mk-2206, a novel allosteric inhibitor of akt, synergizes...

Preclinical Development

MK-2206, a Novel Allosteric Inhibitor of Akt, Synergizes withGefitinib against Malignant Glioma via Modulating BothAutophagy and Apoptosis

Yan Cheng1, Yi Zhang1,2, Li Zhang2, Xingcong Ren1, Kathryn J. Huber-Keener1, Xiaoyuan Liu2,Lei Zhou2, Jason Liao1, Heike Keihack3, Li Yan3, Eric Rubin3, and Jin-Ming Yang1

AbstractGefitinib, a smallmolecule inhibitor of the epidermal growth factor receptor tyrosine kinase, has been shown

to induce autophagy as well as apoptosis in tumor cells. Yet, how to use autophagy and apoptosis to improve

therapeutic efficacy of this drugagainst cancer remains to be explored.We reportedhere thatMK-2206, apotent

allosteric Akt inhibitor currently in phase I trials in patients with solid tumors, could reinforce the cytocidal

effect of gefitinib against glioma. We found that cotreatment with gefitinib and MK-2206 increased the

cytotoxicity of this growth factor receptor inhibitor in the glioma cells, and the CompuSyn synergism/

antagonismanalysis showed thatMK-2206 acted synergisticallywith gefitinib. The benefit of the combinatorial

treatment was also shown in an intracranial glioma mouse model. In the presence of MK-2206, there was a

significant increase in apoptosis in glioma cells treatedwith gefitinib.MK-2206 also augmented the autophagy-

inducing effect of gefitinib, as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of

autophagy by silencing of the key autophagy gene, beclin 1 or 3-MA, further increased the cytotoxicity of this

combinatorial treatment, suggesting that autophagy induced by these agents plays a cytoprotective role.

Notably, at 48 hours following the combinatorial treatment, the level of LC3-II began to decrease but Bim was

significantly elevated, suggesting a switch fromautophagy to apoptosis. On the basis of the synergistic effect of

MK-2206 on gefitinib observed in this study, the combination of these two drugs may be utilized as a new

therapeutic regimen for malignant glioma. Mol Cancer Ther; 11(1); 154–64. �2011 AACR.

Introduction

Activation of epidermal growth factor receptor (EGFR),a cell surface protein belonging to the ErbB receptortyrosine kinase family, exerts stimulatory effects on anumber of oncogenic signaling cascades, such as RAS,phosphoinositide 3-kinase (PI3K), and mitogen-activatedprotein kinase pathways, thereby promoting prolifera-tion, growth, and survival of tumor cells (1). On the basisof the essential role of the EGFR-initiated signaling intumor development and progression, this receptor tyro-

sine kinase has been actively pursued as a therapeutictarget for cancer treatment (2). Thus far, EGFR inhibitorssuch as gefitinib, cetuximab, and erlotinib have alreadybeen approved as targeted therapies for treating patientswith lung cancer, colon cancer, breast cancer, and someother types of malignancies (3).

Malignant gliomas are the most common and aggres-sive type of primary brain tumors in humans. Despiteoptimal treatment with surgery, chemotherapy, and radi-ation therapy, the prognosis of this disease remains poor.Thus,more effective therapeutic interventions are urgent-ly needed to improve the treatment of this malignancy.Because mutations or aberrant expression of EGFR isfrequent in this type of cancer, use of inhibitors of EGFRis believed to be an effective therapeutic intervention forpatients with malignant gliomas (4, 5). Indeed, EGFRinhibitors such as lapatinib have been reported to exhibitinhibitory effects on proliferation andmigration of gliomacells and to activate apoptosis (6). Gefitinib (Iressa,ZD1839), another selective inhibitor of EGFR tyrosinekinase, has also been shown to possess inhibitory effectson both cell viability and invasion in malignant gliomasthat have amplification of EGFR (7). In addition, inhibitorsof EGFR such as erlotinib andgefitinib have been reportedto inhibit proliferation of tumor-initiating cells in humanglioma (8). Nevertheless, in the reported clinical trials,

Authors' Affiliations: 1Department of Pharmacology and The PennState Hershey Cancer Institute, The Pennsylvania State UniversityCollege of Medicine and Milton S. Hershey Medical Center, Hershey;2Department of Pharmacology, School of Pharmacy, SoochowUniversity, Jiangsu Province, China; and 3Merck & Co. Inc., NorthWales, Pennsylvania

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors: Jin-Ming Yang, Penn State College of Medicine,Department of Pharmacology and The Penn State Hershey Cancer Insti-tute, CH74, 500University Drive, P.O. Box 850, Hershey, PA 17033. Phone:717-531-1630; Fax: 717-531-0011; E-mail: [email protected]; and YanCheng, E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0606

�2011 American Association for Cancer Research.

