Role of Survivin in EGFR Inhibitor Induced Apoptosis in ... · Cancer Res; 70(24); 10402–10. 2010 AACR. Introduction Survivin is a member of the inhibitor of apoptosis (IAP) family

Tumor and Stem Cell Biology

Role of Survivin in EGFR Inhibitor–Induced Apoptosis inNon–Small Cell Lung Cancers Positive for EGFR Mutations

Kunio Okamoto1, Isamu Okamoto1, Wataru Okamoto1, Kaoru Tanaka1, Ken Takezawa1,Kiyoko Kuwata1, Haruka Yamaguchi1, Kazuto Nishio2, and Kazuhiko Nakagawa1

AbstractThe molecular mechanism by which epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR-TKI)

induce apoptosis in non–small cell-lung cancer (NSCLC) cells that are positive for activating mutations of theEGFR remains unclear. In this study, we report the effects of the EGFR-TKI gefitinib on expression of theantiapoptotic protein survivin that have functional consequences in EGFR mutation–positive NSCLC cells.Immunoblot analysis revealed that gefitinib downregulated survivin expression, likely through inhibition of thePI3K-AKT signaling pathway, in NSCLC cells positive for EGFR mutation. Stable overexpression of survivinattenuated gefitinib-induced apoptosis and also inhibited the antitumor effect of gefitinib in human tumorxenografts. Furthermore, the combination of survivin overexpression with inhibition of the gefitinib-inducedupregulation of the proapoptotic protein BIM attenuated gefitinib-induced apoptosis to a greater extent thaneither approach alone. Our results indicate that downregulation of survivin plays a pivotal role in gefitinib-induced apoptosis in EGFR mutation–positive NSCLC cells. Furthermore, they suggest that simultaneousinterruption of the PI3K-AKT-survivin and MEK-ERK-BIM signaling pathways is responsible for EGFR-TKI–induced apoptotic death in these cells. Cancer Res; 70(24); 10402–10. �2010 AACR.

Introduction

Survivin is a member of the inhibitor of apoptosis (IAP)family of proteins and has been shown to inhibit caspasesand to prevent caspase-mediated cell death (1–3). Survivin isabundant in many types of cancer cells but not in thecorresponding normal cells (4, 5). In nonmalignant prolif-erating cells, the expression of survivin is regulated in a cellcycle-dependent manner (6, 7). The upregulation of survivinexpression in tumors does not seem to be dependent solelyon the cell cycle, however, given that it occurs in tumor cellsthat are not actively cycling (4, 8, 9). Indeed, growth factorshave been found to regulate survivin expression in endothe-lial cells and neuroblastoma cells (10, 11). Although expres-sion of survivin has been demonstrated in non–small cell-lung cancer (NSCLC; refs. 12–14), the mechanism by whichsuch expression is regulated in NSCLC cells has remainedunknown.

The epidermal growth factor receptor (EGFR) is a receptortyrosine kinase that is abnormally amplified or activated in avariety of tumors including NSCLC, and it has therefore beenidentified as an important target in cancer treatment (15–17).Inhibitors of the tyrosine kinase activity of EGFR (EGFR-TKI),which compete with ATP for binding to the tyrosine kinasepocket of the receptor, have been extensively studied inpatients with NSCLC (18, 19). Several prospective clinicaltrials have revealed marked antitumor activity of EGFR-TKIsin NSCLC patients with EGFR mutations. The therapeuticbenefit of these drugs is much greater than that historicallyobserved with standard cytotoxic chemotherapy for advancedNSCLC. NSCLC cells with EGFRmutations manifest activationof the PI3K (phosphatidylinositol 3-kinase)–AKT and MEK–ERK (extracellular signal-regulated kinase) signaling pathwaysunder the control of EGFR, and exposure of such cells toEGFR-TKIs blocks signaling by both pathways and inducesapoptosis (20–22). The precise molecular mechanism bywhich EGFR-TKIs induce apoptosis has remained unclear,however. We have therefore now examined the effect of theEGFR-TKI gefitinib on survivin expression as well as furtherinvestigated the mechanism of gefitinib-induced apoptosisin EGFR mutation–positive NSCLC cells.

