ets2 mediated tumor suppressive function and …...identify aberrant signaling pathways and...

Human Cancer Biology

ETS2 Mediated Tumor Suppressive Function and METOncogene Inhibition in Human Non–Small Cell Lung Cancer

Mohamed Kabbout1, Melinda M. Garcia1, Junya Fujimoto1, Diane D. Liu4, Denise Woods1, Chi-Wan Chow1,Gabriela Mendoza1, Amin A. Momin5, Brian P. James6, Luisa Solis3, Carmen Behrens1, J. Jack Lee4,Ignacio I. Wistuba1,2, and Humam Kadara1

AbstractPurpose: The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer.

We analyzed the transcriptomeof lung adenocarcinomas andnormal lung tissue by expressionprofiling and

found that ETS2 was significantly downregulated in adenocarcinomas. In this study, we probed the yet

unknown functional role of ETS2 in lung cancer pathogenesis.

Experimental Design: Lung adenocarcinomas (n¼ 80) and normal lung tissues (n¼ 30) were profiled

using the AffymetrixHumanGene 1.0 ST platform. Immunohistochemical (IHC) analysis was conducted to

determine ETS2 protein expression in non–small cell lung cancer (NSCLC) histologic tissue specimens (n¼201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank

and Kaplan–Meier tests. RNA interference and overexpression strategies were used to assess the effects of

ETS2 expression on the transcriptome and on various malignant phenotypes.

Results: ETS2 expression was significantly reduced in lung adenocarcinomas compared with normal

lung (P < 0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in

NSCLC (P ¼ 0.009, HR ¼ 1.89) and adenocarcinoma (P ¼ 0.03, HR ¼ 1.86). Moreover, ETS2 was found

to significantly inhibit lung cancer cell growth, migration, and invasion (P < 0.05), and microarray and

pathways analysis revealed significant (P < 0.001) activation of the HGF pathway following ETS2

knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET

expression significantly attenuated (P < 0.05) cell invasion mediated by ETS2-specific siRNA. Further-

more, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration, and

invasion.

Conclusion(s):Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis

through inhibition of the MET proto-oncogene. Clin Cancer Res; 19(13); 3383–95. �2013 AACR.

IntroductionLung cancer remains the leading cause of cancer-related

deaths in the United States and worldwide (1). Non–small cell lung cancer (NSCLC) represents the majority of

diagnosed lung cancer cases and is associated with arelatively poor 15% overall 5-year survival rate (2).Understanding the molecular profiles of NSCLC as wellas elucidating the roles of oncogenes and tumor suppres-sors in the development of this malignancy is expected toidentify aberrant signaling pathways and molecular tar-gets for therapy.

V-ets erythroblastosis virus E26 oncogene homolog 2(ETS2) belongs to the ETS family of transcription factorsand controls gene expression by binding to numerousgenes with GGA(A/T) ETS response elements (ERE)thus impacting a broad spectrum of cellular functionsincluding proliferation, differentiation, migration, trans-formation, and apoptosis (3–5). During embryonicdevelopment, ETS2 was shown to insure proper devel-opment of the trophoblast (6) and to regulate endothelialcell survival during embryonic angiogenesis (7). More-over, ETS2 was reported to be activated in response toextracellular mitogenic signaling mediated by the Rasoncogene (8) in murine fibroblasts, to maintain telome-rase gene expression in breast cancer cells (9) and,

Authors' Affiliations: Departments of 1Thoracic/Head and Neck MedicalOncology, 2Translational Molecular Pathology, 3Pathology, 4Biostatistics,5Bioinformatics, and 6Experimental Therapeutics, The University of TexasMD Anderson Cancer Center, Houston, Texas

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

I.I. Wistuba and H. Kadara contributed equally to this work.

Corresponding Authors: Ignacio I. Wistuba, Departments of TranslationalMolecular Pathology and Thoracic/Head and Neck Medical Oncology, TheUniversity ofTexasMDAndersonCancerCenter,Houston,TX77030.Phone:713-563-9184; Fax: 713-792-0309; E-mail: [email protected];and Humam Kadara, Department of Thoracic/Head and Neck MedicalOncology, The University of Texas MD Anderson Cancer Center, Hous-ton, TX 77030. Phone: 713-745-3186; Fax: 713-792-0309; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-0341

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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notably, to exhibit both tumor-promoting and -suppres-sive effects in different types of carcinomas (10–15).However, ETS2 expression and function in human lungcancer is still unknown.

Our current lung adenocarcinoma expression profilingefforts revealed marked downregulation of ETS2 transcriptexpression in lung tumors compared with paired normallung tissues prompting us to examine ETS2 function inlung cancer pathogenesis. In this study, we found that lowETS2 immunohistochemical protein expression was sig-nificantly associated with shorter time to recurrence inNSCLC. Moreover, we found that ETS2 negatively regu-lates cellular growth, migration and invasion, MET onco-gene phosphorylation and activation, as well as HGF-mediated signaling. Our findings reveal for the first timea potential tumor-suppressive role for ETS2 in lung cancerpathogenesis that is, in part, mediated by the inhibition ofMET oncogenic signaling.

Materials and MethodsNSCLC frozen tissue specimens and tissue microarrays

All human tissues were obtained from the MD AndersonCancer Center Lung Cancer Specialized Program ofResearch Excellence (SPORE) tissue bank (Houston, TX)and had been classified using the 2004 World HealthOrganization classification system as described before(16). All specimens were obtained from patients, whounderwent surgery at the same institution from 2003 to2005, under a protocol that was approved by the MDAnderson Cancer Center Institutional review board.Detailed clinical and pathologic information was availablefor most of these cases and included patients’ demographicdata, smoking history (never smokers or ever smokers,patients who had smoked at least 100 cigarettes in their

lifetime), and pathologic tumor–node–metastasis (TNM)staging.

A collection of 80 lung adenocarcinomas and 30 non-tumoral paired tissues were snap-frozen and preserved inliquid nitrogen for total RNA extraction and microarrayprofiling. For each tissue sample, the percentage of malig-nant tissue was calculated and the cellular composition ofspecimens was determined by histologic examination(J. Fujimoto) following hematoxylin–eosin (H&E) stain-ing. All malignant samples retained contained more than40% tumor cells.