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Therapeutics

Mol Cancer Ther; 11(1) January 2012154

on August 9, 2019. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst November 4, 2011; DOI: 10.1158/1535-7163.MCT-11-0606

http://mct.aacrjournals.org/

gefitinib only showed modest antiglioma efficacy (9).More recently, it was shown that, although gefitinib canreach the brain tumors in high concentrations and effi-ciently dephosphorylate EGFR, this drug is not sufficientto repress the activity of the pathway (10). It is likelythat single molecular targeted therapy may not beenough to control the complex pathogenesis of glioblas-toma, and that targeting multiple signaling molecules ofthe EGFR-initiated pathways may produce better antitu-mor effects.TheAkt serine/threonine kinase family, which consists

of Akt1, Akt2, and Akt3, is a central component of the PI3kinase pathway, and aberrant activation ofAkt is found tobe common in malignant glioma (11, 12). The activity ofAkt is closely regulated by the EGFR signaling and hasinhibitory effects on apoptosis. Recently, inhibiting Akthas been shown to possess autophagy-inducing effect inaddition to apoptogenic effect (13), so as do the EGFRinhibitors including gefitinib (14, 15). Autophagy is acatabolic process that degrades cytoplasmic componentsvia the lysosomal machinery. Induction of autophagy canpromote either cell survival or cell death, depending ondifferent circumstances (16). In light of the critical role ofAkt in the EGFR-initiated pathway, we sought to deter-mine whether dual inhibitions of EGFR and Akt by cor-responding inhibitors would reinforce the antigliomaefficacy of EGFR inhibitors, and what roles autophagywould play in determining the antitumor efficacy of thecombinatorial therapy. It has been reported that the novelsmallmolecule allosteric inhibitor ofAkt,MK-2206,whichis being tested both in preclinical settings and clinicaltrials as an anticancer agent, can synergistically enhancethe antitumor efficacy of some conventional chemother-apeutic drugs andmolecular targeted agents in preclinicalmodels of lung cancer, ovarian cancer, and breast cancer(17–19). In this study, we focused on testing whethercombining MK-2206 with gefitinib would improve theantiglioma activity of this EGFR inhibitor, andhow induc-tion of autophagy would affect apoptotic cell deathcaused by dual inhibitions of EGFR and Akt. We foundthat MK-2206 synergistically enhanced the cytocidaleffects of gefitinib on glioma cells, and the mechanism ofthis synergism involved the Akt inhibition–mediatedmodulation of apoptosis and autophagy in tumor cellstreated with the targeted therapies. The results of thisstudy recommend that that combined administration ofgefitinib and MK-2206 should be considered as a newtherapeutic tactic that warrants clinical investigation fortreating patients with malignant brain tumors.

Materials and Methods

Cell lines and cultureThe human glioblastoma cell lines, T98G, LN229, and

U87MG were purchased from American Type CultureCollection. These glioma cell lines all express EGFR. T98Gcells were cultured in Ham’s F-10: DMEM (10:1) medium,and LN229, U251, and U87MG cells were cultured in

Dulbecco’s Modified Eagle’s Medium (DMEM) medium.These media were supplemented with 10% FBS, 100units/mL of penicillin, and 100 mg/mL of streptomycin.Cells were maintained at 37�C in a humidified atmo-sphere containing 5% CO2/95% air. All cell cultures weremonitored routinely and found to be free of contamina-tion byMycoplasma or fungi. All cell lines were discardedafter 3 months and new lines propagated from frozenstocks.

Reagents and antibodiesMK-2206 was a gift from Merck & Co. Inc. Gefitinib

was purchased from LC Laboratories. 3-MA, BafilomycinA1 and MTT were purchased from Sigma. Antibodiesto LC-3, Akt, Bim, survivin, phospho-mTOR, mTOR,phospho-S6 kinase (Ser371), S6 kinase, phospho-EGFR,and EGFR, were purchased from Cell Signaling Techno-logies. Antibodies to Beclin 1 and a-tubulin were pur-chased from Santa Cruz. All cell culture media werepurchased from Invitrogen. Western blot reagents wereobtained from Pierce Biotechnology.

