2004). In
general, these mRNAs usually contain long 5 UTR regions with secondary struc-
tures. Hence besides mTOR inhibitors other methods of targeting this pathway have
been envisioned. These methods included splicing of a long 5 UTR upstream of a
thymidine kinase suicide gene which enhanced its translation within cells over-
expressing eIF4E (Siegele etal. 2008). Such eIF4E-targeted suicide gene therapy
resulted in significantly improved DFS and OS in a mouse minimal residual soft-
tissue HNSCC metastasis model (Siegele etal. 2008). In addition, treatment of the
HNSCC cells with eIF4E-specific ASOs reduced expression of eIF4E, as well as
lowered protein expression of its downstream targets, such as VEGF and FGF2 and
FGF2 (DeFatta etal. 2000). Antisense eIF4E treatment attenuated the oncogenic
properties of HNSCC cells, as demonstrated by decreased growth in soft agar, in-
creased contact inhibition and loss of tumorigenicity in nude mice (DeFatta etal.
2000). Thus, while in its infancy, development of direct eIF4E targeting strategies
represent a promising clinical strategy for HNSCC.
25.5Translation Machinery in Therapeutic Resistance

and Radioresistance
Surgery followed by radiotherapy is the main treatment modality for head and
neck cancers (Haddad and Shin 2008). Chemotherapy is often administered to
act as a radiosensitizer that helps increase survival in locally advanced disease
(Cohen etal. 2004). Using HNSCC cell lines with different levels of eIF4E expres-
sion, it was demonstrated that expression levels of eIF4E positively correlate with
radioresistance (Nathan etal. 2004b). Solid tumors that are more than 1mm3 in size
commonly have regions of hypoxia that result from the rapid growth of tumors cells
(Rockwell 1997). There are numerous studies demonstrating that hypoxic cancer
cells are resistant to radiotherapy due to upregulation of HIF-1 that subsequent-
ly induces expression of VEGF and FGF2 (Artman etal. 2010; Hosokawa etal.
2012; Moeller etal. 2005; Sasabe etal. 2007; Semenza 2004; Schwartz etal. 2011).
Upregulated expression of VEGF and FGF2 promotes survival of cancer cells and
adjacent endothelial cells in various types of cancer, including HNSCC (Moeller
etal. 2005; Semenza 2004). It has been established that translation initiation of
HIF-1, VEGF and FGF2 is controlled by the eIF4F complex (Harada etal. 2009;
Konicek etal. 2008). siRNA-mediated silencing of eIF4E in head and neck cancer
cell line UMSCC22B led to a decrease in the expression of cyclin D1, FGF2 and
VEGF and inhibited cell growth. Interestingly, a combination of siRNA against

eIF4E and cisplatin treatment showed synergistic inhibition of colony formation
suggesting that overexpression of eIF4E in HNSCC can contribute to cellular re-
sistance to chemotherapy (Oridate etal. 2005). Thus targeting eIF4E directly or
indirectly might improve effects of currently available therapeutic modalities in
HNSCC.
The overexpression of eIF3a, one of the core subunits of the eIF3 complex, has
been reported to lead to malignant transformation in some cellular models (Zhang
etal. 2007). This subunit is found to be overexpressed in squamous cell carcinoma
of the oral cavity (Spilka etal. 2012). Although eIF3a demonstrated oncogenic
properties in vitro (Zhang etal. 2007; Saletta etal. 2010), surprisingly high expres-
sion of this protein correlates with a significantly better prognosis in oral squamous
cell carcinoma patients (Spilka etal. 2012). There was no correlation between eIF3a
expression status and therapy response rates in patients receiving radiotherapy, but
the patients demonstrating high expression of this factor responded significantly
better to platinum-based chemotherapy (Spilka etal. 2012). This effect can possibly
be explained by eIF3a involvement in the modulation of DNA repair. High expres-
sion of eIF3a was shown to lead to a reduced DNA repair and increased apoptosis
(Liu etal. 2011; Spilka etal. 2012).
Phosphorylation of eIF2 is upregulated during endoplasmic reticulum stress.
Agents that induce eIF2 phosphorylation, such as proteasome inhibitor PS-341
(Bortezomib) or histone deacetylase inhibitor suberoylanilide hydroxamic acid
(SAHA), can enhance cisplatin sensitivity of HNSCC and may potentially be used
to overcome cisplatin resistance in head and neck cancer (Fribley etal. 2006; Suzuki
etal. 2009).
Studies elucidating the role of eIF4E, eIF2 and eIF3 in the sensitivity of HNSCC
to radiotherapy and chemotherapy are limited.
25.6Components of Translation Machinery

as Biomarkers
One of the earliest recognized biomarkers predicting recurrence was overexpres-

sion of the translation initiation factor eIF4E in histologically tumor-free surgical
margins of HNSCC patients (Nathan etal. 1997). Several studies observed that
eIF4E is overexpressed (sometimes as much as 24-fold) in almost all of HNSCC
samples versus normal head and neck tissue specimens (Nathan etal. 2002; Nathan
etal. 1999; Nathan etal. 1997). As previously mentioned, elevated levels of eIF4E
in tumor margins that are devoid of microscopic tumor are an independent predic-
tor of local recurrence (Nathan etal. 1997). Whereas histological grading often
failed to predict prognosis, eIF4E levels at the margins served as a marker of lo-
cal recurrence. In HNSCC tumor-free margins the overexpression of eIF4E was an
independent risk factor for local recurrence. Patients with eIF4E-positive margins
had a sevenfold increased risk of recurrence compared to patients with negative
eIF4E margins, independent of tumor size, nodal status, histological grade and tu-
mor site (Nathan etal. 1999). eIF4E expression was significantly associated with
the increasing grades of dysplasia but recurrence correlated with eIF4E expression
and not the grade of dysplasia in the margins (Nathan etal. 1999). This is important
in head and neck where due to a long-term exposure of tissue to a carcinogen (such
as tobacco) margins are often dysplastic. In addition to eIF4E, its regulators could
serve as HNSCC disease markers. For example, the elevated ratio of eIF4E mRNA
to 4E-BP1 mRNA in HNSCC patient tumor samples was also shown to correlate
significantly with increased disease recurrence (Sunavala-Dossabhoy etal. 2011).
Importantly, it was shown in the preclinical setting that the antiproliferative effect
of mTOR inhibition can be significantly affected by eIF4E/4E-BP ratio. An increase
in the eIF4E/4E-BP ratio, either due to overexpression of eIF4E or loss of 4E-BPs,
dramatically limits the sensitivity of cancer cells to mTOR-targeted treatment (Alain
etal. 2012). Therefore it is critically important to evaluate the eIF4E/4E-BP ratio as
a predictive marker of response to mTOR inhibitors in clinical trials.
In order to identify reliable biomarkers for clinical application in the manage-
ment of head and neck cancer patients, expression of proteins within the PI3K/AKT/
mTOR pathway was studied in tissue samples of HNSCC patients using IHC and
Western blot analysis. Western blot analysis demonstrated that expression of phos-
pho-AKT and phospho-mTOR was significantly higher in cancer patient tumors
compared to noncancerous oral mucosa samples (Clark etal. 2010). Phospho-mTOR
and phospho-4E-BP1 expression (determined by IHC) were found to be the most
promising biomarkers (Clark etal. 2010). At that, phospho-mTOR expression was
81.9% sensitive and 100% specific in differentiating HNSCC from noncancerous
mucosa, whereas phopsho-4E-BP1 expression was 50% sensitive and 95.5% spe-
cific. Furthermore, in agreement with earlier studies by our group, expression of
phospho-mTOR and phospho-4E-BP1 detected by Western blot analysis was found
to be higher in patient junctional zones (advancing edge of the tumor) compared to
the main tumor mass. Junctional zones are the more active, rapidly dividing regions
of the tumor with highly increased expression of the components of the transla-
tion machinery, which might represent the best regions for the biomarker analysis
of the tumor (Clark etal. 2010). Among other biomarkers, various components of
the translation machinery and its regulators (such as mTOR, S6K1, rpS6, 4E-BP1,
eIF4E and eIF4G) are being evaluated in clinical trials of mTOR inhibitors for pa-
tient stratification as predictive markers of sensitivity to novel therapeutics (Raza
etal. 2011; Nguyen etal. 2012; Bozec etal. 2013; Fury etal. 2013).
Studies during the last two decades clearly demonstrated that aberrant translation
and its control is a hallmark of head neck squamous cell carcinoma with virtually
all cases demonstrating PI3K/AKT/mTOR pathway-driven upregulation of eIF4E
expression and activity. Regulation of translation is a very complex process and the
exact mechanism of its control is yet to be understood. Nevertheless, it is becom-

ing increasingly clear that eIF4E is a key proto-oncogene that has a critical role
in head and neck cancers. Targeting this protein and its regulatory pathways can
potentially improve survival and be employed as therapy for the high-risk patients
with HNSCC. Furthermore, as discussed in this book, other translation factors are
emerging as important determinants in cancer biology. Further studies of the com-
ponents of the translation machinery, such as eIF4E, and their regulation in head
and neck cancers should prove to be promising.
Acknowledgments This work was supported by the National Institute of Health Grant
R01CA102363 (to C.O.N.).
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Chapter 26
Breast Cancer
Armen Parsyan, Ana Maria Gonzalez-Angulo, Dimitrios Zardavas,

Martine Piccart and Sarkis Meterissian
Contents
26.2eIF4E as a Proto-oncogene in Breast Cancer 516
26.3Expression Levels of eIF4E in Breast Cancer 519
26.4Expression Level of eIF4E as a Clinical Marker 520
26.5Introduction to Regulation of eIF4E Activity and 4E-BPs in Breast Cancer 521
26.6Signaling Pathways Controlling 4E-BPs and eIF4E Activity in Breast Cancer 522
26.7eIF4E and Therapeutic Resistance 523
26.8mTOR, Translation and Breast Cancer 524
26.8.1Combination with Endocrine Therapy 525
26.8.2Combination with HER2 or EGFR/HER2 Inhibitors 525
26.8.3Combination with Conventional Chemotherapy 526
26.8.4Other Strategies of Targeting the mTOR/4E-BPs/eIF4E Axis 526
26.8.5Translation-Related Aspects of Sensitivity and Resistance
to mTOR Inhibitors 527
26.9Direct Targeting of eIF4E and eIF4F 534
26.10eIF4G as a Marker of Highly Proliferative and Metastatic Breast Cancer 535
26.11eIF4A and Its Regulatory Protein Tumor Suppressor PDCD4 536
26.12The PDCD4/eIF4A Axis in Chemoresistance and Therapeutic Targeting 537
26.13The eIF3 Complex and Its Components in Breast Cancers 538
26.14eIF2, Its Regulation and Targeting in Breast Cancer 539
26.15Other Translation Initiation Factors 540
26.16Elongation and Termination 540
References 542
A.M.Gonzalez-Angulo
Department of Breast Medical Oncology and Department of Systems Biology,
The University of Texas MD Anderson Cancer Center, Houston, TX, USA
D.Zardavas M.Piccart
Institut Jules Bordet, Universit Libre de Bruxelles (ULB), Brussels, Belgium
S.Meterissian
Abstract Breast cancer remains one of the leading causes of cancer morbidity and
mortality. Despite significant advances in treatment of breast cancer a substantial
proportion of women affected by this disease succumb to it. Survival of patients
with advanced disease, chemoresistant tumors or a suboptimal response to endo-
crine therapy is significantly shortened. Hence, further understanding of disease
pathogenesis is required to enhance the arsenal of approaches to cure this deadly ail-
ment. Recent advances in biochemistry, molecular cell biology and cancer research
highlighted the importance of dysregulation of protein synthesis, translation, in the
development and progression of tumors. This dysregulation appears to take place at
an early stage of translation, called translation initiation, that is a highly controlled
and rate-limiting step of the protein synthesis. In this chapter we summarize decades
of knowledge accumulated in regards to the role of translation and its regulation
in the development and progression of breast cancer. We then extensively discuss
applications of this knowledge in diagnosis and treatment of breast cancer.
26.1Introduction
Breast cancer is a leading cause of mortality and morbidity in developed and devel-
oping countries (Benson and Jatoi 2012; CCS 2013; CDC/NCI 2013; CRUK 2010a;
Ferlay etal. 2008, 2010). It is the most common cancer diagnosed among women
worldwide, and in countries like Canada (CCS 2013), the UK (CRUK 2010a) and
the USA (CDC/NCI 2013). In recent years, an estimated 1.4million women across
the world are being annually diagnosed with breast cancer (Ferlay etal. 2008, 2010).
Breast cancer is the most common cause of cancer-related mortality among women
worldwide (Ferlay etal. 2008) and the second cause of cancer-related mortality in
industrialized countries, such as Canada, the UK and the USA (CCS 2013; CDC/
NCI 2013; CRUK 2010b). Despite significant advances in the diagnosis, treatment
and prevention of breast cancer, its incidence continues to increase (Benson and
Jatoi 2012; DeSantis etal. 2011). Thus, further understanding of the etiology and
pathogenesis of breast cancer is imperative to develop better preventive and cura-
tive modalities.
The current range of strategies to tackle breast cancer include preventive mo-
dalities (such as mammographic screening); surgical treatment (which remains the
mainstay of therapy for early stage disease); neoadjuvant or adjuvant chemothera-
peutic approaches (including, doxorubicin, epirubicin, taxanes, cyclophosphamide
and 5-fluorouracil (5-FU)); radiotherapy, and endocrine therapy with selective estro-
gen receptor modulators (such as tamoxifen, raloxifene and toremifene), aromatase
inhibitors (such as letrozole, anastrozole and, exemestane) and fulvestrant (Cardoso
etal. 2012; Carlson etal. 2012; Theriault etal. 2013). Targeted therapies incorpo-
rate anti-HER2 receptor antibodies trastuzumab (Herceptin) and pertuzumab; dual
EGFR (HER1) and HER2 kinase inhibitor lapatinib; mTOR inhibitors (everolimus)
and others (see Cardoso etal. 2012; Mohamed etal. 2013).
Substantial advances in the molecular understanding of breast cancer have been
made in recent years, leading to novel clinical approaches to diagnose and treat this
26 Breast Cancer 515
disease based on molecular information (see reviews Cadoo etal. 2013; Eroles etal.
2012). In fact, breast cancer can be considered as a vanguard in cancer research
from which lessons are to be learned and applied to other cancers. While describing
the complexities of molecular milieu of breast cancers is beyond the objectives of
this chapter, it is worth mentioning a few molecular events that lead to and sustain
breast cancers, and that are relevant for our presentation.
The term breast cancer refers to a range of neoplastic conditions. Important for
this discussion is to delineate two major histological types of breast cancer: ductal
(that arises from ductal components of the breast tissue) and lobular (that arises
from lobules). These can in turn be divided into in situ carcinomas (DCIS and LCIS
respectively) and invasive carcinomas. However, alongside its histological features,
more and more attention in the clinical management of breast cancer is being paid
to the molecular profile of specific cancers. This includes estrogen/progesterone
receptor (ER/PR) status that is positive in almost 7080% of breast cancers and that
bears prognostic and therapeutic values (Vollenweider-Zerargui etal. 1986). Almost
30 years ago, one of the first pieces of evidence supporting the clinical relevance
of specific molecular alterations in cancer was reported, showing that the human
epidermal growth factor receptor 2 (HER2/neu) is amplified in almost a quarter of
breast cancer patients, correlating with higher relapse rates and worsened survival
(Slamon et al. 1987). HER2/neu is a member of the ERBB-like oncogene family re-
lated to the EGFR/ERBB family. Currently, the ER/PR, HER2 and Ki-67 (a nuclear
protein that serves as a marker of cell proliferation) status is used to classify breast
cancers for prognostication and therapeutic purposes (Goldhirsch etal. 2011). Other
molecular markers and multigene signature assays have been proposed, with some
of them, such as the Oncotype DX assay, being used in the clinical management of
breast cancer patients (Colombo etal. 2011; Cronin etal. 2004; Haibe-Kains etal.
2008; Paik etal. 2004; Prat etal. 2010).
To further emphasize the molecular complexities of breast cancer, it is worth
noting that this disease can be genetically categorized to familial (hereditary) and
non-familial (sporadic) breast cancer. High-risk hereditary breast cancer occurs in
approximately 10% of the cases and another 25% of patients present with a fam-
ily history (likely representing low-risk genes) (Margolin etal. 2006). Hereditary
genetic alterations that lead to breast cancer include breast cancer, early onset 1 and
2 genes (BRCA1/2) and genetic conditions, such as Cowden (PTEN), Li-Fraumeni
(TP53), Peutz-Jeghers (LKB1/STK11), Bloom (BLM) and Lynch (MSH2, MLH1)
syndromes; hereditary diffuse gastric cancer (CDH1); ataxia teleangiectasia (ATM);
Fanconis anemia (BRCA2) and others (reviewed in Bogdanova etal. 2013; Lynch
etal. 2008).
Within this complexity an important observation ensues that some of the ge-
netic aberrations and molecular patterns (involving p53, PIK3CA, AKT, PTEN and
LKB1) in breast cancer are associated with oncogenic signaling by the PI3K/AKT/
mTOR and MAPK pathways. Within the MAPK pathway, the RAS/RAF/MEK/
ERK cascade appears to play an important role in breast cancer. Most notably,
these pathways are activated by RTKs, including EGFR (HER1), HER2, IGF-1R,
FGF receptor (FGFR) and others (Lo etal. 2006; Polyak and Metzger Filho 2012).
Some of the components that either constitute a part of the signaling pathways or
intrinsically regulate them are often mutated in breast cancers. Those include TP53
(mutated in 37% of breast cancers), PIK3CA (36%), MAP3K1 (8%), PTEN (3%)
and AKT1 (2%) (Cancer Genome Atlas 2012). Such molecular aberrations lead to
subsequent activation of oncogenic signaling in breast cancer.
As described in this book, these pathways converge on regulation of the transla-
tion machinery. For example, the PI3K/AKT/mTOR cascade, once activated, results
in phosphorylation of inhibitory eIF4E-binding proteins, 4E-BPs. Phosphorylation
of 4E-BPs leads to their dissociation from eIF4E and activation of cap-dependent
translation (see Chaps.4 and 15). MAPK signaling results in control of eIF4E by di-
rect phosphorylation (see Chaps.4 and 17). Notably, these pathways regulate other
aspects of translation. However, the key hub appears to be represented by proto-
oncogene eIF4E. Other components of the translation machinery are involved in
breast cancer (see Table26.1). The following discussion will dissect the role of the
translation machinery and its control in breast cancer.
26.2eIF4E as a Proto-oncogene in Breast Cancer
eIF4E is a key component of the translation initiation machinery and a proto-onco-

gene (see Chap.4). It has been proposed to be a central regulator of tumor forma-
tion, growth, invasion and metastasis and to play a key role in the development of
various types of malignancies (Lazaris-Karatzas etal. 1990, 1992; Zimmer etal.
2000). The contribution of eIF4E to malignant transformation and progression has
been studied and reported over the past decades. eIF4E overexpression has been
demonstrated in various cancers including breast (see Part IV).
eIF4E appears to be a fundamental factor for the development and progression of
breast malignancy. Overexpression of eIF4E in primary HMECs imparts cells with
proliferative and survival autonomy, stimulating protumorigenic states and impair-
ing apoptosis (Avdulov etal. 2004; Larsson etal. 2007). On the other hand, deple-
tion of eIF4E impairs cancerous properties of different breast cancer cell subtypes,
including ER/PR/HER2 triple-negative cells, and sensitizes them to the cytotoxic
effects of chemotherapeutic agents, such as cisplatin, doxorubicin, paclitaxel and
docetaxel (Dong etal. 2009; Nathan etal. 1997; Soni etal. 2008; Zhou etal. 2011).
eIF4E contributes to malignancy in general and breast cancer in particular by selec-
tively enabling translation of a limited pool of mRNAs that encode key proteins in-
volved in cellular growth, angiogenesis, survival and tumorigenesis, such as cyclin
D1, c-MYC and VEGF (DeFatta etal. 1999; Kleiner etal. 2009; Larsson etal. 2007;
Nasr etal. 2013; Scott etal. 1998b; Zhou etal. 2006). The levels of eIF4E in breast
cancer positively correlate with the levels of expression of these proteins (Byrnes
etal. 2006; Dong etal. 2009; Kleiner etal. 2009; Norton etal. 2004; Scott etal.
1998b; Yang etal. 2007; Zhou etal. 2006). As mentioned previously (see Chap.4),
the defining feature of these mRNAs is the complexity of their 5 UTRs. The more
structured they are, the more they require eIF4E for an efficient translation. Interest-
ingly, together with the aforementioned prosurvival and protumorigenic messages,
Table 26.1 Translation initiation factors implicated in breast cancer biology.
Translation factor Functiona Role in breast cancer (BC)
eIF1 Prepares 40S ribosome for mRNA loading eIF1 is involved in start codon selection. Little is known about involvement of
and promotes scanning with eIF1A eIF1 in the malignant process. Functional genetic screens with subsequent
evaluation of the clinical relevance of identified genes with respect to tumor
aggressiveness and tamoxifen resistance in ER-positive patients revealed that in
26 Breast Cancer
patients with advanced disease there was a significant association with clinical
benefit and PFS for eIF1 mRNA levels (van Agthoven etal. 2010).
eIF2 and its subunit eIF2 binds and recruits initiator tRNA to the eIF2 is a heterotrimeric protein that forms a ternary complex with GTP and the ini-
40S ribosome tiator Met-tRNA. The -subunit represents the main target of phosphorylation,
and, therefore, can be considered the regulatory subunit of eIF2. It is a down-
stream target of PKR and PERK that are implicated in mammary development
and proliferation. See text for more details.
eIF2B and its subunit A five-subunit GTP exchange factor for eIF2 This proteins is not studied well in cancer biology, however, there is a hypothesis
that helps to regenerate eIF2-TC that increased levels of eIF2B counteract inhibitory effects of increased eIF2
phosphorylation on translation in BC cells (Kim etal. 2000) and in cancer in
general (see Chap.9). A reduction of expression of the subunit of eIF2 leads
to marked decrease in BC cell growth rate in culture, colony formation in soft
agar, and tumor progression (Gallagher etal. 2008).
eIF3a; eIF3h Subunits of the eIF3 complex that has vari- Expression levels of eIF3a and eIF3h have been found elevated in BC. Decreasing
ous functions, including recruitment of eIF3a or eIF3h expression reverses BC cell malignant growth phenotype. See
mRNA to 40S text for more details.
eIF3e A subunit of eIF3 High tumor grade significantly correlates with an elevated cytoplasmic eIF3e level
in BC cells (Grzmil etal. 2010). It is required for BC cell invasion and prolifera-
tion (Grzmil etal. 2010), and is an important determinant of HIF-2 (hypoxia
inducible factor) dependent angiogenesis and cancer formation (Chen etal.
2007). It might also be important for developing chemoresistance to tamoxifen.
See text for more details.
eIF3f A subunit of eIF3 Decreased expression of eIF3f has been found in breast and other cancers, suggest-
ing that it might act as a negative regulator of translation and be associated with
ribosomal RNA degradation (Shi etal. 2006).
517
518
Translation factor Functiona Role in breast cancer (BC)

eIF4A A helicase component of the eIF4F complex Silvestrol and hippuristanol that inhibit eIF4A activity exhibit antiproliferative
effects in BC models and delay the onset of associated pulmonary metastasis
(Cencic etal. 2009; Nasr etal. 2013). The overexpression of oncogenic MUC1-
C in BC cells is inhibited by blocking eIF4A with silvestrol and CR-131-B (Jin
etal. 2013).
eIF4B Stimulates helicase activity of eIF4A There is only indirect evidence suggesting that eIF4B plays a role in BC. 14-3-3
is a tumor suppressor that is often lost in BCs acts as a regulator of mitotic
translation through its direct mitosis-specific binding to eIF4B (Wilker etal.
2007).
eIF4E Cap-binding component of the eIF4E Increased expression levels and activation of eIF4E are linked to the development
complex of BC. Control by 4E-BPs and phosphorylation leads to alterations of tumori-
genic potential. See text for more details.
eIF4G Scaffolding component of the eIF4F complex eIF4G overexpression is described in highly metastatic inflammatory BC and
advanced highly proliferative BC. See text for more details.
4E-BPs Bind to eIF4E and thus inactivate it in response 4E-BPs act as tumor suppressors. 4E-BPs activation and increased expression are
to mTOR signaling linked to the pathogenesis of BC. See text for more details.
PDCD4 Binds eIF4A and inhibits its function Decreased levels of PDCD4 are implicated in antiapoptotic effects and develop-
ment of chemoresistance. See text for more details.
a
See Part I and List of Abbreviations for details.
BC - breast cancer.
A. Parsyan et al.
a differential regulation of ER isoforms by 5 UTRs in cancer has been reported,

suggesting a role of eIF4E-dependent translational regulation of ER in breast cancer
(Smith etal. 2010).
26.3Expression Levels of eIF4E in Breast Cancer
eIF4E is overexpressed in breast cancer and its expression levels are almost 10
times higher in cancerous than in benign breast tissues and cells (Anthony etal.
1996; Byrnes etal. 2006; Kerekatte etal. 1995; Li etal. 1997, 1998; Norton etal.
2004; Sorrells etal. 1998). eIF4E overexpression in breast cancer can be due to
gene amplification and/or increase of eIF4E mRNA transcription (Anthony etal.
1996; Scott etal. 1998a; Sorrells etal. 1998, 1999). Indeed, in breast cancers, not
only the eIF4E protein but also mRNA levels are found elevated, in agreement with
gene amplification or transcriptional upregulation (Anthony etal. 1996; Pettersson
etal. 2011; Scott etal. 1998b; Sorrells etal. 1998). Transcription of eIF4E is con-
trolled by c-MYC, which, in turn, is regulated at the translational level by eIF4E
(see Chap.4). Elevated levels of c-MYC are reported in breast cancers and hence
could, at least in part, contribute to the observed increased expression of eIF4E
(Kleiner etal. 2009; Spandidos etal. 1989).
Increased levels of eIF4E protein expression appear to positively correlate with
the level of malignancy of the tumor and the type of the tumor. Compared to ad-
vanced stages of breast cancer, where the level of eIF4E expression can increase as
much as 50 times of that in nonmalignant cells, its expression in DCIS is on average
increased by 2.5 times (Li etal. 1997).
Overexpression of eIF4E within the tumor itself might not be homogeneous,
reflecting various levels of malignancy of the tumor cells in various parts of the
neoplasm. In intraductal carcinoma, the highest levels (average 17.4-fold) of the
eIF4E protein are found in the tumor core and decrease towards the periphery of
the tumor (DeFatta etal. 1999; Sorrells etal. 1999). The highest concentration of
eIF4E is found in islets of viable cells in the center of poorly vascularized DCIS,
where a hypoxic state exists (DeFatta etal. 1999). Hypoxia increases eIF4E expres-
sion, most likely giving cells a critical advantage to survive hypoxia and marking
transition toward the vascular phase of cancer progression (DeFatta etal. 1999)
(see Chap.20). Increase of eIF4E in hypoxia was linked to HIF-1 via HREs of the
promoter of the former (Yi etal. 2013).
In invasive ductal carcinoma, eIF4E was found to be markedly increased in
vascularized malignant ductules, whereas necrotic and avascular sites displayed
significantly lower levels, suggesting a role of eIF4E in the tumor vascularization
(Nathan etal. 1997). Increased expression of eIF4E in breast cancer likely plays
important etiologic and pathogenetic roles. For example, siRNA-mediated silencing
of eIF4E in MDA-MB-231 triple-negative breast cancer cells results in cell cycle
arrest, suppression of colony formation, inhibition of cell motility and sensitization
to chemotherapeutic treatment (Zhou etal. 2011). Similarly, silencing of eIF4E with
shRNA induces apoptosis of breast carcinoma cells and enhances their sensitivity to
cisplatin (Dong etal. 2009).
26.4Expression Level of eIF4E as a Clinical Marker
An increase in the level of eIF4E correlates with clinicopathologic determinants of

breast cancer. Its overexpression positively correlates with poorer clinical outcomes
and thus could be used as a prognostic marker (Byrnes etal. 2006; Flowers etal.
2009; Heikkinen etal. 2013; Holm etal. 2008; Li etal. 2002, 1997; McClusky etal.
2005; Zhou etal. 2006). First studies into levels of eIF4E and clinical outcomes
showed that patients with breast cancer who had a high level of eIF4E expression
in their tumors experienced higher recurrence rates and increased mortality when
compared to the group with a relatively low eIF4E expression level (Li etal. 1997).
Subsequent studies confirmed that patients whose breast cancer overexpresses
eIF4E, had significantly worse OS and DFS, higher cancer-related mortality and
higher rates of cancer recurrence than patients whose tumors had lower degrees of
eIF4E expression (Byrnes etal. 2006; Heikkinen etal. 2013; Holm etal. 2008; Li
etal. 1998; McClusky etal. 2005; Zhou etal. 2006). Similarly, patients with triple-
negative breast cancer, whose cancer expressed high levels of eIF4E, had greater
rates of cancer recurrence and cancer-related mortality than those expressing lower
levels (Flowers etal. 2009).
As high as a seven-fold increase in the relative risk for recurrence and cancer-
related death has been reported among patients with stage 1 to 3 breast cancer ex-
pressing high levels of eIF4E (Li etal. 2002). A high eIF4E expression level is a
factor of cancer recurrence independent of tumor size, tumor grade, nodal disease,
ER/PR status and menopausal status (Byrnes etal. 2006; Holm etal. 2008; Kleiner
etal. 2009; Li etal. 2002, 1998; McClusky etal. 2005).
Higher eIF4E expression appears to positively correlate with angiogenesis and
vascular invasion of breast cancer cells, as well as recurrence after neoadjuvant
chemotherapy (Hiller etal. 2009; Zhou etal. 2006). Breast cancer patients whose
tumors had lower degrees of eIF4E expression after neoadjuvant chemotherapy had
lower cancer recurrence compared to those who did not (Hiller etal. 2009). A more
recent study confirmed these findings in a large population of breast cancer pa-
tients. In 1085 IHC-stained breast tumors, a high level of eIF4E protein expression
was significantly associated with features of aggressive tumor phenotype (such as
advanced grade, negative hormone receptor status and high expression of Ki-67)
and worse cancer-specific survival (Heikkinen etal. 2013). In a multivariate analy-
sis a high eIF4E level was an independent prognostic factor and predicted survival
after treatment with anthracycline therapy. Thus eIF4E can be used as a prognostic
marker of breast cancer recurrence and survival, as well as a potential therapeutic
target (for review see (Meric-Bernstam 2008b)). Evaluation of eIF4E levels and
activity can be a useful tool in identifying patients at high risk of distant metastases
(Giusiano etal. 2011) and could be employed in stratifying DCIS lesions according
to their malignant potential (DeFatta etal. 1999).
26.5Introduction to Regulation of eIF4E Activity

and 4E-BPs in Breast Cancer
A pathogenic role of eIF4E in breast cancer is not only defined by the level of its
expression and abundance, but also by its activation. Activity of eIF4E is regulated
by its binding proteins 4E-BPs, which are controlled by the PI3K/AKT/mTOR sig-
naling pathway (see Chaps.4 and 15). It is also regulated by direct phosphorylation
by MNK 1 and 2 via the MAPK pathway (see Chaps.4 and 17). mTOR-dependent
phosphorylation of translational repressor 4E-BP1 (one of the main isoforms of
4E-BPs) leads to its dissociation from eIF4E and activation of the latter, thereby
causing an increase in the formation of the eIF4F complex and promotion of cap-
dependent translation. This activation has a specifically higher effect on structured
mRNAs that encode prosurvival, antiapoptotic, proangiogenesis and protumorigen-
ic signals. On the other hand, direct phosphorylation of eIF4E leads to a decreased
association of 4E-BPs with the former and, hence, its activation.
As discussed previously, overexpression of eIF4E in mammary cells stimulates a
pro-oncogenic state. However, expression of phosphorylation site mutant 4E-BP1,
which constitutively binds and inactivates eIF4E, suppresses the tumorigenic effects
of the latter (Avdulov etal. 2004). Loss of these 4E-BP1 phosphorylation site mutants,
and thus re-activation of eIF4E, is accompanied by spontaneous reversion to a ma-
lignant phenotype. On the other hand, expression of phosphorylated 4E-BP1, which
leads to activation of eIF4E in breast tissue, is associated with malignant progression
and adverse prognosis (Armengol etal. 2007). Together with their phosphorylation
status, the level of 4E-BPs in the cell is important for tumor control. Overexpression
of 4E-BPs, in accord with the suggested tumor suppressor role for these proteins,
leads to cell cycle arrest and phenotypic reversion of some transformed cells (Jiang
etal. 2003; Pons etal. 2011). Thus, eIF4E activation is an essential component of the
malignant phenotype in breast cancer (Armengol etal. 2007; Avdulov etal. 2004),
and the role of 4E-BPs in pathogenesis of the disease is an important one.
4E-BP1, that is detected in both HER2-positive and HER2-negative breast can-
cers, has been shown to be one of the main factors associated with prognosis, grade
of malignancy and endocrine resistance (Karlsson etal. 2013; Rojo etal. 2007).
Phosphorylated 4E-BP1 is highly abundant in poorly differentiated tumors, and
its abundance correlates with tumor size, presence of lymph node metastases, and
locoregional recurrences (Rojo etal. 2007). A recent study of a large cohort of breast
cancer patients showed that 4E-BP1 mRNA expression correlates significantly with
a poor outcome (Karlsson etal. 2013). The 4E-BP1 protein has been shown to be
an independent prognostic factor, especially in PR-expressing cancers, and also to
be associated with a poor response to endocrine treatment in the ER/PR-positive
group (Karlsson etal. 2013). Thus, 4E-BP1 can be utilized as a new clinical marker
for prognosis and response to endocrine therapy in breast cancer (Karlsson etal.
2013). Since the influence of eIF4E on cancer survival is modulated substantially
by 4E-BPs, analysis of combined eIF4E, 4E-BPs and phosphorylated 4E-BPs might
give greater prognostic insights than that of eIF4E alone (Coleman etal. 2009).
26.6Signaling Pathways Controlling 4E-BPs and eIF4E

Activity in Breast Cancer
4E-BP1 appears to be a channeling point at which different upstream oncogenic

alterations within the signaling pathways, such as the mTOR cascade, converge
and transmit their proliferative signals, modulating protein translation (Rojo etal.
2007). Activation of mTOR leads to phosphorylation of 4E-BPs and their dissocia-
tion from eIF4E, activating the latter.
However, it has been described that some human carcinomas present a high level
of phosphorylated 4E-BP1 that is not always associated with high levels of activated
mTOR, suggesting the role of other kinases. One study using a breast carcinoma
cell line examined 48 kinases that could be involved in 4E-BP1 phosphorylation
and stability (Pons etal. 2012). That study identified several kinases affecting 4E-
BP1 stability (LRRK2, RAF-1, p38, GSK3, AMPK and PKA catalytic subunits
(PKAC and PKAC)) and 4E-BP1 phosphorylation (CDK1, PDK1, SRC, PKC1,
PAK2, p38, PKC and CaMKK). Another study reported a novel mechanism of
4E-BP1 regulation at Thr41 and Thr50 sites that appear to be essential for its inacti-
vation and cell proliferation, and that are phosphorylated by casein kinase 1 (CK1)
which is highly expressed in breast cancers (Shin etal. 2014). However, the role of
these kinases in regulation of 4E-BP1 activity requires further validation. Until then,
the PI3K/AKT/mTOR cascade ranks as a main regulator of the activity of 4E-BPs.
The mTOR pathway is activated in up to 76% of primary and metastatic breast
cancers (Akcakanat etal. 2008; Rojo etal. 2007). Suppression of mTOR activity, as
well as levels and activity of the downstream translational regulators, such as eIF4E,
delayed breast cancer progression, onset of associated pulmonary metastasis, can-
cer cell invasion and migration (Nasr etal. 2013). The degree of phosphorylation of
AKT, mTOR and 4E-BP1 increases progressively from normal breast epithelium to
hyperplasia to tumor invasion and independently correlates with DFS (Zhou etal.
2004). Activation of this pathway in cancer can take place by various mechanisms
(see Chaps.15 and 16), which are beyond the scope of this discussion. However,
it is important to mention that two major factors implicated in breast cancer malig-
nancy, RTKs (such as HER2 and EGFR) and hormone receptors (such as ER and
PR), play an important role in activation of the mTOR signaling. Not surprisingly,
levels of phosphorylated AKT, mTOR, and 4E-BP1 are positively associated with
HER2 overexpression (Rojo etal. 2007; Zhou etal. 2004). Similarly, an estrogen,
17- estradiol (E2), rapidly increases the phosphorylation of mTOR and its down-
stream targets (such as 4E-BP1) via TSC2 and RHEB (Yu and Henske 2006). In
addition, long-term estrogen deprivation causes hypersensitivity of MCF-7 breast
cancer cells to the mitogenic effect of E2, which is associated with activation of
MAPK and PI3K pathways (Yue etal. 2003). These findings provide a mechanism
by which factors implicated in breast cancer biology orchestrate this disease via
signaling cascades that converge on the translation machinery.
Another, albeit less-studied, pathway that regulates eIF4E activity is the MAPK
pathway that phosphorylates eIF4E at serine 209 through MNK1/2 (see Chap.17).
In fact, the combined deficiency of MNK1 and MNK2 delays tumor development
(Ueda etal. 2010). Regulation of eIF4E by phosphorylation might play a role in
breast cancer development and progression. Phosphorylation of eIF4E leads to
lower affinity for 4E-BP1 and thus the activation of the former. In breast cancer
models, high levels of phosphorylated eIF4E may counteract the tumor suppressor
effect of 4E-BP1 (Pons etal. 2011). Phosphorylated eIF4E protein is detectable in
some breast tumor samples, but is below a detection limit in a subset of specimens
(Wheater etal. 2010). Those cell lines that exhibit increased levels of phosphorylat-
ed eIF4E were also sensitive to pharmacological MNK inhibition. Thus, assessing
the phosphorylation state of eIF4E in breast cancer could be a valuable biomarker
for the identification of tumors responsive to the MAPK pathway inhibition. Impor-
tantly, phosphorylation of eIF4E appears to be necessary for oncogenic transforma-
tion while being dispensable for normal development (Hou etal. 2012). Hence,
pharmacologic targeting by the MAPK pathway inhibitors may provide a non-toxic
and effective strategy for anticancer therapy in breast malignancies.
In summary, eIF4E and 4E-BPs appear to play an important role in breast can-
cer development and progression. Their expression and phosphorylation levels can
serve as useful prognostic markers for assessing recurrence risk and survival in
breast cancer, as well as represent markers for targeted therapies.
26.7eIF4E and Therapeutic Resistance
The role of translation initiation in conferring resistance to various breast cancer

treatments should be clearly recognized. An important role in therapeutic resistance
is attributed to the levels and activity of eIF4E, which is controlled by the PI3K/
AKT/mTOR and MAPK pathways.
Elevated eIF4E and TLK1B levels might also be associated with resistance to
doxorubicin treatment that induces DNA breaks (Byrnes etal. 2007a). TLK1B (that
facilitates the repair of DNA breaks and that is associated with an increased risk for
cancer recurrence after adjuvant radiation therapy) has been suggested to be regu-
lated by eIF4E via the structured 5 UTR (Byrnes etal. 2007b; Norton etal. 2004).
While no direct evidence implicated eIF4E in resistance to endocrine therapy,
the mTOR and MAPK pathways have been closely linked to therapeutic resistance
to antihormonal treatment (for review see Hasson etal. 2013; Xu and Sun 2010).
Increased expression of eIF4E renders cells resistant to the anti-HER2 anti-
body trastuzumab-mediated decrease in cell proliferation and rescues breast cancer
xenografts from the anticancer effects of trastuzumab (Zindy etal. 2011). Moreover,
the level of eIF4E expression negatively correlates with the clinical therapeutic re-
sponse to a trastuzumab regimen (Zindy etal. 2011). The effect could be explained
by the fact that trastuzumab, cetuximab and erlotinib decrease the formation of the
eIF4F complex in breast cancer. In this setting, increased expression of eIF4E shifts
the balance towards eIF4F formation and translational activation. Formation of the
eIF4F complex may be a critical determinant of response to anticancer therapy that
targets HER2 and EGFR by agents, such as trastuzumab, cetuximab, lapatinib and
erlotinib (Zindy etal. 2011).
Increased eIF4E levels could also be associated with resistance to radiotherapy,
via aforementioned regulation of TLK1B (Wolfort etal. 2006).
Thus, assessment of the levels and activity of eIF4E, as well as its upstream regu-
lators, such as 4E-BPs, might be important in predicting responses to chemotherapy
and radiotherapy, as well as hormonal and targeted therapy. Since the activation
of the mTOR/eIF4E axis is implicated in chemoresistance, the use of mTOR in-
hibitors could be justified in combination with other chemotherapeutic agents in
patients with reduced sensitivity to chemotherapy. This topic is further discussed in
the subsequent text.
26.8mTOR, Translation and Breast Cancer
mTOR inhibitors, which possess immunosuppressive properties were initially used

in the posttransplantation setting, but their use in cancer treatment is expanding.
Everolimus (RAD001) and temsirolimus (CCI-779) are FDA-approved and are be-
ing used in the setting of metastatic RCC (Figlin etal. 2012), metastatic PaNETs
(Yao etal. 2011), some types of astrocytomas and in advanced hormone receptor
positive, HER2-negative breast cancers in combination with exemestane (Baselga
etal. 2012; NCI/NIH 2013).
Evidence from extensive preclinical research efforts has demonstrated the poten-
tial usefulness of these agents in the setting of breast cancer, thus leading to their
clinical assessment (Del Bufalo etal. 2006; Mosley etal. 2007; Shor etal. 2008).
mTOR inhibitors have been assessed in clinical trials as a monotherapy for women
with heavily pretreated metastatic breast cancer and, despite good safety profiles,
limited anticancer efficacy was observed (Ellard etal. 2009; Maass etal. 2013).
Temsirolimus was assessed as a monotherapy for heavily pretreated patients with
metastatic breast cancer, showing some clinical activity with an objective response
rate (ORR) of 9.2% (Chan etal. 2005).
Subsequently, mTOR inhibitors were tested as parts of combinational regimens.
Since activation of the mTOR/eIF4E axis is implicated in treatment resistance, the
majority of efforts in combination therapy are centered on studying mTOR inhibi-
tors together with other therapeutic modalities, namely endocrine treatment (in pa-
tients with endocrine refractory disease), targeted therapy (in cases of trastuzumab
and lapatinib resistance) or chemotherapeutic agents (in patients with reduced
sensitivity to chemotherapy).
26.8.1Combination with Endocrine Therapy
In the setting of hormone receptor-positive HER2-negative breast cancer, an abun-

dance of preclinical evidence supports the activation of the PI3K/AKT/mTOR
signaling pathway as a mediator of endocrine resistance. Ligand-independent ac-
tivation of ER by mTORC1 has been reported (Yamnik etal. 2009). In addition,
multiple RTKs, such as EGFR, HER2 and IGF-1R, leading to PI3K/AKT/mTOR
pathway activation, have been shown to induce endocrine resistance in hormone
receptor-positive breast cancer cells (Juncker-Jensen etal. 2006; Massarweh etal.
2008; Miller etal. 2011). However, a phase III study that assessed the combination
of temsirolimus with letrozole as first-line treatment for postmenopausal women
with metastatic luminal breast cancer stopped prematurely due to futility (Chow
etal. 2006). On the other hand, everolimus combined with letrozole showed prom-
ising antitumor activity in a phase Ib study for letrozole-pretreated metastatic hor-
mone receptor-positive HER2-negative breast cancer patients (Awada etal. 2008).
This combination was also tested in the neoadjuvant setting in a phase II randomized
study, resulting in a significant improvement of clinical response rate (Baselga etal.
2009). Subsequently, the landmark randomized phase III clinical trial, BOLERO-2,
showed that the addition of everolimus to the aromatase inhibitor exemestane is an
effective treatment option, improving PFS in postmenopausal women with hormone
receptor-positive advanced breast cancer (Baselga etal. 2012; Beaver and Park
2012). Preliminary OS analysis showed no significant difference in OS between the
treatment arms (25.4% with placebo versus 32.2% with everolimus, hazard ratio of
0.77; 95% confidence intervals of 0.571.04) (Piccart etal. 2013).
Lastly, everolimus has been assessed in combination with tamoxifen in a phase
II clinical trial of 111 patients with hormone receptor-positive, HER2-negative
metastatic breast cancer resistant to aromatase inhibitors. This study randomized
patients to receive tamoxifen monotherapy or tamoxifen plus everolimus (Bachelot
etal. 2012) and, the combination therapy resulted in a significant improvement
of PFS and OS. Of note, more pronounced antitumor activity of the everolimus
and tamoxifen combination was seen among patients with secondary endocrine re-
sistance. Other clinical studies of everolimus combination with hormonal therapy
are underway (see clinicaltrials.gov NCT01805271, UNIRAD and NCT01674140,
S1207).
26.8.2 Combination with HER2 or EGFR/HER2 Inhibitors
In trastuzumab-refractory HER2-overexpressing breast cancer, mTOR inhibitors

rapamycin and ridaforolimus (AP23573, MK-8669) increased lapatinib sensitivity
in cells with poor response to lapatinib alone, leading to synergistic antiprolifera-
tive effects (Gayle etal. 2011). Inhibition of mTOR results in clinical benefit and
disease response in patients with trastuzumab-resistant HER2-overexpressing met-
astatic breast cancer, as evidenced by a clinical trial that evaluated the efficacy of
the combination of trastuzumab and everolimus in HER2-overexpressing metastatic

breast cancer that progressed on trastuzumab-based therapy (Morrow etal. 2011).
In patients with metastatic breast cancer pretreated with trastuzumab, everolimus
with paclitaxel and trastuzumab showed encouraging antitumor activity (Andre etal.
2010). In this phase I/II study, a clinical benefit rate of 34% was achieved among
47 enrolled patients. Another phase Ib clinical trial assessed everolimus combined
with weekly vinorelbine and trastuzumab, showing encouraging antitumor activity
in heavily pretreated patients with HER2-overexpressing metastatic breast cancer
that progressed on trastuzumab (Jerusalem etal. 2011). The results of a phase III tri-
al (BOLERO-3) that randomized 572 trastuzumab-resistant HER2-positive breast
cancer patients to receive trastuzumab and vinorelbine in combination with either
everolimus or placebo were recently presented (ORegan etal. 2013). The addition
of everolimus resulted in a statistically significant, although clinically modest, pro-
longation of PFS (7 months versus 5.78 months, hazard ratio of 0.78, p=0.0067),
with the data for OS being still immature.
26.8.3Combination with Conventional Chemotherapy
In preclinical studies everolimus has been shown to potentiate the cytotoxicity of

conventional chemotherapeutic agent carboplatin and enhance the treatment effi-
cacy for breast cancer, suggesting that the combination of mTOR inhibitors and car-
boplatin is a promising treatment approach for breast cancer that can be evaluated
in clinical trials (Liu etal. 2011a). Thus combination of conventional chemothera-
peutic agents with mTOR inhibitors appears to be a potential strategy to overcome
resistance and enhance effects of anticancer therapy.
26.8.4Other Strategies of Targeting

the mTOR/4E-BPs/eIF4E Axis
Another medication that is currently being investigated for cancer treatment in gen-
eral and breast cancer in particular is metformin, a biguanine, and a widely used
antidiabetic drug (for review see Belda-Iniesta etal. 2011). Metformin activates the
AMPK pathway, a major sensor of the energy status of the cell and an inhibitor of
mTOR. In breast cancer MCF-7 cells, metformin inhibits translation initiation asso-
ciated with mTOR inhibition and a decrease in the phosphorylation of the S6K and
4E-BP1 (Dowling etal. 2007). Together with AMPK, LKB1 and TSC2 might be
involved in the mechanism of action of metformin (Dowling etal. 2007). Diabetic
patients with breast cancer receiving metformin and neoadjuvant chemotherapy
have been shown to have higher pathologic complete response rate than diabetics
not receiving metformin (Jiralerspong etal. 2009). A recent randomized trial pre-
sented biomarker evidence for antitumor effects of metformin in women with breast
cancer and provided support for therapeutic trials of metformin in breast cancer
(Hadad etal. 2011). Of note, in this window of opportunity trial, administration

of metformin for two weeks was associated with significant Ki-67 reduction and
mTOR pathway downregulation. Clinical trials are underway to assess effects of
metformin in treating breast cancer either as a single agent or combined with other
agents (Esteva etal. 2013; Martin-Castillo etal. 2010) (also see clinicaltrials.gov).
A list of potential breast cancer chemotherapeutic targets and approaches that
potentially target the mTOR/4E-BPs/eIF4E axis are presented in Table26.2.
26.8.5 T
ranslation-Related Aspects of Sensitivity
and Resistance to mTOR Inhibitors
mTOR inhibitors repress translation initiation by decreasing the phosphorylation of