For NSCLC tissue microarray analysis (TMA), weobtained archived formalin-fixed paraffin-embedded(FFPE) samples from surgically resected lung cancer speci-mens from the lung cancer tissue bank. The tissue micro-array analyzed in this study was composed of 201 NSCLCtumor specimens [135 lung adenocarcinomas and 66 squa-mous cell adenocarcinomas (SCC); Supplementary TableS1]. After histologic examination of NSCLC specimens, theNSCLC TMAs were constructed by obtaining three 1 mmdiameter cores from each tumor at 3 different sites (periph-ery, intermediate, and central tumor sites). The TMAs wereprepared with a manual tissue arrayer (Advanced TissueArrayer ATA100, Chemicon International) as describedpreviously (16).

Cell cultureLung cancer cell lines were either originally purchased

from the American Type Culture Collection (ATCC; H441,H2291, H3255, H1299, H1693, H522, H1792, H23, andH2009) or were obtained from Dr. Adi Gazdar (Universityof Texas Southwestern, Dallas, TX) (DFC1024, HCC4006,H2228, H1650, andH1944) and were grown in Dulbecco’smodified Eagle medium-F12 low glucose medium supple-mented with 10% FBS and maintained in humidified5% CO2 incubator. Information on the smoking status ofpatients from whom the cell lines were isolated wasobtained from the ATCC or from Dr. Adi Gazdar. All celllines used in the study were authenticated by short tandemrepeat DNA fingerprinting using the PowerPlex 16 HSSystem (Promega). For MET inhibition studies, lung cancercells were treated with the MET tyrosine kinase inhibitor,PHA-665752 (SelleckBio) at a 100 nmol/L final concentra-tion. For treatment with hepatocyte growth factor (HGF)and activation of MET, cells were washed twice with PBS,serum-deprived overnight, and then treated with HGF(EMD Millipore), at a final concentration of 50 ng/mL inserum-free cell culture medium.

Total RNA isolationTotal RNA was isolated from cells using the RNeasy kit

from Qiagen according to the manufacturer’s instructions.Lung adenocarcinoma andnormal lung tissue sampleswerehomogenized usingOmni plastic disposable probes and anOmni (TH-115) homogenizer (Omni International) for 1minute on dry ice after which total RNA was isolated usingTrizol reagent according to the manufacturer’s instructions.Total RNA was quantified using the Nanodrop 1000

Translational RelevanceThe expression and role of ETS2, a transcription factor

with various important cellular and molecular func-tions, in NSCLC biology and pathogenesis is stillunknown. In this study, we found that the ETS2 geneexhibited downregulated expression in lung adenocar-cinomas compared with normal lung following micro-array profiling and reduced expression of its codingprotein product was an independent predictor of shortertime to recurrence in human NSCLC. Moreover, wefound that ETS2 negatively regulated cell growth, migra-tion, and invasion, in part, through suppression of METphosphorylation and activation and inhibited HGF-mediated oncogenic signaling. Our findings point to anadditional level of intracellular pathway control of aber-rant MET oncogene activation, through ETS2 tumorsuppressor function, in human NSCLC and that maypossibly be used as a marker for selection of patients forMET-targeted therapy.

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Clin Cancer Res; 19(13) July 1, 2013 Clinical Cancer Research3384




spectrophotometer (Thermo Scientific). RNA quality wasassessed based on RNA integrity numbers generated by theAgilent Bioanalyzer 2000 (Agilent) according to the man-ufacturer’s instructions.

Microarray processing and analysisTotal RNA was isolated from lung adenocarcinomas

(n ¼ 80) and adjacent normal lung tissues (n ¼ 30). RNAwas also isolated from lung cancer cells transfected withscrambled siRNA and ETS2-specific siRNA (3 replicateseach, n ¼ 6). RNA samples were analyzed by microarrayexpression profiling using the Affymetrix Human Gene1.0 ST platform (Affymetrix) according to the manufac-turer’s instructions. Briefly, in vitro transcription andcDNA target preparation were conducted using the WTexpression kit (Life Technologies). A total of 2.5 mg offragmented and labeled cDNA was generated using theAffymetrix GeneChip WT Terminal Labeling and ControlsKit and hybridized onto Human Gene 1.0 ST arraysaccording to the manufacturer’s instructions (Affymetrix).Arrays were washed, stained, and processed using Affy-metrix GeneChip Fluidics Station 450 systems after whichthey were imaged using Affymetrix GeneChip Scanner3000 7G for subsequent generation of raw data (�CELfiles). Microarray data were submitted to the Gene Expres-sion Omnibus and were MIAME compliant. Lung tumorsand normal lung tissue were deposited under seriesGSE43458 (samples GSM1062765-GSM1062874) andarray data of transfected cells were submitted under seriesGSE43459 (samples GSM1062875-GSM1062880). Rawdata were normalized using Robust Multichip Array andlog2 transformed using BRB-ArrayTools v 4.3.0 developedby Dr. Richard Simon and the BRB-ArrayTools Develop-ment Team (Biometric Research Branch, Rockville, MD;ref. 17). Genes significantly differentially expressedbetween lung adenocarcinomas and normal lung tissueswere selected based on a false discovery rate (FDR)threshold of less than 0.001 and a 1.5-fold changebetween the 2 groups (n ¼ 2,665 transcripts). Genesdifferentially expressed between H441 lung cancer cellstransfected with ETS2-specific siRNA compared with cellstransfected with scrambled siRNA were selected on thebasis of a P < 0.01 (n ¼ 1,816 transcripts; SupplementaryTable S2). Functional pathway analysis was conductedusing the commercially available software Ingenuity Path-ways Analysis (IPA) according to the manufacturer’sinstructions.

Transfection of siRNA and expression vectorssiRNAs against ETS2 and MET as well as scrambled

(control) siRNA were synthesized by a proprietary designas SMARTpool siRNA (Dharmacon, Thermoscientific).Knockdown of ETS2 or MET expression was conductedusing Lipofectamine RNAiMAX (Life Technologies) accord-ing to the manufacturer’s instructions. ETS2 overexpressionwas achieved using an ETS2 cDNA clone (Origene)inserted into a Cytomegalovirus plasmid pCMV-XL5 vec-tor. Cells were transfected with the expression vectors

using Lipofectamine 2000 (Life Technologies) accordingto the manufacturer’s instructions. For HGF treatmentexperiments, transfections were conducted one daybefore overnight incubation with serum-free cell culturemedium.

