Cancer Gene Therapy (2013) 20, 101108

& 2013 Nature America, Inc. All rights reserved 0929-1903/13

www.nature.com/cgt

ORIGINAL ARTICLE

RNA interference targeting human FAK and EGFR suppresses

human non-small-cell lung cancer xenograft growth in nude mice
C Li1,3, X Zhang1,3, L Cheng1, L Dai1, F Xu1, J Zhang1, H Tian1, X Chen1, G Shi1, Y Li1, T Du1, S Zhang1, Y Wei1 and H Deng1,2
Transfection of plasmid vectors coexpressing short hairpin RNA (shRNA) for focal adhesion kinase (FAK) and epidermal growth
factor receptor (EGFR) signicantly inhibited protein level of FAK and EGFR. Knockdown of FAK and EGFR expression signicantly
inhibited cell proliferation and induced cell apoptosis of A549 lung cancer cells in vitro. In A549 subcutaneous xenograft model,
mice treated for 3 weeks with plasmid that coexpresses FAK and EGFR shRNA had signicantly smaller tumors than those in control
mice (Po0.01). FAK and EGFR dual silencing also signicantly decreased microvessel density, tumor cell proliferation and increased
the level of apoptosis in tumor cells. Moreover, administration with plasmid that coexpresses FAK and EGFR shRNA signicantly
inhibited the A549 experimental lung metastases. Collectively, our data suggest that the dual inhibition of FAK and EGFR by using
plasmid vector-based RNA interference might be a novel therapeutic approach to the treatment of human NSCLC.
Cancer Gene Therapy (2013) 20, 101108; doi:10.1038/cgt.2012.91; published online 18 January 2013
Keywords: focal adhesion kinase; epidermal growth factor receptor; RNA interference; non-small-cell lung cancer
cationic liposome

INTRODUCTION
Lung cancer has emerged as a leading cause of cancer-related
mortality in the world.1,2 Lung cancers are divided into small-cell
lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) based
on their histology and cellular origin. It has been reported that
NSCLC constitutes about 8085% of all lung cancers and is the
most common cause of death in men and second only to breast
cancer in woman.3 The majority of NSCLC patients are diagnosed
with late-stage disease and most of those diagnosed with earlystage disease will develop recurrence, experiencing metastatic
disease.4 The median survival of a patient with advanced or
metastasis NSCLC is approximately 68 months and chemotherapy provides only minimal gains in overall survival, resulting in a
dismal 5-year survival rate of o15%.2 Current therapies are
ineffective, thus new approaches are needed to improve the
therapeutic ratio.
Metastasis is a multistep process in which primary tumor cells
are re-established at distant organ sites and develop into
secondary tumors. It accounts for the majority of death among
patients with solid tumors.5 The process of metastasis requires a
disseminating cancer cell to survive an environment that actively
promotes apoptosis. Thus, for a cancer cell to metastasize
effectively, it must possess survival signals that suppress
apoptosis. One survival signal that has been shown to modulate
apoptotic signaling is focal adhesion kinase (FAK).6 FAK, a nonreceptor protein tyrosine kinase, localizes to regions of the cell
that attach to the extracellular matrix, the focal adhesions. FAK
activity is regulated by extracellular matrix receptors and integrins,
and implicated in several cellular processes, such as proliferation,
apoptosis, motility and invasion.7 FAK is overexpressed and/or
constitutively activated in various malignant tumors, including

tumors derived from breast, ovary, head and neck.810 It has also
been reported that FAK was overexpressed in NSCLC tissues
compared with its surrounding non-neoplastic tissue.11 Moreover,
recent report has demonstrated that downregulation of FAK using
small interfering RNA (siRNA) stimulates the apoptosis of cultured
NSCLC A549 cells.12
The epidermal growth factor receptor (EGFR) is a member of a
family of growth factor receptor tyrosine kinases, which includes
EGFR (ErbB-1), HER2/neu (ErbB-2), HER3 (ErbB-3) and HER4
(ErbB-4).13 Among the four kinds of subtype, EGFR is expressed
in a majority of NSCLC.14,15 It has been a promising target for the
development of therapeutic agents. However, clinical trial ndings
and research have revealed that targeted EGFR monotherapy
confronts the complexity of alternative survival pathways, which
may overcome targeted inhibition in cancer cells.16 Recent reports
have suggested that FAK serves to integrate EGFR signals upon
EGF induction, promoting tumor cell motility and invasion.17,18
Loss of FAK from the focal adhesions can inhibit EGFR signaling at
the cell membrane and transmit a proapoptotic signal.19
Moreover, inhibition of both FAK and EGFR signaling pathways
cooperatively induces death receptor-mediated apoptosis in
cultured human breast tumor cells.20 Based on these
observations, we therefore hypothesized that the dual inhibition
of FAK and EGFR signaling pathways might be more benecial in
the treatment of NSCLC.
RNA interference (RNAi) is a specic way of post-transcriptional
gene silencing by a specic double-stranded RNA molecules,
which is homologous to the sequence of the target gene. RNAi
technology is now widely used as a potential therapeutic strategy
because of its high efcacy and specicity in downregulating
gene expression.21 Compared with chemically or enzymatically

1
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, Peoples Republic of China and 2Laboratory Animal Center, Sichuan University,
Chengdu, Sichuan, Peoples Republic of China. Correspondence: Professor H Deng, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Keyuan Road 4,
No. 1, Gaopeng Street, Chengdu, Sichuan 610041, Peoples Republic of China.
E-mail: denghongx@scu.edu.cn
3
These authors contributed equally to this work.
Received 4 September 2012; revised 29 November 2012; accepted 10 December 2012; published online 18 January 2013

