Enhanced Glycolysis Supports Cell Survival in EGFR ... · enhancement of glycolysis is critical for EGFR stability. EGFR knockdown signiﬁcantly decreased levels of glycolytic path-way

Metabolism and Chemical Biology

Enhanced Glycolysis Supports Cell Survival inEGFR-Mutant Lung Adenocarcinoma by InhibitingAutophagy-Mediated EGFR DegradationJi Hye Kim1, Boas Nam1, Yun Jung Choi2, Seon Ye Kim2, Jung-Eun Lee2,Ki Jung Sung2,Woo Sung Kim3, Chang-Min Choi3,4, Eun-Ju Chang1,Jae Soo Koh5, Joon Seon Song6, Shinkyo Yoon7, Jae Cheol Lee4,7,Jin Kyung Rho4, and Jaekyoung Son1

Abstract

Oncogenic EGFR is essential for the development andgrowth of non–small cell lung cancer (NSCLC), but theprecise roles of EGFR in lung cancer metabolism remainunclear. Here, we show that EGFR mutation-mediatedenhancement of glycolysis is critical for EGFR stability. EGFRknockdown significantly decreased levels of glycolytic path-way intermediates via transcriptional regulation of glycolyticgenes. EGFR mutation-enhanced glycolysis was requiredfor fueling the tricarboxylic acid cycle, a critical componentof EGFR stability. Nonsustained ATP production enhancedreactive oxygen species accumulation and subsequent

JNK-mediated activation of autophagy, which in turn induc-ed EGFR degradation. Our data show that EGFR-mutantNSCLCs require EGFR mutation-enhanced glycolysis tomaintain EGFR stability. This pathway may serve as anattractive therapeutic target for EGFR-mutant NSCLCs.

Significance: Enhanced glycolysis by EGFR mutationis required for maintaining EGFR levels via inhibition ofJNK-induced autophagy. This provides a promising rationalefor use of JNK activators in patients with EGFR-mutatedNSCLC. Cancer Res; 78(16); 4482–96. �2018 AACR.

IntroductionApproximately 15% to 20% of lung adenocarcinomas

harbor EGFR mutations, and this incidence is higher amongEast Asian patients (1, 2). EGFR mutations are generally morecommon among female and in those with no history ofsmoking (3). Conventional chemotherapy and radiotherapyare used to treat inoperable lung cancer, but primary oracquired resistance to conventional chemotherapy and radio-

therapy remains as a significant impediment to successfullung cancer therapy.

Activating EGFR mutations such as exon 19 deletion (del19)and exon 21 L858R are predominant among lung adenocarci-nomas and have strong sensitivity to EGFR-tyrosine kinaseinhibitors (TKI); conversely, T790Mmutation leads to acquiredresistance to first- and second-generation EGFR-TKIs (e.g., gefi-tinib, erlotinib, and afatinib; refs. 4–6). Various mechanismsof acquired EGFR-TKI resistance have been reported (7, 8), andnotably, T790M mutation occurs in approximately half ofthe patients with TKI-resistant EGFR-mutated tumors. Recently,several third-generation EGFR-TKIs have been developed tocircumvent T790M-mediated resistance and were proven tobe effective in patients with T790M-mutated tumors afterfirst-line EGFR-TKI treatment failure (9, 10). However, as withthe first- and second-generation EGFR-TKIs, third-generationEGFR-TKIs also elicit resistance mechanisms such as smallcell transformation, point mutation (C797S), or FGFR1 geneamplification (11, 12). Thus, a new therapeutic approach isneeded to overcome the acquired resistance to first- or third-generation EGFR-TKIs.

Metabolic reprogramming, a hallmark of cancer, has recentlygained interest as area of cancer therapy research. Cancer cellsmust undergo increases in genomic and protein content as wellas lipid mass to maintain continuous proliferation (13). Thus,in order to meet the demands of enhanced proliferation,metabolic requirements of cancer cells differ considerably fromthose of normal cells. To obtain sufficient amount of metabo-lites required for enhanced proliferation, cancer cells rewiretheir metabolic pathways (14). Recently, accumulating

1Department of Biomedical Sciences, Asan Medical Center, AMIST, Universityof Ulsan College of Medicine, Seoul, South Korea. 2Asan Institute for LifeSciences, Asan Medical Center, University of Ulsan College of Medicine,Seoul, South Korea. 3Department of Pulmonology and Critical Care Medicine,Asan Medical Center, University of Ulsan College of Medicine, Seoul, SouthKorea. 4Department of Convergence Medicine, Asan Medical Center, Uni-versity of Ulsan College of Medicine, Seoul, South Korea. 5Department ofPathology, Korea Cancer Center Hospital, Korea Institute of Radiological andMedical Sciences, Seoul, South Korea. 6Department of Pathology, AsanMedical Center, University of Ulsan College of Medicine, Seoul, South Korea.7Department of Oncology, Asan Medical Center, University of Ulsan Collegeof Medicine, Seoul, South Korea.

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

J.H. Kim and B. Nam contributed equally to this article.

Corresponding Authors: Jaekyoung Son, University of Ulsan College ofMedicine, 88, Olympic-ro 43-gil, Seoul 138-786, South Korea. Phone: 82-2-3010-4184; E-mail: [email protected]; and Jin Kyung Rho,[email protected]

doi: 10.1158/0008-5472.CAN-18-0117

�2018 American Association for Cancer Research.

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evidence has indicated that oncogenic signaling pathwaysmediate metabolic reprogramming (15). Particularly, the onco-genic EGFR signaling pathway plays a critical role in cancerglucose (Glc) metabolism regulation (16). In glioblastoma, theactivating EGFRvIII mutation enhances glycolytic flux by pro-moting glycolytic gene expression via the Myc/Max pathway(17). EGFR is required for intracellular Glc level maintenancethrough sodium/glucose cotransporter 1 (SGLT1) interactionsand stabilization (18). A recent study on EGFR-mutantlung adenocarcinoma showed that EGFR signaling plays animportant role in aerobic glycolysis, and a combination ofGlc metabolism inhibitor and EGFR-TKIs overcame T790M-mediated resistance (19). In addition, several studies describedimportant roles of AKT in Glc metabolism within oncogenicEGFR signaling. AKT activation leads to increased Glc meta-bolism in tumor cells (20, 21), and AKT mediates translocationof the Glc transporter GLUT1 or GLUT4 to the plasma mem-brane (22). Thus, the oncogenic EGFR signaling pathway playsan important role in Glc metabolism; however, the precisemetabolic role of EGFR mutation and particularly the effectsof EGFR mutation-mediated metabolic reprogramming ontumor growth and survival have yet to be clearly described.

Here, we demonstrate that EGFR mutation enhances glyco-lytic flux to supply fuel for the tricarboxylic acid (TCA) cycle.Failure to sustain Glc-derived ATP production in mitochondriaresulted in reactive oxygen species (ROS)-mediated JNKactivation, leading to induction of apoptosis via autophagy-induced EGFR degradation. Thus, EGFR stability requires themaintenance of proper mitochondrial functions via EGFRmutation-enhanced glycolytic flux, providing an appropriatetherapeutic rationale for EGFR-mutant NSCLC.

