caspase-independent cell death is involved in the …...2013/01/22 · cancer therapy: preclinical...

Cancer Therapy: Preclinical

Caspase-Independent Cell Death Is Involved in the NegativeEffect of EGF Receptor Inhibitors on Cisplatin in Non–SmallCell Lung Cancer Cells

Hirohito Yamaguchi1, Jennifer L. Hsu1,4,5, Chun-Te Chen1,3,4, Ying-Nai Wang1,4,5, Ming-Chuan Hsu1,Shih-Shin Chang1,3, Yi Du1,3, How-Wen Ko1, Roy Herbst2, and Mien-Chie Hung1,3,4,5

AbstractPurpose: Results of multiple clinical trials suggest that EGF receptor (EGFR) tyrosine kinase inhibitors

(TKI) exhibit negative effects on platinum-based chemotherapy in patients with lung cancer with wild-type

(WT) EGFR, but the underlying molecular mechanisms are still uncertain. Studies that identify the

mechanism of how TKIs negatively affect patients with WT EGFR are important for future development

of effective strategies to target lung cancer. Thus, we returned to in vitro study to investigate and determine a

possible explanation for this phenomenon.

Experimental Design: We investigated the effects of TKIs and cisplatin on caspase-independent cell

death (CID) and the role of CID in the efficacy of each drug and the combination. Furthermore, we studied

the mechanism by which EGFR signaling pathway is involved in CID. Finally, on the basis of the identified

mechanism, we tested the combinational effects of cisplatin plus suberoylanilide hydroxamic acid (SAHA)

or erastin on CID.

Results: We found that gefitinib inhibited cisplatin-induced CID but not caspase-dependent apoptotic

cell death. In WT EGFR cells, gefitinib not only inhibited CID but also failed to induce apoptosis, therefore

compromising the efficacyof cisplatin. InhibitionofEGFR-ERK/AKTbygefitinibactivates FOXO3a,which in

turn reduces reactiveoxygen species (ROS) andROS-mediatedCID. Toovercome this,we showed that SAHA

and erastin, the inducers of ROS-mediated CID, strongly enhanced the effect of cisplatin in WT EGFR cells.

Conclusion: TKI-mediated inhibition of CID plays an important role in the efficacy of chemotherapy.

Moreover, FOXO3a is a key factor in the negative effects of TKI by eliminating cisplatin-induced ROS. Clin

Cancer Res; 1–10. �2012 AACR.

IntroductionLung cancer is a leading cause of cancer-related death in

the United States. More than 70% of patients with lung

cancer diagnosed at advanced stage, and those patientsare treated primarily with platinum-based chemotherapy(1). Recently, the EGF receptor (EGFR) tyrosine kinaseinhibitors (TKI) such as gefitinib or erlotinib have showneffectiveness in blocking tumor growth and increasedsurvival rate. Preclinical studies showed that gefitinibenhances the efficacy of cytotoxic drugs (2, 3). However,several large-scale phase III clinical trials that were con-ducted in the United States to test the combination ofTKIs and chemotherapy in randomly selected patientswith lung cancer (4–6) failed when patient groups thatreceived TKIs and chemotherapy did not show any benefitin the overall survival rate compared with chemotherapyalone (7). Surprisingly, several studies showed that sen-sitivity of patients with lung cancer to gefitinib correlatedwith EGFR mutations in which patients who had mutant(mt) but not those with wild-type (WT) EGFR showedresponse to gefitinib (8, 9). Subsequently, data analysis ofEGFR mutation status from clinical trials indicated thatTKIs might even induce a negative or antagonistic effectwhen administered with chemotherapeutic drugs inpatients with WT EGFR, whereas additive effects wereobserved in patients with mt EGFR (7).

Authors' Affiliations: Departments of 1Molecular and Cellular Oncologyand 2Thoracic/Head and Neck Medical Oncology, The University of TexasMD Anderson Cancer Center; 3Graduate School of Biomedical Sciences,The University of Texas, Houston, Texas; 4Center for Molecular Medicineand Graduate Institute of Cancer Biology, China Medical University and5Asia University, Taichung, Taiwan

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

J.L. Hsu and C.-T. Chen contributed equally to this work.

Current address for R. Herbst: Department of Medical Oncology, YaleUniversity School of Medicine, New Haven, CT.

In memoriam of Mr. Nan-Tu Haung for his courageous fight against lungcancer.

