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

Survivin is a member of the inhibitor of apoptosis (IAP) family of proteins and has been shown to inhibit caspases and to prevent caspase-mediated cell death (1–3). Survivin is abundant in many types of cancer cells but not in the corresponding normal cells (4, 5). In nonmalignant proliferating cells, the expression of survivin is regulated in a cell cycle-dependent manner (6, 7). The upregulation of survivin expression in tumors does not seem to be dependent solely on the cell cycle, however, given that it occurs in tumor cells that are not actively cycling (4, 8, 9). Indeed, growth factors have been found to regulate survivin expression in endothelial cells and neuroblastoma cells (10, 11). Although expression of survivin has been demonstrated in non–small cell-lung cancer (NSCLC; refs. 12–14), the mechanism by which such expression is regulated in NSCLC cells has remained unknown.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is abnormally amplified or activated in a variety of tumors including NSCLC, and it has therefore been identified 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 kinase pocket of the receptor, have been extensively studied in patients with NSCLC (18, 19). Several prospective clinical trials have revealed marked antitumor activity of EGFR-TKIs in NSCLC patients with EGFR mutations. The therapeutic benefit of these drugs is much greater than that historically observed with standard cytotoxic chemotherapy for advanced NSCLC. NSCLC cells with EGFR mutations manifest activation of the PI3K (phosphatidylinositol 3-kinase)–AKT and MEK–ERK (extracellular signal-regulated kinase) signaling pathways under the control of EGFR, and exposure of such cells to EGFR-TKIs blocks signaling by both pathways and induces apoptosis (20–22). The precise molecular mechanism by which EGFR-TKIs induce apoptosis has remained unclear, however. We have therefore now examined the effect of the EGFR-TKI gefitinib on survivin expression as well as further investigated the mechanism of gefitinib-induced apoptosis in EGFR mutation–positive NSCLC cells.

The human NSCLC cell lines PC9, HCC827, NCI-H1975 (H1975), A549, and H1299 were obtained from American Type Culture Collection. The NSCLC line PC9/ZD was obtained as described previously (23). All cells were cultured under a humidified atmosphere of 5% CO₂ at 37°C in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum. Gefitinib was obtained from Kemprotec, U0126 and LY294002 were from Cell Signaling Technology and BEZ235 and AZD6244 were from ShangHai Biochempartner.

Cells 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 sodium pyrophosphate, 1 mmol/L phenylmethylsulfonyl fluoride, and leupeptin (1 μg/mL). The protein concentration of the cell lysates was determined with the use of the Bradford reagent (Bio-Rad), and equal amounts of protein were subjected to SDS-PAGE on a 7.5% gel. The separated proteins were transferred to a nitrocellulose membrane, which was then exposed to 5% nonfat dried milk in PBS for 1 hour at room temperature before incubation overnight at 4°C with primary antibodies. Rabbit polyclonal antibodies to human phosphorylated EGFR (pY1068), to XIAP, to phosphorylated and total AKT, to phosphorylated and total ERK, to poly(ADP-ribose) polymerase (PARP), to caspase-3, and to BIM were obtained from Cell Signaling Technology; those to survivin were from Santa Cruz Biotechnology; those to cIAP-1 were from R&D Systems; and those to β-actin were from Sigma. Mouse monoclonal antibodies to EGFR were obtained from Invitrogen. All antibodies were used at a 1:1,000 dilution, with the exception of those to β-actin (1:200). The nitrocellulose membrane was then washed with PBS containing 0.05% Tween 20 before incubation for 1 hour at room temperature with horseradish peroxidase-conjugated goat antibodies to rabbit (Sigma) or mouse (Santa Cruz Biotechnology) immunoglobulin G. Immune complexes were finally detected with chemiluminescence reagents (Perkin-Elmer Life Science).

Cells were plated at 50% to 60% confluence in 6-well plates or 25-cm² flasks and then incubated for 24 hours before transient transfection for the indicated times with small interfering RNAs (siRNA) mixed with the Lipofectamine reagent (Invitrogen). The siRNAs specific for AKT (AKT-1, 5′-CCAGGUAUUUUGAUGAGGA-3′; AKT-2, 5′-CAACCGCCAUCCAGACUGU-3′), survivin (survivin-1, 5′-GAAGCAGUUUGAAGAAUUA-3′; survivin-2, 5′-AGAAGCAGUUU GAAGAAUU-3′), or BIM (BIM-1, 5′-GGAGGGUAUUUUUGAAUAA-3′) mRNAs as well as corresponding scrambled (control) siRNAs were obtained from Nippon EGT.