Apoptosis assayApoptosis was determined by flow cytometric analysis

of Annexin V and 7-AAD staining. Briefly, 100 mL ofGuava Nexin reagent (Millipore) was added to 1 � 105

cells (in 100mL), and the cellswere then incubatedwith thereagent for 20minutes at room temperature in the dark.Atthe end of incubation, the samples were analyzed by aGuava EasyCyte Plus FlowCytometry System (Millipore).

Western blotCells were lysed in the M-PER mammalian protein

extraction reagent (Thermo Scientific) supplementedwith a protease inhibitor cocktail (Roche) at room tem-perature for 5 minutes, followed by centrifugation at14,000 � g for 10 minutes. Protein concentrations of celllysates were measured using the Bio-Rad DC assayreagent (Bio-Rad). Proteins (20–40 mg) were resolvedby SDS-PAGE and then transferred to polyvinylidenedifluoride (PVDF) membrane (Bio-Rad). The PVDFmembranes were incubated with the respective antibo-dies in 3% BSA/TBST at 4�C for overnight, followed byincubation with secondary antibody at room tempera-ture for 1 hour. Protein signals were detected byenhanced chemiluminescence method following themanufacturer’s protocol.

Short interfering RNA transfectionShort interfering RNA (siRNA) targeting Beclin 1 was

prepared by Dharmacon Research. Scrambled siRNA wasused as a control. Transfection of siRNAwas done accord-ing to the manufacturer’s protocol. Briefly, cells in expo-nential phase of growthwereplated in 6-well tissue cultureplates at 1 � 105cells per well, grown for 24 hours, thentransfected with siRNA using Oligofectamine and OPTI-MEM I–reduced serum medium. The concentrations ofsiRNAs were chosen based on dose–response studies.

Synergistic Antitumor Effect of MK-2206 and Gefitinib

www.aacrjournals.org Mol Cancer Ther; 11(1) January 2012 155




Cellular viability assayCell viability wasmeasured byMTT assay. Briefly, cells

were plated at 5 � 103 cells per well in 96-well tissueculture plates and subjected to different treatments at37�C for 72 hours in a humidified atmosphere containing5% CO2/95% air. At the end of treatments, MTT wasadded to each well and incubated for another 4 hours.The formazan product was dissolved in dimethyl sulfox-ide (DMSO) and read at 570 nm on a Victor3 Multi Labelplate reader (PerkinElmer).

Animal experimentsSix-week-old male BALB/c nude mice were used for

intracerebral implantation of glioma cells. Briefly, humanglioma cells LN229 (1 � 105 cells in 15 mL of DMEMmedium) were injected into the brains at 4 mm depthunder anesthesiawith chloralic hydras (4%, 2mL/kg, i.p.).Three days after tumor cell implantation, mice wererandomly divided into 4 groups (15 mice per group).Treatments were begun on day 4, according to the follow-ing regimens: gefitinib, 80mg/kg, orally, for 5 consecutivedays; MK-2206, 100 mg/kg, orally, 3 times per week for 2weeks; gefitinib þ MK-2206, given as same as above;control, 10% DMSO in saline, orally, given as same asabove. Animal body weight and physical signs were mon-itoreddaily during the experiments. Themicewerehousedin a temperature-controlled and light-controlled environ-ment in the animal facility. At day 17 postinoculation, themice were euthanized, and the brains were fixed in 10%buffered formalin, embedded in paraffin and then stainedwith hematoxylin-eosin (H&E). The animal experimentswere approved by the Institutional Animal Care and UseCommittee of our university.

Statistical analysisThe differences between treatments were analyzed

using a 2-sample t test. The survival curves of thetumor-bearing mice subjected to different treatmentswere estimated using Kaplan–Meier method and com-pared by log-rank statistic analysis.