4E-BP1 and increasing interaction of 4E-BP1 with eIF4E. The molecular mecha-
nism of action of rapalogs provides insight into attenuated antitumor activity seen
in the clinical setting.
Increased phosphorylation of mTOR and 4E-BP1 has been found associated with
sensitivity to mTOR inhibitor rapamycin (Zhou etal. 2004). Depletion of 4E-BP1
from breast cancer cells leads to abrogation of the rapamycin effect (Averous etal.
2008). Heterogeneous effects of rapamycin on breast cells could also depend on
carcinogen-induced variations of 4E-BP1 isoforms with varying degrees of 4E-BP1
phosphorylation, as shown in rat models (Takabatake etal. 2011). However, this has
not yet been conclusively proven in human models.
Additionally, eIF4E activity has been found to be a significant predictor of ra-
pamycin sensitivity, with higher eIF4E activity being indicative of increased resis-
tance (Satheesha etal. 2011). An increase in the eIF4E/4E-BP ratio dramatically
limits the sensitivity of cancer cells to mTOR inhibitors (Alain etal. 2012). Thus,
the eIF4E/4E-BP ratio, as opposed to eIF4E or 4E-BPs alone, has been suggested to
be a better predictive marker for therapeutic response to mTOR inhibitors.
mTOR inhibitors block only the mTORC1 complex, showing a higher affinity
for certain substrates than for others, thus not thoroughly abrogating PI3K signaling
(see Chaps.15 and 16 and (Choo and Blenis 2009)). Rapalogs do not completely
inhibit the 4E-BP/eIF4E axis, but rather affect S6K, whereas a newer generation
mTOR inhibitors affect both axes (see Chap.15). Various feedback mechanisms,
such as paradoxical compensatory AKT activation have been shown to counteract
therapeutic effects of mTOR inhibition by rapalogs (see Chap.15) (Del Bufalo etal.
2006; Noh etal. 2004; OReilly etal. 2006; Shor etal. 2008). While the use of
everolimus resulted in phosphorylating activation of AKT, addition of LY294002, a
PI3K inhibitor, suppressed the activity of the PI3K/AKT/mTOR axis and mitigated
the AKT activation feedback (Chen etal. 2013). Everolimus and dual PI3K/mTOR
inhibitor BEZ235 (NVP-BEZ235) can also be synergistically combined to inhibit
mTOR pathway activation, cell proliferation and tumor growth in various cancers,
including breast cancer (Nyfeler etal. 2012). The feedback AKT activation due to
mTOR inhibition was prevented by higher doses of BEZ235 (Serra etal. 2008).
Table 26.2 Various compounds implicated to affect translation and show anticancer effects in breast cancer.
528
Compound Proposed translation target/ Proposed effect/s References

mechanism
15d-PGJ(2)(cyclopentenone prostaglan- eIF2 Likely indirectly inhibits translation initiation of Campo etal. (2002)
din 15-deoxy-(12,14)-PGJ(2)) Increases phosphorylation of cyclin D1 mRNA in MCF-7 cells. The D-group
eIF2 cyclins play a key role in the progression of cells
through the G1 phase of the cell cycle. Downregu-
lation of cyclin D1 protein expression leads to
growth arrest in the G0/G1 phase of the cell cycle.
All-trans-retinoic acid and interferon PKR All-trans-retinoic acid and IFN- act synergistically Shang etal. (1998)
(IFN) All-trans-retinoic acid upregulates to inhibit growth of ER+ or ER- BC cells. PKR
expression of PKR in BC cells downregulates c-MYC that leads to inhibition of
cell growth.
Am580 PDCD4 PDCD4 overexpression results in apoptosis of BC Afonja etal. (2004) and
(RAR-selective agonist; Am580, RAR Induces PDCD4 expression in a cells (see text). Wen etal. (2007)
pan-agonists, retinoids) wide variety of BC cell lines
Antrocin AKT/mTOR It is a potent antiproliferative agent in metastatic BC. Rao etal. (2011)
(a small molecule from Antrodia Behaves as an AKT/mTOR dual Induces dose-dependent cell death by apoptosis,
camphorate) inhibitor Suppresses the phosphorylation of AKT and its
downstream effectors, mTOR, GSK3, and NF-B.
Bortezomib and MG132 (26S protea- eIF2 Inhibition of the 26S proteasome causes a rapid Yerlikaya and DoKudur
some inhibitors) Phosphorylation of eIF2 by HRI decrease in the protein synthesis rate due to phos- (2010)
phorylation of eIF2. In 4T1 BC cells it was found
that both of the inhibitors caused rapid phosphory-
lation of eIF2.
Curcuminoids (Demethoxycurcumin 4E-BP1, mTOR, AMPK DMC demonstrated the most potent cytotoxic effects Shieh etal. (2013)
(DMC), curcumin, and bisdeme- Proposed to activate AMPK on triple-negative breast cancer cells, while
thoxycurcumin (major forms of and lead to inhibition of the nonmalignant cells were unaffected.
curcuminoids found in the rhizomes mTOR/4E-BP1 axis and down-
of turmeric)) regulation of translation
D-glucosamine (a precursor in the eIF4B and rpS6 Inhibits activity of p70S6K and the proliferation of Oh etal. (2007)
biochemical synthesis of glycosylated Decreases phosphorylation of MDA-MB-231 cells. In addition, enhances the
A. Parsyan et al.
proteins and lipids) p70S6K, and its downstream growth inhibitory effects of rapamycin.
substrates rpS6 and eIF4B
mechanism
Elemene and Ili (13,14-bis(cis-3,5- mTOR/4E-BP1 and S6K Shows potent antitumor activities via inhibition of Ding etal. (2013)
dimethyl-1-piperazinyl)--elemene) Likely inhibits mTOR mTOR in BC cells, leading to decrease in phos-
phorylated p70S6K1 and 4E-BP1 levels.
Fulvestrant ((ICI-182780, Faslodex) PDCD4 PDCD4 overexpression results in apoptosis of BC Afonja etal. (2004)
26 Breast Cancer
antiestrogen) Induces PDCD4 expression in a cells.

wide variety of BC cell lines
Gemini compounds ([Gemini-23-yne- AKT In MCF-7 cells these compounds cause inhibition of OKelly etal. (2006)
26,27-hexafluoro-D3] 1, 25-dihy- Inhibits dephosphorylation of clonal growth. They decrease the rate of the protein
droxy-21-(3-hydroxy-3- methylbutyl) AKT that leads to inhibition synthesis and increase association of 4E-BP1 with
vitamin D3) of mTOR and subsequently eIF4E (by inhibition of the AKT-mTOR pathway).
decreased phosphorylation of This represents a novel mechanism by which
4E-BP1 and S6K vitamin D3 analogs may modulate expression
and activity of proteins involved in cancer cell
proliferation.
Hippuristanol (a small molecule found eIF4A Suppresses eIF4A activity and delays BC progres- Nasr etal. (2013)
in the coral Isis hippuris) Inhibits activity of eIF4A sion, cell invasion and migration and onset of
associated pulmonary metastases.
Honokiol (naturally occurring neolignan mTOR, AMPK Honokiol treatment results in inhibition of breast Nagalingam etal.
abundant in Magnolia. It is a small- Downregulates the expression tumorigenesis in vitro and in vivo. Inhibits growth (2012) and Park
molecule polyphenol that is widely and phosphorylation of c-SRC, in ER- MDA-MB-231 cells and causes cell cycle etal. (2009)
known for its anti-inflammatory, EGFR and AKT, leading to arrest in G0/G1 phase and induction of apoptotic
antithrombosis, and antioxidant the inactivation of the mTOR cell death. The honokiol-induced cell cycle arrest
properties, and more recently, for its signaling, including 4E-BPs is correlated with the expression of cyclin D1,
protective function in the pathogenesis and p70S6K (Park etal. 2009). cyclin E and c-MYC. Increases AMPK phosphory-
of carcinogenesis) Increases AMPK phosphoryla- lation and activity, and thus inhibits phosphoryla-
tion and activity tion of S6K and 4E-BP1. Increases expression and
cytoplasmic translocation of tumor-suppressor
LKB1 in BC cells.
Metformin (biguanine, antidiabetic eIF4E-BP1, mTOR, S6K The effects of metformin are likely mediated via Dowling etal. (2007)
agent) Acts through AMPK and inhibits activation of AMPK, which regulates cellular
529
translation initiation associated energy metabolism. Inhibits growth of cancer cells

with mTOR inhibition and BC cells through the activation of AMPK.
530

mechanism
Nitric oxide (NO) inhibitors mTOR/4E-BPs/eIF4E NO was reported to dramatically increase prolifera- Pervin etal. (2007)
Likely inhibits the mTOR pathway tion and total protein synthesis in MDA-MB-231
and MCF-7 cells, likely through activation of
mTOR and its downstream targets, 4E-BPs and
p70S6K.
OSU-03012 (a small molecule derivative PERK, eIF2 This compound, together with lapatinib, enhances West etal. (2013)
of the COX-2 inhibitor celecoxib) cell killing via the Nck1/eIF2 complex that could
represent a novel target for treatment of metastatic
BC.
Paclitaxel (taxane and a known mitotic 4E-BP1 Induces hyperphosphorylation of 4E-BP1 in (Greenberg and Zim-
inhibitor and anticancer drug that Increases activity of eIF4E by pro- MDA-MB-231, which reduces its association mer 2005)
stabilizes microtubules and interferes moting hyperphosphorylation with eIF4E, but does not alter the expression and
with their normal breakdown during and release of 4E-BP1 through a phosphorylation of eIF4E. Hyperphosphorylation
cell division) CDK1-dependent mechanism of 4E-BP1 correlates with G2/M accumulation and
with an increase in the phosphorylation of CDK1
substrates.
Pentamidine (aromatic diamine for treat- eIF4F, eIF2 In MDA-MB-231 breast cancer cells pentamidine Jung etal. (2011)
ment of protozoan infections) Likely acts on translation via suppressed global protein translation, accompanied
inhibition of HIF-1 (hypoxia by the reduction of the eIF4F complex and induc-
inducible factor) tion of eIF2 phosphorylation.
Phenethyl isothiocyanate (a natural mTOR/4E-BP1 axis This compound effectively blocked HIF-1 RNA Cavell etal. (2012)
dietary phytochemical) mTOR inhibition, likely via TSC2 translation in MCF-7 BC cells, and this was associ-
TSC2 ated with reduced phosphorylation of 4E-BP1 and
p70S6K.
Platycodin D (a natural compound found PERK, eIF2 Induces apoptotic cell death in various carcinoma Yu and Kim (2012)
in Platycodon grandiflorum) Phosphorylation of PERK and cells. Activates MAPK. Causes activation of the
eIF2 endoplasmic reticulum stress response and phos-
phorylation of PERK and eIF2.
A. Parsyan et al.
mechanism
Pomolic acid (triterpenoid isolated from AMPK, mTOR, S6K, 4E-BP1 Appears to inhibit the growth of cancer cells. Inhibits Youn etal. (2012)
Licania pittieri) Activates AMPK. Inhibits mTOR, cell proliferation and induces sub-G1 arrest. Acts
S6K, 4E-BP1 by decreasing expression of fatty acid synthase and
26 Breast Cancer
decreasing acetyl-CoA carboxylase activation and

incorporation of [(3)H]acetyl-CoA into fatty acids.
In addition, inhibits key enzymes involved in pro-
tein synthesis such as mTOR, S6K, and 4E-BP1.
Poncirus trifoliate (Rutaceae mTOR/4E-BP1 Inhibits growth in ER- MDA-MB-231 cells and Chung etal. (2011)
fruit, potential compound is Inactivates mTOR and its down- causes cell cycle arrest in G0/G1 phase likely
25-methoxyhispidol A (25-MHA), a stream signal molecules, such as through downregulation of cyclin D1, CDK4,
triterpenoid) 4E-BPs and S6K CDK2, cyclin A, phosphorylated retinoblastoma
protein (pRB), and induction of CDK inhibitor
p21 (WAF1/Cip1) protein. Suppresses activation
of c-SRC/EGFR/AKT signaling, and consequently
leads to inactivation of mTOR.
PUFA (polyunsaturated fatty acids, -3) eIF2 Inhibition of eIF2 leads to inhibition of translation Aktas and Halperin
Ca2+ depletion mediated phos- initiation that accounts for the anticancer activity (2004)
phorylation of the eIF2 of PUFAs. Studies suggest negative correlation
between consumption of fish and incidence of BC.
In vitro and animal models, studies indicate that
PUFAs inhibit proliferation of cancer cells and
growth of tumors. Present at high concentrations in
marine animals.
Reishi (Ganoderma lucidum mushroom) eIF4G, eIF4E/4E-BP axis Selectively inhibits BC cell viability but does not Martinez-Montemayor
Decreases expression of eIF4G. affect the viability of noncancerous mammary etal. (2011); Suarez-
Likely acts via mTOR pathway epithelial cells. Inhibits cell invasion. In inflamma- Arroyo etal. (2013)
inhibition tory BC cells SUM-149 Reishi reduced expression
of eIF4G levels coupled with increased levels of
eIF4E bound to 4E-BPs with consequential protein
531
synthesis reduction and tumor growth.

532

mechanism
Ribavirin (antiviral medication) eIF4E At clinically relevant concentrations, ribavirin Pettersson etal. (2011)
Reduces expression of eIF4E reduces cell proliferation and suppresses clono-
targets genic potential. This effect correlates with reduced
expression of important eIF4E targets. Effects
in different cells might vary. The mechanism of
eIF4E targeting is unclear.
Silibinin (Flavonoid) mTOR/eIF4F Displays antiproliferative activity in MCF-7 BC Lin etal. (2009)
Concentration-dependent reduc- cells. Elicits decrease in polysome content and
tion of global protein synthesis translation of cyclin D1 mRNA. Inhibits the mTOR
associated with reduced levels signaling pathway by acting upstream of TSC2.
of eIF4F
Silvestrol (cyclopenta[b]benzofuran, eIF4A/eIF4F Exhibits significant anticancer activity in human BC Cencic etal. (2009);
from the fruits and twigs of Aglaia Impairs the ribosome recruitment xenograft models. It is associated with increased Nasr etal. (2013)
foveolata) apoptosis, decreased proliferation and inhibi-
step of translation initiation by
affecting the composition of tion of angiogenesis. Targeting translation by
eIF4F silvestrol leads to selective inhibition of weakly-
initiating mRNAs. Suppression of eIF4A activity
by silvestrol delays BC progression, cell invasion
and migration and onset of associated pulmonary
metastases.
TZD18 (the peroxisome proliferator- eIF2 TZD18 treatment leads to the activation of endoplas- Zang etal. (2009)
activated receptor / dual ligand) Induces phosphorylation of PERK mic reticulum stress response and induces growth
and eIF2. Activates endoplas- arrest and apoptosis in BC cells. TZD18 induces
mic reticulum stress-induced phosphorylation of PERK and eIF2. Activates
pathways MAPK pathway.
Zilongjin (Chinese herbal medicine) eIF3i, eIF1A Suggested to have anticancer activity. Mechanism is Tian etal. (2010)
Has broad effect on non-transla- unclear.
tion factors as well
Zolendronic acid PERK, eIF2. Causes endoplasmic reticulum stress and activates the Lan etal. (2013)
A. Parsyan et al.
Inhibits the mTOR pathway. PERK/eIF2/CHOP pathway to induce REDD1

Activates PERK expression and to inhibit the mTOR pathway.
See List of Abbreviations; BCbreast cancer.
BEZ235 inhibits the activation of AKT, rpS6 and 4E-BP1 in breast cancer cells
with a superior antiproliferative activity to that of everolimus (Serra etal. 2008).
Thus, combined inhibition of PI3K and mTOR may further improve therapy in
breast cancer (see (Hernandez-Aya and Gonzalez-Angulo 2011)). However, while
PI3K inhibition could address the issue of feedback activation of AKT in mTORC1
inhibition, resistance to BEZ235 reproduced in laboratory is mediated via eIF4E
amplification and elevation of 5 cap-dependent protein translation (Ilic etal. 2011).
Another way to abrogate the rapamycin-induced activation of AKT is to com-
bine rapamycin therapy with lapatinib, a dual EGFR/HER2 kinase inhibitor. In
triple-negative breast cancer cells, EGFR inhibition abrogated the expression of
rapamycin-induced activation of AKT (Liu etal. 2011b). In addition, lapatinib tar-
gets EGFR that is commonly overexpressed in triple-negative breast cancer. Finally,
mTOR inhibition appears to reverse resistance to EGFR inhibitors (Liu etal. 2011b).
While lapatinib or rapamycin alone are not effective in triple-negative breast cancer,
their combination results in significant synergy and greater cytotoxicity and antip-
roliferative activity than the single agents alone (Liu etal. 2011b).
As mentioned previously, activation of AKT in cancer cells exposed to mTOR
inhibitors likely contributes to its attenuated antitumor activity. Thus, strategies to
impede or abolish AKT activation in a context of mTOR-targeted therapies are prom-
ising. Indeed, in breast cancer cells the use of geldanamycin derivative and HSP90
inhibitor tanespimycin (17-allylamino-17-demethoxygeldanamycin (17-AAG)) ab-
rogates AKT activation and potentiates mTOR inhibition by rapamycin (Roforth and
Tan 2008). Rapamycin and tanespimycin combination results not only in an effi-
cient inhibition of mTOR but also of MAPK signaling, a pathway that is otherwise
not inhibited by rapamycin. This leads to enhanced antiproliferative activity in both
MCF-7 and MDA-MB-231 breast cancer cells (Roforth and Tan 2008).
Inhibition of the 4E-BP/eIF4E axis could be augmented by dual mTORC1 and
mTORC2 inhibitors (see Chaps.4 and 15). In contrast to rapamycin, investigational
drug MLN0128 (a potent and selective small molecule active-site TORC1/2 kinase
inhibitor) appeared to be superior in blocking mTOR signaling, as evidenced by
potent inhibition of cell proliferation and growth in xenograft models and decreased
phosphorylation of AKT, rpS6 and 4E-BP1 (Gokmen-Polar etal. 2012).
Other approaches are being studied to overcome feedback activation of AKT
upon mTOR inhibition in breast cancer. These include antrocin, which acts as an
AKT/mTOR dual inhibitor and has recently been shown to be a very potent antipro-
liferative agent in metastatic breast cancer (Rao etal. 2011); D-glucosamine, which
enhances the growth inhibitory effects of rapamycin (Oh etal. 2007); NO inhibitors,
which decrease proliferation and total protein synthesis in both MDA-MB-231 and
MCF-7 cells, likely via mTOR/4E-BPs-p70S6K (Pervin etal. 2007); and others
(see Table26.2).
Interestingly, radiotherapy also appears to boost the effects of rapamycin on
mTOR inhibition. In MCF-7, both radiation and rapamycin share molecular targets
and induce similar physiologic responses, causing changes in mitochondrial me-
tabolism, development of autophagy and an overall decrease in cell survival (Paglin
etal. 2005). Both treatments lead to dephosphorylation of mTOR downstream
effectors 4E-BP1 and p70S6K, but also decrease the level of eIF4G. Radiation-
induced inactivation of the mTOR pathway appears to be an underlying mechanism
of radiation-induced autophagy in human breast cancer (Paglin etal. 2005; Paglin
and Yahalom 2006).
26.9Direct Targeting of eIF4E and eIF4F
As discussed previously, eIF4E depletion inhibits breast cancer cell growth.

Importantly, unlike rapamycin and its analogs that target eIF4E indirectly, direct
targeting of eIF4E is not associated with AKT activation (Soni etal. 2008). Indeed,
the 4E-BPs/eIF4E axis is the convergence point of oncogenic signaling and hence
might represent an ultimate therapeutic target. Inhibition of eIF4E is a potential
breast cancer therapeutic strategy that may be especially promising against specific
molecular subtypes and in metastatic, as well as primary tumors (Chu etal. 2007;
Pettersson etal. 2011). eIF4E knockdown inhibits growth of cells with varying
total and phosphorylated 4E-BP1 levels and inhibits rapamycin-insensitive, as well
as rapamycin-sensitive cell lines (Soni etal. 2008; Yellen etal. 2011). eIF4E has
been shown to be a useful target not only for chemosensitization but also radiosen-
sitization in breast cancer (Yang etal. 2012; Zhou etal. 2011). Silencing of eIF4E
enhances chemosensitivity by sensitizing MDA-MB-231 cells to cisplatin, doxo-
rubicin, paclitaxel and docetaxel (Zhou etal. 2011). Targeting eIF4E with survivin
promoter-driven shRNA also leads to downregulation of eIF4E in breast carcinoma
cells (but not in normal human mammary epithelial cells), induces their apoptosis
and enhances their sensitivity to cisplatin (Dong etal. 2009). Thus, antisense chem-
istry represents one of the approaches for direct targeting of eIF4E.
Novel peptidomimetic strategies to abolish the assembly of the eIF4F complex
are being developed (Brown etal. 2011; Chen etal. 2012; Zhou etal. 2012). 4EGI-1,
a peptidomimetic which disrupts eIF4G/eIF4E interaction and thus eIF4F complex
formation, has been shown to inhibit translation initiation to preferentially abrogate
translation of mRNAs coding for oncogenic proteins, and to inhibit proliferation of
human cancer cells (Chen etal. 2012). 4EGI-1 strongly inhibited growth of human
breast and melanoma cancer xenografts with a good toxicity profile (Chen etal. 2012).
Another mimetic based on the eIF4E-binding peptide has been synthesized showing a
potent inhibition of cap-dependent translation (Brown etal. 2011).
Since targeted delivery of small peptides and ASOs might represent a challenge,
novel approaches are also being developed to deliver these compounds to targeted
tissues. Targeted delivery of eIF4E ASOs using self-assembled nanostructures re-
sulted in a good uptake by breast cancer cells and an efficient knockdown of eIF4E
(Gangar etal. 2013).
Cap-dependent translation involves interaction of eIF4E with the N(7)-meth-
ylated guanosine cap structure on the 5 end of the mRNA (see Chap. 2). Thus,
compounds that can interfere with this interaction could be proven potent in treat-
ment of cancers with overexpressed or activated eIF4E. Such a compound, 7-benzyl
guanosine monophosphate (7Bn-GMP), is a potent antagonist of eIF4E cap-binding,
however, it is not readily permeable by the cell. This led to the development of a
more permeable molecule, a tryptamine phosphoramidate prodrug of 7Bn-GMP,
4Ei-1, that inhibits eIF4E function by both antagonizing eIF4E cap-binding and ini-
tiating eIF4E proteasomal degradation in breast and lung cancer cell lines, as well
as leads to chemosensitization to gemcitabine (Li etal. 2013).
The fact that breast cancer cells overexpress eIF4E could be used to devise
suicide gene based therapies (DeFatta etal. 2002a, b; Mathis etal. 2006). Such
a system, using cancer-specific targeting of an adenovirus-delivered herpes sim-
plex virus thymidine kinase suicide gene with a modified complex 5 UTR to limit
efficient translation to cells expressing high levels of eIF4E, has been developed
(Mathis etal. 2006). Indeed, enhanced cancer cell-specific gene expression and
reduced normal tissue gene expression of the suicide gene led to an increase in the
specificity of cytotoxic effects.
In AML patients, antiviral drug ribavirin has been shown to target eIF4E activity
correlating with clinical responses (Assouline etal. 2009; Kraljacic etal. 2011). Its
effects, at least partially, relate to the reduction of eIF4E expression. Similarly, in
breast cancer, ribavirin reduces cell proliferation and suppresses clonogenic potential
in eIF4E-dependent manner (Pettersson etal. 2011). Additionally, ribavirin triphos-
phate, a metabolite of ribavirin, has been demonstrated to bind to the cap-binding site
of eIF4E and to induce conformational changes of the latter, representing potential for
ribavirin-based novel drug designs targeting eIF4E (Volpon etal. 2013). Interestingly,
ribavirin, likely at the level of transcriptional control, restores expression of ER1
(alone or in combination with suberoylanilide hydroxamic acid) and hence confers
sensitivity of breast cancer cells to hormonal therapy (Sappok and Mahlknecht 2011).
Thus, ribavirin and its analogs can be of an interest in targeting breast cancer.
26.10eIF4G as a Marker of Highly Proliferative

and Metastatic Breast Cancer
The unique pathogenic properties of inflammatory breast cancer ensue, at least in

part, from overexpression of the translation initiation factor eIF4G1, one of the main
isoforms of eIF4G and the scaffolding component of the eIF4F complex (Silvera
etal. 2009; Silvera and Schneider 2009) (see Chap.7). Rather than developing as a
solid tumor, inflammatory breast cancer generates rapidly metastasizing tight clus-
ters of cancer cells called cell emboli, which represent cancer cells held together
by increased membrane expression of E-cadherin (Alpaugh etal. 2002; Mahooti
etal. 2010). eIF4G1 probably reprograms the protein synthetic machinery to cap-
independent IRES-mediated (see Chaps.3, 7 and 19) translation of mRNAs that
code for proteins promoting inflammatory breast cancer cell survival and formation
of tumor emboli (Braunstein etal. 2007; Silvera etal. 2009; Silvera and Schneider
2009). Overexpression of eIF4G1 in inflammatory breast cancer leads to a specific
increase in the translation of IRES-containing mRNAs, such as p120 catenin, which
mediates E-cadherin retention at the cell surface, and VEGF, which accounts for
high levels of angiogenesis and resistance to hypoxia (Silvera and Schneider 2009).
It has been demonstrated that a majority of large advanced breast cancers over-
express eIF4G and 4E-BP1 (Braunstein etal. 2007). Hyperphosphorylation of
4E-BP1 together with increased levels of eIF4G has also been reported in breast
cancers (Avdulov etal. 2004). Overexpressed eIF4G together with 4E-BP1 prob-
ably orchestrate a hypoxia-activated switch from cap-dependent to cap-independent
mRNA translation promoting increased tumor angiogenesis and growth. Accord-
ing to this model, 4E-BP1 inhibits cap-dependent mRNA translation, while eIF4G
overexpression increases selective translation of IRES-driven mRNAs coding for
proteins facilitating angiogenesis and tumor growth (Braunstein etal. 2007). In-
deed, high coexpression levels of phosphorylated 4E-BP1 and eIF4G correlate with
a significantly high tumor proliferation rate (Rojo etal. 2007). Thus, eIF4G might
represent a marker that plays a role in highly metastatic inflammatory or highly
proliferative advanced breast cancers.
eIF4G1 might also be an important component of the DNA damage response
to ionizing radiation. In breast cancer, such DNA damage response is mediated by
high levels of eIF4G1 that selectively increases translation of mRNAs involved in
cell survival, antiautophagic and antiapoptotic responses and DNA damage response
(Badura etal. 2012). These include survivin, HIF1A, XIAP, GADD45A, TP53,
BRCA1/2, PARP and ATM (ataxia telangiectasia mutated) and others, some of which
could be translated via IRES. Reduced expression of eIF4G1 (but not its homolog
eIF4G2), while conferring little reduction in the overall protein synthesis, sensitizes
cells to DNA damage by ionizing radiation, delays resolution of DNA damage and
induces cell death by both apoptosis and autophagy (Badura etal. 2012).
26.11eIF4A and Its Regulatory Protein Tumor

Suppressor PDCD4
eIF4A is the helicase component of a trimeric eIF4F complex. The role of eIF4A
in cancer in general and in breast cancer pathogenesis in particular is unclear (see
Chap.5). However, several studies into eIF4A binding partners and eIF4A inhibitors
silvestrol and hippuristanol suggest that it might represent an important hub in the
pathogenesis of breast cancer. In human breast cancer xenograft models, silvestrol
exhibits significant anticancer activity that is associated with increased apoptosis,
decreased proliferation and inhibition of angiogenesis (Cencic etal. 2009). In a
recent study, it has been shown that suppression of eIF4A activity by silvestrol or
hippuristanol delays breast cancer progression, prevents cell invasion and migration
and delays the onset of associated pulmonary metastases (Nasr etal. 2013).
PDCD4 is a bona fide tumor suppressor that functions by binding to and inhib-
iting the activity of eIF4A (see Chap.6). It is being evaluated as a target for che-
motherapy. Decreased PDCD4 expression levels correlate with breast cancer cell
prosurvival effects, invasion, aberrant cell-cycling and poor prognosis in hormone
receptor-positive breast cancer (Afonja etal. 2004; Bourguignon etal. 2009; Goke
etal. 2004; Jansen etal. 2004; Meric-Bernstam etal. 2012; Nieves-Alicea etal.
2009; Santhanam etal. 2010; Wen etal. 2007).
The regulation of PDCD4 expression in cancer in general and in breast can-

cer in particular takes place via various mechanisms (see Chap.6). One of such
mechanisms, studied in the context of breast cancer, takes place via methylation of
PDCD4 by PRMT5 that causes accelerated tumor growth (Powers etal. 2011) (see
Chap.6).
However, one of the major mechanisms, especially in cancer, includes
PDCD4 protein downregulation by activation of miR-21 that promotes transfor-
mation in breast cancer models (Frankel etal. 2008; Lu etal. 2008; Zhu etal.
2008). miR-21 is upregulated in various types of breast cancers and is associated
with decreased PDCD4 expression, high tumor grades, loss of hormone recep-
tor expression, positive lymph node status and shortened DFS and OS (Baffa
etal. 2009; Hafez etal. 2012; Ota etal. 2011; Walter etal. 2011). Activation
of miR-21 in breast cancers involves various pathways including the PKC-
Nanog signaling pathway (Bourguignon etal. 2009), DNA damage response
(Niu etal. 2012), estradiol-to-ER activation (Wickramasinghe etal. 2009) and
others. N
otably, stimulation of HER2/neu signals via the MAPK pathway to in-
duce miR-21, downregulating PDCD4 and facilitating cell invasion (Huang etal.
2009). Anti-HER2 antibody trastuzumab induces PDCD4 expression, suggesting
that PDCD4 may play a central role in growth inhibition in HER2-positive breast
cancer cells (Afonja etal. 2004). Thus, this represents yet another mechanism
by which HER2/neu contributes to breast cancer pathogenesis via regulation of
translation.
26.12The PDCD4/eIF4A Axis in Chemoresistance

and Therapeutic Targeting
PDCD4 tumor suppressor appears to play an important role in conferring resistance

to breast cancer treatment (Jansen etal. 2004). Downregulation of PDCD4 poten-
tially activates eIF4A, which leads to upregulation of inhibitors of apoptosis and
MDR1 (multidrug-resistant protein 1) that plays a key role in chemotherapy resis-
tance (Afonja etal. 2004; Bourguignon etal. 2009). Stable suppression of PDCD4
expression significantly reduces the sensitivity of breast cancer cells to chemothera-
peutic agents while increased PDCD4 expression positively correlates with the anti-
tumor activity of geldanamycin and tamoxifen (Jansen etal. 2004).
eIF4A inhibitors silvestrol and hippuristanol have significant anti-breast cancer
effects associated with increased cells death, decreased proliferation, inhibition of
angiogenesis and delayed onset of associated pulmonary metastasis (Cencic etal.
2009; Nasr etal. 2013). Their role in various cancers is being evaluated at the pre-
clinical level.
PDCD4 expression and decreased proliferation of breast cancer cells can be in-
duced by inhibition of miR-21 (Frankel etal. 2008). Thus, strategies to upregulate
expression of PDCD4 via inhibition of its regulatory miRNAs in breast cancer are
being developed. One such approach included the development of an inhibitor of
DICER-mediated biogenesis of miR-21, AC1MMYR2, which reversed EMT and
suppressed tumor growth and progression by a mechanism involving upregulation

of miR-21 targets, such as PTEN, PDCD4 and RECK (Shi etal. 2013).
Other mechanisms of PDCD4 induction in breast cancer have been reported (see
Table26.2), including the retinoic acid receptors (RAR) -selective agonist Am580
and RAR pan-agonists, as well as antiestrogen fulvestrant (Afonja etal. 2004).
26.13The eIF3 Complex and Its Components

in Breast Cancers
Among other translation initiation factors that are implicated in the pathogenesis
of breast cancer is the multisubunit eIF3 protein complex that plays a fundamental
role in translation initiation and contains at least thirteen nonidentical subunits (see
Chap.8 and Hinnebusch 2006; Sun etal. 2011). Enhanced activity of eIF3 as a
whole and its component proteins separately is thought to contribute to tumorigen-
esis (Hershey 2010; Zhang etal. 2007). Four eIF3 subunits have been studied in
breast cancer (Table26.1). In this disease, elevated levels of eIF3a, eIF3e and eIF3h
have been reported, and their depletion significantly reversed cell proliferation. On
the other hand, eIF3f is suggested to act as a negative regulator of breast cancer
proliferation (Shi etal. 2006).
The expression level of eIF3a (p170) has been found elevated in breast and other
cancers (Bachmann etal. 1997; Chen and Burger 1999, 2004; Dellas etal. 1998;
Lin etal. 2001; Pincheira etal. 2001). Decreasing eIF3a expression significantly
reverses the breast cancer cell malignant growth phenotype (Dong etal. 2004). It
has been shown that genetic variations in eIF3a are significantly associated with an
altered risk of breast cancer (Olson etal. 2010).
eIF3h is often amplified and highly expressed in breast cancer (Nupponen etal.
1999, 2000; Savinainen etal. 2004, 2006). siRNA silencing of eIF3h significantly
inhibits the growth of various breast cancer cell lines, suggesting that eIF3h regu-
lates cell growth and viability, and that its overexpression may provide a growth
advantage to cancer cells (Savinainen etal. 2006; Zhang etal. 2008).
Initially, eIF3e was proposed to act as a tumor suppressor since its reduction
was observed in 37% of samples in one study (Marchetti etal. 2001). However, the
majority of later reports suggests that eIF3e promotes mammary carcinogenesis,
invasion and metastasis (Chen etal. 2007; Gillis and Lewis 2013; Grzmil etal.
2010). Decreased eIF3e expression causes EMT in breast epithelial cells, negatively
affecting invasive and migratory properties and suggesting the role of this subunit
in breast cancer metastasis (Gillis and Lewis 2013). eIF3e is also involved in the
DNA damage response and is required for function of ATM and BRCA1 in DNA
repair (Morris etal. 2012). As presented earlier in this chapter, both of these pro-
teins are involved in breast cancer biology. Since its depletion leads to the reduction
of cancer cell invasion and proliferation, eIF3e has been proposed to function by
positively regulating translation of genes involved in cell proliferation, invasion
and apoptosis (Grzmil etal. 2010). Elevated eIF3e levels have also been reported to
correlate significantly with a high tumor grade.
Interestingly, eIF3e (INT6) was first identified as a common integration site for
MMTV (a milk-transmitted retrovirus) in mouse mammary tumors. Insertion of
MMTV into INT6 results in a mutated allele that encodes a shortened INT6 mRNA
and protein that contributes to the neoplastic transformation of the mammary epi-
thelial cells (Rasmussen etal. 2001). Such truncated eIF3e can possibly cause a
shift from cap-dependent to cap-independent translation, activating a tumorigenic
program by increasing synthesis of IRES-driven prosurvival and proangiogenic fac-
tors, such as XIAP, c-MYC and CYR61 (Chiluiza etal. 2011). The existence of
analogous mechanism in human mammary carcinomas has not been described.
eIF3e might also be important for resistance to breast cancer treatment, since
increased levels of eIF3e have been found to be significantly associated with PFS
upon tamoxifen treatment for recurrent breast cancer (Umar etal. 2009). Hence, the
use of eIF3e as a biomarker in breast cancer has been proposed (Traicoff etal. 2007;
Umar etal. 2009).
26.14 e IF2, Its Regulation and Targeting

in Breast Cancer
eIF2 mediates the effects of various external stimuli on protein synthesis and appears
to be involved in cancer pathogenesis (see Chap.9). As discussed in Chap.9, its role
in cancer, while very suggestive, is surrounded by controversial findings. We refer
the reader to that chapter of this book for better understanding of the complexities
of the eIF2 involvement in cancer. However, in general, activation of eIF2 or
increase in its level are expected to facilitate protein synthesis and hence tumori-
genesis. Direct studies of the role of eIF2 in breast cancer are limited. Expression
of eIF2 is increased markedly in both benign and malignant neoplasms, suggesting
that elevated expression of eIF2 may contribute to tumor initiation and progres-
sion, but is not sufficient for establishing a malignant phenotype (Rosenwald etal.
2003). On the other hand, phosphorylation of eIF2 by various stress-responsive
kinases inhibits translation. In MCF-7 breast cancer cells, the excessive phosphory-
lation of eIF2 decreased survival of cancer cells (Tuval-Kochen etal. 2013). In
this system, inhibitor of eIF2 dephosphorylation salubrinal (a GADD34/PP1C
inhibitor) increased senescence and induced cell death (Tuval-Kochen etal. 2013).
In breast cancer, eIF2 phosphorylation appears to be modulated by HER2
RTK that is frequently amplified in human breast cancers (Sequeira etal. 2009).
Forced phosphorylation of eIF2 can antagonize HER2-dependent dysregulation
of mammary acinar morphogenesis. This is yet another example by which HER2
might utilize the translation machinery in breast cancer. Compounds that stabilize
phospho-eIF2 levels, such as salubrinal, may be effective in treating HER2-positive
cancers without severely disrupting normal tissue function and structure (Sequeira
etal. 2009). Various other, most likely less specific, eIF2-targeting compounds are
listed in Table26.2.
Thus, targeting eIF2 might be a promising strategy in treating breast can-
cer. Direct eIF2-targeting strategies are being developed. Novel peptidomimetic
s trategies to abolish the assembly of translation complexes, such as eIF4F and eIF2-
TC have been described (Brown etal. 2011; Chen etal. 2012; Zhou etal. 2012).
Such a small molecule inhibitor, #1181, which targets the eIF2-TC by phosphory-
lating eIF2, was shown to inhibit translation initiation and proliferation of human
cancer cells in breast cancer and melanoma models (Chen etal. 2012).
eIF2 kinases include HRI, GCN2, PKR and PERK (see Chap.9). However,
their role in cancer pathogenesis vis--vis regulation of eIF2 is rather poorly un-
derstood. Knockdown of PKR expression has no effect on cell proliferation under
normal growth conditions, but leads to significantly lower sensitivity to doxorubicin,
compared to control cells (Bennett etal. 2012). In addition, in breast cancer cell
lines with decreased PKR expression the rate of eIF2 phosphorylation following
treatment with doxorubicin was delayed, suggesting importance of PKR/eIF2 sig-
naling in conferring sensitivity to chemotherapeutic agents. Hence, approaches to
promote PKR expression/activation and eIF2 phosphorylation may be beneficial
for the treatment of breast cancer (Bennett etal. 2012). Overall, however, PKR ap-
pears to be elevated and/or activated in breast cancer (Bennett etal. 2012; Haines
etal. 1996; Kim etal. 2000; Nussbaum etal. 2003; Savinova etal. 1999). Interest-
ingly, PKR expression has also been found to be increased in precancerous stages of
mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that
PKR expression may be upregulated by the process of tumorigenesis (Bennett etal.
2012). Finally, the PERK pathway might be responsive to adhesion-regulated signals
and is essential for proper acinar morphogenesis and prevention of mammary tumor
formation (Sequeira etal. 2007). Thus, deficiencies in PERK signaling could lead to
an abnormally increased proliferation of the mammary epithelium and to tumorigen-
esis. However, the direct role of eIF2 in these perturbations requires investigations.
26.15Other Translation Initiation Factors
Several other factors, such as eIF4B, eIF2B and eIF1, have been associated with
or indirectly linked to the pathogenesis of breast cancer (Table26.1). However,
the precise role of these proteins in mammary tumorigenesis is unclear and further
studies of these and other translation initiation factors in breast cancer are required.
26.16Elongation and Termination
The role of elongation, termination and ribosome recycling (the other three steps
of translation) in cancer in general and in breast cancer in particular is much less
understood than that of the initiation step (see Chap.12). A limited number of stud-
ies are available, generally providing indirect or associative information on the
role of these steps of translation in breast cancer biology (Goncalves etal. 2005;
Malta-Vacas etal. 2009; Vislovukh etal. 2013).
A recent report suggests that eEF2 is a novel target of PI3K in the MDA-
MB-231 metastatic breast cancer cell line that facilitates cell migration (Niu etal.
2013). eEF2 is a known phosphorylation target of eEF2K that regulates the rate of
elongation (see Chap.12). eEF2K levels were shown to be increased in hormone
receptor-positive breast cancer, associating with poor prognosis (Meric-Bernstam
etal. 2012). Importantly, silencing of eEF2K suppresses growth and sensitizes
breast tumors to doxorubicin (Tekedereli etal. 2012). The role of eEF2K in breast
epithelial cells and cancer is also linked to inhibition of the protein synthesis in
hypoxia via inhibition of mTOR (Connolly etal. 2006). Inhibition of the expression
of eukaryotic elongation factor eEF1A2 by miR-663 and miR-744 has been report-
ed to slow down MCF-7 breast cancer cells proliferation (Vislovukh etal. 2013).
Eukaryotic release factor eRF3a/GSPT1 (G1 to S phase transition 1) 12-GGC a llele,
that is present in 5.1% of breast cancer-affected versus 0% of control subjects, has
been reported to increase susceptibility for breast cancer development (Malta-Vacas
etal. 2009). Patients with the 12-GGC allele overexpress eRF3a/GSPT1 in tumor tis-
sues relative to the normal adjacent tissues. However, no difference in the activity of
the eRF3a proteins encoded by the various eRF3a/GSPT1 alleles was d etected.
Breast cancer remains one of the leading causes of morbidity and mortality among
women worldwide. Despite significant advances in our understanding of cancer
in general and breast cancer in particular, this disease continues to remain one of
the top killers of women and its incidence is increasing. Thus, a refined and more
detailed look at the pathogenesis of the disease is required to devise novel and
more effective treatments. This chapter summarizes decades of research into the
development, progression and treatment of breast cancer from the perspective of
dysregulated translation. Current evidence indicates that one of the molecular hubs
in breast cancer etiology and pathogenesis is the trimeric eIF4F complex, consist-
ing of eIF4E, eIF4G and eIF4A, which is regulated by the PI3K/AKT/mTOR and
MAPK signaling cascades. eIF4E and its binding proteins, 4E-BPs, appear to be
important determinants of breast cancer biology. eIF4G appears to be a factor that is
required in the pathogenesis of inflammatory breast cancer and probably advanced
breast carcinomas. As follows from the studies of tumor suppressor PDCD4, the
eIF4A/PDCD4 axis appears to be involved in the pathogenesis and development of
chemoresistance in breast cancer. Research into these molecules might be particu-
larly fruitful, since signaling pathway regulation affects the eIF4A/PDCD4 axis,
and disruptions of signaling pathways are shown to be critical in breast cancer.
Various components of the translation machinery can be used as prognostic and
therapeutic targets. Indeed, increased eIF4E, rpS6, phosphorylated 4E-BP1, eEF2K
and decreased PDCD4 are associated with poor prognosis in breast cancer (Meric-
Bernstam 2008a, 2012).
Other hubs of dysregulation of translation in breast malignancies include the

eIF3 multiprotein complex and eIF2. The latter is subject to complex regulation
by various stress stimuli-responsive kinases, such as PKR, HRI, GCN2 and PERK.
Many of these pathways that converge on translation initiation can be targeted by
available investigational targeted compounds. Furthermore, the translation initia-
tion machinery can be directly utilized in disease diagnosis and prognosis, defining
treatment and its likelihood of success.
We are just starting to understand that the translation initiation machinery and
its control are important components of cancer pathogenesis in general, and that
specific disruptions in the control of translation initiation could lead to the devel-
opment of different types of neoplastic processes (Mathews etal. 2007). Current
research strongly supports the pivotal role that the translation machinery plays in
breast cancer pathogenesis, breast cancer therapeutic resistance and radioresistance.
Thus, developing novel therapies targeting this machinery in breast cancer has a
strong rationale.
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Chapter 27
Cancers of the Respiratory System
Armen Parsyan and Karen L. Reckamp
Contents
27.2eIF4E and 4E-BPs in Lung Cancers 559
27.2.1Expression of eIF4E 559
27.2.2Phosphorylation of eIF4E 560
27.2.3Activation of eIF4E via 4E-BPs and mTOR 561
27.2.4Expression of 4E-BPs 561
27.2.5Activity of 4E-BPs 561
27.3Indirect Targeting of the Translation Machinery in Lung Cancers 562
27.3.1Translation and Therapeutic Resistance 563
27.4Direct Targeting of the eIF4E/4E-BP Axis in Lung Cancer Therapeutics 563
27.5eIF3 in Lung Cancer 564
27.6PDCD4 in Lung Cancer 565
27.7eIF2 in Lung Cancer 566
27.8Other Translation Factors in Lung Cancer 567
27.9Translation in Mesothelioma 567
References 569
Abstract Malignancies of the respiratory system are represented by very prevalent

and lethal lung cancer. Lung cancer is the leading cause of cancer deaths worldwide.
Thus, developing effective targeted therapies that exploit our expanding knowl-
edge of the molecular biology of the disease is essential. Among these, activated
oncogenic signaling and its downstream target, the translation machinery, appear
to be key players in the etiology and pathogenesis of lung cancer. Various hubs of
translation appear to be involved, including proto-oncogene eIF4E, its suppressor
A.Parsyan()
K.L.Reckamp
Department of Medical Oncology and Therapeutics Research,
City of Hope Comprehensive Cancer Center, Duarte, CA, USA
558 A. Parsyan and K. L. Reckamp
proteins 4E-BPs, tumor suppressor PDCD4 and others. Based on the knowledge
of the pathogenic role of these proteins, elegant therapeutic strategies are being
developed that directly target these proteins. Here, we will focus our attention on
aberrations in the translation machinery, its regulation and therapeutic targeting in
lung cancer. We will also discuss interesting findings regarding the role of the trans-
lation machinery in mesothelioma of the pleura.
27.1Introduction
There are various types of cancers that originate in the respiratory system. How-
ever, only a few of them are studied in relation to translation and its regulation. The
best-studied cancer of the respiratory system is lung cancer, which has the highest
cancer incidence (excluding non-melanoma skin cancers) with estimated 1.5mil-
lion people diagnosed worldwide annually (Ferlay etal. 2008, 2010). While more
than half of these cases occur in the developing world, it is still a common cancer in
industrialized countries, ranking second in Canada (CCS 2013) and the UK (CRUK
2010) and third in the USA (CDC/NCI 2013). It is the most frequent cause of cancer
mortality in industrialized countries and worldwide, estimated to be responsible for
nearly 1.4million of annual cancer deaths (Ferlay etal. 2008).
Another rare type of cancer of the respiratory system is malignant pleural meso-
thelioma, commonly associated with exposure to asbestos. While being a relatively
rare pathological entity, it has been studied vis--vis translation with interesting
results that will also be discussed in this chapter.
One of the etiologic factors of lung cancer is tobacco smoking. A causative link
between smoking and lung cancer was established more than 60 years ago (Doll
and Hill 1950). Since then, other environmental and genetic factors were found to
participate in the pathobiology of lung cancers (Powell etal. 2013). For the sake
of this discussion, lung cancers could be conceptualized as non-small-cell carci-
nomas (NSCLC) and the less common, small-cell carcinomas (SCLC). NSCLC is
represented by adenocarcinoma and squamous and large cell carcinomas in order of
frequency (Travis etal. 2013).
Treatment of these cancers, as evidenced by the extremely high mortality rates, is
far from optimal. The arsenal includes surgical interventions, chemotherapy with var-
ious agents, radiotherapy and targeted therapy (reviewed in Lwin etal. 2013; Nana-
Sinkam and Powell 2013; Savas etal. 2013; Villaruz etal. 2013). In the rising era of
targeted therapy in oncology, it appears that treatment strategies based on knowledge
of molecular alterations provide options for better outcomes with decreased toxicity.
Hence, understating molecular targets of therapy and prevention in lung cancer is
of paramount importance. Mutations, amplifications and other aberrations that were
identified in lung cancers include KRAS, EGFR, BRAF, MET, MEK, HER2, TP53,
PTEN and other MAPK and PI3K pathway proto-oncogenes (reviewed in Cooper
etal. 2013)). From this long list of aberrations, a pattern emerges that suggests that
lung cancers originate and sustain in a milieu of altered signaling, such as PI3K/AKT/
mTOR and MAPK cascades that converge to control the ranslation machinery (see
PartII). Hence in this chapter we will attempt to provide a comprehensive summary
of our knowledge of the translation machinery in lung cancers.
27 Cancers of the Respiratory System 559
27.2eIF4E and 4E-BPs in Lung Cancers
27.2.1 Expression of eIF4E
eIF4E is a proto-oncogene and an important regulator of translation that plays key