Materials and MethodsCell culture

EGFR WT (A549 and H1299) and EGFR-mutant NSCLC cells(HCC827 and H1975) were purchased from the ATCC. ThePC-9 cell line was a kind gift from Dr. Kazuto Nishio (NationalCancer Center Hospital, Tokyo, Japan) and has been previouslycharacterized (23, 24). PC-9/GR (gefitinib-resistant cell line)and PC-9/ER (erlotinib-resistant cell line) cells were establishedas part of a previous study (25). All cells were maintainedat 37�C in humidified air with 5% CO2 and in RPMI1640medium supplemented with 10% FBS, 100 U/mL penicillin,and 100 mg/mL streptomycin (all from Thermo Scientific). Thecell lines were used within 10 to 15 passages from the initialexpansion and freeze-down, routinely tested for Mycoplasmacontamination, and authenticated by short tandem repeat(STR) DNA profiling as described previously before the study(26). For glucose or glutamine deprivation, RPMI1640 withoutglucose (R1383) or without glutamine (R0883) was obtainedfrom Sigma-Aldrich.

Cell proliferation assayCells were plated in 24-well plates (density: 2,000 cells/well).

For nutrient deprivation, cells were plated in complete cul-ture media (10 mmol/L glucose, 2 mmol/L Gln), which wasexchanged with Glc or Gln-free medium the following day.Media was not changed throughout the course of the exper-iment. At the indicated time intervals, cells were fixed in 10%formalin and stained with 0.1% crystal violet. The dye

was extracted with 10% acetic acid, and relative prolifera-tion was determined according to the optical density (OD)at 595 nm.

Reagents and antibodiesAnisomycin (1290) and SP600125 (1496) were obtained

from Tocris Bioscience, and methyl-pyruvate (371173), rote-none (R8875), dimethyl a-KG (349631), BPTES (SML0601),and 2DG (D6134) were purchased from Sigma-Aldrich. Anti-bodies to AKT (9272), p-AKT (4060), cleaved PARP (9541),cleaved-caspase-3 (9661), ERK (9102), and p-JNK (4668) werepurchased from Cell Signaling Technology; antibodies tob-actin (sc-47778), EGFR (sc-03), p-EGFR (sc-12351), p-ERK(sc-7383), and JNK (sc-7345) were obtained from Santa CruzBiotechnology.

Glucose consumption and lactate production assayCells were plated in six-well plates (2 � 105 cells/well). Media

was not changed throughout the experimental course and wascollected at the indicated time intervals. Glucose and lactateconcentrations in media were measured using the YSI 2300 STATPlus GlucoseLactate Analyzer.

Extracellular acidification rate and oxygen consumption ratemeasurement

Extracellular acidification rate (ECAR) and oxygen consump-tion rate (OCR) were measured with an XF24 extracellular fluxanalyzer (Seahorse Bioscience). Briefly, cells were plated in a24-well Seahorse plate and cultured at 37�C with 5% CO2;medium was replaced the following day with unbufferedDMEM, and cells were incubated at 37�Cwithout CO2 for 1 hour.For OCR measurement, oligomycin, FCCP, and rotenone wereadded to final concentrations of 2, 5, and 2 mmol/L, respectively.For ECAR measurements, glucose, oligomycin, and 2DG wereadded to final concentrations of 10 mmol/L, 1 mmol/L, and20 mmol/L, respectively.

MetabolomicsCells were grown to �60% confluence in growth media

(RPMI1640, 10% FBS) on 10-cm dishes in biological tripli-cates. After 24 hours, cells were harvested using 1.4 mL of coldmethanol/H2O (80/20, v/v) after sequential PBS and H2Owashes, and lysed by vigorous overtaxing; 100 mL of 5 mmol/Linternal standard was added. Metabolites were liquid–liquidextracted from the aqueous phase after adding chloroform. Theaqueous phase was dried via vacuum centrifugation, and thesample was reconstituted with 50 mL of 50% methanol priorto LC/MS-MS analysis. The LC/MS-MS system was equippedwith an Agilent 1290 HPLC (Agilent Technologies), Qtrap 5500(ABSciex), and reverse phase column (Synergi fusion RP 50 �2 mm). A 3 mL volume was injected into the LC/MS-MS systemand ionized with a turbo spray ionization source. Multiplereaction monitoring was used in negative ion mode, andthe extracted ion chromatogram (EIC), corresponding to thespecific transition for each metabolite was used for quantita-tion. The area under the curve of each EIC was normalizedto that of the internal standard EIC. The peak area ratio ofeach metabolite to the internal standard was normalizedusing protein amount in a sample, and then was used forrelative comparison.

The Role of Glycolysis in Maintaining EGFR Stability

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Quantitative reverse transcription PCRTotal RNA was extracted using TRIzol (Invitrogen). cDNA

was synthesized from 2 mg of total RNA, using oligo-dT andMMLV HP reverse transcriptase (Epicentre) according to themanufacturer's instruction. Quantitative reverse transcriptionPCR (qRT-PCR) was performed on an AriaMax Real-Time PCRinstrument (Agilent Technologies) using the SYBR detectionprotocol. cDNA was calculated by the comparative Ct method,with 18S ribosomal RNA as a control. PCR reactions wereperformed in triplicates.

ROS quantificationTo determine cytoplasmic ROS, cells were incubated with

20,70-dichlorodihydrofluorescein diacetate (DCFDA, 5 mmol/L;Invitrogen) for 30 minutes. Excess DCFDA was removed bywashing cells twice with PBS at room temperature; labeled cellswere trypsinized, rinsed, and resuspended in PBS. The oxida-tion of DCFDA to the highly fluorescent 20,70-dichloro-fluores-cein (DCF), which is proportionate to ROS generation, wasanalyzed by flow cytometry. To analyze mitochondrial ROS,the cells were then incubated with 5-mmol/L MitoSOX reagent(Thermo Scientific) for 10 minutes at 37�C and trypsinized,washed with PBS, and then resuspended in 200 mL of PBS.Stained cells were then quantified and analyzed on a flow cyto-meter (BeckmanCoulter). Excitation wavelength was 510 nm,and emission wavelength was 580 nm.

Apoptosis quantitationApoptotic cell death was detected using an Annexin-V/FITC

assay. Cells were harvested by trypsinization, washed with PBS,and resuspended in Annexin-V binding buffer (10 mmol/LHEPES, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2) contain-ing Annexin-V FITC and propidium iodide (PI). Stained cells werequantified and analyzed on a flow cytometer (BeckmanCoulter).

Western blot analysisCells were lysed in RIPA lysis buffer containing protease inhib-

itor cocktail (Thermo Scientific); lysate concentrations wereassayed using a BCA assay (Thermo Scientific). Equal amountsof lysates were mixed with Laemmli loading dye and boiledfor 10 minutes. Lysates were subjected to SDS-PAGE, and sepa-rated proteins were transferred to PVDF membranes (EMDMilli-pore). Membranes were blocked in Tris-buffered saline (TBS)containing 5% nonfat dry milk and 0.1% Tween 20 (TBS-T) priorto overnight incubation with primary antibody at 4�C; afterwashing with TBS-T, blots were exposed to appropriate horserad-ish peroxidase-conjugated secondary antibodies for 1 hour. Pro-teins–antibody complexes were visualized on Kodak X-ray filmusing an enhanced chemiluminescence (ECL) detection system(Thermo Scientific).