Corresponding Author: Mien-Chie Hung, Department of Molecular andCellular Oncology, Unit 108, The University of Texas MD Anderson CancerCenter, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3668; Fax: 713-794-3270; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-2621

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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Research. on May 8, 2020. © 2013 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 23, 2013; DOI: 10.1158/1078-0432.CCR-12-2621

http://clincancerres.aacrjournals.org/

Studies that identify the mechanism of how TKIsnegatively affect patients with WT EGFR will likely beimportant for future development of effective strategiesto target lung cancer. Thus, we return to in vitro study toinvestigate and determine a possible explanation for thisphenomenon. EGFR TKIs exhibit distinct responses inWT EGFR and mt EGFR lung cancer cells: they induceapoptotic (caspase-dependent) cell death in lung cancercells expressing mt EGFR (10) but not in those expressingWT EGFR (11). Cisplatin, a commonly used drug fortreating lung cancer, can induce cell death via caspase-dependent (CDD; apoptosis) or caspase-independentcell death (CID) pathway (12, 13) regardless of EGFRmutation status. Because we discovered that gefitinibactually inhibits CID independently of EGFR mutation,we hypothesized that the absence of active TKI-inducedapoptosis in WT EGFR cells concurrent with gefitinib-induced inhibition of CID might negatively impact thetherapeutic benefit of cisplatin. Here, we identified apotential mechanism for TKI-mediated inhibition of CIDand provided, at least in part, an explanation to why theclinical trials of combination of TKIs and chemothera-peutic drugs have failed in lung cancer patients with WTEGFR.

Materials and MethodsDetection of cell death

To determine viability, we stained the cells with Try-pan blue dye (Fig. 1D and Supplementary Figs. S1B andS2A) and counted at least 200 to 300 cells under micro-scope. All experiments were carried out in triplicate andrepeated several times. To determine the long-term via-bility, the cells were seeded in 6-well plates at about 50%confluency and treated with the indicated reagents. Themedium was changed 4 to 5 days later and furthercultured for 10 days. The living cells were then stainedwith crystal violet.

ReagentsCaspase inhibitor z-VAD-fmk was purchased from

Axxora. Cisplatin, N-acetyl-L-cysteine (NAC), U0126, anderastin were obtained from Sigma. Suberoylanilide hydro-xamic acid (SAHA) was synthesized as described previously(14). AKT inhibitor (AKTi), MK2206, was obtained fromSelleck Chemicals. The anti-Bax 6A7, phospho-ERK, extra-cellular signal–regulated kinase (ERK), Mn-superoxidedismutase (SOD), and catalase antibodies were purchasedfrom Santa Cruz Biotechnology and anti-Bax, AKT sub-strate, AKT, FOXO3a, phosho-FOXO3a (Thr32), GSK3b,phosphor-GSK3b (Ser9) antibodies from Cell SignalingTechnology.

Cell cultureAll cell lines used in this research were obtained

from American Type Culture Collection and maintainedin MDEM/F112 medium supplemented with 10% FBSand antibiotics. No further authentication was conducted.Transient and stable transfections were conducted byelectroporation.

Plasmid and siRNAMyc-tagged Bcl-XL expression plasmid, pcDNA3-myc-

Bcl-XL, was described previously (14). The active form ofAKT and mitogen-activated protein/extracellular signal–regulated kinase (MEK), FOXO3a mutant, WT, and GFP-FOXO3a expression plasmid were described previously(15). siRNAs against FOXO3a were purchased from Sigma,and siRNAs for apoptosis inducing factor (AIF), calpain,Mn-SOD, and catalase were purchased from Dharmacon.

Animal modelH1299 or A549 cells stably expressing Bcl-XL and lucif-

erase was used for xenograft experiment. In brief, 5 � 106

(H1299) or 2 � 106 (A549) cells were intrathoracicallyinjected into the chest of nude mice. Drug treatments werestarted on day 29. Cisplatin (1 mg/kg) was injected intra-peritoneally once per week, whereas gefitinib (100 mg/kg)was injected by oral injection 5 times per week. Total2 treatment cycles were conducted. Luciferase activity,which represents tumor volume, was monitored by the InVivo Imaging System. Each treatment group contains 10(for H1299) or 20 (for A549) mice.

ROS measurementCellular reactive oxygen species (ROS) levels were deter-

mined by using H2DCFDA (Molecular Probe). Briefly, cellstreatedor untreatedwithdrugswere trypsinizedandwashedwith PBS and then incubated in PBS containing 10ng/mLofH2DCFDA for 10 minutes at room temperature. Fluores-cence signal were measured using flow cytometry.