roles in tumor transformation, progression and metastasis (see Chap.4). Higher
expression levels of eIF4E in various types of lung cancers are consistently re-
ported and, importantly, they positively correlate with worsening clinicopathologic
parameters and survival (Khoury etal. 2009; Seki etal. 2002, 2010; Wang etal.
2009; Yang etal. 2007). Moreover, siRNA-mediated silencing of eIF4E inhibits the
growth and invasion of NSCLC cells, suggesting a causative link between levels of
eIF4E and lung cancer progression (Li etal. 2012).
Using NSCLC cell lines and human tissue array, eIF4E expression was found
to be significantly elevated in cancerous tissues compared to normal ones (Li etal.
2012). Western blot analysis showed that eIF4E expression in adenocarcinomas
was 3.47.4-fold higher than in a normal lung and that its expression progressively
increased from a less to a more malignant phenotype in both tumor and stromal
components (Seki etal. 2002). Consistently, it was found that eIF4E mRNA and
protein expression (by IHC) was higher in lung adenocarcinoma tissues and cell
lines, compared to normal lung tissues and nontransformed cells (Wang etal. 2009).
IHC analysis of eIF4E using 143 tissue samples showed that eIF4E levels in the
tumor and surrounding stroma cells were elevated in bronchioloalveolar carcinoma,
atypical adenomatous hyperplasia and early and overt mixed subtypes (Seki etal.
2002). While eIF4E was found to increase in adenocarcinoma, some reports suggest
that it is only rarely overexpressed in squamous cell carcinoma types (Comtesse
etal. 2007; Rosenwald etal. 2001).
eIF4E asserts its function in tumorigenesis via selective increase in translation
of mRNAs that encode for prosurvival, proproliferative and proangiogenic pro-
teins, such as cyclin D1 and VEGF (see Chap.4). These proteins, together with
eIF4E, are found to be overexpressed in NSCLC. Yang etal. (2007) provided
evidence that eIF4E, VEGF and cyclin D1 proteins are elevated in NSCLC (81,
82 and 29% respectively), and that the elevated eIF4E levels moderately and
significantly correlated with VEGF and cyclin D1. Notably, in this study only
29% of NSCLC samples had elevated cyclin D1 expression levels, compared
to 81% that had elevated eIF4E levels. This observation somewhat agrees with
that of another study which, using an IHC and Western blot analysis of NSCLC
samples, reported 91% of eIF4E positivity, compared to only 63% for cyclin
D1 (Khoury etal. 2009). While differences in populations studied and assay
sensitivities could contribute to the discrepancies, overall these data suggest that
mechanisms other than translation contribute to the regulation of cyclin D1 in
lung cancers. Along with aforementioned eIF4E-sensitive messages, eIF4E, via
the RAS oncoprotein, appears to control translation of the Vb regulatory subunit
of cytochrome c oxidase that is required for cancer cell growth in A549 lung
adenocarcinoma (Telang etal. 2012).
Thus, eIF4E expression is important in lung cancer progression. Moreover,

eIF4E overexpression is associated with clinicopathologic parameters. Significant
correlations were observed between the higher levels of eIF4E and poorer cell dif-
ferentiation, higher pathological and clinical stages, a higher incidence of metasta-
sis and cancer-related death (Wang etal. 2009). This trend was sustained in a multi-
variate analysis. A significantly lower 5-year survival rate was observed in patients
with higher eIF4E protein expression in lung adenocarcinoma samples compared
to those with lower eIF4E expression (Wang etal. 2009). Similar results have been
reported in other studies. The 10-year survival rate of patients with pathological
stage I lung adenocarcinoma was significantly lower for patients with high IHC-
defined eIF4E expression than for patients with low eIF4E expression (Seki etal.
2010). The prognostic value of eIF4E was further strengthened by using 4E-BP1
expression levels. It is a negative regulator of eIF4E and its downregulation would
be expected to lead to activation of eIF4E. Indeed, in patients with high eIF4E ex-
pression, the 10-year survival rate was decreased when low 4E-BP1 expression was
observed (Seki etal. 2010). In this study, alongside older age and higher stage, high
eIF4E expression and low 4E-BP1 expression have been identified as factors for
unfavorable survival in stage I lung adenocarcinoma (Seki etal. 2010).
27.2.2 Phosphorylation of eIF4E
eIF4E is regulated by phosphorylation and binding by 4E-BPs. Both of these

mechanisms are controlled by signaling cascades (see Chaps.15 and 17). Recent
studies have shown that MNK-mediated eIF4E phosphorylation is required for the
oncogenic function of eIF4E (see Chap.17). Phosphorylation of eIF4E leads to
increased cap-binding of the eIF4F complex and levels of eIF4E-sensitive mRNAs,
such as c-MYC and MCL-1 (Li etal. 2010). Phosphorylation of eIF4E is also nega-
tively regulated by PP2A through direct dephosphorylation of MNK and eIF4E (Li
etal. 2010). In certain types of malignancies (such as lung, gastric and colorectal
cancers), phosphorylated eIF4E staining is significantly higher in the early stage
(T1) than in the late stage (T3) disease, suggesting that it plays a critical role in
cancer development, particularly in early stages of tumorigenesis (Fan etal. 2009).
Interestingly, increased levels of eIF4E and enhanced phosphorylation of eIF4E are
found in differentiating epithelial lung tumor cell lines (Walsh etal. 2003).
Phosphorylated eIF4E levels are found to be elevated in various types of lung
cancer (Fan etal. 2009; Yoshizawa etal. 2010). A tissue microarray analysis con-
taining 300 NSCLC samples showed overall activation of pro-oncogenic signaling
(mTOR, AKT, ERK1/2 etc.) and increased levels of phosphorylated eIF4E in ap-
proximately 40% of samples (Yoshizawa etal. 2010). OS in NSCLC patients was
significantly shorter in cases with increased phosphorylated eIF4E and AKT alone
and in combination. Those patients who exhibited an activated AKT/mTOR/eIF4E
axis had significantly shorter survival compared with the survival of all cases. Mul-
tivariate analysis showed that phosphorylated eIF4E overexpression is an indepen-
dent prognostic factor for NSCLC (Yoshizawa etal. 2010).
27.2.3 Activation of eIF4E via 4E-BPs and mTOR
A second major mechanism of regulation of eIF4E activity is via the PI3K/AKT/

mTOR signal transduction pathway that converges on eIF4E-binding proteins, 4E-
BPs, of which three isoforms exist (see Chaps.4 and 15). mTOR phosphorylates
4E-BPs, thus facilitating their dissociation from eIF4E and allowing the latter to
form the eIF4F complex. Thus activity of eIF4E could be controlled by the level
of expression of 4E-BPs and their phosphorylation/activity downstream of mTOR.
27.2.4 Expression of 4E-BPs
The 4E-BP family of proteins has been proposed to operate as guardians of a transla-
tional control checkpoint in lung tumor defense (Kim etal. 2009). Targeted disrup-
tion of dysregulated cap-dependent translation by increased recombinant expression
of 4E-BP1 or by a dominant-active mutant of this protein abrogates tumorigenicity
and enhances cell death in NSCLC, as well as effects of chemotherapeutic agents,
such as gemcitabine (Jacobson etal. 2006). Decreased levels of 4E-BP1 mRNA
and protein in NSCLC have been reported (Liu etal. 2012; Seki etal. 2010) and,
moreover, were associate with a decreased 10-year survival in patients with stage I
adenocarcinoma (Seki etal. 2010).
A causal link between 4E-BP1 and tobacco smoking in lung cancer has been pro-
posed. In a series of elegant experiments using 4E-BP1 and 4E-BP2 KO mice, an
increased sensitivity to tumorigenesis compared to wild-type controls was observed
using carcinogen from tobacco (4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone
(NNK)) (Seki etal. 2010). The 4E-BP-deficient state, likely via decreased avail-
ability for a negative control of the proto-oncogene eIF4E, creates pro-oncogenic
genome-wide skewing of the molecular landscape that includes translational acti-
vation of the cytochrome p450 enzyme isoform that activates NNK into mutagenic
metabolites (Kim etal. 2009).
27.2.5 Activity of 4E-BPs
The role of 4E-BPs in cancers is also signified by their phosphorylation via acti-
vated mTOR signaling. Furthermore, increased phosphorylation of 4E-BP1 is asso-
ciated with a poor prognosis in adenocarcinoma (Trigka et al. 2013). As evidenced
in other chapters of this book, the PI3K/AKT/mTOR pathway is upregulated in a
number of human cancers. Activation of the PI3K/AKT/mTOR pathway is a rela-
tively common event in NSCLC (less so in SCLC), occurring via various mecha-
nisms, such as increased mTOR expression, increased phosphorylation of mTOR
and AKT, activation of EGFR, loss of PTEN, PIK3CA mutations, high expression
of IGF-1R and others (Dobashi etal. 2011; Han et al. 2006; Liu etal. 2012; Trigka
etal. 2013; Yoshizawa etal. 2010). Among various lung cancer subtypes, up to
90% mTOR activation is found in adenocarcinoma (Dobashi etal. 2011). JNK is

involved in the etiology of NSCLC and has been shown to represent an alternative
pathway regulating translation via mTOR, with inhibition of JNK resulting in inhi-
bition of 4E-BP1 phosphorylation and a decrease in formation of the cap-dependent
translation complex, eIF4F (Patel etal. 2012). The importance of 4E-BPs in lung
cancer etiology and pathogenesis is further emphasized by evidence that the tumor
suppressor p53 protein, which plays a fundamental role in cancer biology, appears
to work via dephosphorylation and accumulation of the translational inhibitor 4E-
BP1, leading to an increase in the degree of inhibition of eIF4E and subsequently,
cap-dependent translation (Tilleray etal. 2006).
27.3Indirect Targeting of the Translation Machinery

in Lung Cancers
Activation of the mTOR pathway represents an attractive target via various phar-
maceutical agents, such as mTOR inhibitors, represented by rapamycin and its
analogs (rapalogs). In lung cancer cells rapamycin inhibits mTOR and suppresses
the phosphorylation of p70S6K and 4E-BP1 (Sun etal. 2005). However, it also
concurrently increased the phosphorylation of both AKT and eIF4E, attenuating
its own negative effects on cell growth. Since activation of AKT in this context
appears to be dependent on PI3K, use of rapamycin with the PI3K inhibitor
LY294002 had enhancing anticancer effects (Sun etal. 2005). Indeed, combin-
ing mTOR inhibitors with PI3K inhibition using compounds such as BEZ235
(Nyfeler etal. 2012), PF-04691502 (Yuan etal. 2011) and NYP-BKM120 (pan
PI3K inhibitor) (Ren etal. 2012) represents more effective antitumor approach
in lung cancer.
Furthermore, the combination of MEK inhibition by selumetinib and mTORC1
and 2 inhibition with AZD8055 resulted in increased apoptosis and increased anti-
tumor efficacy in an adenocarcinoma model (Holt etal. 2012). In a phase I trial, the
combination of neratinib (EGFR/HER2 TKI) with temsirolimus showed clinical ef-
ficacy in patients with HER2 mutated NSCLC (Gandhi etal. 2014).
While the discussion on pharmacologic inhibition of signaling cascades is be-
yond the scope of this chapter, in lung cancers, other novel compounds that target
signaling and hence have downstream effects on the translation machinery are be-
ing developed and tested. These include a novel oral pan-class I PI3K inhibitor
SAR245408 (XL147) (Shapiro etal. 2013) and metformin that asserts its func-
tion via AMPK (Storozhuk etal. 2013). Various other, less-studied, compounds
are claimed to inhibit the mTOR pathway and hence act via translational control in
lung cancers. Some of these include yuanhuadine from Daphne genkwa (Hong etal.
2011), dietary flavonoid fisetin (Khan etal. 2012), l-type amino acid transporter
1 inhibitor BCH (Imai etal. 2010), a new tubulin inhibitor MG-2477 (Viola etal.
2012), folate antimetabolite pemetrexed (Rothbart etal. 2010), (19Z)-HCA from
the marine sponge Chondrosia corticata (Bae etal. 2013) and 6-Shogaol, an active
constituent of dietary ginger (Hung etal. 2009). All the aforementioned compounds
have an indirect effect on the translational apparatus via signaling.
The development of novel compounds targeting the translation machinery di-
rectly via various mechanisms, such as silencing of eIF4E and inhibition of its in-
teraction with eIF4G by peptidomimetics is underway and will be discussed below.
27.3.1 Translation and Therapeutic Resistance
The translation machinery likely also plays a key role in acquired resistance to
various chemotherapeutical agents and targeted therapies in lung cancer, includ-
ing, among others, microtubule-stabilizing agent discodermolide (Chao etal. 2011),
docetaxel (Ramalingam etal. 2013), EGFR TKIs and cetuximab (Dobashi etal.
2011; Iida etal. 2013; Patel etal. 2013) and others. Rapamycin, both in general
and within the context of lung cancers, triggers the activation of survival pathways
contributing to drug resistance (see Chap.15). The resistance is mediated, at least in
part, by the eIF4E/4E-BPs axis. EGFR is found to be overexpressed or phosphory-
lated in approximately 40% of lung carcinomas, often with concomitant activation
of the mTOR pathway (Dobashi etal. 2011). In a large proportion of patients with
NSCLC, response to EGFR TKI targeted therapies, such as erlotinib and gefitinib,
is suboptimal. In this circumstance, activation of AKT and ERK1/2 has been re-
ported, resulting in eIF4F complex formation in erlotinib-resistant but not erlotinib-
sensitive cells (Patel etal. 2013). Activation and increased expression of eIF4E and
cap-dependent translation have been linked to the acquired resistance to erlotinib (Li
etal. 2012). The AKT activation (together with its substrates eIF4E and rpS6) feed-
back mechanism, has also been linked to resistance to the anti-EGFR monoclonal
antibody cetuximab (Iida etal. 2013). Resistance to EGFR targeting agents has been
shown to be responsive to targeting the eIF4F complex by various means, including
mTOR inhibitors (Ishikawa etal. 2013; Li etal. 2012). Indeed, temsirolimus and
everolimus helped to overcome resistance to EGFR TKIs in EGFR-mutant lung
cancer cells via inhibition of mTOR and subsequent phosphorylation of p70S6K
and 4E-BP1 (Ishikawa etal. 2013). In a phase II trial of everolimus and gefitinib,
62 patients received the combination with a response rate of 13%, including 2 out
of 2 patients with the KRAS G12F mutation (Price etal. 2010).
27.4Direct Targeting of the eIF4E/4E-BP Axis in Lung

Cancer Therapeutics
It follows from the aforementioned that indirect targeting of translation mechanisms

via signaling pathways may lead to various degrees of success in the treatment
of lung cancer as a single or combined therapy to tackle the resistance. How-
ever, approaches to target translation factors directly might prove more effective
(see Chap.14). These approaches are still making their first steps from in vitro stud-
ies towards preclinical evaluations.
Such strategies include the use of ASO against eIF4E or small-molecule inhibi-
tors to disrupt eIF4F formation (Patel etal. 2013). They were successfully used in
the context of attempting to enhance sensitivity to erlotinib (Patel etal. 2013).
Another approach included the use of recombinant adeno-associated virus
4E-BP1 delivery to K-RAS-LA1 lung cancer model mice (Chang etal. 2013). Im-
portant from a pharmacological perspective, such delivery is possible using aero-
sols. Long-term repeated delivery of 4E-BP1 effectively reduced tumor progression
and cancer cell proliferation by suppression of cap-dependent protein expression of
FGF2 and VEGF (Chang etal. 2013).
Yet another novel approach is to antagonize eIF4E cap-binding capabilities by
various compounds such as 7Bn-GMP or its derivatives with better pharmacologi-
cal profiles (such as tryptamine phosphoramidate prodrug of 7Bn-GMP, 4Ei-1) (Li
etal. 2013). In pharmacological studies, results demonstrated that 4Ei-1 is likely
to inhibit translation initiation by eIF4E, both, by antagonizing eIF4E cap-binding
and initiating eIF4E proteasomal degradation (Li etal. 2013). 4Ei-1 also resulted in
chemosensitization of breast and lung cancer cells to gemcitabine.
Development of anti-eIF4E peptides and other peptidomimetic design strategies
is underway (Brown etal. 2011; Fan etal. 2010; Zhou etal. 2012). One such small
molecule, 4EGI-1, inhibits cap-dependent translation by disrupting eIF4E/eIF4G as-
sociation and thus formation of the eIF4F complex, and was shown to inhibit growth
and induce apoptosis in human lung cancer cells (Fan etal. 2010). 4EGI-1 reduced
the levels eIF4E-sensitive messages, such as cyclin D1 and HIF-1, and augmented
tumor necrosis factor-related apoptosis-inducing ligand TRAIL-induced apoptosis
independent of the cap-dependent translation mechanism (Fan etal. 2010).
Other compounds have also been tested in lung cancer with various effects on
the translation machinery. Such compounds include curcumin that appears to affect
levels of expression and activation of eIF4E and eIF2 in lung adenocarcinoma
cells. (Chen etal. 2010).
27.5eIF3 in Lung Cancer
eIF3 is a large multisubunit complex whose role in disease is not well understood,
partly due to the complexity of its organization (Chap.8) One of the core and larg-
est subunits of eIF3 is eIF3a (p170) which is overexpressed in all types of human
lung cancer compared to normal tissues (Pincheira etal. 2001). Thus, while the eIF3
complex appears to facilitate translation and hence hypothetically would be ex-
pected to induce protumorigenic state, it appears that some of its components, like
eIF3a in the aforementioned example, might behave like tumor suppressors. Further
studies elucidated that p170 mRNA is expressed at higher levels in adult proliferat-
ing tissues (such as bone marrow) and tissues during development (such as fetal
tissues). This study suggests that p170 and eIF3 in general may be important factors
for cell proliferation, development, and tumorigenesis (Pincheira etal. 2001). De-
creasing eIF3a (p170) expression in the human lung cancer cell line H1299 and
breast cancer cell line MCF-7 significantly reversed their malignant growth pheno-
type (Dong etal. 2004). However, reduction of eIF3a expression only resulted in
25% decrease in global protein synthesis compared to controls, suggesting that it
may regulate the translation of a subset of mRNAs (Dong etal. 2004).
Mutations or aberrant levels of eIF3 subunits have been associated with response
to platinum-based chemotherapy in lung cancer patients (Xu etal. 2013a, b; Yin
etal. 2011). Specifically, eIF3a expression was found to be associated with chemo-
sensitization in lung cancer patients (Yin etal. 2011). eIF3a knockdown increased,
while overexpression decreased, the cellular resistance to cisplatin and anthrocy-
cline anticancer drugs (also having an effect on DNA repair activity and expression
of DNA repair proteins) (Yin etal. 2011). Three mutations of eIF3a have been
found in a screen of 771 lung cancer patients who received platinum-based therapy,
including 11279G>A in intron 6, Arg438Lys in exon 9, 29671G>A in intron 15,
with minor allele frequency of 0.16, 0.18 and 0.16, respectively (Xu etal. 2013b).
Patients with rs3740556A allele of eIF3a tended to have a favorable prognosis after
treatment with platinum-based chemotherapy (Xu etal. 2013b). Compounds that
target eIF3a in lung cancer, such as pyridine-2(1H)-one derivatives, have been sug-
gested to inhibit translation initiation (Zhu etal. 2013). However, understanding
of the mechanism of action of these mutations and the role of eIF3a in cancer in
general and in lung cancer in particular is lacking (see Chap.8).
eIF3e (INT6, p48) is ubiquitously expressed in adult tissues and early on in em-
bryonic development (Marchetti etal. 2001). Reduced expression of this protein
(frequently associated with loss of heterogeneity at its locus) has been seen in 37%
of breast cancer and 31% of NSCLC samples (Marchetti etal. 2001). Low levels
of eIF3e mRNA expression were also reported to significantly predict OS and DFS
in stage I NSCLC (Buttitta etal. 2005). Among other subunits of eIF3, eIF3h gene
coamplification with MYC was shown to significantly improve response and sur-
vival of NSCLC patients treated with the EGFR TKI, gefitinib (Cappuzzo etal.
2009). The pathophysiologic relevance of these findings is unclear and requires
more studies to determine the importance of eIF3 subunits vis--vis cancer biology.
27.6PDCD4 in Lung Cancer
The tumor suppressor PDCD4 may perform a critical function in the regulation of
lung cancer cell proliferation (Hwang etal. 2007, 2010; Jin etal. 2006). In some
types of lung cancer, mainly adenocarcinomas, decreased PDCD4 expression is ob-
served, especially in pathologically or clinically advanced stages (Chen etal. 2003;
Goke etal. 2004). IHC analysis of 124 primary carcinomas comprising all sub-
types demonstrated that PDCD4 protein expression has been widely lost in tumor
samples (83%) and negatively related to poor prognosis, higher grade and disease
stage (Chen etal. 2003). While PDCD4 expression was linked to tumor grade in
adenocarcinoma, no relationship between PDCD4 expression and clinicopathologic

parameters has been established in squamous cell carcinoma (Chen etal. 2003), and
some authors do not even find decrease of PDCD4 protein levels (Kalinichenko
etal. 2008). Together with the protein level, loss of PDCD4 mRNA expression
has been reported in human lung cancer cell lines and in lung tumor specimens
(Chen etal. 2003; Kalinichenko etal. 2008). Decrease in PDCD4 mRNA levels
was significantly associated with high-grade adenocarcinomas. The mechanism of
decreased PDCD4 mRNA in lung cancers has not been established, but could range
from gene methylation to changes in posttranscriptional control. As evidenced from
other chapters, a canonical regulator of PDCD4 protein expression is miR-21 that
downregulates levels of this tumor suppressor. miR-21 overexpression in human
squamous cell lung carcinoma was reported using miRNA microarray, and has been
associated with poor patient prognosis (Gao etal. 2011). However, how this finding
correlates with the PDCD4 protein expression is unclear, especially for squamous
cell type, where the role of PDCD4 is debatable. miR-182 has been linked to control
PDCD4 (Wang etal. 2014). miR-182 was upregulated, whereas PDCD4 was down-
regulated in lung adenocarcinoma cell lines, and the downregulation of the former
inhibited cell growth and invasion (Wang etal. 2014). While further understanding
of PDCD4 and its regulation in lung cancers is warranted, it appears that targeting
this protein might represent an effective strategy against lung cancer. Using a gene
carrier, urocanic acid-modified chitosan, PDCD4 was delivered as a nasal aerosol
into K-RAS-null lung cancer model mice and facilitated apoptosis, inhibited path-
ways important for cell proliferation, and efficiently suppressed pathways impor-
tant for tumor angiogenesis (Jin etal. 2006).
27.7 eIF2 in Lung Cancer
eIF2 represents an important hub of translational control sensitive to various stress

stimuli (see Chap.9). The role of this protein in cancer is somewhat controversial
and is under scrutiny. In lung cancer models and consistent with its regulation,
eIF2 has been implicated in the response to endoplasmic reticulum stress and un-
folded protein responses to various carcinogens, such as cigarette smoke (Jorgensen
etal. 2008; Kenche etal. 2013; Park etal. 2013). Together with mTOR signaling,
eIF2 regulatory pathways were reported altered using transcriptomic analysis in
a lung cancer model of B6C3F1 mice with spontaneous lung tumors (Pandiri etal.
2012). While there are data indicating overexpression of eIF2 in a subset of bron-
chioloalveolar carcinomas, it appears to be much less expressed or overexpressed in
squamous cell carcinomas (Comtesse etal. 2007; Rosenwald etal. 2001).
It is probably the level of eIF2 activity (as evidenced by its level of phosphory-
lation) that bears important pathogenic implications in lung cancer. Phosphorylation
of eIF2 is expected to lead to a decrease in translation and hence antitumorigenic
effects. Indeed, lung cancer patients presenting with increased levels of phosphory-
lated PKR (one of the kinases that regulates eIF2, see Chap.9) and eIF2 have
significantly longer median survival than those with relatively little phosphorylated
PKR or eIF2 (He etal. 2011). PKR and phosphorylated PKR and eIF2 were found
to be strong independent prognostic markers for overall NSCLC patient survival
(He etal. 2011). Thus, retaining the ability to signal via eIF2 and to phosphorylate
the latter provides a survival advantage.
Hence, strategies to increase phosphorylation of eIF2 are valid in terms of
tackling lung cancer (Chen etal. 2010). One such approach includes adenoviral-me-
diated overexpression of the melanoma differentiation-associated gene 7 (Ad-mda7)
that induces apoptosis in a wide range of cancer cells via induction and activation
of PKR and leads to phosphorylation of the eIF2 and the induction of apoptosis in
lung cancer cells (Pataer etal. 2002).
27.8Other Translation Factors in Lung Cancer
Frequent amplification of EIF4G on 3q27.1 in squamous cell carcinoma of the lung

is observed and significantly associates with the advanced locally-invasive T3 stage
(Comtesse etal. 2007). Interestingly, the eukaryotic translation initiation factors
eIF4AI, eIF2B and eIF4B, as well as the poly(A)-binding protein PABP were found
to be overexpressed in all lung cancer specimens (Comtesse etal. 2007). Overex-
pression (by IHC and Western blot) of the eIF4G1 protein was detected in up to
72% of squamous cell lung carcinomas compared to respective normal lung tissues
(Bauer etal. 2001, 2002). Interestingly, 15% of patients with squamous cell lung
cancer had serum antibodies against eIF4G, compared to none in control patients
without known tumor (Bauer etal. 2001).
Limited and indirect evidence exists regarding the involvement of other transla-
tion factors, such as eIF4A (Comtesse etal. 2007; Lai etal. 2012), eIF4B (Comtesse
etal. 2007; Liang etal. 2013; Shen etal. 2011), eEF1 (Byun etal. 2009) in the
biology of lung cancer and resistance to therapy. Among these, elongation factor
eEF2 was reported as highly expressed in lung adenocarcinoma, compared to neigh-
boring normal tissues (Chen etal. 2011). Using Western blot, IHC and immunofluo-
rescence microscopy authors reported that high eEF2 expression is associated with
higher incidence of early tumor recurrence and a significantly worse prognosis.
Silencing of eEF2 increased mitochondrial elongation, cellular autophagy and cis-
platin s ensitivity (Chen etal. 2011).
27.9Translation in Mesothelioma
Malignant mesothelioma (referred here as mesothelioma) is a rare form of can-

cer that develops from cells of the mesothelium, most commonly from pleura, and
iscommonly caused by exposure to asbestos. This type of cancer deserves special
attention since recent developments suggest the importance of the 4E-BPs/eIF4E

hub of translation and its targeting. Both PI3K/AKT/mTOR and MAPK pathways
are activated in mesothelioma cells, correlating with poor survival in a subset of
patients and depending on 4E-BPs and eIF4E downstream of these pathways (Ce-
dres etal. 2012; Grosso etal. 2011; Jacobson etal. 2006; Varghese etal. 2011).
Indeed, activated 4E-BP1 represses tumorigenesis and IGF-1-mediated activation
of the eIF4F complex in mesothelioma (Jacobson etal. 2006). In a mouse xeno-
graft model, mesothelioma cells expressing the dominant-active 4E-BP1 (A37/
A46) repressor protein showed abrogated tumorigenicity compared to controls.
Hence, targeting the translation machinery in mesothelioma appears to be a tempt-
ing strategy, including the use of mTOR inhibitors (Hartman etal. 2010; Hoda etal.
2011). However, a direct targeting of the translation machinery has also recently
been reported to be effective in mesothelioma. Targeting the eIF4F complex via
eIF4E with eIF4E-specific antisense oligonucleotide (4EASO) has been assessed
in mesothelioma cells, showing decrease of the eIF4E level and of the formation of
the eIF4F complex by 4EASO, compared to control, and resulting in a decrease in
proliferation of mesothelioma cells (Jacobson etal. 2013). eIF4E knockdown also
resulted in a decreased expression of antiapoptotic and proproliferative proteins, as
well as enhanced chemosensitivity (Jacobson etal. 2013).
This chapter presents a substantial body of evidence implicating the participation

of translation and its regulatory mechanisms in lung cancer. Likely, this involve-
ment is not only limited to the causation of lung cancer but also to feedback adjust-
ments to other causative drivers and overall maintenance of the disease. The eIF4F
complex activation and upregulation appear to represent etiologic and pathogenetic
mechanisms. eIF4E, the part of the eIF4F complex together with eIF4A and eIF4G,
is not only found to be overexpressed, but also activated in lung malignancies. This
activation takes place via various signaling mechanisms, where mTOR likely plays
a crucial role. Its activation signals to 4E-BPs that releases inhibitory function on
eIF4E and activates translation of prosurvival, protumorigenic and proangiogenic
signals. These proteins are also linked to various clinicopathologic parameters,
making them useful as diagnostic, prognostic and treatment response assessment
markers. Other components of eIF4F might also be involved. PDCD4 tumor sup-
pressor, which regulates eIF4A function, is most likely involved in the biology of
lung cancer. The roles of other proteins, such as eIF3 subunits, eIF2 and elongation
factors, still require further in-depth studies. Overall, 4E-BPs/eIF4E and PDCD4/
eIF4A axes appear to play a more prominent role in the pathogenesis of adenocar-
cinoma types of lung cancers versus squamous cell carcinomas. The reason for this
distinction is unclear. A small but very suggestive body of evidence exists that also
implicates the 4E-BPs/eIF4E axis and upstream dysregulated signaling in malig-
nant mesothelioma. Very little information is available in relationship to translation
and small-cell carcinomas.
Importantly, this information is already being used to devise novel approaches

for targeting cancers of the respiratory system. These include not only agents that
target the translation machinery indirectly via signaling (such as mTOR inhibi-
tors), but also directly regulating its components. These strategies include various
modes of delivery to overexpress tumor suppressors, such as 4E-BPs and PDCD4;
silencing of proto-oncogene eIF4E with antisense technologies and peptidomimet-
ics that disrupt the formation of the eIF4F complex. These strategies could be em-
ployed not only for systemic but also for local treatments. Hence, research into
translation and its regulation in lung cancer is evolving and revealing important
clinical and pharmacological cues, which can lead to better outcomes for these
deadly diseases.
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mechanism study of pyridin-2(1H)-one derivatives as regulators of translation initiation factor
3A. Arch Pharm 346:654666
Chapter 28
Gastric and Esophageal Cancers
Armen Parsyan and Lorenzo Ferri
Contents
28.2Levels of eIF4E Expression 577
28.3eIF4E Activation and 4E-BPs 578
28.4eIF4E Axis-Based Therapeutics 579
28.5mTOR Inhibitors 581
28.6PDCD4 582
28.7eIF3 584
28.8Other Translation Initiation Factors 585
28.9Translation Elongation and Termination 585
References587
Abstract Malignancies of the foregut, including, gastric and esophageal cancers,

carry a heavy healthcare burden. Gastric cancer is one of the leading causes of can-
cer-related mortality worldwide. While surgical resection remains the cornerstone
of curative intent treatment, most patients present with locally-advanced or meta-
static disease, requiring systemic therapy. The high toxicity and variable response
rates of standard cytotoxic systemic chemotherapy leave much room for improve-
ment in the management of patients with these devastating diseases. The foundation
for tackling these cancers lies in our understanding of molecular events underly-
ing their etiology and pathogenesis. The appreciation of the role of translation and
its regulation in gastric and esophageal cancers is growing and, while still in its
A.Parsyan() L.Ferri
L.Ferri
Division of Thoracic Surgery, Department of Surgery, Faculty of Medicine,
576 A. Parsyan and L. Ferri
infancy, research in this area highlights that various hubs of translation are involved.
Namely, dysregulation in expression and activity of proto-oncogene eIF4E and its
inhibitory mTOR responsive molecules, 4E-BPs, are found in these types of cancer.
Other components of the translation machinery, such as tumor suppressor PDCD4,
also appear to play an important role in gastric and esophageal cancers. The role
of these factors, their regulation, and therapeutic targeting in upper gastrointestinal
tract malignancies will be further detailed in this chapter.
28.1Introduction
Gastric adenocarcinoma is the second most common cause of cancer-related mor-

tality worldwide, contributing to almost 750,000 annual deaths (Ferlay etal. 2008,
2010). It ranks as the forth most commonly diagnosed cancer in the world. Slightly
lagging behind in its letality, incidence and prevalence is esophageal cancer (Fer-
lay etal. 2008). Prevalence and incidence of gastric and esophageal cancers vary
between geographic regions, with substantially higher proportions (7080%) found
in the developing world, mainly in Eastern Asia (gastric cancer) and South Africa
(esophageal cancer). Meanwhile in countries like the USA these cancers do not
even constitute the top-ten of the most incident and deadly malignancies (CDC/
NCI 2013). The arsenal of current treatment for gastric and esophageal cancers con-
sists of surgical treatment, including endoscopic resection, chemotherapy (5-FU,
cisplatin, capecitabine, docetaxel etc.), radiation therapy and targeted therapies (for
review see Cidon etal. 2013; Strong etal. 2013; Takahashi etal. 2013).
Major causes of gastric cancer are thought to be due to the Helicobacter py-
lori infection and diet; while esophageal cancer is caused by smoking and alcohol
(squamous type) or reflux disease and excess body weight (adenocarcinoma type)
(Freedman etal. 2007; Lochhead and El-Omar 2008; Renehan etal. 2008). Molecu-
lar underpinnings of mechanisms by which these and other environmental stimuli
promote gastric and esophageal cancers are not fully understood. However, activa-
tion or overexpression of VEGF, EGFR, HER2/neu, FGFR, hepatocyte growth fac-
tor (HGF) and c-MET are consistently linked to both cancers and represent founda-
tions for developing targeted therapies (Cho 2013; Kordes etal. 2014). Based on
these molecular alterations, a range of targeted therapies is being developed and
used in gastric and esophageal cancers (for reviews see Cho 2013; Cidon etal.
2013; Takahashi etal. 2013). These include anti-EGFR antibodies (e.g. cetuximab),
EGFR TKIs (e.g. erlotinib and gefitinib), HER2 inhibitors and TKI (trastuzumab
and lapatinib), VEGFR TKI (e.g. sorafenib and sunitinib), HGF/c-MET and FGFR
pathway targeting (tivantinib, rilotumumab and dovitinib), and mTOR inhibitors
(everolimus and other rapalogs).
It is notable for this discussion that the MAPK (including its RAS/RAF/MEK/ERK
branch) and even more so the PI3K/AKT/mTOR pathways are found activated in gas-
tric and esophageal cancers, correlating with poor prognosis and metastatic disease
(Chao etal. 2012; Hou etal. 2007; Prins etal. 2013; Yeh etal. 2011; Yu etal. 2009).
As we learned from previous chapters, these pathways converge on translational
28 Gastric and Esophageal Cancers 577
control. Moreover, some of the factors involved in upper gastrointestinal tract ma-
lignancies, such as VEGF, are sensitive to translational control. This chapter will
explore growing evidence as to the role of the translation machinery and its regula-
tion and pharmacologic targeting in gastric and esophageal cancers.
28.2Levels of eIF4E Expression
Research of the translation machinery, despite being well-advanced in cancers of

breast, prostate, colon and rectum, is at its infancy in malignancies of the foregut.
Albeit limited, the major focus of research in translation in gastric and esophageal
cancers has pivoted around the mTOR/4E-BPs/eIF4E axis. The expression level of
a translation initiation factor and proto-oncogene eIF4E is significantly higher in
gastrointestinal malignant tumors compared with normal tissue (Chen etal. 2004;
Liang etal. 2013; Martin etal. 2000; Salehi and Mashayekhi 2006). Using IHC and
Western blotting, eIF4E overexpression was found to be as high as in 74% of gas-
tric adenocarcinoma specimens where marked expression of eIF4E was present not
only in the malignant cells but also in the stromal cells of the vascular endothelium
of the tumor (Chen etal. 2004), suggesting a role of eIF4E in angiogenesis in gastric
cancers. Consistent with this notion, a significant correlation was found between
marked (more than 7-fold) eIF4E overexpression and cancer vascular invasion.
However, in this study, the degree of eIF4E expression appeared to be independent
of the invasion depth of the tumor, lymph node metastasis, Lauren classification,
Borrmann types and Helicobacter pylori infection. Notably, the prognosis for gas-
tric cancer patients with marked overexpression of eIF4E was worse (Chen etal.
2004). A pathogenetic role of the eIF4E overexpression in gastric cancers is signi-
fied by the findings that the siRNA-mediated silencing of eIF4E and thus reduction
of its expression blocks the proliferation of gastric cancer cells (Liang etal. 2013).
Similar results of increased expression of eIF4E were reported in esophageal can-
cers (Hsu etal. 2011; Salehi and Mashayekhi 2006). In both esophageal squamous
cell carcinoma and adenocarcinoma samples from 99 patients, an average 13-fold
increased expression of eIF4E levels was observed in malignant tissues compared to
adjacent normal tissues by Western blotting (Salehi and Mashayekhi 2006). A higher
expression of eIF4E correlated with advanced stages of esophageal carcinoma. An-
other study reported IHC-detected increase of eIF4E levels in almost 40% of gastric
cancers (Hsu etal. 2011). Increased expression of eIF4E protein was found to be as-
sociated with an increase in its mRNA levels. Interestingly, this study also reported
a significant positive association between the eIF4E and MDM2 expression, which
is an important negative regulator of the p53 tumor suppressor (Hsu etal. 2011).
Extrapolating from these studies, it appears that the eIF4E protein has a patho-
biological significance in gastric and esophageal cancers where it is often found
overexpressed. The overexpression of eIF4E in these cancers is likely a reflection
of an increased transcription of the eIF4E mRNA, likely by c-MYC oncogene (see
Chap.4). Indeed, in gastric cancers MYC was found to promote transcription of
MCL-1 and eIF4E genes (Labisso etal. 2012).
28.3eIF4E Activation and 4E-BPs
Control of eIF4E activity takes place via its binding partners, 4E-BPs or by direct
phosphorylation (see Part II). Activating phosphorylation of eIF4E is achieved by
MNK1/2 through the activation of the MAPK pathway (see Chap.17). Although
earlier studies did not find significant differences in the relative amount of the phos-
phorylated form of eIF4E between gastric cancer tissue and normal control samples
(Martin etal. 2000; Salehi and Mashayekhi 2006), more recent investigations have
consistently reported a marked proportional increase of phosphorylated eIF4E in
tumorous compared to corresponding normal tissues (Fan etal. 2009; Liang etal.
2013; Yang etal. 2013). Increase in eIF4E phosphorylation was found to be posi-
tively associated with an early stage (T1) disease (Fan etal. 2009), while its in-
volvement in lymph node metastasis is somewhat controversial (Fan etal. 2009;
Yang etal. 2013).
One of the major mechanisms that control the activity of eIF4E is via the mTOR
pathway that signals to 4E-BPs and phosphorylates them. Once phosphorylated,
4E-BPs dissociate from eIF4E, thus making it available for the formation of the
eIF4F complex and initiation of translation (see Chap.4). The levels and/or activity
of 4E-BPs (the major isoform of which is 4E-BP1) can thus control the effects of
eIF4E. Within this context, 4E-BPs could be conceptualized as tumor suppressors;
hence, their levels in cancers would be expected to be low. Interestingly, a few ear-
lier studies reported increased expression of 4E-BP1 and its dephosphorylation in
cancerous versus normal gastric tissues (Martin etal. 2000; Salehi and Mashayekhi
2006). However, more and more recent reports point on findings opposite to the
aforementioned earlier reports and suggest activation of the mTOR pathway and
suppression of the inhibitory function of 4E-BPs on eIF4E (Jiao etal. 2013; Kamata
etal. 2007; Lang etal. 2007; Sun etal. 2014; Yang etal. 2013). In a recent study,
significantly lower expression of mTOR and 4E-BP1 proteins was found in gastric
adenocarcinoma tissues compared to adjacent normal gastric mucosa (Yang etal.
2013). Moreover, a marked proportional increase of phosphorylated mTOR, signi-
fying activation of the mTOR cascade, was reported in tumor tissues (Yang etal.
2013). Furthermore, yet another recent study described activation of the mTOR
pathway in gastric cancers (Sun etal. 2014). Indeed, phosphorylated mTOR and
phosphorylated 4E-BP1 were found in 71% and 68% of the human gastric cancer
tissues tested, respectively (compared to a significantly lower level of phosphoryla-
tion of these proteins found in adjacent nonmalignant (50% and 58%) and relatively
distant normal tissues (45% and 29%)) (Sun etal. 2014). Phosphorylated 4E-BP1
overexpression was reported to have an inverse correlation with median survival
time (Jiao etal. 2013). Under specific circumstances, activation of AKT and the
mTOR/S6K and the mTOR/4E-BP1 axes appears to be mediated by CXCL12, a
chemokine that has been shown to play an important role in the development of
peritoneal carcinomatosis from gastric carcinoma (Yasumoto etal. 2006). Activa-
tion of S6K and 4E-BP1 in this milieu led to enhancements of metastatic properties,
such as MMP production, increased cell migration and cell growth in peritoneal
disseminated gastric cancer NUGC4 cells (Hashimoto etal. 2008). In this model,
administration of the mTOR inhibitor rapamycin drastically inhibited migration and

induced programmed cell death and autophagic cell death (Hashimoto etal. 2008).
The mTOR pathway is also found activated in both esophageal squamous cell
carcinoma and adenocarcinoma, where high expression of phosphorylated mTOR
or phosphorylated 4E-BP1 has been associated with poor oncologic outcomes (Chao
etal. 2012; Hou etal. 2007; Prins etal. 2013; Yeh etal. 2011). The effect of an in-
crease in phosphorylated 4E-BP1 on survival was significant only in patients with
relatively early stage 1 or 2 disease (Yeh etal. 2011). In another study of 60 early
stage T1 or T2 esophageal squamous cell carcinoma patients, the 5-year disease-spe-
cific survival of patients with a high phosphorylated 4E-BP1 (by IHC and Western
blotting) level was also found to be significantly lower than that of patients with low-
er levels (Chao etal. 2012). Moreover, high levels of phosphorylated 4E-BP1 after
chemoradiotherapy were shown to be a predictor for loco-regional recurrence and
worse survival in esophageal squamous cell carcinoma patients (Chao etal. 2012).
In one study the levels of phosphorylated 4E-BP1 were not found to meaningfully
correlate with age, gender, preoperative concurrent chemoradiotherapy, tumor grade,
TNM status or stage of the disease (Yeh etal. 2011).
Thus, although somewhat controversial, there is ample recent data to support the
notion that eIF4E proto-oncogene is activated in both esophageal and gastric can-
cer, either via phosphorylation of the protein or via disinhibitory phosphorylation of
4E-BPs. This activation of eIF4E takes place via oncogenic signaling mechanisms.
While more studies are required, activation of the 4E-BP/eIF4E axis in gastric and
esophageal cancers appears to bear important clinicopathologic, diagnostic, prog-
nostic and, as we shall see in the upcoming section, therapeutic value.
28.4eIF4E Axis-Based Therapeutics
Various strategies to target eIF4E and 4E-BPs in cancer are discussed in Chaps.4
and 14. Since, as described earlier, the PI3K/AKT/mTOR/4E-BPs/eIF4E signaling
axis appears to be activated in gastric and esophageal cancers, studies of inhibitors
of the pathway, such as mTOR inhibitors, were performed in preclinical settings.
Rapamycin alone, or even more so in combination with PI3K inhibitor LY294002,
markedly inhibited gastric cancer cell proliferation via reduction of phosphorylation
of translational regulators p70S6K and 4E-BP1 and induction of G1 cell cycle arrest
(Sun etal. 2014). Similarly, in esophageal squamous cell carcinoma, rapamycin and
siRNA against mTOR rapidly inhibit expression of mTOR and the phosphorylation
of its major downstream effectors, p70S6K and 4E-BP1, leading to cell cycle ar-
rest in G0/G1 phase (Hou etal. 2007). Overexpression of 4E-BP1 in gastric cancer
cells using an adenovirus vector enhanced rapamycin inhibitory effect on prolif-
eration and tumor growth (Mishra etal. 2009), consistent with a report that low
eIF4E/4E-BPs ratio is protective for drug resistance in cancers (Alain etal. 2012).
Phosphorylation of 4E-BP1 was suggested to be a predictive biomarker of everoli-
mus sensitivity in gastric cancer (Nishi etal. 2013). Basal phosphorylation levels of
4E-BP1 were significantly higher in everolimus-sensitive gastric cancer cell lines

than in those that were everolimus-resistant. Everolimus may also have a positive
effect in controlling gastric cancer, as in cases with peritoneally disseminated dis-
ease, where the mTOR pathway and 4E-BP1 are activated (Taguchi etal. 2011).
In a model of metastatic gastric cancer with activation of the AKT/mTOR/4E-BPs
axis, rapamycin drastically inhibited migration and induced both programmed and
autophagic cell death (Hashimoto etal. 2008). A novel dual PI3K/mTOR inhibi-
tor NVP-BEZ235 (BEZ235), alone and in combination with nanoparticle albumin-
bound (nab)-paclitaxel, inhibited cell proliferation in an experimental gastric cancer
model via modulation of the PI3K/mTOR pathway (Zhang etal. 2013a).
However, pharmacologic targeting of mTOR to suppress eIF4E function often
leads to feedback activation of other signaling mechanisms that may diminish an
expected pharmacologic efficacy (see Chap.15). eIF4E functions in translation
within a trimeric eIF4F complex consisting of two other proteins, eIF4A helicase
and eIF4F scaffolding protein (see Chap.2). It appears that direct targeting of eIF4E
alone or as a component of the eIF4F complex might represent a more efficient
strategy to overcome feedback activation (Issaenko etal. 2012). One such exam-
ple stems from a study of the ERBB2-amplified esophageal adenocarcinoma cells
(Issaenko etal. 2012). In these cells targeting of the eIF4F complex directly by
a constitutively active form of 4E-BP1 triggered apoptosis and decreased colony
formation and cell proliferation. In contrast, targeting eIF4F indirectly via pharma-
cologic targeting of mTOR by rapamycin only modestly inhibited malignant phe-
notype, likely due to the feedback activation of other oncogenic pathways. Another
approach to directly target eIF4F is to silence expression of their components using
ASO technology. siRNA silencing of eIF4E inhibits tumor cell growth in a gastric
cancer model, thereby demonstrating a therapeutic potential (Liang etal. 2013).
While rapamycin and rapalogs were found to be effective in gastric cancer mod-
els, they also appear to increase sensitivity to chemotherapeutic agents when given
in combination (Kamata etal. 2007; Nishi etal. 2013; Shigematsu etal. 2010;
Sun etal. 2008; Taguchi etal. 2011). In gastric cancer cell lines, where the mTOR
pathway appears to be activated, as evidenced by phosphorylation of p70S6K and
4E-BP1, it appears to be involved in mechanisms of resistance to conventional che-
motherapeutic agents, emphasizing the important potential of using inhibitors of
the pathway together with conventional chemotherapeutics (Kamata etal. 2007;
Xu etal. 2012). Rapamycin has been shown to enhance the effects of docetaxel and
5-FU on a panel of gastric carcinoma cells (TMK-1, MKN-28, MKN-45 and MKN-
74) by a factor of up to 4. In combination with docetaxel, rapamycin decreased
the phosphorylation of 4E-BP1 (Shigematsu etal. 2010). Rapamycin significantly
intensified the cytotoxic action of cisplatin in -fetoprotein-producing gastric car-
cinoma (AFPGC), a highly malignant variant of gastric cancer that appears to be
resistant to multiple chemotherapeutic agents, including cisplatin (Kamata etal.
2007). Rapamycin was also shown, in the mTOR/4E-BP1 dependent manner, to
enhance an otherwise moderate anticancer effects of the chemotherapeutic agent
5-aza-2deoxycytidine (also known as decitabine) on gastric cancer cell lines MKN-
45 and SGC-7901 (Sun etal. 2008). Furthermore, rapamycin was found to synergize
strongly with the proteasome inhibitor bortezomib to inhibit growth of esophageal

adenocarcinoma cells (Issaenko etal. 2012).
Various other compounds targeting the translation machinery have been studied
in gastric and esophageal carcinoma. However, the specificity of these effects on
the translation machinery needs further investigations. For example, initial studies
have suggested that matrine from Sophora flavescens could inhibit tumor growth
and induce apoptosis in various cancer cells in vitro. One study found that ma-
trine, likely via ERK1/2, inhibited activity of eIF4E through dephosphorylation of
4E-BP1 in a dose- and time-dependent manner in gastric cancer MKN-45 cells
(Jiang et al. 2007). Another compound, 13,14-bis(cis-3,5-dimethyl-1-piperazinyl)-
elemene (IIi), a novel elemene derivative, shows potent antitumor activities at least
partly via inhibition of mTOR in various cancers (Li etal. 2013; Zhang etal. 2013b;
Zhang etal. 2013c). IIi was found to be cytotoxic to a wide spectrum of human
cancer cells in culture, including gastric cancer cells lines MKN-45 and SGC-7901
(Ding etal. 2013). IIis effects at least in part appear to be conferred by decreased
levels of phosphorylated p70S6K1 and 4E-BP1 (Ding etal. 2013). Perifosine, an
alkylphospholipid that exhibited antitumor activity in a series of cancer types, in-
hibited the growth of gastric cancer cells via the downregulation of eIF4E and levels
of phosphorylated eIF4E (Liang etal. 2013).
28.5mTOR Inhibitors
Everolimus (RAD001), an oral inhibitor of mTOR, alone or in combination with a

chemotherapeutic agent, has been shown to have good safety and efficacy profile
against gastric cancer in phase I/II studies (Doi etal. 2010; Lee etal. 2013; Wer-
ner etal. 2013). While detailing the results of these trials is outside of the scope
of this book, it is worth mentioning the results of a recently-published phase III
randomized, double-blind GRANITE-1 study of everolimus for previously-treated
advanced gastric cancer (Ohtsu etal. 2013). This study, which enrolled 656 pa-
tients, concluded that, compared to the best supportive care, everolimus did not
significantly improve OS for advanced gastric cancer that had progressed after one
or two lines of previous systemic chemotherapy. Although overall a negative trial,
this study showed that everolimus significantly reduced the risk of progression by
34%. It is possible that the use of this mTOR inhibitor would be most effective in
a subset of gastric cancer patients, ideally those that harbor high eIF4E-expressing
tumors or those with the activated 4E-BP/eIF4E axis. This type of analysis was not
performed in the GRANITE-1 trial.
While numerous trials are registered in the clinicaltrials.gov database, looking at
mTOR inhibitors alone or in combination, another phase III randomized, double-
blind trial evaluating paclitaxel with and without everolimus in patients with gastric
carcinoma after prior chemotherapy (AIO-STO-0111) is underway (see clinicaltrials.
gov and Cho 2013). Further studies of these compounds in gastric and esophageal
cancers will most likely establish their role in the management of these conditions.
28.6PDCD4
PDCD4 is a tumor suppressor with a large potential for therapeutic targeting