Quantitation of intracellular ATPIntracellular ATP concentrations were measured using an

ATP Colorimetric/Fluorometric Assay Kit (Biovision Incorpo-rated) according to the manufacturer's instructions. Briefly,cells were lysed in 100 mL of ATP assay buffer; 50 mL ofsupernatants were collected and added to a 96-well plate. Toeach well, 50 mL of ATP assay buffer containing ATP probe, ATPconverter, and developer were added. Absorbance was mea-sured at 570 nm.

Xenograft studiesFemale SCID mice were purchased from Charles River

Laboratories. All experimental procedures were approved bythe Institutional Animal Care and Use Committee of AsanInstitute for Life Sciences (protocol 2015-14-241). For eachanimal, 2 � 106 cells mixed with Matrigel (BD Biosciences)were injected into the flank. Five mice per group were treatedwhen the tumor volumes reached 50 to 100 mm3 with ani-somycin (5 mg/kg, intratumoral, 5 days a week) or 2DG(500 mg/kg, intraperitoneal, 5 days a week) for 2 weeks. Thelength (L) and width (W) of each tumor were measured usingcalipers, and the tumor volume (TV) was calculated as TV ¼(L � W2)/2. To evaluate EGFR-related signaling in xenografttumors, tumors were harvested after 3 days of drug treatment,lysed, and analyzed with Western blotting.

Lentiviral-mediated shRNA targetsThe following RNAi Consortium clone IDs for shRNAs were

used in this study: shEGFR-1 (TRCN0000195303) and shEGFR-2(TRCN0000298822).

Tissue microarraysA tissue microarray (TMA) block was made from lung speci-

mens of lung adenocarcinoma that were surgically resectedbetween June 2009 and May 2016 at Asan Medical Center,Seoul, South Korea. These TMA block included 244 subjectscontaining 126 EGFR-mutant and 118 wild-type for EGFRmutation. The EGFR-mutant group included specimens withmutations in exon 19 or exon 21 only, except mutations inexon 18 or exon 20. The EGFR mutation status within exons18 to 21 was analyzed by direct DNA sequencing using anautomatic ABI PRISM 3100 Genetic Analyzer (Applied Biosys-tems) until August 2015, and thereafter the PNAClamp EGFRMutation Detection Kit with PNA-mediated PCR clampingmethod. Medical records of study subject were retrospectivelyreviewed at April 2017. The study design was approved bythe Institutional Review Board of Asan Medical Center, whichwaived the requirement for informed consent due to the retro-spective nature of the analysis (project identification number2016-0752). However, all study subjects had been providedinformed consent for utilization of extracted lung for studyafter surgical resection.

IHC and statistical analysisIHC staining was done using a specific primary antibody

including P-JNK (1:100; 700031; ThermoScientific) and EGFR(1:200; 28-0005; ThermoScientific). IHC data were made bypathologists at Asan Medical Center and Korea Cancer CenterHospital. Chi-square test was used to evaluate the differencesbetween positive and negative expression of P-JNK and EGFR.

Statistical analysisData are presented as mean � standard deviation. All compar-

isons were analyzed using unpaired Student t test.

ResultsOncogenic EGFR-mediated enhanced glycolysis is requiredfor maintaining EGFR levels

EGFR mutations are critical for clinical responses and pro-longed survival of TKI-treated patients with non–small cell lung

Kim et al.

Cancer Res; 78(16) August 15, 2018 Cancer Research4484




cancer (NSCLC; refs. 1, 24, 27), but the importance of EGFRmutation in lung cancer metabolism is unknown. To examinethe functional role of EGFR mutation in NSCLC metabolism, wefirst investigated the effect of EGFR on Glc metabolism viatargeted LC/MS-MS metabolomic analysis of EGFR-mutantNSCLCs. Consistent with a recent study showing that EGFR-TKItreatment decreases glycolysis metabolites (16), we observed thatEGFR knockdown led to a significant decrease in glycolysismetabolites (Fig. 1A). Oncogenic signaling components such asMyc and HIF-1 mediate metabolic reprogramming via transcrip-tional regulation (28), and EGFR signaling pathway inhibitionleads to decreased levels of GLUT1 and hexokinase 2mRNA (16).Accordingly, EGFR knockdown reduced glycolytic gene expres-sion at the transcription level (Fig. 1B; Supplementary Fig. S1),and also reduced Glc uptake and lactate production in EGFR-mutant NSCLCs (Fig. 1C and D). EGFR knockdown alsodecreased extracellular acidification rate (Fig. 1E), suggesting thatEGFR mutation enhances glycolytic flux through transcriptionalregulation.

To further define the function of EGFR in glycolysis regu-lation, we compared Glc uptake and lactate production in EGFR-mutant NSCLCs with those in EGFR-WT NSCLCs. As shownin Fig. 1F and G, EGFR-mutant NSCLCs exhibited significantlyelevated Glc uptake and lactate production compared withEGFR-WT NSCLCs, and the relative changes in Glc uptake andlactate production increased gradually over time. Consistentwith these data, EGFR-mutant NSCLCs had a significantly higherextracellular acidification rate compared with EGFR-WTNSCLCs(Fig. 1H); this finding indicates that EGFR-mutant NSCLCs havea higher glycolytic rate. We then tested whether enhancedglycolysis is needed for growth support in EGFR-mutantNSCLCs. Glc deprivation markedly decreased EGFR-mutantNSCLC cell proliferation (Supplementary Fig. S2A and S2B),but had minimal effects on EGFR-WT NSCLCs (SupplementaryFig. S2C and S2D). To confirm the requirement of aerobicglycolysis for EGFR-mutant NSCLC survival, we examined celldeath in response to Glc deprivation. Consistent with the growthcurve, significant apoptotic cell death was triggered in EGFR-mutant NSCLCs following Glc deprivation (SupplementaryFig. S2E and S2F), whereas Glc deprivation did not sensitizeEGFR-WT NSCLCs to apoptosis (Supplementary Fig. S2G andS2H). Consistently, 2DG treatment, but not BPTES, promotedapoptosis only in EGFR-mutant NSCLCs (SupplementaryFig. S2I–S2L). For further confirmation of the importance ofaerobic glycolysis for the survival of EGFR-mutant NSCLCs, weassessed the ability of EGFR-mutant NSCLC cells to grow in vivoas a xenograft. As shown in Fig. 1I–K, 2DG treatment robustlydiminished the growth of EGFR-mutant NSCLCs compared withthat of EGFR-WT NSCLCs. These data suggest that EGFR muta-tionmediates Glc metabolism reprogramming via transcription-al regulation to promote tumor survival.