Targeting FAK and EGFR for treatment of lung cancer

C Li et al

102
synthesized small siRNA, plasmid vector-mediated shRNA
expression system is less expensive, more easy and efcient in
suppressing the expression of targeted genes, further expanding
the utility of RNAi.22 In this study, we developed a bicistronic
plasmid vector-expressing shRNA directed against both FAK and
EGFR under the control of a human U6 promoter. We show that
RNAi driven by these shRNA-based plasmid constructs can
effectively silence FAK and EGFR gene expression at the protein
level. Functional analyses revealed that the abrogation of both
FAK and EGFR expression strongly inhibited proliferation and
induced apoptotic cell death in vitro. Furthermore, administrating
with these shRNA-based plasmids encapsulated by 1,2bis(oleoyloxy)-(trimethylammonio) propane-cholesterol (DOTAPchol) cationic liposome via tail vein signicantly inhibited the
growth of established subcutaneous A549 xenografts and
experimental lung metastasis.
MATERIALS AND METHODS
Vector construction
The pGensil-2/U6 parental vector (Genesil Biotechnology Company,
Wuhan, China) was used for DNA vector-based RNAi synthesis. Small
interfering oligonucleotide specic for FAK (50 -GATCCGCCACCTGGGCCAG
TATTATTTCAAGACGATAATACTGGCCCAGGTGGTTTTTTGAATTCA-30 ) and for
EGFR (50 -GATCCGCACAGTGGAGCGAATTCCTTTCAAGACGAGGAATTCGCTC
CACTGTGTTTTTTGTCGACA-30 ) were synthesized and annealed.23,24 An FAKEGFR RNAi plasmid vector that expresses shRNA for both FAK and EGFR
under the control of a human U6 promoter was constructed by inserting
pairs of the annealed DNA oligonucleotide specic for FAK at the HindIII
site and EGFR at BamHI site sequentially into the pGensil-2/U6 vector
(p-shFE). Also, shRNA expression vectors for FAK (p-shFAK) and EGFR
(p-shEGFR) singly were constructed. Oligonucleotide sequences of HK-shRNA,
which have no homology with any of the mammalian sequence, were
designed as negative control (p-shHK). All of the constructs were veried by
DNA sequencing. Plasmids were extracted using EndoFree Plasmid Giga kits
(Qiagen GmbH, Hilden, Germany) from DH5a Escherichia coli transformants
and stored at  20 1C before use. The concentration was determined by
measuring A260/A280 ratio using ultraviolet spectrophotometry.

Cell culture and transfection

blocked with 0.5% non-fat milk for 1 h at room temperature, membranes

were incubated with mouse anti-FAK (1:200 dilution; Santa Cruz
Biotechnology, Santa Cruz, CA, USA) or mouse anti-EGFR (1:1000 dilution;
Cell Signaling Technology, Beverly, MA, USA) antibody in TBS/T
(Tris-buffered saline and Tween-20) overnight at 4 1C. The blots were
labeled with horseradish peroxidase-conjugated anti-mouse secondary
antibody, and visualized with enhanced chemiluminescence detection
system (Pierce Biotech, Rockford, IL, USA). Equal loading was normalized by
glyceraldehyde 3-phosphate dehydrogenase.

Assessment of apoptosis in vitro

Cell apoptosis was analyzed by Hoechst 33258 staining (Apoptosis-Hoechst
Staining Kit; Beyotime Biotechnology, Haimen, China) 72 h after transfection. Briey, cells were immersed in 0.5 ml of methanol for 15 min, followed
by rinsing twice with PBS. Then, cells were stained with 1 mg ml  1 Hoechst
33258 compounds in a dark chamber at room temperature for 10 min
and again rinsed twice with PBS. Cells were analyzed by uorescence
microscopy using excitation and emission wavelengths of 350 and 460 nm,
respectively. The apoptotic cells are featured as pyknotic and fragmented
nuclei emitting intense uorescence.
Quantitative evaluation of cellular apoptosis was performed by owcytometric analysis. Briey, both of the oated cells and the attached
cells were collected 72 h after transfection and washed twice with cold
PBS. Cells were stained with 50 mg ml  1 propidium iodide plus 0.1% Triton
X-100, and collected on a ow cytometer (Becton Dickinson, San Jose, CA,
USA). Data were analyzed using the BD FACSDiva software (BD Biosciences,
Franklin Lakes, NJ, USA).

Cationic liposome and preparation of plasmid/lipoplexes

Cationic liposome was prepared as multilamellar vesicles for in vivo use as
described previously.25 Briey, DOTAP from Avanti Polar Lipids (Alabaster,
Shelby County, AL, USA) and cholesterol (Sigma) were mixed in a 1:1 molar
ratio, dried down in round-bottomed tubes, and then rehydrated in 5%
glucose solution by heating at 50 1C for 6 h. For in vivo injection, plasmid/
lipoplexes were prepared immediately before injection by gently mixing
cationic lipids with plasmid DNA at a ratio of 25 mg total cationic liposome
to 5 mg plasmid DNA, to a nal concentration of 25 mg plasmid DNA per ml
in a sterile solution of 5% glucose in water.

In vivo transfection efciency in subcutaneous tumors

The human lung adenocarcinoma cell line, A549 cell line, was obtained
from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells
were cultured in RPMI-1640 medium supplemented with 10% heatinactivated fetal bovine serum, 20 mM L-glutamine, 100 U ml  1 of penicillin
G sodium and 100 mg ml  1 of streptomycin. Cells were maintained in a
humidied atmosphere containing 5% CO2 at 37 1C. Cell transfection was
carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA)
according to the manufacturers construction. At 72 h after transfection,
cells were used for cell proliferation assays, immunoblot analysis, ow
cytometry analysis and Hoechst 33258 staining.

To determine the transfection efciency of the complexes in NSCLC in vivo,

nude mice were injected with A549 tumor cells (2  106/100 ml)
subcutaneously on the right ank. When the tumors reached
200300 mm2 in size, a single dose of DOTAP-chol/DNA complex (5 mg of
p-EGFP (enhanced green orescent protein)-N1; BD Biosciences Clontech,
Palo Alto, CA, USA) was injected intravenously. At 48 h after injection, mice
were euthanized and tumors were removed, frozen in OCT compound and
sectioned on a freezing microtome (Leica, Glattbrugg, Switzerland) at
10 mm. Green uorescence was visualized and photographed directly
under a uorescence microscope.

Cell proliferation assay

Tumor growth and treatments in vivo

Viability of cells 72 h after transfection was evaluated using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma,
St Louis, MO, USA). MTT was added to the culture medium in each well of a
96-well plate at a concentration of 500 mg ml  1, and the plates were
incubated for 4 h at 37 1C. Acidic isopropyl alcohol (0.04 N HCl/isopropyl
alcohol) was immediately added to all wells and mixed vigorously so that
the dark blue crystals dissolved effectively. Absorbance was measured at
570 nm.