ResultsAntagonistic effect of TKI on cisplatin inWT EGFR lungcancer cells

To determine the combinational effects of EGFR TKIs andchemotherapeutic drugs in vitro, we first treated lung cancer

Translational RelevanceEGF receptor (EGFR) tyrosine kinase inhibitors (TKI)

have been used to treat various cancers and shownexcellent clinical results, especially in patients with lungcancer with mutant EGFR. However, results from mul-tiple clinical trials support the notion that TKIs exhibitnegative effects on platinum-based chemotherapy inpatients with lung cancer with wild-type EGFR, andthe underlying molecular mechanisms are still uncer-tain. Here, we show a possible mechanism by whichFOXO3a-mediated attenuation of caspase-independentcell death (CID) plays an important role in the negativeeffects of TKI. Moreover, we also showed that reactiveoxygen species–inducible drugs enhance cisplatin-induced CID. Thus, this study provides the possibleexplanation of the failure of the previous clinical trialsas well as a novel insight into future cancer therapy.

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Clin Cancer Res; 2013 Clinical Cancer ResearchOF2




cells harboring either mt EGFR (H3255, PC9) or WT EGFR(A549,H1299)with cisplatin, gefitinib, or the combinationand measured the caspase-3 activity, which is indicative ofapoptosis. As shown in Fig. 1A, cisplatin induced apoptosisin both mt EGFR and WT EGFR cells, whereas gefitinibinduced apoptosis only in mt EGFR cells. As expected, cellgrowth of WT EGFR cells was not significantly affectedunder gefitinib treatment (Supplementary Fig. S1). Thedifferential response to cisplatin and gefitinib was furthervalidated by long-term drug treatment (Fig. 1B). Cisplatinnearly eliminated viable cells as effectively as gefitinib inmtEGFR cells; however, only cisplatin treatment eliminatedthe majority of cells with WT EGFR but not gefitinib.

Interestingly, we observed residual cells after the combina-tional treatment only inWT EGFR cells (Fig. 1B), suggestingthat the TKImight have also reversed the cell-killing effect ofcisplatin in WT EGFR lung cancer cells. These results areconsistent with the data from the previous clinical trials inwhich addition of EGFR TKI to chemotherapy yieldeddisappointing outcomes in patient with WT EGFR (7), andthus prompted us to further investigate the underlyingmechanisms.

The role of CID in cisplatin-induced cell deathOur data from above indicate that gefitinib reduces the

killing effect of cisplatin without any significant effects on

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Figure 1. Gefitinib induces apoptotic cell death (CDD) in only mt EGFR lung cancer cells, whereas cisplatin induces both CDD and CID in WT EGFR and mtEGFR lung cancer cells. A, H3255 (mt EGFR), PC9 (mt EGFR), H1299 (WT EGFR), and A549 (WT EGFR) lung cancer cells were treated with 50 mmol/L ofcisplatin (Cis) and/or 5 mmol/L of gefitinib (Gef) for 24 hours and subjected to caspase assay (n ¼ 3; �, P < 0.01; #, P < 0.05). In A549 and H1299 cells,there is nosignificant differencebetweenCis andCisþGef. B, the samecells used inAwere treatedwith 50mmol/Lof cisplatin (Cis) and/or 5mmol/L of gefitinib(Gef), and the long-term effects of drug combination were determined as described in Materials and Methods. The relative densities of cells(from the left) treatedwith cisplatin (Cis) or ciplatin plus gefitinib (CisþGef) are shown on the right (n¼ 3). C, A549 cells were treatedwith 50 mmol/L of cisplatinin the presence or absence of 50 mmol/L of z-VAD-fmk, and the viability was determined by Trypan blue dye exclusion assay (left, n ¼ 3). The cellularmorphology of A549 cells treated for 72 hours (right). Right, the cells stained with Trypan blue dye. Arrows indicate typical apoptotic cell death, whereasarrowheads indicate CID. D, A549 control (Neo) or Bcl-XL stable (Bcl-XL) cells were treatedwith 50 mmol/L of cisplatin. The viability was determined by Trypanblue dye exclusion assay (top, n ¼ 3). Caspase activity was determined by caspase assay (bottom, n ¼ 3).