Materials and Methods

Cell culture and reagentsThe human NSCLC cell lines PC9, HCC827, NCI-H1975

(H1975), A549, and H1299 were obtained from American TypeCulture Collection. The NSCLC line PC9/ZD was obtained as

Authors' Affiliations: Departments of 1Medical Oncology and 2GenomeBiology, Kinki University School of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka, Japan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Isamu Okamoto, Department of Medical Oncol-ogy, Kinki University School of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511, Japan. Phone: 81-72-366-0221; Fax: 81-72-360-5000; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-2438

�2010 American Association for Cancer Research.

CancerResearch

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described previously (23). All cells were cultured under ahumidified atmosphere of 5% CO2 at 37�C in RPMI 1640medium (Sigma) supplemented with 10% fetal bovine serum.Gefitinib was obtained from Kemprotec, U0126 and LY294002were from Cell Signaling Technology and BEZ235 andAZD6244 were from ShangHai Biochempartner.

Immunoblot analysisCells were washed twice with ice-cold PBS and then lysed in

a solution containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 2.5 mmol/L sodiumpyrophosphate, 1 mmol/L phenylmethylsulfonyl fluoride, andleupeptin (1 mg/mL). The protein concentration of the celllysates was determined with the use of the Bradford reagent(Bio-Rad), and equal amounts of protein were subjected toSDS-PAGE on a 7.5% gel. The separated proteins were trans-ferred to a nitrocellulose membrane, which was then exposedto 5% nonfat dried milk in PBS for 1 hour at room temperaturebefore incubation overnight at 4�C with primary antibodies.Rabbit polyclonal antibodies to human phosphorylated EGFR(pY1068), to XIAP, to phosphorylated and total AKT, tophosphorylated and total ERK, to poly(ADP-ribose) polymer-ase (PARP), to caspase-3, and to BIM were obtained from CellSignaling Technology; those to survivin were from Santa CruzBiotechnology; those to cIAP-1 were from R&D Systems; andthose to b-actin were from Sigma. Mouse monoclonal anti-bodies to EGFR were obtained from Invitrogen. All antibodieswere used at a 1:1,000 dilution, with the exception of those tob-actin (1:200). The nitrocellulose membrane was thenwashed with PBS containing 0.05% Tween 20 before incuba-tion for 1 hour at room temperature with horseradish perox-idase-conjugated goat antibodies to rabbit (Sigma) or mouse(Santa Cruz Biotechnology) immunoglobulin G. Immune com-plexes were finally detected with chemiluminescence reagents(Perkin-Elmer Life Science).

Gene silencingCells were plated at 50% to 60% confluence in 6-well plates

or 25-cm2 flasks and then incubated for 24 hours beforetransient transfection for the indicated times with smallinterfering RNAs (siRNA) mixed with the Lipofectaminereagent (Invitrogen). The siRNAs specific for AKT (AKT-1,50-CCAGGUAUUUUGAUGAGGA-30; AKT-2, 50-CAACCGC-CAUCCAGACUGU-30), survivin (survivin-1, 50-GAAGCA-GUUUGAAGAAUUA-30; survivin-2, 50-AGAAGCAGUUU-GAAGAAUU-30), or BIM (BIM-1, 50-GGAGGGUAUUUUU-GAAUAA-30) mRNAs as well as corresponding scrambled(control) siRNAs were obtained from Nippon EGT.

Annexin V binding assayThe binding of Annexin V to cells was measured with the

use of an Annexin-V-FLUOS Staining Kit (Roche). Cells wereharvested by exposure to trypsin-EDTA, washed with PBS, andcentrifuged at 200 � g for 5 minutes. The cell pellets wereresuspended in 100 mL of Annexin-V-FLUOS labeling solution,incubated for 10 to 15 minutes at 15�C to 25�C, and then

analyzed for fluorescence with a flow cytometer (FACSCali-bur) and Cell Quest software (Becton Dickinson).

Cell cycle analysisCells were harvested, washed with PBS, fixed with 70%

methanol, washed again with PBS, and stained with propi-dium iodide (0.05 mg/mL) in a solution containing 0.1%Triton X-100, 0.1 mmol/L EDTA, and RNase A (0.05 mg/mL).The stained cells were then analyzed for DNA content witha flow cytometer and Modfit software (Verity SoftwareHouse).