The binding of Annexin V to cells was measured with the use of an Annexin-V-FLUOS Staining Kit (Roche). Cells were harvested by exposure to trypsin-EDTA, washed with PBS, and centrifuged at 200 × g for 5 minutes. The cell pellets were resuspended in 100 μL 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 (FACSCalibur) and Cell Quest software (Becton Dickinson).

Cells were harvested, washed with PBS, fixed with 70% methanol, washed again with PBS, and stained with propidium 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 with a flow cytometer and Modfit software (Verity Software House).

A full-length cDNA fragment encoding human survivin was obtained from HCC827 cells by reverse transcription and PCR with the primers survivin-forward (5′-GCGGCCGCGGCGGCATGGGTGCCCCGACGTTG-3′) and survivin-reverse (5′-GGATCCTCAATCCATGGCAGCCAGCTGCTCG-3′). The amplification product was verified by sequencing after its cloning into the pCR-Blunt II-TOPO vector (Invitrogen). The survivin cDNA was excised from pCR-Blunt II-TOPO and transferred to the pQCXIH retroviral vector (Clontech). Retroviruses encoding survivin were then produced and used to infect PC9 and HCC827 cells as described (24). Cells stably expressing survivin were then isolated by selection with hygromycin at 300 μg/mL (Invivogen).

All animal studies were performed in accordance with the Recommendations for Handling of Laboratory Animals for Biomedical Research compiled by the Committee on Safety and Ethical Handling Regulations for Laboratory Animal Experiments, Kinki University (Osaka, Japan). The ethical procedures followed conformed to the guidelines of the United Kingdom Coordinating Committee on Cancer Prevention Research. Tumors cells (5 × 10⁶) were injected subcutaneously into the axilla of 5- to 6-week-old female athymic nude mice (BALB/c nu/nu; CLEA Japan). Treatment was initiated when tumors in each group of 6 mice achieved an average volume of 200 to 400 mm³. Treatment groups consisted of vehicle control and gefitinib (10 or 25 mg/kg). Gefitinib was administered by oral gavage daily for 4 weeks, with control animals receiving a 0.5% (w/v) aqueous solution of hydroxypropylmethylcellulose as vehicle. Tumor volume was determined from caliper measurements of tumor length (L) and width (W) according to the formula LW²/2. Both tumor size and body weight were measured twice per week.

Quantitative data are presented as means ± SE from 3 independent experiments or for 6 animals per group unless indicated otherwise. The significance of differences in the percentage of Annexin V-positive cells was evaluated with the unpaired 2-tailed Student's t test. P < 0.05 was considered statistically significant.

We first examined the effects of the EGFR-TKI gefitinib on the expression of IAP family members in a subset of NSCLC cell lines (PC9, HCC827, PC9/ZD, H1975, A549, and H1299) by immunoblot analysis (Fig. 1). PC9 and HCC827 cells harbor an EGFR allele with an activating mutation, whereas A549 and H1299 cells express wild-type EGFR and PC9/ZD and H1975 cells harbor an EGFR allele with both an activating mutation and a mutation (T790M) that confers resistance to EGFR-TKIs. In PC9 and HCC827 cells, gefitinib induced the dephosphorylation of EGFR and reduced the abundance of survivin in a concentration-dependent manner. In contrast, in cells expressing wild-type EGFR or harboring the T790M resistance mutation, gefitinib did not affect the phosphorylation level of EGFR or the expression of survivin. The expression of other IAP family members, including XIAP and cIAP-1, was not substantially affected by gefitinib in any of the cell lines examined. These data thus showed that gefitinib downregulated survivin expression in NSCLC cells with an activating mutation of EGFR.

To identify the signaling pathway (or pathways) responsible for downregulation of survivin by gefitinib, we examined the effects of specific inhibitors of MEK (U0126 and AZD6244) and PI3K (LY294002 and BEZ235) in EGFR mutation–positive NSCLC cells (PC9 and HCC827). Each of the PI3K inhibitors reduced the abundance of survivin, whereas the MEK inhibitors had no such effect (Fig. 2A), suggesting that the regulation of survivin expression is mediated by PI3K rather than by MEK in EGFR mutation-positive NSCLC cells. Given that the protein kinase AKT is an important downstream 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 different siRNAs specific for AKT mRNA (AKT-1 and AKT-2 siRNA) resulted in downregulation of survivin expression in both PC9 and HCC827 cells (Fig. 2B). These results thus suggested that gefitinib might regulate survivin expression through inhibition of the PI3K-AKT signaling pathway in EGFR mutation–positive NSCLC cells.