EGFR-mutant NSCLCs, which depend on EGFR for growthand survival, rely more strongly on EGFR signaling than doEGFR-WT NSCLCs (29–31). Given the importance of Glcmetabolism in growth and survival, we speculated that Glcmetabolism might be needed for EGFR signaling in EGFR-mutant NSCLCs. We therefore tested whether nutrient starva-tion would affect EGFR signaling in this condition. Glutamine(Gln) deprivation, which had no effect on growth or survival,did not significantly affect EGFR signaling, whereas Glc depri-vation markedly decreased EGFR levels in a time-dependent

manner and inhibited phosphorylation of the EGFR signalingcomponents AKT and ERK (Fig. 1L). Consistent with our results(Supplementary Fig. S2E and S2F), PARP and caspase-3 werecleaved only upon Glc deprivation (Fig. 1L). BPTES treatmenthad no effect on EGFR signaling, whereas 2DG treatmentresulted in robust reduction of EGFR levels and inhibition ofEGFR signaling in EGFR-mutant NSCLCs, thereby leading toapoptosis (Fig. 1M). Given that Glc metabolism inhibitionled to a robust apoptotic cell death, we speculated that Glcmetabolism inhibition might suppress other receptor tyrosinekinases (RTK). We thus examined phosphorylation and thetotal level of other RTKs upon Glc metabolism inhibition.As shown in Fig. 1N, glucose deprivation or 2DG treatmentsignificantly decreased IGF1R phosphorylation and marginallydecreased MET phosphorylation; conversely, the total formof either did not change in response to glucose deprivation or2DG treatment, indicating that the combined inhibition ofactivation of RTKs may activate profound apoptotic cell death.Taken together, these data demonstrate that EGFR mutation-mediated enhanced glycolysis supports EGFR-mutant NSCLCssurvival by maintaining EGFR levels.

Glucose as TCA cycle fuel is essential for survival of EGFR-mutant NSCLCs

Glc and Gln are the major sources of energy and biosynthesisin proliferating tumor cells (32); cells convert Glc for use inanabolic processes, whereas Gln, an alternative energy source,is used to fuel the TCA cycle (33). Blocking Gln metabolism asa source of TCA cycle fuel impairs tumor growth (34). Based onour previous observation that Glc, but not Gln, deprivationsignificantly reduced growth and survival, we suspected thatunlike other tumors, EGFR-mutant NSCLCs might utilize Glcas a source of carbon fuel for the TCA cycle. We first examinedATP levels in EGFR-mutant NSCLCs in the absence of either Glcor Gln. We observed that ATP levels were not significantlyaffected by Gln deprivation, but markedly reduced by Glcdeprivation (Fig. 2A). To validate the essentiality of Glc formitochondrial ATP production, oxygen consumption rateswere measured. As shown in Fig. 2B, oxygen consumptiondecreased significantly upon Glc deprivation, compared withGln deprivation. To explore the direct effect of nutrient dep-rivation on levels of TCA cycle intermediates, we performed ametabolic analysis. BPTES treatment had no significant inhib-itory effect on TCA cycle intermediates, whereas 2DG treatmentsignificantly decreased the levels of TCA cycle intermediates(Supplementary Fig. S3A). We next examined whether EGFR isessential for mitochondrial metabolic function and energylevel maintenance via increased glycolytic flux. EGFR knock-down led to significant decreases in the oxygen consumptionrate as well as TCA intermediate levels (Fig. 2C; SupplementaryFig. S3B). These results suggest that EGFR-mediated enhancedglycolysis is a major source of carbon for TCA cycle in EGFR-mutant NSCLC.

To test whether the Glc metabolism inhibition-inducedcell death results from insufficient maintenance of mitochon-drial ATP production, we attempted to rescue Glc meta-bolism inhibition-induced cell death by supplementing thecells with either pyruvate or a-ketoglutarate, which providessubstrates for the TCA cycle. We observed that both pyruvateand a-ketoglutarate supplementation rescued EGFR-mutantNSCLCs from Glc deprivation-induced apoptosis (Fig. 2D






and E). Consistently, 2DG treatment-induced apoptotic celldeath was reversed significantly upon pyruvate or a-ketoglu-tarate supplementation (Fig. 2F and G). In addition, we found

that both pyruvate and a-ketoglutarate supplementation ledto a significant recovery of ATP levels, which were reduced byGlc deprivation (Supplementary Fig. S3C).

Figure 1.

EGFR-mutant NSCLCs rely more heavily on aerobic glycolysis than do EGFR-WT NSCLCs. A, Glycolysis metabolite pools in EGFR-mutant NSCLCwere analyzed via LC/MS-MS. Western blot analysis confirmed EGFR knockdown. Error bars, SD of triplicate wells from a representative experiment.B, Summary of changes in glycolytic enzyme mRNA levels upon EGFR knockdown. The gene names for enzymes exhibiting significant changes arehighlighted in gray bold. C and D, EGFR-mutant NSCLCs expressing control (shGFP) or EGFR-targeting shRNA were plated in complete media;glucose uptake and lactate production were measured over time using a YSI 2300 STA Plus Glucose–Lactate Analyzer. Error bars, SD of triplicatewells from a representative experiment. E, Real-time glycolytic rates were determined using an extracellular flux analyzer. PC9 cells expressing control(shGFP) or EGFR-targeting shRNAs were sequentially treated with glucose (10 mmol/L), oligomycin (1 mmol/L), and 2DG (20 mmol/L). Error bars,SD of triplicate wells from a representative experiment. F and G, EGFR-WT and EGFR-mutant NSCLCs were plated in complete media; glucose uptake andlactate production were measured over time using a YSI 2300 STA plus Glucose–Lactate Analyzer. Error bars, SD of triplicate wells from a representativeexperiment. H, Real-time glycolytic rates were determined using an extracellular flux analyzer. Cells were sequentially treated with glucose (10 mmol/L),oligomycin (1 mmol/L), and 2DG (20 mmol/L). Error bars, SD of triplicate wells from a representative experiment. I–K, SCID mice bearingestablished PC9, H1975, and A549 tumor cell xenografts were treated with 2DG (Materials and Methods). Tumor volumes were calculated on indicated days.Arrows, drug treatment cessation. L, EGFR-mutant NSCLCs were plated in complete media that was replaced the following day with glucose or glutamine-free medium, incubated for another 24 hours, and immunoblotted with indicated antibodies. M, Cells were treated with 2DG (10 mmol/L) or BPTES(10 mmol/L) for up to 48 hours and immunoblotted with indicated antibodies. N, EGFR-mutant NSCLCs were plated in complete media that wasreplaced the following day with glucose-free medium or treated with 2DG (10 mmol/L) for 24 hours and immunoblotted with indicated antibodies.G6P, glucose-6-phosphate; FBP, fructose 1,6-bisphosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; LAC, lactate.� , P < 0.05; ��, P < 0.01.

Kim et al.





To further define the functional role of Glc metabolism inEGFR level maintenance, we attempted to see if the reducedEGFR level resulting from Glc metabolism inhibition can be

restored by pyruvate supplementation. As shown in Fig. 2Hand I, pyruvate supplementation led to a significant recoveryof EGFR levels reduced by the inhibition of Glc metabolism

Figure 2.