Results

Gefitinib simultaneously induces apoptosis andautophagy in human glioma cells

Apoptosis and autophagy are the 2 cellular processeslikely to alter efficacy of a therapeutic agent. In this study,we observed that treatment of the human glioma cell linesLN229 and T98G with gefitinib, which decreased thephosphorylation of p-EGFR (Fig. 1B), triggered both apo-ptosis and autophagy in a dose-dependent manner, asevidenced by increases in the number of cells withAnnexin V staining (Fig. 1C), an indicator of apoptosis,and in the amount of LC3 II (Fig. 1D), a specific marker ofautophagy. We also showed that in the presence of bafi-lomycin A1 (an inhibitor of lysosomal protease), theaccumulation of LC3 II was more abundant, indicatingan increase in autophagic flux (Fig. 1D). Stimulatory effect

of low concentration (0.5 mmol/L) but inhibitory effect ofhigh concentrations (1 and 5mmol/L) of gefitinib onEGFRphosphorylation was also observed by others (20). Treat-ment with gefitinib also caused a dose-dependentdecrease in the phosphorylation of Akt (Fig. 1E), whichindicates an inhibition of Akt activity. These results implythat activation of apoptosis and autophagy in response togefitinib treatment could be a part of cellular processesthat determine the cytocidal efficacy of this EGFRinhibitor.

The Akt inhibitor, MK-2206, synergisticallyincreases the cytocidal effect of gefitinib on humanglioma cells

As gefitinib showed an inhibitory effect on Akt activity(Fig. 1E), and this actionmight account, at least in part, forits proapoptotic effect,wenext tested inLN229, T98G, andU87MG human glioma cell lines whether combining anAkt inhibitor with gefitinib would enhance the cytocidalefficacy of this EGFR inhibitor. Figure 2 shows that thenovel allosteric small molecule Akt inhibitor MK-2206, ata noncytotoxic concentration (0.5 mmol/L), inhibited theactivation of Akt (Fig. 2B) and increased the cytocidalactivity of gefitinib against the glioma cells (Fig. 2C–E). Inthe presence of MK-2206, the IC50 values of gefitinib weredecreased by approximately 2- to 3-fold (Table 1).Although the IC50 values of gefitinib in the presence ofMK-2206 were still in the mmol/L ranges, these concen-trations are achievable in the brain tumor tissues, asgefitinib has a high ability to penetrate into solid tumortissues, including glioma (21–23), and gefitinib concen-tration in tumor tissues can be 10- to 40-fold higher thanthat in blood (21–26). Analysis of the effects of the drugcombination using the CompuSyn synergism/antago-nism analysis software (ComboSyn, Inc.) informed thatMK-2206 acted synergistically with gefitinib in producingcytocidal activity. The combination indexes obtainedwere all below 1.0 (range: 0.16–0.85; Table 2), indicatingthat synergism varies between strong and moderate (27).

MK-2206 augments both apoptogenic andautophagenic effects of gefitinib at early stages butswitches autophagy to apoptosis at late stages of thetreatment

To investigate the mechanisms underlying the syner-gistic cytocidal effect between gefitinib and MK-2206,we compared apoptotic and autophagic activity in cellstreated with gefitinib alone or with combination ofgefitinib and MK-2206. As shown in Fig. 3A, cotreat-ment with gefitinib and MK-2206 for 60 hours increasedthe percentage of cells with Annexin V staining, ascompared with treatment with gefitinib alone, indicat-ing an increase in apoptosis. Augmentation of apoptosisby this combined drug treatment was further evidencedby a downregulation of the antiapoptotic protein, sur-vivin, and an upregulation of the proapoptotic protein,Bim (Fig. 3B). Autophagic activity, as determined byWestern blot of LC3 II and microscopic observation of

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Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics156




GFP-LC3 localization, was also higher in the cellscotreated with gefitinib andMK-2206, as compared withthose treated with gefitinib alone (Fig. 3C and D),indicating that MK-2206 also augments the gefitinib-induced autophagy.To delineate the relationship between autophagy and

apoptosis activated by gefitinib andMK-2206, we extend-ed our observation of these 2 cellular processes to 96hours.We found that activation of autophagy by gefitinib,as indicated by the levels of LC3 II, lasted up to 96 hours(Fig. 4A); by contrast, in the presence of MK-2206, au-tophagy levels reached a peak at 48 hours, but began todecline thereafter (Fig. 4B). Notably, with the decrease ofautophagy (LC3 II) after 48 hours, the level of the proa-poptotic protein Bim was further increased (Fig. 4B). Incontrast, no detectable increase in the Bim level wasobserved in the cells treated with gefitinib alone

(Fig. 4A). Elevations of Bim in the tumor cells subjectedto combinatory treatment were accompanied by increasesin apoptotic cell death (Fig. 4C). The importance of Bim ininduction of apoptosis was further shown in the experi-ments showing that knockdownofBimby siRNAreducedapoptotic rate in those treated cells (Fig. 4D). These resultssuggested that excessive autophagy promoted by MK-2206 may trigger a switch from autophagy to apoptoticcell death.