To investigate whether downregulation of survivin by gefitinib is related to gefitinib-induced apoptosis, we transfected PC9 or HCC827 cells with 2 independent siRNA specific for survivin mRNA (survivin-1 and survivin-2 siRNAs). Depletion of survivin resulted in generation of the cleaved forms of both caspase-3 and PARP in both cell lines (Fig. 3A). Staining with Annexin V also revealed that the proportion of apoptotic cells was markedly increased by transfection with the survivin siRNAs (Fig. 3B). In addition, depletion of survivin resulted in an increase in the size of the sub-G₁ (apoptotic) cell population, as revealed by flow cytometry (Fig. 3C). These data suggested that downregulation of survivin induces apoptosis in EGFR mutation–positive NSCLC cells.

To examine further the role of survivin in gefitinib-induced apoptosis, we established PC9 and HCC827 sublines (PC9S7, PC9S8, HCC827S6, and HCC827S7) that stably overexpress survivin as a result of retroviral infection. The abundance of survivin in these sublines was substantially greater than that in cells infected with the empty virus (PC9-Mock and HCC827-Mock; Fig. 4A). In addition, gefitinib markedly reduced the level of survivin expression in PC9-Mock and HCC827-Mock cells but not in the corresponding sublines overexpressing survivin (Fig. 4B). Immunoblot analysis of the cleaved forms of caspase-3 and PARP (Fig. 4B) as well as staining with Annexin V (Fig. 4C) also revealed that overexpression of survivin resulted in marked inhibition of gefitinib-induced apoptosis. Examination of the effect of gefitinib on cell cycle distribution revealed that gefitinib increased the proportion of cells in G₀–G₁ phase and reduced that in S phase at 24 hours in a manner independent of survivin overexpression (Fig. 4D). The survivin-overexpressing sublines, however, showed a smaller time-dependent increase in the size of the sub-G₁ cell population than did cells infected with the empty virus. These results thus further indicated that downregulation of survivin by gefitinib contributes to the proapoptotic action of this drug in EGFR mutation–positive NSCLC cells.

To investigate whether the antitumor effect of gefitinib on EGFR mutation–positive NSCLC cells might be affected by survivin overexpression in vivo, we injected HCC827-Mock cells or cells of the survivin-overexpressing subline HCC827S7 into nude mice for elicitation of the formation of solid tumors. When the tumors became palpable (200–400 mm³), mice were divided into 3 groups and treated with vehicle (control) or gefitinib at a daily dose of 10 or 25 mg/kg by oral gavage for 4 weeks. Gefitinib treatment at either dose eradicated tumors in mice injected with HCC827-Mock cells (Fig. 5A and C). In contrast, tumors in mice injected with survivin-overexpressing cells were not eradicated by gefitinib even at the dose of 25 mg/kg per day, although tumor growth was partially inhibited by gefitinib in a dose-dependent manner (Fig. 5B and C). These results showed that survivin overexpression inhibits the antitumor effect of gefitinib on EGFR mutation–positive NSCLC cells in vivo.

Survivin overexpression did not completely eliminate gefitinib-induced apoptosis in PC9 and HCC827 cells, suggesting that other signaling pathways might contribute to this process. Induction of the proapoptotic BH3-only protein BIM has been found to be important for EGFR-TKI–induced apoptosis in EGFR mutation–positive lung cancers, and inhibition of the EGFR-MEK-ERK signaling pathway is required for BIM induction (25–27). We therefore examined whether survivin overexpression in combination with specific inhibition of BIM induction results in an additive antiapoptotic effect in EGFR mutation–positive NSCLC cells. We transiently transfected survivin-overexpressing sublines of PC9 or HCC827 cells with an siRNA specific for BIM mRNA. Transfection with the BIM siRNA specifically inhibited the induction of BIM expression by gefitinib in both mock-infected and survivin-overexpressing sublines (Fig. 6A). Staining with Annexin V further revealed that the combination of survivin overexpression and attenuation of BIM induction resulted in a greater level of inhibition of gefitinib-induced apoptosis than that observed with either approach alone (Fig. 6B). These data were confirmed with a second BIM siRNA to rule out off-target effects (Supplementary Fig. 1). These results thus suggested that both survivin downregulation and BIM induction contribute independently to gefitinib-induced apoptosis in EGFR mutation–positive NSCLC cells.