(Chap.6). The role of PDCD4 in esophageal and gastrointestinal cancers is being
increasingly studied, showing that PDCD4 is an essential component of the etiology
and pathogenesis of these cancers (Cao etal. 2012; Guo etal. 2013; Ma etal. 2013;
Tu etal. 2013). Lower expression of PDCD4 protein and mRNA in gastric adeno-
carcinoma and both histologic subtypes of esophageal cancer (compared to normal
respective tissues) has been shown by numerous studies (Cao etal. 2012; Fassan
etal. 2010a, b, 2012; Guo etal. 2013; Hiyoshi etal. 2009; Ma etal. 2013; Mishra
etal. 2009; Motoyama etal. 2010; Tu etal. 2013).
Asides from levels of expression of PDCD4, its proportional nuclear versus cy-
toplasmic distribution may be associated with the gastric and esophageal adenocar-
cinomas (Fassan etal. 2012; Kakimoto etal. 2011). While PDCD4 participation in
tumorigenesis can occur via different mechanisms (see Chap.6), in gastric cancer
cells, this protein may act via enhancing cellular sensitivity to TRAIL-induced (tu-
mor necrosis factor-related apoptosis induced ligand) apoptosis by downregulating
expression of FLIP (a negative regulator of apoptosis), and by inhibiting the PI3K/
AKT signaling pathway (Wang etal. 2010a, b).
Levels of PDCD4 correlate with various clinicopathologic factors, making it a
putative prognostic and diagnostic marker in gastric cancers. Using Western blot
analysis and IHC, PDCD4 was shown to be significantly downregulated in gastric
cancer specimens as compared to noncancerous tissues (Cao etal. 2012; Guo etal.
2013; Ma etal. 2013). Loss or reduced PDCD4 expression were observed in over
one third (79/215) of gastric cancer specimens (Cao etal. 2012). Expression of
PDCD4 mRNA in gastric cancer tissues was significantly lower than in non-cancer
tissues, and low PDCD4 mRNA expression correlated with size, depth, lymph node
metastasis, venous invasion, advanced stage, and poor clinical prognosis (Guo etal.
2013; Motoyama etal. 2010). Decrease in PDCD4 protein expression is associ-
ated with higher grade and advanced stage (Guo etal. 2013; Ma etal. 2013). The
relationship between PDCD4 protein expression and clinicopathologic features of
patients with pT2 stage gastric cancer was examined in a recent study (Guo etal.
2013). In pT2 stage gastric cancers, decreased PDCD4 expression was associated
with a reduction in cumulative survival rate of gastric cancer patients (Guo etal.
2013). However, in another study that found downregulation of PDCD4 expression
in gastric cancers, altered PDCD4 expression was not significantly associated with
the clinicopathologic parameters, including tumor differentiation, location, lymph
node metastasis and OS (Cao etal. 2012). Thus, while PDCD4 is consistently re-
ported to be downregulated in a subset of gastric cancers, its role in clinicopatho-
logic prognostication still requires further studies.
Similar findings of PDCD4 downregulation are reported for esophageal malig-
nancies. The IHC expression of PDCD4 protein in 63 esophageal adenocarcinomas
and 48 squamous cell carcinomas and paired non-cancerous samples showed de-
creased PDCD4 expression in cancerous tissues (Fassan etal. 2010a). A significant
inverse correlation was found between nuclear PDCD4 expression and tumor stage,
pT, nodal metastasis, and perivascular and perineural invasion (Fassan etal. 2010a).
Moreover, PDCD4 expression predicted the patient outcome in esophageal cancers,
and its nuclear expression status was associated with a longer DFS and OS (Fassan
etal. 2010a). Interestingly, a progressive decrease in PDCD4 expression, but also
specific patterns of distribution of the protein within the cell, are observed from
preneoplastic to neoplastic lesions of the esophagus (Fassan etal. 2010b). PDCD4
IHC expression was assessed in 88 biopsy samples with non-intestinal or intestinal
columnar metaplasia, various grades of intraepithelial neoplasia or Barretts adeno-
carcinoma (Fassan etal. 2010b). PDCD4 expression levels decreased progressively
and significantly with the progression of the phenotypic changes occurring during
Barretts carcinogenesis (Fassan etal. 2010b). Normal basal squamous epithelial
cells had strong PDCD4 nuclear staining and weak-to-moderate cytoplasmic stain-
ing. Compared with histologically proven normal esophageal mucosa, PDCD4 ex-
pression was decreased in 25 endoscopic esophageal mucosal resection samples
performed for squamous intraepithelial or squamous intramucosal neoplasia (Fas-
san etal. 2012).
While various factors were reported to play none-to-minimal (such as genetic
mutations of PDCD4 gene) or mild-to-moderate roles (such as promoter hyper-
methylation) (Cao etal. 2012) in downregulation or dysregulation of PDCD4 ex-
pression in gastric and esophageal cancers, it appears that silencing of this protein
expression by miR-21 plays a fundamental role in cancer biology. miR-21 was
significantly upregulated in gastric cancers, correlating with the decreased levels
of PDCD4 expression, advanced tumor stage, lower degree of differentiation and
presence of local lymphatic node metastasis and remote metastasis (Tu etal. 2013).
Indeed, downregulation of PDCD4 expression in gastric cancers is most likely to
be a reflection of upregulated miR-21 expression. miR-21 overexpression has been
reported in almost 70% of gastric cancers with a significant inverse correlation
between miR-21 and PDCD4 protein and mRNA expression (Cao etal. 2012; Mo-
toyama etal. 2010; Tu etal. 2013). Induction of miR-21 in gastric cancers was
shown to be associated with the reactive oxygen species and oxidative stress (Tu
etal. 2013). A role of zinc deficiency in pathogenesis of esophageal carcinoma
through the induction of specific miRNAs, such as miR-21 and the downregulation
of their targets, has been suggested (Alder etal. 2012).
miR-21 also regulates proliferation and invasion in esophageal squamous cell
carcinoma (Hiyoshi etal. 2009). miR-21 expression was investigated in normal
esophageal epithelial samples matched with esophageal squamous cell carcinoma
samples and esophageal squamous cell carcinoma cell lines (TE6, TE8, TE10,
TE11, TE12, TE14 and KYSE30). Of 20 paired samples, 18 cancer tissues overex-
pressed miR-21 in comparison to their respective matched normal epitheliums. In-
terestingly, a significantly high expression of miR-21 was observed in samples from
the patients with lymph node metastasis or venous invasion. miR-21 expression
was also significantly upregulated in high-grade intraepithelial neoplasia or Bar-
retts adenocarcinoma, compared to non-cancerous mucosa (Fassan etal. 2010b).
Thus, a pattern emerges by which increasing levels of miR-21 associate with wors-
ening pathological parameters in esophageal carcinoma. Expectedly, this increase
in miR-21 expression inversely correlates with PDCD4 expression (Fassan etal.

2012; Hiyoshi etal. 2009). Moreover these findings are not just correlative, since
anti-miR-21-transfected cells increased PDCD4 protein expression without chang-
ing the PDCD4 mRNA level and increased a luciferase reporter activity containing
the PDCD4 3 UTR construct (Hiyoshi etal. 2009). However, it is important to bear
in mind that miR-21 targets, together with PDCD4, also include tumor suppressor
PTEN and putative metastatic suppressor RECK (reversion-inducing-cysteine-rich
protein with kazal motifs). Hence some effects of miR-21 are likely mediated not
only by downregulating PDCD4, but by those proteins too. Nevertheless, target-
ing miR-21 in cancers in general and gastro-esophageal cancers in particular might
be a promising anticancer strategy. AC1MMYR2, an inhibitor of DICER-mediated
biogenesis of miR-21, was shown to reverse EMT and suppress tumor growth and
progression, likely via upregulated expression of PTEN, PDCD4, and RECK in
gastric cancer cells, but also in glioblastomas and breast cancer (Shi etal. 2013).
28.7eIF3
eIF3a, a subunit of a multiprotein eIF3 complex whose exact role in cancer is still
debated mostly due to a complex subunit organization, appears to be implicated in
gastric and esophageal cancer biology (see Chap.8).
eIF3a may play some role in development and differentiation, with the decreased
eIF3a expression being a prerequisite of intestinal epithelial cell differentiation (Liu
etal. 2007). eIF3a expression has been found elevated in fetal tissues compared to
postnatal tissues. Whereas the expression of eIF3a in the intestine, stomach, and
lung stops abruptly on the 18th day of gestation, its expression, albeit at lower lev-
els, persists throughout gestation in the liver, heart and kidneys (Liu etal. 2007).
Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, is drasti-
cally decreased prior to differentiation. Recombinant eIF3a overexpression inhib-
ited Caco-2 differentiation and RNAi silencing of eIF3a-promoted Caco-2 differ-
entiation (Liu etal. 2007), signifying a role of this protein in differentiation events
of intestinal cells.
eIF3a (p150) has been studied by IHC and by Western blotting in patient-derived
esophageal squamous-cell carcinoma samples revealing a positive correlation be-
tween levels of its expression and the state of differentiation (Chen and Burger
1999). Tumors with high levels of eIF3a expression were more likely to be well-
differentiated cancers, have fewer metastases and a significantly better OS than
those with low levels of eIF3a expression. Multivariate analysis showed that eIF3a
is an independent factor for predicting patient survival, suggesting that this protein
can be used as a biomarker for prognosis (Chen and Burger 1999). Expression of
eIF3a (p150) was assessed by IHC in 102 and by Western blotting in 14 gastric car-
cinoma samples, revealing a close association between eIF3a expression levels and
the clinicopathologic parameters of gastric cancer (Chen and Burger 2004). Simi-
larly, high eIF3a expression has been more commonly seen in well-differentiated,
non-metastatic tumors at early stages of disease (Chen and Burger 2004). While
eIF3a appears to represent an interesting clinicopathologic marker, this seemingly

protective role of eIF3a in esophageal and gastric cancers, as described above, is un-
clear. Being a factor that promotes translation, increase of eIF3a as a part of the eIF3
complex, would expect to facilitate prosurvival and protumorigenic state. However,
it also appears from the aforementioned data that eIF3a plays an important role in
the intestinal cell differentiation, where its increase associates with increased dif-
ferentiation, while its loss would facilitate worsening of pathological parameters.
Thus, experimental and clinical data suggest that eIF3a could play a role of a tumor
suppressor in cancers of the upper gastrointestinal tract.
28.8Other Translation Initiation Factors
The role of other translation factors in gastric and esophageal cancers is much
less studied and understood. However, there are anecdotal reports and indications
that other components of the translation machinery are involved. For example, in
a prospective, high-throughput transcriptional profiling study to assess signature
of acquired chemoresistance to cisplatin and 5-FU combination chemotherapy in
gastric cancer, the most highly represented functional category in the acquired re-
sistance signature included eukaryotic translation initiation factors (eIF4B, eIF3d,
eIF3e, eIF3f and eIF3h), together with AKT1 and ribosomal protein mRNAs (RPS6,
RPL13, RPL14, RPL15, RPL18, RPL29 , RPL3, RPL30 , RPL4, RPS11, RPS19 and
RPS9) (Kim etal. 2011). eIF2 has been implicated in gastric cancers but has been
poorly studied (Lobo etal. 2000). The levels of unphosphorylated and phosphory-
lated eIF2, as well as the percentage of phosphorylated factor over the total, were
found to be significantly higher in gastric and colorectal tumors compared with
normal tissues from the same patients (Lobo etal. 2000) (see Chap.9 for the role
of eIF2 in cancer).
28.9Translation Elongation and Termination
Data on the role of elongation factors in gastric cancers are available from anecdotal
reports, while almost no data exist in this area in esophageal cancers. An isolated
study of the protein and mRNA levels of the A1, B, B or B subunits of eEF1,
has reported that levels of expression of eEF1 subunits in cancer could be misbal-
anced and that the disintegration of eEF1B could be an important sign of cancer
development (Veremieva etal. 2011). Interestingly, it has also been reported that the
individual subunits can exist separately from the eEF1B complex.
eEF2 protein has been found to be overexpressed in 93% and 92% of gastric
cancers and CRCs respectively (Nakamura etal. 2009). No mutations of the gene
were identified. shRNA-mediated knockdown of eEF2 was found to inhibit growth
of two gastric cancer cell lines (AZ-521 and MKN-28) and one colon cancer cell
line (SW620) by inducing G2/M arrest and inactivating AKT and a G2/M regulator
CDK1. Recombinant expression of eEF2 in gastric cancer cells significantly en-

hanced the cell growth through promotion of G2/M progression in cell cycle, ac-
tivated AKT and CDK1, and inactivated eEF2K and enhanced tumorigenicity in a
mouse xenograft model (Nakamura etal. 2009).
While translation termination is generally not considered a major step of the
translation process involved in tumorigenesis, there are studies that investigate the
role of eukaryotic release factors, such as eRF1 and eRF3, in gastric cancer (Brito
etal. 2005; Malta-Vacas etal. 2005; Malta-Vacas etal. 2009). It is hard to draw
definitive conclusion from these mostly anecdotal reports. An isolated report sug-
gests that the eRF3 transcript is differentially overexpressed according to the gastric
cancer histological type (Malta-Vacas etal. 2005). Gene expression levels of eRF3/
GSPT1 in 25 gastric tumor biopsies and adjacent non-neoplastic mucosa were ana-
lyzed, revealing that the eRF3 transcript was overexpressed in 8 of the 12 intesti-
nal type carcinomas, but only in 1 out of 10 diffuse type carcinomas (Malta-Vacas
etal. 2005). Another study from the same group suggested that the GGC expansion
might have a potential role in regulating eRF3/GSPT1 expression and/or changing
the protein function that can be involved in gastric cancer pathogenesis (Brito etal.
2005). Overexpression of eRF3/GSPT1 was not associated with increased transla-
tion rates because the upregulation of eRF3/GSPT1 was not found to correlate with
the increased eRF1 levels (Malta-Vacas etal. 2005). The biological or clinical rel-
evance of these findings is unclear.
While there are more questions than answers in the realm of translational regulation
in gastric and esophageal cancers, the birds eye view on the current findings in this
area delineates the importance of the dysregulation of the translational landscape
in the biology of these forms of malignancy. In contrast to other tumor types, gas-
tric cancers appear to present with dysregulation in three out of four major steps
of translation: initiation, elongation and termination. Of these three, the initiation
step is the most studied and potentially the most involved in the development and
progression of not only gastric, but also esophageal carcinomas. Within translation
initiation, the control of the eIF4F complex is a main etiologic and pathogenetic
battlefield. As detailed in the other chapters of this book, the eIF4F complex is a
trimeric complex that consists of the eIF4E cap-binding protein, the eIF4G scaffold-
ing protein and the eIF4A helicase, and is a subject of regulation by major oncogen-
ic signaling pathways, such as PI3K/AKT/mTOR and MAPK cascades. In cancers
discussed in this chapter, eIF4E is often found overexpressed or activated directly
or via inhibition of 4E-BPs via signaling cascades. Activation of eIF4E, which is
a proto-oncogenic molecule, leads to a selective upregulation of prosurvival, an-
tiapoptotic and protumorigenic signals (see Chap.4). On the other hand, tumor
suppressor PDCD4, which negatively regulates eIF4A component of eIF4F (see
Chap.6), is often found downregulated in gastric and esophageal cancers. Thus,
tumorigenic signals in a multidirectional manner converge on the control of the

eIF4F complex to upregulate the translation machinery. Our understanding of the
role of other components of the translation machinery is still in its infancy. How-
ever, available pieces of information strongly suggest that eIF4E, 4E-BPs, eIF3 and
PDCD4 can be further explored and used as potential diagnostic, prognostic and
therapeutic targets in gastric and esophageal cancers.
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Chapter 29
Colorectal Cancers
Armen Parsyan, Nathaniel Robichaud and Sarkis Meterissian
Contents
29.2Translation and Colorectal Cancers 595
29.2.1eIF4E and 4E-BPs 595
29.2.2PDCD4 597
29.2.3eIF3 598
29.2.4eIF2 599
29.2.5eIF5A 600
29.2.6Other Translation Factors 600
29.3Translation and Colorectal Cancer Therapy 600
References 604
AbstractColorectal cancers (CRCs) are malignancies with high incidence and

mortality. Due to the introduction of screening programs leading to early diagno-
sis and treatment, a decrease in mortality from these cancers is being observed.
However, therapeutic options for CRC, especially in advanced disease, are limited
and require improvements. Development of targeted therapies based on the under-
standing of molecular alterations is a promising strategy. Our knowledge of the
molecular environment that contributes to CRC is expanding. As research has pro-
gressed, we have come to realize that the protein biosynthesis pathway and its trans-
lational apparatus contribute significantly to cancer development and progression in
Division of General Surgery, Department of Surgery,
N.Robichaud
Department of Biochemistry and Rosalind and Morris Goodman
S.Meterissian
general, and to CRC in particular. Among the various steps of protein synthesis,
dysregulation of the process of translation initiation appears to play a key role in
CRC. This chapter summarizes our knowledge of the role of translation and its
regulatory mechanisms in CRC development and progression, and discusses mod-
ern therapeutic approaches targeting the translation machinery.
29.1Introduction
Colorectal cancers (CRCs) are the third most common group of cancers and the
fourth most common cancer-related cause of death in the world (Ferlay etal. 2008;
Ferlay etal. 2010). Similarly, in industrialized countries it is generally among the
top five causes of cancer-related mortality and morbidity (CCS 2013; CDC/NCI
2013; Ferlay etal. 2008). The implementation of screening strategies and improve-
ments in treatment have resulted in a trend towards the decrease of the morbidity
and mortality associated with this disease (Edwards etal. 2010). Current clinical
management of CRC includes a multidisciplinary approach that incorporates sur-
gery, radiation, chemotherapy and targeted therapy (Engstrom etal. 2005). Chemo-
therapy regimens are recommended for advanced metastatic disease and in adjuvant
and neoadjuvant settings. A number of therapeutic agents are used in CRC or meta-
static CRC, such as 5-FU, leucovorin, irinotecan, oxaliplatin and capecitabine. Dif-
ferent combinations of these drugs, with or without a targeted therapy agent, have
been shown to improve the outcomes in metastatic CRC (for review see (Benson
etal. 2013a, b; Edwards etal. 2012; Peeters and Price 2012). Targeted therapy
options include monoclonal antibodies, such as bevacizumab (Avastin, an inhibitor
of VEGF-A) and cetuximab (anti-EGFR), and other molecules, such as regorafenib
(multiple RTK inhibitor) and aflibercept (anti-VEGF agent) (for reviews see (Ben-
son etal. 2013a, b; Cheng etal. 2013)). While adding targeted therapeutic agents
to chemotherapeutic regimens in the treatment of patients with metastatic CRC is
effective, the promise of those in the adjuvant setting is less straightforward, and
several clinical trials have reported failures to improve clinical outcomes (Van Loon
and Venook 2011). Hence, it is recommended that the existing paradigm for the se-
lection of agents in the adjuvant treatment of CRC be reconsidered (Van Loon and
Venook 2011). In light of this, improvements in the efficacy of current medications
to treat both metastatic and nonmetastatic CRC warrant further research efforts.
Achieving better therapies for CRC requires a refined understanding of the biology
of these cancers.
CRCs can arise due to genetic mutations and chromosomal instability, which
occur in either a hereditary or sporadic manner. While these are outside of the
scope of our discussion, it is worth noting that a large proportion of these aberra-
tions involves oncogenic pathways converging on the translation machinery. These
pathways are largely represented by the MAPK and PI3K/AKT/mTOR cascades
that include components and regulators strongly associated with the pathobiol-
ogy of CRC, such as PIK3CA, K-RAS, BRAF, PTEN, RTKs and others (Bhalla
etal. 2013; Cancer Genome Atlas 2012; Shao etal. 2004; Wong and Ma 2014).
29 Colorectal Cancers 595
Mutations of KRAS and BRAF that result in activation of MAPK signaling (namely
its RAS/RAF/MEK/ERK branch) are frequently found in CRCs (Cancer Genome
Atlas 2012). Activating mutations of PIK3CA and inactivating mutations or de-
creased function of PTEN are also often found in CRC, leading to the activation of
the PI3K/AKT/mTOR pathway (Cancer Genome Atlas 2012; Parsons etal. 2005).
More so, coactivation of both pathways also occurs in CRC (Cancer Genome Atlas
2012). These events lead to the activation of oncogenic signaling which converges
on downstream effectors, such as the translation machinery.
29.2Translation and Colorectal Cancers
An early report using a CRC model showed that global alterations in translation
play an important role in cancer progression to metastasis (Provenzani etal. 2006).
An analysis of the transcriptional and translational profiles in CRC revealed distinct
molecular signatures involving the program of cell proliferation for both profiles,
while the apoptosis and protein synthesis programs were affected mainly at the level
of translation (Provenzani etal. 2006). These effects were proposed to be largely
mediated via eIF4E and its negative regulators, 4E-BPs.
29.2.1 eIF4E and 4E-BPs
eIF4E is one of the most studied translation factors. It is implicated in cancer biol-
ogy in general and in CRC in particular (see Chap. 4). It is a proto-oncogene that
functions as a part of the trimeric eIF4F complex, together with the eIF4A helicase
and the eIF4G scaffolding protein. eIF4E binds to a special structure at the very 5
end of the mRNA called the cap and thus assists in the recruitment of the mRNA to
the 40S ribosomal subunit (see Chaps.2 and 4). The paradigm of the involvement
of eIF4E in physiology and cancer is currently based on the observation that some
mRNAs that are responsible for cell survival, angiogenesis, proliferation and growth,
are more sensitive to eIF4E than others (see Chap.4). These mRNAs, called eIF4E-
sensitive, include cyclin D1, c-MYC, VEGF and MMP-9 and contain extensive sec-
ondary structures at the 5 UTR (see Chap.4). This paradigm is generally supported
by the studies in CRCs (Provenzani etal. 2006).
eIF4E is also a hub for regulation by different signaling cascades, including
MAPK and PI3K/AKT/mTOR. The latter pathway controls the 4E-BPs, which,
when phosphorylated by an activated mTOR, dissociate from eIF4E and facilitate
translation. Thus, the 4E-BPs act as tumor suppressors. For a detailed discussion
regarding the aforementioned hubs of translation and their regulation, please refer
to Chaps. 4, 15, and 17. eIF4E is a central regulator of tumor formation, growth,
invasion and metastasis and plays a key role in the development of various types of
cancers (Lazaris-Karatzas etal. 1990; Lazaris-Karatzas etal. 1992; Zimmer etal.
2000).
As described in other chapters (see Part IV) of this book, eIF4E overexpression
has been demonstrated in breast, prostate, bladder, cervix, lung and many other
malignancies. High eIF4E levels are linked to changes in cellular morphology, en-
hanced proliferation and induction of cellular transformation, tumorigenesis and
metastasis. Blocking eIF4E expression using ASOs or overexpression of the inhibi-
tory 4E-BPs suppresses cellular transformation, tumor growth, tumor invasiveness
and metastasis (see Chap.4). Not s urprisingly, in CRC, eIF4E protein and mRNA
levels are found to be elevated (Berkel etal. 2001; Martin etal. 2000; Rosenwald
etal. 1999; Xi etal. 2008; Yang etal. 2007). In some reports approximately 72%
of colon tumors have increased eIF4E protein levels (Yang etal. 2007). The level
of eIF4E expression is significantly linked to the histological type of the lesion,
suggesting a relation between eIF4E expression and the adenoma-cancer sequence
in the colon; the lowest level of eIF4E expression is found in normal colon tis-
sue, whereas its highest level is observed in colorectal adenocarcinomas (Berkel
etal. 2001). Colonic carcinomatous lesions have 43 times higher chance of having
a high level of the eIF4E expression when compared with the normal tissue (Berkel
etal. 2001). This increase in eIF4E expression is often accompanied by elevation
of eIF4E-sensitive cyclin D1 and VEGF levels, which moderately but significantly
correlate with the level of eIF4E in CRC (Rosenwald etal. 1999; Yang etal. 2007).
As mentioned previously, the role of eIF4E in tumorigenesis is also determined
by its activation via signaling pathways (Bhalla etal. 2013; Cancer Genome Atlas
2012; Shao etal. 2004; Wong and Ma 2014). In CRC cells with coexistent activa-
tion of the PI3K/AKT/mTOR and MAPK signaling, inhibition of both pathways
showed a significantly superior activity in decreasing cell migration and invasion,
as compared to inhibiting each of these pathways separately (She etal. 2010; Ye
etal. 2014). Importantly, these effects were largely determined by the state of the
translation machinery at the 4E-BPs/eIF4E axis. Silencing eIF4E or expressing a
dominant-active 4E-BP1 (that is the main isoform of 4E-BPs) mutant in CRC cells
have been shown to inhibit their migratory, invasive and metastatic properties (Ye
etal. 2014). On the other hand, overexpression of eIF4E or s ilencing 4E-BP1 re-
duced CRC cell dependence on ERK and AKT signaling (She etal. 2010; Ye etal.
2014). These studies show that 4E-BP1 is a key effector of the oncogenic activation
of AKT and ERK signaling in CRC. In addition to the dysregulated activity of the
4E-BPs, the expression levels of these proteins are also important for CRC biology.
Expression of 4E-BP1 is found elevated in most gastrointestinal cancers, including
CRC (Martin etal. 2000). An inverse correlation between 4E-BP1 elevation and N
and M stages has been reported, showing a significantly higher elevation of 4E-BP1
in node-negative patients, as well as in patients without distant metastasis (Martin
etal. 2000).
While the phosphorylation of 4E-BP1 is controlled by mTOR, the mechanism
underlying the dephosphorylation of 4E-BP1 remains unclear. Recently, PPM1G
has been shown to be a phosphatase of 4E-BP1 and a regulator of CRC cell growth
(Liu etal. 2013). An interplay between phosphatases and translation in CRC has
also been recently investigated in other studies. PH domain and leucine-rich repeat
protein phosphatases (PHLPPs) are negative regulators of AKT that behave like
tumor suppressors in CRC via mTOR- and 4E-BP- dependent downregulation of
mRNA translation (Liu etal. 2011a). Interestingly, the regulation of PHLPP expres-
sion has been proposed, at least in part, to occur via translational control involving
4E-BP1 (Wen etal. 2013).
Another mechanism of eIF4E regulation involves its phosphorylation by MNKs
via the MAPK cascade (see Chap.17). An earlier report observed decreased levels
of phosphorylated eIF4E in CRC (Martin etal. 2000). However, a more recent
study showed a significantly higher expression of phosphorylated eIF4E in CRC
tissues compared to their corresponding adjacent normal tissues (Fan etal. 2009).
The latter finding would be more consistent with the robust observations of aber-
rantly activated MAPK signaling in CRC that would lead to an increase in phos-
phorylation and activity of eIF4E via MNKs (Bhalla etal. 2013; Cancer Genome
Atlas 2012; Shao etal. 2004; Wong and Ma 2014). Interestingly, the levels of phos-
phorylated eIF4E were significantly higher in the early stage (T1) than in the late
stage (T3) of the disease (p<0.05), suggesting a critical role in cancer development,
particularly at the early stages.
29.2.2 PDCD4
PDCD4 is a bona fide tumor suppressor that regulates the activity of the eIF4A
helicase, which is a part of the eIF4F translation initiation complex (see Chap.6).
PDCD4 protein expression is often downregulated in CRC, which is associated with
upregulation of its negative regulator miR-21 (Baffa etal. 2009; Chang etal. 2011;
Horiuchi etal. 2012; Ma etal. 2013; Muppala etal. 2013). Western blot, reverse
transcription PCR, and IHC show decreased levels of PDCD4 in CRCs compared
to noncancerous tissues (Ma etal. 2013). PDCD4 expression is s ignificantly lower
in moderately or poorly differentiated cancers than in well-differentiated ones (Ma
etal. 2013). The reduced PDCD4 mRNA expression is associated with poor prog-
nosis in CRC patients with Dukes stage B, C and D, and is linked to negative regu-
lation by miR-21 in each tumor stage (Horiuchi etal. 2012). Similar observations
have been reported previously, linking low PDCD4 protein and mRNA levels to high
expression of miR-21. These expression patterns have been shown to a ssociate with
worsening pathology in the adenoma-carcinoma progression (Chang etal. 2011;
Fassan etal. 2011; Mudduluru etal. 2007; Shibuya et al., 2010; Yamamichi etal.
2009). Together with decreased levels of PDCD4 protein and mRNA expression
in the pathogenesis of CRC, PDCD4 cellular localization also appears important
(Goke etal. 2004). Its nuclear loss is implicated in CRC biology. While normal
colonic cells and hyperplastic polyps feature strong PDCD4 nuclear immunostain-
ing, a significantly lower PDCD4 nuclear expression is observed in dysplasias and
invasive CRC (Fassan etal. 2011).
As mentioned previously, miR-21 plays a substantial role in regulating PDCD4.
High levels of miR-21 are observed in the specimens of CRC associated with de-
creased expression of its target genes, including PDCD4 (Chang etal. 2011; Ferraro
etal. 2014; Muppala etal. 2013). High miR-21 expression has been reported to
be significantly associated with venous invasion, liver metastasis and tumor stage
(Shibuya etal. 2010). OS and DFS of patients with high miR-21 expression are
significantly worse than those of patients with low miR-21 expression (Shibuya
etal. 2010). In CRC cell lines, PDCD4 is also found to be negatively regulated by
miR-4995p, promoting cellular invasion and metastasis via the less studied regula-
tory mechanism (Liu etal. 2011b).
High miR-21 with low PDCD4 protein expression is often found in metastatic
disease and predicts the presence of metastasis (Baffa etal. 2009; Ferraro etal.
2014; Yamamichi etal. 2009). The role of decreased PDCD4 expression in the de-
velopment of metastasis involves the promotion of EMT (Ferraro etal. 2014; Wang
etal. 2008; Wang etal. 2013). PDCD4 knockdown in HT-29 colon cancer cells
results in a fibroblast-like transition, including a reduction of E-cadherin expression
and the activation of -catenin/Tcf and AP-1-dependent transcription (Wang etal.
2008). PDCD4 also negatively affects cancer cell lymphovascular invasion in CRC
(Leupold etal. 2007). Some of the other mechanisms of action of PDCD4 in CRC
biology include suppression of cancer progression by downregulating transcription
of MAP4K1 with consequent inhibition of c-JUN activation and AP-1-dependent
transcription (Yang etal. 2006) and repression of transcription of the mitosis-
promoting factor CDK1 via upregulation of p21 (Waf1/Cip1) (Goke etal. 2004).
Downregulation of PDCD4 is also implicated in inflammatory bowel disease-asso-
ciated carcinogenesis (Ludwig etal. 2013). In this context, the downregulation of
PDCD4 is often associated with an increase in its negative regulator miR-21.
Thus, in CRC the miR-21/PDCD4 axis has a potential to be used as a prog-
nostic marker and therapeutic target. In CRC, curcumin shows promising potential
for anticancer therapy and has been implicated in the control of miR-21 expres-
sion by its transcriptional downregulation, thus inhibiting invasion and m etastasis
in CRC (Mudduluru et al., 2007). Another compound, resveratrol (trans-3,4,
5-trihydroxystilbene, a natural antioxidant), has been shown to downregulate var-
ious miRNAs and to increase levels of tumor suppressors, such as PDCD4 and
PTEN (Tili etal. 2010). Other potential strategies, which target PDCD4 but are yet
to be studied in CRC, are described in other chapters of this book, such as Chap. 6.
29.2.3eIF3
The multisubunit translation initiation factor eIF3 is a pivotal player in protein syn-
thesis (see Chap.8). In general, the differential expression of its subunits might lead
to abnormal cell growth and proliferation, while their downregulation results in an-
tiproliferative effects (Goh etal. 2011; Haybaeck etal. 2010; Liu etal. 2007; Wang
etal. 2012). eIF3a is a large core subunit of eIF3 that has been suggested to play a
regulatory role in mRNA translation (see Chap.8). Decreased eIF3a expression has
been observed to take place prior to cell differentiation (Liu etal. 2007). Increase
of eIF3a expression inhibited differentiation of Caco-2 colon cancer cells, while its
silencing promoted differentiation (Liu etal. 2007). Silencing of another large and
core eIF3 subunit, eIF3b, in colon cancer cells results in decreased proliferation and
increased apoptosis (Wang etal. 2012). eIF3m, one of the poorly-characterized
non-core subunits of eIF3, is overexpressed in colon cancer cells and tissues and
is proposed to regulate tumorigenesis-related messages (Goh etal. 2011). In the
human colon cancer cell line HCT116, siRNA-mediated silencing of eIF3m im-
pedes cell proliferation and cell cycle progression and leads to cell death (Goh etal.
2011). While the eIF3 complex appears to be implicated in CRC biology, the role
of individual eIF3 subunits and the eIF3 multiprotein complex as a whole in this
cancer requires better understanding and more detailed studies.
29.2.4 eIF2
The function of eIF2 and controversies surrounding this protein in cancer biology
are discussed in detail in Chap. 9. Translation initiation factor eIF2 promotes trans-
lation and is essential for mediating the effects of stress stimuli on protein synthesis
(see Chap.9). It appears that expression of eIF2 is increased in both benign and ma-
lignant neoplasms of colonic epithelium (Rosenwald etal. 2003). This suggests that,
in CRC, elevated eIF2 may contribute to tumor initiation and progression, but is not
sufficient for establishing a malignant phenotype (Rosenwald etal. 2003).
eIF2 is negatively regulated by phosphorylation via various kinases that re-
spond to stress stimuli, such as HRI, GCN2, PKR and PERK (see Chap.9).
Phosphorylation of eIF2 by activated kinases leads to a decrease in the rate of
protein synthesis and an expected decrease in cancer cell proliferation. Silencing of
PKR leads to increased human colon cancer cell proliferation and resistance to anti-
cancer agents (Yoon etal. 2009). PKR has also been reported to be a target of 5-FU
which activates the former in a p53-independent manner, inducing phosphorylation
of eIF2 and apoptotic cell death (Garcia etal. 2011). PERK has also been impli-
cated in CRC biology as a component of the endoplasmic reticulum stress response
(Ranganathan etal. 2008).
Various agents are thought to possess anticancer properties by inducing phos-
phorylation of eIF2 via activation of PKR, such as the nonsteroidal anti-inflamma-
tory drug (NSAID) indomethacin (Brunelli etal. 2012) and Candida spongiolide,
a novel polyketide from a marine sponge (Trisciuoglio etal. 2008). Others activate
PERK, such as arctigenin (Kim etal. 2010) and NSAID tolfenamic acid (Zhang
etal. 2013). A derivative of the anti-inflammatory drug mesalamine (2-methoxy-
5-amino-N-hydroxybenzamide, or 214) has been shown to be a potent inhibi-
tor of CRC proliferation via activation of eIF2 kinase 3 and phosphorylation of
eIF2 (Stolfi etal. 2010). eIF2 and eEF2 might also be inhibited by temsirolimus
(CCI-779) in micromolar but not nanomolar concentrations, resulting in a marked
decline in global protein synthesis and disassembly of polyribosomes (Shor etal.
2008). In several human cancer cell types, including colon carcinoma, resveratrol
(a natural polyphenolic compound found in several plants) induced endoplasmic
reticulum stress response and phosphorylation of eIF2 leading to antiproliferative
and proapoptotic effects (Park etal. 2007).
While these studies provide evidence of the involvement of eIF2 in CRC, the
exact model of its involvement in cancers in general is debated and warrants further
investigations (see Chap.9 for discussion).
29.2.5eIF5A
The function of eIF5A in translation initiation is still unclear (see Chap.10). It has
been proposed to facilitate the formation of the first peptide bond by stabilizing the
aa-tRNA in the P-site and thus to function at the junction of translation initiation
and elongation (see Chap.10). eIF5A is especially interesting, since it contains
an unusual (for higher eukaryotes) amino acid hypusine. Hypusine is essential for
eukaryotic cell proliferation (Park etal. 1997). This specific modification makes
this protein a unique diagnostic and therapeutic target. The expression levels of
eIF5A have been reported to be upregulated in CRC tissues compared to normal
colorectal tissues, correlating with poor prognosis in early-onset CRC (Tunca etal.
2013). eIF5A is thought to play a role in the regulation of cell proliferation and
apoptosis (Caraglia etal. 2013). Overexpression of eIF5A or a mutant of eIF5A that
is unable to be hypusinated induces apoptosis in colon carcinoma cells, indicating
that non-hypusinated eIF5A possesses proapoptotic functions (Taylor etal. 2007). A
proteomics analysis indicates that the conformationally restricted polyamine analog
PG-11047, which impedes eIF5A hypusination, results in decreased general protein
synthesis and growth suppression of HCT116 colon cancer cells (Ignatenko etal.
2009). Thus eIF5A represents a potential specific therapeutic target in CRC.
29.2.6Other Translation Factors
Other translation factors are implicated in CRC biology, but the reports are anec-
dotal and further studies are needed to evaluate their possible roles and mechanisms
of action. These include translation initiation factor eIF6 and elongation factor
eEF2. eIF6 is a protein that is essential for ribosome biosynthesis and assembly (see
Chap.11). It has been implicated in the selective inhibition of the WNT signaling
pathway in adenomatous polyposis coli (APC)-mutant colon cancer cells, which
express high levels of active -catenin (Ji etal. 2008). eEF2 is an elongation factor
that is implicated in tumorigenesis (see Chap.12). eEF2 is overexpressed in gas-
trointestinal cancers cells, including the colon cancer cell line SW620; it appears to
promote G2/M progression and enhance cell growth (Nakamura etal. 2009).
29.3Translation and Colorectal Cancer Therapy
CRC therapy targeting translation initiation is a relatively new concept, and, as

such, inhibitors of this process have yet to receive approval for clinical use. One
notable exception is 5-FU, which was introduced in the 1950s and is currently a
part of standard regimens such as FOLFOX (leucovorin, 5-FU and oxaliplatin) and
FOLFIRI (leucovorin, 5-FU and irinotecan). Its role in blocking protein synthesis
was originally overlooked. However, it was subsequently demonstrated that 5-FU
affects translation by at least two distinct mechanisms. Firstly, 5-FUs target protein
thymidylate synthase has RNA-binding properties and normally suppresses

translation of a subset of mRNAs, including its own mRNA and that encoding tu-
mor suppressor p53 (Chu etal. 1991; Ju etal. 1999; Xi etal. 2006). Treatment with
5-FU relieves this inhibition and results in translational upregulation of thymidylate
synthase, p53 and many other proteins involved in processes such as protein syn-
thesis and cell cycle regulation (Kudo etal. 2010; Tai etal. 2004). Secondly, 5-FU
leads to activation of PKR, phosphorylation of eIF2, inhibition of protein synthe-
sis and apoptotic death (Garcia etal. 2011). Most of these findings were obtained
using human colon cancer cell lines and imply that translational control is directly
relevant to 5-FU-based treatment regimens in patients with CRC. Nonetheless, it is
important to keep in mind that 5-FU has pleiotropic effects, including nucleotide
imbalance and incorporation into RNA and DNA, which all have significant impact
on its cytotoxicity (An etal. 2007; Houghton etal. 1995; Kufe and Major 1981;
Longley etal. 2003).
Targeted therapies specifically designed to inhibit the translation machinery
have, so far, focused mostly on upstream regulators of the cap-binding protein
eIF4E. Specifically, protein synthesis-targeting preclinical research and clinical tri-
als in CRC have been vastly dominated by studies of the mTOR kinase, its inhibitor
rapamycin and derivatives (rapalogs). These compounds have received much atten-
tion in CRC, as various components of the mTOR pathway, including mTORC1,
mTORC2, AKT1, AKT2 and others are elevated and/or activated in these cancers
(Fujishita etal. 2008; Gulhati etal. 2011; Johnson etal. 2010). While rapalogs
display favorable characteristics in cell culture, such as induction of apoptosis and
inhibition of proliferation, migration and invasion of CRC cells (Fujishita etal.
2008; Gulhati etal. 2011; OReilly etal. 2011), their effectiveness as a stand-alone
treatment in CRC patients has proved limited (Altomare and Hurwitz 2013; Ng
etal. 2013). However, preclinical studies have demonstrated cooperative antitu-
mor effects using rapalogs in combination with cisplatin, doxorubicin or paclitaxel
(OReilly etal. 2011). There are currently several clinical trials assessing the effica-
cy of rapalogs in combinations with other chemotherapeutic drugs, especially in ad-
vanced CRCs or as a second-line treatment (see clinicaltrials.gov). One such phase
IIb trial in combination with VEGF receptor inhibitor tivozanib showed promising
results, as 50% of patients with refractory, metastatic CRC displayed stable disease
(Wolpin etal. 2013). Another phase I trial in combination with cetuximab and iri-
notecan resulted in ~50% stable disease and 16% response rate (Myre etal. 2011).
Despite the promising effects of rapalogs as anticancer agents, there are various
mechanisms that lead to resistance, including rapamycin-insensitive targets, loss
of feedback inhibition and uninhibited mTORC2 (see Chap.15). This resistance
prompted the development of various other agents, among which are dual ATP-
competitive mTORC1 and mTORC2 inhibitors, also known as active-site mTOR
inhibitors (asTORi). In a large panel of CRC cell lines, asTORi, such as BEZ235,
PP242 and WYE354, displayed broader anti-CRC activity than rapamycin (Zhang
and Zheng 2012). A novel dual mTORC1/C2 inhibitor AZD-2014 showed dramat-
ic suppression of CRC cell growth and activation of autophagy, but not apoptosis
(Huo etal. 2014). In mice, AZD-2014 oral administration significantly inhibited the
growth of HT-29 cell xenografts and improved animal survival (Huo etal. 2014).
Another agent, OSI-027, a selective and potent dual inhibitor of mTORC1/C2,

showed a robust antitumor activity in several human xenograft models represent-
ing various tissue types (Bhagwat etal. 2011). Moreover, in COLO-205 and GEO
colon cancer xenograft models, OSI-027 displayed superior efficacy compared to
rapamycin (Bhagwat etal. 2011). This compound has passed phase I clinical trials
and is reported to be well-tolerated (Tan etal. 2010).
Another strategy to overcome feedback activation of AKT upon mTOR inhi-
bition in CRC is the use of dual PI3K-mTOR inhibitors, such as NVP-BEZ235.
This compound has been found to inhibit growth and induce apoptosis in three-
dimensional cultures of colon carcinoma tissue, as well as to decrease viability and
tumor growth in CRC cell lines and xenografts (Hernlund etal. 2012; Roper etal.
2011), although it has not reached the clinical studies phase yet in CRC. Another
dual-specificity inhibitor, PF-05212384, has entered clinical testing in combina-
tion with irinotecan or FOLFIRI in metastatic CRC, as well as in combination with
docetaxel, cisplatin or dacomitinib in advanced solid tumors (see clinicaltirals.gov
NCT01925274, NCT01937715 and NCT01920061).
One pitfall of dual-specificity PI3K/mTOR inhibitors is that their use can result
in hyperactivation of MAPK signaling and resistance to treatment. This observation
has led to the hypothesis that more efficient mTOR inhibition and anticancer effects
in CRC could also be achieved via combined use of inhibitors of both pathways.
In support of this notion, synergistic effects were observed in CRC cell lines using
combinations of MEK (AZD6244 and PD032590) and PI3K inhibitors (GDC-
0941) or dual mTOR/PI3K NVP-BEZ235 (Haagensen etal. 2012). Such a combi-
nation with the MEK inhibitor MSC1936369B and the PI3K inhibitor SAR245409
is entering clinical testing in locally advanced or metastatic solid tumors (including
CRC) (see clinicaltrials.gov NCT01390818).
The mTOR and MAPK pathways converge on eIF4E to regulate translational
control and depend on the interplay between this initiation factor and its inhibitory
4E-BPs (She etal. 2010). Thus, resistance to the PI3K/AKT/mTOR and MAPK
pathway inhibitors can also be mediated by the level and/or activity of 4E-BP1
(Dilling etal. 2002; She etal. 2010; Ye etal. 2014). Accordingly, colon carcinoma
cells with intrinsic rapamycin resistance have low 4E-BP/eIF4E ratios (Dilling etal.
2002). Upon recombinant overexpression of 4E-BPs in HCT8 CRC cell line, sen-
sitivity to rapamycin is dramatically increased, emphasizing that the 4E-BP and
eIF4E content and ratio are important determinants of rapamycin function and resis-
tance (Dilling etal. 2002). Moreover, this mechanism of resistance at the 4E-BPs/
eIF4E axis would apply to all aforementioned strategies and has been described as
a general mechanism of resistance to mTOR inhibitors (Alain etal. 2012a; Alain
etal. 2012b). Indeed, resistance to asTORi, such as PP242, has been commonly re-
ported in cancer cell lines of colonic origin and has been correlated with incomplete
inhibition of phosphorylation of 4E-BP1 (Ducker etal. 2014). In one study, almost
40% of CRC cell lines were intrinsically resistant to dual mTORC1/2 inhibitors,
correlating with mTOR-independent 4E-BP1 phosphorylation (Zhang and Zheng
2012). Even knockdown strategies of the upstream regulators of the pathways, such
as PIK3CA and KRAS, depend on 4E-BPs/eIF4E, with decreased 4E-BP1 phos-

phorylation levels correlating with anticancer effects (Valentino etal. 2012).
Considering that eIF4E overexpression and 4E-BP1 hyperphosphorylation are
common in CRC (Berkel etal. 2001; Martin etal. 2000; Rosenwald etal. 1999;
Xi etal. 2008; Yang etal. 2007; Zhang etal. 2009), the use of inhibition strategies
directly targeting 4E-BPs and/or eIF4E, rather than upstream signaling pathways,
is promising. These approaches are being developed and tested in various cancers,
such as ASOs that reduce eIF4E expression. In fact, the eIF4E ASO ISIS183750 is
currently being tested in CRC patients in combination with irinotecan (clinicaltrial.
gov, NCT01675128). Other compounds have been developed which target various
parts of the translation machinery, such as the cap-eIF4E interaction, the eIF4E/eI-
F4G interaction, eIF4A activity, PDCD4 expression, eIF2 phosphorylation, eIF5A
hypusination and others (see respective chapters in Part I). However, the majority of
these inhibitors have yet to be extensively tested in the models of CRC. A few no-
table findings encourage further studies in this direction. For example, an inhibitor
of the eIF4E/eIF4G interaction, 4EGI-1, has been reported to induce apoptosis in
HCT116 cells (Matassa etal. 2013). In addition, treatment with the eIF4A inhibitor
pateamine A results in decreased proliferation in several colon cancer cell lines and
in DLD-1 colon cancer cell xenografts (Kuznetsov etal. 2009). Furthermore, cerco-
sporamide, a MNK inhibitor that effectively blocks eIF4E phosphorylation both in
vitro and in vivo, has been reported to induce apoptosis and suppress proliferation
of HCT116 colon carcinoma cells, as well as inhibit their growth in soft agar and
subcutaneous xenografts (Konicek etal. 2011).
Another compound that deserves a mention is metformin. It is an antidiabetic
agent that appears to be a promising drug for various types of cancers. Metformin ac-
tivates AMPK and inhibits the mTOR pathway (Aljada and Mousa 2012). In chemi-
cal carcinogen-induced colorectal carcinogenesis, metformin significantly reduces
aberrant crypt foci (ACF) formation and modestly inhibits colonic polyp formation
(Hosono etal. 2010). It also suppresses colonic epithelial proliferation (Hosono
etal. 2010; Zakikhani etal. 2008) and selectively suppresses the tumor growth of
HCT116 colon cancer cell xenografts (Buzzai etal. 2007). Metformin is currently
entering several clinical trials, including a phase II, randomized double-blind trial
investigating its potential as a preventive modality in patients with familial adeno-
matous polyposis, as well as studies evaluating combinations of metformin with iri-
notecan and 5-FU-based therapies in refractory or advanced CRC (see clinicaltrials.
gov, NCT01725490, NCT01926769, NCT01941953 and NCT01930864).
Other reported anticancer approaches to translational inhibition in CRC include
use of patellazoles (Richardson etal. 2005); phenethyl isothiocyanate (a constituent
of many edible cruciferous vegetables) (Hu etal. 2007); curcumin (a natural poly-
phenol product of the plant Curcuma longa) (Johnson etal. 2009); proteasome in-
hibitor MG-132 (Wu etal. 2009); siRNA-mediated gene silencing of mTOR (Zhang
etal. 2009); enzastaurin (Dumstorf etal. 2010); aspirin (Din etal. 2012); synthetic
cannabinoids (Sreevalsan and Safe 2013); and the antimalarial compound dihydro-
artemisinin (DHA) (Odaka etal. 2014). However, specificity of their effect on the
translation machinery and/or anticancer efficacy needs further studies.
CRC are common and lethal forms of cancers that are often characterized by aber-
rations leading to oncogenic signaling. One of the major downstream targets of
these pathways is the translation machinery. As follows from this chapter, aberrant
expression or function of this machinery itself and dysregulation of upstream sig-
naling transduction pathways play important roles in the etiology and pathogenesis
of CRC. The most notable hub of translation that takes part in CRC biology is repre-
sented by the eIF4E proto-oncogene and its negative regulators, the 4E-BPs. Over-
expression and activating phosphorylation of eIF4E, as well as inactivating phos-
phorylation and downregulation of 4E-BPs, are common themes in CRC. Hence,
using the 4E-BPs/eIF4E axis as a diagnostic and prognostic marker and therapeutic
target is warranted.
Other translation factors, albeit less studied, show evidence of participation in
CRC pathogenesis. One of the better understood members of this group is PDCD4, a
tumor suppressor and a negative regulator of the eIF4A helicase. Downregulation of
PDCD4, frequently associated with upregulation of miR-21, is commonly observed
in CRC. The eIF4A/PDCD4 axis presents interesting parallels to the eIF4E/4E-BPs
axis, since both are components of the eIF4F complex that are inhibited by an inter-
acting protein, the function of which is dysregulated in CRC in a pattern following
the adenoma-carcinoma sequence. Moreover, the abnormal function of these pro-
teins is associated with tumor invasion and metastasis. They also appear to play an
important role in resistance to chemotherapeutic and targeted agents.
Another translation factor that deserves attention in CRC therapy is eIF5A,
which possesses a unique hypusine modification. Novel targeting strategies of this
protein might represent promising therapeutic alternatives. The eIF3 complex and
the eIF2 protein, both of which are required for translation initiation, merit further
studies in CRC, since current evidence provides a preliminary support for their in-
volvement in the biology of these malignancies.
In conclusion, the translation initiation step is critically involved in the develop-
ment and progression of CRC, and its clinical and pharmacological targeting will
likely prove fruitful.
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Chapter 30
Hepatic, Pancreatic and Biliary Cancers
Jennifer A. Sanders and Philip A. Gruppuso
Contents
30.1.1Liver Cancers 612
30.1.2Biliary Cancers 613
30.1.3Pancreatic Cancers 613
30.2Translation in HPB Cancers 614
30.3Viral Hepatitis, Translation and HPB Cancers 617
30.4Translational Regulation in HPB Cancers 618
30.4.1PI3K/AKT/mTOR Pathway 618
30.4.2MAPK Pathway 621
30.5Translation in Therapeutic Resistance 622
References 623
AbstractHepato-pancreato-biliary (HPB) cancers, which are among the most

lethal of malignancies, often show alterations in the expression of components of
the translation machinery and the regulation of their function. Changes in trans-
lation initiation factors, elongation factors and ribosomal proteins and RNA have
been demonstrated across the range of HPB cancers. Viruses that are causative
for hepatocellular carcinoma (HCC) have been shown to directly interact with the
translation apparatus. All forms of HPB cancer, including not only HCC but also
cholangiocarcinoma (CCA) and pancreatic ductal adenocarcinoma (PDAC), have
been associated with altered signal transduction pathways involved in the control
of translation. Understanding the mechanisms accounting for the dysregulation of
translation in HPB cancers has led to the identification of novel biomarkers, prog-
nostic indicators, and preventive and chemotherapeutic treatment strategies. Fur-
ther developments in this area have the potential to improve the outcome for these
devastating disorders.
P.A.Gruppuso() J.A.Sanders
Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI, USA
e-mail: philip_gruppuso@brown.edu
612 J. A. Sanders and P. A. Gruppuso
30.1Introduction
Hepato-pancreato-biliary (HPB) cancers are among the most lethal of malignancies.