EGFR TKIs Inhibit Caspase-Independent Cell Death

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the caspase activity, suggesting that other caspase-indepen-dent mechanism may be involved in the antagonistic effectof TKI on cisplatin in WT EGFR cells. Because it has beenshown that chemotherapeutic drugs induce CID (16–19),we hypothesized that cisplatin also induces CID in additionto apoptosis in lung cancer cells, whereas gefitinib inhibitsCID. To test this hypothesis, we first measured cisplatin-induced CID inWT EGFR cells by blocking apoptosis with acaspase inhibitor (z-VAD-fmk). When we treated A549 andH1299 cells with cisplatin (Fig. 1C and Supplementary Fig.S2A), a majority of the cells exhibited typical apoptosismorphology including membrane blebbing, apoptoticbody formation, and cell shrinkage (Fig. 1C, right, arrows).However, in the presence of z-VAD-fmk, which inhibitsapoptosis, cells treated with cisplatin were eventually killedwithout any apoptotic morphologic changes as shown byTrypan blue dye, which stains all dead cells, suggesting CIDhad occurred (Fig. 1C, right). In some cells treated withcisplatin alone, we observed similar CID characteristicswithout any apoptoticmorphologic changes (Fig. 1C, right,arrowheads), indicating both apoptosis and CID hadoccurred.

To mimic the effect of z-VAD-fmk, we also establishedBcl-XL overexpressing stable clones in WT EGFR cells toblock cisplatin-induced apoptotic signaling. Indeed, cis-platin-induced caspase activation was completely inhib-ited in A549 Bcl-XL stable line (Fig. 1D, bottom). Aftercisplatin treatment, the Bcl-XL stable cells eventuallyunderwent CID, which is similar to the treatment ofcisplatin plus z-VAD-fmk (Fig. 1D, top and Supplemen-tary Fig. S2B). We also obtained similar results usingH1299 cells (Supplementary Fig. S2C). Furthermore, weconfirmed the HMGB1 was released from the cells toextracellular environment after cisplatin treatment (datanot shown), which is the characteristic of caspase-inde-pendent necrosis (19). Therefore, the CID induced bycisplatin is likely necrosis. Collectively, these results indi-cate that cisplatin induces both apoptosis and CID in lungcancer cells and that CID remains intact when apoptosisis compromised.

The effects of TKI on cisplatin-induced CIDNext, we examined the effects of TKIs on CID induced by

cisplatin.We treatedH1299 and A549 cells with cisplatin orcisplatin plus gefitinib in the presence or absence of z-VAD-fmk and compared the effects of gefitinib on cisplatin-induced cell death. We did not observe any effects ofgefitinib alone on cell viability and caspase activation inthese cells (data not shown). However, when we addedgefitinib to cells pretreated with cisplatin and z-VAD-fmk,we observed increased cell viability (Fig. 2A). When apo-ptosis is inhibited by z-VAD-fmk inmt EGFR cells, CID wasalso attenuated by gefitinib (Supplementary Fig. S3A).Likewise, whenwe overexpressed Bcl-XL to block apoptosis,we found that gefitinib inhibited cisplatin-induced celldeath more strongly in Bcl-XL stable cells than control cells(Fig. 2B). Similar results were observed when we examinedthe long-term effect of these drugs (Fig. 2C) and when we

used erlotinib, another EGFR TKI, instead of gefitinib(Supplementary Fig. S3B and S3C). To further validatethe negative effects of TKI on cisplatin in vivo, we estab-lished H1299 and A549 Bcl-XL/luciferase stable cell linesand inoculated the cells into the lung of nude mice. Theanimals were then treated with cisplatin or gefitinib or thecombination, and then the tumor volumes were moni-tored by a bioluminescent imaging system. As shownin Fig. 2D, gefitinib alone did not inhibit tumor growthin H1299-Bcl-XL or in A549-Bcl-XL (Supplementary Fig.S4) tumors compared with cisplatin. These results areconsistent with that from in vitro study (Fig. 1). Impor-tantly, the effects of cisplatin on these tumors growthwere significantly attenuated by the addition of gefitinib(Fig. 2D and Supplementary Fig. S4), further indicatingthat EGFR TKIs have negative effects on cisplatin-inducedantitumor activity.

Themolecular mechanisms of TKI-mediated inhibitionof CID

Next, to explain the underlyingmolecularmechanisms inTKI-mediated inhibition of CID, we first analyzed theactivity of 2 major EGFR downstream molecules, ERK andAKT, in the presence of cisplatin. Consistent with theprevious study (20), our data also showed that EGFR, ERK,and AKT were activated by cisplatin (Fig. 3A and Supple-mentary Fig. S5). The addition of TKIs was able to reversethe cisplatin-enhanced AKT/ERK activation (Fig. 3A andSupplementary Fig. S5), which suggests that activation ofEGFR-AKT/ERK pathway plays a role in cisplatin-inducedCID. To validate this, we treated the cells with cisplatin inthe presence of MEK or AKTi. Inhibition of either MEK/ERK or AKT-attenuated CID induced by cisplatin in Bcl-XLstable A549 (Fig. 3B) or H1299 cells (Fig. 3C and Sup-plementary Fig. S6). In contrast, overexpression of activeform of MEK or AKT promoted CID induced by cisplatin(Fig. 3D). Thus, these results indicate that EGFR down-stream ERK/AKT signaling increased cisplatin-inducedCID.