EGFR-TKIs induce marked clinical responses in patients with NSCLC positive for activating mutations of EGFR (1–3). In vitro experiments have shown that EGFR-TKIs induce a substantial level of apoptosis in NSCLC cell lines expressing mutant EGFRs (4). However, the key downstream mediators of EGFR-TKI–induced apoptosis in EGFR mutation–positive cells have remained unidentified. We have now found that gefitinib downregulated survivin expression in EGFR mutation-positive NSCLC cells but not in NSCLC cells expressing wild-type EGFR or EGFR with the T790M resistance mutation. With the use of specific PI3K inhibitors and siRNAs specific for AKT mRNA, we further showed that the downregulation of survivin expression by gefitinib is likely mediated through inhibition of PI3K-AKT signaling. Human epidermal growth factor receptor 2 (HER2)–targeting agents such as lapatinib and trastuzumab were previously found to induce downregulation of survivin through inhibition of the PI3K-AKT pathway in breast cancer cells positive for HER2 amplification (28, 29). Given that downregulation of survivin through inhibition of the PI3K-AKT pathway was induced by EGFR-TKIs in EGFR mutation–positive NSCLC cells and by HER2-targeting agents in breast cancer cells positive for HER2 amplification, the expression of survivin is likely dependent on PI3K-AKT signaling that operates downstream of receptor tyrosine kinases and is essential for cell survival. This hypothesis is further supported by the observation that transfection of EGFR mutation–positive NSCLC cells with an siRNA specific for EGFR mRNA resulted in marked inhibition of survivin expression, whereas transfection of cells expressing wild-type EGFR had no such effect (Supplementary Fig. 2). The PI3K-AKT pathway has been implicated in the regulation of survivin expression by cytokines, growth factors, and chemotherapeutic drugs (8, 10, 30). Although no direct correlation has been established between downregulation of survivin and inhibition of EGFR signaling, these previous findings support the notion that inhibition of the EGFR-PI3K-AKT pathway contributes to downregulation of survivin expression by EGFR-TKIs in EGFR mutation–positive NSCLC cells.

Survivin has been implicated in resistance of cancer cells to apoptosis, although the effect of survivin expression on gefitinib-induced apoptosis in EGFR mutation–positive NSCLC cells has not previously been examined. We have now shown that survivin overexpression inhibited gefitinib-induced apoptosis in such cells. Inhibition of the PI3K-AKT and MEK-ERK pathways was previously found to account for much of the proapoptotic activity of EGFR-TKIs in EGFR mutation–positive NSCLC cells (31). We further found that overexpression of survivin resulted in inhibition of apoptosis induced by a combination of PI3K and MEK inhibitors in such cells (Supplementary Fig. 3). Increased AKT activity as a result either of the loss of PTEN or of expression of a constitutively active form of AKT was previously found to be associated with a reduced sensitivity to EGFR-TKIs in EGFR mutation–positive NSCLC cells (32). However, the principal molecular target underlying the response to inhibition of PI3K-AKT signaling by EGFR-TKIs has remained to be elucidated. In the present study, we show that the sensitivity of EGFR mutation–positive NSCLC cells to EGFR-TKIs depends, at least in part, on survivin downregulation through inhibition of the PI3K-AKT pathway. In our xenograft model, we showed that survivin overexpression inhibited the antitumor effect of gefitinib on EGFR mutation–positive NSCLC cells. The extent of the clinical benefit of EGFR-TKIs varies among NSCLC patients harboring activating EGFR mutations, and the efficacy of these drugs is limited by either de novo resistance or resistance acquired after the onset of therapy (33). Although several mechanisms of acquired resistance have been described, it remains of clinical concern that molecular markers for prediction of de novo resistance to these drugs have not been well delineated (23, 34–38). It will therefore be of interest to determine whether increased survivin expression in tumors is clinically useful as a negative predictive marker of sensitivity to EGFR-TKIs in patients with EGFR mutation–positive NSCLC.

Our observations revealed that survivin overexpression did not completely abolish gefitinib-induced apoptosis, suggesting that another proapoptotic regulator activated after EGFR inhibition might contribute to EGFR-TKI–induced apoptotic cell death. Previous studies have shown that gefitinib induces BIM expression via inhibition of the MEK-ERK pathway and that BIM induction plays a key role in EGFR-TKI–induced apoptosis in EGFR mutation–positive NSCLC cells (25–27). We have now shown that inhibition of both survivin downregulation and BIM induction attenuated gefitinib-induced apoptosis to a greater extent than did inhibition of either process alone. The recent preclinical study showing that the combination of a PI3K inhibitor and a MEK inhibitor, but neither agent alone, induced substantial growth inhibition in EGFR mutation–positive NSCLC cells (31) supports the notion that both the PI3K-AKT-survivin and MEK-ERK-BIM pathways contribute independently to gefitinib-induced apoptosis in such cells (Fig. 7).

In conclusion, we have shown that the EGFR-TKI gefitinib downregulated survivin expression, likely through inhibition of PI3K-AKT signaling, and that this effect plays a key role in gefitinib-induced apoptosis. Moreover, we found that survivin downregulation and BIM induction are independently required for EGFR-TKI–induced apoptosis. Our results thus show that simultaneous upstream interruption of the PI3K-AKT-survivin and MEK-ERK-BIM pathways mediates EGFR-TKI–induced apoptosis.

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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.