Glycolysis Inhibition Sensitizes Non Small Cell Lung ...The secondary EGF receptor (EGFR) T790M is...

Cancer Therapeutics Insights

Glycolysis Inhibition Sensitizes Non–Small Cell Lung Cancerwith T790M Mutation to Irreversible EGFR Inhibitors viaTranslational Suppression of Mcl-1 by AMPK Activation

Sun Mi Kim1,5, Mi Ran Yun5, Yun Kyoung Hong5, Flavio Solca4, Joo-Hang Kim2,3, Hyun-Jung Kim5, andByoung Chul Cho2,3

AbstractThe secondary EGF receptor (EGFR) T790M is the most common mechanism of resistance to revers-

ible EGFR-tyrosine kinase inhibitors (TKI) in patients with non–small cell lung cancer (NSCLC) with

activating EGFR mutations. Although afatinib (BIBW2992), a second-generation irreversible EGFR-TKI,

was expected to overcome the acquired resistance, it showed limited efficacy in a recent phase III

clinical study. In this study, we found that the inhibition of glycolysis using 2-deoxy-D-glucose (2DG)

improves the efficacy of afatinib in H1975 and PC9-GR NSCLC cells with EGFR T790M. Treatment

with the combination of 2DG and afatinib induced intracellular ATP depletion in both H1975 and PC9-

GR cells, resulting in activation of AMP-activated protein kinase (AMPK). AMPK activation played a

central role in the cytotoxicity of the combined treatment with 2DG and afatinib through the inhibition

of mTOR. The alteration of the AMPK/mTOR signaling pathway by the inhibition of glucose metab-

olism induced specific downregulation of Mcl-1, a member of the antiapoptotic Bcl-2 family, through

translational control. The enhancement of afatinib sensitivity by 2DG was confirmed in the in vivo

PC9-GR xenograft model. In conclusion, this study examined whether the inhibition of glucose

metabolism using 2DG enhances sensitivity to afatinib in NSCLC cells with EGFR T790M through

the regulation of the AMPK/mTOR/Mcl-1 signaling pathway. These data suggest that the combined

use of an inhibitor of glucose metabolism and afatinib is a potential therapeutic strategy for the

treatment of patients with acquired resistance to reversible EGFR-TKIs due to secondary EGFR T790M.

Mol Cancer Ther; 12(10); 2145–56. �2013 AACR.

IntroductionThe EGF receptor (EGFR), a member of the HER family

of receptor tyrosine kinases, mediates cell proliferation,angiogenesis, invasion, and metastasis (1, 2). Aberrantexpression of EGFR is frequently observed in multipletumor types and is known to have a strong oncogenicpotential (3, 4).

First-generation EGFR-tyrosine kinase inhibitors (TKI)such as gefitinib and erlotinib reversibly bind to the ATPcleft within the EGFR kinase domain to block autophos-phorylation of EGFR (5). Although these EGFR-TKIswere shown to be effective in patients with advancednon–small cell lung cancer (NSCLC) harboring EGFR-activating mutations such as small in-frame deletions inexon 19 or the L858R missense mutation in exon 21,patients almost always develop resistance to these agents,most commonly through the acquisition of a secondaryT790Mmutation in EGFR exon 20 (6). To date, there is nostandard therapeutic option for patients with acquiredresistance to reversible EGFR TKIs due to acquisition ofEGFR T790M (7).

Afatinib (BIBW2992) is one of the second-generationirreversible EGFR-TKIs. In recent preclinical studies, afa-tinib was shown to have antitumor activity in NSCLCswith the EGFR T790M in vitro and in vivo. On the basisof these results, afatinib is expected to be a standardtherapeutic option for patients with NSCLCs with EGFRT790M (8–10). However, afatinib was more than 100-fold less potent in NSCLC cells harboring EGFR T790Mmutation than in NSCLC cells with activating EGFRmutation (11). It also showed limited efficacy in a recent

Authors' Affiliations: 1Brain Korea 21 Project for Medical Sciences;2Institute for Cancer Research, Yonsei Cancer Center, and 3Departmentof Internal Medicine, Yonsei University College of Medicine, Seoul, Repub-lic of Korea; 4Department of Pharmacology, Boehringer Ingelheim Austria,Vienna, Austria; and 5JEUK Institute for Cancer Research, JEUK Co., Ltd.,Gumi-City, Kyungbuk, Republic of Korea

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

Corresponding Authors: Byoung Chul Cho, Yonsei Cancer Center, Divi-sion of Medical Oncology, Department of Internal Medicine, Yonsei Uni-versity College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul,Republic of Korea. Phone: 82-2-2228-8126; Fax: 82-2-393-3652; E-mail:[email protected]; and Hyun-Jung Kim, JEUK Institute for CancerResearch, JEUK Co., Ltd., Gumi-City, Kyungbuk, Republic of Korea.Phone: 82-2-2228-0869; Fax: 82-2-393-3652; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-1188

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 2145

on June 19, 2021. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 24, 2013; DOI: 10.1158/1535-7163.MCT-12-1188

http://mct.aacrjournals.org/

phase III clinical study suggesting the necessity ofdeveloping a new strategy to improve the efficacy ofafatinib (12).

In 1924, OttoWarburg proposed that most cancer cellspreferentially use glycolysis to generate ATP ratherthan undergo oxidative phosphorylation (OXPHOS),regardless of the availability of oxygen (13). BecauseATP production through aerobic glycolysis is less effec-tive than that through OXPHOS, cancer cells maintain ahigh rate of glycolysis to generate sufficient ATPs forrapid cell proliferation (14). Increased aerobic glycolysishas been recently discussed as a potential hallmark ofcancer and is considered a possible therapeutic targetfor treatment of cancers (14, 15). Indeed, recent studiesshowed that targeting glycolysis induces cell death andsensitizes cancer cells to chemotherapeutic agents orradiotherapy in different types of cancer (16–21). Todate, there is no published study showing that targetingglycolysis potentiates the sensitivity of NSCLC cells toEGFR TKIs.

AMP-activated protein kinase (AMPK) is the majorenergy sensor kinase and is activated by the increaseof the intracellular AMP/ATP ratio, which is a goodindicator of energetic stress. The critical function ofAMPK is to phosphorylate a number of downstreamtargets that switch metabolism of the cell toward cata-bolic instead of biosynthetic pathways (22, 23). mTOR,one of the targets of AMPK, is known to promote cellgrowth and proliferation through the regulation ofprotein translation by direct interaction with p70 ribo-somal S6 kinase (p70-S6K) and eukaryotic initiationfactor 4E-binding protein 1 (4E-BP1; ref. 24). Previousstudies showed that AMPK activation or mTOR inhibi-tion mediates cellular cytotoxicity in a variety of cancertypes (25–27).