The majority of hepatobiliary malignancies are represented by HCC and cholangio-
carcinoma (CCA) (Vasilieva etal. 2012). The vast majority of pancreatic tumors
are adenocarcinomas with pancreatic ductal adenocarcinoma (PDAC) constitut-
ing more than two-thirds of all pancreatic cancers (Sauerland etal. 2009). Another
pancreatic malignancy that will be discussed in the next chapter is PaNET. Unless
amenable to surgical resection, PDAC is almost uniformly fatal, having a 5-year
survival rate of 5% (Ferrone etal. 2012).
30.1.1 Liver Cancers
Primary liver cancers, the most common being HCC, usually arise in the setting of
chronic liver disease and cirrhosis. Risk factors for HCC include HBV and HCV
infection, aflatoxin exposure, alcohol abuse, and obesity (Trevisani etal. 2008).
Genomic alterations in HCC are heterogeneous and include loss of the tumor sup-
pressor p53, and gains in the oncogenes c-MYC and -catenin. Alterations in insu-
lin signaling, the WNT/-catenin pathway, and the MAPK/ERK signaling cascade
are also thought to play a role in HCC (Trevisani etal. 2008). In many cases of liver
disease, liver specific stem cells, termed progenitor cells, are activated (Lowes etal.
1999; Roskams 2006). These progenitor cells arise from the terminal bile ductules
and are capable of differentiating into hepatocytes or biliary cells. Progenitor cells
can serve as targets for carcinogenesis, resulting in the formation of the progenitor
subtype of HCC (Durnez etal. 2006; Yang etal. 2008, 2010). This subtype repre-
sents approximately 2850% of HCC and is more aggressive and genetically dis-
tinct from non-progenitor HCC (Roskams 2006).
Liver cancer is the sixth most commonly diagnosed cancer worldwide with wide
variation in region-specific incidence rates due to the prevalence of HBV and HCV
infection, aflatoxin exposure, and other risk factors (Sauerland etal. 2009; Trev-
isani etal. 2008). Though the high incidence rate of liver cancer has been stable
in Southeast Asia and sub-Saharan Africa where HBV is endemic, the incidence is
increasing in the United States and Europe due to rising rates of HCV, alcohol abuse
and obesity (Center and Jemal 2011). Long-term survival is very low owing to the
advanced stage of the disease at diagnosis, tumor heterogeneity and lack of effective
treatment options. For HCC, the 5-year survival rate is 15% with a 3-year recur-
rence rate of 5060% (Soong etal. 2011). Curative treatment options for patients
diagnosed at earlier stages include surgical resection, liver transplantation, ablation
and transarterial chemoembolization (Sauerland etal. 2009). However, these treat-
ments are not suitable for patients with impaired liver function or advanced disease,
nor are they very effective. Thus preventive and therapeutic strategies targeting
the underlying etiology and the molecular changes within the tumor are attractive
alternatives. For advanced HCC, sorafenib, a small molecule inhibitor of RAF and
30 Hepatic, Pancreatic and Biliary Cancers 613
tyrosine kinases that targets vascular VEGF and PDGF, is the only systemic agent
currently approved. Sorafenib has been found to increase survival by about three
months (Chang etal. 2007; Llovet etal. 2008; Wilhelm etal. 2004).
30.1.2Biliary Cancers
The second most frequent primary liver tumor originates from the biliary epithelial
cells. CCA can arise from biliary system located both within (intrahepatic) and
outside the liver (extrahepatic). Similar to HCC, the incidence rate of CCA is ris-
ing worldwide (Blechacz and Gores 2008; Vasilieva etal. 2012). The leading risk
factors for the development of CCA include primary sclerosing cholangitis, par-
asitic infestation of the liver, and medical imaging-related exposure to thorotrast
(Vasilieva etal. 2012; Yachimski and Pratt 2008). Many of the same factors de-
scribed above that contribute to the development of HCC are also risk factors for
CCA (Sauerland etal. 2009; Vasilieva etal. 2012; Yachimski and Pratt 2008). The
development of biliary malignancies is often associated with late presentation, such
as an obstruction of the biliary flow. Similar to HCC, the 5-year survival rate for
patients with CCA is exceedingly low (510%) due to late diagnosis and lack of
treatment options (Blechacz and Gores 2008). Surgical resection and liver trans-
plantation are the only potentially curative therapeutic options since tumor response
to various chemotherapeutic agents is poor (Blechacz and Gores 2008).
Molecular diagnosis of CCA has proven to be difficult since biopsy is gener-
ally possible only for intrahepatic tumors, which account for only 10% of cases
(Vasilieva etal. 2012). Mutations in KRAS, p53 dysfunction and loss of p16 have
been identified in CCA (Boberg etal. 2000; DeHaan etal. 2007; Gonda etal. 2012).
The cytokine, IL-6, has also been shown to be important in the proliferation and
survival of CCA cells (Kobayashi etal. 2005; Okada etal. 1994). Targeted thera-
peutic approaches for CCA are in the earliest stages of development. The current
focus is on the identification of distinct diagnostic biomarkers that would increase
the chance of early diagnosis in individuals at high-risk for CCA, thus allowing for
surgical resection or liver transplantation (Yachimski and Pratt 2008).
30.1.3Pancreatic Cancers
Pancreatic cancer, including the most common form, PDAC, is also classified as a
biliary tract cancer. Like the malignancies discussed above, the majority of patients
with PDAC are diagnosed when the disease is locally advanced or metastatic. Treat-
ment for these patients is palliative and includes chemotherapy, radiation therapy,
and the TKI erlotinib, an EGFR inhibitor (Cinar and Tempero 2012; Sauerland
etal. 2009). Compared to other hepatobiliary cancers, little is known about the
risk factors or molecular mechanisms underlying PDAC. Epidemiologic studies
have identified cigarette smoking and alcohol abuse as the main risk factors, while
genetic studies have identified a large number of mutations that affect signaling
pathways (Lowenfels and Maisonneuve 2005). These alterations include the onco-
gene K-RAS, the tumor suppressors p53 and p16, and changes in TGF- and WNT/
NOTCH signaling (Jones etal. 2008; Korc 2003; Matthaios etal. 2011). Our slowly
evolving understanding of the pathogenesis of pancreatic malignancy accounts for
the limited arsenal of treatment options for this deadly condition.
30.2Translation in HPB Cancers
The lack of effective treatment options for HPB cancers has led to intense interest in
understanding the molecular pathogenesis of these malignancies. As presented else-
where in this book, numerous cancers are associated with dysregulation of transla-
tion. Our understanding of the role of translation in HPB malignancies is, however,
limited. In this chapter, we, for the first time, will attempt to summarize our knowl-
edge on the role of the translation machinery in HPB cancers. We will focus on
the components of the translational apparatus and its control via major signaling
pathways in HPB cancers. We will also discuss potential use of components of the
translation machinery as prognostic markers and targets for novel therapeutics.
Chronic inflammation and oxidative stress are risk factors for primary liver cancers
(McGlynn and London 2011). Hepatic carcinogenesis may occur as a result of muta-
tions that are acquired during the rapid cell turnover that occurs under these condi-
tions (Sauerland etal. 2009). However, many cancer cells exhibit high rates of protein
synthesis that, in contrast to normal cells, may be uncoupled from cell proliferation
(Schuhmacher etal. 1999; Stanners etal. 1979). These high rates of protein synthesis
can attenuate apoptosis (Barna etal. 2008) and promote the translation of specific genes
that promote dysregulation of cell growth (Silvera etal. 2010). Such mechanisms are
relevant to HPB cancers, in which enhanced expression of the proto-oncogene c-MYC
has been associated with transformation of liver progenitor cells and HCC progression
(Braun etal. 1989; Kaposi-Novak etal. 2009; Sinha etal. 1989). c-MYC, in turn, is a
key factor in promoting the dysregulation of translation and its uncoupling from cell
proliferation (Barna etal. 2008; Schuhmacher etal. 1999).
Further evidence for a causative role of abnormal translation in HPB cancer
comes from the identification of mutations in ribosomal proteins and factors in-
volved in ribosome biogenesis (ribosomopathies) that correlate with increased can-
cer risk (reviewed in Narla and Ebert 2010). In regard to HPB cancers, a spectrum of
ribosomal disorders have been found to be associated with mouse models of HCC,
human HCC cell lines and, most recently, biopsy samples from patients with HCC
(Shenoy etal. 2012). In fact, the expression of ribosomal protein rpL36 has been
found to be an independent prognostic marker for survival in HCC and has been
proposed as a key factor in early-stage hepatic carcinogenesis (Song etal. 2011).
Additional evidence that dysregulation of translation is causal for HPB can-
cers has come from studies on signal transduction in chronic liver disease and in
early-stage lesions. Growth factor activation of the PI3K/mTOR pathway results in
stimulation of eIF4F formation and the translation of cap-dependent mRNA (Topi-

sirovic and Sonenberg 2011). These mRNAs encode proteins involved in cell cycle
progression and survival (Mills etal. 2008). The expression and activity of signal-
ing pathways, such as, mTOR, MAPK, and c-MYC that regulate the components of
the initiation complex serve as prognostic indicators for HPB cancers (Chung etal.
2009; Kaposi-Novak etal. 2009; Li etal. 2012; Villanueva etal. 2008). Impairment
of eIF4F formation and translation of cap-dependent mRNAs is being pursued as
a therapeutic target for a wide variety of cancers including HPB (Bhat etal. 2013;
Mishra etal. 2009; Schnitzbauer etal. 2010; Schoniger-Hekele and Muller 2010).
Alterations in the expression and activity of components of the translation
machinery have been found to play critical roles in tumor development, inva-
sion and metastasis (see Stumpf and Ruggero (2011) and Parts I and IV of this
book). Table30.1 enlists alterations of the translation machinery in HPB cancers.
Among translation initiation factors, the role of eIF4E in cancer is particularly well
established (Ruggero etal. 2004; Wendel etal. 2007) (see Chap.4). Its expression
is elevated in a large number of human malignancies, including HCC, CCA and
PDAC (Mishra etal. 2009; Wang etal. 2012; Wehbe etal. 2006). It has been shown
that eIF4E is overexpressed in the majority of human liver cancers compared to sur-
rounding non-cancerous tissue (Wang etal. 2012). In addition, overexpression of
this protein is correlated with cancer recurrence (Wang etal. 2012). Similar to HCC,
eIF4E expression was found to be elevated in PDAC compared to normal ductal
cells (Mishra etal. 2009). However, no significant correlation between the expres-
sion level of eIF4E and tumor grade, lymphatic invasion, lymph node metastasis or
OS has been demonstrated for PDAC (Mishra etal. 2009).
Another component of the translation machinery, eIF3, is a multisubunit pro-
tein complex that has been implicated in tumorigenesis, with some of its subunits
behaving to promote cancer development and others acting as tumor suppressors
(see Chap.8) (Silvera etal. 2010). The example of the latter is the f subunit of the
eIF3 complex, overexpression of which in PDAC cells inhibits translation, cellular
growth, and proliferation, and induces apoptosis (Shi etal. 2006). In accordance
with these findings, Doldan etal. (2008) reported that eIF3f protein expression was
decreased in PDAC owing to allelic loss of the eIF3f gene. Overexpression of eIF3h
and eIF3i have both been observed in HCC (Okamoto etal. 2003; Wang etal. 2013).
While the role of eIF3h remains obscure, eIF3i has been shown to interact with
AKT1. This interaction prevents the dephosphorylation of AKT by PP2A, leading
to constitutive activation of AKT1 signaling (Wang etal. 2013). Administration
of a specific AKT1 inhibitor to eIF3i-overexpressing HCC cells suppressed eIF3i
tumorigenesis both in vitro and in vivo (Wang etal. 2013).
Alterations in the initiation factor eIF5A, which facilitates formation of the first
peptide bond during translation initiation (see Chap.10), have also been associated
with HCC and PDAC (Lee etal. 2010b; Shek etal. 2012). Humans express two iso-
forms of eIF5A. The predominant form, eIF5A1, is expressed in most mammalian
cells, while eIF5A2 is expressed in specific tissues such as brain and testes. eIF5A
is synthesized as an inactive precursor that undergoes a unique posttranslational
modification of a specific lysine residue referred to as hypusination (see Chap.10).
Table 30.1 Translation machinery in HPB cancers.

Function Protein Expression/ Cancer type Reference
Activity level
Translation initiation eIF4E Increase HCC, PDAC, Shuda etal. (2000),
factors CCA Ruggero etal.
(2004), Wehbe etal.
(2006), Wendel etal.
(2007), Mishra etal.
(2009), Wang etal.
(2012)
eIF3f Increase/ PDAC Shi etal. (2006),
Decrease Doldan etal. (2008)
eIF3h Increase HCC Okamoto etal. (2003)
eIF3i Increase HCC Wang etal. (2013)
eIF5A Increase HCC, PDAC Lee etal. (2010a), Shek
etal. (2012)
eIF4AI Increase HCC Shuda etal. (2000)
Elongation factors eEF2 Increase HCC Shuda etal. (2000)
eEF1A1 Increase HCC Shuda etal. (2000),
Grassi etal. (2007)
eEF1A2 Increase HCC, PDAC Grassi etal. (2007),
Cao etal. (2009)
Translation-regulating c-MYC Increase HCC, CCA, Kaposi-Novak etal.
signal transduction PDAC (2009), Voravud
components etal. (1989), Toku-
moto etal. (2005),
He etal. (2012)
mTORC1 Increase HCC, CCA Villanueva etal.
(RAPTOR) (2008), Chung etal.
(2009), Lee etal.
(2012)
mTORC2 Increase HCC Villanueva etal. (2008)
(RICTOR)
ERK Increase HCC Ito etal. (1998), Li
etal. (2012)
RACK1 Increase HCC Ruan etal. (2012)
In patients with HCC, increased expression of eIF5A1 and eIF5A2 have been as-
sociated with an increased number of tumor nodules and venous infiltration, respec-
tively (Lee etal. 2010b). Therefore, eIF5A2 has been proposed as a marker of tumor
invasiveness in HCC. Suppression of eIF5A2 by siRNA or inhibition of hypusina-
tion by a small molecular compound, N1-guanyl-1,7-diaminoheptane (GC7), inhib-
ited the cell growth and migration of HCC cells in vitro, suggesting that targeting
eIF5A2 could be a potential treatment for HCC (Lee etal. 2010b; Tang etal. 2010).
eIF6 (see Chap.11) has recently been shown to be the only initiation factor linking
external stimuli with the 60S ribosomal subunit (Gandin etal. 2008). eIF6 haplo-
insufficient mice show a defect in hepatocyte proliferation (Gandin etal. 2008).
Overexpression of eIF6 has been observed in various tumors and while mutations
in eIF6 may play a role in HPB cancers, no direct evidence has yet emerged (Benelli
etal. 2012; Rosso etal. 2004; Sanvito etal. 2000).
eEF1A is also involved in the development of different cancers (Browne and

Proud 2002). eEF1A is encoded by two actively transcribed genes, EEF1A1 and
EEF1A2. In humans, the eEF1A1 protein is ubiquitously expressed whereas
eEF1A2 expression is normally confined to heart, brain, and muscle (Knudsen etal.
1993). In addition to its role in polypeptide elongation, eEF1A has been assigned
roles in cytoskeletal modification, apoptosis, targeting of proteins for degradation,
and cellular proliferation (Grassi etal. 2007; Gross and Kinzy 2005; Talapatra etal.
2002). EEF1A mRNA levels are increased in human HCC compared to normal liver
and its level of expression correlated with cell growth and differentiation stage in
HCC cell lines (Grassi etal. 2007; Shuda etal. 2000). The eEF1A2 protein is elevat-
ed in pancreatic carcinoma (Cao etal. 2009). Moreover, overexpression of eEF1A2
promotes cell growth, survival, and invasion in experimental models and is associ-
ated with increased tumor cell proliferation and angiogenesis in a nude mouse xeno-
graft model (Cao etal. 2009). Stable eEF1A2 overexpression in a mouse model of
peritoneal metastasis revealed that eEF1A2 can upregulate MMP-9 expression and
activity through induced activation of AKT signaling (Xu etal. 2013). However, the
exact mechanism by which eEF1A2 promotes cell proliferation, survival or AKT
signaling remains unknown.
Shuda etal. (2000) found that the expression of a number of translation fac-
tors including eIF4AI, eIF4E, eEF2 and ribosomal proteins rpLP0 and rpL9 were
elevated in primary HCC tissues. Furthermore, the upregulation of these mRNAs
was correlated with histological grading. These results suggest that the specific ac-
tivation and upregulation of protein translation is coordinated during liver carcino-
genesis. The expression level of these factors may provide a promising strategy for
diagnosis and staging of HCC and pancreatic cancer.
30.3Viral Hepatitis, Translation and HPB Cancers
Chronic hepatitis infections initially lead to liver inflammation followed by cirrhosis

and HCC. During viral infection and cellular stress, cap-dependent protein transla-
tion is shut down and viral mRNAs that are translated under these circumstances
contain an IRES (Pestova etal. 2001). During persistent HCV infection translation
of viral mRNAs coexists with host cell translation resulting in the maintenance of
the host cells and, therefore, viral replication (Huang etal. 2012). One mechanism
by which HCV and HBV sustain host cell translation is through the upregulation
of RNA polymerase I and II-dependent transcription of rRNA, that in turn results
in increased ribosome biogenesis (Aufiero and Schneider 1990; Kao etal. 2004;
Raychaudhuri etal. 2009; Wang etal. 1998). Genome wide profiling of mRNA
translation during HCV infection revealed that the expression of genes involved in
mRNA splicing, translation, protein metabolism, and the mTOR pathway were al-
tered during HCV infection (Colman etal. 2013). HCV has been shown to modulate
host cell translation through regulation of eIF4E by NS5A, a nonstructural HCV pro-
tein (He etal. 2001). NS5A has also been implicated in viral replication, cell cycle
regulation and modulation of the response to antiviral agents such as IFN (Pineiro
and Martinez-Salas 2012). Infection of a human hepatoma cell line with HCV led
to NS5A-dependent, mTOR-mediated phosphorylation of eIF4E by NS5A (George
etal. 2012). In addition, HCV infection and NS5A expression both augmented as-
sembly of the cap-binding complex, resulting in enhancement of cap-dependent
translation while global protein synthesis was not altered. Furthermore, NS5A was
found to be directly associated with the cap-binding complex, suggesting that this
viral protein was actively involved in the regulation of host translation (George etal.
2012). The consequences of NS5A-mediated regulation of the cap-binding complex
for HCV IRES-dependent translation, cap-dependent host cell translation, and host
IRES 5 TOP translation are unclear. Given the role of mTOR and eIF4E in tumori-
genesis, their activation by NS5A may be a contributing factor to the development of
HCC. NS5A has also been shown to inhibit PKR, resulting in decreased phosphory-
lation of eIF2 and stimulation of protein synthesis (He etal. 2001). However, it is
uncertain whether HCV inhibition of PKR maintains global host protein translation
or favors translation of viral mRNAs through cap-independent processes. It is also
unknown whether inhibition of PKR is involved in the development of HCC. These
findings suggest that, in addition to promoting oncogenesis from chronic inflamma-
tion, HBV and HCV may promote hepatic carcinogenesis by influencing host cell
translation leading to enhanced protein synthesis and cellular proliferation.
Another aspect of viral modulation of translation in HCC is the relationship
between specific 40S ribosomal proteins and the IRES-dependent translation of
HCV mRNAs (Hertz etal. 2013; Huang etal. 2012; Landry etal. 2009). Ribosom-
al proteins rpS25 and rpS6 are required for HCV translation and propagation, but
are dispensable for global protein synthesis and cap-dependent translation of host
mRNAs (Hertz etal. 2013; Huang etal. 2012). Using mass spectrometry, it has been
shown that ribosomes involved in HCV mRNA translation show a ribosomal RNA
methylation pattern that differs from ribosomes bound to host cell mRNA (Yu etal.
2005). This observation indicates a relationship between ribosome heterogeneity
and IRES-dependent translation of viral mRNA. It also indicates a likely role for
ribosomal heterogeneity in viral-mediated hepatic carcinogenesis.
30.4Translational Regulation in HPB Cancers
30.4.1 PI3K/AKT/mTOR Pathway
The signaling pathways that sense diverse extracellular stimuli, such as redox
stress, energy status, nutrient availability and growth factors, integrate these sig-
nals to directly control ribosome production and translation. This is particularly
the case for the liver, which is a central metabolic regulator that has a capacity to
increase its mass in response to metabolic stress. One of the primary pathways in-
volved in the regulation of ribosome biogenesis, protein synthesis and cell growth
and proliferation is the PI3K/AKT/mTOR pathway (see Chaps.15 and 16). mTOR
is a component of two functionally distinct complexes, mTORC1 and mTORC2

(Zoncu etal. 2011) (see Chap.15). PI3K and AKT feed into mTORC1, which also
functions as a nutrient sensing mechanism for the cell and is sensitive to rapamycin.
A key mechanism by which mTORC1 controls cell growth and proliferation
is by regulating translation initiation through its downstream substrates, 4E-BPs
and S6Ks. mTORC1 has also been shown to regulate the translation of 5 TOP
mRNAs that encode ribosomal proteins. In addition to these well described targets,
mTORC1 has a role in regulating translation elongation by indirectly inhibiting
eEF2K. It also contributes to the regulation of ribosomal RNA synthesis via its abil-
ity to modulate RNA polymerase I activity (Wang and Proud 2011).
Activation of the PI3K/AKT/mTOR pathway is an established oncogenic event
for a wide variety of human cancers (see Chap.15). An integrated genomics ap-
proach in a large cohort of patients revealed mTOR pathway activation in 50%
of HCC cases (Villanueva etal. 2008). In the majority of these cases, the aberrant
activation resulted from changes in IGF and EGF signaling rather than from muta-
tions in PTEN, PI3K, or members of the mTOR complex (Villanueva etal. 2008).
Patients with mTOR activation had less differentiated tumors and a higher inci-
dence of recurrence, suggesting that activation of this pathway could be used as a
prognostic indicator for HCC.
Activation of the PI3K/AKT/mTOR signaling pathway has also been reported in
intra- and extrahepatic CCA. Herberger etal. found that survival was significantly
decreased for patients with phospho-mTOR positive malignant biliary tract tumors
compared to patients with phospho-mTOR negative tumors (Herberger etal. 2007).
A recent study of 221 patients with extrahepatic CCA found that low PTEN expres-
sion and decreased PTEN/phospho-AKT1 and PTEN/phospho-mTOR ratios were
poor prognostic factors, although there was no significant difference in survival rate
based on the level of phosphorylated AKT1 or phosphorylated mTOR (Chung etal.
2009). In contrast, another study of 101 patients with intrahepatic CCA revealed
that hyperphosphorylation of mTOR was associated with well to moderately dif-
ferentiated tumors, lack of metastasis, and improved survival (Lee etal. 2012). In a
small prospective pilot study of 9 patients with advanced intrahepatic CCA, treat-
ment with the mTOR inhibitor sirolimus was well tolerated and induced temporary
partial remission or stabilization of disease (Rizell etal. 2008). These limited data
suggest that mTOR is involved in the pathogenesis of CCA.
Relative to mTORC1, what is known about the regulation and role of mTORC2
is very limited. The main substrates for mTORC2 are AKT and SGK1 (Garcia-
Martinez and Alessi 2008; Sarbassov etal. 2005). Until recently, the only functional
role assigned to this complex was regulation of the actin cytoskeleton (Jacinto etal.
2004). More recent studies indicate that the functional role of mTORC2 includes
regulation of whole-body metabolic homoeostasis through the control of hepatic
glucose and lipid metabolism (Hagiwara etal. 2012; Lamming etal. 2012). Over-
expression of the mTORC2 component RICTOR occurs in HCC and is correlated
with early recurrence, suggesting a possible role of mTORC2 in hepatocarcinogen-
esis (Villanueva etal. 2008). More recent studies link mTORC2 with translation
control through the expression of genes encoding ribosomal proteins (Lamming

etal. 2013).
There are, at present, over 500 open clinical trials studying the use of mTOR
inhibitors as therapeutic agents for cancer (www.clinicaltrials.gov). The first de-
scribed mTOR inhibitor, rapamycin, was found to have immunosuppressant and
anticancer activity in vivo (Eng etal. 1984; Vezina etal. 1975). Rapamycin and its
derivatives, commonly referred to as rapalogs, are being actively studied as thera-
peutics for HPB cancers (recently reviewed by Bhat etal. 2013). In a number of
studies, rapamycin and its analogs have been shown to inhibit tumor growth, vascu-
larization, and invasion (Shirouzu etal. 2010; Stelzer etal. 2010; Wang etal. 2009).
However, in clinical trials of advanced cancers, including HCC, only a small subset
of patients show a response to rapamycin, and that response is typically of mini-
mal clinical significance (Rini 2010; Schoniger-Hekele and Muller 2010; Subbiah
etal. 2010). A recently completed phase I/II trial of everolimus in advanced HCC
patients, the majority of which had received prior systemic therapy, confirmed the
safety of the drug. However, PFS at 24 weeks was observed in only two of the pa-
tients (8%) who were included in the phase II portion of the trial (Zhu etal. 2011).
The efficacy of rapamycin and its analogs may be limited because of its inability
to fully inhibit 4E-BP1 function (Guertin and Sabatini 2009). In addition, these
inhibitors only target mTORC1, allowing for a potential escape mechanism through
mTORC2-mediated activation of AKT, which can in turn lead to proliferative and
prosurvival signaling (OReilly etal. 2006). However, other studies have shown
that prolonged rapamycin treatment results in disruption of the mTORC2 complex
and decreased AKT signaling (Lamming etal. 2012; Sarbassov etal. 2006). An-
other explanation for the minimal effect of mTOR inhibition on HCC growth is that
the mTOR pathway may not be a driver of tumor growth in advanced HCC. Several
animal studies provide support for the theory that mTOR is a critical driver of car-
cinogenesis during early HCC, but not advanced liver cancer (Stelzer etal. 2010).
In studies using a murine model of hereditary tyrosinemia, the rapamycin analog,
everolimus (RAD001), suppressed the proliferation of hepatocytes with DNA dam-
age resulting in delayed tumor development (Buitrago-Molina etal. 2009).
Given the limited efficacy of rapamycin, the development and use of kinase
inhibitors that target both mTORC1 and mTORC2 has received much attention.
These kinase inhibitors, examples of which include Torin 1 and pp242, have been
shown to more completely inhibit cell proliferation and cap-dependent translation
under conditions where rapamycin had no effect (Feldman etal. 2009; Thoreen
etal. 2009).
Given the limited therapeutic potential of rapalogs and mTOR inhibitors, there
has been considerable focus on the broad issue of rapamycin resistance. Resis-
tance to the antiproliferative effects of rapamycin has been attributed to a broad
array of mechanisms ranging from reduced expression of the rapamycin binding
protein, FKBP, to null mutations in CDK inhibitors (Hosoi etal. 1998). Our own
work in the rodent indicates that there are at least two downstream loci for rapamy-
cin resistance in the developing fetal liver, cell cycle progression that we have found
to occur at the level of cyclin E-dependent kinase activity (Gruppuso etal. 2011)
and translation control (Gruppuso etal. 2008). These observations suggest that the
resistance of HCC and other HPB cancers to rapamycin and its analogs may reflect
the emergence during carcinogenesis of cell cycle and translation control mecha-
nisms that function independently of mTOR signaling.
Chronic liver diseases, which are risk factors for HCC and CCA, are charac-
terized by ductular reaction or the activation of progenitor cells (Roskams 2006).
These cells can differentiate into either hepatocytes or biliary epithelial cells. Sev-
eral studies have shown that a subset of HCC are positive for progenitor cell mark-
ers (Durnez etal. 2006; Yamashita etal. 2008; Yang etal. 2010) and a majority of
small-cell dysplastic foci, the earliest premalignant lesion known in humans, consist
of progenitor cells and intermediate hepatocytes providing a strong argument for a
progenitor cell origin of these tumors (Roskams 2006). Progenitor marker-positive
HCCs are unique in their genetic signature and are associated with a higher rate of
recurrence and lower OS compared to progenitor marker-negative tumors (Ander-
sen etal. 2010; Lee etal. 2006). In our own studies using a rodent model of pro-
genitor-derived HCC (Sanders etal. 2012), we found that mTORC1 signaling was
activated during the formation of preneoplastic foci, but waned during later stages
of tumor development. Administration of rapamycin during the first three weeks
of foci development was effective in markedly reducing focal lesion burden at ten
weeks. Our data suggest that mTOR inhibition may be an effective chemopreven-
tive strategy for primary and recurrent HCC.
30.4.2 MAPK Pathway
The mitogen activated protein kinase (MAPK) pathway regulates critical cellular
processes including proliferation, differentiation and survival (see Chap.17). The
overexpression of components of this pathway has been shown to contribute to
tumorigenesis, tumor progression, and metastasis in HPB cancers (Collisson etal.
2012; Ito etal. 1998; Menakongka and Suthiphongchai 2010; Whittaker etal. 2010).
Hyperactivation of the pathway occurs through a variety of mechanisms, including
mutations in RAS, overexpression of growth factors, and inactivating mutations in
the negative MAPK pathway regulators, RKIP and Sprouty (Whittaker etal. 2010).
The MAPK pathway regulates the MAPK-interacting kinases MNK1 and MNK2.
The MNKs bind to eIF4G and phosphorylate eIF4E at serine 209, a site that has
been shown to be required for the oncogenic activity of eIF4E in vivo (Wendel etal.
2007). Phosphorylation of eIF4E was positively associated with tumor grade and
correlated with a worse prognosis and earlier disease onset in patients with PDAC
(Adesso etal. 2012). In some model systems, phosphorylation of eIF4E has been
shown to correlate with increased and decreased global protein synthesis (Topi-
sirovic and Sonenberg 2011). It has also been proposed that phospho-eIF4E selec-
tively stimulates the translation of a subset of mRNAs coding for proteins involved
in carcinogenesis, including MCL-1, MMP-3, MMP-7 and SMAD2 (De Benedetti
and Graff 2004; Grzmil etal. 2011; Mills etal. 2008; Wendel etal. 2007).
30.5Translation in Therapeutic Resistance
The ability of cancer cells to rapidly regulate protein synthesis and the translation of
specific mRNAs has been shown to result in resistance to many standard therapies.
Gemcitabine is the current standard systemic therapy for advanced CCA and PDAC
(Cinar and Tempero 2012). However, treatment responses of both tumors are poor
(Ferrone etal. 2012). In vitro studies using several PDAC cell lines showed that
treatment with gemcitabine induced phosphorylation of eIF4E through activation of
the MNK pathway (Adesso etal. 2012). This response was specific as treatment did
not induce AKT phosphorylation. Similar results were obtained with cisplatin, sug-
gesting that activation of eIF4E may be a general feedback loop resulting in resis-
tance to chemotherapeutic treatment (Adesso etal. 2012; Altman etal. 2010). Direct
inhibition of MNK activity using the chemical inhibitor, MNK-I, sensitized cells to
gemcitabine-induced apoptosis (Adesso etal. 2012). These studies suggest that the
MNK/eIF4E axis represents a prosurvival escape pathway utilized by pancreatic
carcinoma cells. eIF4E has also been shown to regulate the sensitivity of CCA cells
to gemcitabine (Wehbe etal. 2006). Treatment of these cells with the small molecule
pifithrin-, a p53 inhibitor and modulator of p53-independent intracellular signal-
ing, resulted in increased phosphorylation of eIF4E at serine 209 through activation
of p38 MAPK and increased sensitivity to gemcitabine (Wehbe etal. 2006).
The scaffold protein, RACK1, acts as a bridge for many protein kinases thus
playing an important role in signal transduction, cellular growth, and differentiation
(McCahill etal. 2002). RACK1 has also been shown to be part of the 40S ribosomal
subunit (Nilsson etal. 2004; Sengupta etal. 2004). RACK1 is overexpressed in
many cancers, including HCC where its localization to the ribosome has been shown
to mediate chemoresistance in vitro and tumor growth in vivo (Ruan etal. 2012).
Ribosomal RACK1 in concert with protein kinase C II (PCKII) phosphorylate
eIF4E leading to the preferential translation of mRNAs involved in cellular growth
and survival. Inhibition of PCKII or eIF4E depletion suppresses RACK1-mediated
chemoresistance, providing further evidence for the role of translation in modulating
the response of tumors to therapeutic agents (Ruan etal. 2012).
Our current knowledge suggests that translation and its control are cornerstones of
HPB cancer biology and efficacy of treatment. Dysregulated translation in HPB
tumors allows for an increase in protein synthesis, leading to enhanced cellular
growth and proliferation. The enhanced translation of specific mRNAs that are
advantageous for cell survival and adaptation to environmental changes can lead
to disease progression and alterations in therapeutic response. The role of specific
signaling pathways and the mechanisms that drive translation of specific mRNAs
warrant further investigation, especially in defined types and stages of cancer. There
are also a number of translation factors that have been identified as playing im-
portant roles in the development and progression of other types of cancer (Silvera
etal. 2010) whose role has not been investigated in HPB cancer. Because changes
in the translation apparatus are associated with therapeutic resistance, combining
novel targeted with standard therapies may significantly increase the efficacy of
treatments for HPB cancers. However, for these targeted therapies to be of maximal
clinical benefit, patient subgroups will have to be defined by histological and mo-
lecular assessment. Further studies into the translation machinery and its control in
HPB cancers will no doubt lead to the identification of novel biomarkers, prognos-
tic indicators, and preventive and chemotherapeutic treatment strategies.
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Chapter 31
Pancreatic Neuroendocrine Tumors
Mamatha Bhat, Peter Metrakos, Santiago Ramon y Cajal, Nahum Sonenberg

and Tommy Alain
Contents
31.2mTOR and Downstream Translation Targets in PaNETs 633
31.3Translation Dysregulation in PaNETs 635
31.4Current Chemotherapeutics for PaNETs 637
References 640
Abstract In two landmark phase III trials, the drugs everolimus and sunitinib dem-
onstrated remarkable efficacy against pancreatic neuroendocrine tumors (PaNETs).
Everolimus acts as a specific inhibitor of the mTOR complex 1 (mTORC1), which
is often found to be hyperactivated in PaNETs. Sunitinib is a multitargeted inhibitor
of RTKs, such as PDGFR, FGFR and VEGFR. These receptors are under frequent
stimulation in PaNETs and result in the activation of the mTOR signaling pathway.
When hyperactive, mTOR prompts a dysregulation in translation, especially at
T.Alain()
Childrens Hospital of Eastern Ontario Research Institute, Department of Biochemistry,
Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
e-mail: tommy@arc.cheo.ca
M.Bhat N.Sonenberg
Department of Biochemistry, Rosalind and Morris Goodman Cancer Research Centre,
M.Bhat
Faculty of Medicine, Division of Gastroenterology, Department of Medicine,
P.Metrakos
Department of Anatomy and Cell Biology and Hepatopancreatobiliary and Transplant Research
Unit, Division of General Surgery, Department of Surgery, Faculty of Medicine,
S.Ramon y Cajal
Department of Pathology, Vall dHebron University Hospital, Autonoma University
of Barcelona, Barcelona, Spain
632 M. Bhat et al.
the initiation step of translation, and thus drives a tumorigenic program. Limiting
the activity of mTOR with small molecular inhibitors can therefore prevent the
dysregulated translation of mRNAs encoding factors with oncogenic or metastatic
functions. Further strategies directly targeting translation initiation factors might be
therapeutically effective in PaNETs. In this chapter, we review the common molec-
ular and biochemical alterations in PaNETs and how they can lead to dysregulated
translation. We also discuss novel therapeutic approaches vis--vis translation and
its regulation in PaNETs.
31.1Introduction
Pancreatic neuroendocrine tumors (PaNETs) represent the second most common

malignancy of the pancreas, with a five-year survival rate of only 40% (Kloppel
etal. 2004; Oberg 2010). The annual incidence of PaNETs is 1 per 100,000 individ-
uals, and has been gradually increasing with improved detection of asymptomatic
malignancies during cross-sectional imaging such as computed tomography per-
formed for other purposes (Yao etal. 2008; Zerbi etal. 2010). Given their endocrine
origin, PaNETs may secrete a variety of peptide hormones, including gastrin, insu-
lin, glucagon and vasoactive intestinal peptide, which leads to a variety of clinical
features at presentation according to the particular hormonal syndrome produced.
For instance, a patient with an insulinoma PaNET would typically present with in-
termittent hypoglycemia, a gastrinoma with severe peptic ulcer disease and possibly
diarrhea, and a VIPoma with watery diarrhea, hypokalemia (low serum potassium
levels), and hypochlorhydria (low serum chloride levels) (Zerbi etal. 2010).
On the other hand, 75% of PaNETs are non-functional, leading to a delayed
clinical presentation with symptoms of local compression (Vagefi etal. 2007). Ad-
ditionally, many PaNETs may be benign, as evidenced by autopsy studies (Kimura
etal. 1991). Nevertheless, it is the malignant PaNETs that present to medical atten-
tion (Vagefi etal. 2007). While most cases of PaNET are sporadic, some occur in
the context of a hereditary endocrine syndrome such as multiple endocrine neopla-
sia, neurofibromatosis type I, and von Hippel Lindau syndrome. In fact, 80100%
of individuals with multiple endocrine neoplasia type 1 (MEN1) syndrome develop
PaNETs (Jensen etal. 2008). The diagnosis of PaNETs is made on the basis of
conventional imaging techniques, such as computed tomography and magnetic
resonance imaging, along with measurement of serum chromogranin levels. Ad-
ditionally, somatostatin-receptor scintigraphy may be used as an imaging modality
more specific to PaNETs, based on the high expression of somatostatin receptors
by most of these tumors. Two principal classification systems are used to catego-
rize PaNETs, including that of the WHO and TNM classification system by the
American Joint Committee on Cancer (AJCC). The WHO classification has very
good prognostic value, and is based on tumor size, proliferative capacity and angio/
perineural invasion (Heitz etal. 2004). Using these criteria, this system makes a
distinction between those tumors that are well versus poorly differentiated, as well
31 Pancreatic Neuroendocrine Tumors 633
as those with benign versus malignant behavior. The TNM staging system classifies
PaNETs according to size, lymph node metastasis and distant metastases, and also
has good prognostic value (Pape etal. 2008; Rindi etal. 2006). However, prospec-
tive validation of these classification systems and comparison of validity have not
been performed. It has therefore not been possible to implement a single classifica-
tion system as a standard (Ehehalt etal. 2009).
Given the indolent nature of PaNETs, these tumors are often diagnosed well
beyond criteria of resectability. Furthermore, most patients with advanced stage
disease have liver metastases. Somatostatin analogs such as octreotide are often
used to manage symptoms of hormonal excess secondary to peptide hypersecre-
tion by PaNETs. Because patients are frequently diagnosed with unresectable tu-
mors or with extensive metastatic disease, limited therapies are available. There
is therefore a clear need for a deeper understanding of the molecular features that
regulate cell proliferation, survival and metastasis in PaNETs, and this has been
the focus of recent studies. The identification of key mutations present in these
tumors, which included prominent mutations in DAXX (death domain-associated
protein), ATRX (X-linked mental retardation and -thalassemia syndrome protein),
as well as MEN1 (multiple endocrine neoplasia 1), brought novel avenues in the
understanding of this disease and how to target it (Kloppel etal. 2004). Additional
candidate genes have included RAR-, hMLH1, RASSF1, HER2/neu, cyclin D1,
TP53, and various tyrosine kinase receptors (Zikusoka etal. 2005). Importantly, a
dependence of PaNETs on a hyperactivated mTOR pathway was also demonstrated
(Jiao etal. 2011). With multiple well-known and novel drugs aimed at limiting the
hyperactivation of mTOR in cancer, this driver of malignancy has appropriately be-
come an attractive and amenable therapeutic target against PaNETs. The evidence
from mTOR hyperactivation and targeting in PaNETs is highly suggestive of the
role of dysregulation of translation control, since the PI3K/AKT/mTOR pathway
converges on translation initiation machinery. Additionally, elevated phosphoryla-
tion of the translation factors eIF4E, 4E-BPs and increased activity of S6Ks in Pa-
NETs further support aberrant translation control in these tumors.
31.2mTOR and Downstream Translation