Migration and invasion assaysTranswell cell migration was quantified by seeding cells

(n ¼ 8 � 104) in serum-free medium onto the top layer of24-well BD BioCoat 8.0 mm PET membrane inserts (BDBiosciences). Cells (n ¼ 8 � 104) in serum-free mediumwere seeded onto the top layer of 24-well BD BioCoatMatrigel invasion chambers (BD Biosciences) for quantita-tive assessment of cell invasion. After 48 hours,migrating orinvading cells were washed with PBS, fixed with 10%formalin, and stained with 0.5% crystal violet then countedusing bright-field microscopy. For HGF treatment experi-ments, a concentration of 50 ng/mL of HGF in serum-freemedium was supplied to the top layer. Qualitative assess-ment of cell migration was conducted by the wound-heal-ing assay in which cells were scratchedwith a 200 mL pipettetip after which the wound was monitored for closure. Allconditions were conducted in 3 replicates.

Trypan blue exclusion and cell countCells were seeded in 3 replicate wells for each experimen-

tal condition at a density of 3� 104 cells per well in 12-wellplates and then transfected the followingdaywith siRNAsoroverexpression vectors. Seventy-two hours after transfec-tion, cells were washed with 1� PBS, trypsinized, mixedwith 0.4% Trypan blue solution (Sigma Aldrich) and thencounted using the Reichert Bright-Line Hemacytometer(Hausser Scientific) by Trypan blue exclusion principle.

Anchorage-dependent and -independent colonyformation assays

For anchorage-dependent colony formation on plastic,cells were seeded at a density of 150 cells per well in 6-well plates. Plates were monitored for colony develop-ment for 2–3 weeks under a phase-contrast microscopeafter which cells were washed with 1� PBS, fixed with10% formalin, and stained with 0.5% crystal violet beforecounting. All experiments were carried out in triplicatesand Student t test was used to test for statistical signifi-cance. Cells were assessed for anchorage-independentcolony formation by growth on soft agar. Cells wereseeded (1 � 104/well) in 2 mL culture medium contain-ing of 0.3% (w/v) of granulated agar (Difco, BD Bios-ciences). Cell-agar suspensions were plated on a 2 mLbottom layer composed of 0.6% (w/v) of agar. Both layersof soft agar were supplemented with 20% FBS. All con-ditions were conducted in triplicates and colonies werescored 2 to 3 weeks after cell seeding.

Western blot analysisCell monolayers were washed twice with PBS, har-

vested, and lysed with ice-cold radioimmunoprecipita-tion assay buffer (Sigma-Aldrich) after which protein

ETS2 Tumor Suppressor in Human NSCLC

www.aacrjournals.org Clin Cancer Res; 19(13) July 1, 2013 3385




lysates (20 micrograms) were subjected to SDS- PAGEand Western blotting. The antibodies used for immuno-blotting included those raised against ERK1/2, phosphor-ylated-ERK1/2 (THR202/TYR204), MET, phosphorylated-MET (Y1234/Y1235; Cell Signaling Technology), ETS2(Santa Cruz Biotechnology), and b-actin (loading con-trol; Sigma-Aldrich). Antibody binding was detected byenhanced chemiluminescence (Amersham BiosciencesCorp.). Band intensities were quantified relative to inten-sity of b-actin using ImageJ software.

Phospho-MET ELISALevels of phosphorylated MET (Y1234/Y1235) were

quantified 48 hours after knockdown of ETS2 expressionusing the phospho-MET(Y1234/Y1235) PathScan sand-wich ELISA (Cell signaling Technology) according to themanufacturer’s instructions.

Quantitative real-time PCRA total of 1 mg of RNA was reverse-transcribed using

the High Capacity RNA-to-cDNA kit (Life Techno-logies) according to the manufacturer’s instructions anddiluted in nuclease-free water. Quantitative real-time PCR(qRT-PCR) was conducted using predesigned TaqManexpression assays for ETS2 (Hs00232009_m1), b-ACTIN(Hs99999903_m1), MET (Hs01565584_m1), RAB3B(Hs01001137_m1), SERPINB5 (Hs00985285_m1), ZEB1(Hs00232783_m1), SNAI2 (Hs00950344_m1), andSPDEF (Hs01026050_m1; Life Technologies) on an ABI7300 Real-Time PCR System (Applied Biosystems). Rela-tive quantification was calculated using the 2�DDCT relativequantification method.

ImmunohistochemistryImmunohistochemical (IHC) analysis was conducted on

4 mm FFPE histologic sections using purified rabbit poly-clonal primary antibodies raised against ETS2 (1:1000dilution, clone RB24588, AP9846c, Abgent) and phos-pho-MET (Y1234/Y1235) (1:250 dilution, catalog numberAF2480; R&D systems). Antigen retrieval was carried outusing the Dako Target Retrieval System at a pH of 6 (Dako).Intrinsic peroxidase activity was blocked by 3% methanoland hydrogen peroxide for 15 minutes and serum-freeprotein block from Dako was used for 30 minutes forblocking nonspecific antibody binding. Slides were thenincubated with antibodies against ETS2 and phospho-MET(Y1234/Y1235) at room temperature for 65 and 90 min-utes, respectively. After 3 washes in TBS, slides were thenincubated for 30 minutes with Dako Envisionþ Dual Linkat room temperature. Following 3 additional washes, slideswere incubated with Dako chromogen substrate for 5 min-utes and were counterstained with hematoxylin for another5 minutes. FFPE pellets from lung cancer cell lines display-ing positive ETS2 and phospho-MET (Y1234/Y1235)expression were used as positive controls for antibodyoptimization, whereas samples and whole-section tissuespecimens processed similarly, except for the omission ofthe primary antibodies, were used as negative controls.