Suppression of autophagy enhances the efficacy ofcombined treatment with gefitinib and MK-2206

As autophagy can play either a prosurvival or prodeathrole under various stressful conditions, we nextwanted tounderstand how activation of autophagy affected viabil-ity of glioma cells treated with gefitinib alone or withthe combination of gefitinib and MK-2206. In these

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Figure 1. Simultaneous induction of apoptosis and autophagy and inhibition of Akt by gefitinib in human glioma cells. A, chemical structure of gefitinib.B, LN229 and T98G cells were treated with gefitinib for 24 hours; the levels of p-EGFR and EGFR were examined by Western blot. Tubulin was used as aloading control. C, LN229 and T98G cells cultured in medium supplemented with 10% FBS were treated with gefitinib for 60 hours, and apoptosis wasexamined bymeasuring Annexin V staining using flow cytometry. D, LN229 and T98G cells were treated with gefitinib for 24 hours in the absence or presenceof Bafilomycin A1, and the levels of LC3 were examined by Western blot. Tubulin was used as a loading control. E, LN229 and T98G cells were treated withgefitinib for 48 hours, and the levels of Akt and p-Akt were measured by Western blot. Tubulin was used as a loading control.






experiments, we first suppressed autophagy by silenc-ing the expression of Beclin 1, a key autophagy-regu-latory gene, and then determined the viability of tumorcells subjected to the treatments. Figure 5A and B showthat inhibition of autophagy via transfection with aBeclin 1–targeted siRNA further decreased viability ofthe cells treated with the combinatorial treatment,as compared with transfection with a nontargetingRNA. The reduced cellular viability seen in Fig. 5A andB seems to result from apoptosis, as suppression of

autophagy by silencing Beclin 1 further increased apo-ptotic cell death rate of the cells subjected to the com-binatorial treatment (Fig. 5C and D). The effect ofautophagy on cytocidal effect of the combinatorial treat-ment was further verified through use of the autophagyinhibitor, 3-MA (1 mmol/L; Supplementary Fig. S1Aand S1B). These results suggest that induction of

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Figure2. Effects of gefitinib onviability of gliomacells in the absenceor presenceofMK-2206. A, chemical structure ofMK-2206.B, LN229andT98GcellsweretreatedwithMK-2206 for 24 hours, and the levels of p-Akt and Akt were examined byWestern blot. Tubulin was used as a loading control. C–E, LN229, T98G,and U87MG human glioma cells cultured in medium supplemented with 10% FBS were treated with a series of concentrations of gefitinib for 72 hours in theabsenceor presenceof 0.5mmol/L ofMK-2206. At the endof treatments, the cell viabilitywasmeasuredbyMTTassay. Eachbar represents themean�SEof 3experiments. �, P < 0.05; ��, P < 0.01.

Table 1. IC50 values of gefitinib in the absenceor presence of 0.5 mmol/L of MK-2206

IC50 (mmol/L)

Cell line Gefitinib GefitinibþMK-2206

LN229 7.9 2.5T98G 12.7 4.9U87MG 17.8 5.9

Table 2. Combination indices for gefitinib andMK-2206 combination therapy, as computed byCompuSyn

Combination index

Cell line ED50 ED75 ED90

LN229 0.46 0.26 0.16T98G 0.46 0.34 0.23U87MG 0.85 0.37 0.18

NOTE: CI < 1.0 suggests synergy.

Cheng et al.





autophagy serves as a compensatory mechanism intumor cells in response to the targeted therapies.