Herein, we examined whether the inhibition of glycol-ysis using 2-deoxy-D-glucose (2DG) enhances sensitivityto afatinib in NSCLC cells with EGFR T790M. Combinedtreatment with 2DG and afatinib showed significant anti-tumor activity through the downregulation of Mcl-1 viathe alteration of the AMPK/mTOR signaling pathway inthose cells. These data suggest that the combined use of aninhibitor of glycolysis and afatinib is a potential thera-peutic strategy for the treatment of patients with acquiredresistance to reversible EGFR-TKIs due to secondaryEGFR T790M.

Materials and MethodsCell culture

The NCI-H1975 cells (EGFR L858R/T790M) werepurchased from the American Type Culture Collectionand were not authenticated. The PC9-GR cells (EGFRdelE746_A750/T790M) were provided by Lee JC (KoreaInstitute of Radiological and Medical Science, Seoul,Republic of Korea). Existence of EGFR T790M mutationin PC9-GR cells was identified by direct sequencing.Both cells were maintained in RPMI-1640 supplementedwith 10% FBS. Culture methods for normal human

bronchial epithelial (NHBE) cells and MRC5 can befound in Supplementary Methods. Cell culture mediaand supplements were obtained from HyClone.

Reagents and antibodiesAfatinib was provided by Boehringer Ingelheim

Pharma (Boehringer Ingelheim PharmaGmbH&CoKG).AICAR (5-aminoimidazole-4-carboxamide 1-b-D-ribofur-anoside) and cycloheximide were obtained from Sigma.Anti-b-actin antibody was purchased from Santa CruzBiotechnology, and all other antibodies were purchasedfrom Cell Signaling. All other reagents were purchasedfrom Calbiochem.

Cell viability assayAfter incubationwith drugs for 72 hours, 0.5mg/mL of

MTT (Amresco) was added to the medium. Formazancrystals in viable cells were solubilized with 100 mLdimethyl sulfoxide (DMSO). The optical density of theMTT formazan product was read at 565 nm on a micro-plate reader. All experiments were carried out intriplicate.

Analysis of cell death using Annexin V/propidiumiodide staining

Annexin V/propidium iodide (PI) double staining wasused according to the manufacturer’s instructions (BDPharmigen). Briefly, cells were incubated with AnnexinV/PI in 1� binding buffer for 15 minutes and then ana-lyzed by flow cytometry (BD Biosciences). Data wereprocessed using WinMDI 2.9 software (Salk Institute).

Intracellular ATP assayIntracellular ATP was determined with an ATP col-

orimetric assay kit according to the manufacturer’sinstructions (Abcam). Briefly, after centrifugation(13,000 rpm, 5 minutes, 4�C), cell lysates were incubatedwith ATP reaction mixture for 30 minutes. The opticaldensity of the mixture in each well was read at 570 nmon a microplate reader. The ATP concentration wascalculated from standard curve and normalized againstcell numbers.

Lactate production assayLactate production was measured with a lactate

assay kit according to the manufacturer’s instructions(Biovision). Briefly, after centrifugation (13,000 rpm,15 minutes, 4�C), cell culture media were diluted inlactate assay buffer and mixed with lactate reactionmixture for 30 minutes. The optical density of themixture in each well was read at 570 nm on a micro-plate reader. The lactate concentration was calculatedfrom a standard curve and normalized against cellnumbers.

Transient transfectionpUseAkt-CA (myristoylated constitutively active Akt)

plasmidwas kindly obtained fromLee JC (Korea Institute

Kim et al.

Mol Cancer Ther; 12(10) October 2013 Molecular Cancer Therapeutics2146




of Radiological and Medical Science, Seoul, Republic ofKorea). Transfections were carried out with Lipofecta-mine 2000 reagent according to the manufacturer’sinstructions (Invitrogen). Briefly, cells were transfectedwith 1.5 mg/well of DNA for 6 hours with transfectionreagent and replaced with fresh growth medium. After24 hours, cells were treated with drugs for further experi-ments. The method for siRNA transfection can be foundin Supplementary Methods.

Western blot analysisCell lysateswere prepared as previously described (28).

Equal amounts of proteinwere fractionatedbySDS-PAGEand then transferred onto a nitrocellulose membrane(BioRAD). Membranes were blocked with 5% skim milkand incubated with the appropriate primary antibody at4�C overnight. Proteins were detected using horseradishperoxidase (HRP)-conjugated secondary antibodies andECL chemiluminescence detection system (Amersham-Pharmacia Biotech).

Methyl7-GTP sepharose 4B pull-down assayAbout 450 mg of cell lysateswas incubatedwith 50 mL of

methyl7-GTP sepharose 4B beads (Amersham Bios-ciences) for 2 hours at 4�C. The beads were washed andthen boiled in 2� sample buffer. After SDS-PAGE reso-lution, the association of 4E-BP1 with eIF4E was detectedby Western blotting.

Quantitative reverse transcription PCRQuantitative reverse transcription PCR (qRT-PCR) was

carried out on a 7500 Real-Time PCR System (AppliedBiosystems) using the SYBRGreendetectionprotocol. Theprimers used for real-time PCR are as follows: Mcl-1, (F)50-GGACATCAAAAACGAAGACG-30; (R) 50-GCAGCT-TTCTTGGTTTATGG-30; Bcl-2, (F) 50-ATGTGTGTGGA-GAGCGTCAACC-30; (R) 50-TGAGCAGAGTCTTCAGA-GACAGCC-30; b-actin, (F) 50-CTGGAACGG-TGAAGGT-GACA-30; (R) 50-AAGGGACTTCCTGTAACAATGCA-30.

Xenograft studiesFemale athymic BALB-c/nu mice were obtained from

Orient Bio at 5 to 6weeks of age. All mice were handled inaccordance with the Animal Research Committee’sGuidelines at Yonsei University College of Medicine andall facilities are approved by AAALAC (Association ofAssessment and Accreditation of Laboratory AnimalCare). Mice were injected subcutaneously with PC9-GRcells (5 � 106). When tumor volumes reached approxi-mately 70 mm3, mice were randomly allocated intogroups of 6 animals to receive either vehicle control,afatinib alone, 2DG alone, or afatinib and 2DG together.Afatinib was suspended in 0.5% (w/v) methylcellulosecontaining 0.4% Tween-80 and administered orally bygavage at 5 mg/kg on a once-daily dosing schedule. 2DGwas dissolved in saline and administered by intraperito-neal injection at a daily dose of 500 mg/kg. Tumor sizewas measured every 2 days using calipers. The average

tumor volume in each group was expressed in mm3 andcalculated according to the equation for a prolate spher-oid: tumor volume ¼ 0.523 � (large diameter) � (smalldiameter)2.