Survival of EGFR-mutant NSCLCs requires glucose as a carbon source for the TCA cycle. A, PC9 cells were plated in a complete media that wasreplaced the following day with glucose or glutamine-free medium, incubated for another 24 hours, and assayed for intracellular ATP. Error bars, SDof triplicate wells from a representative experiment. B, Oxygen consumption rates were measured with an extracellular flux analyzer. PC9 cells wereplated in a complete media that was replaced the following day with glucose or glutamine-free medium. Cells were sequentially treated with oligomycin(2 mmol/L), FCCP (5 mmol/L), and rotenone (2 mmol/L). Error bars, SD of triplicate wells from a representative experiment. C, TCA metabolite pools in PC9 cellsexpressing control (shGFP) or EGFR shRNAs (shEGFR) were analyzed via LC-MS/MS. Error bars, SD of triplicate wells from a representative experiment. D and E,EGFR-mutant NSCLCs were plated in complete media that was replaced the following day with glucose-free medium supplemented with either methylpyruvate (MPYR; 7 mmol/L) or a-ketoglutarate (AKG; 7 mmol/L), incubated for another 24 hours, and assayed for cell death by Annexin V/PI staining andflow cytometry. Error bars, SD of three separate experiments. F and G, EGFR-mutant NSCLCs were treated with 2DG (10 mmol/L) in the presence ofeither MPYR (7 mmol/L) or AKG (7 mmol/L) and assayed for cell death by Annexin V/PI staining and flow cytometry. Error bars, SD of three separateexperiments. H, Cells were plated in complete media that was replaced the following day with MPYR-supplemented (7 mmol/L) glucose-free medium,incubated for another 24 hours, and immunoblotted with indicated antibodies. I, Cells were treated with 2DG (10 mmol/L) and MPYR (7 mmol/L) for 24 hoursand immunoblotted with indicated antibodies. NS, not significant.� , P < 0.05; �� , P < 0.01.






via Glc deprivation or 2DG treatment, and also restored theAKT and ERK phosphorylation. In the presence of pyruvate, thecleavage of PARP and caspase-3 upon Glc deprivation or 2DGtreatment was inhibited as well. Our results show that Glc, asTCA cycle fuel, is indispensable for sustaining EGFR levels inEGFR-mutant NSCLCs.

To confirm the importance of Glc-derived ATP production inmitochondria for EGFR level maintenance, we assessed EGFRlevels following mitochondrial respiratory chain inhibitionusing the complex I inhibitor rotenone. Consistent with ourresults in Fig. 2, inhibition of mitochondrial ATP productionwith rotenone robustly decreased EGFR levels in both dose- andtime-dependent manners (Supplementary Fig. S4A and S4B).Given the importance of the mitochondrial respiratory chain inEGFR level maintenance, we speculated that excess pyruvatemight not rescue the reduced EGFR levels following rotenonetreatment, because the mitochondrial respiratory chain cannotoperate under complex I inhibition. Indeed, we observed thatexcess pyruvate could not rescue the decreased EGFR levels afterrotenone treatment (Supplementary Fig. S4A and S4B). Simi-larly, pyruvate supplementation could not rescue EGFR-mutantNSCLCs from rotenone-mediated apoptosis (SupplementaryFig. S4C and S4D). Taken together, these data demonstratethat mitochondrial ATP production is critical for maintainingEGFR levels.

JNK activation inhibits EGFR-mutant NSCLC cell survival viareduced EGFR levels

We further investigated the downstreammechanism by whichGlc-derived ATP production supports EGFR-mutant NSCLCsurvival. JNK mediates apoptosis and cell death in response toenvironmental stress (35), but the mechanisms by which JNKactivation induces tumor cell death remain unclear. Interesting-ly, JNK was strongly activated in EGFR-mutant NSCLCs in atime-dependent manner following Glc deprivation, but not Glndeprivation (Fig. 3A). To examine whether Glc deprivation-mediated JNK activation was due to inhibited mitochondrialATP production, we assessed JNK activation upon rotenonetreatment. As shown in Fig. 3B, rotenone treatment significantlyinduced JNK activation in both dose- and time-dependentmanners. We next examined whether ATP depletion-mediatedJNK activation decreases EGFR levels. Consistent with ourprevious results, treatment with JNK-activator anisomycin sig-nificantly reduced EGFR levels, inhibited AKT and ERK phos-phorylation, and led to PARP and caspase-3 cleavage in a dose-dependent manner (Fig. 3C). Anisomycin did not trigger apo-ptotic cell death in EGFR-WT NSCLCs (Fig. 3D and E), whereasanisomycin-induced JNK activation led to significant inductionof apoptosis in EGFR-mutant NSCLCs in a dose-dependentmanner (Fig. 3F and G) and robustly diminished growth inEGFR-mutant NSCLCs tumor (Fig. 3H and I) via decreasedEGFR levels (Supplementary Fig. S5). These data are consistentwith our observation that Glc deprivation induced apoptotic celldeath in EGFR-mutant NSCLCs only. We further examined theeffects of JNK inhibition on Glc deprivation-induced apoptosisin EGFR-mutant NSCLCs to verify the requirement for JNKactivation. Treatment with SP600125, a JNK inhibitor, signifi-cantly rescued cells from Glc deprivation-induced apoptosis(Fig. 3J and K). Consistently, JNK inhibition significantlyblocked the Glc deprivation-mediated reduction in EGFR levels(Fig. 3L). These results suggest that the ATP depletion-mediated

JNK activation triggers apoptotic cell death in EGFR-mutantNSCLCs by reducing EGFR levels.

ROS induces JNK-mediated reduction of EGFR levelsATP production is a major function of the mitochondria,

which provides a source of cellular ROS (36), and inhibition ofmitochondrial respiration leads to excessive ROS. Consideringthe previous studies that showed that ROS activates MAPK (37–39) and that ROS can induce JNK activation (40, 41), wespeculated that the Glc deprivation-mediated mitochondrialATP depletion might lead to a significant increase in ROS levelsand JNK activation. Indeed, we observed that Glc metabolisminhibition by Glc deprivation, 2DG treatment, or rotenonetreatment led to significant increases in both cytoplasmic andmitochondrial ROS levels (Fig. 4A and B). Also, hydrogenperoxide (H2O2) treatment activated apoptosis in EGFR-mutant NSCLCs in a dose-dependent manner (Fig. 4C).

To determine whether ROS accumulation would result inthe JNK-mediated reduction of EGFR levels, we tested theeffects of ROS on EGFR signaling. H2O2 treatment markedlyreduced the EGFR levels, inhibited the AKT and ERK phos-phorylation, and cleaved the PARP and caspase-3 in a dose-dependent manner (Fig. 4D). In addition, H2O2 activated JNKin a dose-dependent manner as well (Fig. 4E), indicating thatROS can activate apoptosis via JNK-mediated reductionof EGFR levels. To confirm that the JNK-mediated reductionof EGFR levels was indeed due to ATP depletion-inducedROS production, we assessed apoptotic cell death upon Glcdeprivation, 2DG, or rotenone treatment in the absence orpresence of N-acetyl-L-cysteine (NAC). NAC significantly res-cued cell death caused by Glc deprivation, 2DG, or rotenonetreatment (Fig. 4F and G). Conversely, NAC did not rescueanisomycin-induced cell death (Supplementary Fig. S6A), sug-gesting that JNK is a downstream target of ROS. NAC supple-mentation led to a significant recovery of EGFR levels and AKTand ERK phosphorylation following Glc deprivation, 2DG, orrotenone treatment; PARP and caspase-3 cleavage upon Glcdeprivation, 2DG, or rotenone treatment was significantlyinhibited by NAC (Fig. 4H–J). Importantly, JNK activationinduced by Glc deprivation, 2DG, or rotenone treatment wascompletely inhibited by NAC (Fig. 4K–M). Conversely, NACcould not recover anisomycin-decreased EGFR signaling orEGFR levels (Supplementary Fig. S6B) or inhibit anisomy-cin-induced JNK activation (Supplementary Fig. S6C). Glucosemetabolism may affect ROS levels through other mechanismssuch as the imbalanced redox status. Verifying that inhibitionof Glc-derived ATP generation in mitochondria is the majorreason for ROS upregulation, we tested whether Glc metabo-lism inhibition via Glc deprivation or 2DG treatment couldaffect NADPþ/NADPH ratios through a compromised pentosephosphate pathway. Indeed, Glc metabolism inhibition had nosignificant effect on NADPþ/NADPH ratios (data not shown),indicating that ATP depletion-mediated ROS generation in-duces JNK-mediated reduction of EGFR levels.