Regulation of autophagy in glioma cells cotreatedwith gefitinib and MK-2206 is mediated via themTOR-S6 kinase pathwayTo explore the signaling pathway involved in the

modulation of autophagy by combinatorial treatmentwith gefitinib and MK-2206, we examined the activitiesof mTOR and S6 kinase, 2 components downstream ofthe EGFR–PI3 kinase–Akt cascade with known roles inthe regulation of autophagy in response to cellularstress. As shown in Fig. 5E and F, in comparison withtreatment with gefitinib alone, the combinatorial treat-ment with gefitinib and MK-2206 markedly decreased

the levels of phospho-mTOR and phospho-S6 kinase,indicating a deactivation of the mTOR–S6 kinase path-way. Because inhibition of mTOR and S6 kinase isknown to induce autophagy, the decreases in the activ-ity of this pathway by dual inhibition of EGFR and Aktmay account for the activation of autophagy by gefitiniband MK-2206, as shown in Fig. 3C.

Combinatorial treatment with gefitinib and MK-2206 produces a stronger antitumor activity in anintracranial glioma mouse model

To determine the in vivo relevance of the above obser-vations, we tested the therapeutic benefits of the combi-natorial treatment with gefitinib and MK-2206 in anorthotopic glioma mouse model in which the LN229

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Figure 3. Effects ofMK-2206 on the gefitinib-induced apoptosis and autophagy. A, LN229 and T98G cells were treatedwith various concentrations of gefitinibfor 60 hours in the absence or presence of 0.5 mmol/L of MK-2206. At the end of treatment, apoptosis was examined by measuring Annexin V staining usingflow cytometry. Each bar represents the mean � SE of 3 experiments. �, P < 0.05; ��, P < 0.01. B, LN229 and T98G cells were treated with variousconcentrations of gefitinib for 24 hours in the absence or presence of 0.5 mmol/L of MK-2206. The levels of survivin and Bimwere examined byWestern blot.Tubulinwasused as a loading control. C, LN229and T98Gcellswere treatedwith gefitinib for 24 hours in the absence or presenceof 0.5mmol/LMK-2206, andthe levels of LC3 were examined by Western blot. Tubulin was used as a loading control. D, LN229 and T98G cells were transfected with a GFP-LC3plasmid and then treated with gefitinib for 24 hours in the absence or presence of 0.5 mmol/L of MK-2206. At the end of treatment, the cells were examinedby fluorescence microscopy. Bars are the quantification of the percentage of cells with 10 or more GFP-LC3 puncta. At least 100 cells were scoredin each treatment. ��, P < 0.01, t test.






human glioma cells were implanted intracranially. Inthese experiments, the tumor-bearing mice were eithertreated with vehicle, gefitinib (80 mg/kg) or MK-2206(100 mg/kg) alone, or with combination of the 2drugs. Figure 6A shows that as compared with gefitinibtreatment alone, coadministration of this EGFR inhibi-tor with MK-2206 showed a better therapeutic bene-fit, as indicated by a significant increase in the survivalof the tumor-bearing mice (P ¼ 0.0155). The greaterantitumor effect of this combinatorial treatment thangefitinib alone was also evidenced by histologic exam-inations of the brain tissues on the day 17 followingtumor inoculations. As shown in Fig. 6B, althoughgefitinib and MK-2206 individually showed inhibitoryeffects on tumor growth in comparison with vehicle,the brain tissues of the tumor-bearing mice treated with

the combination of gefitinib and MK-2206 containedeven smaller tumor masses and less glioma cells ascompared with those treated with gefitinib or MK-2206 alone, suggesting that the prolonged survival ofthe mice receiving combination therapy might be attri-buted to the less brain damage caused by glioma.

Discussion

The EGFR-initiated pathway is considered an attractivetherapeutic target in malignant glioma due to its frequentdysregulation in this disease. Thus, small-molecule inhi-bitors of EGFR tyrosine kinase such as gefitinib are beingevaluated as antiglioma therapies (28). Also, it has beennoticed that sensitivity of tumor cells to gefitinib is closelycorrelated to their dependence on Akt activation for

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Figure 4. Prolonged cotreatment with gefitinib andMK-2206 promotes the switch from autophagy to apoptosis. A, LN229 and T98G cells were treated with 5mmol/L of gefitinib for different time periods, and the levels of LC3 and Bim were examined by Western blot. Tubulin was used as a loading control. B,LN229 andT98Gcellswere treatedwith 5mmol/L gefitinib for different timeperiods in the absence or presenceof 0.5mmol/L ofMK-2206, and the levels of LC3and Bim were examined by Western blot. Tubulin was used as a loading control. C, LN229 and T98G cells were treated with 5 mmol/L of gefitinib fordifferent periods of time in the absence or presence of 0.5 mmol/L of MK-2206. Apoptosis was examined by measuring Annexin V staining using flowcytometry. D, LN229 and T98G cells were transfected with a Bim siRNA or a nontargeting siRNA and then treated with gefitinib for 60 hours in the absenceor presence of 0.5 mmol/L of MK-2206. Apoptosis was examined by measuring Annexin V staining using flow cytometry.