Immunostaining intensity and reactivity were examined byan experienced pathologist (J. Fujimoto) using a lightmicroscope under a � 20 magnification objective. ETS2expression was quantified using a 4-value intensity score (0,none; 1, weak; 2, moderate; and 3, strong) and the percent-age (0%–100%)of the extent of reactivity. Afinal expressionscore was obtained by multiplying the intensity and reac-tivity extension values (range, 0–300; ref. 16). For analysisof phosphorylated MET expression, sections with membra-nous immunoreactivity were considered positive for phos-pho-MET expression as previously described (18, 19) andphospho-MET stainingwas quantifiedusing a score range of0 to 3þ (20).

Statistical analysisANOVA and Student t test were used to test for statis-

tical significance among different groups in the in vitroexperiments. Statistical analysis of the IHC data was firstsummarized using standard descriptive statistics and fre-quency tabulations. Associations of ETS2 IHC proteinexpression alone or in combination phospho-MET(Y1234/Y1235) with patient outcome (time to recur-rence) was estimated using the Kaplan–Meier methodand compared among groups by log-rank statistical tests.Multivariate Cox proportional hazard models wereapplied to assess the effects of ETS2 and/or phospho-MET IHC expression on time to recurrence and adjustedfor tumor stage. All computations were carried out in SAS9.3 and S-plus 8.2.

ResultsETS2 expression is significantly lower in lungadenocarcinomas relative to normal lung

We conducted gene expression profiling of lung adeno-carcinomas (N ¼ 80) and matched normal lung (N ¼ 30)and identified, using an FDR threshold of 0.1% and a 1.5-fold change cutoff, 2,665 transcripts that were significantlydifferentially expressed between lung adenocarcinomasand normal lung. Further analysis from our current expres-sionprofiling efforts revealed significantly decreased expres-sion of the ETS2 transcription factor transcript in lungadenocarcinomas compared with normal lung tissues (P <10�7; Fig. 1A). In silico analysis of a publicly availablemicroarray dataset (21) corroborated our findings andsimilarly showed a significant decrease in ETS2 expressionin NSCLC tumors compared with adjacent normal lung.qRT-PCR analysis confirmed the ETS2 decreased expression(P¼ 0.003; Fig. 1B). Moreover, ETS2 expression was highlycorrelated between genemicroarray andqRT-PCR in apanelof normal lung and lung adenocarcinoma tissues (R¼ 0.72,P < 0.001; Fig. 1C). Analysis of ETS2 expression in our geneexpression microarray dataset in the context of variousclinicopathologic features revealed that ETS2 was signifi-cantly lower in smoker compared with never-smoker ade-nocarcinomas (P < 0.001; Supplementary Fig. S1A) whichwas confirmed in a subset of adenocarcinoma tumors byqRT-PCR (P < 0.001; Supplementary Fig. S1B). Similarly,

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ETS2 expression, assessed by qRT-PCR, was lower in estab-lished NSCLC cell lines previously derived from smokerscompared with those isolated from never-smoker patients(P ¼ 0.05; Supplementary Fig. S2). These findings showthat ETS2 mRNA expression is reduced in lung adeno-carcinomas relative to normal lung, in particular, in smokerlung tumors.

Decreased ETS2 immunohistochemical expression isassociated with shorter time to recurrence in NSCLCpatientsETS2 is a canonical transcription factor that affects a

broad spectrum of genes by binding to ERE sequences inthe genome thereby affecting variousmolecular and cellularfunctions (3, 5). Our findings on reduced ETS2 transcriptexpression in lung tumors prompted us to examine the IHCexpression of ETS2 protein in association with patientoutcome that has not been characterized before. We ana-lyzed ETS2 expression in a NSCLC (n ¼ 201) TMA (135adenocarcinomas and 66 SCCs) derived from patients whodid not receive neoadjuvant or adjuvant treatment. Repre-sentative photomicrographs of low (left) and high (right)ETS2 IHC expression are depicted in Supplementary Fig. S3.Association of ETS2 IHC expression with patient clinicaloutcome was conducted following dichotomization ofpatients into groups with relatively higher or lower ETS2expression than the IHC score of 55, which was determinedby the martingale residual. Low ETS2 expression predictedshorter time to recurrence in all-stages (Fig. 2A) and stage-I(Fig. 2B) NSCLC (both P ¼ 0.004 of the log-rank test).Moreover, relatively low ETS2 expression predicted shortertime to recurrence in all-stages (P¼ 0.03; Fig. 2C) and stage-I (Fig. 2D; P ¼ 0.046) lung adenocarcinoma but not inlung SCC. Importantly, multivariate Cox proportionalhazard models showed that, after adjusting for stage, rela-tively lower ETS2 IHC protein expression was an indepen-dent predictor of shorter time to recurrence in NSCLC

(P ¼ 0.009, HR ¼ 1.89, 95% confidence interval 1.17–3.06) and in lung adenocarcinoma (P ¼ 0.03, HR ¼ 1.86,95% confidence interval 1.05–3.31). Our findings on theassociation of low ETS2 immunohistochemical expressionwith shorter time to recurrence in NSCLC points to apotential tumor suppressor function for ETS2 in humanlung cancer.

ETS2 inhibits lung cancer cell growth, migration, andinvasion

We then sought to test the role of ETS2 in NSCLCpathogenesis. H441 lung cancer cells transfected withsiRNA specific to ETS2 exhibited significantly reduced ETS2transcript compared with cells transfected with controlscrambled siRNA (P < 0.05) (Fig. 3A). Expression of ETS2protein was also reduced by 0.6-fold, albeit less than thatobserved for the transcript, following RNA interference (Fig.3A).Moreover,ETS2knockdown significantly increased cellgrowth (1.55-fold) evidenced by Trypan blue exclusion(Fig. 3B, right) count as well as augmented cell migration(Fig. 3B, left) and invasion (Fig. 3B,middle) by 4.4-fold and6.2-fold, respectively (all P < 0.05). Conversely, ETS2 over-expression in H1299 lung cancer cells, with a relatively lowbasal expression of the gene (Fig. 3C), significantlydecreased cell migration (Fig. 3D, left) and invasion (Fig.3D, middle) by 2.6-fold and proliferation by 1.5-fold (Fig.3D, right; all P < 0.05).Moreover, overexpression of ETS2 inH1299 cells significantly suppressed anchorage-dependent(1.8-fold) and –independent (2.1-fold) colony formationevidenced by growth on plastic and soft agar, respectively(P < 0.05; Supplementary Figs. S4A and S4B). Importantly,siRNA-mediated knockdown of ETS2 expression nearlycompletely rescued cells from cell growth inhibition medi-ated by overexpression of the gene suggesting that theobserved cell growth inhibition was ETS2-mediated (Sup-plementary Fig. S4C). It is worthwhile to mention that thefold changes in cellular migration and invasion following

