The Identification of Oncogenes

How molecular methods partnered with genetics to identify many of the genes involved in cancer

An oncogene is an altered form (allele) of a normal cellular gene (proto-oncogene). It is operationally defined as a regulatory gene with dominant transforming properties. Oncogenes were first discovered through their association with specific retroviruses, and this form of an oncogene is called a v-oncogene (v-onc). The first oncogene was discovered from the Avian Sarcoma Virus and was called src. Proto-oncogenes function in signal transduction, cell cycle regulation, or cell differentiation. Normal expression of proto-oncogenes is critical for normal tissue differentiation and growth. There are also tumor suppressor genes, which are recessive genes that restrain cell proliferation. These will be discussed anon.

Oncogenes Identified from Retroviruses

Acutely Transforming Retroviruses Carry v-oncs v-onc
src myc erb A, erb B myb ets rel H-ras K-ras abl raf fos fms fes sis

Virus
Rous sarcoma virus Avian myelocytomatosis virus Avian erythroblastosis virus Avian myeloblastosis virus Avian erythroblastosis virus Avian reticuloendotheliosis virus Harvey rat sarcoma virus Kirsten murine sarcoma virus Abelson murine leukemia virus Murine sarcoma virus Mouse osteosarcoma virus Feline sarcoma virus Feline sarcoma virus Simian sarcoma virus

Host
Chicken Chicken Chicken Chicken Chicken Turkey Rat Mouse Mouse Mouse Mouse Cat Cat Monkey

From LSU Health Sciences Center

These oncogenes were found as the carcinogenic gene of each tumor virus
As with the V-SRC gene, the relevant segment of DNA was unnecessary for virus replication As with V-SRC, removal of the oncogene greatly reduced the virus tumorigenicity Unlike V-SRC, the sequence of the gene did not necessarily tell the investigator anything useful about the gene product And what did all this have to do with human cancer, anyway

IN A DIFFERENT APPROACH Human oncogenes were first discovered by a different method

Discovery of Oncogenes as Dominant Transforming Genes by Shotgun Cloning -ras from bladder carcinoma -met oncogene from hepatocarcinoma -hst oncogene from stomach carcinoma -erb B oncogene from breast carcinoma

From www.orst.edu/instruction

LM of cell culture with a focus

The use of ALU sequences as markers for human DNA allowed Weinberg to recover genes that might have contributed to the human tumor with which he started. The fact that the human DNA could cause tumors in mice was encouraging that the gene in hand was basic and important to tumor biology. First gene so cloned was the RAS gene. This was identified as a small, GTP-binding protein involved in the signaling pathways of human cells.
From www.orst.edu/instruction

A SURPRISING BREAKTHROUGH In the 1970s it was shown that virally encoded oncogenes were almost identical to genes found in normal cells
V-SRC was a virtually identical to corresponding sequences in chickens, turkeys, ducks, quails, and emus V-RAS was similar to sequences in mice, rats, and people, just like the RAS just cloned from human tumors A concept emerged: oncogenes are mutated forms (or perhaps simply over-expressed forms) of a normal gene Call the normal gene a proto-oncogene and its mutated, carcinogenic counterpart an oncogene

Oncogenes must be dominant

Weinbergs shotgun cloning showed that oncogenes were oncogenic in cells whose genotype included the wild-type allele; thus, the mutants were dominant Proto-oncogenes could be seen as normal inducers of cell cycle progression; the dominant, carcinogenic alleles were gainof-function alleles of these genes

This is distinct from Rb, where the oncogenic mutation was a LOF allele
The biology of retinoblastoma showed us that it normally serves as a negative regulator of cell cycle progression The oncogenic mutation of Rb was a loss of function allele. It was recessive and required a loss of heterozygosity to produce a phenotype Such genes have come to be called tumor suppressors

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What Kinds of Genes are Mutated in Cancer? Oncogenes and Tumor Suppressors
Oncogenes are genes that positively regulate the cell cycle (move it forward) - dominant mutations in proto-oncogenes cause excess cell proliferation Tumor Suppressors are genes that negatively regulate the cell cycle - recessive LOF mutations in these genes also cause excess cell proliferation, but they generally need homozygosity for their phenotype to be expressed Presumably, there exist gain-of-function alleles of tumor suppressors, but they are not oncogenic, merely CELL LETHAL, so there are no progeny

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The signal transduction pathway is a major source of proto-oncogenes

src

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The signal transduction pathway is a major source of proto-oncogenes.