ImmunohistochemistrySacrificed tumors were fixed, embedded in paraffin,

and sectioned (4mm). Tissue sectionsweredeparaffinized,soaked in ethanol, and incubated in 3% H2O2 for 10minutes after microwave treatment in 0.01mol/L sodiumcitrate buffer (pH 6.0). After incubation in 1% bovineserum albumin (BSA) in PBS for 10minutes, sectionswereincubated overnight at 4�Cwith amonoclonalmouse anti-PCNA (1:300 dilution), a monoclonal rabbit anti-p-AMPKa-T172 (1:100 dilution), a monoclonal rabbit anti-p-mTOR-S2448 (1:100 dilution), and a monoclonal rabbitanti-Mcl-1 (1:100 dilution). After incubation with perox-idase-conjugated secondary antibody, peroxidase activitywas revealed using diaminobenzidine.

Statistical analysisIn vitro results are expressed as mean � SD and in vivo

results are expressed asmean� SE. The Student t test wasconducted to determine statistically significant differ-ences between groups, and P < 0.05 was consideredstatistically significant.

ResultsInhibition of glycolysis enhances afatinib sensitivityin NSCLC cells with EGFR T790M mutations

We first determined whether inhibition of glucosemetabolism could enhance afatinib-induced cytotoxicityusingMTT assay. To block glycolysis, we used 2DG. 2DGis a nonmetabolizable form of glucose and is known as ablocker of the first rate-limiting step in glycolysis (29).Structures of afatinib and 2DG are depicted in Fig. 1A.Treatment with 2DG decreased cell viability in a dose-dependent manner and significantly enhanced sensitivityto afatinib in both H1975 and PC9-GR cells (Fig. 1B).Treatment with 2DG also increased cell death inducedby afatinib (Fig. 1C).

As cancer cells, but not normal cells, are strictly depen-dent on glycolysis for their energy supply, we testedwhether the inhibition of glycolysis induces selectivecancer cell cytotoxicity. As shown in Supplementary Fig.S1, in NSCLC cells, including H1975 and PC9-GR, cellgrowth was inhibited in a time-dependent manner bytreatment with 2DG or afatinib alone. Combined treat-mentwith both agentsmarkedly inhibited cell growth in atime-dependent manner and decreased cell numbersbelow those on day 0 (control), indicating cell death. Incontrast to cancer cells, cell growth in normal cells, includ-ing NHBE and MRC5, was slightly inhibited in a time-dependent manner by treatment with 2DG or afatinibalone. The combined inhibitory effect on cell growth wasmuch less than that observed in cancer cells. These datasuggest that the combination of 2DG and afatinib hascancer cell–selective cytotoxicity.

Inhibition of Glycolysis Enhances Sensitivity to Afatinib

www.aacrjournals.org Mol Cancer Ther; 12(10) October 2013 2147




Combined treatment of 2DG and afatinib hamperscancer cell metabolism and induces ATP depletion

To verify whether 2DG treatment blocked glycolysis,we conducted an intracellular ATP assay and lactate (aproduct of aerobic glycolysis) assay. Treatment of 2DGalone led to marked reduction in both intracellular ATPlevel and lactate production in both H1975 and PC9-GRcells. Interestingly, the treatment of afatinib alone alsodecreased intracellular ATP content and lactate levels inboth cells. Combined treatment of 2DG and afatinibmarkedly induced ATP depletion and reduced lactateproduction (Fig. 2A and B). These data show that thecombination of 2DG and afatinib effectively inhibits glu-cose metabolism in both H1975 and PC9-GR cells.

Next, we examined how afatinib interferes with theglucose metabolism in cancer cells. Several lines of evi-dence are suggesting that the PI3K/Akt signaling path-way positively regulates glycolysis (30–32). Afatinib is

well known to have an inhibitory effect on Akt activitythrough the blockade of the ErbB family (11). Therefore,we tested whether Akt is involved in afatinib-inducedinhibition of glycolysis. As shown in Fig. 3A and B, Aktwas markedly inactivated by treatment of afatinib alonebut not 2DG alone in both H1975 and PC9-GR cells.Induction of constitutive Akt activation by the forcedexpression of myr-AKT abrogated the inhibitory effect ofafatinib alone and the combinatorial treatment of 2DGandafatinib on Akt activation and ATP production in bothcells. These results suggest that blockade of glycolysis byafatinib ismediated through the inhibition of Akt activity.

Cytotoxicity by the combination of 2DG and afatinibismediated by the regulation ofAMPK/mTOR/Mcl-1signaling pathway

AMPK is known to be activated by stimuli thatincrease the cellular AMP/ATP ratio (22, 23). Therefore,

A OH OO

ON

N

N

F

Cl

HN

HN

OH

HOHO

O

B

C

2-Deoxy-D-Glucose (2DG)

2DG (mmol/L)

2DG

Annexin V

Pro

pid

ium

iod

ide

CON

0 1 2 5

2DG (mmol/L)

0 1 2 5

H1975

***** ***

*** ****

* *

Cel

l via

bili

ty (

% o

f co

ntr

ol)

120

100

80

60

40

20

0

Cel

l via

bili

ty (

% o

f co

ntr

ol)

120

100

80

60

40

20

0

PC9-GR

H1975

PC9-GR

Afatinib (BIBW2992)

Afatinib2DG +

afatinib

10 nmol/L afatinib

DMSO

100 nmol/L afatinib

Figure 1. 2DG sensitizes NSCLCcells with EGFR T790M to afatinib.A, structures of 2DG and afatinib(BIBW2992). B, cells wereincubated with indicated drugsfor 72 hours. Cell viability wasdetermined by MTT assay.�, P < 0.05; ��, P < 0.01;��,P < 0.001. C, cells were treatedwith 2 mmol/L 2DG, 100 nmol/Lafatinib, or combination of 2agents for 24 hours. Deadcells were assessed by AnnexinV/PI staining and fluorescence-activated cell-sorting (FACS)analysis.

Kim et al.





we examined whether ATP depletion induced by 2DGand afatinib could activate AMPK. In both H1975 andPC9-GR cells, the combination of 2DG and afatinibinduced marked activation of AMPK (Fig. 4A and Sup-plementary Fig. S2A). The treatment with compound C,an AMPK inhibitor, prevented the reduction of cellviability induced by combined treatment with 2DGand afatinib in H1975 and PC9-GR cells (Fig. 4B andSupplementary Fig. S2B), suggesting the mediation ofAMPK activation in the enhanced cytotoxicity of thiscombination. To confirm the possibility that AMPKactivation mediates cytotoxicity of these tumor cells,we used AICAR, an AMPK activator. In H1975 and PC9-GR cells, treatment with AICAR significantly decreasedcell viability in a dose-dependent manner (Fig. 4C andSupplementary Fig. S2C). These results show that thecombination of 2DG and afatinib induces cytotoxicitythrough AMPK activation in EGFR T790M–harboringNSCLC cells.Several reports have suggested that the maintenance

of antiapoptotic Bcl-2 family proteins is critical for sur-vival under metabolic stress (33, 34). We therefore exam-