Figure 1. ETS2 transcript is significantly decreased in lung adenocarcinomas compared with normal lung tissue. Lung adenocarcinomas (n¼ 80) and normallung tissues (n ¼ 30) were profiled using the Human Gene 1.0 ST platform as detailed in the Materials and Methods section. A, ETS2 normalizedexpression in adenocarcinomas (ADC) and normal lung. B, relative ETS2mRNA expression was assessed by qRT-PCR, normalized to that of b-ACTIN andquantified using the 2�DDCT relative quantification method as detailed in the Materials and Methods section. P values indicate statistical significance, by theStudent t test, of difference inETS2 expression betweenADCs and normal lung. C, statistical correlation ofETS2 expression quantifiedbymicroarray profilingand qRT-PCR; R, Pearson correlation coefficient.






knockdown or overexpression of ETS2were greater than thenoted differences in cell growth. These findings suggest atumor suppressor function for the ETS2 gene in vitro inhuman lung cancer.

Global gene expression analysis downstream of ETS2knockdown reveals modulation of key pathways inlung cancer cells

To gain insights into the mechanisms of ETS2 tumorsuppressor function, we sought to compare and contrast thetranscriptome of cells transfected with scrambled siRNAand ETS2-specific siRNA. Gene expression profiling, usingthe Affymetrix Human Gene 1.0 ST platform, identified1,816 transcripts that were significantly differentiallyexpressed, based on a P < 0.01 threshold, in H441 lungcancer cells with ETS2 knockdown compared with theircontrol counterparts (Fig. 4A; Supplementary Table S2).Moreover, functional analysis of the genes using IPA,revealed that ETS2 knockdown modulated key pathwaystypically activated in cancer, in particular, HGF, integrin,tissue factor, semaphoring, and mitogen-activated proteinkinase (MAPK) signaling (all P < 0.001; Fig. 4B). Notably,the pathway analysis revealed that HGF signaling was thetop modulated canonical pathway following ETS2 knock-down (P < 10–5; Fig. 4B) and that an HGF-mediated geneinteraction network was among the top significant genenetworks following topological arrangement of the differ-entially expressed genes by IPA (Supplementary Fig. S5). Inaddition, we observed significant induction of genesinvolved inepithelialmesenchymal transition (EMT; ref. 22;e.g., zinc finger E-box binding homeobox 1/ZEB1 and snailhomolog 2/SNAI2), GTPases (e.g., RAB3B), known ETS2

targets (ref. 23; e.g., serpin peptidase inhibitor clade Bmember 5/SERPINB5), and members of the ETS family(ref. 5; e.g., SAM pointed domain containing ets transcrip-tion factor/SPDEF) all ofwhichwere confirmedbyqRT-PCRanalysis following selection by a combination of statisticalcriteria and pathways analysis (Fig. 4C). Our global tran-scriptome analysis further points to a tumor suppressorfunction by ETS2 in human lung cancer cells.

ETS2 suppresses MET activation and HGF signaling inhuman lung cancer cells

Our expression profiling analysis showed that HGF sig-naling was the top significant modulated pathway follow-ing siRNA-mediated ETS2 knockdown in lung cancer cells.As HGF is known to function as ligand for theMET receptoroncogene (24, 25), we sought to determine the effects ofETS2 knockdown on the latter tyrosine kinase receptor.Western blotting analysis showed that siRNA-mediatedknockdown of ETS2 expression increased phosphorylatedlevels of MET by 1.2-fold (Fig. 5A). Enzyme-linked immu-nosorbent assay for phosphorylated MET levels similarlyrevealed an increase in MET phosphorylation at Y1234/Y1235 following ETS2 knockdown (Supplementary Fig.S6). Conversely, ETS2 overexpression suppressed METphosphorylation which was rescued by cotransfection ofETS2-targeting siRNA (Supplementary Fig. S7). Important-ly, cotransfection of H441 lung cancer cells with siRNAtargeting MET (Fig. 5B) abrogated cell migration (Fig. 5C,top), invasion (Fig. 5C, middle) and, to a less pronouncedeffect, cell growth (Fig. 5C bottom; P < 0.05) mediated byknockdown of ETS2 expression alone (Fig. 5B and C).Moreover, we examined the expression of genes we had

Figure 2. Reduced ETS2 IHCexpression is associated withshorter time to recurrence inpatients withNSCLC. ETS2 proteinexpression was analyzed byimmunohistochemistry in aNSCLCTMA composed of 201 FFPEhistologic tissue specimens (135lung adenocarcinomas and 66SCCs). Differences in time torecurrence between patientsstratified by ETS2 IHC expression(ETS2high:�55, blue; ETS2low: <55,black) were statistically assessedby the log-rank test and Kaplan–Meier survival probability methodand depicted for all-stages (A) andstage-I (B) patients with NSCLC aswell as in all-stages (C) and stage-I(D) adenocarcinoma patients. E,number of censored patients ineach subgroup; N; number ofpatients in each group based onrelatively higher or lower ETS2 IHCexpression.

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identified to be induced by ETS2 knockdown (Fig. 4C)in cells transfected with siRNAs targeting both ETS2 andMET. Cotransfection of cells with MET-specific siRNA sig-nificantly attenuated the induction of SNAI2, RAB3B,SPDEF, and SERPINB5 mediated by knockdown of ETS2alone (all P < 0.05) as well as reduced, albeit not significant,expression of the ZEB1 gene (Fig. 5D). These data show thatETS2 suppresses key features of the lung cell malignantphenotype, at least in part, through inhibition of the METoncogene.