Growth factors: sis, fgf, tgf- Receptors: erbB-1, neu, kit, ros Non-receptor kinase: src, abl GTP-binding: ras Ser/Thr kinases: raf TXN-factors: myc, fos, jun src

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What Classes of Genes can be Oncogenes?

The cellular form of RAS contributes to Receptor-Mediated Signal Transduction

SOS, a GEF

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RAS has been studied extensively, in part because of its role in cancer, and in part because proteins like it are widely used in signaling biology
GTPaseActivating Protein: accelerates turning RAS OFF GTPExchange Factor: accelerates turning RAS ON Active RAS binds to down-stream kinases and turns them on

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RAS is in a pathway for cell growth control

i.e. EGF-Receptor

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Mutations can activate ras in the absence of the normal Growth Factor, making the pathway always on

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-activated ras has been identified as a v-onc -activated ras also identified in bladder cancer
Inactive Pi GAP GTP GEF

Active
Adapted from

GDP

http://peo.cshl.org/~mangone/medbench/last_ras.htm

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Activating ras Mutations:

-Mutations that destroy GTPase activity, or prevent GAP binding (residues 12, 13, 59, 61) -Mutations that weakly bind nucleotide or cause high release rate (residues 116 and 119) Inactive
GDI

Pi GAP

GTP GEF

Active
Adapted from

GDP

http://peo.cshl.org/~mangone/medbench/last_ras.htm

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Other ras-like proteins have been identified as proto-oncogenes, but this is a complicated problem, because:

- ras family members also activated other signal transduction pathways involving

several other signal molecules, including PI3, DAG, Ca++, and cAMP. -There are at least 140 small GTP-binding proteins in the human genome. -There are 160 identified GAP (GTPase-activating proteins inactivates G-protein) genes. -There are 150 identified GEF (GTP-exchage factor activates G-protein) genes. -GDI proteins (GTP dissociation inhibitor) are also numerous suppresses G-protein activation.

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v-oncs are Mutated Proto-oncogenes

c-oncs Regulate Cell Growth and Cell Cycle
c-onc
src myc erb A, erb B myb ets rel ras abl raf fos fms fes sis

Cellular Function
Activator of ras-dependent Signal Transduction G1 Transcription Factor EGF Receptor G1 Transcription Factor G1 Transcription Factor G1 Transcription Factor Signal Transduction Tyrosine Kinase; Activates G1 Transcription ras-dependent Signal Transduction G1 Transcription Factor G1 Transcription Factor G1 Transcription Factor G1 Transcription Factor

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v-erbB Expresses a Truncated EGF-Receptor Which is ALWAYS on, regardless of [EGF]

v-

http://www.wwnorton.com/cdly/cells/fig11_4.gif

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This truncated EGF-R signals in absence of EGF

s rc

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Overexpression of EGF-R (i.e. her2/neu) is oncogenic

But note that there are several places in this pathway where one could imagine or invent inhibitors to the hyperproliferation that the oncogene induces. These are the targets of many novel cancer therapies
http://www.prous.com/journals/dnp/sample/html/dn130325/dn130325.html

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Myc is also a target of the RAS pathway

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Growth factors also stimulate our old pals, the CDKs needed for progression

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Carcinogenesis is largely the mutation of clearly identified components of normal cell cycle regulation
Many components of the cell cycle regulation pathways are proto-oncogenes Their GOF mutants can be oncogenes Many other components of the cell cycle regulation pathways are tumor suppressors Their LOF mutants can be oncogenic We begin to see why carcinogenesis requires many mutations to be completed

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Another pathway for retroviral oncogenesis

Retroviruses Lacking v-oncs can Activate c-oncs
Host Chromosome Viral Genome
LTR gag pol

Proto-oncogene

env

LTR

LTR

gag

pol

env

LTR

Promoter insertion

Enhancer insertion

Retroviruses can Transform by Insertional Mutagenesis -insertion of retroviruses into proto-oncogene locus -LTR promoter or enhancers activate c-onc expression