Autophagy is required for JNK-mediated reduction of EGFRlevels

We next investigated the mechanisms by which JNK regu-lates the EGFR expression. Either 2DG or anisomycin treat-ment had no significant effect on EGFR transcriptional level(data not shown). Our previous work demonstrated that

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activation of autophagy leads to EGFR degradation, which inturn induces apoptosis (42). Thus, we tested whether JNKactivates autophagy, which leads to EGFR degradation. Cor-respondingly, Glc deprivation, 2DG, or anisomycin treatmentthat significantly reduced EGFR levels resulted in a significantincrease in LC3-II levels (Fig. 5A). As further confirmationof this increase in autophagic activity, we used GFP-LC3reporter to examine the recruitment of LC3 into autophago-

somes. As shown in Fig. 5B, the number of GFP-LC3 punctaprofoundly increased upon Glc deprivation, 2DG, or aniso-mycin treatment compared with that in cells cultured innormal conditions. To further confirm the role of JNK inGlc deprivation-mediated autophagy activation, we assessedLC3-II levels following Glc metabolism inhibition with orwithout SP600125. The increase in LC3-II levels induced byGlc deprivation or 2DG treatment was significantly inhibited

Figure 3.

Sustained JNK inactivity is required for EGFR stability. A, EGFR-mutant NSCLCs were plated in complete media that was replaced the following daywith glucose or glutamine-free medium, incubated for another 24 hours, and immunoblotted for JNK and P-JNK. B, EGFR-mutant NSCLCs were treatedwith rotenone and immunoblotted for JNK and P-JNK. C, EGFR-mutant NSCLCs were treated with anisomycin for 24 hours and immunoblotted withindicated antibodies. D–G, EGFR-mutant and WT NSCLCs were treated with anisomycin for 24 hours and assayed for cell death by Annexin V/PIstaining and flow cytometry. Error bars, SD of three separate experiments. H and I, SCID mice bearing established PC9 and H1975 tumor cell xenograftswere treated with anisomycin (Materials and Methods). Tumor volumes were calculated on indicated days. Arrows, drug treatment cessation. J and K,EGFR-mutant NSCLCs were cultured in complete or glucose-free medium with or without SP600125 (15 mmol/L) and assayed for cell death by Annexin V/PIstaining and flow cytometry. Error bars, SD of three separate experiments. L, EGFR-mutant NSCLCs were cultured in complete or glucose-free mediumwith or without SP600125 (15 mmol/L) for 24 hours and immunoblotted for EGFR. ANS, anisomycin; SP, SP600125. ��, P < 0.01.






by JNK inhibition (Fig. 5C), indicating that activated JNKinduces autophagy activation.

To test whether activated autophagy could induce EGFRdegradation, we examined the effect of autophagy activator

on EGFR levels. As shown in Fig. 5D, trehalose, which isknown as an autophagy activator (43), significantly inducedEGFR degradation in a dose-dependent manner. Moreover,the reduction in EGFR levels caused by Glc deprivation, 2DG,

Figure 4.

ROS induced JNK-mediated-EGFRturnover. A and B, EGFR-mutantNSCLCs were plated in completemedia that was replaced the followingday with glucose-free medium,incubated or treated with 2DG(10 mmol/L) or rotenone (5 mmol/L)for 24 hours, and subjected to DCFDAassay (A) or MitoSox Red assay (B).C–E, EGFR-mutant NSCLCs weretreated with H2O2 at indicated dosesfor 24 hours and assayed for cell deathby Annexin V/PI staining and flowcytometry (C), or immunoblottedwith indicated antibodies (D and E).F–M, EGFR-mutant NSCLCs wereplated in complete media that wasreplaced the following day withNAC-supplemented (4 mmol/L)glucose-free medium, incubated ortreated with either 2DG (10 mmol/L)or rotenone (5 mmol/L) for 24 hours inthe absence or presence of NAC(4 mmol/L), and assayed for celldeath by Annexin V/PI staining andflow cytometry (F and G), orimmunoblotted with indicatedantibodies (H–M). Error bars, SDof three separate experiments.� , P < 0.05; �� , P < 0.01.

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Figure 5.

Autophagy induces EGFR degradation. A, EGFR-mutant NSCLCs were plated in complete media that was replaced the following day with glucose-freemedium or treated with either 2DG (10 mmol/L) or anisomycin (5 mmol/L) for 24 hours and immunoblotted with indicated antibodies. B, PC9 cellsexpressing GFP-LC3 were plated in complete media that was replaced the following day with glucose-free medium or treated with either 2DG (10 mmol/L)or anisomycin (5 mmol/L) for 24 hours and analyzed for LC3 dots. C, EGFR-mutant NSCLCs were plated in complete media that was replaced thefollowing day with glucose-free medium or treated with 2DG (10 mmol/L) for 24 hours in the absence or presence of SP600125, and immunoblottedwith indicated antibodies. D, EGFR-mutant NSCLCs were treated with trehalose (TRE) at indicated doses for 24 hours and immunoblotted with indicatedantibodies. E, EGFR-mutant NSCLCs were plated in complete media that was replaced the following day with glucose-free medium or treated with2DG (10 mmol/L) for 24 hours in the absence or presence of chloroquine and immunoblotted with indicated antibodies. F, EGFR-mutant NSCLCswere treated with anisomycin (5 mmol/L) for 24 hours in the absence or presence of chloroquine and immunoblotted with indicated antibodies. G, EGFR-mutant NSCLCs expressing a control (shGFP) or ATG7 shRNAs (shATG) were plated in the complete medium, which was replaced with glucose-free mediumor treated with 2DG (10 mmol/L) for 24 hours and immunoblotted with indicated antibodies. H, EGFR-mutant NSCLCs expressing a control (shGFP) or ATG7shRNAs (shATG) were treated with anisomycin (5 mmol/L) for 24 hours and immunoblotted with indicated antibodies. CQ, chloroquine.






or anisomycin treatment was dramatically inhibited bychloroquine, a potent autophagy inhibitor (Fig. 5E and F).Consistent with these results, Glc deprivation, 2DG, or ani-somycin treatment did not reduce EGFR levels upon ATG7knockdown (Fig. 5G and H), suggesting that functionalautophagy is required for JNK-mediated EGFR degradation.Given that autophagy is indispensable for Glc deprivation-mediated EGFR degradation, we speculated that auto-phagy inhibition might suppress Glc deprivation-inducedapoptotic cell death. Indeed, PARP and caspase-3 cleavageupon Glc deprivation, 2DG, or anisomycin treatment wasrobustly inhibited by chloroquine (Supplementary Fig. S7Aand S7B). Consistently, PARP and caspase-3 cleavage wasdramatically blocked upon ATG7 knockdown (SupplementaryFig. S7C and S7D). Thus, these results demonstrate that acti-vated JNK mediates autophagy activation, which in turninduces EGFR degradation.