Establishment of cells stably overexpressing survivinA full-length cDNA fragment encoding human survivin was

obtained from HCC827 cells by reverse transcription and PCRwith the primers survivin-forward (50-GCGGCCGCGGCGGC-ATGGGTGCCCCGACGTTG-30) and survivin-reverse (50-GGA-TCCTCAATCCATGGCAGCCAGCTGCTCG-30). The ampli-fication product was verified by sequencing after its cloninginto the pCR-Blunt II-TOPO vector (Invitrogen). The survivincDNA was excised from pCR-Blunt II-TOPO and transferred tothe pQCXIH retroviral vector (Clontech). Retroviruses encod-ing survivin were then produced and used to infect PC9 andHCC827 cells as described (24). Cells stably expressing survivinwere then isolated by selection with hygromycin at 300 mg/mL(Invivogen).

Growth inhibition assay in vivoAll animal studies were performed in accordance with the

Recommendations for Handling of Laboratory Animals forBiomedical Research compiled by the Committee on Safetyand Ethical Handling Regulations for Laboratory AnimalExperiments, Kinki University (Osaka, Japan). The ethicalprocedures followed conformed to the guidelines of the Uni-ted Kingdom Coordinating Committee on Cancer PreventionResearch. Tumors cells (5� 106) were injected subcutaneouslyinto the axilla of 5- to 6-week-old female athymic nude mice(BALB/c nu/nu; CLEA Japan). Treatment was initiated whentumors in each group of 6 mice achieved an average volume of200 to 400mm3. Treatment groups consisted of vehicle controland gefitinib (10 or 25 mg/kg). Gefitinib was administered byoral gavage daily for 4 weeks, with control animals receiving a0.5% (w/v) aqueous solution of hydroxypropylmethylcelluloseas vehicle. Tumor volume was determined from caliper mea-surements of tumor length (L) and width (W) according to theformula LW2/2. Both tumor size and body weight were mea-sured twice per week.

Statistical analysisQuantitative data are presented as means � SE from 3

independent experiments or for 6 animals per group unlessindicated otherwise. The significance of differences in thepercentage of Annexin V-positive cells was evaluated with theunpaired 2-tailed Student's t test. P < 0.05 was consideredstatistically significant.

Role of Survivin in EGFR-TKI–Induced Apoptosis

www.aacrjournals.org Cancer Res; 70(24) December 15, 2010 10403



Results

Gefitinib downregulates survivin expression in EGFRmutation-positive NSCLC cell lines

We first examined the effects of the EGFR-TKI gefitinib onthe expression of IAP family members in a subset of NSCLCcell lines (PC9, HCC827, PC9/ZD, H1975, A549, and H1299) byimmunoblot analysis (Fig. 1). PC9 and HCC827 cells harbor anEGFR allele with an activating mutation, whereas A549 andH1299 cells express wild-type EGFR and PC9/ZD and H1975cells harbor an EGFR allele with both an activating mutationand a mutation (T790M) that confers resistance to EGFR-TKIs. In PC9 and HCC827 cells, gefitinib induced the dephos-phorylation of EGFR and reduced the abundance of survivin ina concentration-dependent manner. In contrast, in cellsexpressing wild-type EGFR or harboring the T790M resistancemutation, gefitinib did not affect the phosphorylation level ofEGFR or the expression of survivin. The expression of otherIAP family members, including XIAP and cIAP-1, was notsubstantially affected by gefitinib in any of the cell linesexamined. These data thus showed that gefitinib downregu-lated survivin expression in NSCLC cells with an activatingmutation of EGFR.

Inhibition of the PI3K-AKT pathway results in survivindownregulation in EGFR mutation–positive cells

To identify the signaling pathway (or pathways) respon-sible for downregulation of survivin by gefitinib, we exam-

ined the effects of specific inhibitors of MEK (U0126 andAZD6244) and PI3K (LY294002 and BEZ235) in EGFR muta-tion–positive NSCLC cells (PC9 and HCC827). Each of thePI3K inhibitors reduced the abundance of survivin, whereasthe MEK inhibitors had no such effect (Fig. 2A), suggestingthat the regulation of survivin expression is mediated byPI3K rather than by MEK in EGFR mutation-positive NSCLCcells. Given that the protein kinase AKT is an importantdownstream target of PI3K, we examined whether the PI3K-dependent survivin expression is also dependent on AKT.Depletion of AKT by transfection of cells with 2 differentsiRNAs specific for AKT mRNA (AKT-1 and AKT-2 siRNA)resulted in downregulation of survivin expression in bothPC9 and HCC827 cells (Fig. 2B). These results thus suggestedthat gefitinib might regulate survivin expression throughinhibition of the PI3K-AKT signaling pathway in EGFRmutation–positive NSCLC cells.