ined whether the combined treatment with 2DG andafatinib affects the expression levels of antiapoptoticBcl-2 family proteins such as Mcl-1, Bcl-2, and Bcl-xL.Treatment with 2DG alone or afatinib alone decreasedMcl-1 levels, and a combination of 2DG and afatinibsynergistically reduced Mcl-1 levels in PC9-GR cells,whereas Bcl-2 or Bcl-xL levels were not affected by thetreatment with 2DG and afatinib, alone or in combination(Supplementary Fig. S2D). To determine whether themaintenance of Mcl-1 is important for cell survival, weexamined whether Mcl-1 knockdown using Mcl-1 target-ing siRNA (siMcl-1) reduces cell viability in H1975 andPC9-GR cells. As shown in Supplementary Fig. S3A,efficient Mcl-1 knockdown was shown by Western blotanalysis. An MTT assay showed that treatment withsiMcl-1 alone induced a significant decrease in cellgrowth, similar to that observed for treatment with thecombination of 2DG and afatinib, in both H1975 and PC9-GR cells (Supplementary Fig. S3B). These data suggestthat Mcl-1 downregulation by the combined treatment of2DG and afatinib is critical for the growth inhibition ofEGFR T790M–harboring NSCLC cells.

A

B

2DG

Cont

rol

H1975

AT

P (

% o

f co

ntr

ol)

AT

P (

% o

f co

ntr

ol)

120

100

80

60

40

20

0

Lac

tate

(%

of

con

tro

l)

120

100

80

60

40

20

0

Lac

tate

(%

of

con

tro

l)

120

100

80

60

40

20

0

120

100

80

60

40

20

0

PC9-GR

H1975 PC9-GR

Afat

inib

2DG

+ af

atin

ib2D

G

Cont

rol

Afat

inib

2DG

+ af

atin

ib

2DG

Cont

rol

Afat

inib

2DG

+ af

atin

ib2D

G

Cont

rol

Afat

inib

2DG

+ af

atin

ib

Figure 2. Combined treatment of2DG and afatinib synergisticallyinhibits glycolytic metabolism. Aand B, cells were treated with 2mmol/L 2DG, 100 nmol/L afatinib,or combined treatment of 2 agents.After 48 hours, ATP or lactate levelwas measured. ��, P < 0.01;��, P < 0.001.






Next, we examined whether Mcl-1 downregulation ismediated via alteration of the AMPK/mTOR signalingpathway upon the treatment of H1975 and PC9-GR cellswith 2DG and afatinib. As shown in Fig. 4D and Supple-mentaryFig. S2E, combined treatment of 2DGandafatinibsynergistically induced AMPK activation and Mcl-1downregulation in both cells. In addition, we found thatmTOR inhibitionwas accompaniedupon the combinationof 2DG and afatinib. Both mTOR inhibition and Mcl-1downregulation by 2DG and afatinib were dramaticallyrestored in the presence of compound C. In both cancercells, AICAR treatment inhibited mTOR and decreasedMcl-1 levels in a dose-dependent manner (Fig. 4E andSupplementary Fig. S2F). In addition, treatment withrapamycin, an mTOR inhibitor, reduced Mcl-1 levels inH1975 and PC9-GR cells (Fig. 4F and Supplementary Fig.S2G). These findings indicate that AMPK activation andmTOR inhibition upon glycolysis block by combinedtreatment of 2DG and afatinib results in the downregula-tion of Mcl-1, but no other Bcl-2, antiapoptotic members.

Mcl-1 is downregulated through a translationalmechanism upon glycolysis inhibition by combinedtreatment with 2DG and afatinib

As shown in Supplementary Fig. S4,Mcl-1 mRNA levelwas not affected by the treatment with 2DG or afatinib,alone or in combination, indicating that Mcl-1 levels werenot regulated at the transcriptional level. As it is wellknown that the central mechanism of Mcl-1 regulation isubiquitin-mediated proteasomal degradation (35, 36),we therefore monitored Mcl-1 protein levels in the pres-ence of MG132, a proteasome inhibitor. In both H1975and PC9-GR cells, the relative changes of Mcl-1 levelsafter MG132 treatment were not different among thetreatments with 2DG or afatinib, alone or in combination,although it seemed that Mcl-1 protein level in the com-bination treatment of both reagents was lower than inalone treatment withMG132 (Fig. 5A and SupplementaryFig. S5A). Also, the half-life of Mcl-1 by treatment of 2DG,afatinib, or the combination of two agents was not accel-erated in the presence of cycloheximide, an inhibitor of

A

B

H1975

MOCKMOCK

myr-AKT

MOCK myr-AKT

myr-AKT

MOCKmyr-AKT

AT

P (

% o

f co

ntr

ol)

140

120

100

80

60

40

20

0

AT

P (

% o

f co

ntr

ol)

140

120

100

80

60

40

20

0

PC9-GR

H1975

PC9-GR

2DG

2DG

Cont

rol

Afat

inib

Afatinib

p-Akt

Akt(long exposure)

Akt(short exposure)

ββ-Actin

2DG

Afatinib

p-Akt

Akt(long exposure)

Akt(short exposure)

ββ-Actin

2DG

+ af

atin

ib

2DG

Cont

rol

Afat

inib

2DG

+ af

atin

ib

Figure 3. Afatinib blocks ATPproduction via the inhibition of Aktactivity. A and B, at 24 hoursposttransfection of mock or myr-Akt vectors, cells were treated with2 mmol/L 2DG, 100 nmol/Lafatinib, or combination with 2agents. After 48 hours, cellswere harvested for ATPproduction assay or Westernblot analysis. ��, P < 0.001.

Kim et al.





protein biosynthesis (Fig. 5B and Supplementary Fig.S5B). Taken together, these results indicate that the sig-nificant reduction of Mcl-1 levels by the combinationtreatment of 2DG and afatinib was neither due to tran-scriptional nor due to posttranslational regulation.Next, we further examined whether Mcl-1 downregu-

lation by 2DG and afatinib was controlled at the transla-tional level in H1975 and PC9-GR cells. The possibility ofMcl-1 downregulation by translational inhibition wassupported by following experiment using methyl7-GTPsepharose 4Bbeads,which resemble the 50 cap structure ofmRNA.As shown in Fig. 5C and Supplementary Fig. S5C,themethyl7-GTP pull-down assay showed that binding ofthe translational suppressor 4E-BP1 was significantlyincreased by treatment with the combination of 2DG andafatinib and markedly decreased by the addition of com-

poundC. In addition, p70S6K,which regulates translationinitiation factors and ribosome biosynthesis (37, 38), wasalmost completely inhibited by the combined treatment ofboth drugs and the blockade of AMPK by compound Cpartially restored the activity of p70S6K (Fig. 5D). Theseresults suggest that translational repression by the com-bination of 2DG and afatinib occurs cooperatively via thetranslational inhibition of 4E-BP1 and downregulation ofp70S6K activity in AMPK-dependent manner. To verifythat the inhibition of glycolysis specifically blocks Mcl-1translation, we monitored the polysome distribution ofMcl-1 upon the treatment with 2DG or afatinib, alone orin combination, in PC9-GR cells. As shown in Supple-mentary Fig. S5D, the amount ofMcl-1mRNA associatedwith polysomes was markedly reduced by the combina-tion of 2DG and afatinib. Under the same condition, the