Western blot
A549 cells were harvested and rinsed twice with phosphate-buffered saline
(PBS) 72 h after transfection. Cell extracts were prepared with RIPA lysis
buffer (50 mM Tris, 150 mM NaCl, 1% Triton, 0.5% deoxycholate, 2 mM
ethylenediaminetetraacetic acid) supplemented with 100  cocktail
(Sigma) and cleared by centrifugation at 12 000 g at 4 1C for 30 min. Total
protein concentration was measured with the BCA assay. Cell extracts that
contained equal total protein were subjected to 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were
transferred to polyvinylideneuoride membranes (Invitrogen). After being
Cancer Gene Therapy (2013), 101 108

In A549 xenograft model, 2  106 A549 cells were injected subcutaneously

into the right ank regions of athymic nude (nu/nu) mice (68 weeks).
After a week, when the tumors could be palpable, the animals were
randomly assigned to ve independent groups of ve mice and subjected
to systemic treatment with one of the following treatments: 5% glucose,
p-shHK/lipoplexes, p-shFAK/lipoplexes, p-shEGFR/lipoplexes and p-shFE/
lipoplexes. Plasmids and DOTAP-chol complex in 200 ml of 5% glucose
were injected into the mouse tail vein three times a week (on Monday,
Wednesday and Friday). Tumor diameters were measured once every
3 days during the treatment period. Tumor volume was estimated using
the formula: tumor volume (mm3) length (mm)  (width (mm))2  1/2.
The weight, appetite and behavior of the mice were observed. Mice
were euthanized after 12 times of treatment and dissected tumors were
weighed. The tumors and viscera were examined grossly and microscopically stained with hematoxylin and eosin (H&E).
An experimental A549 lung metastasis model was used to study the
effects of FAK-EGFR shRNAs on the development of metastases. Briey,
athymic nude (nu/nu) mice (68 weeks) were inoculated with A549 cells
(2  106) in 200 ml PBS via tail vein injection. Pulmonary experimental
metastatic tumor colonies were formed 710 days after inoculation. Then,
& 2013 Nature America, Inc.

Targeting FAK and EGFR for treatment of lung cancer

C Li et al

102
synthesized small siRNA, plasmid vector-mediated shRNA
expression system is less expensive, more easy and efcient in
suppressing the expression of targeted genes, further expanding
the utility of RNAi.22 In this study, we developed a bicistronic
plasmid vector-expressing shRNA directed against both FAK and
EGFR under the control of a human U6 promoter. We show that
RNAi driven by these shRNA-based plasmid constructs can
effectively silence FAK and EGFR gene expression at the protein
level. Functional analyses revealed that the abrogation of both
FAK and EGFR expression strongly inhibited proliferation and
induced apoptotic cell death in vitro. Furthermore, administrating
with these shRNA-based plasmids encapsulated by 1,2bis(oleoyloxy)-(trimethylammonio) propane-cholesterol (DOTAPchol) cationic liposome via tail vein signicantly inhibited the
growth of established subcutaneous A549 xenografts and
experimental lung metastasis.
MATERIALS AND METHODS
Vector construction
The pGensil-2/U6 parental vector (Genesil Biotechnology Company,
Wuhan, China) was used for DNA vector-based RNAi synthesis. Small
interfering oligonucleotide specic for FAK (50 -GATCCGCCACCTGGGCCAG
TATTATTTCAAGACGATAATACTGGCCCAGGTGGTTTTTTGAATTCA-30 ) and for
EGFR (50 -GATCCGCACAGTGGAGCGAATTCCTTTCAAGACGAGGAATTCGCTC
CACTGTGTTTTTTGTCGACA-30 ) were synthesized and annealed.23,24 An FAKEGFR RNAi plasmid vector that expresses shRNA for both FAK and EGFR
under the control of a human U6 promoter was constructed by inserting
pairs of the annealed DNA oligonucleotide specic for FAK at the HindIII
site and EGFR at BamHI site sequentially into the pGensil-2/U6 vector
(p-shFE). Also, shRNA expression vectors for FAK (p-shFAK) and EGFR
(p-shEGFR) singly were constructed. Oligonucleotide sequences of HK-shRNA,
which have no homology with any of the mammalian sequence, were
designed as negative control (p-shHK). All of the constructs were veried by
DNA sequencing. Plasmids were extracted using EndoFree Plasmid Giga kits
(Qiagen GmbH, Hilden, Germany) from DH5a Escherichia coli transformants
and stored at  20 1C before use. The concentration was determined by
measuring A260/A280 ratio using ultraviolet spectrophotometry.

Cell culture and transfection

blocked with 0.5% non-fat milk for 1 h at room temperature, membranes

were incubated with mouse anti-FAK (1:200 dilution; Santa Cruz
Biotechnology, Santa Cruz, CA, USA) or mouse anti-EGFR (1:1000 dilution;
Cell Signaling Technology, Beverly, MA, USA) antibody in TBS/T
(Tris-buffered saline and Tween-20) overnight at 4 1C. The blots were
labeled with horseradish peroxidase-conjugated anti-mouse secondary
antibody, and visualized with enhanced chemiluminescence detection
system (Pierce Biotech, Rockford, IL, USA). Equal loading was normalized by
glyceraldehyde 3-phosphate dehydrogenase.

Assessment of apoptosis in vitro

Cell apoptosis was analyzed by Hoechst 33258 staining (Apoptosis-Hoechst
Staining Kit; Beyotime Biotechnology, Haimen, China) 72 h after transfection. Briey, cells were immersed in 0.5 ml of methanol for 15 min, followed
by rinsing twice with PBS. Then, cells were stained with 1 mg ml  1 Hoechst
33258 compounds in a dark chamber at room temperature for 10 min
and again rinsed twice with PBS. Cells were analyzed by uorescence
microscopy using excitation and emission wavelengths of 350 and 460 nm,
respectively. The apoptotic cells are featured as pyknotic and fragmented
nuclei emitting intense uorescence.
Quantitative evaluation of cellular apoptosis was performed by owcytometric analysis. Briey, both of the oated cells and the attached
cells were collected 72 h after transfection and washed twice with cold
PBS. Cells were stained with 50 mg ml  1 propidium iodide plus 0.1% Triton
X-100, and collected on a ow cytometer (Becton Dickinson, San Jose, CA,
USA). Data were analyzed using the BD FACSDiva software (BD Biosciences,
Franklin Lakes, NJ, USA).

Cationic liposome and preparation of plasmid/lipoplexes

Cationic liposome was prepared as multilamellar vesicles for in vivo use as
described previously.25 Briey, DOTAP from Avanti Polar Lipids (Alabaster,
Shelby County, AL, USA) and cholesterol (Sigma) were mixed in a 1:1 molar
ratio, dried down in round-bottomed tubes, and then rehydrated in 5%
glucose solution by heating at 50 1C for 6 h. For in vivo injection, plasmid/
lipoplexes were prepared immediately before injection by gently mixing
cationic lipids with plasmid DNA at a ratio of 25 mg total cationic liposome
to 5 mg plasmid DNA, to a nal concentration of 25 mg plasmid DNA per ml
in a sterile solution of 5% glucose in water.