Knockdown of survivin expression induces apoptosis inEGFR mutation–positive cells

To investigate whether downregulation of survivin by gefi-tinib is related to gefitinib-induced apoptosis, we transfectedPC9 or HCC827 cells with 2 independent siRNA specific forsurvivin mRNA (survivin-1 and survivin-2 siRNAs). Depletionof survivin resulted in generation of the cleaved forms of bothcaspase-3 and PARP in both cell lines (Fig. 3A). Staining withAnnexin V also revealed that the proportion of apoptotic cellswas markedly increased by transfection with the survivin

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Figure 1. Effects of gefitinib on the expression of IAP family proteins in human NSCLC cells. PC9, HCC827, PC9/ZD, H1975, A549, or H1299 cellswere incubated in complete medium and in the presence of the indicated concentrations of gefitinib for 24 hours. Cell lysates were then preparedand subjected to immunoblot analysis with antibodies to phosphorylated (p) or total forms of EGFR, AKT, or ERK, to survivin, to XIAP, to cIAP-1, or tob-actin (loading control). Data are representative of 3 independent experiments.

Okamoto et al.

Cancer Res; 70(24) December 15, 2010 Cancer Research10404



siRNAs (Fig. 3B). In addition, depletion of survivin resulted inan increase in the size of the sub-G1 (apoptotic) cell popula-tion, as revealed by flow cytometry (Fig. 3C). These datasuggested that downregulation of survivin induces apoptosisin EGFR mutation–positive NSCLC cells.

Overexpression of survivin inhibits gefitinib-inducedapoptosis in EGFR mutation–positive cells in vitroTo examine further the role of survivin in gefitinib-

induced apoptosis, we established PC9 and HCC827 sublines(PC9S7, PC9S8, HCC827S6, and HCC827S7) that stably over-express survivin as a result of retroviral infection. Theabundance of survivin in these sublines was substantiallygreater than that in cells infected with the empty virus (PC9-Mock and HCC827-Mock; Fig. 4A). In addition, gefitinibmarkedly reduced the level of survivin expression in PC9-Mock and HCC827-Mock cells but not in the correspondingsublines overexpressing survivin (Fig. 4B). Immunoblot ana-

lysis of the cleaved forms of caspase-3 and PARP (Fig. 4B) aswell as staining with Annexin V (Fig. 4C) also revealed thatoverexpression of survivin resulted in marked inhibition ofgefitinib-induced apoptosis. Examination of the effect ofgefitinib on cell cycle distribution revealed that gefitinibincreased the proportion of cells in G0–G1 phase andreduced that in S phase at 24 hours in a manner independentof survivin overexpression (Fig. 4D). The survivin-overex-pressing sublines, however, showed a smaller time-depen-dent increase in the size of the sub-G1 cell population thandid cells infected with the empty virus. These results thusfurther indicated that downregulation of survivin by gefiti-nib contributes to the proapoptotic action of this drug inEGFR mutation–positive NSCLC cells.

Overexpression of survivin inhibits the antitumor effectof gefitinib on EGFR mutation–positive cells in vivo

To investigate whether the antitumor effect of gefitinib onEGFR mutation–positive NSCLC cells might be affected bysurvivin overexpression in vivo, we injected HCC827-Mockcells or cells of the survivin-overexpressing subline HCC827S7into nude mice for elicitation of the formation of solid tumors.When the tumors became palpable (200–400 mm3), mice weredivided into 3 groups and treated with vehicle (control) orgefitinib at a daily dose of 10 or 25 mg/kg by oral gavage for 4weeks. Gefitinib treatment at either dose eradicated tumors inmice injected with HCC827-Mock cells (Fig. 5A and C). Incontrast, tumors in mice injected with survivin-overexpres-sing cells were not eradicated by gefitinib even at the dose of25 mg/kg per day, although tumor growth was partiallyinhibited by gefitinib in a dose-dependent manner (Fig. 5Band C). These results showed that survivin overexpressioninhibits the antitumor effect of gefitinib on EGFR mutation–positive NSCLC cells in vivo.