Targets in PaNETs
The PI3K/AKT/mTOR signaling pathway (hereinafter mTOR pathway) is critical

in tumorigenesis, being frequently hyperactivated in many types of cancers (Guertin
and Sabatini 2007; Hay and Sonenberg 2004) and often being found to be an impor-
tant determinant in cancer progression. Indeed, in PaNETs, the mTOR pathway is
hyperactivated due to the frequent loss of the TSC2 and PTEN genes (both suppres-
sors of the mTOR pathway), and also by the high expression of fibroblast growth fac-
tor 13 (FGF13) (Fig.31.1a) (Capdevila etal. 2011; Jiao etal. 2011; Kasajima etal.
2011; Missiaglia etal. 2010). mTOR hyperactivation in PaNETs is associated with
high proliferative activity on tumor histology (Kasajima etal. 2011). The mTOR
634 M. Bhat et al.
Fig. 31.1 mTOR pathway and translation initiation in PaNETs. a The mTOR pathway is hyper-
active in PaNETs. The PI3K/AKT signaling cascade culminates on mTOR to regulate a number
of cellular processes including cell growth and proliferation through the control of translation
initiation. Integration of several different inputs act to activate or inhibit mTOR activity. Pharma-
cological inhibitors of this pathway are indicated in red. For a detailed review of this pathway see
Chap.15. b Expression of mTOR, eIF4E and 4E-BP1 by IHC in PaNET samples demonstrating
the upregulation and activation of these factors in this disease. c Activation of translation initiation
by mTOR. When activated by nutrients, hormones, and growth factors, mTOR phosphorylates
two major targets, 4E-BPs and S6Ks. Phosphorylated (inactivated) 4E-BPs dissociate from eIF4E.
eIF4E can then recruit other initiation factors to form the eIF4F complex, which acts to unwind
the secondary structures of mRNAs and to recruit the ribosome. Phosphorylation and activation
of S6Ks lead to their dissociation from eIF3 and phosphorylation and activation of eIF4B. These
multiple interactions culminate to enhance cap-dependent translation.
pathway components are overexpressed in well-differentiated PaNETs (Kasajima

etal. 2011). Whole exome sequencing of sporadic PaNETs has revealed mutations
along the PI3K/AKT/mTOR pathway in 15% of PaNETs, with PI3K, PTEN and
TSC2 being the most common genes affected (Jiao etal. 2011). However, when
one also looks at expression of genes along this pathway, there are further altera-
tions detected in most PaNETs (Missiaglia etal. 2010). Reflecting hyperactivation
of the mTOR pathway, high expression levels of phospho-mTOR, eIF4E, 4E-BP1
and S6K1, as assessed by IHC, have also been correlated with shorter patient sur-
vival (Heetfeld and Koch 2013) (Fig.31.1b). Intensity of immunohistochemical
expression of phospho-mTOR has also been found to significantly correlate with
clinicopathologic features including tumor size, vascular and extrapancreatic inva-
sion, lymph node/distant metastasis, mitotic count, and TNM staging and the WHO
classification (Komori etal. 2013). Although most PaNETs are sporadic, these tu-
mors are also known to arise in a hereditary manner in individuals with MEN1 and
tuberous sclerosis (Toumpanakis and Caplin 2008). Correspondingly, deletion of
Men1 leads to hyperactivation of the mTOR pathway in animal models (Wang etal.
2011). Additionally, low expression of either TSC2 or PTEN, or high expression
of FGF13, correlates with PaNET aggressiveness, a higher proliferation index, and
the presence of liver metastasis at diagnosis or follow-up (Missiaglia etal. 2010).
These findings strongly implicate mTOR hyperactivation in PaNET tumorigenesis.
The serine/threonine kinase mTOR integrates extra- and intracellular signals to
effect growth, proliferation and protein synthesis. mTOR exists in two complexes:
mTORC1 and mTORC2 (Fig.31.1a and Chap.15). mTORC2 phosphorylates AKT
and SGK1 to regulate cytoskeletal organization. mTORC1 phosphorylates 4E-BPs,
S6Ks and other substrates to promote cell growth and proliferation, predominantly
by enhancing cap-dependent translation initiation (Guertin and Sabatini 2007; Hay
and Sonenberg 2004). mTORC1 exerts its functions on translation through the 4E-
BPs, which when phosphorylated (inactivated) by mTORC1, dissociate from eIF4E
and can no longer repress eIF4E, resulting in enhanced cap-dependent translation
(Fig.31.1c and Chap.4) (Gingras etal. 1999). mTORC1 also affects translation by
phosphorylating S6Ks, which modulate the activity of helicases through PDCD4
and eIF4B (Hay and Sonenberg 2004). S6Ks regulate cell growth while cell prolif-
eration is primarily controlled by 4E-BPs (Dowling etal. 2010). Importantly, both
4E-BPs and S6Ks are known to be highly phosphorylated in PaNETs and to be criti-
cal for the malignant progression of this disease (Capdevila etal. 2011; Kasajima
etal. 2011; Missiaglia etal. 2010). This finding brings direct relevance to dysregu-
lated translation control in the pathogenesis of PaNETs.
31.3Translation Dysregulation in PaNETs
In eukaryotes, the translation of most mRNAs is initiated in a cap-dependent man-

ner. The cap structure, m7GpppN is present at the 5 terminus of all eukaryotic
mRNAs transcribed in the nucleus (Gingras etal. 1999) (see Chap.2). eIF4E is the
636 M. Bhat et al.
factor that binds to the cap structure and recruits the mRNA to the ribosome as part
of the eIF4F complex which also comprises eIF4A, an RNA helicase that unwinds
the secondary structure within the 5 UTR of the mRNA, and eIF4G, a large scaf-
folding protein (Fig.31.1c and Chap.4). PDCD4 curtails cap-dependent translation
by inhibiting the helicase function of eIF4A and interfering with the interaction
between eIF4A and eIF4G (see Chap.6). In addition, eIF4G interacts with the 40S
ribosomal binding factor eIF3 and PABP. The formation of the eIF4F complex is
one of the most important rate-limiting steps in regulating translation initiation. In
this process, the activity of eIF4E is critical as for once, it is the least abundant of
all the initiation factors (Duncan etal. 1987; Hiremath etal. 1985; Lazaris-Karatzas
etal. 1990). Moreover, eIF4E function is tightly regulated by the translation repres-
sors 4E-BPs, which compete with eIF4G for binding to eIF4E and thereby sequester
eIF4E from the translation machinery (Pause etal. 1994). These events respond to
extra- and intracellular stimuli, such as mitogens, hormones, growth factors, energy
and oxygen status, to promote the translation of a subset of mRNAs that encode
proteins important for the physiological processes of proliferation, cell growth,
apoptosis and differentiation.
mRNA translation is the most energy-consuming process in the cell, thereby
requiring stringent control to maintain proper cellular homeostasis (Ma and Blenis
2009). Loss of translation regulation in cells or tissues is indeed attributed to the
development and progression of several conditions including neurological diseases
and cancer (Sonenberg and Hinnebusch 2009). The dysregulation of translation
through loss of eIF4E control frequently occurs in cancer. For instance, following
phosphorylation by mTORC1, the 4E-BPs detach from eIF4E, leaving it free to
initiate translation of most mRNAs in the cell (Gingras etal. 1999). Hyperactiva-
tion of the mTOR pathway results in a dysregulation of translation control that is
considered one of its main oncogenic drivers (Laplante and Sabatini 2012). The bal-
ance of translation is particularly shifted towards those key mRNAs that potentiate
tumorigenesis. Therefore, these regulatory mechanisms act upon eIF4E to ensure
proper translational control and rate of protein expression. In the absence of such
regulation, cells would be pushed into a proliferative state leading to potentially del-
eterious consequences (Koromilas etal. 1992; Sonenberg and Hinnebusch 2009).
Furthermore, in various cancers, eIF4E is overexpressed, and its high expression
levels correspond with poor prognosis (Sonenberg and Hinnebusch 2009). Transla-
tion can be further dysregulated in many human malignancies via an increase in the
phosphorylation of eIF4E (Furic etal. 2010; Mamane etal. 2007). Phosphoryla-
tion of eIF4E on serine 209 by the MAPK-integrating kinases MNK1 and MNK2
is required and plays a critical role in the tumorigenesis of multiple cancers (see
Chap.17) (Furic etal. 2010). There are currently ongoing clinical trials targeting
eIF4E levels in cancer by antisense RNA, shutting down its activity using eIF4E/
eIF4G interaction impeding molecules, or blocking its elevated phosphorylation us-
ing pharmacological inhibitors of MNKs. Importantly, both eIF4E expression and
its phosphorylation are elevated in PaNETs (Capdevila etal. 2011; Kasajima etal.
2011; Missiaglia etal. 2010). Considering the seminal roles of these processes in
tumorigenesis, it is anticipated that elevated protein synthesis and dysregulated con-
trol of translation factors play major roles in PaNETs development and progression,
and could represent critical therapeutic targets.
PaNETs are highly vascular tumors, with significantly upregulated expression
of VEGF, PDGF, FGF, IGF-1, and transforming growth factor and (Ehehalt
etal. 2009). mRNAs with long and structured 5 UTRs, which encode proteins that
promote cell proliferation, angiogenesis, and survival (e.g. cyclins, c-MYC, VEGF
and BCL-XL), are particularly sensitive to eIF4E levels and activity (Dowling etal.
2010; Koromilas etal. 1992; Mamane etal. 2007). Importantly, the translation of
these factors is under the control of eIF4E, making the suppression of translation
initiation a highly plausible target in PaNETs. Limiting the activity of mTOR with
small molecular inhibitors, or directly targeting the activity of eIF4E could prevent
translation of these mRNA-encoding factors with potentially oncogenic or meta-
static functions, potentially improving the efficacy of current therapeutics.
31.4Current Chemotherapeutics for PaNETs
Until recently, the only agent approved for treatment of advanced stage PaNETs
was streptozocin, although the extent of its antitumor effect has been unclear (Ober-
stein etal. 2012). Streptozocin appeared to have an effect principally on tumors
with an intermediate mitotic rate (Ki-67 levels 320%), with overall response rates
of 2060% (Kouvaraki etal. 2004). Well-differentiated PaNETs had very poor re-
sponse to this standard first-line chemotherapy due to their low proliferation rate
(less than or equal to 2%).
Two recent phase III trials documented an increased efficacy of everolimus and
sunitinib against PaNETs, thereby changing the landscape of chemotherapy for this
disease (Raymond etal. 2011; Yao etal. 2011). These agents revolutionized the
paradigm of PaNET treatment by doubling PFS as compared to placebo. Everoli-
mus acts as an allosteric inhibitor of the mTOR complex 1 (mTORC1), which is
often found to be hyperactivated in PaNETs (Capdevila etal. 2011; Kasajima etal.
2011; Missiaglia etal. 2010). Sunitinib, on the other hand, is a multitargeted inhibi-
tor of RTKs, such as PDGFR, FGFR, and VEGFR. These receptors are under fre-
quent stimulation in PaNETs and result particularly, in the activation of the mTOR
signaling pathway (Pavel 2013; Raymond etal. 2012). Therefore, controlling the
dysregulation of mTOR through targeted pharmacological inhibition has proved to
be an effective therapeutic strategy in PaNETs.
In the phase III trial of sunitinib (Delbaldo etal. 2012), patients with advanced
PaNETs were randomized to sunitinib 37.5mg daily versus placebo groups. The
patients in the sunitinib arm received treatment for a median duration of 4.6 months,
the sunitinib group had a significantly longer median PFS of 11.4 versus 5.5 months.
In the phase III trial of everolimus, 410 patients with advanced low- or intermedi-
ate-grade PaNETs were randomized to everolimus 10mg daily versus placebo. The
everolimus group had a 65% decrease in risk of disease progression or death as
compared to placebo group. The patients on everolimus had a median PFS of 11.0
638 M. Bhat et al.
months as compared to 4.6 months with placebo. Additionally, there was evidence
of objective partial response on follow-up imaging among 5% of patients on evero-
limus versus 2% on placebo. On the basis of these pivotal trials, both sunitinib and
everolimus gained approval from the FDA for treatment of advanced PaNETs. A
more recent phase II study assessed the allosteric mTOR inhibitor temsirolimus in
progressive neuroendocrine tumors, with 64% achieving disease control (Duran
etal. 2006). Another phase II trial combining everolimus with the monoclonal an-
tibody bevacizumab (Avastin), which targets VEGFR, demonstrated promise with
44% PFS at 6 months (Hobday etal. 2013).
By directly blocking or impinging upon mTOR activity, these inhibitors thus
demonstrate attractive efficacy against PaNETs. However, everolimus and sunitinib
as broad-based monotherapies still show limitations in PaNETs and other cancers
(Guertin and Sabatini 2007; Yao etal. 2011). Although they inhibit angiogenesis,
they do not cause dramatic tumor shrinkage, and at best only prolong survival of the
patients. It is thought that there may be an adaptive resistance that develops against
these agents, a phenomenon that has been observed with antiangiogenic agents as
a group. Importantly, everolimus has only limited inhibitory effects on shutting
dysregulated translation in PaNETs, being an allosteric inhibitor of mTORC1 that
cannot completely abolish phosphorylation of the 4E-BPs (Dowling etal. 2010;
Hsieh etal. 2012; Thoreen and Sabatini 2009). Furthermore, mTORC2 is not inhib-
ited by allosteric mTOR inhibitors, which results in a feedback loop that activates
AKT. This inability to efficiently suppress translation of mRNAs encoding proteins
with proliferative and oncogenic functions may be a considerable factor in limit-
ing treatment efficacy (Chiu etal. 2010; Choo etal. 2008; Dowling etal. 2010;
Thoreen and Sabatini 2009), and may also significantly contribute to the develop-
ment of resistance to therapies. Therefore, the crucial effect of dysregulated transla-
tion downstream of mTOR in PaNETs, and the factors involved that can predict the
therapeutic success of mTOR inhibition, should be important focuses of research.
Overexpression or activating/inactivating mutations in molecules downstream of
membrane receptors targeted by sunitinib (e.g. loss of the tumor suppressors PTEN
or TSC2) also may account for the lack of significant effect (Makhov etal. 2012).
The elevated expression and increased phosphorylation of eIF4E in cancer may
further promote the translation of mRNA-encoding proteins involved in disease
progression and resistance to therapy (Alain etal. 2012; Furic etal. 2010; Pyronnet
etal. 1999; Sonenberg and Hinnebusch 2009).
In the general treatment of malignancies, de novo and acquired resistance to
mTOR inhibitors have been reported, with mutations detected along the PI3K/
AKT/mTOR pathway. Rapamycin treatment of small intestinal NETs has been as-
sociated with an increase in phospho-AKT expression, representative of an escape
phenomenon (Hill etal. 2010). Phospho-AKT expression in tumor biopsies before
and after treatment with everolimus and octreotide has also been correlated with
patients PFS (Meric-Bernstam etal. 2012). Innovative strategies are needed to ad-
vance the management of patients affected by PaNETs, who currently have very
limited treatment options. The dysregulation of translation represents a novel thera-
peutic target that has become amenable with the recent characterization of advanced
molecular inhibitors, and the current understanding of their functional outputs. As

of August 2014, there are 9 ongoing clinical trials of patients with PaNETs involv-
ing the use of everolimus in combination with another chemotherapeutic agent and
two trials involving temsirolimus and another chemotherapeutic agent.
Recently, several asTORi have been characterized. These inhibitors target the ac-
tivity of both mTORC1 and mTORC2, and exhibit stronger inhibitory effects than
those of rapamycin or everolimus on mTORs downstream effectors, as well as on
cell growth, proliferation, and survival (Dowling etal. 2010; Feldman etal. 2009;
Thoreen and Sabatini 2009). This dual inhibition would help prevent the escape
phenomenon through AKT, and most importantly may provide increased inhibi-
tion of translation of factors involved in proliferation and survival of this disease.
Another strategy would be to concomitantly use PI3K/AKT and mTOR inhibitors
in combination. NVP-BEZ235, a new investigational agent that performs dual sup-
pression of PI3K and mTOR, has been shown to induce apoptosis of PaNET cell
lines more effectively than inhibitors that target single molecules along the pathway
(Serra etal. 2008). Such dual suppression has been shown to inhibit both horizontal
and vertical feedback loops through AKT induced by prior treatment with everoli-
mus (Serra etal. 2008; Yao etal. 2008). There is currently an ongoing clinical trial
investigating such dual target inhibition, which may be able to better avoid develop-
ment of resistance. However, there is a potential risk of increased toxicity with dual
target inhibition, and the clinical trials will give a better appreciation as to whether
such drug combinations can be adequately tolerated by patients. In addition, the
specific inhibitor cercosporamide targeting MNKs and eIF4E phosphorylation, as
well as drugs aimed at reducing the elevated levels/activity of eIF4E, are currently
under extensive scrutiny as cancer therapeutics. These compounds represent impor-
tant pharmacological tools to characterize the definite roles that translation dysregu-
lation plays on PaNETs progression, and how it can modulate response to therapy.
mTOR inhibition has emerged as a valid strategy to treat PaNETs, with conclusive
clinical data on its efficacy. This treatment results in downregulation of translation
initiation, an important step in the protein synthesis that feeds malignant cell prolif-
eration. The asTORi and dual PI3K/mTOR inhibitors have the potential to further
optimize the potency of mTOR inhibition in PaNETs and should be further evalu-
ated in preclinical models of the disease. One deterrent to research into PaNETs has
been the lack of clinically representative and validated cell lines and mouse models,
which in turn is due to the heterogeneous nature of PaNETs. Although most are non-
functional, 25% secrete various hormones that affect tumor behavior. Human cell
lines that have been used for the study of PaNETs include the following: BON cells
derived from carcinoid (Evers etal. 1994), QGP-1 derived from somatostatinoma
(Iguchi etal. 1990), and CM cells representing insulinoma (Jonnakuty and Gragnoli
2007). BON cells specifically were used to investigate everolimus, with evidence of
640 M. Bhat et al.
antiproliferative effect (Zitzmann etal. 2007). Additionally, there are various mouse
cell lines that have been used to test out various therapies for PaNETs. With respect
to animal models, the main approaches used have been transgenic oncogene expres-
sion under the insulin promoter (Power etal. 1987), and heterozygous or homozy-
gous deletions of Men1 resulting in an insulinoma or glucagonoma (Crabtree etal.
2001). Other genetic models for PaNETs have included glucagon receptor deletion
and Vhl deletion (Babu etal. 2013). The RIP-Tag2 mouse model develops multi-
stage islet cell tumors using the simian virus 40 large T antigen under the control of
an insulin promoter. This model has also been used extensively, although its verac-
ity in representing PaNETs has been questioned (Modlin etal. 2008). Appropriate
models and additional basic research and clinical trials using effective suppressors
of the mTOR pathway and dysregulated translation control, hold a great deal of
promise for the therapy of patients with advanced PaNETs.
Acknowledgements The authors thank Dr. Armen Parsyan for critical reading of the chapter.
M. Bhat is a recipient of a CIHR fellowship for health professionals. This manuscript was funded
by a grant from the Cancer Research Society and the Carcinoid NeuroEndocrine Tumour Society
of Canada.
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Chapter 32
Gynecologic Cancers
Armen Parsyan and Susana Banerjee
Contents
32.2Ovarian Cancer and Translation 647
32.3Endometrial Cancer and Translation 647
32.4Cervical Cancer and Translation 648
32.5Novel Therapeutic Approaches to Target Translation Machinery in
Gynecologic Cancers 649
32.6PDCD4 in Gynecologic Cancers 650
32.7Other Translation Factors in Gynecologic Cancers 651
References 652
Abstract Despite major advances in understanding, prevention and treatment of

gynecologic cancers, they still remain a common cause of morbidity and mortality
for women. New insights into the molecular aspects of etiology and pathogenesis
have identified involvement of multiple oncogenic signaling pathways. Transla-
tional regulation is an important mechanism of tumorigenesis. In this chapter, the
important roles of translation factors, such as proto-oncogene eIF4E and its inhibi-
tory binding proteins, 4E-BPs, as well as tumor suppressor PDCD4 are discussed
in the context of gynecologic cancers. An outline of targeted therapies addressing
aberrant function of these factors and their regulation is also presented.
A.Parsyan()
S.Banerjee
Gynaecology Unit, The Royal Marsden National Health Service (NHS) Foundation Trust,
London, UK
646 A. Parsyan and S. Banerjee
32.1Introduction
The majority of gynecologic malignancies are ovarian (including fallopian tube and
primary peritoneal), uterine (endometrial) and cervical cancers. Vaginal and vulvar
cancers are very rare. While in industrialized countries ovarian and endometrial
cancers are the most prevalent, cervical cancer is the most frequently diagnosed
malignancy in low human development index countries and by far the most com-
mon gynecologic malignancy worldwide (CDC/NCI 2013; Ferlay etal. 2008; Fer-
lay etal. 2010). Ovarian cancer is the leading cause of cancer mortality amongst
gynecologic cancers in industrialized countries (CDC/NCI 2013; Weiderpass and
Labreche 2012).
Our understanding of the molecular etiology and pathogenesis of gynecologic
cancers is improving, and key research advances have led to substantial improve-
ments in the management and OS rates-these improvements are partly due to the
recognition that the majority of cancer cases are HPV-related and preventable
through screening and vaccination programs (Banerjee and Kaye 2011; Banerjee
and Kaye 2013; Group F.I.S. 2007; Suh etal. 2013; Weigelt and Banerjee 2012).
The discovery of distinct molecular pathways associated with individual gyneco-
logic cancers has led to the development of clinical trial strategies assessing a wide
variety of targeted agents. Of the many potential targets, signaling pathways that
control translation have been implicated in the development and progression of
gynecologic malignancies. For example, the PI3K/AKT/mTOR cascade that con-
verges on control of the translation machinery is reported to be aberrant in more
than 80% of endometrioid endometrial cancers (Cheung etal. 2011; Vivanco and
Sawyers 2002). This pathway has also been reported to be activated in cervical and
ovarian tumors. Details of PI3K/AKT/mTOR pathway mechanisms are discussed
in Chap.15. To summarize, the activation of mTOR leads to the phosphorylation
of 4E-BPs, resulting in their dissociation from eIF4E and activation of the latter
(see Chap.4). Activation of eIF4E leads to increased translation of prosurvival,
protumorigenic and proangiogenic signals, such as VEGF, cyclin D1 and others
(see Chap.4). In addition to mTOR signaling, several other molecules, known to
be regulated by or involved in translation, have been reported to be potential thera-
peutic targets in gynecologic cancers. For example, angiogenesis plays an important
role in ovarian cancer, and VEGF, which is in part regulated by translation, is a key
component of this process. Targeting VEGF with the monoclonal antibody, beva-
cizumab (Avastin), has been shown to significantly improve survival in advanced
ovarian cancer and is a part of standard of care in many centers (Perren et al. 2011;
Banerjee and Kaye 2012, 2013). The development of clinical trials evaluating the
targeting of the translation machinery is imminent. In this chapter, we will summa-
rize the current information on the role of translation in gynecologic cancers.
32 Gynecologic Cancers 647
32.2Ovarian Cancer and Translation
Aberrations of the PI3K/AKT/mTOR pathway have been reported in ovarian can-

cer. These reports suggest significant differences in the expression of some of the
mTOR-related tumor suppressors and proto-oncogenes, which could be associated
with the pathogenesis of ovarian cancer (Laudanski etal. 2011). Multiple genetic
alterations are found within the PI3K/AKT/mTOR pathway in ovarian carcinoma,
such as overexpression of the p110 catalytic subunit of PI3K (68%), AKT1
(19%) or AKT2 (12.5%), the loss of PTEN (27%), mutations in the PIK3CA (12%)
or KRAS (10%) (De Marco etal. 2013). While various events, as described above,
may explain activation of the pathway upstream of mTOR, the downstream effects
of its activation converge on the translation machinery, namely the 4E-BPs/eIF4E
axis.
An activated mTOR/4E-BPs/eIF4E axis is found in various types of ovarian
cancers and in some types it correlates with chemoresistance and poor prognosis
and clinicopathologic markers (Castellvi etal. 2006; Harasawa etal. 2011; Lau-
danski etal. 2011; No etal. 2011; Noske etal. 2008). One study examining 107
ovarian lesions observed an overexpression of phosphorylated mTOR (47%) and
eIF4E (56%) (Noske etal. 2008). Using tissue microarrays for 129 ovarian epithe-
lial tumors, including serous and mucinous cystadenomas; serous and mucinous
borderline tumors; serous and mucinous carcinomas; endometrioid and clear cell
carcinomas, it has been found that phosphorylated 4EB-P1, p70S6K and rpS6 were
expressed significantly more often in malignant tumors (Castellvi etal. 2006). In
patients with ovarian carcinoma, significant expression of phosphorylated 4E-BP1
was associated with high-grade tumors and a poor prognosis, regardless of other
oncogenic alterations upstream (Castellvi etal. 2006). Increased phosphorylation
of 4E-BP1 was also reported to be associated with poor differentiation and higher
mitotic rate (Noske etal. 2008). Studies of ascites samples from patients have sug-
gested a potential for modulation of this pathway to overcome resistance to chemo-
therapy in ovarian cancer. In one report, phosphorylated p70S6K levels were sig-
nificantly higher in cells from 37 of 61 patients who did not respond to subsequent
chemotherapy (Carden etal. 2012).
32.3Endometrial Cancer and Translation
In preclinical studies direct inhibition of eIF4E has been shown to reduce cell
growth in endometrial adenocarcinoma cells (Choi etal. 2011). IHC showed ex-
pression of eIF4E in 58.1% of endometrial cancer specimens. While eIF4E is a
ubiquitously expressed, this proportion is likely a reflection of a moderate sensitiv-
ity of the IHC technique used, which allowed detection of the protein only in the
samples with high or overexpressed levels of eIF4E. The positivity of eIF4E was
significantly more frequent in more advanced stage III/IV tumors (Choi etal. 2011).
Together with signs of overexpression of eIF4E in endometrial cancers, there is also
an evidence of activation of the PI3K/AKT/mTOR pathway via 4E-BP1 to eIF4E in
various types of endometrial cancers (Darb-Esfahani etal. 2009; Li etal. 2008; Rice
etal. 2006). As mentioned previously, activation of this cascade has been reported
in as many as 80% of endometrial cancers with associated aberrations of com-
ponents and regulators of the pathway. Notably, activated mTOR and increase in
phosphorylated 4E-BP1 levels in endometrial adenocarcinoma are associated with
the worse stage and grade (Darb-Esfahani etal. 2009). The ability of the mTOR in-
hibitor, rapamycin, to suppress the growth of endometrial cancer cells has also been
demonstrated and linked to 4E-BP1/eIF4E axis suppression via mTOR (although
non-translation-related effects of this compound in suppressing tumor growth have
also been reported, such as reduced transcription of hTERT (Bae-Jump etal. 2010;
Darb-Esfahani etal. 2009; Shafer etal. 2010; Zhou etal. 2003). Rapamycin was
also found to potentiate the effects of chemotherapeutic agents, such as paclitaxel
in endometrial cancer cells through inhibition of cell proliferation and induction of
apoptosis (Shafer etal. 2010).
32.4Cervical Cancer and Translation
eIF4E expression was reported to be associated with histopathologic grades in cer-

vical neoplasia: eIF4E expression gradually increased with worsening histopatho-
logic grade (from low-grade cervical intraepithelial neoplasia (CIN) to high-grade
CIN to invasive squamous cell carcinomas) correlating with enhanced expression
of eIF4E mRNA in tumor versus normal tissues (Lee etal. 2005). Another study
also reported a progressive increase in eIF4E staining intensity with worsening cer-
vical pathology (Matthews-Greer etal. 2005). While c-MYC proto-oncogene was
most often linked to increased transcription of eIF4E, there are likely other specific
mechanisms that exist in cervical carcinoma. HPV is the main driver linked to the
etiology and pathogenesis of cervical neoplasia. The HPV protein, E6, was found to
induce transcription of eIF4E and thus promote proliferation and migration of cer-
vical cancer (Wang etal. 2013a). However, whether E6 induces eIF4E transcription
directly or indirectly is largely unknown. Another HPV protein, oncoprotein E7,
shows enhanced expression during differentiation via an increase in its translation,
associated with phosphorylation of 4E-BP1 in HPV-infected cells (Oh etal. 2006).
Thus a pattern emerges by which HPV upregulates cellular proto-oncogene eIF4E
that in turn assists in the production of viral proteins.
An increase in eIF4E activation via mTOR/4E-BPs is also reported in various
types of cervical cancer and is associated with worse prognosis (Faried etal. 2008).
AKT/mTOR was found to be activated in adenocarcinoma of the cervix and cor-
related with poor prognosis. Preclinical studies suggested that pretreatment with ra-
pamycin increased cisplatin-induced apoptosis (Benavente etal. 2009; Faried etal.
2008). Increased levels of 4E-BP1 also were shown to predict poorer outcomes
following postoperative radiotherapy (Benavente etal. 2009).
32.5Novel Therapeutic Approaches to Target Translation

Machinery in Gynecologic Cancers
A full review of the therapeutic strategies addressing oncogenic signaling is outside

the scope of this chapter. For more details we refer the reader to reviews written on
these topics (see Banerjee and Kaye 2011; Banerjee and Kaye 2013; Glaysher etal.
2013; Myers 2013). Nevertheless, it is worth introducing novel approaches target-
ing the translation machinery. These strategies include use of mTOR inhibitors,
such as everolimus and ridaforolimus (Colombo etal. 2013; Gozgit etal. 2013;
Harasawa etal. 2011; Mabuchi etal. 2007; Rico etal. 2012); PI3K/mTOR dual in-
hibitors, such as BEZ235 and PF-04691502 (Mazzoletti etal. 2011; Montero etal.
2012; Sheppard etal. 2013) and others.
There are several agents that target the PI3K/AKT/mTOR pathway that have en-
tered clinical trials of patients with endometrial cancer. For example, temsirolimus,
an intravenous mTOR inhibitor, was evaluated as a single agent in phase II stud-
ies of women with recurrent/advanced endometrial cancer. Interestingly, response
rates were greater in chemotherapy-naive patients (partial response in 14%, stable
disease in 69%) compared to those who had previously received chemotherapy (PR
4%, SD 58%) (Oza etal. 2011). Response rates were independent of PTEN muta-
tions, ribosomal protein rpS6 expression levels and phosphorylation of AKT and
mTOR. In a phase II study of the oral mTOR inhibitor everolimus clinical benefits,
defined as complete or partial response or prolonged stable disease after 8 or more
weeks, have also been reported in 21% of patients with endometrioid endometrial
cancer (Slomovitz etal. 2010). Another oral mTOR inhibitor ridaforolimus has also
shown promising results in a phase II trial with partial response in 11% of patients
and disease stabilization in 18% (Colombo etal. 2013). Combination strategies
with compounds targeting mTOR/PI3K/AKT and chemotherapy, hormonal and an-
tiangiogenic therapy are entering clinical trials stage. Preliminary results of a phase
II trial of everolimus and letrozole in patients with recurrent endometrial carcinoma
showed encouraging results (Slomovitz etal. 2011).
In cervical cancer, a phase II study of temsirolimus as monotherapy reported
modest activity with disease stabilization in 58% of patients and partial response in
3% (Tinker etal. 2013).
It is important to point out that mTOR inhibition by rapamycin and rapalogs
has been shown to lead to feedback activation of other pathways and hence have
a decreased effect on suppressing an activated protumorigenic translation pro-
gram. Various mechanisms of this feedback activation are described in Chap.15
and other chapters of this book (see PartIV Chapters). Briefly, mTOR assembles
into two distinct complexes: mTORC1 that is highly responsive to rapamycin and
mTORC2 that is not. When discussing mTOR activation we often imply activa-
tion of the mTORC1 complex. Inhibition of mTORC1 by rapalogs could still keep
the mTORC2/4E-BPs/eIF4E axis active. In various gynecologic malignancies, a
selective activation of mTORC2 and rebound activation of AKT upon treatment
with rapamycin or rapalogs has been observed, and a stronger inhibition of the 4E-
BPs/eIF4E axis has been achieved using strategies targeting both PI3K and mTOR
(Mazzoletti etal. 2011; Shen etal. 2010). Hence, new-generation mTOR inhibitors,
such as PI3K/mTOR inhibitors and dual TORC1/TORC2 inhibitors or compounds
targeting the translation machinery directly (4E-BPs or eIF4E) could be potentially
more effective (see Chap.15).
Drugs targeting the MAPK pathway (such as MEK inhibitors) that converges
on activating phosphorylation of eIF4E (see Chap.17) have entered clinical trials
of low grade serous ovarian cancer, a rare subtype of ovarian cancer in which this
pathway has been reported to be activated (Givant-Horwitz etal. 2003). A phase II
study of the MEK1/2 inhibitor, selumetinib, has shown promising activity (Farley
etal. 2013).
The potentially important role of the 4E-BP/eIF4E hub of translation in ovarian
cancer therapeutics has been shown by studies of peptides that bind eIF4E and thus
inhibit tumor growth. A preclinical study examined a 4E-BP-based peptide fused
to an analog of gonadotropin-releasing hormone (GnRH) to facilitate an uptake by
GnRH-positive tumor cells and found that it prevented eIF4E from binding eIF4G,
and inhibited cap-dependent translation (Ko etal. 2009). Treatment with this fusion
peptide reduces intraperitoneal tumor burden in mice (Ko etal. 2009).
Another strategy is to silence eIF4E by siRNAs. Indeed, downregulation of
eIF4E by siRNA in cultured endometrial carcinoma cells showed promising antitu-
mor effects (Choi etal. 2011). Similar strategies to control expression of translation
factors directly are further discussed below.
32.6PDCD4 in Gynecologic Cancers
Tumor suppressor PDCD4 that regulates eIF4A plays a role in ovarian cancer. Its
expression in ovarian cancer cell lines (SKOV3, 3AO, and CAOV3) significantly
inhibits proliferation and cell cycle progression (G1 arrest), induces apoptosis, in-
hibits cell migration and invasion and decreases the colony-forming capacity of
ovarian cancer cells in vitro and tumorigenic capacity in mice (Wei etal. 2012;
Wei etal. 2009b). PDCD4 also appears to improve the sensitivity of ovarian can-
cer cells to cisplatin and carboplatin (but not cyclophosphamide, etoposide, or
paclitaxel) chemotherapy, enhancing cisplatin-induced apoptosis by mainly acti-
vating the death receptor pathway (Zhang etal. 2010). Compared to the normal
ovarian tissues or benign serous cystadenomas, PDCD4 expression is found to be
lost or significantly lower in serous cystadenocarcinomas, significantly associat-
ing with higher pathological grade and poorer disease-specific survival of patients
(Wang etal. 2008). Another study presented similar findings, where lower levels of
PDCD4 expression correlated with shorter DFS in borderline and malignant ovarian
specimens (Wei etal. 2009a).
Recent reports suggest that in human ovarian carcinomas and in human lung
adenocarcinoma cells miR-182 might be able to promote cell growth, invasion,
and chemoresistance by downregulating PDCD4 (Wang etal. 2014; Wang etal.
2013b). While miR-182 might indeed be a novel regulator of PDCD4, this pro-
tein in various cancers is often found downregulated via miR-21 (see Chap.6).
This appears true not only for ovarian but also other gynecologic malignancies.
Downregulation of miR-21 using anti-miR short hairpin RNA increased expres-
sion of PDCD4 in OVCAR3 ovarian carcinoma cells and dramatically increased
apoptotic cell death and decreased cell proliferation, invasion and migration (Lou
etal. 2011). Inhibition of miR-21 expression by berberine was reported to upregu-
late PDCD4 to modulate the sensitivity of cisplatin in the ovarian cancer cells (Liu
etal. 2013). miR-21 has been found overexpressed in cervical cancer, correlat-
ing with worsening clinical diagnosis (Deftereos etal. 2011). Inhibition of miR-
21 in HeLa cervical cancer cells caused profound reduction of cell proliferation,
and increased the expression of the tumor suppressor gene PDCD4 (Yao etal.
2009). PDCD4 is significantly less expressed with worsening of cervical lesions;
it is lower in women with invasive cervical carcinoma in comparison to those
with cervical intraepithelial neoplasia or carcinoma in situ (Deftereos etal. 2011).
In endometrial cancer, miR-21 was found overexpressed, but no associated de-
crease in PDCD4 levels was reported compared to normal endometrial specimens
(Torres etal. 2011).
Thus PDCD4 tumor suppressor appears to be important for development, pro-
gression and chemosensitivity in various gynecologic malignancies via, at least in
part, its regulation by miRNAs. Thus, approaches targeting miRNAs represent po-
tential therapeutic strategies.
32.7Other Translation Factors in Gynecologic Cancers
Involvement of other translation factors in cancer of the female reproductive sys-

tem is much less studied and understood. Only anecdotal reports exist in relation
to other translation factors, such as eIF3, DHX29 and eIF6 in ovarian, endometrial
or cervical cancers (Benelli etal. 2012; Dellas etal. 1998; Lomnytska etal. 2010;
Parsyan etal. 2009). It is notable that expression of eIF3a was observed decreasing
from low to high grade CIN and to invasive cervical carcinomas, and significantly
correlating with poorer clinical outcomes (Dellas etal. 1998). eIF6 was reported
to regulate cell migration and invasion of ovarian cancer cells, modulated by the
receptor NOTCH1 that is involved in embryonic development, cell differentiation
and neoplasia (Benelli etal. 2012). eIF6 expression is upregulated and associated
with reduction in DFS in ovarian serous carcinoma (Flavin etal. 2008).
Evidence towards the role of dysregulated translation apparatus in various types of

gynecologic cancers is forthcoming. Activation of a key oncogenic pathway, PI3K/
AKT/mTOR, has been consistently reported. More importantly for our discussion,
the effects of alterations of this and other pro-oncogenic transduction pathways

in cancer biology are increasingly recognized to depend on components of the
translation machinery. Among these, the overexpression and activation of the pro-
to-oncogenic molecule eIF4E are reported in gynecologic cancers. 4E-BPs that
inhibit eIF4E are key molecules communicating oncogenic signaling by mTOR to
eIF4E. Of note, dysregulation of these molecules has been associated with vari-
ous clinicopathologic parameters, and thus these proteins have a potential to serve
as prognostic markers and therapeutic targets. Interestingly, components of the
translation machinery, namely eIF4E, appear to be intrinsically linked to the patho-
genesis of HPV infection that is one of the main etiological factors of cervical
cancer. Another translational regulator is tumor suppressor PDCD4. This protein
via various mechanisms, but most commonly miR-21 regulation, appears down-
regulated in some cancers of the female reproductive system and has been associ-
ated with clinicopathologic parameters. The role of other translation factors is not
well studied but it appears that the translation machinery in general is dysregulated
in cervical, ovarian and endometrial cancers. Further studies into the roles of these
components of protein synthesis should confer valuable information on the under-
standing and treatment of gynecologic cancers.
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Chapter 33
Prostate Cancer
Nina Seitzer, Markus Reschke, John G. Clohessy and Pier Paolo Pandolfi
Contents
33.2Prostate Cancer Genetics 658
33.3The Impact of Aberrant Oncogenic Signaling Pathways on Translation 658
33.4Translation Initiation Factors in Prostate Cancer Biology 661
33.4.1eIF4F 662
33.4.2eIF3 662
33.4.3eIF2 663
33.4.4Other Translation Factors 664
33.5Aberrant Expression of the Ribosomal Complex in Prostate Cancer 664
33.6Targeting the Ribosomal Machinery for Therapy in Prostate Cancer 665
References 668
AbstractProstate cancer is a common cause of male mortality and morbidity.

While our knowledge of the biology of this cancer is growing, the arsenal of thera-
peutic strategies still remains limited, requiring further understanding of this dis-
ease. Recent advances in molecular research indicate that prostate cancer develops
and progresses in a milieu of mutations and aberrations that lead to the dysregu-
lation of major signaling pathways. In prostate cancer, these oncogenic signaling
cascades converge on the translation machinery, which involves the proto-oncogene
eIF4E, the eIF3 and eIF2 complexes, as well as ribosomal biogenesis. Here we dis-
cuss the involvement of the translation machinery in prostate cancer and highlight
recent strategies that aim to target this machinery for prostate cancer therapy.
P. P.Pandolfi ()N.Seitzer M.Reschke

Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine
e-mail: ppandolf@bidmc.harvard.edu
J. G. Clohessy
Preclinical Murine Pharmacogenetics Facility, Cancer Research Institute, Beth Israel Deaconess
Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center,
658 N. Seitzer et al.
33.1Introduction
Prostate cancer is one of the most common cancers in men accounting for 29% of
all new detected cancer cases per year and ranking third overall in terms of mor-
tality worldwide (Siegel etal. 2012). In the US alone, roughly 220,000 new cases
of prostate cancer are detected each year and 29,000 patients die from the disease
(Tannock etal. 2004). The treatment of prostate cancer can range from surgical
removal to radiation therapy, depending on the clinical characteristics of the disease
(Carter etal. 2007; Walsh etal. 2007). Recurrent and metastatic disease are treated
with chemotherapy and/or androgen depletion (Tannock etal. 2004); however, there
is a paucity of treatment options for advanced prostate cancer patients and an ur-
gent need for the development of successful strategies to improve patient outcomes
(Drake 2010).
It is now well-accepted that altered protein synthesis marks a key event in can-
cer and that the malignancy of cells is closely linked to the translation machinery
(Silvera etal. 2010). In prostate cancer, the dysregulation of translational control
becomes more and more appreciated as a crucial driver of malignancy. Thus, this
chapter discusses the dysregulation of translation factors, their impact on malig-
nancy and their potential use in prostate cancer therapy.
33.2Prostate Cancer Genetics
Prostate cancer shows a surprisingly low incidence of mutation compared to other

cancer types and is defined by the dysregulation of a selected number of potent on-
cogenes and tumor suppressors (Grasso etal. 2012). The most frequent alterations
include deletions of tumor suppressors, such as NKX3.1 , PTEN, RB1 and TP53 and
amplifications of proto-oncogenes such as MYC (Grasso etal. 2012; Taylor etal.
2010). Recent studies have further broadened the mutational spectrum by the iden-
tification of alterations in the WNT and androgen receptor (AR) signaling pathways
(Grasso etal. 2012; Taylor etal. 2010).
Strikingly, key genetic alterations in prostate cancer, such as loss of PTEN or
amplification of MYC are known to critically impact oncogenic signaling pathways
that ultimately dysregulate the translation machinery and protein biosynthesis (Ma
and Blenis 2009; Ruggero 2009; Silvera etal. 2010; van Riggelen etal. 2010).
33.3The Impact of Aberrant Oncogenic Signaling

Pathways on Translation
Oncogenic signal transduction pathways are key modulators of the translation ma-
chinery (Silvera etal. 2010; van Riggelen etal. 2010) (Fig.33.1). In prostate can-
cer, the PI3K/AKT/mTOR pathway is the most frequently altered signaling cascade
33 Prostate Cancer 659
MYC
Fig. 33.1 Aberrant signaling pathways in prostate cancer impacting protein biosynthesis. The
PI3K signaling pathway is dysregulated in more than 50% of all prostate cancer cases mainly due
to the genetic loss of PTEN. Subsequently, elevated PI3K signaling leads to hyperactived mTOR
that in turn leads to increased protein synthesis. On the other hand, c-MYC is a frequently ampli-
fied oncogene in prostate cancer. c-MYC impacts translation on multiple levels mainly due to its
function as a transcription factor. Here, c-MYC, among others, increases the expression of rRNA,
ribosomal proteins and initiation factors.
(Taylor etal. 2010). Besides rather rare PI3K and AKT mutations, hyperactiva-
tion of the PI3K/AKT/mTOR signaling hub almost exclusively results from loss of
PTEN function (Di Cristofano etal. 1998; Engelman 2009; Wu etal. 1998) leading
to a downstream activation of mTORC1, a key translational regulator. This hyper-
activation of mTOR is seen in almost 100% of all human prostate cancer cases, and
genetically engineered mouse models clearly underscore the importance of mTOR
in prostate cancer initiation (Furic etal. 2010; Guertin etal. 2009; Nardella etal.
2009; Taylor etal. 2010). As discussed in previous chapters, mTOR activity criti-
cally impacts protein biosynthesis through the upregulation of cap-dependent trans-
lation (Fig.33.1). In turn, phosphorylation and inactivation of 4E-BPs by mTOR
activates the translation initiation factor eIF4E to accelerate the translational initia-
tion process (Ma and Blenis 2009). Specifically, the current paradigm of translation
activation via eIF4E promotes the preferential translation of mRNAs with struc-
tured 5 UTRs and these mRNAs also appear to play pivotal roles in tumor cell
proliferation, and metastasis (Hsieh etal. 2012).
In addition to phosphorylation of 4E-BPs, mTOR is known to activate translation
initiation via the phosphorylation of S6K and its downstream targets (Ma and Blenis
2009). Amongst others, S6K regulates the activity of PDCD4, a known tumor sup-
pressor in prostate cancer, and of the initiation factor eIF4B (Ma and Blenis 2009).
The phosphorylation of PDCD4 by S6K serves as a signal for the SCF/-TRCP
complex to bind and mark PDCD4 for degradation (see Chap.6). Ubiquitinylated
PDCD4 is then degraded by the proteasome, thereby releasing the initiation factors
eIF4A and eIF4G to increase cap-dependent translation (Dorrello etal. 2006). On
the other hand, S6K phosphorylates eIF4B leading to its interaction with eIF4A and
eIF4G for full activation of translation (Dennis etal. 2012). In addition, mTOR in-
creases the overall protein synthesis by stimulating ribosome biogenesis as well as
transcription of rRNA (Ma and Blenis 2009). Taken together, dysregulated mTOR
activity is a key event in prostate cancer connecting aberrant signaling to altered
translation.
Similar to the PI3K/AKT/mTOR pathway, the MAPK pathway is altered in
prostate cancer, albeit less frequently (Silvera etal. 2010; Taylor etal. 2010). The
MAPK pathway ultimately converges on translational control through ERK in
mTOR-dependent and mTOR-independent manners. First, activation of ERK leads
to mTOR dependent upregulation of translation through phosphorylation of TSC2,
a negative regulator of mTOR. This phosphorylation leads to the dissociation of the
TSC1/2 complex that in turn impairs the ability of TSC2 to inhibit mTOR (Ma etal.
2005). ERK further regulates translation independent of mTOR through the activa-
tion of the serine/threonine kinases MNK 1 and 2. Activated MNK 1 and 2 in turn
accelerate translation initiation and protein biosynthesis through the phosphoryla-
tion of their downstream target eIF4E (Ma and Blenis 2009; Pyronnet etal. 1999;
Waskiewicz etal. 1997).
Together with mTOR and MAPK signaling, c-MYC is yet another key driver of
prostate cancer, as demonstrated by several in vitro and in vivo studies (Ellwood-
Yen etal. 2003; Thompson etal. 1989; Williams etal. 2005; Zhang etal. 2000). The
c-MYC function in prostate cancer is commonly fueled by the amplification of the
MYC gene locus. As a transcription factor, c-MYC regulates a variety of biological
processes in the cell including translation (Bouchard etal. 2001; Dominguez-Sola
etal. 2007; Eilers and Eisenman 2008; Frank etal. 2001; Knoepfler etal. 2006; Koh
etal. 2010; Pelengaris and Khan 2003; van Riggelen etal. 2010), through an ability
to promote both translational initiation as well as ribosome biogenesis (Fig.33.1).
In particular, c-MYC activity not only increases the expression of initiation factors
Fig. 33.2 Aberrant translational initiation in prostate cancer. Typical alterations in the initiation
complex include downregulated phosphorylation of eIF2, elevated expression of eIF3 compo-
nents and eIF4E, a member of the eIF4F complex. eIF4E is further implicated by its increased
availability and activity due to the downregulation of its negative regulator 4E-BP1 by mTOR.
Alterations in translational initiation lead to the increased synthesis of protumorigenic proteins.
(e.g. eIF4E, eIF4A, eIF4G) but also activates the transcription of rRNA through
chromatin remodeling and the recruitment of required cofactors (Frank etal. 2001;
Silvera etal. 2010). c-MYC also modulates ribosome biogenesis by elevating the
expression of auxiliary factors involved in RNA processing, ribosome assembly and
the transport of mature ribosomal subunits into the cytoplasm (Coller etal. 2000;
Kim etal. 2000; Schlosser etal. 2003). Furthermore, c-MYC can increase the rate
of translation of its target genes by promoting the methylation of the mRNA cap
structure. This methylation protects mRNA against degradation and is essential for
successful eIF4E binding and cap-dependent translation. Interestingly, the expres-
sion levels of most c-MYC target genes seem to be increased via efficient cap-
methylation and not simply by the transcriptional activity of c-MYC. However, the
exact mechanism by which c-MYC promotes cap-methylation is not fully defined
yet (Cole and Cowling 2009).
Thus, c-MYC, one of the most powerful prostate cancer oncogenes, regulates the
translational process and its components at multiple levels, thereby clearly linking
aberrant protein biosynthesis to prostate tumorigenesis.
33.4Translation Initiation Factors in Prostate Cancer