A B

C D

E

F

H1975

H1975

Compound C (–)

Compound C

Compound C (+)

Cel

l via

bili

ty (

% o

f co

ntr

ol)

120

100

80

60

40

20

0C

ell v

iab

ility

(%

of

con

tro

l) 120

100

80

60

40

20

0

H1975

H1975

H1975– 1 5

– 10 100

H1975

0 1 5AICAR (mmol/L)

Rapamycin (nmol/L)

AICAR (mmol/L)

2DG

2DG

Cont

rol

Afat

inib

Afatinib

p-AMPK

AMPK

ββ-Actin

2DG

Afatinib

p-AMPK

p-mTOR

mTOR

Mcl-1

AMPK

ββ-Actin

p-AMPK

p-mTOR p-mTOR

mTOR mTOR

Mcl-1

AMPK

ββ-ActinMcl-1

ββ-Actin

2DG

+ af

atin

ib

Figure 4. Cell cytotoxicity bycombined treatment with 2DG andafatinib is mediated through theregulation of AMPK/mTOR/Mcl-1signaling pathway. A, cells weretreated with 2 mmol/L 2DG, 100nmol/L afatinib, or combinationwith 2 agents for 24 hours, andthen harvested. B, cells werepretreated with 1 mmol/Lcompound C for 1 hour, and thenincubated with 2mmol/L 2DG, 100nmol/L afatinib, or combinationwith 2 agents for 72 hours. C, aftertreatment with AICAR for 72 hours,cell viability was determined byMTT assay. D, cells werepretreated with 1 mmol/Lcompound C for 1 hour and thenincubated with 2mmol/L 2DG, 100nmol/L afatinib, or combinationwith 2 agents for 24 hours. E and F,cells were treated with AICAR orrapamycin for 24 hours and thenharvested. ��, P < 0.001.






polysomedistribution of a controlmRNA(b-actin) orBcl-2was largely unaffected compared with Mcl-1. Takentogether, these results indicate that among the Bcl-2 fam-ily, Mcl-1 is specifically downregulated at the translation-al level by combined treatment of 2DG and afatinib.

The addition of 2DG synergistically enhancesantitumor activity of afatinib in PC9-GR xenograftmodels

To examine the antitumor activity of combination ther-apy with 2DG and afatinib, athymic nude mice bearingPC9-GR implanted xenografts were treated with control,2DG, afatinib, or a combination of both agents. Afatinibmonotherapy for 30 days delayed tumor growth com-pared with control. 2DG monotherapy did not showsignificant antitumor activity. Notably, the combinationof 2DG with afatinib resulted in significant tumor regres-sion (Fig. 6A). The synergistic antitumor activity of thecombined treatment of 2DG and afatinib was also con-firmed by immunohistochemical (IHC) staining for pro-

liferating cell nuclear antigen (PCNA), a marker for cellproliferation (Fig. 6B). Consistent with in vitro observa-tions, staining for p-AMPKwas clearly enhanced, where-as staining for mTOR and Mcl-1 was markedly reducedupon combined administration of 2DG and afatinib. Tak-en together, our data obtained by both in vitro and in vivoexperiments suggest that glucose metabolism is an attrac-tive therapeutic target for enhancement of afatinib sus-ceptibility in NSCLCs with the EGFR T790M mutation.

DiscussionIn this study, we identified that glycolysis inhibition

by treatment of 2DG potentiates sensitivity to afatinib inNSCLC cells harboring EGFR T790Mmutation. The com-bined treatment of 2DG and afatinib altered the AMPK/mTORpathway through the induction ofmetabolic stress.Interestingly, we showed that upon glycolysis inhibition,the AMPK/mTOR pathway controlled Mcl-1 levels nei-ther through a transcriptional nor through a posttransla-tional mechanism but rather by controlling its translation.

A

B

C

D

H1975

H1975

H1975

M7-GTPsepharose

TCL

H1975

Compound C

Compound C

Rel

ativ

e M

cl-1

leve

ls

2.5

2

1.5

1

0.5

0

Rel

ativ

e M

cl-1

leve

ls

1.2

1

0.8

0.6

0.4

0.2

0

2DG

2DG

0 3

0 1

Control

Afatinib

Afatinib

MG132

MG132 (h)

CHX (h)

Mcl-1

ββ-Actin

2DG

Afatinib

CHX

Mcl-1

ββ-Actin

2DG

Afatinib

2DG

Afatinib

4E-BP1

4E-BP1

eIF4E

eIF4Ep70S6K

p-p70S6K

ββ-Actin

ββ-Actin

2DG + afatinib

2DGControl

Afatinib2DG + afatinib

Figure 5. Downregulation of Mcl-1by the combined treatment of 2DGand afatinib is occurred throughthe blockade of cellular translation.A and B, cells were treated with 2mmol/L 2DG, 100 nmol/L afatinib,or combinationwith 2 agents for 24hours and then treated with 20mmol/L MG132 for 3 hours or 100mg/mL cycloheximide (CHX) for 1hour. Quantification of Mcl-1 wasnormalized to b-actin and resultswere expressed as ratio of Mcl-1level to control (nontreated). C,cells were pretreatedwith 1 mmol/Lcompound C for 1 hour and furtherincubated with 2mmol/L 2DG, 100nmol/L afatinib, or the combinationof 2 agents for 24 hours.After methyl7-GTP pull-downassay, the association between4E-BP1 and eIF4Ewas revealed byWestern blot analysis. Total celllysates (TCL) were used as inputcontrol. D, H1975 was pretreatedwith 1 mmol/L compound C for1 hour and further incubated with2 mmol/L 2DG, 100 nmol/Lafatinib, or the combination of 2agents for 24 hours. Cell lysateswere fractionated in SDS-PAGEgel and phospho- and totalp70S6K were immunoblotted.b-Actin was used as a loadingcontrol.

Kim et al.