Effect of attenuation of BIM induction on gefitinib-induced apoptosis in EGFR mutation–positive cellsoverexpressing survivin

Survivin overexpression did not completely eliminate gefi-tinib-induced apoptosis in PC9 and HCC827 cells, suggestingthat other signaling pathways might contribute to this pro-cess. Induction of the proapoptotic BH3-only protein BIM hasbeen found to be important for EGFR-TKI–induced apoptosisin EGFRmutation–positive lung cancers, and inhibition of theEGFR-MEK-ERK signaling pathway is required for BIM induc-tion (25–27). We therefore examined whether survivin over-expression in combination with specific inhibition of BIMinduction results in an additive antiapoptotic effect in EGFRmutation–positive NSCLC cells. We transiently transfectedsurvivin-overexpressing sublines of PC9 or HCC827 cells withan siRNA specific for BIM mRNA. Transfection with the BIMsiRNA specifically inhibited the induction of BIM expressionby gefitinib in both mock-infected and survivin-overexpres-sing sublines (Fig. 6A). Staining with Annexin V furtherrevealed that the combination of survivin overexpressionand attenuation of BIM induction resulted in a greater levelof inhibition of gefitinib-induced apoptosis than that observedwith either approach alone (Fig. 6B). These data were

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Figure 2. Effects of inhibition of MEK or PI3K signaling pathways onsurvivin expression in EGFR mutation–positive NSCLC cells. A, PC9 orHCC827 cells were incubated in the absence (control, 0.1% dimethylsulfoxide) or the presence of gefitinib (1 mmol/L), LY294002 (20 mmol/L),BEZ235 (0.2 mmol/L), U0126 (20 mmol/L), or AZD6244 (0.2 mmol/L) for24 hours, after which cell lysates were prepared and subjected toimmunoblot analysis with antibodies to phosphorylated (p) or totalforms of AKT or ERK, to survivin, or to b-actin. B, Cells were transfected (ornot) with 2 different AKT (AKT-1 or AKT-2) or scrambled (control)siRNAs for 48 hours, lysed, and subjected to immunoblot analysis withantibodies to AKT, to survivin, or to b-actin. All data are representativeof 3 independent experiments.





confirmed with a second BIM siRNA to rule out off-targeteffects (Supplementary Fig. 1). These results thus suggestedthat both survivin downregulation and BIM induction con-tribute independently to gefitinib-induced apoptosis in EGFRmutation–positive NSCLC cells.

Discussion

EGFR-TKIs induce marked clinical responses in patientswith NSCLC positive for activating mutations of EGFR (1–3). Invitro experiments have shown that EGFR-TKIs induce a sub-stantial level of apoptosis in NSCLC cell lines expressingmutant EGFRs (4). However, the key downstream mediators

of EGFR-TKI–induced apoptosis in EGFR mutation–positivecells have remained unidentified. We have now found thatgefitinib downregulated survivin expression in EGFR muta-tion-positive NSCLC cells but not in NSCLC cells expressingwild-type EGFR or EGFR with the T790M resistance mutation.With the use of specific PI3K inhibitors and siRNAs specific forAKT mRNA, we further showed that the downregulation ofsurvivin expression by gefitinib is likely mediated throughinhibition of PI3K-AKT signaling. Human epidermal growthfactor receptor 2 (HER2)–targeting agents such as lapatiniband trastuzumab were previously found to induce downre-gulation of survivin through inhibition of the PI3K-AKT path-way in breast cancer cells positive for HER2 amplification

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Figure 3. Effect of survivin depletion on apoptosis in EGFR mutation–positive NSCLC cells. A, PC9 or HCC827 cells were transfected (or not) with2 different survivin (survivin-1 or survivin-2) or scrambled (control) siRNAs for 48 hours, after which cell lysates were prepared and subjected to immunoblotanalysis with antibodies to survivin, to PARP, to caspase-3, or to b-actin. Bands corresponding to the cleaved (cl) forms of caspase-3 and PARP areindicated. Data are representative of 3 independent experiments. B, cells were transfected with survivin or scrambled siRNAs for 72 hours, afterwhich the proportion of apoptotic cells was determined by staining with fluorescein isothiocyanate–conjugated Annexin V and propidium iodide followed byflow cytometry. Data are means � SE from 3 independent experiments. *, P < 0.05 versus the corresponding value for cells transfected with thescrambled siRNA. C, cells were transfected with survivin or scrambled siRNAs for 48 hours, fixed, stained with propidium iodide, and analyzed for cell cycledistribution by flow cytometry. Data are means of triplicates from representative experiments that were repeated 3 times.

Okamoto et al.