ETS2 suppresses HGF-mediated signaling in lungcancer cellsAs we found that ETS2 suppresses cellular migration and

invasion through inhibition of MET phosphorylation andactivation, we sought to examine the role of ETS2 in HGF-mediated signaling. Following an overnight serum starva-tion, H441 lung cancer cells were treated with HGF (50ng/mL final concentration) for one hour. HGF treatment

increased ETS2 protein expression by 1.3-fold in H441 cells(Fig. 6A). The increase in ETS2 protein was also noted inadditional lung cancer cell lines (Supplementary Fig. S8).Moreover, and as shown in previous studies (26, 27), HGFtreatment also increased phosphorylation of bothMET andextracellular-regulated kinase (ERK1/2) MAPK (Fig. 6A) inH441 lung cancer cells which was alleviated at later treat-ment time points (Supplementary Fig. S8A) as previouslyreported for earlier HGF treatment and ligand stimulationstudies (28, 29). Furthermore, expression of ETS2 proteinand of phospho-ERK1/2 and phospho-MET was attenuatedby cotreatment of cells with the MET tyrosine kinase inhib-itor PHA-665752 (100 nmol/L final concentration; Fig. 6Aand Supplementary Fig. S8B).

We then sought to assess the relevance of ETS2 to HGFsignaling in lung cancer cells. RNA interference-mediatedknockdown of ETS2 expression significantly augmentedHGF-induced cell migration (1.9-fold) and invasion (1.7-fold; P<0.05; Fig. 6B, right). These effects were concomitant

Figure 3. ETS2 expression is a negative regulator of cellular migration, invasion, and proliferation. qRT-PCR (top) and Western blotting analyses (bottom) ofETS2 mRNA and protein expression, respectively, were conducted 48 hours after transfection of H441 cells with scrambled (control) siRNA andsiRNA-targeting ETS2 (A) and of H1299 cells with control and ETS2 overexpression vectors (C). ETS2 expression was normalized to that of the b-ACTINhousekeeping gene and quantified relative to the expression in cells transfected with control siRNA. ForWestern blotting, 20 mg of total protein from sampleswere analyzed by SDS-PAGE as detailed in the Materials and Methods section. Membranes were stained with polyclonal antibody against ETS2 and with ab-ACTIN monoclonal antibody to ensure equal protein loading. Analysis of Transwell cell migration (left) and invasion (middle) was conducted in thetransfected H441 (B) and H1299 (D) cells using BD BioCoat Transwell inserts or inserts with Matrigel, respectively. Cells that migrated or invaded after48 hours to the bottom side of the inserts were fixed, stained, and quantified, as described in the Materials and Methods section, relative to number of cellstransfected with control siRNA. Growth of cells (right, B and D) was assessed by the Trypan blue exclusion method as mentioned before. All assays arerepresentative of 3 independent experiments. �, P <0.05 assessed by the Student t test.






with increased MET phosphorylation in HGF-treated cellstransfected with ETS2-specific siRNA (1.8-fold with respectto control cells) which was higher than that observed intreated cells transfected with control siRNA (1.5-fold withrespect to control cells; Fig. 6B, left). Furthermore, we alsofound that, similar to knockdown of ETS2 alone (P < 0.05),HGF treatment also significantly increased the expressionof the SNAI2, RAB3B, SPDEF, ZEB1, and SERPINB5 genes(all P < 0.05; Fig. 6C). It is worthwhile to mention thatHGF treatment also significantly increased ETS2 mRNAlevels (P < 0.05; Fig. 6C). Importantly, the expression ofthe aforementioned genes was significantly higher in HGF-treated cells transfected with ETS2-specific siRNA comparedwith similarly treated cells thatwere transfectedwith controlsiRNA (P < 0.05; Fig. 6C). The observed additive effect of thecombination of HGF treatment and ETS2 knockdown onMET phosphorylation was also confirmed in an additionallung cancer cell line (H1944; Supplementary Figs. S9A andS9B). While HGF treatment alone increased phospho-METprotein, as evidenced by Western blotting, by 61-fold,phosphorylated MET protein was increased by 98-fold

following HGF treatment in cells with knockdown of ETS2(Supplementary Fig. S9B). This effect was concomitantwith increased cell growth (1.6-fold), rather than invasion,compared with HGF-treated cells with control siRNA(Supplementary Fig. S9C). Moreover and conversely, over-expression of ETS2 in H1299 lung cancer cells decreasedHGF-induced phosphorylated MET levels and decreasedHGF-induced cell migration evidenced by the wound-heal-ing assay (Supplementary Fig. S9D). Our findings suggestthat ETS2 may impact dissimilar phenotypic and antima-lignant effects downstream of HGF in different lung cancercell lines. Interestingly, differential antimalignant pheno-typic effects by theMET inhibitor, PHA-665752, in differentcancer cell lines were also previously noted in the originalreport by Christensen and colleagues (26). Our findingssuggest that ETS2 inhibits HGF-mediated oncogenic signal-ing in a negative-feedback manner.

It is worthwhile to mention that we also analyzed phos-phorylated MET IHC expression (Supplementary Fig. S10)along with that of ETS2 in association with patient time torecurrence. Interestingly, patientswithNSCLC (P¼ 0.01) or

Figure 4. Global changes in lung cancer cell transcriptome following knockdown of ETS2 expression. H441 lung cancer cells were transfected with controlsiRNA and ETS2-specific siRNA (3 replicates each). Total RNA was isolated 48 hours after transfection and was analyzed using the Human Gene 1.0 STplatform, as detailed in the Materials and Methods section, to compare and contrast the transcriptome of H441 cells transfected with control siRNAand ETS2-specific siRNA. A, heatmap depicting 1,816 transcripts that were significantly differentially expressed between cells transfected with scrambled(blue) and ETS2-specific (red) siRNA based on a P < 0.01. Rows and columns represent transcripts and samples, respectively. Upregulated anddownregulated gene expression is indicated by red and green colors, respectively. B, functional pathway analysis of the differentially expressed genes wasconducted using the IPA commercially available software. Statistically significant modulation (indicated by the inverse log of P values) of the top5 over-represented canonical pathways following ETS2 knockdown and predicted by IPA is depicted. C, confirmation of microarray profiling by qRT-PCRanalysis of RAB3B, SPDEF, ZEB1, SNAI2, and SERPINB5 genes in H441 lung cancer cells transfected with control and ETS2-specific siRNA. Expressionchanges are depicted relative to cells transfected with control siRNA. �, P values <0.05 assessed by the Student t test.

