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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
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Research. mct.aacrjournals.org Downloaded from
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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
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Published OnlineFirst July 24, 2013; DOI:
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http://mct.aacrjournals.org/
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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
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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
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ol)
120
100
80
60
40
20
0
Cel
l via
bili
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% o
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120
100
80
60
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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.
Mol Cancer Ther; 12(10) October 2013 Molecular Cancer
Therapeutics2148
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Research. mct.aacrjournals.org Downloaded from
Published OnlineFirst July 24, 2013; DOI:
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http://mct.aacrjournals.org/
-
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
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AT
P (
% o
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Lac
tate
(%
of
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H1975 PC9-GR
Afat
inib
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+ af
atin
ib2D
G
Cont
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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.
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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
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P (
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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.
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Therapeutics2150
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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.
Inhibition of Glycolysis Enhances Sensitivity to Afatinib
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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.
Mol Cancer Ther; 12(10) October 2013 Molecular Cancer
Therapeutics2152
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-
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.
Inhibition of Glycolysis Enhances Sensitivity to Afatinib
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-
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.
Mol Cancer Ther; 12(10) October 2013 Molecular Cancer
Therapeutics2154
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-
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.
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