Next, we investigated which downstream signalingmole-cules of ERK/AKT are involved in cisplatin-induced CID.Previously, it has been shown that FOXO3a is a commontarget of AKT and ERK (15, 21) and that FOXO3a protectscells from oxidative stress (22, 23). Phosphorylation ofFOXO3a by AKT induces the nuclear exclusion of FOXO3aand inhibits its transcriptional activity (21). Our data indi-cated that the basal level of AKT was unable to phosphor-ylate FOXO3a or induce FOXO3a nuclear exclusion (Fig.4A). However, we observed increased phosphorylation andnuclear exclusion of FOXO3a under cisplatin treatment(Fig. 4A) that is most likely through cisplatin-enhancedEGFR and AKT activation (Fig. 3A). In contrast, cisplatin-induced FOXO3a nuclear exclusion was reversed by gefi-tinib treatment (Fig. 4A). Thus, to determine if FOXO3aplays a role in cisplatin-induced CID, we establishedH1299 GFP-FOXO3a stable cell lines and observedtheir response to cisplatin in the presence of z-VAD-fmk(Fig. 4B). As shown in Fig. 4B, CID was attenuated by

Yamaguchi et al.





overexpression of GFP-FOXO3a. We also observed nucle-ar exclusion of GFP-FOXO3a after cisplatin treatment,which was reversed by gefitinib (Fig. 4B, right). In con-trast, when FOXO3a was knocked down using 2 specificsiRNAs for FOXO3a, cisplatin-induced CID was enhanced(Fig. 4C). The mutant FOXO3a (TM) in which 3 phos-phorylation sites (21) by AKT were substituted withalanine protected cells from cisplatin-induced CID moreeffectively than did WT FOXO3a. However, in the pres-ence of TKI, the protective effect of both WT FOXO3a andmutant FOXO3a are similar, suggesting that TKI-mediat-ed dephosphorylation of FOXO3a is critical for the neg-ative effect of TKI on cisplatin (Fig. 4D). Collectively, theresults suggest that cisplatin enhances EGFR/AKT kinaseactivity and that the activated EGFR/AKT further inacti-vates FOXO3a via phosphorylation.

The role of ROS in cisplatin-induced CIDAs mentioned earlier, it has been shown that FOXO3a

protects cells from oxidative stress. Both catalase and Mn-SOD, which are known to reduce cellular ROS level, arecritical FOXO3a targets for FOXO3a-mediated cytoprotec-tive effect from ROS (17, 18). Because cisplatin is alsoknown to induce ROS (24, 25), which is induced by varietyof stress such as etoposide, arsenic trioxide, TNF, andhistone deacetylase inhibitor (HDACi; refs. 16, 26–28), wehypothesized that ROS might be involved in cisplatin-induced CID. Indeed, ROS was upregulated in response tocisplatin, (Fig. 5A), and the addition of NAC, an antioxi-dant, inhibited CID but not apoptosis induced by cisplatin(Fig. 5B and C and Supplementary Fig. S7). Moreover, wefound that gefitinib decreased cisplatin-induced ROS (Fig.5A), indicating that TKI inhibits cisplatin-induced CID by

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Figure 2. TKI inhibits CID induced by cisplatin. A, A549 or H1299 cells were treated with 50 mmol/L of cisplatin (Cis) or cisplatin plus 5 mmol/L of gefitinib(Cis þ Gef) in the presence or absence of 50 mmol/L of z-VAD-fmk (z-VAD). Cellular morphology of A549 and H1299 cells at 72 hours treatment (left). Theviability was determined by Trypan blue dye exclusion assay (right, n¼ 3). B, A549 Bcl-XL stable cells (Bcl-XL) or empty vector–transfected control cells (Neo)were treated with cisplatin (Cis) or cisplatin plus gefitinib (Cis þ Gef), and the viability was determined by Trypan blue dye exclusion assay (n ¼ 3). C,H1299 and A549 Bcl-XL and their control cells were treated with cisplatin (Cis) or cisplatin plus gefitinib (Cis þ Gef), and the long-term effects of drugswere determined as described in Materials and Methods. The relative cell densities are shown on the right (n ¼ 3). D, the effects of cisplatin, gefitinib,and their combination on H1299 Bcl-XL/luciferase tumor growth in vivo were investigated by orthotopic lung cancer xenograft model. The graph showsluciferase activity, which represents tumor sizes, at day 50 after cancer cell inoculation. Tumor size of cisplatin/gefitinib-treated group (Cis þ Gef) issignificantly larger than that of cisplatin alone (Cis; n ¼ 10; �, P < 0.01).