In vivo transfection efciency in subcutaneous tumors

The human lung adenocarcinoma cell line, A549 cell line, was obtained
from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells
were cultured in RPMI-1640 medium supplemented with 10% heatinactivated fetal bovine serum, 20 mM L-glutamine, 100 U ml  1 of penicillin
G sodium and 100 mg ml  1 of streptomycin. Cells were maintained in a
humidied atmosphere containing 5% CO2 at 37 1C. Cell transfection was
carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA)
according to the manufacturers construction. At 72 h after transfection,
cells were used for cell proliferation assays, immunoblot analysis, ow
cytometry analysis and Hoechst 33258 staining.

To determine the transfection efciency of the complexes in NSCLC in vivo,

nude mice were injected with A549 tumor cells (2  106/100 ml)
subcutaneously on the right ank. When the tumors reached
200300 mm2 in size, a single dose of DOTAP-chol/DNA complex (5 mg of
p-EGFP (enhanced green orescent protein)-N1; BD Biosciences Clontech,
Palo Alto, CA, USA) was injected intravenously. At 48 h after injection, mice
were euthanized and tumors were removed, frozen in OCT compound and
sectioned on a freezing microtome (Leica, Glattbrugg, Switzerland) at
10 mm. Green uorescence was visualized and photographed directly
under a uorescence microscope.

Cell proliferation assay

Tumor growth and treatments in vivo

Viability of cells 72 h after transfection was evaluated using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma,
St Louis, MO, USA). MTT was added to the culture medium in each well of a
96-well plate at a concentration of 500 mg ml  1, and the plates were
incubated for 4 h at 37 1C. Acidic isopropyl alcohol (0.04 N HCl/isopropyl
alcohol) was immediately added to all wells and mixed vigorously so that
the dark blue crystals dissolved effectively. Absorbance was measured at
570 nm.

Western blot
A549 cells were harvested and rinsed twice with phosphate-buffered saline
(PBS) 72 h after transfection. Cell extracts were prepared with RIPA lysis
buffer (50 mM Tris, 150 mM NaCl, 1% Triton, 0.5% deoxycholate, 2 mM
ethylenediaminetetraacetic acid) supplemented with 100  cocktail
(Sigma) and cleared by centrifugation at 12 000 g at 4 1C for 30 min. Total
protein concentration was measured with the BCA assay. Cell extracts that
contained equal total protein were subjected to 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were
transferred to polyvinylideneuoride membranes (Invitrogen). After being
Cancer Gene Therapy (2013), 101 108

In A549 xenograft model, 2  106 A549 cells were injected subcutaneously

into the right ank regions of athymic nude (nu/nu) mice (68 weeks).
After a week, when the tumors could be palpable, the animals were
randomly assigned to ve independent groups of ve mice and subjected
to systemic treatment with one of the following treatments: 5% glucose,
p-shHK/lipoplexes, p-shFAK/lipoplexes, p-shEGFR/lipoplexes and p-shFE/
lipoplexes. Plasmids and DOTAP-chol complex in 200 ml of 5% glucose
were injected into the mouse tail vein three times a week (on Monday,
Wednesday and Friday). Tumor diameters were measured once every
3 days during the treatment period. Tumor volume was estimated using
the formula: tumor volume (mm3) length (mm)  (width (mm))2  1/2.
The weight, appetite and behavior of the mice were observed. Mice
were euthanized after 12 times of treatment and dissected tumors were
weighed. The tumors and viscera were examined grossly and microscopically stained with hematoxylin and eosin (H&E).
An experimental A549 lung metastasis model was used to study the
effects of FAK-EGFR shRNAs on the development of metastases. Briey,
athymic nude (nu/nu) mice (68 weeks) were inoculated with A549 cells
(2  106) in 200 ml PBS via tail vein injection. Pulmonary experimental
metastatic tumor colonies were formed 710 days after inoculation. Then,
& 2013 Nature America, Inc.

Targeting FAK and EGFR for treatment of lung cancer

C Li et al

103
p-shFE/lipoplexes, p-shHK/lipoplexes or 5% glucose were administered
systemically to animals by intravenous injection for three times within a
week, respectively. At 2 weeks after the last injection, the animals were
euthanized, and their lung tissues were excised. Tumor colonies on lung
surfaces were counted under a dissecting microscope without the
knowledge of the treatment groups, and the lung tissues were sectioned
for further pathological and immunohistochemical analysis.
Animal studies were performed in accordance with the Institutional
Animal Care and Treatment Committee of Sichuan University (Chengdu,
Peoples Republic of China).

Immunostaining
Expression of FAK, EGFR, proliferating cell nuclear antigen (PCNA) and
CD31 was performed with mouse anti-human FAK antibody (Santa Cruz
Biotechnology), mouse anti-human EGFR antibody (Cell Signaling Technology), mouse anti-human PCNA antibody (Santa Cruz Biotechnology) and
goat anti-mouse CD31 antibody (Santa Cruz Biotechnology), respectively.
Tumor sections (35 mm) of parafn-embedded were mounted on
3-aminopropyltriethoxysilane (APES)-coated glass slides. Sections were
deparafnized in xylene, treated with a graded series of alcohol (100, 95
and 80% ethanol/double-distilled H2O (v v  1), and rehydrated in PBS
(pH 7.4). Antigen retrieval was carried out by heating for 5 min in a pressure
cooker with 0.1 mol l  1 citrate buffer (pH 6.0). Endogenous peroxide was
blocked with 3% H2O2 in methanol for 5 min. After PBS washes, slides were
blocked with 5% normal goat or rabbit serum in PBS for 15 min at room
temperature followed by incubation with primary mouse anti-FAK (1:100),
anti-EGFR (1:50), anti-PCNA (1:100) or anti-CD31(1:100) antibody in blocking
solution overnight at 4 1C. All slides were subsequently incubated with a 1:
200 dilution of biotin-conjugated goat anti-mouse or rabbit anti-goat
secondary antibody for 40 min at 37 1C and streptavidinbiotin complex at
37 1C for 40 min. The immunoreaction was visualized by using diaminobenzidine peroxide solution and cellular nuclei were counterstained with
hematoxylin. All specimens were evaluated using Olympus BX600
microscope and Spot Fiex camera. Control samples exposed to secondary
antibody alone showed no specic staining.
To quantify mean vessel density (MVD), 10 random 0.159-mm2 elds at
 100 nal magnication were examined for each tumor (one slide per
mouse, eight slides per group) and the number of microvessels per eld
was counted by two investigators in a blinded manner. A single
microvessel was dened as a discrete cluster or a single cell stained
positive for CD31, and the presence of a lumen was required for scoring as
a microvessel. To quantify proliferation index, the percentage of PCNApositive cells was counted in 10 random 0.159-mm2 elds at  100
magnication.
To detect apoptotic cells in tumor tissues, terminal deoxynucleotidyl
transferase dUTP nick-end labeling (TUNEL) assay using a DeadEnd
Fluorometric TUNEL System (Promega, Madison, WI, USA) was performed
following the manufacturers protocol. Cell nuclei with dark green
uorescent staining were dened as TUNEL-positive nuclei. TUNEL-positive
nuclei were monitored by uorescence microscope. To quantify TUNELpositive cells, the number of green uorescence-positive cells was counted
in 10 random 0.011-mm2 elds at  200 magnication.