Therefore, our results show a novel mechanism for thesensitization to irreversible EGFR-TKIs linking glucosemetabolism to Mcl-1 downregulation. In addition, thisstudy provides a rationale for the combined use of aninhibitor of glucose metabolism with irreversible EGFR-TKIs in the treatment of NSCLCs with secondary EGFRT790M.Given that the acquisition of EGFR T790M is a

main mechanism of acquired resistance to reversibleEGFR-TKIs in patients with NSCLCs with activatingEGFR mutations, it is important to develop new ther-apeutic strategies to overcome the EGFR T790M–medi-ated acquired resistance (7). Because afatinib showeda strong preclinical antitumor activity in NSCLCs

harboring EGFR T790M, it was expected to overcomeEGFR T790M–mediated acquired resistance in the clin-ic (8–10). Disappointingly, a recent phase III study ofafatinib failed to show overall survival benefit inpatients with acquired resistance to reversible EGFR-TKIs (12). The population of the study was enriched forpatients who were sensitive to reversible EGFR-TKIs,indicating that a significant proportion of the enrolledpatients originally harbored the activating EGFR muta-tion. Given that the EGFR T790M mutation accountsfor about 50% of acquired resistance mechanism to re-versible EGFR-TKIs, a considerable number of the pati-ents enrolled in the study might have EGFR T790M.These results suggest that the development of new

A

B

Tum

or

volu

me

(mm

3 )

700

600

500

400

300

200

100

0

500 mg/kg 2DG (n = 6)

0 2 4 6 8 10 12 14 16 18Days

20 22 24 26 28 30

Control (n = 6)

5 mg/kg afatinib (n = 6)500 mg/kg 2DG + 5 mg/kg afatinib (n = 6)

2DGCON

H&E

PCNA

p-AMPK

p-mTOR

Mcl-1

Afatinib 2DG + afatinib

Figure 6. 2DG increases theantitumor activity by afatinib inPC9-GR tumor xenograft model.A, indicated drugs wereadministered daily to mice bearingPC9-GR xenografts. Datarepresent mean � SE. ��, P <0.001 versus control; ###, P <0.001 versus 2DG;þþþ,P < 0.001versus afatinib. B, five days afterdrug treatment, tumors weresacrificed for IHC analysis.






therapeutic options is needed to improve the efficacy ofafatinib in patients with EGFR T790M. Herein, we firstfound that the use of a glycolysis inhibitor, 2DG, sensi-tizes NSCLC cells harboring EGFR T790M to afatinibthrough AMPK-dependent Mcl-2 downregulation,suggesting that targeting of glycolysis is an effectivetherapeutic option to overcome the limited efficacy ofafatinib in NSCLCs with EGFR T790M.

In our study, enhanced cell cytotoxicity by the com-bined treatment of 2DG and afatinib was mediated bythe reduction of intracellular ATP production, resultingin AMPK activation and mTOR inhibition. Interestinglyenough, afatinib alone decreased intracellular ATP inboth H1975 and PC9-GR cells through the inactivationof Akt. Akt has been known to regulate glycolysis atmultiple steps of glucose metabolism. Akt increasesglucose uptake through translocation of glucose trans-porter from the cytoplasm into the plasma membrane(39). Furthermore, Akt regulates the activities of phos-phofructokinase II involved in the glycolytic pathway(40). Akt activation also increases glycolysis through theactivation of hexokinase, a key rate-limiting step (30,31). Moreover, it was shown that Akt activationswitches the metabolism of cancer cells from mitochon-drial oxidative phosphorylation to aerobic glycolysis,thus increasing the dependency on aerobic glycolysisfor persistent growth and survival (32). To our knowl-edge, this is the first report to date showing that EGFR-TKIs hamper glycolysis leading to reduction of ATPproduction.

Herein, although 2DG alone decreased cell growth inboth H1975 and PC9-GR cells in vitro, 2DG monother-apy did not affect tumor growth in vivo compared withcontrols. These data are consistent with a previousreport by Maschek and colleagues (17). 2DG is a glucoseanalogue that competes with glucose for cellular uptake(41). In in vitro studies, the addition of 2DG is sufficientto inhibit glucose metabolism due to the limited avail-ability of glucose in culture media. In contrast, glucoseis continuously supplied from the blood circulation tothe tumor region in in vivo settings. Nonetheless, com-bined treatment with 2DG and afatinib in the in vivoxenograft model showedmore potent antitumor activitythan that in in vitro. Although cancer cells exhibitincreased glycolysis and depend more on this pathwayfor ATP generation, the inhibition of glycolysis alone isnot sufficient to effectively kill the malignant cells, likemonotherapy with glycolysis inhibitors including 2DGdo not show antitumor activity in in vivo xenograftstudies (17, 42). It has been suggested that ATP deple-tion should reach certain thresholds to trigger cell deathby apoptosis or necrosis (43). Therefore, combinationtherapies with a glycolysis inhibitor and drugs thatblock enzymes regulating the glycolysis pathway areexpected to be much more effective. As afatinib inhibitsAkt, which regulates the activity of phosphofructoki-nase II, cotreatment of 2DG can enhance the antitumoractivity of afatinib. Plus, as hypoxic cells rely solely on

the glycolysis pathway for ATP production, tumor cellsin the hypoxic inner tumor of an in vivo xenograft modelcan be more severely affected by the combined treat-ment of 2DG and afatinib compared with tumor cellscultivated at normoxia. Several studies show that gly-colysis inhibitors are particularly effective againsttumor cells under hypoxic conditions (44). Although2DG monotherapy did not show antitumor activity in invivo study, combined treatment with 2DG and afatinibresulted in potent antitumor activity, suggesting thatthis combination therapy could be more effective thanafatinib alone in a clinical setting.

Bcl-2 family proteins are known to be key regulatorsof apoptotic cell death. Overexpression of antiapoptoticBcl-2 family members such as Bcl-2, Bcl-xL, and Mcl-1has been identified in a number of cancer types and istherefore considered as a therapeutic target for cancertreatment (45, 46). Recent reports have shown thatapoptosis by metabolic stress is mediated by the down-regulation of Mcl-1 (33, 34). Consistently, we found thatMcl-1–specific downregulation at the translational levelby the inhibition of mTOR played a key role in meta-bolic stress–induced cytotoxicity upon the combinedtreatment of 2DG and afatinib in NSCLC cells withEGFR T790M. Although mTOR regulates general pro-tein synthesis through repression of 4E-BP1 (47), whydid the combined treatment of 2DG and afatinib spe-cifically downregulate the translation of Mcl-1, amongBcl-2 family members in our study? Several groupsreported that, once translated, Mcl-1 has a faster turn-over rate than other antiapoptotic Bcl-2 family membersdue to the rapid degradation through the ubiquitin-dependent pathway (35, 36, 48, 49). To consistentlymaintain the basal protein level, Mcl-1 should have arapid translation. For that reason, the blockade of trans-lation by mTOR inhibition could induce selective down-regulation of Mcl-1 among Bcl-2 family members. Con-sistent with our data, recent studies showed that Mcl-1is specifically regulated at the translational level in anmTOR-dependent manner, suggesting that Mcl-1 mightplay a critical role in cell cytotoxicity induced by diversestimuli leading to inactivation of mTOR (33, 50).