Cheng et al.





survival and proliferation (29), and this suggests thatinhibiting Akt may serve as an approach to improvingthe antitumor efficacy of EGFR inhibitors. In this study,we evaluated whether or not combining the novel allo-steric Akt inhibitor, MK-2206, with gefitinib couldenhance the antiglioma activity of this targeted therapy.Our results showed that gefitinib in combination withMK-2206 produced a synergistic effect against gliomacells (Fig. 2) and enhanced the antitumor activity in anorthotopic glioma mouse model (Fig. 6). It was reportedthat the responsiveness to EGFR inhibitors is associatedwith the coexpressions of EGFRvIII and functionalPTEN in glioma cells (30), and that downstream inhi-bition of the PI3K pathway can be combined with EGFRinhibitors to promote responsiveness in patients withPTEN-deficient tumors (31). Indeed, the synergisticeffect reported here was observed not only in the glioma

cells harboring wild-type PTEN (LN229 cells) but also inthe tumor cells with PTEN mutant (T98G cells) or PTENnull (U87MG cells). PTEN is a tumor suppressor thatacts as a critical negative regulator of PI3K–Akt–mTORsignaling axis. Loss of PTEN is one of the most commongenetic lesions and occurs in 60% to 80% of malignantgliomas. A previous study suggests that glioblastomamultiforme (GBM) patients who have high levels ofEGFR expression and low levels of phosphorylated Akthad better response to EGFR inhibitor erlotinib treat-ment than those with low levels of EGFR expression andhigh levels of phosphorylated Akt (32), but these resultshave not been confirmed in larger studies. Although thesynergistic effect reported here may have potentialimpacts in the treatment of GBM, we are aware thatthis synergism was observed only in 3 glioma cell linesand in a mouse glioma model; whether this synergistic

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action is truly unaffected by PTEN status would needfurther preclinical and clinical investigation. In addi-tion, successful treatment of malignant glioma maydepend on tailoring cocktails of targeted drugs to indi-vidual patients. For instance, combinations of temozo-lomide with inhibitors of mTOR, PI3K, Akt, or withreticulum stress inhibitors have been proposed (33).

The synergismbetweenMK-2206 and gefitinib seems tobe associated with the modulation of autophagy andapoptosis in the cells subjected to the combinatory treat-ment of gefitinib and MK-2206 (Fig. 3 and Fig. 4). Apo-ptosis and autophagy are the 2 cellular processes knownto affect efficacies of numerous cancer therapeutic agents.To explore the mechanism underlying the synergismbetween gefitinib and MK-2206 in killing tumor cells, weexamined the activity of both apoptosis and autophagy inthe glioma cells subjected to combinatorial treatment ofthe 2 agents. We observed that gefitinib simultaneouslyinducedapoptosis andautophagy (Fig. 1CandD) and thatin the presence ofMK-2206, both of apoptosis and autoph-agywere increased in the tumor cells treatedwithgefitinibfor 48 hours. As autophagy may contribute to either cellsurvival or cell death, we then assessed the role of autop-

hagy in determining the efficacy of the treatments. Wefound that suppression of autophagy significantlyenhanced the cytocidal activity of the combinatorial treat-ments, suggesting that induction of autophagy is a com-pensatory response to therapeutic stress. Interestingly, 48hours after the cotreatments, autophagic activity began todecrease but apoptosiswas further activated (Fig. 4B), andthis shift did not occur in the glioma cells treated withgefitinib alone (Fig. 4A). Cotreatment of gefitinib withMK-2206 also caused a reduction of antiapoptotic protein,survivin, but led to an elevation of proapoptotic protein,Bim (Fig. 3B). It has been reported that Bim is required forapoptosis induced by certain targeted therapy in cancercells with oncogenic EGFR (34) and that the downregula-tion of survivin plays a pivotal role in the gefitinib-induced apoptosis (35). Thus, our results are consistentwith those reported observations. More importantly, ourstudy suggests that Akt inhibitors such as MK-2206 mayplay a therapeutically beneficial role in promoting func-tional switch from autophagy to apoptosis, and thisswitch may account for the synergistic effect of MK-2206 on cytocidal activity of gefitinib. Evidence of aswitch from autophagy to apoptosis was previously