Statistical analysis
Data are expressed as the means.d. Statistical analysis was performed by
Students t-test for comparing two groups and by analysis of variance for
multiple group comparisons. Po0.05 was considered statistically signicant. The Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA)
was used for all statistical analyses.

RESULTS
Effect of FAK-EGFR silencing in vitro
To examine whether the plasmid encoding FAK-EGFR shRNAs
could simultaneously inhibit FAK and EGFR expression in A549
lung cancer cells by transient transfection, A549 lung cancer cells
were transfected with p-shFAK, p-shEGFR, p-shFE or control
p-shHK using Lipofectamine 2000. As shown in Figure 1, analysis
of the shRNA-transfected cells for FAK and EGFR expression via
western blot demonstrated a specic reduction in protein levels
for each gene relative to the p-shHK-transfected cells or mock
cells. However, the RNAi effect was slightly more with the shRNA
& 2013 Nature America, Inc.

Figure 1. In vitro silencing of focal adhesion kinase (FAK) and

epidermal growth factor receptor (EGFR) expression. Western blot
analysis of FAK and EGFR after transfection of A549 cells with
p-shFAK, p-shEGFR, p-shFE or control p-shHK.

vector simultaneously targeting FAK and EGFR. No effects of RNAi

were observed on the expression of GAPDH, which was used as an
internal control for specicity and loading at protein levels. In
addition, p-shHK-transfected cells also showed that RNAi-directed
FAK and EGFR knockdown is specic.
Knockdown of FAK and EGFR expression inhibits cell proliferation
and induces apoptosis
To access the potential effects of RNAi-mediated FAK and EGFR
silencing on cell proliferation and survival, MTT analysis was
performed 72 h after transfection with shRNA specic to FAK and
EGFR. RNAi-targeting against FAK or EGFR had a relatively low
inhibitory effect. In contrast, a dramatic reduction in proliferation
was observed with RNAi simultaneously targeting FAK and EGFR
(Figure 2a). Hoechst 33258 staining resulted in morphological
changes in p-shFAK-, p-shEGFR- or p-shFE-tranfected cells
(Figure 2b: brightly blue-uorescent condensed nuclei (intact or
fragmented), decreased cell volume, blebbing and condensation
of nuclear chromatin, nuclear fragmentation and apoptotic
bodies). However, these changes were less prominent in
p-shHK-transfected or control cells. In addition, the sub-G1
population estimated from the number of apoptotic cells by ow
cytometry showed that apoptotic cells increased in p-shFAK(23.3%) and p-shEGFR-transfected (32.0%) cells compared with
p-shHK-transfected cells (11.3%). However, treatment with p-shFE
dramatically increased the percentage of apoptotic cells in
contrast with p-shFAK- or p-shEGFR-treated cells (42.1 versus
23.3%, 42.1 versus 32.0%) (Figure 2c).
In vivo transfection efciency of plasmid DNA/liposome complexes
Before the in vivo treatment, we examined the biodistribution of
p-shFE/lipoplexes in vivo. A plasmid encoding EGFP was used to
further determine the ability of extruded DOTAP-chol liposome to
effectively transfect and deliver plasmid DNA into subcutaneous
A549 tumor xenografts. At 48 h following a single tail vein
injection of EGFP-N1, plasmid DNA encapsulated in DOTAP-chol
liposomes. Green orescence was observed in A549 tumor
sections. In contrast, no green orescence was observed in
control group treated with 5% glucose (Figure 3). And the
approximate transfection efciency in tumor xenografts was 30
40%.
Knockdown of FAK and EGFR suppresses A549 tumor growth and
metastasis in vivo
By having established the signicant effects of FAK-EGFR knockdown by RNAi in A549 cells in vitro and determined the in vivo
efciency of DOTAP-chol delivery system, we then asked whether
FAK-EGFR shRNA would also suppress the growth and metastasis
of A549 tumor in nude mice. As shown in Figure 4a, although
p-shFAK/lipoplexes and p-shEGFR/lipoplexes signicantly inhibited the A549 tumor growth compared with control groups
(Po0.05), but the degree of inhibition was less than that in
p-shFE/lipoplexes-treated group (Po0.01). Tumor weight was also
Cancer Gene Therapy (2013), 101 108

Targeting FAK and EGFR for treatment of lung cancer

C Li et al

104

Figure 2. In vitro focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) silencing inhibits A549 cell proliferation and induces
apoptosis. (a) Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay 72 h after transfection.
(b) At 72 h after transfection, A549 cells in six wells were examined by fluorescent microscopy after Hoechst staining (  100).
(A) Untreated; (B) p-shHK; (C) p-shFAK; (D) p-shEGFR; and (E) p-shFE. (c) The percentage of apoptosis estimated from the sub-G1 cell fraction.
(A) Untreated; (B) p-shHK; (C) p-shFAK; (D) p-shEGFR; and (E) p-shFE. DNA fluorescence histograms of Hoechst-stained cells were representative
of three independent experiments.

Figure 3. Expression of green fluorescent protein in tumors. (Right)

Tumor cells with green fluorescence were present in the mice
injected with p-EGFP-N1/lipoplexes. (Left) No fluorescence was
found in tumors from mice treated with 5% glucose. Original
magnification,  400.

measured at the termination of the animal experiment

(Figure 4b). Mean tumor weight was 0.120.03, 0.140.03,
0.040.01, 0.210.07 and 0.220.06 g in p-shFAK/lipoplexes,
p-shEGFR/lipoplexes, p-shFE/lipoplexes, p-shHK/lipoplexes and
untreated group, respectively. Although p-shFAK/lipoplexes and
Cancer Gene Therapy (2013), 101 108

p-shEGFR/lipoplexes did not inhibit tumor growth completely, the

weights of the tumor from these treatment groups were smaller
than tumors from the control treatment groups (Po0.05). A
drastic reduction in tumor weight was observed in mice treated
with p-shFE/lipoplexes (Figure 4b; Po0.01). Immunohistochemical
analysis for protein levels in tumor samples conrmed that the
tumors treated with p-shFE/lipoplexes had signicantly decreased
FAK and EGFR levels (Figure 4c).
Next, an experimental A549 metastatic human lung cancer
model (established by intravenous injection of tumor cells) was
used to explore the potential of p-shFE/lipoplexes in suppressing
the development of lung metastases in nude mice. The development of A549 pulmonary metastases was inhibited signicantly,
and the numbers of metastatic tumor colonies found on the
surfaces of lungs from mice inoculated with A549 cells were
reduced by B60% in animals treated with p-shFE/lipoplexes
compared with those in control treatment groups (Figures 5a and
b). H&E staining also showed that lungs treated with p-shFE/
lipoplexes contained only a few small nodules; most of the lung
was free of tumor. In contrast, metastasis nodules occupied most
of the lung tissue from control groups (Figure 5c).
Statistical analysis revealed no signicant difference between
the p-shHK/lipoplexes group and the untreated group (P40.05),
& 2013 Nature America, Inc.