Biology
As extensively discussed in this book, the control of protein synthesis primarily oc-
curs at the initiation step of translation. Accordingly, dysregulation of translation in
cancer mainly occurs at the initiation step, and several key players in this process
are specifically targeted for dysregulation in prostate cancer including the eIF4F,
eIF3 and eIF2 complexes (Fig.33.2).
33.4.1eIF4F
The mRNA-bound eIF4F complex facilitates cap-dependent mRNA translation and

is the best characterized complex of translational regulation to date (Sonenberg and
Hinnebusch 2009). Activity of the eIF4F complex is tightly regulated and its dysreg-
ulation is strongly linked to cancer initiation and progression (Silvera etal. 2010).
The eIF4F complex contains three major components: eIF4E, eIF4G and eIF4A
(Silvera etal. 2010). Although eIF4G and eIF4A are generally implicated in cancer,
the most extensive research efforts have concentrated on eIF4E (Comtesse etal. 2007;
Eberle etal. 1997). An analysis of human patient data revealed that increased expres-
sion and phosphorylation of eIF4E can be associated with decreased survival and
higher Gleason score, respectively (Graff etal. 2009; Furic etal. 2010). Furthermore,
the intensity of total and phospho-eIF4E gradually increases from normal prostate to
prostatic intraepithelial neoplasia and from hormone-sensitive to hormone-resistant
prostate cancer, thereby associating eIF4E not only with tumor progression, but also
with hormone resistance (Furic etal. 2010). In line with these findings on human
patient samples, eIF4E induces cell survival after androgen withdrawal and exposure
to chemotherapeutic drugs in vitro (Andrieu etal. 2010). Here, HSP27 chaperones
eIF4E upon cellular stress and ensures continued translational initiation through the
inhibition of proteasomal degradation of eIF4E (Andrieu etal. 2010).
The general dependence of prostate cancer on eIF4E availability is highlighted
by in vitro studies in which oligonucleotides targeting eIF4E show growth-sup-
pressive activity in prostate cancer cell lines (Graff etal. 2007). More importantly,
elegant in vivo studies validate eIF4E activity as a crucial mediator of prostate can-
cer progression. Namely, knock-in mice that express a nonphosphorylatable eIF4E
mutant are resistant to PTEN loss-induced prostate cancer. This deficiency in eIF4E
activity primarily affects the efficient translation of a distinct subset of protumori-
genic mRNAs including angiogenic factors (Furic etal. 2010). In keeping with
these findings, Furic and others thereby show a qualitative correlation between the
disease progression and eIF4E activity that is not simply based on elevated prolif-
eration due to a global increase in translation (De Benedetti and Graff 2004; Yang
etal. 2007; Furic etal. 2010).
It must be remembered that the phenotypes induced by eIF4E are highly depen-
dent on its availability that is tightly regulated by 4E-BPs. Hypophosphorylation of
4E-BP1 leads to its activation and sequestration of eIF4E. This results in the inhibi-
tion of the cap-dependent translation initiation complex by removing eIF4E from
eIF4G (Silvera etal. 2010). Indeed, prostate cancer is characterized by high levels
of hyperphosphorylated and thereby inactive 4E-BP1 (Graff etal. 2009). However,
the levels of 4E-BPs in prostate cancer generally fail to serve as prognostic marker
highlighting eIF4E as a more robust prognostic indicator, as well as a promising
therapeutic target (Coleman etal. 2009).
33.4.2eIF3
During translation initiation, the 43S PIC joins with the mRNA-bound eIF4F com-
plex to form the 48S initiation complex (Sonenberg and Hinnebusch 2009). The
crucial mediator in bridging both complexes is the eIF3 complex, that consists of
over a dozen of subunits (Dong and Zhang 2006) (see Chap.8). Several in vitro
studies now highlight eIF3h as a relevant player in prostate cancer. The overex-
pression of eIF3h in normal prostatic epithelial cells leads to higher proliferation
and clonogenic ability. Vice versa, the loss of eIF3h markedly reduces malignancy
(Savinainen etal. 2006; Zhang etal. 2008). Remarkably, eIF3h is located on chro-
mosome 8q, a genomic region frequently amplified in human cancer. eIF3h gain
could be further associated with poor cancer-specific survival in prostate carcino-
mas (Saramki etal. 2010). Additionally, high eIF3h expression levels correlate
with Gleason score and advanced tumor stage (Nupponen etal. 2000; Savinainen
etal. 2004).
33.4.3 eIF2
The role of the initiation factor 2 (eIF2) in cancer in general is controversial,

largely due to a lack of data on this topic. However, the fact that this protein serves a
downstream target of various signaling cascades makes it an attractive candidate for
further studies. As previously mentioned in this book (see Chap.9), eIF2 is an es-
sential component of a ternary complex that consists of eIF2, guanine triphosphate
(GTP) and the methionyl initiator Met-tRNAi. This complex is indispensable for
translation initiation that is catalyzed by GTP bound to eIF2. Upon phosphoryla-
tion, eIF2 acts as a competitive inhibitor for eIF2, another subunit of the EIF2
complex. Consequently, eIF2 is inhibited in its activity as GEF that mediates the
exchange of GDP to GTP at every round of translation initiation. This inhibitory
effect finally leads to a block of global protein biosynthesis (Sonenberg and Hin-
nebusch 2009).
Although eIF2 per se does not appear to be overexpressed in prostate cancer,
its phosphorylation is highly dependent on typical environmental stresses and, more
importantly, on the activity of classical prostate cancer signaling pathways (Mounir
etal. 2009; Overcash etal. 2013; Sonenberg and Hinnebusch 2009). First, the eIF2
phosphorylation pathway was demonstrated to be downstream of PTEN. Loss of
functional PTEN in melanoma cells results in decreased levels of phosphorylated
eIF2, thereby stalling translation. On the other hand, overexpression of either wild-
type or phosphatase-deficient PTEN in prostate cancer cells induces phosphoryla-
tion of eIF2 by activating PKR, but not other eIF2 phosphorylation pathways.
Concordantly, the tumor suppressive activity of PTEN in MEFs was highly com-
promised by either the lack of PKR or phosphorylation-defective eIF2 (Mounir
etal. 2009). However, the mechanism by which PTEN activates PKR is not fully
understood. These data clearly highlight the cooperation of the tumor suppressive
activity of PTEN and translation initiation that seems to be independent of PTENs
phosphatase activity (Mounir etal. 2009). Besides PTEN, androgen receptor activ-
ity also seems to affect protein synthesis through eIF2. Stimulation of prostate
cancer cells with the metabolite of androgen, dihydrotestosterone (DHT), induces
eIF2 phosphorylation and blocks global translation. Although DHT is known to in-
duce eIF2 indirectly through oxidative stress, Overcash etal. demonstrate that this
phosphorylation can also be androgen receptor-dependent. The full mechanism of
AR dependent eIF2 phosphorylation, however, still needs to be determined (Over-

cash etal. 2013). As mentioned above, environmental stresses can also influence
eIF2 activity. DU145 prostate cancer cells cultured under hypoxic conditions show
rapid phosphorylation of eIF2. A comparison of the translatome and transcriptome
of these cells validates the translational upregulation of genes as a key component
of acute hypoxia and dominant over transcriptional control, which seems to be more
important during a prolonged hypoxic exposure (van den Beucken etal. 2011).
33.4.4Other Translation Factors
Despite a clear body of evidence for the impact of translation factors in prostate
cancer, the majority of these factors are barely characterized. For instance, although
the translation initiation factors eIF4A, eIF4B and eIF4G are all associated with
cancer cell growth and survival, their causal roles in prostate cancer are still not
fully understood (Cencic etal. 2009; Ren etal. 2013; Renner etal. 2007). Besides
translational initiation, the process of elongation has also been implicated in pros-
tate cancer progression (see Chap.12). For example, the elongation factor eEF1 is
overexpressed in prostate cancer tissue samples and correlates with reduced metas-
tasis-free survival and OS after surgery (Liu etal. 2010). Along these lines, down-
regulation of EF1 in prostate cancer cell lines impairs various biological processes
including cell proliferation, invasion and migration (Zhu etal. 2009).
In addition, the elongation factor eEF2 seems to be implicated in a protective
response to chemotherapeutic agents. Upon doxorubicin treatment, the prolifera-
tion of PC3 prostate cancer cell lines decreases. This cell cycle arrest is in part due
to elevated eEF2 phosphorylation that subsequently stalls translation (White etal.
2007). Accordingly, suppression of eEF2 expression by RNAi induces an apoptotic
response in vitro (Wullner etal. 2008). Despite these very exciting preliminary re-
sults, the field of prostate cancer clearly needs robust research efforts to ultimately
uncover the impact of translation factors in tumor progression specifically at the
step of translational elongation and termination.
33.5Aberrant Expression of the Ribosomal Complex

in Prostate Cancer
Like translation factors, alterations in ribosomal proteins are found in prostate can-
cer; however their role in prostate cancer biology is poorly defined. So far, ribosom-
al proteins are mainly implicated in prostate cancer because of their dysregulated
expression levels. A variety of ribosomal proteins are known to be overexpressed
in prostate cancer (RPL19, RPL7a, RPL37, RPL23a, RPS14) (Bee 2006; Vaarala
etal. 1998). Furthermore, a prostate cancer metastasis gene signature composed of
17 genes has been identified, where four out of the eight upregulated genes were
components of the translation machinery (Ramaswamy etal. 2002).
In particular, high expression of rpL19 correlated with reduced OS (Bee 2006)

and the knockdown of rpL19 decreased the aggressiveness of human prostate can-
cer cell lines (Bee etal. 2011). Along these lines, the ribosomal protein rpS2 has
also been found to be overexpressed in both prostate cancer cell lines as well as
tumor specimens and furthermore seems to be important for the proliferation and
survival of cancer cell lines (Ohkia 2004; Wang etal. 2009).
In summary, these results suggest that the alteration of the ribosomal complex
itself may potentially impact prostate cancer biology similar to the dysregulation of
translation factors.
33.6Targeting the Ribosomal Machinery for Therapy

in Prostate Cancer
Extensive research efforts are underway to pharmacologically inhibit the transla-

tion machinery (see Chap.4, 14, 15, and 17). Importantly, both in vitro and in vivo
studies using genetically engineered mouse models have already demonstrated that
the selective impairment of the translational process can suppress prostate tumori-
genesis (Furic etal. 2010; Zhu etal. 2009). Therefore, targeting of the translation
machinery in prostate cancer promises to be a useful strategy to improve patient
outcome.
Due to its central role in translation initiation, the eIF4F complex is a very prom-
ising drug target and currently under extensive investigation. The eIF4F complex
can either be targeted directly or indirectly via signaling nodes that converge on
eIF4F. So far, attempts to directly inhibit the eIF4F complex encompass the use of
either ASOs, small molecules or chemical/botanical compounds (Balabanov etal.
2007; Cencic etal. 2009; Graff etal. 2008; Konicek etal. 2008; Moerke etal. 2007).
To date, the benefit of pharmacological inhibition of the eIF4F complex in prostate
cancer is not fully understood. Only the botanical compound silvestrol, that spe-
cifically inhibits eIF4A, was shown to efficiently inhibit protein synthesis in PC3
prostate cancer cells; however the ability of silvestrol to inhibit the in vivo growth
of cancer cells has thus far only been assessed in a breast cancer xenograft model
(Cencic etal. 2009).
To date, the most straightforward strategy in therapeutic targeting of the ribosom-
al machinery is clearly the inhibition of aberrant signaling cascades that impinge on
translational control. Amongst these nodes, mTOR represents the most prominent
and successful example (Fig.33.3). As previously discussed in this book, mTOR is
found in two distinct complexes: mTORC1 and mTORC2 (see Chap.152.1). The
best-characterized compounds that target the mTOR pathway include Rapamycin,
PP242/INK128, Torin 1 and WYE-132. Unlike Rapamycin, that is an allosteric in-
hibitor of mTORC1, the ATP-site inhibitors PP242/INK128, Torin 1 and WYE-132
target both mTORC1 and mTORC2 (Faivre etal. 2006; Feldman etal. 2009; Hsieh
etal. 2012; Silvera etal. 2010; Thoreen etal. 2009; Yu etal. 2010). Although sev-
eral studies show a growth inhibitory effect of mTOR inhibitors on prostate cancer
Fig. 33.3 Impairment of

mTOR function inhibits
protein synthesis. The most
characterized mTOR inhibi-
tors include Torin 1, rapamy-
cin and its analogs, WYE-132
and PP242/INK128. Upon
inhibitor treatment, phos-
phorylation of mTOR down-
stream targets (S6K, AKT
and 4E-BP1) gets normalized
in malignant cells. Hypo-
phosphorylation of 4E-BP1
leads to its activation and in
turn sequestration of eIF4E.
Sequestration of eIF4E leads
to disruption of the eIF4F
complex and the decrease in
translation of protumorigenic
mRNAs.
cell lines in vitro, a study by Hsieh and colleagues provided the most compelling
preclinical assessment of mTOR inhibition in prostate cancer in vivo (Hsieh etal.
2012; Yu etal. 2010). They reported highly significant data on the in vivo efficacy
of PP242/INK128, a next-generation, clinical-grade inhibitor derived from PP242.
They were able to show that while rapamycin only blocks the activation of S6K,
PP242/INK128 inhibits all three downstream targets of mTOR, namely AKT, 4E-BP
and S6K, and is therefore a more powerful inhibitor towards translational inhibition
(Hsieh etal. 2012). Strikingly, in vivo administration of PP242/INK128 restored
4E-BP and S6K phosphorylation to wild-type levels in a PTEN-deficient setting of
prostate cancer. In contrast, everolimus (RAD001), a rapalog, could only restore
S6K phosphorylation as predicted by their in vitro data. In line with these observa-
tions, administration of PP242/INK128 resulted in a 50% decrease of neoplastic
lesions accompanied by decreased proliferation and increased apoptosis, exceeding
by far the effects seen with RAD001 treatment in mice. Most importantly, PP242/
INK128 inhibited the progression towards an invasive phenotype, and a reduction
in the number of metastatic lesions. These data could be explained by decreased
translation (but not transcription) of specific mTOR-regulated proinvasion genes
(Hsieh etal. 2012; Nardella etal. 2009; Thoreen etal. 2012). This provides sup-
port to the fact that aberrant translation is important for prostate cancer progression
and validates mTOR, and indirectly the translation machinery, as a highly relevant
target for prostate cancer therapy.
Besides mTOR, AMPK also represents an important target for the treatment of
prostate cancer. AMPK is a cellular energy sensor and regulates cellular energy
homeostasis (Hardie and Hawley 2001). Importantly, activation of AMPK leads to
the phosphorylation of TSC2 that inhibits the translational regulators S6K and 4E-
BP1 (Inoki etal. 2003; Corradetti etal. 2004). Thus, pharmacological activation of
AMPK and subsequent inhibition of protein synthesis could be an effective means
for the treatment of prostate cancer. Indeed, metformin and phenformin, two well-
characterized AMPK activators, have been shown to exert antineoplastic activity in
prostate cancer (Clements etal. 2011; Caraci etal. 2003) suggesting AMPK activa-
tion as a promising strategy for the treatment and prevention of prostate cancer.
As mentioned above, the c-MYC oncogene predominantly drives prostate cancer
initiation and strongly affects the translation machinery (Ellwood-Yen etal. 2003;
Koh etal. 2010; Ruggero 2009; van Riggelen etal. 2010). However, being a tran-
scription factor, c-MYC has been considered an undruggable target until recently.
The Bradner lab, however, has now developed a highly innovative strategy to target
c-MYC via BET family proteins that are required for c-MYC-mediated transcrip-
tion as well as expression (Delmore etal. 2011; Zuber etal. 2011). These efforts re-
sulted in the development of a specific BET-family inhibitor that was shown to hold
in vivo efficacy in blood cancer. Thus, BET inhibition could represent an important
strategy to inhibit c-MYC expressing solid tumors like prostate cancer (Delmore
etal. 2011; Zuber etal. 2011).
Although ribosomal proteins are not generally considered to be suitable as drug
targets, rpS2 was recently reported as a novel therapeutic target in prostate cancer
using a very innovative approach (Wang etal. 2009). To target ribosomal proteins
for therapy, a ribozyme-like DNAZYM-1P 1023-motif oligonucleotide was de-
veloped to knock down rpS2 expression. Strikingly, DNAZYM-1P inhibited cell
growth and induced apoptosis in malignant prostate cells with minor effects on the
proliferation of normal cell lines (Wang etal. 2009).
In summary, while targeting of the translation machinery for cancer therapy is
still in its infancy, there is already compelling evidence that aberrant cancer as-
sociated protein biosynthesis represents a valuable target for therapy and that the
direct or indirect targeting of translation displays efficacy in vivo. The available data
also demonstrate that the pharmacologic targeting of central oncogenic signaling
pathways in prostate cancer results in the inhibition of protein biosynthesis, which
certainly contributes to the observed drug response.
We are far from the understanding of how the ribosome translates cancer and how
this process and its contributing factors really differ in malignant cells. As men-
tioned previously in this chapter, the prostate cancer research field lacks clearly
defined roles for translation initiation, elongation and termination factors in the
pathogenesis of cancer, and significant efforts are required to better understand the
importance of these proteins in this context. Beyond this, future research will aim
to translate these findings to the clinic, and will include efforts to effectively em-
ploy translation factors for therapeutic targeting, clinical prognostication and diag-
nostics.
Furthermore, there is an unmet need to understand the full magnitude of ribopro-

teome complexity that includes far more components than translation factors and
ribosomal proteins. One central question, which needs to be addressed, relates as
to what other proteins (beyond translation factors and ribosomal proteins) directly
impact protein biosynthesis. A recent publication on ribosome composition by Re-
schke etal. has started to address this critical point, and demonstrates that quanti-
tative mass spectrometry has not only the ability to uncover novel components of
the riboproteome, but also those that differ between cancer cells and normal tissues
(Reschke etal. 2013). This approach could also be applied to patient samples or
integrated into in vivo studies. Importantly, such analyses could uncover new regu-
lators that can subsequently be validated for their relevance in the aberrant transla-
tional process underlying tumorigenesis.
In conclusion, the differential composition of the cancer riboproteome in gener-
al, and specifically in prostate cancer, is an exciting topic for future research efforts.
In turn, research focused on aberrant translation will surely provide innovative op-
portunities and targets to be integrated in future clinical applications for cancer
patients in the years to come.
Acknowledgments We thank Pandolfi lab members for helpful discussions and critical reading of
the manuscript. M.R. was supported from the German Academy of Sciences Leopoldina (Leopol-
dina Research Fellowship grant number: LPDS 200927). P.P.P acknowledges support from NIH
grants CA141496 and CA082328.
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Chapter 34
Cancers of the Urinary System
Armen Parsyan, Emmanuel Seront and Jean-Pascal Machiels
Contents
34.2Bladder Cancer and Translation 674
34.3Renal Cancer and Translation 676
References678
Abstract The role of translation and its regulation in cancers of the urinary system
is poorly studied and understood, and is limited to investigations of the activity of
eIF4E and its binding proteins (4E-BPs) in bladder cancer and, to a lesser extent,
RCCs. The lack of studies on this topic is surprising given the established role of
the mTOR signaling pathway, which converges on the translation machinery, in the
pathobiology and therapeutic targeting in RCC. Nevertheless, the available body
of evidence suggests involvement of eIF4E overexpression and activation via the
mTOR/4E-BPs module in the biology of cancers of the urinary system. This chap-
ter discusses the current state of our knowledge on alterations of the translation
machinery in these malignancies.
34.1Introduction
A key effector of signaling cascades is a eukaryotic translation initiation factor

eIF4E that is a bona fide proto-oncogene regulated via phosphorylation and inhibi-
tory binding of 4E-BPs (see Chap.4). Overexpression or activation of eIF4E lead
A.Parsyan()
McGill University, Montreal, Quebec, Canada
E.Seront J.-P.Machiels
Medical Oncology, Cliniques Universitaires Saint Luc, Brussels, Belgium
to enhanced translation of protumorigenic proteins, such as VEGF, HIF-1, cyclin

D1 and others (see Chap.4). One of the key regulators of eIF4E activity is the
PI3K/AKT/mTOR pathway, an important signaling cascade which, when dysregu-
lated could be implicated in pathogenesis of some human malignancies, including
renal and bladder cancers. As this pathway is critically involved in cell prolifera-
tion, survival and angiogenesis it serves as a good target for therapy of renal (Cho
etal. 2010; Shuuin and Karashima 2009) and urinary bladder cancers (Korkolopou-
lou etal. 2012; for review see Ching and Hansel 2010). As evidenced from other
chapters of this book the mTOR pathway cascades down to regulate translation via
control of S6K and 4E-BPs and thus eIF4E. Since excellent reviews are available
regarding the role of mTOR signaling and its pharmacological inhibition in cancers
of urinary system (see Ching and Hansel 2010; Cho 2013), in this chapter, we will
attempt to present events downstream of this and other signaling pathways, which
lead to dysregulation of the translation machinery in these malignancies. Unfortu-
nately, the translation machinery in cancers of the urinary system is studied rela-
tively poorly despite its role in mediating oncogenic signaling.
34.2Bladder Cancer and Translation
The main body of evidence describing the role of the translation machinery in can-
cers of the urinary system comes from the studies of bladder cancers and RCC and
the 4e-BPs/eIF4E axis. Only anecdotal reports exist in relation to bladder cancers
and other translation factors, such as PDCD4 (Baffa etal. 2009) and eIF4G2 (Buim
etal. 2005) that present only associative findings and hence require further investi-
gations. Compared to normal bladder tissues, expression of eIF4E was found to be
higher in the most common type of bladder cancer, transitional cell or urothelial car-
cinoma (Crew etal. 2000). Moreover, silencing of elF4E expression by siRNA leads
to significantly reduced cell migration and invasion of bladder cancer cells com-
pared to those of the control (Kyou Kwon etal. 2014). In this cancer eIF4E may be
involved in translational regulation of VEGF and might have a role as a prognostic
factor. Increased levels of eIF4E correlate with overexpression of VEGF (Crew etal.
2000), which is itself associated with aggressiveness of bladder cancer and is an in-
dependent prognostic indicator in invasive urothelial carcinoma, showing significant
association with DFS and OS (Bochner etal. 1995; Crew etal. 1997). Translation
of VEGF, an important factor mediating tumor angiogenesis, has been shown to be
eIF4E-sensitive (see Chap.4) (Kevil etal. 1996; van der Velden and Thomas 1999).
4E-BP1 is a main isoform of eIF4E-BPs, which via phosphorylation by activated
mTOR, regulates activity of eIF4E. When dephosphorylated, these proteins bind eIF4E
and sequester it away from the formation of the eIF4F complex and thus inhibit trans-
lation. Decreased levels of 4E-BPs or their phosphorylation by mTOR would lead to
activation of the eIF4E cap-dependent translation. Indeed, evidence suggests that 4E-
BP1 is a potential new target molecule and stratification marker for anticancer therapy
in urothelial carcinoma (Nawroth etal. 2011). In almost one third of bladder urothe-
lial carcinomas 4E-BP1 levels have been found undetectable by a tissue microarray
analysis (Schultz etal. 2010). However, somewhat unexpectedly, overall significantly
34 Cancers of the Urinary System 675
higher levels of 4E-BP1 expression in bladder urothelial carcinoma compared with

benign urothelium were found (Schultz etal. 2010). 4E-BP1 expression did not cor-
relate with pathological or TNM staging, however a trend towards a lower expression
levels in noninvasive bladder urothelial carcinoma was observed when compared with
higher pathological stage (Schultz etal. 2010). These findings are hard to reconcile in
light of the known function of 4E-BPs described above and need further corrobora-
tion. However, they could also point out that the functional activity of 4E-BPs in these
cancers is more important than the level of 4E-BPs expression.
Indeed, components of the PI3K/AKT/mTOR pathway including phosphorylat-
ed 4E-BP1 are reported altered, mutated or upregulated in bladder carcinoma and
upper urinary tract urothelial carcinoma compared to normal urothelium (Fahmy
etal. 2013; Korkolopoulou etal. 2012; Munari etal. 2013; Nawroth etal. 2011;
Platt etal. 2009; Schultz etal. 2010; Sun etal. 2011). These alterations include func-
tional loss of PTEN (reported in 49% of bladder cancers); activating mutations of
PI3K (27%); TSC1 (13%); activating mutation of AKT (5%) and others (Platt etal.
2009). 4E-BP1 is directly regulated by the PI3K/AKT/mTOR cascade, activation of
which can result in phosphorylation of 4E-BPs and subsequent activation of eIF4E
and upregulation of protein translation. Accordingly, an inverse relationship was
established between phosphorylated 4E-BP1 and histological grade or T category
in bladder cancers (Korkolopoulou etal. 2012). Increase in phosphorylated 4E-BP1
is also more common in high-grade and high-stage bladder tumors (Sun etal. 2011).
Hence, pharmacological inhibition of the mTOR pathways may represent a
promising strategy in bladder cancer. Indeed, various mTOR inhibitors, such as
rapamycin and rapalogs (Kyou Kwon etal. 2014; Schedel etal. 2011; Seager etal.
2009), mTORC1/C2 inhibitors OSI-027 or PP242 (Becker etal. 2013) and the dual
PI3K/mTOR inhibitor NVP-BEZ235 (Li etal. 2013) showed efficacy in animal
and in vitro models. A phase II clinical trial of mTOR inhibitor temsirolimus as a
second-line treatment in metastatic urothelial carcinoma showed poor activity after
the failure of a platinum-based therapy (Gerullis etal. 2012). A phase II study as-
sessed the safety and efficacy of the orally available mTOR inhibitor everolimus in
advanced transitional cell carcinoma after the failure of a platinum-based therapy
and showed a clinical activity (Seront etal. 2012). In this clinical study, loss of
PTEN was found to be associated with a decreased efficacy of everolimus, contrast-
ing with preclinical models that showed that PTEN loss could enhance sensitivity to
mTOR inhibitors through a sustained activation of signaling downstream of PI3K,
rendering tumor cells more dependent on this pathway for survival and prolifera-
tion (Neshat etal. 2001). This clinical observation highlighted the role of the mTOR
inhibition-induced AKT activation in the development of resistance to mTOR in-
hibitors. PTEN deficiency was shown to be associated with reduced sensitivity to
everolimus in bladder cancer through the unhampered feedback loop driving PI3K/
AKT activation (Seront etal. 2013). In another phase II study of everolimus in a
metastatic urothelial cancer, this rapalog was found to possess meaningful antican-
cer activity in a subset of patients with an advanced urothelial cancer (Milowsky
etal. 2013). Other clinical studies are underway in regards to the inhibition of the
PI3K/AKT/mTOR pathway in bladder cancer (see clincialtrials.gov).
Other compounds were also suggested to inhibit translation via the mTOR/4E-BP1
axis and showed effective anticancer activity, such as Rhodiola rosea and its bioactive
components, salidroside (Liu etal. 2012) and gartanin, a naturally occurring xan-
thone in mangosteen juice (Liu etal. 2013).
Thus, as it stands from a handful of studies in the area, eIF4E appears to contrib-
ute to bladder cancers via its overexpression and/or activation by mTOR-dependent
phosphorylation of 4E-BPs, but not by the downregulation of 4E-BPs expression
levels. As such, activity of 4E-BPs as defined by their phosphorylation state might
be an important prognostic factor of the malignancy. While no strategies directly
targeting components of the translation machinery for treatment of bladder cancer
have been attempted, research in this area should lead to promising findings.
34.3Renal Cancer and Translation
Among kidney cancers, renal cell carcinoma (RCC) is by far the most common
one. However, the involvement of the translation machinery in this cancer is poorly
understood due to lack of studies. The mTOR pathway that is linked to dysregula-
tion of translation is activated in kidney cancers and this activation serves as a target
for currently approved therapy using mTOR inhibitors, temsirolimus and everoli-
mus, in advanced RCC (for review see (Huber etal. 2011; Pal and Quinn 2013)).
Pharmacological inhibition of mTOR was shown to act via 4E-BP1 and p70S6K,
emphasizing that the state of the translation machinery is key in these cancers (Cho
etal. 2010; Zhang etal. 2013).
Activation of the mTOR pathway in renal cancer could occur at least in part by
mutations within components of this pathway, which could include a constitutively
activating point mutation (R2505P) of mTOR (Sato etal. 2010) or mutations of
TSC1 and TSC2 (Guo etal. 2012; Henske 2004). TSC1 and TSC2 participate in
mTOR signaling and lead to the development of tumors of various organs, including
kidneys (reviewed in Astrinidis and Henske 2005). Tumors from tuberous sclero-
sis patients and the Eker rat model of tuberous sclerosis expressed elevated levels
of phosphorylated mTOR and its effectors, such as p70S6K, rpS6, 4E-BP1 and
the component of the eIF4F translation initiation complex, eIF4G (Kenerson etal.
2002). In the Eker rat model, inhibition of mTOR by rapamycin was associated with
an induction of apoptosis and reduction in cell proliferation (Kenerson etal. 2002).
In addition to activation of mTOR and hence its downstream effector eIF4E proto-
oncogene, elevated levels of the latter were reported in RCC (Gemmill etal. 2005).
Temsirolimus and everolimus are currently approved for treatment of metastatic
RCC. In a large phase III study, temsirolimus was investigated in 626 patients with
previously untreated metastatic RCC with poor prognosis (Hudes etal. 2007). These
patients were randomized to receive either IFN-, temsirolimus or a combination of
temsirolimus plus IFN-. Median survival was superior in the temsirolimus alone
arm compared with IFN- (10.9 versus 7.3 months respectively, p=0.008), while
the combination arm was not superior to IFN- (8.4 versus 7.3 months respectively,
p=0.7). The median PFS was statistically superior for temsirolimus-alone and tem-
sirolimus-combination arms compared to IFN- (3.8 and 3.7 versus 1.9 months
respectively, p<0.001) (Hudes etal. 2007).
The RECORD-1 study randomized 416 patients with metastatic renal clear cell
carcinoma who had experienced a disease progression after previous therapy (main-
ly TKIs) to receive either everolimus or placebo (Motzer etal. 2008). This study
was terminated early after the results of interim analysis showed that PFS was sig-
nificantly longer in the everolimus group than in the placebo group. The study was
then unblinded and patients who were receiving placebo were offered everolimus.
In the final analysis, the median PFS was longer in the everolimus group compared
to placebo group (4.9 months versus 1.9 months, p<0.0001). The median OS was
similar in the two groups (14.8 months in patients randomized to everolimus versus
14.4 months in patients randomized to placebo, p=0.162), with the insignificant
result likely due to the aforementioned crossover between the groups. Indeed, a
post hoc exploratory analysis that accounted for the confounding effects of cross-
over showed that everolimus was associated with a 1.9 month longer survival than
placebo. It is interesting to note that in these two trials evaluating clinical efficacy
of mTOR inhibitors, the response rate is very low, not exceeding 10%, suggesting
that mTOR inhibitors control the disease mainly due to a cytostatic effect (Hudes
etal. 2007; Motzer etal. 2008).
Among other components of the translation machinery, tumor suppressor
PDCD4 expression was significantly decreased in RCCs, compared with normal
renal tissues, and found to be significantly associated with metastasis, tumor T-stage
and tumor grade (Li etal. 2012). Low PDCD4 expression was related to a decrease
in the mean OS (Li etal. 2012). PDCD4 expression was related to an increased sen-
sitivity to geldanamycin in UO-31 renal cancer cells (Jansen etal. 2004).
Data on the role of the translation machinery in bladder and kidney cancers are
very scarce. There are only a handful of studies, which are however suggestive
that alterations in eIF4E and 4E-BPs expression and/or activity may play a role
in the pathobiology of these malignancies. Given an established involvement of
the PI3K/AKT/mTOR pathway in RCC, it is expected that more research on the
role of the downstream effectors of this cascade and translation will lead to novel
important discoveries of the etiology, pathogenesis and therapy of RCC, as well
as other cancers of the urinary system. The roles of other factors, such as PDCD4
and eIF2 are surfacing in relation to lung, colorectal, breast and other cancers,
and the studies of these factors in urinary tract malignancies would likely lead to
promising results.
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Index
12-O-tetradecanoylphorbol-13-acetate 4E-BPs, 53, 55, 79, 80, 83, 84, 86, 87, 89, 92,
(TPA), 143, 145 94, 9698, 165, 182, 281, 286, 310,
13-HOA,447 312, 314, 316319, 327, 330, 347, 350,
14-3-3, 311, 355 364, 368, 379, 384, 387, 388, 402, 412,
17- estradiol, 522 423, 424, 439, 474, 490, 491, 493, 494,
18S, 268, 269, 273 516, 521, 522, 524, 533, 541, 560, 561,
197/15,136 568, 578, 579, 586, 596, 619, 638, 660,
28S, 268, 269, 273 662, 674676
2-methoxy-5-amino-N-hyroxynezamide,210 4E-BPs KO, 316
4-(methylnitrosamino)-I-(3-pyridyl)-1- 4EGI, 55, 98, 168, 286, 428, 477, 564
butanone (NNK), 561 4EHP, 83, 424
40S ribosomal subunit, 312, 403 4Ei-1, 98, 428, 535, 564
40S ribosome, 243, 291, 405 4E-T,423
40S ribosomal subunit, 17, 18, 56, 125, 126, 5.8S, 268, 269
164, 165, 174176, 179, 181, 188, 196, 5-fluorouracil,514
380, 475, 595, 622 5-FU, 576, 580, 585, 594, 599, 600
40S ribosome, 9, 17, 18, 20, 21, 23, 26, 40, 5q syndrome, 270, 471
41, 55, 56, 117, 126, 138, 139 60S ribosomal subunit/60S ribosome, 10, 27,
43S PIC, 280 41, 164, 174, 175, 234, 235, 271, 470,
43S PIC formation, 403 616
43S PIC, 16, 18, 19, 2123, 40, 45, 76, 117, 60S ribosome joining, 15
118, 126, 139, 176, 196, 203, 477, 662 7,12-dimethylbenz(a)anthracene
43S PIC formation, 17, 26 (DMBA),145
47S, 268, 269 7-benzyl-GMP,287
48S complex, 118 7Bn-GMP, 534, 535, 564
4E1RCat, 286, 428 7-methyl-guanosine cap, 15
4E2RCat,286 9.2.27PE,442
4EASO, 284, 568
4E-BP1, 79, 80, 8284, 88, 9598, 167, 168, A
283, 286, 312, 313, 317, 324, 325, -catenin, 427
347, 384, 403, 423, 439, 446, 455457, -fetoprotein-producing gastric carcinoma
461, 474, 479, 490, 503, 521523, 527, (AFPGC),580
533, 534, 536, 541, 561, 563, 564, 568, A172,493
578581, 596, 602, 634, 661, 662, 667, A375,442
674676 A431,488
4E-BP1/2 DKO, 122, 314 A549, 255, 559
4E-BP2, 79, 80, 312, 347, 388 ABCE1, 26, 41
4E-BP3, 79, 312 ABL, 124, 294
DOI 10.1007/978-94-017-9078-9, Springer Science+Business Media Dordrecht 2014
682 Index
ABT-737, 286, 288, 442, 477 AP23573, 503, 525

AC1MMYR2, 537, 584 Aplidine,292
Actinomycin D, 272 Apoptosis, 21, 55, 57, 60, 125, 127, 137, 149,
Activating protein 1 (AP-1), 141, 144, 145, 165, 184, 224, 228, 229, 256, 317, 318,
149, 151, 152 328, 378, 384, 399, 428, 536, 617, 676
Activating transcription factor 4 (ATF4), 48, Apoptosis signaling regulating kinase 1
58, 200, 203, 204, 404 (ASK1),456
Active-site TOR inhibitor (asTORi), 325, 368, Apoptotic protease activating factor 1
601, 602, 639 (APAF-1),410
Acute promyelocytic leukemia, 236, 250, Aptamer, 257, 258
270, 293 Arctigenin,599
Adenocarcinoma, 251, 367, 559, 561, 562, Arg110, 136, 138
568, 577 Arg157,75
Adenoma-carcinoma, 597, 604 Argonaute, 52, 248
Adenomatous polyposis coli (APC), 600 Aromatase inhibitor, 329, 514, 525
Adenosylmethionine decarboxylase Aryl hydrocarbon receptor nuclear translocator
(AMD1),227 (ARNT),426
Adjuvant, 329, 514, 594 A-site, 10, 11, 18, 19, 2325, 126, 243,
ADP ribosylation, 257 244,292
Afinitor,503 ASO ISIS 183750, 603
Aflibercept,594 Asp253,138
AGC/AGC kinase, 312, 346, 354, 355 Asp418, 138, 139
AKT, 91, 143, 150, 200, 253, 310312, 325, Aspartate aminotransferase (AST), 290
356, 357, 383, 387, 455, 457, 501, 527, Aspirin,603
533, 560, 562, 563, 585, 601,638 Astrocytic tumor, 489, 490
AKT1, 187, 356, 615 Astrocytomas, 488, 489, 493, 524
AKT2, 500, 601 Ataxia teleangiectasia mutated (ATM), 185,
Alanine aminotransferase (ALT), 290 515, 536, 538
All-trans-retinoic acid, 250 ATG13, 310, 311
Am580,538 ATPase activity, 55, 122, 137, 350
Amino acid response element (AARE), 203 ATP-competitive, 317, 325, 347, 491, 601
Aminoacyl-tRNA (aa-tRNA), 11, 2325, 59, ATRX,633
243, 245, 248, 257, 600 AUF1,82
AML, 121, 142, 206, 207, 270, 271, 290, 293, AUG initiation codon, 19
327, 470, 473, 474, 476, 477 AU-rich element (ARE), 82
AMP-activated protein kinase (AMPK), 311, Autophagosome,385
316, 327, 347, 354357, 479, 526, 562, Autophagy, 385, 533
603, 666, 667 Avastin, 594, 638
Anaplastic large cell lymphoma, 270 AZ-521, 253, 585
Anastrozole,514 AZD-2014,601
Angiogenesis, 78, 87, 120, 124, 146, 152, AZD6244, 325, 326, 602
318, 327, 422, 425, 426, 438, 490, 501, AZD8055, 98, 325, 562
516, 520, 595, 617, 637 AZD-8055,491
Angiosarcoma, 319, 458, 460
Angiotensin II, 365 B
Antiangiogenic agent, 638 -catenin, 91, 92, 285, 600, 612
Antiapoptotic factor, 77, 81, 98, 456, 469 -lysylation, 225
Antiestrogen, 142, 538 -lysyl-lysine, 225
Anti-HER2 receptor, 514 -thalassemia, 201
Antihormonal treatment, 523 -TRCP, 315
Antisense oligonucleotides (ASO), 83, 98, B16F10, 257, 440
100, 318, 445, 477, 534, 564, 580, 596, B6 melanoma, 285
603 BAD, 355, 357
Antrocin,533 Bag1,274
Index 683
Barretts adenocarcinoma, 583 CaMKK,522

Base-pairing, 10, 22, 77 cAMP/cyclic AMP, 145, 198
B-cell acute lymphoblastic leukemia, 325 Cancer stem cell, 422, 426428
B-cell lymphoma (BCL), 123, 472, 474 Cancers of the urinary system, 674, 677
B-cell lymphoma 2 (BCL-2), 50, 92, 123, Cap recognition, 15, 74
124, 284, 318, 355, 380, 444 Cap-binding protein (CBP), 15, 74, 116, 351,
family, 168, 294, 408 396, 586
family:inhibitor, 121, 286, 288 Cap-competitive homolog, 83
B-cell lymphoma extra large (BCL-XL), 45, Cap-dependent translation, 15, 49, 53, 57, 83,
77, 79, 82, 87, 140, 144, 151, 284, 355, 124, 127, 139, 140, 165, 168, 286288,
380, 384, 408, 456, 469, 492,637 320, 325, 327, 379, 380, 382, 385, 398,
BCR-ABL1,124 401, 405, 407, 425, 428, 472, 475,
BEAM,445 516, 521, 534, 561564, 618, 620, 635,
BEZ235, 98, 99, 326, 328, 443, 527, 562, 580 660662, 674
BH-3 mimetic, 442 Capecitabine, 576, 594
Biliary cancer, 612, 613, 619 Carbonic anhydrase type II (CA II), 147, 149
Biomarker, 58, 95, 98, 150, 330, 358, 506, Carboplatin, 99, 436, 503
526, 584, 623 Carcinoma in situ, 515, 651
BJAB cell, 401 Cardio-esophageal carcinoma, 251
BKM120, 326, 369 Casein kinase, 123
BKM162,326 Caspase, 399, 402, 404, 406, 492
Bladder cancer, 227, 674676 Caspase-dependent, 401, 442
Block of proliferation 1 (BOP1), 273 CAT,140
BOLERO-2, 329, 525 Catalogue of Somatic Mutations in Cancer
BOLERO-3, 328, 526 (COSMIC),88
Bon-1 cell, 148 CBP20,74
Bone, CBP20/80,287
cancer,457 CBP80,351
marrow failure, 271, 468, 469, 471 CCAAT-enhancer-binding proteins (C/
Bortezomib, 209, 326, 442, 581 EBP), 46, 200, 203
Bouvardin,257 CCI-779, 257, 321, 456, 524, 599
BPSL1549,288 CCT020312,211
BRAF, 206, 248, 294, 383, 436, 437, 439, 594 CCT, 352
BRAFV600E, 206, 437, 439, 441, 443 CD44, 317, 427
Brain tumor, 58, 205, 488493 CD95L,290
BRCA1,538 CDC25C, 123, 402
BRCA1/2, 515, 536 CDK inhibitor, 620
BRCA2,515 CDK11p46, 180, 185
Breakpoint cluster region(BCR), 124 CDK4, 380, 444, 454
Breast cancer, 87, 88, 92, 9597, 146, 167, CDK4/6 inhibitor, 444
168, 208, 252, 257, 317, 320, 388, CDKN2A, 48, 437, 444
514516, 519521, 523, 526, 527, 533, cDNA, 137, 139, 145, 146, 149
535, 536, 538541, 584, 665 Cell cycle, 61, 87, 148, 180, 249, 381383,
BT474,318 386, 438, 586, 621
BTdCPU, 211, 291 Cell death, 254, 257, 288, 318, 381, 405, 599
Burkitts lymphoma, 84, 127 Cellular transformation, 120, 205, 236, 319,
476, 596
C Cephalotaxine,292
C2C12, 137, 289 Cercosporamide, 98, 285, 367, 479, 603
C6 glioblastoma, 488 Cervical cancer, 84, 143
Ca2+/calmodulin, 249, 250 Cetuximab, 98, 168, 503, 523, 563, 576, 594
Caco-2, 584, 598 C-FAG, 165, 399
CAD,353 c-FLIP, 168, 290
CaMKII,425 CGP57380, 285, 367, 479, 489
684 Index
Chaperone, 212, 662 CXCL12, 141, 144