Kabbout et al.





adenocarcinoma (P ¼ 0.02) with relatively lower ETS2expression and higher phosphorylated MET membraneimmunoreactivity exhibited significantly shortest time torecurrence compared with other patient subgroups, in par-ticular those with higher ETS2 and higher phosphorylatedMET expression (Supplementary Fig. S11A–S11D). Thesedata further suggest thatETS2 suppressionofMEToncogenesignaling is operative in human lung cancer.

DiscussionOur expression profiling efforts revealed significant

downregulation of the ETS2 transcription factor in lung

adenocarcinomas compared with normal lung tissues. Wequestioned the relevance of ETS2 downregulation toNSCLC biology, given the important cellular and molec-ular roles this transcription factor plays (3, 5), and foundthat low ETS2 protein was predictive of shorter time torecurrence and the gene was a negative regulator oflung cancer cell growth, migration, and invasion and ofHGF/MET oncogenic signaling. It is worthwhile to notethat we had found significant reduced expression of ETS2in smoker compared with never-smoker lung adenocar-cinomas (Supplementary Fig. S1) and, similarly, NSCLCcell lines originally derived from smokers exhibited

Figure 5. ETS2 inhibits cell migration and invasion through suppression of MET phosphorylation and activation. A, H441 lung cancer cells were transfectedwith control and ETS2-specific siRNA and analyzed by Western blotting analysis, as described earlier, for ETS2 and total and phosphorylated (P-MET)MET levels 48 hours following transfection. B, qRT-PCR analysis of ETS2 and MET transcript expression (left) and Western blotting analysis of total andphosphorylated MET protein levels (right) was conducted 48 hours following transfection of H441 cells with control siRNA, siRNA against ETS2 orMET alone, andwith both ETS2- andMET-specific siRNAs. �,P values <0.05 assessed by the Student t test. C, H441 cells transfected as described in Bwereanalyzed for differences in migration (top), invasion (middle), and cell growth (bottom) 48 hours following transfection. Analysis of cellular migration,invasion, and growth, relative to control cells transfected with scrambled siRNA only, was conducted as described earlier in Fig. 3. D, qRT-PCR analysis ofthe expression of RAB3B, SPDEF, ZEB1, SNAI2 and SERPINB5 in cells transfected with ETS2-specific siRNA, MET-specific siRNA, and with bothETS2- and MET-specific siRNAs compared with cells transfected with scrambled siRNA only. �, P values <0.05 indicating statistically significant pairwise(indicated by the dotted connectors) differences. All assays are representative of 3 independent experiments.






reduced ETS2 expression compared with lung cancer celllines isolated from never-smokers. It is worthwhile tomention that in silico analysis of publicly available expres-sion datasets also revealed reduced ETS2 transcriptexpression in smoker adenocarcinomas as well as markeddownregulation of expression of the gene in airways ofphenotypically healthy smokers compared with disease-free nonsmokers (data not shown). In addition andnotably, ETS2 mRNA expression, part of a genomic sig-nature of phosphoinositide 3-kinase (PI3K) pathwayactivation, was decreased in the normal bronchial airwaysof smokers with lung cancer compared with the airways ofdisease-free smokers (30). It is plausible to suggest thatcigarette smoking reduces expression of the ETS2 gene inthe course of NSCLC pathogenesis.

Microarray profiling coupled with functional pathwaysanalysis revealed that ETS2 expression is linked to variouscanonical cancer-associated pathways in lung cancer cells,in particular theHGFpathway.Moreover, qRT-PCR analysisconfirmed the array findings and showed upregulation ofthe ZEB1, SNAI2, RAB3B, SERPINB5, and SPDEF genesfollowing siRNA-mediated knockdown of ETS2 expression

concomitant with elevated cell migration and invasion.ZEB1 and SNAI2 (also known as SLUG) are transcriptionfactors with well-established roles in promotion of EMT invarious physiologic and pathologic conditions (22).RAB3Bis a relatively understudiedRAS familymember andGTPasethat was found to be an important positive regulator ofepithelial cell polarity (31) and of the stemness of adulthuman mesenchymal stem cells (32). SERPINB5, a serpinpeptidase inhibitor, was shown to be a target repressed bymurine Ets2 (23) and was shown to be upregulated inhuman lung tumors compared with normal lung tissues(33) and hypomethylated and elevated in lung SCCs (34).SPDEF (also known as PDEF), amember of the ETS-domainprotein family (5), was shown to be upregulated in mam-mary tumors (35) and to positively control cancer cellmigration and invasion (36). Notably, SPDEF was foundto promote airway epithelial cell (e.g., goblet cell) hyper-plasia, inflammation, and mucus hypersecretion (37). It isalso noteworthy, that HGF signaling, known for its promi-gratory and -invasive effects (27), was the top pathwaysignificantlymodulated followingETS2knockdown in vitro.In addition, ETS2 knockdown significantly augmented

Figure 6. ETS2 inhibits signaling downstream of HGF. A, Western blotting analysis was conducted 1 hour after treatment of serum-starved H441 cells withHGF, 100 nmol/L PHA-665752, and the combination of both HGF and PHA-665752. Protein lysates were immunoblotted with antibodies against ETS2, totaland phosphorylated levels of MET and ERK1/2, as well as with an antibody against b-ACTIN to ensure equal protein loading. B, H441 cells weretransfected with scrambled and ETS2-specific siRNA and serum-starved overnight the following day. Twenty-four hours after serum starvation, transfectedcells were incubated in medium with and without 50 ng/mL HGF for 1 hour for subsequent Western blotting (left) for total and phosphorylated MET proteinlevels or were transferred onto the top layer of 24-well BD BioCoat Transwell inserts or inserts with Matrigel, in the presence or absence of 50 ng/mLHGF, for analysis of cell migration (top right) and invasion (bottom right), respectively. C, qRT-PCR analysis of the expression of the indicated genes in cellstransfected with control and ETS2-specific siRNA and incubated in cell culture medium with and without 50 ng/mL HGF for 48 hours. �, P values <0.05indicating statistically significant pairwise (indicated by the connectors) differences. All assays were conducted in triplicates and are representative of 3independent experiments.

Kabbout et al.