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Other pathways for oncogenesis do not require external genes but the re-arrangement of existing genes
Philadelphia Chromosome
Reciprocal Translocation 95% of CML Patients (Chronic Myelogenous Leukemia) Posses the Philadelphia Chromosome

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Chronic Myelogenous Leukemia

Hyperproliferation of de-differentiated Lymphocytes
Peripheral Blood

Normal
Bone Marrow

CML

95% of CML Patients Possess the Philadelphia Chromosome

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The translocation leads to the misregulation of c-Abl, causing hyperproliferation and CML

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Misregulation of c-Abl in CML

c-Abl is a tyrosine kinase (phosphorylates only tyrosines)
-c-Abl is inhibited by binding to pRb -pRb phosphorylation (by Cdk2) releases c-Abl -nuclear c-Abl promotes cell cycle transcription

Fusion to Bcr causes the misexpression of c-Abl in Myeloid Cells -leads to inappropriately regulated tyrosine kinase activity -causes hyperproliferation and de-differentiation

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Specific Cytogenetic Changes are associated with other cancers as well

Burkitts Lymphoma

From McKinnel et al. The Biological Basis of Cancer

From NCBI http://www.ncbi.nlm.nih.gov/cgi-bin/SCIENCE96/nph-gene?MYC

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c-myc Functions in G1 Transcription Control:

- activates growth-promoting genes - represses growth-arresting genes

From NCBI http://www.ncbi.nlm.nih.gov/cgi-bin/SCIENCE96/nph-gene?MYC

Strong Expression of myc in Lymphoid Cells

-myc expressed from IgH promoter (highly active in lymphoid cells) -leads to inappropriately regulated G1 transcriptional activity -causes hyperproliferation and de-differentiation

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Specific Cytogenetic Changes are Associated with Some Cancers

Amplification of the myc Oncogene

Overexpression of c-myc can occur without chromosomal translocation

DNA myc Locus Double Minute

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How many cancer genes have been identified, and how does this compare to the complete gene set in the human genome?

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How many cancer genes have been identified, and how does this compare to the complete gene set in the human genome? As of 2004, 291 cancer genes have been reported; approximately 1% of the human genome. The most common domain that is encoded by cancer genes is the protein kinase. DNA-binding domains are the second most common.

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What percentage of genes examined show a difference in expression levels when comparing normal versus tumor tissue?

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What percentage of genes examined show a difference in expression levels when compairing normal versus tumor tissue? Approximately 5% of transcripts show some change in expression levels, regardless of tumor type or history.

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Tasmanian Devil Facial Tumor Disease (DFTD)

- First described in 2003, DFTD has devastated the devil population of the Australian island state of Tasmania, with 50% of the population lost. -The devils are riddled with rapidly invasive lesions and tumors that erupt on the tongue, roof of the mouth, gums, and lips. As the disease advances, the lesions spread aggressively throughout the body. Death comes within six months of the onset of the disease. -Disease presents itself as a large cancerous tumor of neuroendrocrine origin around the face and neck, often preventing feeding. Normal devil Diseased devil

http://www.dpiwe.tas.gov.au/inter.nsf/WebPages/LBUN-5QF86G?open

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Tasmania Devil Facial Tumor Disease (DFTD)

What causes the disease? How can the disease be contained?

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Tasmania Devil Facial Tumor Disease (DFTD)

-VIRUS? The high prevalence and rapid spread through the population suggested an infectious agent, probably a virus. Researchers used the Koala leukemia retrovirus and canine venerial sarcoma virus as a model for investigation. However, to date electron microscopic studies and attempts to find reverse transcriptase have produced negative results. Thus, the viral hypothesis is losing favor. - MUTATION? Tumor karyotyping shows similar chromosome rearrangements among animals, suggesting DFTD may be passed animal to animal by implantation of cells during fighting and biting. -Chemical toxins? Of particularly interest are those used in local farming. There are 1,080 candidate compounds under investigation. -The Australian government is attempting to control the disease by quarantine using zoos, and by constructing natural barriers in the wild. They are also trying to develop accurate diagnostic tests to monitor the diseases progression carefully.

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