Targeting FAK and EGFR for treatment of lung cancer

C Li et al

105

Figure 4. In vivo antitumor efficacy of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) silencing in A549
subcutaneous model. (a) Tumor growth curve of A549 subcutaneous model. (b) Tumors weight of A549 subcutaneous model after the final
treatment in each group. (c) Immunostaining of FAK and EGFR in tumor tissue sections. Original magnification,  400; n 5; *Po0.01;
**Po0.01.

thereby suggesting that the injection of the control plasmids had

no obvious side effect on tumor growth.
Knockdown of FAK and EGFR inhibits cell proliferation,
angiogenesis and induced apoptosis in vivo
To determine the biological effects of treatment with FAK and
EGFR shRNA, A549 subcutaneous tumors harvested from mice
following therapy were subjected to imaging measurements for
cell proliferation (PCNA), apoptosis (TUNEL) and MVD (CD31).
Treatment with p-shFAK/lipoplexes or p-shEGFR/lipoplexes
resulted in a modest decrease in MVD compared with the 5%
GS control (Figure 6a; Po0.05), but the greatest decrease in MVD
was noted in the targeted FAK and EGFR groups (72.7% decrease;
Po0.01). There was a 37.5 and 33.3% reduction in the mean
number of PCNA-positive cells in p-shFAK/lipoplexes-treated and
p-shEGFR/lipoplexes compared with the control (Figure 6c;
Po0.05), respectively. The p-shFE/lipoplexes treatment group
produced the largest reduction in PCNA-positive cells (70.8%;
Po0.01). We further performed in situ TUNEL assay to evaluate the
inuence of oncogene knockdown on apoptosis of tumor cells.
We observed an elevated apoptosis rate in tumors of mice
injected with p-shFE/lipoplexes compared with that of tumors
& 2013 Nature America, Inc.

from mice injected with control group and 5% glucose (Figure 6c;
Po0.001). Thus, inhibition of tumor xenograft growth by blocking
the FAK and EGFR pathway might be due to inhibition of tumor
proliferation, neovascularization as well as induction of tumor
apoptosis.
Toxicity observation
The animal weight was measured twice in a week and there were
no signicant differences among the ve groups. No gross
abnormalities were noticed in p-shFAK/lipoplexes- or p-shEGFR/
lipoplexes- or p-shFE/lipoplexes-treated mice. In addition, there
were no signicant pathological alterations in the liver, lung,
kidney, spleen or heart as revealed by H&E histological staining.

DISCUSSION
Despite intensive efforts in the treatment of patients with NSCLC,
the 5-year relative survival rate has not improved substantially.26
Clinical outcomes have reached a plateau with conventional
methods. Based on advances in the knowledge of tumor biology
and mechanisms of oncogenesis, multiple specic molecular
targets for NSCLC treatment have been developed.27 Therefore,
Cancer Gene Therapy (2013), 101 108

Targeting FAK and EGFR for treatment of lung cancer

C Li et al

106

Figure 5. In vivo antitumor efficacy of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) silencing in A549 experimental
metastasis model. (a) Lung metastatic nodules of each group (n 5); (b) average number of lung metastasis, n 5, **Po0.01; (c)
representative images of hematoxylin and eosin (H&E) staining of the lungs from mice of different groups. Original magnification,  100.

Figure 6. In vivo, effects of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) silencing on cell proliferation,
angiogenesis and apoptosis in A549 subcutaneous model. (a) Inhibition of angiogenesis was indicated by CD31 immunostaining. Original
magnification,  200. (b) Tumor tissues were stained with PCNA. The number of cancer cell nuclei that were strongly proliferating cell nuclear
antigen (PCNA) positive was counted as a ratio of immunoreactive-positive cells to the total number of cells counted. Original magnification,
 200. (c) Induction of apoptosis was indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The TUNELpositive cells display dark green nuclei and are observed under a fluorescence microscope, and the percentage of apoptotic cells was
determined as described in the Materials and methods. Original magnification,  400. *Po0.05; **Po0.01; and ***Po0.001.

gene therapy techniques that target various related genes are

gaining popularity.
To date, two approaches to RNAi have been explored:
chemically or enzymatically synthesized siRNA and plasmid
vector-mediated RNAi. Chemically or enzymatically synthesized
siRNA is costly, easily contaminated by RNase and has a relatively
short half-life. However, plasmid vector-mediated shRNA may
express siRNA stably and has a lower immunogenicity in vivo.28
Cancer Gene Therapy (2013), 101 108

Recent studies have also demonstrated the potential of

simultaneous inhibition of two or three genes using a plasmidbased siRNA system. The advantage of using multicistronic
constructs over single constructs is that a single plasmid
molecule can produce multiple RNAi molecules specic to more
than one target, thereby enhancing the RNAi effect and resulting
in higher efciency.29,30 In this study, we used plasmid-based RNAi
to target FAK and/or EGFR genes, which are markedly
& 2013 Nature America, Inc.