HGF-mediated induction of all 5 genes. It is reasonable tosuggest, given the increased expression of the aforemen-tioned genes following ETS2 knockdown, along with theirgiven previous reported promalignant roles, that ETS2functions as a lung tumor suppressor in lung cancer cells,at least in part, by regulation of themigration- and invasion-promoting transcriptome.In this study, we found that ETS2 suppressed HGF-medi-

atedMET phosphorylation and cell migration and invasionin a negative feedback manner. Our findings are in accor-dance with previous reports showing ETS2 negative feed-back regulation of other signaling networks, namely FGFsignaling (38) and phorbol ester-induced MAPK signaling(39) by upregulation of MAPK phosphatases (38, 39).Moreover, a previous study by Cao and colleagues showedmodulation of ETS2 expression and intracellular localiza-tion downstream of HGF/MET pathway activation inmesothelioma cancer cell lines, albeit without functionalassessment of the relevance of increased ETS2 expression(40). Because we also showed that ETS2 basally suppressedMET phosphorylation and activation, it is plausible tosuggest thatETS2 functionsbothupstreamanddownstreamofMET oncogene to inhibit cell migration and invasion. Inan attempt to further probe the relevance of the identifiedMET/ETS2 signaling to human lung cancer, we studied theassociation of both ETS2 and phosphorylated MET IHCprotein expression with NSCLC patient outcome. The com-bination of both decreased ETS2 and increased phosphor-ylatedMET IHCexpression identified a subgroupof patientswith further reduced time to recurrence compared withpatients with high ETS2 and high phosphorylated METprotein or when comparedwith analysis of ETS2 expressionalone. Further studies to confirm the combinatorial effect oflow ETS2 protein and high phosphorylated MET IHCexpression on reduced time to recurrence in lung canceror other malignancies are warranted. It is worthwhile tonote that MET function was shown to be deregulated indifferent types of solid tumors and hematologic malignan-cies by increased transcript expression or copy number gainor amplification (27, 41, 42). It is intriguing to suggest thatETS2 may represent an additional endogenous level ofpathway control of MET activation in NSCLC.It is important to note that ETS2 has been shown to

exhibit both oncogenic and tumor suppressor roles invarious malignancies. For example, ETS2 was shown tohavemitogenic activity downstream of Ras inmurine fibro-blasts (8), mediate transformation and tumorigenicity ofprostate and breast epithelial cells (13, 14), and to beupregulated in breast, thyroid, prostate, and pancreatictumors as well as in hematologic malignancies such asleukemias (4, 43–45).Moreover, loss ofmurine Ets2 expres-sion in tumor-associated macrophages was shown todecrease breast cancermetastasis inmice (23). On the otherhand, various studies have revealed tumor suppressor func-tions and effects mediated by the ETS2 gene. Foos andcolleagues showed that overexpression of full-length Ets2,with intact transactivator function, reversed Ras-inducedNIH3T3 cell transformation (46) and, in a separate study,

Hever and colleagues showed that Ets2was not required forErbB2-mediated cell transformation in vitro (47). In addi-tion, mice modeling Down’s syndrome and colon cancerexhibited reduced intestinal tumor incidence which wasattributed to increased gene dosage of Ets2, located in the21q22.2 chromosomal locus, in Down’s syndrome withtrisomy of chromosome 21 (15). In this context andsimilarly, mice with Ets2-deficient intestinal cells wereshown to develop more colon tumors in response totreatment with carcinogens (12). Additional potentialmechanisms of ETS2-mediated tumor suppression havebeen attributed to suppression of miR-196b–inducedinvasion in gastric cancer cells (11) and promotion ofcell-cycle arrest by negative feedback inhibition of MAPKsignaling following treatment with the phorbol ester PMA(39). Interestingly, overexpression in lung cancer cells ofthe frequently epigenetically inactivated lung tumor sup-pressor RASSF1A was shown to increase ETS2 transcriptexpression concomitant with arrest of cell-cycle progres-sion (48). Furthermore, ETS2 expression was shown to bedownregulated in human lung invasive adenocarcinomascompared with noninvasive bronchioalveolar carcinomas(49). The opposing tumor-repressive and -supportivefunctions attributable to ETS2 in different tumors maybe, in part, related to the dissimilar roles ETS2 mayexhibit in tumors of different cell lineages.

In conclusion, we showed, in this study, reduced expres-sion of ETS2 transcript expression in human lung tumorsrelative to normal lung tissues, association of low ETS2protein expression with shorter time to recurrence inpatients with NSCLC, and inhibition of human lung cancercell invasion, migration, and growth by ETS2, at least inpart, through suppression of MET oncogene phosphoryla-tion and HGF signaling. Our findings provide collectivetestament to a potential tumor suppressor role of the ETS2gene in human NSCLC pathogenesis, in part, throughinhibition of MET oncogene activation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Kabbout, I.I. Wistuba, H. KadaraDevelopment of methodology: M. Kabbout, L.M. Solis Soto, I.I. Wistuba,H. KadaraAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Garcia, D. Woods, G. Mendoza, C. BehrensAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M. Kabbout, J. Fujimoto, D. Liu, A.A.Momin, B.P. James, J.J. Lee, I.I. Wistuba, H. KadaraWriting, review, and/or revision of the manuscript: M. Kabbout,J. Fujimoto, L.M. Solis Soto, I.I. Wistuba, H. KadaraAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Kabbout, D. Woods, C.-W.Chow, I.I. Wistuba, H. KadaraStudy supervision: I.I. Wistuba, H. Kadara

Grant SupportThis work was supported in part by a Lung Cancer Research Foundation

grant (to H. Kadara), Department of Defense (DoD; W81XWH-04-1-0142to I.I. Wistuba), and by UT Lung SPORE P50CA70907 (to I.I. Wistuba) andCancer Center Support Grant CA-16672 from the National Cancer Institute.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked






advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 4, 2013; revised April 17, 2013; accepted May 7, 2013;published OnlineFirst May 9, 2013.

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2013;19:3383-3395. Published OnlineFirst May 9, 2013.Clin Cancer Res Mohamed Kabbout, Melinda M. Garcia, Junya Fujimoto, et al.

Small Cell Lung Cancer−Inhibition in Human Non ETS2 Mediated Tumor Suppressive Function and MET Oncogene

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