Chaperonin,352 CXCR,144
Chemoresistance, 209, 408, 412, 443, 524 CXCR4,144
Chemotherapy, 167, 209, 211, 294, 318, 454, Cyclin B, 148
488, 500, 504, 505, 536, 581, 594,613 Cyclin D1, 45, 55, 78, 92, 125, 147, 149, 182,
Chitosan,566 212, 284, 317, 318, 424, 456, 490, 504,
Chlorotoxin,492 559, 595, 646, 673
Cholangiocarcinoma (CCA), 612, 613, 619, Cyclin D3, 181, 249
621, 622 Cyclin E1, 127
Chondrosarcoma,457 Cyclin-dependent kinase (CDK), 85, 148
CHOP, 203, 209, 212 inhibitor, 148, 249
Chronic lymphocytic leukemia, 121, 127, Cyclin-dependent kinase 2 (CDK2), 147, 249
144, 207, 290, 477 Cyclooxygenase 2 (COX-2), 142
Chronic myeloid leukemia (CML), 209, 293, Cyclophosphamide, 461, 514, 650
474, 479 CYR61, 402, 539
cIAP1, 403, 407 Cytoplasmic polyadenylation-element binding
Cisplatin, 98, 99, 254, 256, 357, 403, 442, proteins (CPEB), 382
503, 516, 534, 576, 601, 622
c-JUN N-terminal kinase (JNK), 145147, D
489, 562 Dabrafenib,436
CK1, 522 Dacarbazine, 436, 444
c-KIT, 436, 454 DAP5, 399, 425
Classical swine fever virus (CSFV), 404 DAP5/p86, 401, 407
Clinicopathologic parameter, 559, 560, 566, DAP5/p97, 401, 407, 408, 410
568, 582, 584 DAP5/p97/NAT1,413
Clotrimazole,210 DAXX, 123, 633
cMA3, 136, 138 DCIS, 515, 519, 520
c-MET, 248, 576 Dcp2,287
c-MYB,149 DDIT3, 457, 459
c-MYC, 45, 7779, 86, 87, 89, 91, 92, 94, DDX3, 117, 127129
124, 146, 147, 166, 182, 236, 284, 318, DDX3X,127
380, 382384, 386, 402, 411, 422, 456, DDX3Y,127
501, 516, 539, 614, 659661 DEAD box, 137
Cobimetinib,443 helicase, 15, 16, 76, 117, 164, 280, 287
Cognate CUG, 48 Deamidation,288
Colon carcinoma, 142, 146, 440, 599, 602 Decoding site, 17, 1922, 45
Colorectal adenocarcinoma, 596 Deforolimus,460
Colorectal cancer (CRC), 94, 125, 144, 150, DENR,404
253, 292, 293, 594599, 601604 Deoxyharringtonine,292
Common deleted region (CDR), 471 Deoxyhypusine, 224, 227, 228
Constitutive photomorphogenic 9 Deoxyhypusine hydroxylase
(COP9), 176, 182 (DOHH), 224,229
Cowden,515 Deoxyhypusine synthase (DHS), 224,
CR-1-31-B,290 228,229
CREB,422 DEPTOR,309
CReP,202 Destructive autophagy, 378, 383, 385
CReP/PPP1r15B,202 D-glucosamine,533
Csde1,274 DHBDC,211
CTX,492 DHX29, 20, 117, 125, 126, 440, 494, 651
Curcumin, 441, 456, 564, 603 DHX9, 128, 129
Curcuminoid,542 DIABLO,408
Cutaneous malignancy, 446 Diamond-Blackfan anemia (DBA), 60, 270,
CXC,144 469, 470
CXC chemokine, 144 Diaryl-oxindole,210
Index 685
DICER,52 eEF2, 24, 54, 59, 243, 244, 246, 249, 253,
Diftitox,257 254, 256258, 275, 316, 423, 541, 567,
Dihydroartemisinin (DHA), 603 585, 599, 664
Dihydrotestosterone (DHT), 663 eEF2-GDP, 10, 243
Discodermolide,563 eEF2K, 59, 249, 254, 257, 275, 316, 351,
Disease-free survival (DFS), 456, 459, 490, 357, 493, 541, 586, 619
504, 520, 522, 537, 565, 583, 598, 651, eEF3,243
674 Eeverolimus, 99, 321323, 329, 460, 491,
DLBCL, 123, 323, 476 503, 525, 526, 563, 580, 620, 637639,
DNA damage response, 167, 311, 383, 675677
536538 EF3, 123, 166
DNAZYM-1P,667 EF4,243
Docetaxel, 98, 150, 442, 503, 516, 534, 580, EF-G,24
602 EFL1,470
Double-stranded RNA (dsRNA), 52, 202, EF-P, 24, 225
291, 403, 448 EF-Tu, 24, 197
Dovitinib,576 EGFR inhibitor, 533
Doxorubicin, 98, 150, 255, 256, 286, 290, EGFR TKI, 563, 565
326, 514, 516, 534 EGFR/ERBB family, 515
D-Penicillamine,442 EGR1,454
DROSHA,52 eIF1, 9, 17, 18, 21, 22, 26, 40, 41, 58, 138,
DRYG,122 139, 175, 179181, 184, 235, 350, 477
DU145,664 eIF1A, 9, 17, 18, 2123, 26, 40, 41, 58, 138,
Dual mTOR/PI3K inhibitor, 325, 326 139, 175, 179, 235, 350
Dyskeratosis congenita, 406, 408, 469, 470 eIF2, 9, 17, 18, 22, 26, 58
Dyskeratosis congenita 1 (DKCL1), 271, subunit,18
274,469 eIF2/GTP/Met-tRNAi, 181, 199, 201, 208
Dyskerin, 270, 271, 408, 469 eIF2B, 10, 22, 196199, 207, 291, 403, 404,
540, 567
E eIF2B, 198
E-MYC,50 eIF2-GDP complex, 10
E2, 141, 142, 522 eIF2-GTP, 22, 54, 58, 175, 291
E2F1,383 eIF2-TC, 10, 18, 2022, 27, 40, 48, 58, 540
E3 ubiquitin ligase, 272, 355 eIF2, 198201, 205, 206, 210, 211, 256,
E3-ligase -1, 151 291, 404, 423, 441, 442, 445, 475, 492,
E6, 84, 648 505, 539, 542, 566568, 585, 599, 601,
E7, 84, 648 663, 677
E-box, 82, 380 dephosphorylation, 202, 209, 210, 539
E-cadherin, 57, 146, 147, 427, 535, 598 eIF2 kinase, 197, 202, 209211, 403, 443,
eEF1A/B, 247 476, 540
eEF1A/GDP, 243, 245, 246 eIF2 phosphorylation, 58, 198, 199, 202,
eEF1A, 24, 40, 41, 59, 243246, 248, 251, 203, 206, 207, 209211, 291, 404, 442,
256, 617 492, 505, 539, 540, 603, 663
eEF1A/GDP,24 eIF2, 18, 197, 198, 663
eEF1A1, 244, 245, 247, 248, 250, 251, 255, eIF2, 18, 197, 198
258 eIF3, 9, 18, 19, 21, 23, 26, 27, 40, 41, 45, 49,
eEF1A2, 244, 245, 247249, 251253, 255, 55, 57, 79, 122, 126, 138, 139, 165,
617 174176, 179, 661
eEF1B, 24, 59, 243247, 250, 251, 585 subunit, 178, 179, 187, 598
eEF1B, 246 eIF3a, 179183, 188, 505, 584, 598, 651
eEF1B, 246, 247 eIF3b, 176, 181, 182, 184, 493, 598
eEF1B, 246 eIF3c, 19, 181, 182, 184, 440, 493
eEF1B, 246 eIF3d, 19, 181, 182
eIF3e, 182, 184, 401, 411, 538, 539, 585
686 Index
eIF3f, 180, 182, 185, 440 eIF5, 10, 17, 18, 22, 23, 26, 41, 175, 181,
eIF3g, 176, 181 197, 350, 405
eIF3h, 180, 182, 185, 186, 538, 585, 615, 663 eIF5A-1, 224, 226, 227, 229
eIF3i, 176, 181, 182, 186, 615 eIF5A-2, 224, 226, 227, 229
eIF3j, 19, 176, 181 eIF5B, 23, 41, 404, 405, 408
eIF3k, 176, 181 eIF6, 23, 234237, 470, 476, 600, 616, 651
eIF3l,18 eIFG2,165
eIF3m, 179, 182, 187, 598 eIFG3,165
eIF4A, 182, 280, 282, 283, 287289, 312, Elf1p,234
315, 396, 462, 474, 475, 477, 502, 536, Elongation, 242244, 246, 247, 250, 258,
541, 567, 636, 660662, 664,665 292, 313, 441, 472, 540, 586, 667
eIF4Ac, 116, 120, 122, 126 Elongation factor, 49, 56, 59, 180, 243, 244,
eIF4Af, 116, 118, 120, 122 247, 250, 254, 258, 477, 568, 585,600
eIF4AI, 117, 137, 281, 283, 287, 440, Elongation factor 3, 123
567,617 Elongation factor 4, 243
eIF4AII, 53, 117, 137, 281, 287, 440 Elongation factor-like 1, 470
eIF4AIII, 117, 137, 287, 289 Endocrine therapy, 514, 521, 523
eIF4B, 118, 122124, 129, 138, 175, 181, Endometrial cancer, 491, 646, 647, 649,
288, 312, 315, 380, 399, 402, 440, 461, 651,652
476, 585, 634, 664 Endoplasmic reticulum, 352, 403, 519, 522,
eIF4E, 525
binding protein, 15, 165, 281, 311, 379, Ca2+ store, 201, 210
402, 423, 516, 561 stress, 168, 201203, 208, 210, 251, 286,
dependent, 84, 91, 92, 286, 382, 535 352, 407, 410, 505, 566, 599
dependent translation, 84 Enzastaurin,603
homologous protein, 83 Ependymomas,488
independent, 405, 412 Epidermal growth factor (EGF), 380
phosphorylation, 81, 88, 92, 9598, 285, Epidermal growth factor receptor
320, 364, 367369, 489, 502, 560, 578, (EGPR), 168, 319, 328, 369, 412, 425,
639 503, 522, 524, 525, 533, 558, 561, 563,
sensitive, 78, 97, 559, 560, 564, 595, 674 576, 613
transporter,423 Epidermal tumor, 446
eIF4E/4E-BP axis, 89, 368 Epigenetic, 140, 293, 386
eIF4E/4E-BPs ratio, 83, 99, 579 Epirubicin,514
eIF4E/eIF4G inhibitor, 286 Epithelial-mesenchymal transition (EMT), 88,
eIF4E/eIF4G interaction, 603, 636 91, 127, 146, 227, 402, 411, 422, 427,
eIF4E1, 83, 422, 424 584, 598
eIF4E2, 83, 412, 425 Epstein-Barr virus, 84
eIF4ES209A, 320, 364 ERBB2,329
eIF4F cap-binding complex, 15, 40, 41, ERCC5,123
50,53 eRF1, 25, 26, 41, 51, 244, 586
eIF4F-associated eIF4A, 378 eRF3, 11, 25, 26, 41, 51, 244, 586
eIF4G, 10, 1517, 19, 27, 40, 41, 44, 53, 55, eRF3a/GSPT1,541
57, 7881, 83, 98, 116, 118, 122124, ERK, 596, 612, 660
139, 164168, 175, 185, 280, 312, 315, Erlotinib, 98, 168, 503, 524, 563
350, 351, 364, 368, 379, 396, 399, 402, Erythroid progenitor, 270, 274, 469
405, 412, 423, 425, 439, 445, 456, 474, E-site, 10, 18, 25, 41, 59, 243
475, 477, 488, 534, 536, 541, 568, 586, Esophageal,
595, 636, 650, 660, 662, 664, 676 cancer, 96, 97, 576, 584
eIF4G1, 79, 165167, 315, 388, 399, 401, carcinoma, 143, 577, 581, 583
410, 425, 534536, 567 Estrogen,522
eIF4G2, 79, 315, 399, 401, 425, 536, 674 Estrogen/progesterone receptor
eIF4H, 17, 55, 117, 118, 122, 124, 125, (ER/PR), 515,520
129,462 Etoposide, 290, 357, 399, 402, 408, 650
Index 687
Eukaryotic release factor (eRF), 25, 41, 244 G1/S, 148, 211, 273, 382
Eukaryotic translation initiation factor, 15, G2 phase, 148
198, 223, 567, 585, 673 G2/M, 253, 256, 382, 440, 585
European Medicines Agency (EMA), 329 G361,442
Ewing sarcoma (Rh1), 456 GADD34,202
Ewings,454 GAP, 22, 311
Exemestane, 323, 329, 514, 524, 525 GAPDH,77
Ezrin,457 Gastric adenocarcinoma, 576, 578, 582
Gastric cancer, 97, 324, 576578, 580586
F Gastrointestinal cancer, 59, 253, 582, 596
F-actin, 245, 456 Gastrointestinal stromal tumor
reorganization,456 (GIST), 454,459
Fanconis anemia, 515 GC7, 228, 616
Farnesyltransferase inhibitors (FTI), 327 GCN2, 291, 403, 443, 448, 476, 540,
FAS, 402, 403, 411 542,599
FAT1, 142, 492 GDC-0941, 328, 602
FAT10,251 Gefitinib, 563, 565, 576
FATC,309 Gemcitabine, 284, 287, 368, 461, 535, 561,
Feedback activation, 324, 325, 460, 494, 503, 564, 622
533, 580, 602, 649 Gemini compounds, 527
Feed-forward loop, 91 GEO cell, 146
Fenretinide,442 GIGYF2,83
FGF,637 GLI,355
receptor,515 Glia,488
FGFR, 576, 637 Glioblastoma multiforme, 84, 329, 488, 489,
pathway,576 492, 493
Fibroblast growth factor (FGF2), 87, 141, Gln339,288
284, 380, 407409, 425, 426, 501, 504, Glu103,75
564 Glu210,138
Fibronectin, 146, 227 Glu249,138
Fibrosarcoma, 89, 460 Gonadotropin-releasing hormone
FIP200, 310, 311 (GnRH),650
FKBP12,309 Growth arrest-specific protein 2 (GAS2), 87
Flavaglines,121 GRP75,352
Flavonoid,562 GSK1059615,369
FLIP, 151, 582 GSK2606414,211
FMS-like tyrosine kinase receptor-3, 290 GSK3, 198, 357
Focal adhesion kinase (FAK), 456 GSK3, 91, 148
FOLFIRI, 600, 602 GSPT1,541
FOLFOX,600 GTP hydrolysis, 10, 2224, 197
Food and Drug Administration (FDA), 321 GTPase, 22, 23, 234, 309, 327
323, 329, 436, 454, 460 Guanine nucleotide exchange factor
Forkhead box O (FOXO), 119, 142 (GEF), 22, 24, 58, 59, 196, 198,
FOS, 145, 203, 380 244248, 311, 663
FRB,309 Gynecologic cancer, 646, 651
Fulvestrant, 514, 538
FUS, 457, 459 H
FUS-DDIT3, 457, 459 H1299, 257, 565
H420,257
G H731,136
-catenin, 427 Hallmarks of cancer, 85, 87
G protein-coupled receptor, 458 Harringtonine,292
G0, 381, 382 HBV, 612, 617, 618
688 Index
HCT116, 255, 285, 440, 599, 600, 603 HSPA9,352

HCT15,257 HT29, 137, 146, 148, 149
HCV, 199, 404, 612, 617 Human immunodeficiency (HIV), 166
HDM2,85 Human papilloma viruses (HPV), 502, 648
Head and neck squamous cell carcinomas Human telomerase reverse transcriptase
(HNSCC), 95, 96, 500505, 507 (hTERT), 87, 384, 648
HEAT, 123, 166, 309 Hypoxic niche, 427
HEAT-1,125 Hypusine, 24, 224, 228, 229, 600
HEAT-2,125
HEK293, 137, 149, 255 I
HeLa, 148, 166, 186, 254, 255, 440, 651 IFN, 84, 181, 207, 321, 617
Hemangiosarcoma,458 IFN regulatory factor 7 (IRF7), 84
Hematological malignancies, 468, 473, 474, IFNAR1,443
476, 477, 479 IFN-, 676
Hematopoietic, 91, 291, 293, 454, 470 IFN-2b, 442
Heme-regulated inhibitor (HRI), 58, 198, 199, IGF-1 receptor (IGF-1R), 353, 412, 425, 458,
201, 202, 208, 211, 291, 403, 448, 540, 461, 515, 525, 561
542, 599 IKK, 149
Hepatitis,617 IKK, 149
Hepatocellular carcinoma (HCC), 58, 120, IL-15, 141, 142, 380
128, 137, 140, 199, 251, 253, 612, IL-2, 141, 142, 257
614622 IL-6, 385, 411, 613
Hepatocyte growth factor (HGF), 576 Imatinib, 124, 151, 209, 325, 436, 454,
Hepatoma, 228, 319 476,477
Hepatoma-derived growth factor (HDGF), 86 Indomethacin,599
Hepato-pancreato-biliary (HPB), 612, 614, INF,443
615, 620, 622 Inherited bone marrow failure syndrome, 468
HepG2,149 Inhibitor of apoptosis (IAP), 81, 406
HER2/neu, 537, 576 Initiation codon selection, 17, 26
HER2-negative, 323, 329, 521, 524, 525 Initiator Met-tRNAiMet, 48
HER2-positive, 328, 521, 526, 537, 539 Initiator tRNA, 10, 20, 22, 175, 404
Herceptin, 142, 514 INK128, 317, 325
HIF-1 and -2, 673 INK4,249
HIF-1, 82, 310, 412, 422, 425427, 456, iNOS,289
504, 519, 564 Insulin, 57, 198, 235, 247, 255, 315, 322, 347,
HIF-1/ARNT, 426 353, 356, 612, 640
HIF-2, 427 Insulin-like growth factor (IGF), 86
HIF-2/RBM4/eIF4E2 complex, 412, 425 IGF-1, 123, 456
High-grade intraepithelial neoplasia, 97, 583 IGF-2, 380, 425, 426
Hippuristanol, 121, 287, 288, 477, 536, 537 INT6, 184, 539, 565
Histidine triad nucleotide binding protein, 287 Integrin,327
HIV-1, 127, 224 Intraductal papillary mucinous
hMSH2,492 carcinoma,149
hnRNP C, 492 Invasive carcinoma, 501, 515
hnRNP K, 82, 185, 438 IRES trans-acting factors (ITAFs), 49, 71, 407
Hodgkins lymphoma, 58, 121 IRES-independent translation, 412, 426
Homoharringtonine (HHT), 292, 293 IRES-mediated,50
Honokiol,527 IRES-mediated translation, 50, 165, 167, 402,
Hormone receptor-positive, 536 404, 405, 407, 410, 411, 469, 472
HPB cancer, 614616, 621, 623 Irinotecan, 461, 594, 600603
HRAS,283 IRS-1, 313, 324, 353
HRE, 424, 519 Isoharringtonine,292
HSP90,533
Index 689
J Macrocytic anemia, 271

JB6 Cl41 model, 447 Macrophage migration inhibitory factor
JUN,203 (MIF),187
JUN family, 145 MAFbx,186
JUNB, 145, 255 Malignant mesothelioma, 236, 567, 568
JUND, 128, 145, 255 Mantle cell lymphoma, 322, 323, 356
MAP kinase kinase kinase 5 (MAP3K5), 456
K MAP4K1, 146, 147, 598
K562,274 MAPK pathway, 165, 326, 379, 403, 439,
Kaposis sarcoma, 84, 458 447, 489, 502, 522, 523, 578, 621,660
Kaposis sarcoma-associated herpesvirus MAPK/RSK pathway, 123
(KSHV), 84, 121, 288, 458 MAPK-interacting kinases (MNKs), 16, 285,
Keratinocyte, 145, 406, 446, 447, 502 320, 502, 621
Ki-67, 149, 515, 527, 637 Matrix metalloproteinases (MMPs), 77,
labeling index, 459 88,578
Kozak consensus sequence, 46 Maturation-promoting complex (MPF), 247
K-RAS/KRAS, 149, 384, 594, 614 MBI-eEF1A, 251, 252
K-RAS-LA1 lung cancer model, 564 MCF-7, 146, 151, 207, 257, 402, 440, 526,
533, 539, 565
L MCL-1, 81, 87, 284, 288, 293, 294, 318, 560,
L1TD1,129 577, 621
Lactate dehydrogenase (LDH), 444, 445 MDA-MB-231, 137, 519, 533, 534, 541
Laminin B1 (LAMB1), 411 MDM2, 85, 272, 355, 470, 577
Laminin-332,446 MDM4,253
Landing pad, 17, 118, 280 Mechanistic target of rapamycin (mTOR), 11,
Lapatinib, 210, 325, 328, 524, 525, 533 49, 53, 57, 80, 82, 89, 91, 96, 99
LARP6,128 Medulloblastoma, 127, 254, 488, 492, 493
LCIS,515 MEK inhibitor, 325, 326, 436, 439, 461,
LEE011,444 602,650
Leiomyosarcoma, 323, 454, 457 MEK/ERK, 82, 89, 199, 366
Letrozole, 514, 525, 649 pathway, 89, 290
Leucovorin, 594, 600 MEK/ERK/MNK,365367
Li-Fraumeni,515 MEK162, 326, 444
Liposarcoma, 454, 457, 459 Melanoma, 48, 56, 80, 120, 126, 205, 208,
Liver kinase B1 (LKB1), 319, 356, 515, 526 229, 257, 326, 435447
LRRK2,522 Melphalan,326
Lung cancer, 58, 97, 163, 167, 169, 227, 287, Meningioma,365
558560, 562568 Merkel cell,
Lung metastasis, 236, 367 carcinoma, 84, 446
LY2275796, 284, 294, 445 polyomavirus, 84, 446
LY294002, 327, 328, 527, 562, 579 Mesalamine,599
Lymph node metastasis, 577, 578, 582, 583, Mesothelioma, 284, 368, 558, 568
615, 633 Messenger ribonucleoprotein particles
Lymphangiogenesis,325 (mRNPs), 51, 76, 83, 351
Lymphoid lineage, 468 Messenger RNA translation, 15, 27, 49, 58,
Lynch,515 76, 164, 183, 206, 225, 316, 317, 404,
Lys159,83 662
Lys162,75 MET,500
Lysosome,385 Metallothionein 2A (MT2A), 187
Metastatic,
breast cancer, 99, 323, 326, 328, 522,
M 524526, 533, 541
m7GpppN, 74, 116, 280, 312, 424, 635 melanoma, 99, 436, 439, 440, 442, 444
MA3, 136, 138 Metformin, 327, 446, 479, 526, 562, 603
690 Index
Methionine, 18, 24, 196, 350 mTOR signaling pathway, 308, 446, 500,
Methionyl-puromycin, 176, 223, 225 521, 525
Methylation, 140, 141, 150, 252, 492, 537, mTOR/4E-BPs/eIF4F axis, 201
566, 661 MUC1-C, 121, 290
Met-tRNAi or Met-tRNAiMet, 164, 174 Multiple endocrine neoplasia, 632
Met-tRNAior Met-tRNAiMet, 18 Multiple endocrine neoplasia See MEN, 632
M-FAG, 165, 399 Multiple endocrine neoplasia type 1
MG-132 or MG132, 603 (MEN1), 632, 633, 635, 640
MicroRNA (miRNA), 52 Multiple myeloma, 55, 209, 211, 286, 293,
MIF4G,166 326, 478
MIF4GD,185 MYC, 50, 60
miR-182, 141, 143, 566, 650 Mycophenolic acid, 272
miR-21, 141144, 149152 Myelodysplasia,270
miR-663, 249, 252, 255, 541 Myelodysplastic syndrome (MDS), 270, 271,
miR-744, 249, 252, 253, 255, 541 468, 470, 471
Mitogen-activate protein (MAP), 146 Myeloid cell, 142
Mitogen-activated protein kinase
(MAPK), 11, 81, 95, 165, 237, 319, N
602, 615, 621, 622, 660 N,N-diarylurea,211
MK-8669, 503, 525 N1-guanyl-diaminoheptane or GC7, 228
MKN-28, 253, 580, 585 NAB2,454
MKN-45,580 NAB2-STAT6,454
MLN0128, 325, 533 Narciclasine,256
MLN51,182 N-cadherin, 146, 427
MMP-9, 317, 595, 617 NDRG1,183
MNK inhibitor, 98, 285, 439, 603 Neoadjuvant, 96, 520, 526
MNK1, 165, 166, 285, 365, 366, 636 Neuroblastoma, 3, 442
MNK1a/1b,365 Neuroendocrine tumors (NETs), 322, 638
MNK2, 165, 166, 285, 320, 365367, 403 Neurofibromatosis 2 (NF2), 493
MNK2a/2b,365 Neurofibromatosis type I, 632
MNK2b, 365, 366 NF45 (nuclear factor 45), 407
MOS,380 N-FAG, 165, 399
Mouse embryonic fibroblasts (MEFs), 199, NF-B, 92, 141, 143, 208, 474
206, 207, 314, 316, 404 NH125,257
Mouse mammary tumor virus (MMTV), 184, NIH, 85, 91, 167, 182185, 292, 321
401, 539 NIH 3T3, 183, 186, 283, 476
MPN domain, 176, 179, 185 Nitric oxide, 201, 530
mRNA recruitment to the ribosome, 15, 16, Nitric oxide See NO, 201
19, 57 NKX3.1,658
MSC1936369B,602 nMA3, 136, 138
mSIN1,309 NO,201
mTOR complex 1 (mTORC1), 19, 80, 237, cytostatic effects of, 208
309314, 316, 317, 319, 320, 324,326 inhibitors of, 530, 533
mTOR complex 2 (mTORC2), 309311, Non-epithelial cadherin, 427
324,353, 478, 503, 601, 619, 620, 635, Non-Hodgkins lymphoma, 58, 87, 93, 94,
665 205, 285, 322, 323
mTOR inhibitor, 84, 98, 99, 318, 324, 327, Non-melanoma skin cancer, 436,
330, 350, 357, 455, 460, 478, 491, 503, 446448,558
506, 524, 527, 562, 581, 602, 620, 649, Nonsense-mediated decay (NMD), 51, 117
675 Non-small-cell carcinomas, 558
mTOR pathway, 48, 84, 91, 200, 308, 313, Nonsteroidal anti-inflammatory drug
321, 323, 328, 330, 353, 446, 456, 461, (NSAID),599
475, 489, 491, 534, 563, 580,633 Nonstructural protein (NS5A), 199, 617, 618
in cancer, 320 Nonstructural protein 5A, 84
Index 691
NOS,289 p70S6 kinase (p70S6K), 346, 440, 491, 534,

NOTCH,427 562, 563, 579, 580, 647, 676
NOTCH1, 91, 651 p70S6K1, 346, 581
NOXA, 168, 286 p85S6K1,346
NRF2,203 PA-1,255
NS-398,142 Paclitaxel, 98, 99, 150, 256, 257, 326, 328,
NSCLC, 96, 100, 205207, 325, 368, 436, 444, 503, 534, 581, 601, 650
559,565 PAIP,44
Nucleolin,269 PAIP1, 44, 181
Nucleolin and nucleophosmin (NPM1), 269, PAIP2A, 44, 45
471, 473 PAIP2B, 44, 45
NUGC4,578 Pancreatic cancer, 422, 440, 612, 613, 617
NVP-BEZ235, 491, 527, 580, 602, 639, 675 Pancreatic ductal adenocarcinoma, 143, 612
Pancreatic neuroendocrine tumor, 321, 632
O PaNET, 321, 322, 612, 632, 633, 635640
Octamer-binding transcription factor 4 Pan-PI3K inhibitor, 328
(OCT4), 128, 427 PARP,536
Oligodendroglial tumors, 365, 489 Pateamine A, 121, 287289
Oligodendrogliomas, 488, 493 Patellazoles,603
Oligonucleotide, 152, 445 PAX3-FOXO1,455
Oligonucleotide/oligosaccharide-binding Paxillin,456
(OB), 126, 197 Pazopanib,454
Omacetaxine mepesuccinate, 292, 293 P-body/P-bodies,51
Oncolytic virus, 199 PC-12,210
ONCOMINE, 126, 440 PC-3/PC3,325
Ontak,257 PCR, 226, 597
Open reading frame, 8, 22, 44 PD032590,602
Open reading frame See ORF, 22 PD-0325901,369
Oral dysplasia, 446 PDAC, 612, 613, 615, 622
ORF, 22, 26, 44, 46, 71, 203 PDCD4D414A,D418A,138
Ornithine decarboxylase (ODC), 45, 77, 78, PDGFR, 425, 454
182, 184, 228, 284, 402, 501 PDGFRA, 412, 459
Overall survival (OS), 320, 321, 388, 444, PDGFR-, 454
490, 491, 504, 537, 615, 674 Pentamidine,530
Peptidyl-transferase center, 292
P Peripheral nerve sheath tumor, 143, 461
p120 catenin, 57, 404, 535 Peripheral vascular epithelioid cell tumors
p130(Cas),456 (PEComas),460
p16, 502, 613 Perivascular epithelioid cell tumor, 323
p16INK4A, 249, 383, 444 PERK, 441443, 448, 540, 599
p21, 87, 598 inhibitor,211
p21waf1/cip1, 128, 147, 148 PERK/eIF2 axis, 211, 476
p27kip, 274, 380 Pertuzumab,514
p27kip1, 85, 183 Peutz-Jeghers,515
p31S6K1,346 PF05212384,369
p38, 81, 365, 366, 403, 622 P-Glycoprotein protein, 291
p38 MAP kinase, 249, 250 PH domain and leucine rich repeat protein
p38 MAPK pathway, 366 phosphatases (PHLPPs), 596
P388, 289, 292 Phenethyl isothiocyanate, 530, 603
p38 kinase, 209 Phorbol 12-myristate 13-acetate, 252
p53, 60, 80, 85, 87, 89, 149, 252, 255, 272, Phosphatase and tensin homolog (PTEN), 89,
273, 355, 470, 577, 614 206, 285, 313, 320, 328, 383, 439, 442,
p70S6 kinase (p70S6K), 455457, 459 488, 492, 493, 515, 561, 594, 635, 659,
663, 675
692 Index
Phospho-4E-BP1, 96, 325, 501, 503, 506 Pomolic acid, 531

Phospho-AKT, 144, 503, 506, 638 Poncirus trifoliate, 531
Phospho-eIF4E, 96, 97, 439, 621, 662 Posttranscriptional control element
Phosphoinositide 3-kinase, 11, 308 (PCE),128
Phosphoinositide 3-kinase See PI3K, 11 PP1,202
Phosphoinositide-dependent kinase 1 PP1C,202
(PDK1), 346, 522 PP1, 352, 357
Phosphorylated eIF2, 198, 200, 202, 203, PP242, 98, 325, 479, 601, 602, 620, 675
209, 210, 404, 442, 493, 585, 663 PPAR coactivator 1 (PGC1), 354, 357
Phosphorylation of eIF4E, 81, 89, 92, 9597, PPAR coactivator 1 (PGC1), 310
285, 365, 366, 403, 502, 523, 560, 618, PPM1G,596
621, 636, 650, 662 PPP1r15A,202
PI3K/AKT/mTOR pathway, 253, 327, 328, PR,522
330, 364, 455, 458, 459, 525 Precursor mRNA, 351
PI3K/mTORC1 axis, 311 Preinitiation complex, 15
PI3K/mTORC1/2 axis, 369 Preinitiation complex See 43S PIC, 15
PI3K-related kinase (PIKK), 308, 309 Premature senescence, 378, 383, 384
PI3K, 11, 91, 309, 319, 491 Pre-mRNA, 51, 351, 365
PI3K inhibitor, 562, 579, 602 Primary human mammary epithelial cell
PI3K/AKT/mTOR pathway, 89, 165, 205, (HMEC), 86, 383, 384, 516
280, 282, 385, 437, 439, 443, 445, 474, Prion,286
490, 503, 561, 595, 618, 647, 660, 677 PRMT,144
PI3K/AKT/mTOR/S6K pathway, 118 Proapoptotic protein, 168, 409
PIK3CA, 89, 92, 328, 458, 500, 515, 561, Pro-B-cell lymphoid cell, 251
594, 603, 647 Progesterone receptor, 515
PIM1, 122, 124, 274, 380, 402, 501 Prognostic marker, 96, 123, 144, 150, 258,
PIM1/2 inhibitor, 124 520, 523, 567, 604, 614, 652
PIM2,122 Programmed cell death 4 (PDCD4), 15, 56,
Pimasertib,443 117120, 122, 125, 129, 136140,
PIWI,52 142150, 315, 441, 447, 475, 492, 502,
PIWI-interacting RNA (piRNA), 52 536, 537, 566, 568, 582, 597, 598, 650,
PKAC,522 651, 660
PKB,308 Progression-free survival (PFS), 321323,
PKC, 165, 310, 312, 522 328, 329, 460, 491, 525, 620, 637,676
PKC, 237 Prohibitin,290
PKC, 143 Proliferator-activated receptor
PKR-like endoplasmic reticulum kinase (PPAR), 210, 310, 311, 354
(PERK), 58, 198200, 202, 208, 210, Proline-rich AKT substrate 40 kDa
211, 291, 403 (PRAS40), 309, 503
Placental growth factor (PGF), 86 Promyelocytic tumor suppressor protein
Platelet-derived growth factor (PDGF), 380, (PML), 384, 388
412, 637 Prostate cancer, 96, 97, 100, 121, 152, 253,
Platycodin D, 530 255, 257, 290, 317, 320, 329, 658,
Poly(A) tail, 8, 15, 16, 44, 71, 164, 185, 382, 660665, 667
396 Prostatic-specific membrane antigen
Poly(A)-binding protein (PABP), 9, 13, 16, (PSMA),257
40, 44, 57, 76, 164166, 175, 396, 401, Proteasomal degradation, 56, 83, 141, 143,
567 151, 153, 408, 447, 535, 564, 662
Polypeptide chain, 1, 10, 11, 25, 243, Proteasome inhibitor, 83, 209, 212, 505,
257,396 581,603
Polypyrimidine tract-binding protein Protein arginine methyltransferase 5
(PTB), 410, 411 (PRMT5), 136, 141, 144, 537
Polyribosome, 381, 387, 599 Protein kinase R (PKR), 58, 198200, 206,
Polyunsaturated fatty acids (PUFA), 531 207, 211, 403, 441, 492, 542, 566,599
Index 693
Protein kinase/protein kinase A (PKA), 198, RAS/RAF/MEK/ERK cascade, 515

249, 250 RAS-related GTPase (RAG), 311
Protein phosphatase, 202, 456 RASSF1A,440
Protein phosphatase 2A (PP2A), 249, Rat-1, 283, 488
403,456 Ratchet model, 20
Protein phosphatase 2A (PP2A), 365 RB,383
Protein phosphatase 2A (PP2A), 123, 166, RBM4, 412, 425
187, 560, 615 R-CHOP,323
Proto-oncogene, 45, 81, 120, 166, 269, 438, Reactive oxygen species (ROS), 447
461, 488, 507, 559, 676 RecA1/RecA2 domain, 126
PROTOR1,309 RECORD-1,677
PROTOR1/2,309 Recycling factor, 14, 26
PrPSc,286 REDD1, 311, 423
P-site, 10, 14, 1820, 22, 23, 25, 41, 243, 600 Regorafenib,594
PUF,248 Regulatory-associated protein of mTOR
Pumilio homolog 2 protein (PUM2), 249 (RAPTOR), 309, 311, 315, 354
Puromycin,176 Reinitiation, 16, 2527, 44, 48, 186, 203
Reishi,531
Q Release factor, 26
QT6,145 Renal cell carcinoma, 151, 320, 321, 356,
491, 524, 674, 676, 677
R Repeat-containing protein 2 (BIRC2), 81, 87
Rabbit reticulocyte lysate, 77, 79, 139, 140 Resistance, 78, 87, 98, 150, 250, 253, 282,
RACK1, 235, 237, 622 294, 327, 328, 330, 357, 411, 494, 505,
RAD001, 321, 503, 524, 581, 666 523, 524, 527, 537, 563, 620
RADIANT-2,322 Resveratrol, 152, 254
RADIANT-4,322 Reticulocytopenia,469
Radiation therapy, 209, 523, 576, 613, 658 Retinoic acid receptor, 538
Radiosensitization, 329, 534 Reversion-inducing-cysteine-rich protein with
Radiotherapy, 98, 208, 209, 257, 329, 421, kazal motifs (RECK), 538, 584
436, 454, 468, 500, 503, 505, 524,533 RGDS peptide, 327
RAF/MEK/ERK, 576, 595 Rhabdomyosarcoma, 80, 323, 455, 456
axis,447 Rhabdomyosarcoma (Rh30), 456
cascade,437 RHO,256
signaling pathway, 290 Ribavirin, 532, 535
RAGULATOR,311 Ribonucleotide reductase M2 subunit
Raloxifene,514 (RRM2),183
Rapalogs, 282, 311, 320, 323325, 330, 457, Ribosomal DNA (rDNA), 268
491, 503, 527, 601, 649, 675 Ribosomal protein, 49, 55, 56, 59, 60, 126,
Rapamycin, 84, 98, 99, 282, 290, 308, 309, 139, 187, 269271, 274
312, 318, 320, 324, 325, 346, 350, 367, Ribosomal protein S6 kinase, 345, 352, 380,
456, 457, 491, 503, 527, 533, 534, 563, 471, 614, 618, 664
579, 580, 601, 620, 621, 639, 648, 649, Ribosomal RNA (rRNA), 59, 60, 126, 185,
665, 676 268, 269, 271273, 353, 470, 659
Rapamycin-insensitive companion of mTOR Ribosome,
(RICTOR), 309, 353, 616 biogenesis, 23, 49, 50, 59, 128, 235, 269,
Rapidly accelerated fibrosarcoma (RAF), 439 273, 274, 352, 468, 471, 473, 479, 614,
RAR pan-agonists, 528 660
RAS, 559, 621 footprint,46
RAS homolog enriched in brain (RHEB), 309, recruitment, 16, 42, 43, 55, 118, 119, 280,
311, 327, 522 312, 424, 532
RAS/MAPK,309 recycling, 8, 11, 25, 26, 41, 396, 540
RAS/MAPK/ribosomal S6 kinase Ribosomopathy, 60, 271
(RSK), 118,357 Rilotumumab,576
694 Index
RNA helicase, 117, 118, 120, 124, 129, 137, S6K1/2, 314, 325
175, 287, 315, 477 S6K2, 60, 140, 144, 314, 346
RNA helicase A (RHA), 128 Salubrinal, 210, 539
RNA hypoxia response element SAR245409, 491, 602
(rHRE), 412,425 Sarcine ricine loop (SRL), 236
RNA interference, 166, 407 Sarcoma, 84, 99, 205, 323, 454, 455, 457, 461
RNA polymerase I, 268, 272, 617, 619 Scaffolding protein, 76, 116, 280, 312, 315,
RNA polymerase II, 269, 617 364, 396, 488, 580, 595, 636
RNA-binding motif protein 19 (RBM19), 273 Scanning, 1618, 2022, 47
RNA-dependent ATPase, 117, 121, 122, 287 Scanning 40S ribosomal subunit, 17
RNAi, 52, 56, 121123, 125, 664 SCF, 475, 660
Rocaglamide, 121, 289, 290 SCF/-TRCP, 475, 660
rpL11, 272, 274 SCLC, 167, 292, 408
rpL12,269 SDS, 271, 470
rpL13,270 Secondary structure, 9, 1517, 20, 45, 76, 78,
rpL19, 664, 665 118, 137, 175, 280, 407, 595
rpL23, 234, 236, 272 SEGA,356
rpL23a, 269, 270, 664 Selective estrogen receptor,
rpL24, 234, 236, 351 modulator,514
rpL26,270 Selumetinib, 443, 461, 562, 650
rpL27,269 Senescence, 2, 60, 86, 89, 381, 383, 384
rpL29, 272, 585 Senescence-associated heterochromatic foci
rpL30, 269, 585 (SAHF),383
rpL35,270 Ser/Thr protein kinase, 309
rpL35A,270 Ser1148,315
rpL36, 270, 614 Ser1188,315
rpL36a,270 Ser1232,315
rpL37,664 Ser174,234
rpL5, 270272, 473 Ser175,234
rpL7,270 Ser1859,353
rpL7a,664 Ser209, 81, 285, 320, 363365, 367, 379,
rpS10,270 403,502
rpS14, 271, 471, 664 Ser209Ala, 285, 366
rpS15a,270 Ser21,248
rpS17,270 Ser235, 234, 235, 237
rpS18,270 Ser239,237
rpS19, 60, 270, 274, 470, 585 Ser2448, 353, 501
rpS24,270 Ser300,248
rpS29,270 Ser359, 250, 316
RPS6KB1 gene, 346 Ser366, 250, 316, 351
RPS6KB2 gene, 346 Ser406,124
rpS7, 270, 272 Ser422, 122124, 315
rpS8, 269, 270 Ser457, 136, 143
rpS9, 272, 585 Ser500,250
RTK, 329, 459, 594 Ser51, 58, 196, 197, 206, 441, 492
RTK inhibitor, 594 Ser595,249
Ser65, 80, 314
S Ser67, 136, 143, 315
S phase, 180, 257, 381, 382, 541 Ser78,316
S6 (rpS6) kinase 1 (S6K1), 55, 56, 59, 60, Ser84,355
143, 201, 313, 314, 346 Serine/threonine kinase 11 (STK11), 319
S6 kinase (S6K), 356 Serum/glucocorticoid regulated kinase 1
S6 kinase (S6K), 311 (SGK1), 312, 619, 635
S6K, 311, 313, 315, 346, 352, 353, 356, 357 Sestrin,311
S6K1 Aly/REF-like target (SKAR), 351 Sex determining region Y-box 2
(SOX2), 427,490
Index 695
SF1126,327 Surgical margin, 93, 96, 99, 501, 504, 505

SF2/ASF,365 Survivin, 45, 87, 318, 380, 443, 444, 534, 536
SGC-7901, 580, 581 inhibitor,444
SH2,200 SW620, 253, 585, 600
Shunting model, 21 Swachman-Bodian-Diamond syndrome
Silibinin,532 protein (SBDS), 234, 270, 271
Silvestrol, 121, 287, 289, 290, 294, 477, 518, SwachmanBodianDiamond syndrome
536, 537, 665 protein (SBDS), 470
Sirolimus, 459, 460, 619 Synovial sarcoma, 455, 457
Skin cancer, 56, 97, 435437, 446, 447 Synthetic cannabinoids, 603
Skin papillomas, 447 Synthetic viability, 386, 387
SLBP-interacting protein 1 (SLIP1), 177, 185
SMAC,408 T
SMAD2, 88, 91, 92, 489, 490, 621 T antigen, 84
Small hairpin RNA (shRNA), 52, 59, 124, T cell, 88, 142, 288, 290
229, 284, 456, 520, 534, 585 T315I,326
Small T (sT), 446 T47D cell, 146
Small-cell carcinomas, 558, 568 T-ALL, 271, 473
SMI-4a,124 Tamoxifen, 151, 329, 514, 517, 525, 537, 539
SNAIL, 88, 92, 127, 146148, 411, 427 Tanespimycin,533
Soft tissue, Targeted therapy/targeted therapies, 454, 524,
leiomyosarcoma,457 558, 594
sarcomas, 454, 460, 461 TAT peptide, 168, 283
Solitary fibrous sarcoma, 454 Taxanes,514
Sorafenib, 290, 461, 576, 613 Taxol, 256, 411
Sp transcription factors, 142 TC,291
Spermidine, 224, 228 T-cell acute lymphoblastic leukemia, 271, 473
Spry1,447 TCL1,290
Squamous cell carcinoma, 94, 96, 128, 167, TEL2,309
436, 446, 500, 505, 506, 566, 568, 577, Telomere, 86, 383
579, 582, 583, 648 Temsirolimus, 257, 318, 321, 322, 330, 456,
of lungs, 567 461, 491, 503, 524, 525, 562, 599, 638,
SRC, 200, 458, 522 649, 675, 676
SS18,455 Terminal oligopyrimidine, 269
SS18-SSX2,455 Termination, 8, 10, 11, 23, 25, 27, 44, 53, 59,
SSX2,455 244, 351, 396, 441, 540, 586, 667
Stable isotope labeling by amino acids Ternary complex, 9, 14, 18, 24, 25, 41, 138,
(SILAC),479 175, 181, 196, 201, 205, 403, 405, 441,
Start codon selection, 46, 517 663
STAT, 200, 459 Ternary complex See TC, 18
Sterol regulatory element binding protein 1 TFEB,310
(SREBP1), 310, 311, 354 TGF, 91, 92
Stress granule, 42, 5153, 289 TGF--receptor-type-II-interacting protein
Stress-activated MAPK-interacting protein 1 (TRIP-1), 182, 187
(SIN1), 353, 357 Thapsigargin, 407, 442, 443
Stromal cell-derived factor 1 (SDF1), 141 Therapeutic resistance, 367, 504, 523, 542,
Structured UTR, 11, 17, 21, 45, 49, 50, 54, 563, 622, 623
56, 77, 116, 120, 123, 280, 315, 462, Thiostrepton,442
523, 637, 660 Thiozolidoneindenones,210
Subependimal giant cell astrocytoma, 320 Thr204,128
Suberoylanilide hydroxamic acid Thr243,237
(SAHA), 505, 535 Thr2446,353
SUCCEED,460 Thr323,128
Sunitinib, 492, 637, 638 Thr37,80
Suppressor of fused (SUFU), 355 Thr389, 201, 346, 347
696 Index
Thr46, 80, 200, 314 Trp56,75

Thr56, 249, 351 Trp73,79
Thr70, 80, 314, 501 TSC1, 322, 356, 490, 675, 676
Thr88,248 TSC1/2, 309, 313, 317, 352
Tif1,289 TSC2, 91, 311, 322, 327, 356, 423, 479, 522,
Tif1/2, 121, 289 526, 635, 660, 676
Tif3,122 TTI1,309
Tif6, 235, 236 TTI1/TEL2,309
Tissue inhibitor of metalloproteinase 2 Tuberous sclerosis complex, 91, 322, 356
(TIMP2), 147, 148 Tuberous sclerosis syndrome, 319
Tivantinib,576 Tumor promoter, 143, 145, 447
TKI, 168, 209, 455, 562, 563, 576, 613 Tumor suppressor, 56, 60, 80, 89, 117, 125,
inhibitor,454 207, 282, 355, 378, 380, 383, 441, 472,
TLK, 98, 523 491, 494, 523, 536, 538, 541, 565, 578,
TLK1B, 98, 523, 524 582, 596, 601, 614, 638, 647, 652, 677
T-lymphoblastic CCRF-CEM, 255 like properties of DDX3X, 128
TMZ, 330, 436, 491 p53, 272, 274
TNF, 141, 144, 145, 168, 208, 289, 355 pathway, 386, 388
TNF-related apopotosis-incuding ligand PDCD4, 136, 144, 145, 151, 152
(TRAIL), 168, 257, 286, 399, Tumor-free margin, 501, 505
402404, 564, 582 TWIST1,148
TNF, 141, 144, 289, 355, 399, 400, 403 Tyr141,248
TNM, 579, 632, 635, 675 Tyr29,248
Tolfenamic acid, 599 Tyrosine kinase inhibitor, 168
Tonsillar squamous cell carcinoma, 502 Tyrosine kinase receptor inhibitor, 98
TOP, 49, 60, 78, 180, 269, 314, 351, 619 TZD18, 210, 532
TOR, 166, 186, 308
Toremifene,514 U
Torin 1, 98, 326, 620, 665, 666 U251, 256, 493
Torisel, 321, 503 U2OS, 255, 457
Tousled-like kinase, 98 U373, 140, 493
TP53, 89, 149, 437, 516, 536, 558, 658 U87, 490, 493
Trametinib, 436, 443 Ubiquitin-like modifier (UBL), 251
Transcription factor, 46, 50, 60, 91, 142, 145, Ubiquitinylation,83
149, 152, 201, 354, 380, 411, 422, 490, ULK1,385
659 ULK1/ATG13/FIP200 complex, 310, 311
Transfer RNA (Initiator tRNA), 9, 18, 22, 23, Ultraviolet A (UVA), 447
76, 225 Ultraviolet radiation (UV), 137, 149, 250, 403
Transfer RNA (tRNA), 10, 18, 25, 26, 58, 59, UMSCC22B,504
243, 405 Unconventional prefoldin RPB5 interactor 1
Transforming growth factor (TGF-), 88, (URI), 352, 357
91, 92, 141, 143, 144, 248, 284, 426, Untranslated region (UTR), 11
489, 614 uORFs, 26, 4648, 58, 71, 186, 203, 204, 404
Transitional cell, 150, 674 Up-frameshift protein 1 (UPF1), 51, 181
Translation initiation, 2, 8, 9, 11, 15, 1823, Upstream binding factor (UBF), 60
26, 116, 126, 127, 139, 204 Upstream open reading frames, 26
Translocation, 10, 24, 59, 148, 243, 244, 249, Upstream open reading frames See uORFs, 26
257, 285, 316, 322, 455, 473 Urokinase-type plasminogen activator receptor
Trastuzumab, 98, 99, 168, 328, 523, 526, 576 (uPAR), 146, 147
TRC8, 181, 186 Urothelial carcinoma, 143, 150, 674, 675
TrkA,183 Uterine leiomyosarcoma
Troglitazone,210 Leiomyosarcoma,460
Trp102,75 UVB,447
Trp166,75
Index 697
V W
Val69,79 White blood cell, 468
Vascular endothelial growth factor WNT, 91, 658
(VEGF), 45, 50, 55, 77, 284, 317, 318, pathway,91
369, 404, 422, 426, 438, 444, 446, 488, signaling, 91, 92, 600
490, 491, 504, 576, 674 WNT/-catenin pathway, 612
Vascular endothelial growth factor A, 84, 87, WYE-125132,491
97 WYE-132, 491, 665, 666
Vascular endothelial growth factor C, 87 WYE354,601
VATPase,311
Vb regulatory subunit of cytochrome c X
oxidase,559 XIAP, 123, 140, 144, 293, 402, 404, 407, 469,
Vemurafenib, 436, 443 539
Vesicular stomatitis virus (VSV), 84 encoding of, 408
Vimentin, 227, 246, 317 XL765, 326, 491
Vincristine,326
Vinorelbine, 328, 526 Y
VIPoma,632 Y box-binding protein 1 (YB-1), 88, 148,
Virus,84 317, 411
encephalomyocarditis virus (EMCV), 140 Ying-Yang 1 (YY1), 310
human immunodeficiency virus type 1 YM155,444
(HIV-1),57
human papilloma viruses (HPV), 84
Oncolytic virus, 84 Z
oncoviruses,84 ZAP-70,248
picornaviruses,165 kinase signaling, 248
poliovirus, 49, 405 Zilongjin,532
reovirus,77 Zinc finger E-box-binding homeobox 2
rhinovirus, 79, 165 (ZEB2),411
Rous sarcoma oncovirus, 458 Zinc finger protein 217(ZNF217), 252
vesicular stomatitis virus (VSV), 84, 199 Zinc-finger-binding protein 89 (ZBP89), 142
von Hippel Lindau syndrome, 632 Zolendronic acid, 532

Translation and Its Role in Cancer

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Translation and Its Role in Cancer

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Translation and Its Regulation in Cancer

Biology and Medicine

Translation and Its

Chapter 10 was created within the capacity of a US governmental employment. US copyright

ISBN 978-94-017-9077-2ISBN 978-94-017-9078-9 (eBook)

Library of Congress Control Number: 2014939521

Springer Science+Business Media Dordrecht 2014

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com).

Part I Translation Machinery in Cancer

2Mechanism of Translation in Eukaryotes 7

3Diverse Mechanisms of Translation Regulation and Their

4eIF4E and Its Binding Proteins 73

5RNA Helicases and Their Cofactors 115

12Translation Elongation 241

14Current and Emerging Therapies Targeting Translation 279

Part II Regulation of Translation by Signaling Pathways in Cancer

15mTOR and Regulation of Translation 307

16Ribosomal Protein S6 and S6 Kinases 345

17eIF4E Phosphorylation Downstream of MAPK Pathway 363

Part III Cell Fate and Translation in Cancer

18Translational Control of Cell Proliferation and Viability in

19Translation and Apoptosis in Cancer 395

20Translation in Cancer at Hypoxia 421

Part IV Translation and Its Regulation by Cancer Types

21Melanoma and Non-Melanoma Skin Cancers 435

23Hematological Malignancies and Premalignant Conditions 467

24Brain Tumors 487

25Head and Neck Cancers 499

26Breast Cancer 513

27Cancers of the Respiratory System 557

28Gastric and Esophageal Cancers 575

29Colorectal Cancers 593

30Hepatic, Pancreatic and Biliary Cancers 611

31Pancreatic Neuroendocrine Tumors 631

32Gynecologic Cancers 645

33Prostate Cancer 657

34Cancers of the Urinary System 673

Michela Abrami Department of Engineering and Architecture, University of

Stefano Biffo Department of Science and Technological Innovation (DISIT),

Ana Maria Gonzalez-Angulo Department of Breast Medical Oncology and

Justin G. Meyerowitz Department of Chemistry and Chemical Biology,

Markus Reschke Cancer Research Institute, Beth Israel Deaconess Cancer

Yoshinori Tsukumo Department of Biochemistry, Rosalind and Morris Goodman

4E-BP eIF4E-binding protein

ARNT Aryl hydrocarbon receptor nuclear translocator

BAD BCL-2 antagonist of cell death

C-FAG C-terminal cleavage fragment of eIF4G

DAP5 Death-associated protein

DEPTOR DEP domain-containing mTOR-interacting protein

E-box Enhancer box

ERCC5  Excision repair cross-complementing rodent repair deficiency,

FAK Focal adhesion kinase

GADD34 Growth arrest and DNA damage-inducible protein (also known as

GMP, GDP, GTP Guanosine mono, di- and triphosphates

H-RAS or HRAS Harvey rat sarcoma viral oncogene homolog

IAP Inhibitor of apoptosis

IGF Insulin-like growth factor

JAK Janus kinase

K-RAS or KRAS Kirsten rat sarcoma viral oncogene homolog

M-FAG M-terminal cleavage fragment of eIF4G

MAP3K5 MAP kinase kinase kinase 5, also see ASK1

N-FAG N-terminal cleavage fragment of eIF4G

P-body Processing body

2Mechanism of Translation in Eukaryotes 7

3Diverse Mechanisms of Translation Regulation and Their

4eIF4E and Its Binding Proteins 73

5RNA Helicases and Their Cofactors 115

12Translation Elongation 241

14Current and Emerging Therapies Targeting Translation 279

15mTOR and Regulation of Translation 307

16Ribosomal Protein S6 and S6 Kinases 345

17eIF4E Phosphorylation Downstream of MAPK Pathway 363

18Translational Control of Cell Proliferation and Viability in

19Translation and Apoptosis in Cancer 395

20Translation in Cancer at Hypoxia 421

21Melanoma and Non-Melanoma Skin Cancers 435

23Hematological Malignancies and Premalignant Conditions 467

24Brain Tumors 487

25Head and Neck Cancers 499

26Breast Cancer 513

27Cancers of the Respiratory System 557

28Gastric and Esophageal Cancers 575

29Colorectal Cancers 593

30Hepatic, Pancreatic and Biliary Cancers 611

31Pancreatic Neuroendocrine Tumors 631

32Gynecologic Cancers 645

33Prostate Cancer 657

34Cancers of the Urinary System 673

ERCC5 Excision repair cross-complementing rodent repair deficiency,

GADD34 Growth arrest and DNA damage-inducible protein (also known as

2.2.1Binding of eIF4F Complex Prepares the mRNA

2.2.2Several Initiation Factors Prepare the 40S Ribosome

2.2.3mRNA Recruitment to 43S PIC

2.2.5Initiation Codon Selection

2.2.660S Ribosome Binding and 80S Ribosome Formation

2.3Translation Elongation and Termination

2.4Ribosome Recycling and Reinitiation

2.5Conclusions and Perspectives

3.2mRNA Sequence and Structure Elements