(28, 29). Given that downregulation of survivin through inhibi-tion of the PI3K-AKT pathway was induced by EGFR-TKIs inEGFR mutation–positive NSCLC cells and by HER2-targetingagents in breast cancer cells positive for HER2 amplification,the expression of survivin is likely dependent on PI3K-AKTsignaling that operates downstream of receptor tyrosinekinases and is essential for cell survival. This hypothesis isfurther supported by the observation that transfection ofEGFR mutation–positive NSCLC cells with an siRNA specific

for EGFR mRNA resulted in marked inhibition of survivinexpression, whereas transfection of cells expressing wild-typeEGFR had no such effect (Supplementary Fig. 2). The PI3K-AKT pathway has been implicated in the regulation of survivinexpression by cytokines, growth factors, and chemotherapeu-tic drugs (8, 10, 30). Although no direct correlation has beenestablished between downregulation of survivin and inhibi-tion of EGFR signaling, these previous findings support thenotion that inhibition of the EGFR-PI3K-AKT pathway

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Figure 4. Effect of survivin overexpression on gefitinib-induced apoptosis in EGFR mutation–positive NSCLC cells in vitro. A, parental (P) PC9 orHCC827 cells or corresponding sublines either stably overexpressing survivin (PC9S7, PC9S8, HCC827S6, and HCC827S7) or infected with the emptyretrovirus (PC9-Mock and HCC827-Mock) were cultured overnight in complete medium, after which cell lysates were prepared and subjected toimmunoblot analysis with antibodies to survivin or to b-actin. B, PC9 or HCC827 isogenic cell lines were incubated in the presence of the indicatedconcentrations of gefitinib for 48 hours, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p) ortotal forms of EGFR, AKT, or ERK, to survivin, to PARP, to caspase-3, or to b-actin. Data in A and B are representative of 3 independent experiments.C, PC9 or HCC827 isogenic cell lines were incubated with gefitinib (0.1 mmol/L) for the indicated times, after which the proportion of apoptotic cells wasdetermined by staining with Annexin V and propidium iodide followed by flow cytometry. Data are means � SE from 3 independent experiments.*, P < 0.05 versus the corresponding value for cells infected with the empty retrovirus. D, PC9 or HCC827 isogenic cell lines were incubated withgefitinib (0.1 mmol/L) for the indicated times and then analyzed for cell cycle distribution by flow cytometry. Data are means of triplicates fromrepresentative experiments that were repeated 3 times.





contributes to downregulation of survivin expression byEGFR-TKIs in EGFR mutation–positive NSCLC cells.

Survivin has been implicated in resistance of cancer cells toapoptosis, although the effect of survivin expression on gefi-tinib-induced apoptosis in EGFR mutation–positive NSCLCcells has not previously been examined. We have now shownthat survivin overexpression inhibited gefitinib-induced apop-tosis in such cells. Inhibition of the PI3K-AKT and MEK-ERKpathways was previously found to account for much of theproapoptotic activity of EGFR-TKIs in EGFR mutation–posi-tive NSCLC cells (31). We further found that overexpression ofsurvivin resulted in inhibition of apoptosis induced by acombination of PI3K and MEK inhibitors in such cells (Sup-plementary Fig. 3). Increased AKT activity as a result either ofthe loss of PTEN or of expression of a constitutively active

form of AKT was previously found to be associated with areduced sensitivity to EGFR-TKIs in EGFR mutation–positiveNSCLC cells (32). However, the principal molecular targetunderlying the response to inhibition of PI3K-AKT signalingby EGFR-TKIs has remained to be elucidated. In the presentstudy, we show that the sensitivity of EGFRmutation–positiveNSCLC cells to EGFR-TKIs depends, at least in part, onsurvivin downregulation through inhibition of the PI3K-AKT pathway. In our xenograft model, we showed that survi-vin overexpression inhibited the antitumor effect of gefitinibon EGFR mutation–positive NSCLC cells. The extent of theclinical benefit of EGFR-TKIs varies among NSCLC patientsharboring activating EGFRmutations, and the efficacy of thesedrugs is limited by either de novo resistance or resistanceacquired after the onset of therapy (33). Although several

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Figure 5. Effect of gefitinib on the growth of EGFR mutation–positive NSCLC cells overexpressing survivin in vivo. A and B, nude mice with tumorxenografts established by subcutaneous injection of HCC827-Mock or HCC827S7 cells, respectively, were treated daily for 4 weeks with vehicle (control)or gefitinib (10 or 25 mg/kg). Tumor volume was determined at the indicated times after the onset of treatment. Data are means � SE of values from6 mice per group. C, representative mice showing tumors at the end of the 4-week treatment period.