Targeting FAK and EGFR for treatment of lung cancer

C Li et al

107
overexpressed in a wide variety of human cancers, including
NSCLC cancer, singly and simultaneously.31,32 Our data indicated
that knockdown of FAK and EGFR expression by RNAi quite
effectively downregulated FAK and EGFR protein expression in the
NSCLC cell line A549 (Figure 1). Although there is a little variation
in the degree of gene silencing among the constructs, all these
gene-specic RNAi plasmids reduced FAK and EGFR expression
substantially compared with mock- or p-shHK-transfected cells.
Moreover, we found that knockdown of both FAK and EGFR
expression cooperatively inhibited cell proliferation and induced
cell apoptosis of A549 lung cancer cells.
To demonstrate whether the observed inhibitory effects of FAK
and EGFR shRNAs on tumor cell growth in vitro could be
reproduced in vivo, we evaluated the efcacy of FAK-EGFR shRNAs
in suppressing tumor growth by injecting p-shFE vectors
encapsulated in DOTAP-chol cationic liposome via tail vein into
A549 tumor xenografts in nude mice. Growth of A549 tumor was
suppressed signicantly by treatments with p-shFE/lipoplexes.
Furthermore, we explored the tumor-suppressing potential of
FAK-EGFR shRNAs in inhibiting experimental metastases in vivo by
systemic administration of p-shFE/lipolexes. The development of
metastases was inhibited effectively by the p-shFE/lipolexesmediated transfer of the FAK-EGFR shRNAs. Similarly, this
combination therapy yielded a higher incidence of apoptosis of
tumor cells, resulting in lower values for MVD and PCNA staining
positive cells compared with the other treatment groups. These
in vivo data are consistent with the in vitro data, further supporting
the roles of FAK-EGFR shRNAs in the treatment of NSCLC.
Gene therapy is a potential strategy for curing cancer and other
diseases, but delivery of therapeutic genes to disseminated tumor
sites has been a major challenge owing to the lack of an efcient
vector delivery system. Although intravenous delivery of naked
plasmids has been applied to block tumor growth in xenograft
models owing to its benets of safety and simplicity, the effect
was limited probably due to the short half-life of the plasmids
in vivo. At present, sufcient evidences have proved that cationic
liposomes are the most extensively investigated non-viral vector
and have been successfully used to deliver genes, proteins,
oligonucleotides and antibiotics in several cell types.3335 Drugs
encapsulated in cationic liposomes have the ability of transporting
through large pores in the tumor vessel walls and are anticipated
to be transported without rapid degradation, which generally do
not exist in normal vessels and appear to be a result of the rapid
angiogenesis occurring in tumors, they make the encapsulated
drugs targetable and localize in tumor sites.36,37 In our lab, we
used DOTAP-chol liposome as the vector deliver system and
demonstrated that it gets the advantage of highly efcient in vitro
and in vivo and is the most characterized system.3840 The choice
of DOTAP-chol liposome mainly depended on several parameter,
such as biodegradation, biodistribution and toxicity.41 The validity
of lipid-based gene delivery is also supported by previous studies
using DOTAP-chol liposomes to deliver tumor suppressor genes
and inhibit tumorigenicity.42,43 Furthermore, DOTAP:chol-FUS1
complex has entered Phase I clinical trials against advanced lung
cancers.44 To avoid the dose-dependent toxicity of DNA/cationic
liposome complexes in vivo, we treated mice at a low dose
(intravenous 5 mg DNA/25 mg liposome per mouse).To ensure the
transfection efcacy, it was evaluated by injecting p-EGFP-N1/
DOTAP-cho liposome complexes intravenously in A549 xenografts
model before the treatment. Our results indicated that a low dose
of pDNA/lipoplexes can efciently deliver pDNA to A549 lung
tumor xenografts. Moreover, we did not nd any side effects in
animals by H&E histological staining.
In summary, our study shows that vector-based dual shRNA
expression system can efciently inhibit FAK and EGFR in vitro and
systematic delivery of p-shFE by cationic liposomes resulted in
reduction of FAK and EGFR expression, leading to decreased
tumor vascularity, proliferation and increased apoptosis in vivo.
& 2013 Nature America, Inc.

These results indicate that simultaneously targeting FAK and EGFR

with shRNAs may have therapeutic benet in the treatment
of NSCLC.

CONFLICT OF INTEREST
The authors declare no conict of interest.

ACKNOWLEDGEMENTS
This work was supported by The National Key Basic Research Program (973 Program)
of China (2012CB917104 and 2010CB529900).

REFERENCES
1 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T et al. Cancer statistics, 2008. CA
Cancer J Clin 2008; 58: 7196.
2 Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin
2005; 55: 74108.
3 Dempke WC, Suto T, Reck M. Targeted therapies for non-small cell lung cancer.
Lung cancer 2010; 67: 257274.
4 Novello S, Le Chevalier T. Chemotherapy for non-small-cell lung cancer. Part 1:
early-stage disease. Oncology 2003; 17: 357364.
5 Mazzocca A, Carloni V. The metastatic process: methodological advances and
pharmacological challenges. Curr Med Chem 2009; 16: 17041717.
6 Kurenova E, Xu LH, Yang X, Baldwin Jr. AS, Craven RJ, Hanks SK et al. Focal
adhesion kinase suppresses apoptosis by binding to the death domain of
receptor-interacting protein. Mol Cell Biol 2004; 24: 43614371.
7 McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC. The role
of focal-adhesion kinase in cancera new therapeutic opportunity. Nature
reviews. Cancer 2005; 5: 505515.
8 Kornberg LJ. Focal adhesion kinase and its potential involvement in tumor
invasion and metastasis. Head Neck 1998; 20: 745752.
9 Oktay MH, Oktay K, Hamele-Bena D, Buyuk A, Koss LG. Focal adhesion kinase as a
marker of malignant phenotype in breast and cervical carcinomas. Hum Pathol
2003; 34: 240245.
10 Sood AK, Cofn JE, Schneider GB, Fletcher MS, DeYoung BR, Gruman LM et al.
Biological signicance of focal adhesion kinase in ovarian cancer: role in migration and invasion. Am J Pathol 2004; 165: 10871095.
11 Carelli S, Zadra G, Vaira V, Falleni M, Bottiglieri L, Nosotti M et al. Up-regulation of
focal adhesion kinase in non-small cell lung cancer. Lung Cancer 2006; 53:
263271.
12 Liu G, Meng X, Jin Y, Bai J, Zhao Y, Cui X et al. Inhibitory role of focal
adhesion kinase on anoikis in the lung cancer cell A549. Cell Biol Int 2008; 32:
663670.
13 Yarden Y. The EGFR family and its ligands in human cancer: signalling mechanisms and therapeutic opportunities. Eur J Cancer 2001; 37: 38.
14 Brabender J, Danenberg KD, Metzger R, Schneider PM, Park J, Salonga D et al.
Epidermal growth factor receptor and HER2-neu mRNA expression in non-small
cell lung cancer Is correlated with survival. Clin Cancer Res 2001; 7: 18501855.
15 Nguyen DM, Schrump DS. Growth factor receptors as targets for lung cancer
therapy. Semin Thorac Cardiovasc Surg 2004; 16: 312.
16 Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor
mutations in lung cancer. Nat Rev Cancer 2007; 7: 169181.
17 Hauck CR, Sieg DJ, Hsia DA, Loftus JC, Gaarde WA, Monia BP et al. Inhibition of
focal adhesion kinase expression or activity disrupts epidermal growth factorstimulated signaling promoting the migration of invasive human carcinoma cells.
Cancer Res 2001; 61: 70797090.
18 Sieg DJ, Hauck CR, Ilic D, Klingbeil CK, Schaefer E, Damsky CH et al. FAK integrates
growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2000; 2:
249256.
19 Jones G, Machado Jr J, Merlo A. Loss of focal adhesion kinase (FAK) inhibits
epidermal growth factor receptor-dependent migration and induces aggregation
of nh(2)-terminal FAK in the nuclei of apoptotic glioblastoma cells. Cancer Res
2001; 61: 49784981.
20 Golubovskaya V, Beviglia L, Xu LH, Earp III HS, Craven R, Cance W. Dual inhibition
of focal adhesion kinase and epidermal growth factor receptor pathways cooperatively induces death receptor-mediated apoptosis in human breast cancer
cells. J Biol Chem 2002; 277: 3897838987.
21 Pai SI, Lin YY, Macaes B, Meneshian A, Hung CF, Wu TC. Prospects of RNA interference therapy for cancer. Gene Therapy 2006; 13: 464477.
22 Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA
interference. Nature 2004; 431: 371378.