blocking ROS production. As mentioned earlier, activationof FOXO3a by gefitinib inhibited CID. Two FOXO3a tar-gets, Mn-SOD and catalase (22, 23), which are known toreduce cellular ROS level, were upregulated by inhibitingEGFR-ERK/AKT signaling (Fig. 5D), and knockdown ofthese FOXO3a targets sensitized the cells to cisplatin-induced CID in the presence of gefitinib (SupplementaryFig. S8). Moreover, knockdown of FOXO3a enhanced cel-lular ROS level in response to cisplatin (Fig. 5E). In H1299Bcl-XL xenograft tumors, cisplatin induced FOXO3a phos-phorylation and decreased the expression of Mn-SOD,which was reversed by gefitinib (Supplementary Fig. S9).Thus, EGFR signaling contributed to cisplatin-induced CID,at least in part, through ROS upregulation by the inhibitionof FOXO3a. In addition to FOXO3a, we also tested the roleof AIF and calpain-1, which are known to function in CID(12, 13, 29, 30), in cisplatin-induced CID by using siRNA.However, knockdown of either AIF or calpain-1 did notattenuate cisplatin-induced CID (data not shown).

Finally, as the above molecular pathway provides a ratio-nale to propose a combination treatment to sensitize cell-killing activity of cisplatin, we tested reagents that enhance

CID through upregulation of ROS in combination withcisplatin. For example, both HDACi and erastin have beenreported to induce CID through upregulation of ROS(27, 31). We confirmed that SAHA, an HDACi, and erastinindeed increased cellular ROS and enhanced cisplatin-inducedROS(Supplementary Fig. S10). Thus,we combinedcisplatin with either HDACi (SAHA) or erastin in A549-Bcl-XL and H1299-Bcl-XL stable cells. Using low dose of cis-platin (25 mmol/L) that kills only 10% cells at 48 hours, weobserved significant synergistic killing effect by combina-tion with SAHA or erastin (Fig. 6A and B). These resultsfurther support our model that CID plays a critical role inthe efficacy of cisplatin in lung cancer cells (Fig. 6C).

DiscussionEGFR TKIs have been shown distinct responses in WT

EGFR and mt EGFR lung cancer cells. In WT EGFR lungcancer cells, EGFR TKIs have almost no effects on cellviability. In contrast, mt EGFR lung cancer cells are quitesensitive to EGFR TKIs and frequently undergo apoptosis inresponse to TKI treatment (11). One potential mechanismby which mt EGFR lung cancer cells undergo apoptosis is

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Figure 3. ERK and AKT are criticaldownstreammolecules of EGFR incisplatin-induced CID. A, H1299Bcl-XL stable cells were treatedwith cisplatin in the presence orabsence of TKIs (Gef: gefitinib, 5mmol/L; Erl: erlotinib, 2 mmol/L) for18 hours. Western blot analysiswas conductedusing the indicatedantibodies. B, A549 Bcl-XL cellswere treated with 50 mmol/L ofcisplatin in the presence of a MEKinhibitor (MEKi: U0126, 2 mmol/L)or AKTi (MK2206, 2 mmol/L). After72 hours, the viability wasdetermined by Trypan blue dyeexclusion assay (n ¼ 3). Thecellularmorphology is shown in theright. C, H1299 Bcl-XL stable cellswere treated with cisplatin orcisplatin plus the MEKi or AKTi,and the long-term effects of drugcombination were determined asdescribed in Materials andMethods. The relative celldensities are shown on the right(n¼ 3). D, H1299 Bcl-XL cells weretransiently overexpressed withactive forms of MEK (CA-MEK) orAKT (Myr-AKT) expressionplasmid or empty vector (Vector)and treated with 50 mmol/L ofcisplatin. The viability wasdetermined by Trypan blue dyeexclusion assay (n ¼ 3). Thecellular morphology is shown onthe right.