In conclusion, the emergence of EGFR T790M muta-tion–mediated acquired resistance poses the greatestunmet medical need in patients with NSCLCs afterprogression on reversible EGFR-TKIs. Here, we showedthat the inhibition of glucose metabolism by 2DGimproves the efficacy of afatinib through the down-regulation of Mcl-1 via the alteration of the AMPK/mTOR signaling pathway in NSCLC cells with EGFRT790M. These data suggest that combined treatmentwith an inhibitor of glucose metabolism and afatinib isa potential therapeutic strategy for treatment of patientswith acquired resistance to reversible EGFR-TKIs due tosecondary EGFR T790M.

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

Kim et al.





Authors' ContributionsConception and design: S.M. Kim, J.H. Kim, B.C. ChoDevelopment of methodology: S.M. KimAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.M. Kim, M.R. YunAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): S.M. Kim, B.C. ChoWriting, review, and/or revision of the manuscript: S.M. Kim, F. Solca,J.H. Kim, H.-J. Kim, B.C. ChoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.M. Kim, Y.K. HongStudy supervision: H.-J. Kim, B.C. Cho

Grant SupportThis work was supported in part by the National Research Foun-

dation of Korea (NRF) funded by the Korea government (MEST;2012R1A2A2A01046927 to B.C. Cho).

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 December 7, 2012; revised June 10, 2013; accepted July 6, 2013;published OnlineFirst July 24, 2013.

References1. Harari PM, AllenGW,Bonner JA. Biology of interactions: antiepidermal

growth factor receptor agents. J Clin Oncol 2007;25:4057–65.2. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat

Rev Mol Cell Biol 2001;2:127–37.3. Hirsch FR, Varella-Garcia M, Bunn PA Jr, Di Maria MV, Veve R,

Bremmes RM, et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number andprotein expression and impact on prognosis. J Clin Oncol 2003;21:3798–807.

4. Rubin Grandis J, MelhemMF, GoodingWE, Day R, Holst VA,WagenerMM, et al. Levels of TGF-alpha and EGFR protein in head and necksquamous cell carcinoma and patient survival. J Natl Cancer Inst1998;90:824–32.

5. Mendelsohn J, Baselga J. Status of epidermal growth factor receptorantagonists in the biology and treatment of cancer. J Clin Oncol2003;21:2787–99.

6. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al.Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib isassociated with a second mutation in the EGFR kinase domain. PLoSMed 2005;2:e73.

7. Riely GJ. Second-generation epidermal growth factor receptor tyro-sine kinase inhibitors in non-small cell lung cancer. J Thorac Oncol2008;3:S146–9.

8. Kwak EL, Sordella R, Bell DW, Godin-Heymann N, Okimoto RA,Brannigan BW, et al. Irreversible inhibitors of the EGF receptor maycircumvent acquired resistance to gefitinib. Proc Natl Acad Sci U S A2005;102:7665–70.

9. Engelman JA, Zejnullahu K, Gale CM, Lifshits E, Gonzales AJ, Shi-mamura T, et al. PF00299804, an irreversible pan-ERBB inhibitor, iseffective in lung cancer models with EGFR and ERBB2 mutations thatare resistant to gefitinib. Cancer Res 2007;67:11924–32.

10. Li D, Ambrogio L, Shimamura T, Kubo S, Takahashi M, Chirieac LR,et al. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective inpreclinical lung cancer models. Oncogene 2008;27:4702–11.

11. Takezawa K, Okamoto I, Tanizaki J, Kuwata K, Yamaguchi H, FukuokaM, et al. Enhanced anticancer effect of the combination of BIBW2992and thymidylate synthase-targeted agents in non-small cell lungcancer with the T790M mutation of epidermal growth factor receptor.Mol Cancer Ther 2010;9:1647–56.

12. Miller VA, Hirsh V, Cadranel J, Chen YM, Park K, Kim SW, et al. Afatinibversus placebo for patients with advanced, metastatic non-small-celllung cancer after failure of erlotinib, gefitinib, or both, and one or twolines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial.Lancet Oncol 2012;13:528–38.

13. Warburg O. On the origin of cancer cells. Science 1956;123:309–14.14. Yeung SJ, Pan J, Lee MH. Roles of p53, MYC and HIF-1 in regulating

glycolysis - the seventh hallmark of cancer. Cell Mol Life Sci2008;65:3981–99.

15. Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer's Achilles'heel. Cancer Cell 2008;13:472–82.

16. GrahamNA, TahmasianM,Kohli B, KomisopoulouE, ZhuM, Vivanco I,et al. Glucose deprivation activates a metabolic and signaling ampli-fication loop leading to cell death. Mol Syst Biol 2012;8:589.

17. Maschek G, Savaraj N, Priebe W, Braunschweiger P, Hamilton K,Tidmarsh GF, et al. 2-deoxy-D-glucose increases the efficacy of

adriamycin and paclitaxel in human osteosarcoma and non-small celllung cancers in vivo. Cancer Res 2004;64:31–4.

18. Singh D, Banerji AK, Dwarakanath BS, Tripathi RP, Gupta JP, MathewTL, et al. Optimizing cancer radiotherapywith 2-deoxy-d-glucosedoseescalation studies in patients with glioblastoma multiforme. Strah-lenther Onkol 2005;181:507–14.

19. Jain VK, Kalia VK, Sharma R, Maharajan V, Menon M. Effects of 2-deoxy-D-glucose on glycolysis, proliferation kinetics and radiationresponse of human cancer cells. Int J Radiat Oncol Biol Phys 1985;11:943–50.

20. Yamada M, Tomida A, Yun J, Cai B, Yoshikawa H, Taketani Y, et al.Cellular sensitization to cisplatin and carboplatin with decreasedremoval of platinum-DNA adduct by glucose-regulated stress. CancerChemother Pharmacol 1999;44:59–64.

21. Priebe A, Tan L, Wahl H, Kueck A, He G, Kwok R, et al. Glucosedeprivation activates AMPK and induces cell death through modula-tion of Akt in ovarian cancer cells. Gynecol Oncol 2011;122:389–95.

22. ShackelfordDB,ShawRJ. The LKB1-AMPKpathway:metabolismandgrowthcontrol in tumour suppression.NatRevCancer 2009;9:563–75.

23. El Mjiyad N, Caro-Maldonado A, Ramirez-Peinado S, Munoz-PinedoC. Sugar-free approaches to cancer cell killing. Oncogene 2011;30:253–64.

24. Corradetti MN, Guan KL. Upstream of the mammalian target ofrapamycin: do all roads pass through mTOR? Oncogene 2006;25:6347–60.

25. Liu H, Scholz C, Zang C, Schefe JH, Habbel P, Regierer AC, et al.Metformin and the mTOR inhibitor everolimus (RAD001) sensitizebreast cancer cells to the cytotoxic effect of chemotherapeutic drugsin vitro. Anticancer Res 2012;32:1627–37.