Cancer Gene Therapy (2013), 101 108

Targeting FAK and EGFR for treatment of lung cancer

C Li et al

108
23 Halder J, Kamat AA, Landen J. CN, Han LY, Lutgendorf SK, Lin YG et al. Focal
adhesion kinase targeting using in vivo short interfering RNA delivery in neutral
liposomes for ovarian carcinoma therapy. Clinical Cancer Res 2006; 12: 49164924.
24 Smith K, Gunaratnam L, Morley M, Franovic A, Mekhail K, Lee S. Silencing of
epidermal growth factor receptor suppresses hypoxia-inducible factor-2-driven
VHL-/- renal cancer. Cancer Res 2005; 65: 52215230.
25 Du XB, Lang JY, Xu JR, Lu Y, Wen YJ, Zhao JM et al. Vesicular stomatitis virus matrix
protein gene enhances the antitumor effects of radiation via induction of
apoptosis. Apoptosis 2008; 13: 12051214.
26 Schiller JH, Harrington D, Belani CP, Langer C, Sandler A, Krook J et al. Comparison
of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J
Med 2002; 346: 9298.
27 Raben D, Helfrich B, Bunn Jr PA. Targeted therapies for non-small-cell lung cancer:
biology, rationale, and preclinical results from a radiation oncology perspective.
Int J Radiat Oncol Biol Phys 2004; 59(Suppl): 2738.
28 de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with
disease: a progress report on siRNA-based therapeuticss. Nat Rev Drug Discov
2007; 6: 443453.
29 Chen SM, Wang Y, Xiao BK, Tao ZZ. Effect of blocking VEGF, hTERT and Bcl-xl by
multiple shRNA expression vectors on the human laryngeal squamous carcinoma
xenograft in nude mice. Cancer Biol Ther 2008; 7: 734739.
30 Pulukuri SM, Gondi CS, Lakka SS, Jutla A, Estes N, Gujrati M et al. RNA interferencedirected knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and
tumorigenicity in vivo. J Biol Chem 2005; 280: 3652936540.
31 Carelli S, Zadra G, Vaira V, Falleni M, Bottiglieri L, Nosotti M et al. Up-regulation
of focal adhesion kinase in non-small cell lung cancer. Lung Cancer 2006; 53(3): 263271.
32 Rusch V, Klimstra D, Venkatraman E, Pisters PW, Langenfeld J, Dmitrovsky E.
Overexpression of the epidermal growth factor receptor and its ligand transforming growth factor alpha is frequent in resectable non-small cell lung cancer
but does not predict tumor progression. Clin Cancer Res 1997; 3: 515522.
33 Jiao X, Wang RY, Qiu Q, Alter HJ, Shih JW. Enhanced hepatitis C virus NS3 specic
Th1 immune responses induced by co-delivery of protein antigen and CpG with
cationic liposomes. J Gen Virol 2004; 85(Part 6): 15451553.

Cancer Gene Therapy (2013), 101 108

34 Sangare L, Morisset R, Omri A, Ravaoarinoro M. Incorporation rates, stabilities,

cytotoxicities and release of liposomal tetracycline and doxycycline in human
serum. J Antimicrob Chemother 1998; 42: 831834.
35 Zelphati O, Szoka Jr FC. Mechanism of oligonucleotide release from cationic
liposomes. Proc Natl Acad Sci USA 1996; 93: 1149311498.
36 Webb MS, Harasym TO, Masin D, Bally MB, Mayer LD. Sphingomyelincholesterol liposomes signicantly enhance the pharmacokinetic and therapeutic
properties of vincristine in murine and human tumour models. Br J Cancer 1995;
72: 896904.
37 Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, Torchilin VP et al. Vascular
permeability in a human tumor xenograft: molecular size dependence and cutoff
size. Cancer Res 1995; 55: 37523756.
38 Simberg D, Weisman S, Talmon Y, Barenholz Y. DOTAP (and other cationic lipids):
chemistry, biophysics, and transfection. Crit Rev Ther Drug Carrier Syst 2004; 21:
257317.
39 Yang Y, Bai Y, Xie G, Zhang N, Ma YP, Chen LJ et al. Efcient inhibition of nonsmall-cell lung cancer xenograft by systemic delivery of plasmid-encoding shorthairpin RNA targeting VEGF. Cancer Biother Radiopharm 2010; 25: 6573.
40 Wan Y, Huang A, Yang Y, Xie G, Chen X, Hu J et al. A vector-based short
hairpin RNA targeting Aurora A inhibits breast cancer growth. Int J Oncol 2010; 36:
11211128.
41 Templeton NS, Lasic DD, Frederik PM, Strey HH, Roberts DD, Pavlakis GN.
Improved DNA: liposome complexes for increased systemic delivery and gene
expression. Nat Biotechnol 1997; 15: 647652.
42 Christensen CL, Gjetting T, Poulsen TT, Cramer F, Roth JA, Poulsen HS. Targeted
cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy
induces small cell lung cancer-specic cytotoxicity and tumor growth delay.
Clinical Cancer Res 2010; 16: 23082319.
43 Lu C, Stewart D, Ji L, Ramesh R, Jayachandran G, Erasmus J et al. Systemic gene
therapy with tumor suppressor FUS1-nanoparticles for recurrent/metastatic lung
cancer. J Clin Oncol 2010; 28: 7582.
44 Lu C, Stewart DJ, Lee JJ, Ji L, Ramesh R, Jayachandran G et al. Phase I clinical trial
of systemically administered TUSC2(FUS1)-nanoparticles mediating functional
gene transfer in humans. PLoS one 2012; 7: e34833.

& 2013 Nature America, Inc.