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through upregulation of BH3-only proapoptotic Bcl-2 fam-ily protein Bim and downregulation of antiapoptotic Bcl-2family protein Mcl-1 as they have been shown to be criticalfor TKI-induced apoptosis in mt EGFR lung cancer cells(10). Both WT EGFR and mt EGFR are known to transducequalitatively distinct signals, such as the AKT and STATsignaling pathways, possibly due to the structural altera-tions within the catalytic pocket affecting substrate speci-ficity or altered interactions with accessory proteins thatmodulate EGFR signaling (11). Therefore, the initial mech-anismof how TKIs affect patients betweenWT EGFR andmtEGFR lung cancer may be EGF-mediated autophosphoryla-tion of multiple tyrosine residues linked to activation ofdistinct downstream effectors. These effectors may regulateBim and Mcl-1 levels in mt EGFR cells but not in WT EGFR

cells, and thus, the inhibition of EGFR inmt EGFR cellsmayinitiate the apoptotic program via Bim/Mcl-1 alteration.Here, we showed EGFR TKI has a negative effect on cisplatinregardless of EGFR status. We have shown that in both WTandmt EGFR cells, gefitinib inhibited cisplatin-induced celldeath, while caspase activity was blocked (Fig. 2A andSupplementary Fig. S3A). Therefore, in mt EGFR cells, TKIsinhibited CID and induced apoptosis, whereas inWT EGFRcells, TKIs only inhibited CID in WT EGFR cells withoutinducing apoptosis (Fig. 6C).

Although we showed here that AKT/ERK-FOXO3a path-ways has negative effects on CID, it is quite possible thatother downstream target of this signaling are also involvedin the negative effects on CID. For example, AKT is thenegative regulator of autophagy, via several pathways such

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Figure 4. FOXO3a plays a critical role in gefitinib-induced resistance to cisplatin. A, H1299 BCl-XL cells treated with cisplatin in the presence or absenceof TKIs (Gef: gefitinib, 5 mmol/L) for 24 hours, and subjected to immunostaining using FOXO3a antibody and 40,6-diamidino-2-phenylindole (DAPI; top).Phospho-FOXO3a (p-Thr 32) was detected in the same sample as used in Fig. 3A by Western blot analysis using the indicated antibodies (right). B, H1299GFP-FOXO3a stable (2 independent clones, 2-2 and 2-3) and control vector–transfected (Neo) cells were treatedwith 50 mmol/L of cisplatin in the presence of50 mmol/L of z-VAD-fmk, and the viability was determined by Trypan blue dye exclusion assay (n ¼ 3). The cellular morphology is shown in the middle.The right fluorescence pictures in the right show the expression and localization of GFP-FOXO3a in H1299 GFP-FOXO3a 2-2 (expression levelsof GFP-FOXO3a in clone 2-2 and 2-3 are similar). GFP-FOXO3a was excluded from the nucleus after cisplatin treatment (top right: Cis) that isinhibited by gefitinib (bottom right: CisþGef). C, A549 Bcl-XL stable cell were transiently transfected with siRNA for FOXO3a (2 different siRNAs, FOXO3a #1or #2) or control siRNA (siCont) and treated with cisplatin. At 48 hours posttransfection, the viability was determined by Trypan blue dye exclusionassay (n ¼ 3). The cellular morphology is shown on the right. D, the effects of WT FOXO3a and AKT-unphosphorylated FOXO3a mutant (TM) oncisplatin-induced CID were investigated. H1299 Bcl-XL cells were transiently transfected with empty vector (empty), WT FOXO3a or unphosphorylatedFOXO3a (TM), and treated with cisplatin in the presence or absence of gefitinib for 72 hours. The viability was determined by Trypan blue dye exclusionassay (n ¼ 3).






as mTOR (32), and autophagy has been shown to havenegative effects on chemotherapy and protect cells fromoxidative stress (33). Thus, the inhibition of AKT by gefi-tinib activates autophagy, which could be involved in thenegative effects on cisplatin. The cytostatic effect of TKIs thatcause cells to arrest at G1-phasemay explain themechanismby which TKI negatively affect cisplatin and reduce the cell-cycle–dependent toxicity of chemotherapy (34). However,in this study, gefitinib hadminimal effect on cell-cycle arrestin the WT EGFR cells used. Thus, cell cycle effects may notplay a major role in the negative effects observed in ourexperimental condition. It is well known that cisplatininduces DNA damage, which links to various cell deathprogram. For example, DNA damage activates p53, whichcan induce apoptosis and necrosis by inducing Bax/pumaand cathepsin, respectively (16, 35). Also, ATM/DNA-PK,which are activated by DNA damage, phosphorylates H2AXin response to DNA damage, and phosphorylated H2AX(g-H2AX) interacts with AIF and induces CID (30). In thisstudy, we did not examine the effect of TKI on thesesignaling pathways. However, we used p53-null H1299cells and showed that TKI inhibited CID in this cell line,

indicating that p53 may not be involved in cisplatin-induced CID. Moreover, we did not observe any significanteffects of AIF knockdown on cisplatin-induced CID (datanot shown). Although these factors may not be involved incisplatin-induced CID or TKI-mediated negative effect onCID, it is still plausible that DNA damage response plays arole in these events. Further systematic studies will berequired to explore these possibilities.