26. Sauer H, Engel S, Milosevic N, Sharifpanah F, Wartenberg M. Activa-tion of AMP-kinase by AICAR induces apoptosis of DU-145 prostatecancer cells through generation of reactive oxygen species and acti-vation of c-Jun N-terminal kinase. Int J Oncol 2011;40:501–8.

27. Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, Kogan S,et al. Survival signalling by Akt and eIF4E in oncogenesis and cancertherapy. Nature 2004;428:332–7.

28. Kim SM, Kim JS, Kim JH, Yun CO, Kim EM, Kim HK, et al. Acquiredresistance to cetuximab ismediated by increased PTEN instability andleads cross-resistance to gefitinib in HCC827 NSCLC cells. CancerLett 2010;296:150–9.

29. Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, et al.Multiparameter metabolic analysis reveals a close link between atten-uated mitochondrial bioenergetic function and enhanced glycolysisdependency in human tumor cells. AmJPhysiol Cell Physiol 2007;292:C125–36.

30. Gottlob K, Majewski N, Kennedy S, Kandel E, Robey RB, Hay N.Inhibitionof early apoptotic eventsbyAkt/PKB isdependent on thefirstcommitted step of glycolysis and mitochondrial hexokinase. GenesDev 2001;15:1406–18.

31. Plas DR, Talapatra S, Edinger AL, Rathmell JC, ThompsonCB. Akt andBcl-xL promote growth factor-independent survival through distincteffects on mitochondrial physiology. J Biol Chem 2001;276:12041–8.

32. Elstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH, Plas DR,et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res2004;64:3892–9.






33. Pradelli LA, BeneteauM, Chauvin C, JacquinMA,Marchetti S,Munoz-Pinedo C, et al. Glycolysis inhibition sensitizes tumor cells to deathreceptors-induced apoptosis byAMPkinase activation leading toMcl-1 block in translation. Oncogene 2009;29:1641–52.

34. Wensveen FM, Alves NL, Derks IA, Reedquist KA, Eldering E. Apo-ptosis induced by overall metabolic stress converges on the Bcl-2family proteins Noxa and Mcl-1. Apoptosis 2011;16:708–21.

35. Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR. Glycogensynthase kinase-3 regulates mitochondrial outer membrane permea-bilization and apoptosis by destabilization of MCL-1. Mol Cell2006;21:749–60.

36. Zhong Q, Gao W, Du F, Wang X. Mule/ARF-BP1, a BH3-only E3ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regu-lates apoptosis. Cell 2005;121:1085–95.

37. Raught B, Peiretti F, Gingras AC, Livingstone M, Shahbazian D,Mayeur GL, et al. Phosphorylation of eucaryotic translation initiationfactor 4B Ser422 is modulated by S6 kinases. EMBO J 2004;23:1761–9.

38. Jastrzebski K, Hannan KM, Tchoubrieva EB, Hannan RD, Pearson RB.Coordinate regulation of ribosome biogenesis and function by theribosomal protein S6 kinase, a keymediator ofmTOR function.GrowthFactors 2007;25:209–26.

39. Rathmell JC, FoxCJ, PlasDR, HammermanPS,Cinalli RM, ThompsonCB. Akt-directed glucose metabolism can prevent Bax conformationchange and promote growth factor-independent survival. Mol Cell Biol2003;23:7315–28.

40. Deprez J, Vertommen D, Alessi DR, Hue L, Rider MH. Phosphorylationand activation of heart 6-phosphofructo-2-kinase by protein kinase Band other protein kinases of the insulin signaling cascades. J BiolChem 1997;272:17269–75.

41. Brown J. Effects of 2-deoxyglucose on carbohydrate metablism:review of the literature and studies in the rat. Metabolism 1962;11:1098–112.

42. Cheng G, Zielonka J, Dranka BP, McAllister D, Mackinnon AC Jr,Joseph J, et al. Mitochondria-targeted drugs synergize with 2-deox-yglucose to trigger breast cancer cell death. Cancer Res 2012;72:2634–44.

43. Lieberthal W, Menza SA, Levine JS. Graded ATP depletion can causenecrosis or apoptosis of cultured mouse proximal tubular cells. Am JPhysiol 1998;274:F315–27.

44. Liu H, Savaraj N, Priebe W, Lampidis TJ. Hypoxia increases tumor cellsensitivity to glycolytic inhibitors: a strategy for solid tumor therapy(Model C). Biochem Pharmacol 2002;64:1745–51.

45. Chonghaile TN, Letai A. Mimicking the BH3 domain to kill cancer cells.Oncogene 2008;27 Suppl 1:S149–57.

46. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647–56.

47. Thoreen CC, Chantranupong L, Keys HR, Wang T, Gray NS, SabatiniDM. A unifying model for mTORC1-mediated regulation of mRNAtranslation. Nature 2012;485:109–13.

48. DingQ,He X, Hsu JM, XiaW, ChenCT, Li LY, et al. Degradation ofMcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumorsuppression and chemosensitization. Mol Cell Biol 2007;27:4006–17.

49. Schwickart M, Huang X, Lill JR, Liu J, Ferrando R, French DM, et al.Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cellsurvival. Nature 2010;463:103–7.

50. Wei G, Twomey D, Lamb J, Schlis K, Agarwal J, Stam RW, et al. Geneexpression-based chemical genomics identifies rapamycin as a mod-ulator of MCL1 and glucocorticoid resistance. Cancer Cell 2006;10:331–42.

Kim et al.





2013;12:2145-2156. Published OnlineFirst July 24, 2013.Mol Cancer Ther Sun Mi Kim, Mi Ran Yun, Yun Kyoung Hong, et al. Suppression of Mcl-1 by AMPK ActivationT790M Mutation to Irreversible EGFR Inhibitors via Translational

Small Cell Lung Cancer with−Glycolysis Inhibition Sensitizes Non

Updated version

10.1158/1535-7163.MCT-12-1188doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2013/07/24/1535-7163.MCT-12-1188.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/12/10/2145.full#ref-list-1

This article cites 50 articles, 18 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/12/10/2145.full#related-urls

This article has been cited by 6 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/12/10/2145To request permission to re-use all or part of this article, use this link


http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-12-1188http://mct.aacrjournals.org/content/suppl/2013/07/24/1535-7163.MCT-12-1188.DC1http://mct.aacrjournals.org/content/12/10/2145.full#ref-list-1http://mct.aacrjournals.org/content/12/10/2145.full#related-urlshttp://mct.aacrjournals.org/cgi/alertsmailto:[email protected]://mct.aacrjournals.org/content/12/10/2145http://mct.aacrjournals.org/

Glycolysis Inhibition Sensitizes Non Small Cell Lung ...The secondary EGF receptor (EGFR) T790M is...

Documents

Transcript of Glycolysis Inhibition Sensitizes Non Small Cell Lung ...The secondary EGF receptor (EGFR) T790M is...