Targeting glucose metabolism overcomes acquired resistanceto EGFR-TKIs

EGFR-TKI-resistant sublines were established in a previousstudy (25, 44, 45). We previously demonstrated that resistancein PC-9/GR and PC-9/ER cells is caused by a secondary T790Mmutation, whereas resistance in HCC827/GR and HCC827/ERcells is mediated by MET and AXL activation, respectively(25, 44, 45). The dependency of EGFR-TKIs-resistant celllines on EGFR signaling varies depending on the mechanismsof acquired EGFR-TKI resistance. We used EGFR knockdownto evaluate the dependence of EGFR-TKI-resistant cell lines onEGFR signaling for proliferation. As shown in Supplement-ary Fig. S8A and S8B, no significant changes were observed inMET or AXL-mediated proliferation of resistant cells (HCC827/GR and HCC827/ER), whereas EGFR knockdown markedlydecreased T790M-mediated proliferation of resistant cell(PC-9/GR and PC-9/ER).

Because Glc deprivation significantly decreased EGFRlevels (Fig. 1L and M), we suspected this condition mightaffect PC-9/GR and PC-9/ER cell proliferation. The prolifera-tion rates of both cell lines were significantly suppressed inresponse to Glc deprivation (Fig. 6A and B), whereas Glcmetabolism inhibition had minimal effect on the prolifera-tion rates of HCC827/GR and HCC827/ER cells (Supplemen-tary Fig. S8C and S8D). Glc metabolism inhibition via Glcdeprivation or 2DG treatment led to significant induction ofapoptosis in cells with EGFR dependency such as PC-9/GRand PC-9/ER cells (Fig. 6C–F).

We next investigated the involvement of JNK activation inGlc deprivation-mediated apoptotic death in PC-9/GR and PC-9/ER cells. Consistent with our previous observations (Fig. 3A),only Glc metabolism inhibition via Glc deprivation or 2DGtreatment markedly activated JNK, resulting in EGFR levelreduction, AKT and ERK phosphorylation inhibition, andPARP and caspase-3 cleavage in both PC-9/GR and PC-9/ERcells (Fig. 6G). The effects of Glc metabolism inhibition onEGFR signaling and apoptosis were also observed with aniso-mycin-mediated JNK activation (Fig. 6H). Anisomycin signif-icantly induced apoptosis in a dose-dependent manner (Fig. 6Iand J). Thus, these data demonstrate that EGFR signaling-dependent EGFR-TKI-resistant cells can overcome acquiredresistance to EGFR-TKIs via the inhibition of Glc metabolismor JNK activation.

Phosphorylated JNK is reduced in NSCLC tissues with EGFRmutations

Based on preclinical data, we hypothesized that in patientswith EGFR-mutant NSCLCs, the activity of JNKmight be reducedto maintain EGFR-dependent tumor growth. To test this hypo-thesis and to evaluate our preclinical data, we examinedthe phosphorylation of JNK and EGFR by IHC in 244 NSCLCtissues containing 126 EGFR-mutants and 118 WT EGFRs. Asshown in Fig. 7A–C, the expression of phosphorylated JNK wasfound to be significantly decreased in EGFR-mutant NSCLCs(in 10 of 126, 7.94%) compared with WT EGFR (in 27 of116, 23.28%). In addition, we observed a reverse correlationbetween phosphorylated JNK and EGFR expression in all tissuesregardless of EGFR mutation (Fig. 7D–F). Thus, these data sug-gest that EGFR-mutant NSCLCs require reduced JNK activity tomaintain EGFR-dependent tumor growth.

Collectively, our data reveal a novel dependence onEGFR-regulated enhanced glycolysis for maintaining EGFRlevels in EGFR-mutant NSCLCs. Disruption of production ofGlc-derived TCA cycle intermediates results in ROS-mediatedJNK activation, leading to induction of autophagy-mediatedEGFR degradation (Fig. 7G).

DiscussionIn this study, we demonstrated that EGFRmutation enhances

glycolytic flux to supply fuel for the tricarboxylic acid (TCA)cycle. Failure to sustain Glc-derived ATP production in mito-chondria resulted in ROS-mediated JNK activation, leadingto induction of apoptosis via autophagy-induced EGFRdegradation.

Glycolytic flux enhanced by EGFR mutation sustains EGFRstability and is thus critical to EGFR-mutant NSCLC survival. Incancer, Glc metabolism reprogramming supports continuousproliferation (46), and many studies have reported that onco-genic signaling pathways mediate metabolic reprogrammingbioenergetics to meet the increased proliferation requirements(15, 47). Accordingly, we have observed that EGFR knockdownsignificantly inhibited the mRNA levels of glycolytic enzymes inEGFR-mutant NSCLCs (Fig. 1B). These transcriptional alterationswere highly concordant with actual changes in metabolism,whereby EGFR knockdown led to significant decreases in gly-colysis metabolite levels, Glc uptake, lactate production, andECAR (Fig. 1A, C, D, and E). EGFR mutation induces aerobicglycolysis in glioblastoma cells, EGFRvIII mutation increasesglycolytic gene expression (17), and EGFR plays a critical rolein maintaining intracellular Glc levels via stabilization of sodi-um/glucose cotransporter 1 (18). EGFR knockdown by siRNAdecreases Glc uptake and lactate production in chondrosarcomacells (48), and EGFR-TKI treatment also reduces glycolytic flux inEGFR-mutant NSCLCs (16). In addition, many studies haveshown that AKT signaling within the oncogenic EGFR signalingpathway is indispensable for increased Glc metabolism in tumorcells (20, 21). These studies support our observation that EGFRmutation mediates Glc metabolism reprogramming via thetranscriptional regulation required for survival.

Although tumor cells normally switch from mitochondrialrespiration to aerobic glycolysis, oxidative phosphorylationmachinery remains functional, and carbon is needed to fuelthe TCA cycle (49). Tumor cells use various nutrient sources tofuel this cycle (28). In our experiment, EGFR-mutant NSCLSs

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utilized Glc rather than Gln for cellular respiration (Fig. 2Aand B) and EGFR knockdown significantly reduced theoxygen consumption rate and TCA intermediate levels (Fig.2C; Supplementary Fig. S3B), indicating the essentiality of

EGFR mutation-mediated enhanced glycolysis for fuelingthe TCA cycle. Intriguingly, inhibition of Glc-derived ATPproduction led to decrease in EGFR levels, which in turnresulted in activation of apoptosis. The addition of pyruvate

Figure 6.