In this study, we focused on EGFR TKIs that are used fortreatment of patients with NSCLC. However, EGFR anti-bodies, for example, cetuximab and panitumumab, whichalso inhibit and downregulate EGFR, have been used forpatients with colon cancer (cetuximab and panitumumab)or head and neck cancer (cetuximab; refs. 36, 37). There-fore, it would be important to further investigate the effectsof EGFR antibodies to chemotherapeutic drugs in thesecancer types.

Identification of the underlying mechanisms in EGFRmutation status-dependent response to TKIs as well as theimpact of CID in chemotherapy led us to 2 drug candidates,SAHA and erastin, that actually maximized the effect ofcisplatin on the basis of the pathways discovered in this

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Figure 5. ROS induced by cisplatinplays a critical role in CID and thenegative effects of gefitinib. A,A549 Bcl-XL stable cells weretreated with cisplatin (Cis) orcisplatin plus gefitinib (Cis þ Gef)for 24 hours, and the cellular ROSlevels were determined using ROSsensitive fluorescence dye (n ¼ 3).B, A549 Bcl-XL stable cells weretreated with 50 mmol/L of cisplatinor cisplatin plus 0.5mmol/L ofNACfor 72 hours, and the viability wasdetermined by Trypan blue dyeexclusion assay (n ¼ 3). Thecellular morphology is shown onthe right. C, A549 or H1299 Bcl-XLstable cells were treated withcisplatin or cisplatin plus NAC, andthe long-term effects of the drugwere determined as described inMaterials and Methods. Therelative densities of cells areshown on the right (n¼ 3). D, A549Bcl-XL stable cells were treatedwith cisplatin in the presence ofgefitinib (Gef), MEKi, or AKTi andsubjected to Western blot analysiswith the indicated antibodies. E,A549 Bcl-XL stable cells weretransfected with control siRNA(siCont) or siRNA for FOXO3a(siFOXO #1) and then treated withcisplatin. After treating cells for24 hours, the cellular ROS levelswere determined using ROSsensitive fluorescence dye (n ¼ 3).

Yamaguchi et al.





study. This study also reiterates the importance of carefulinvestigation in the mechanism involving the killing effectof anticancer drugs in the preclinical stage before they aremoved into clinical trials.

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

Authors' ContributionsConception and design:H. Yamaguchi, C.-T. Chen, R. Herbst, M.-C. HungDevelopment of methodology: H. Yamaguchi, C.-T. ChenAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): H. Yamaguchi, C.-T. Chen, Y.-N. Wang, M.-C.Hsu, S.-S. Chang, H.-W. Ko, R. HerbstAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):H. Yamaguchi, M.-C. Hsu, R. Herbst, M.-C. HungWriting, review, and/or revision of the manuscript: H. Yamaguchi, J.L.Hsu, R. Herbst, M.-C. Hung

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Du, R. Herbst, M.-C. HungStudy supervision: M.-C. Hung

Grant SupportThis work was supported by NIH (CA109311 and CA099031; to M.-C.

Hung) Sister institution Fund of ChinaMedical University andHospital andMDAnderson Cancer Center (to M.-C. Hung), MD Anderson Cancer CenterSupport Grant (CA16672), Private University Grant (NSC99-2632-B-039-001-MY3; to M.-C. Hung), International Research-Intensive Centers ofExcellence in Taiwan (I-RiCE; NSC101-2911-I-002-303; to M.-C. Hung),and Department of Health, Cancer Research Center of Excellence (CRC) inChina Medical University Hospital (DOH101-TD-C-111-005; to M.-C.Hung).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 7, 2012; revised December 12, 2012; accepted December14, 2012; published OnlineFirst January 23, 2013.

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Published OnlineFirst January 23, 2013.Clin Cancer Res Hirohito Yamaguchi, Jennifer L. Hsu, Chun-Te Chen, et al. Cell Lung Cancer Cells

Small−Effect of EGF Receptor Inhibitors on Cisplatin in Non Caspase-Independent Cell Death Is Involved in the Negative

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