Okamoto et al.




mechanisms of acquired resistance have been described, itremains of clinical concern that molecular markers forprediction of de novo resistance to these drugs have not beenwell delineated (23, 34–38). It will therefore be of interest todetermine whether increased survivin expression in tumors isclinically useful as a negative predictive marker of sensitivityto EGFR-TKIs in patients with EGFR mutation–positiveNSCLC.Our observations revealed that survivin overexpression did

not completely abolish gefitinib-induced apoptosis, suggest-ing that another proapoptotic regulator activated after EGFRinhibition might contribute to EGFR-TKI–induced apoptoticcell death. Previous studies have shown that gefitinib inducesBIM expression via inhibition of the MEK-ERK pathway andthat BIM induction plays a key role in EGFR-TKI–inducedapoptosis in EGFRmutation–positive NSCLC cells (25–27). Wehave now shown that inhibition of both survivin downregula-tion and BIM induction attenuated gefitinib-induced apopto-sis to a greater extent than did inhibition of either processalone. The recent preclinical study showing that the combina-tion of a PI3K inhibitor and a MEK inhibitor, but neither agentalone, induced substantial growth inhibition in EGFR muta-tion–positive NSCLC cells (31) supports the notion that boththe PI3K-AKT-survivin and MEK-ERK-BIM pathways contri-

bute independently to gefitinib-induced apoptosis in suchcells (Fig. 7).

In conclusion, we have shown that the EGFR-TKI gefitinibdownregulated survivin expression, likely through inhibition

Figure 6. Effect of thecombination of survivinoverexpression and inhibition ofBIM induction on gefitinib-induced apoptosis in EGFRmutation–positive NSCLC cells.A, cells stably overexpressingsurvivin (PC9S7 and HCC827S6)or infected with the emptyretrovirus (PC9-Mock andHCC827-Mock) were transfectedwith BIM (BIM-1) or scrambledsiRNAs for 24 hours and thenincubated for 24 hours incomplete medium with or withoutgefitinib (0.1 mmol/L). Cell lysateswere then prepared and subjectedto immunoblot analysis withantibodies to survivin, to BIM, or tob-actin. Data are representative of3 independent experiments.B, cells transfected as in (A) wereincubated for 48 hours in theabsence or presence of gefitinib(0.1 mmol/L) and then evaluated forthe proportion of apoptotic cellsby staining with Annexin V andpropidium iodide followed by flowcytometry. Data are means � SEfrom 3 independent experiments.*, P < 0.05 for the indicatedcomparisons.

80

60

40

Survivin overexpression:BIM-1 siRNA:

Gefitinib:

Survivin overexpression:BIM-1 siRNA:

Gefitinib:

Survivin

BIM-EL

BIM-L

BIM-s

Actin

20

0Ann

exin

V-p

ositi

ve c

ells

(%

) 60

40

20

0Ann

exin

V-p

ositi

ve c

ells

(%

)

PC9 HCC827

PC9

–––

– –– –+ + + +

++++ –

––

– –– –+ + + +

++++

–––

– –– –+ + + +

++++

––

+–––

– –– –+ + + +

++++

––

+

HCC827

B

A

Downregulation of survivin BIM induction

Apoptosis

EGFR-TKls

Inhibition of MEK pathwayInhibition of Pl3K pathway

Figure 7. Proposed model for the intracellular signaling underlyingEGFR-TKI–induced apoptosis in EGFR mutation–positive NSCLC cells.





of PI3K-AKT signaling, and that this effect plays a key role ingefitinib-induced apoptosis. Moreover, we found that survivindownregulation and BIM induction are independentlyrequired for EGFR-TKI–induced apoptosis. Our results thusshow that simultaneous upstream interruption of the PI3K-AKT-survivin and MEK-ERK-BIM pathways mediates EGFR-TKI–induced apoptosis.

Disclosure of Potential Conflicts of Interest

No potential conflicts or interest were disclosed.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 07/06/2010; revised 09/11/2010; accepted 10/08/2010; publishedOnline 12/15/2010.

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Okamoto et al.




2010;70:10402-10410. Cancer Res Kunio Okamoto, Isamu Okamoto, Wataru Okamoto, et al.

MutationsEGFRSmall Cell Lung Cancers Positive for −Induced Apoptosis in Non−Role of Survivin in EGFR Inhibitor

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