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Figure 6. Effect of the combinatorial treatment with gefitinib and MK-2206 on tumor growth in an intracranial glioma model. The human glioma cells LN229(1 � 105 cells in 15 mL of DMEM medium) were injected into the brains of 6-week-old male BALB/c nude mice at 4 mm depth under anesthesia withchloralic hydras (4%, 2mL/kg, i.p.). Three days after tumor cell implantation, mice were randomly divided into 4 groups (15 mice per group). Treatments werebegunonday4, according to the following regimens: gefitinib, 80mg/kg, orally, 5 consecutive days;MK-2206, 100mg/kg, orally, 3 timesperweek for 2weeks;gefitinibþMK-2206, same as above; control, 10%DMSO in saline, orally. A, the Kaplan–Meier survival curves, n¼ 10; gefitinibþMK-2206 versus gefitinib,P ¼ 0.0155, log-rank statistic analysis; B, at day 17 after tumor cell implantation, the mice were euthanized, and the brains were fixed in 10% bufferedformalin, embedded in paraffin, and then stained with H&E. The images shown are representative of 5 mice from each group.

Cheng et al.





reported for temozolomide (36), a chemotherapeuticdrug commonly used in treatment of GBM, and target-ing autophagy has been proposed as a new approach totreating apoptosis-resistant GBM (37). In addition, wefound that mTOR–S6K pathway, which locates down-stream of Akt and normally plays a role in suppressingautophagy (38), was involved in the induction of autop-hagy in the cells cotreated with gefitinib and MK-2206,as evidenced by a decrease in the levels of p-mTOR andp-S6K (Fig. 5E and F). These observations were consis-tent with our previous results showing the involvementof mTOR–S6K pathway in altering autophagic responsein tumor cells treated with MK-2206 (39). As mTOR andS6K are downstream effectors of Akt, and their inhibi-tion might cause deinhibition of a negative feedbackloop, how precisely this pathway affects the targetedtherapy might be worth further study.The need and importance for combining gefitinib with

othermolecular targeted agents in treating glioblastoma isalso underscored by a recent phase II study showing thatgefitinib exerts little effects on the activity of the down-stream pathways, despite its inhibitory effect on EGFRphosphorylation (10). Therefore, multitarget based newtherapeutic strategies may be necessary for better sup-pression of the EGFR-initiated oncogenic pathways. It hasnow become increasingly appreciated that manipulatingautophagy and apoptosis may achieve better therapeuticoutcomes in cancer treatment; hence, the molecularmechanisms and pathways involved in the regulation ofthe cross-talk between autophagy and apoptosis, and theapproaches to exploiting these cellular process for kill-

ing cancer cells, are undergoing extensive exploration.The results reported here show that the novel allostericAkt inhibitor, MK-2206, can synergize with gefitinib ininhibiting malignant glioma by promoting a switchfrom autophagy to apoptosis, suggesting that this dualtargeted therapy may make these drugs more usefuland valuable in the treatment of malignant glioma orother types of cancer. Considering that the majority ofhuman malignant gliomas harbor mutations that resultin activation of PI3K/Akt pathway, and that aberrantEGFR signaling is a primary contributor to the patho-genesis of gliomas, our results provide a rationale andbasis for further clinical investigation of the combina-torial use of MK-2206 and gefitinib in the treatment ofthis disease, and thus may have potential impact incancer therapy.

Disclosure of Potential Conflicts of Interest

J-M. Yang received a commercial research grant.

Grant Support

The work was supported by grants from the US Public Health ServiceR01CA135038 and from Merck & Co. Inc.

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 5, 2011; revised September 29, 2011; acceptedNovember 1, 2011; published OnlineFirst November 4, 2011.

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2012;11:154-164. Published OnlineFirst November 4, 2011.Mol Cancer Ther Yan Cheng, Yi Zhang, Li Zhang, et al. and ApoptosisGefitinib against Malignant Glioma via Modulating Both Autophagy MK-2206, a Novel Allosteric Inhibitor of Akt, Synergizes with

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