JNK activation sensitizes EGFR-TKI-resistant NSCLCs to apoptosis. A and B, Cells were plated in complete media that was replaced the following daywith glucose or glutamine-free medium, incubated for another 24 hours, and assayed for cell growth. Error bars, SD of triplicate wells from a representativeexperiment. C and D, Cells were plated in complete media that was replaced the following day with glucose or glutamine-free medium, incubated foranother 24 hours, and assayed for cell death by Annexin V/PI staining and flow cytometry. Error bars, SD of three separate experiments. E and F, Cells weretreated with 2DG (10 mmol/L) or BPTES (10 mmol/L) for 24 hours and assayed for cell death by Annexin V/PI staining and flow cytometry. Error bars,SD of three separate experiments. G, Cells were plated in complete media that was replaced the following day with glucose or glutamine-free medium,or treated with either 2DG (10 mmol/L) or BPTES (10 mmol/L) for 24 hours, and immunoblotted with indicated antibodies. H, Cells weretreated with anisomycin for 24 hours and immunoblotted with indicated antibodies. I and J, Cells were treated with anisomycin for 24 hours andassayed for cell death by Annexin V/PI staining and flow cytometry. Error bars, SD of three separate experiments. � , P < 0.05; ��, P < 0.01.






Figure 7.

Immunohistochemical staining ofP-JNK and EGFR in TMA blocks. Aand D, The representativephotographs of P-JNK (A) and EGFR(D) staining are shown. B and C, Thephosphorylated JNK expression issignificantly reduced in TMA tissueswith EGFR mutation. E and F, Asignificant negative correlationbetween P-JNK and EGFR expressionis shown. G, Model depicting EGFR-regulated aerobic glycolysis in EGFR-mutant NSCLCs used to inhibitautophagy-mediated EGFRdegradation.

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or a-ketoglutarate markedly rescued EGFR-mutant NSCLCsfrom Glc deprivation- or 2DG-mediated apoptosis (Fig. 2D–

G), and pyruvate supplementation markedly inhibited protea-somal EGFR degradation that resulted from Glc metabolisminhibition (Fig. 2H and I). Furthermore, electron transportchain inhibition significantly decreased EGFR levels and acti-vated apoptosis in a manner similar to Glc metabolism inhi-bition (Supplementary Fig. S4A–S4D). Our data show thatpyruvate significantly rescued cells from apoptosis and reducedEGFR levels consequent to Glc metabolism inhibition, but didnot rescue those induced by electron transport chain inhibition;in other words, Glc metabolism inhibition-mediated apoptosisis mediated by mitochondrial electron transport chain dysfunc-tion. In many proliferating cells, glutamine serves to replenishthe TCA cycle (33). Glutamine also contributes to citrate andlipid metabolism through TCA cycle reversal via reductivecarboxylation (28). Thus, the direction of metabolic flow andglutamine utilization varies among different tumors. Amongthe TCA cycle intermediates, only citrate and a-ketoglutaratewere decreased in the presence of BPTES (SupplementaryFig. S3A). Accordingly, EGFR-mutant NSCLCs may use gluta-mine mainly to supply the required acetyl-CoA for lipogenesis.

JNK is activated in response to various extracellular stimuli,including mitogens, growth factors, and environmental stress,and regulates various cellular activities such as gene expression,mitosis, differentiation, proliferation, and cell survival/apopto-sis (50). In this regard, JNK is critical for tumor growth andsurvival. JNK either promotes or inhibits tumorigenesis (51).Thus, the role of JNK in cancer is considered to be dependent ontumor type and genetic context. Our results showed that JNKactivation induced apoptotic cell death, indicating that JNK istumor suppressive in this context. Our IHC results obtainedfrom the tissues of 244 patients with NSCLC show that thepatients with EGFR-mutant NSCLCs have significantly decreasedphosphorylated JNK compared with those with EGFR-WT; thisdata provide further support for the critical role of JNK as atumor suppressor in EGFR-mutant NSCLCs.

EGFR expression at the cell surface is highly regulated byendocytic trafficking mechanism. After ligand-mediated activa-tion and internalization, EGFR is either recycled back to the cellsurface or transported for lysosomal degradation (52). Thus,disruption of this delicate balance is likely to change EGFR levels.Our data show that activated autophagy induced EGFR degrada-tion, suggesting that autophagy activation disrupts the balancebetween recycling to the cell surface and lysosomal degradationvia the fusion of the autophagosome with the lysosome, whichleads to EGFR degradation.

Our results incorporate the clinical meaning of EGFR-TKItherapy. T790M mutation has emerged as the most commonmechanism of acquired resistance to first-generation EGFR-

TKIs. Although second-generation EGFR-TKIs including afati-nib (BIBW2992) and dacomitinib (PF299804) effectivelyinhibit T790M-mediated resistance, they failed in early clinicaltrials (53, 54). Recently, third-generation EGFR-TKIs such asosimertinib (AZD9291) and HM61713 were shown to over-come T790M-mediated resistance; however, osimertinib resis-tance was found to be associated with C797S mutation. In thisstudy, we provide a reliable evidence that the Glc metabolisminhibition is able to overcome T790M-mediated resistanceand that EGFR stability requires the maintenance of propermitochondrial functions via EGFR mutation-enhanced glyco-lytic flux. Thus, glycolysis metabolism regulation may provideattractive strategies for inhibiting EGFR-mutant NSCLCs andovercoming novel mutation-induced EGFR-TKI resistance inpatients with NSCLC.

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

Authors' ContributionsConception and design: J.C. Lee, J.K. Rho, J. SonDevelopment of methodology: K.J. SungAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.H. Kim, B. Nam, Y.J. Choi, S.Y. Kim, J.-E. Lee,K.J. Sung, C.-M. Choi, J.S. Koh, J.S. Song, S. YoonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.H. Kim, B. Nam, Y.J. Choi, S.Y. Kim, J.-E. Lee,K.J. Sung, J.S. Koh, J.C. LeeWriting, review, and/or revision of the manuscript: J.H. Kim, B. Nam,W.S. Kim, J.S. Koh, J.C. Lee, J. SonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.-M. Choi, E.-J. Chang, J.S. Koh, J.C. LeeStudy supervision: C.-M. Choi, J.C. Lee, J. SonOther (preparation of graphic abstract): E.-J. Chang

AcknowledgmentsWe thank the core facilities of Confocal Microscopy Core, Flowcytometry

Core, Metabolomics Core, and the Laboratory of Animal Research at theConvergence Medicine Research Center (CREDIT), Asan Medical Center, forthe use of their shared equipment, services, and expertise. This research wassupported by Basic Science Research Program through the National ResearchFoundation of Korea (NRF), funded by the Ministry of Science, ICT & FuturePlanning (NRF-2016R1C1B2010145 to J. Son), and a grant of the Korea HealthTechnology R&D Project through the Korea Health Industry DevelopmentInstitute (KHIDI), funded by the Ministry of Health & Welfare, Republic ofKorea (grant HI15C0516 to J.K. Rho), and a grant of from the Asan Institute forLife Sciences, Seoul, Korea (2017-605 to J. Son).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 11, 2018; revised May 12, 2018; accepted June 20, 2018;published first June 26, 2018.

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Kim et al.




2018;78:4482-4496. Published OnlineFirst June 26, 2018.Cancer Res Ji Hye Kim, Boas Nam, Yun Jung Choi, et al. DegradationAdenocarcinoma by Inhibiting Autophagy-Mediated EGFR Enhanced Glycolysis Supports Cell Survival in EGFR-Mutant Lung

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