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Preclinical Development

Dual Inhibition of Tumor Energy Pathway by 2-Deoxyglucoseand Metformin Is Effective against a Broad Spectrum ofPreclinical Cancer Models

Jae-Ho Cheong1, Eun Sung Park1, Jiyong Liang1, Jennifer B. Dennison1, Dimitra Tsavachidou4,Catherine Nguyen-Charles1, Kwai Wa Cheng1, Hassan Hall1, Dong Zhang1, Yiling Lu1, Murali Ravoori5,Vikas Kundra5, Jaffer Ajani2, Ju-Seog Lee1, Waun Ki Hong3, and Gordon B. Mills1

AbstractTumor cell proliferation requires both growth signals and sufficient cellular bioenergetics. The AMP-

activatedprotein kinase (AMPK) pathway seemsdominant over the oncogenic signaling pathway suppressing

cell proliferation. This study investigated the preclinical efficacy of targeting the tumor bioenergetic pathway

using a glycolysis inhibitor 2-deoxyglucose (2DG) and AMPK agonists, AICAR andmetformin. We evaluated

the in vitro antitumor activity of 2DG, metformin or AICAR alone, and 2DG in combination either with

metformin or AICAR. We examined in vivo efficacy using xenograft mouse models. 2DG alone was not

sufficient topromote tumor cell death, reflecting the limited efficacy showed in clinical trials.Acombineduse of

2DG and AICAR also failed to induce cell death. However, 2DG and metformin led to significant cell death

associated with decrease in cellular ATP, prolonged activation of AMPK, and sustained autophagy. Gene

expression analysis and functional assays revealed that the selective AMPK agonist AICAR augments

mitochondrial energy transduction (OXPHOS) whereas metformin compromises OXPHOS. Importantly,

forced energy restoration with methyl pyruvate reversed the cell death induced by 2DG and metformin,

suggesting a critical role of energetic deprivation in the underlying mechanism of cell death. The combination

of 2DGandmetformin inhibited tumor growth inmouse xenograftmodels.Deprivation of tumor bioenergetics

bydual inhibition of energypathwaysmight be an effective novel therapeutic approach for a broad spectrumof

human tumors. Mol Cancer Ther; 10(12); 2350–62. �2011 AACR.

Introduction

Proliferation of cancer cells requires oncogenic growthsignals as well as sufficient metabolic energy for biogen-esis of cellular constituents. The "Warburg effect" (1), ametabolic derangement in cancer cells resulting inincreased glucose uptake and glycolysis, provides a selec-

tive advantage to rapidly proliferating tumor cells bysupplying cellular bioenergetics required to supporttumor progression. Clinically, tumors with high glucoseuptake detected by the 2[18F]fluoro-2-deoxy-D-glucose–positron emission tomography (FDG–PET) scan show aworsened outcome (2–4), underscoring the therapeuticpotential of inhibition of glycolysis.

The mammalian target of rapamycin complex 1(mTORC1) is a key downstream effector of the phosphoi-nositide 3-kinase (PI3K)-AKT signaling pathway (5) thatregulates and promotes cell proliferation, growth, andsurvival in many cancer lineages (6–8). The mTORC1complex integrates oncogenic growth signaling with theglycolytic switch (9). The growth-promoting effects ofmTORC1 including protein synthesis and cell-cycle pro-gression are highly energy consuming (10, 11), suggestingthat mTORC1 activity would be dependent on cellularenergy status. Indeed, mTORC1 is directly inhibited byphosphorylation of raptor as a consequence of activationof the AMP-activated protein kinase (AMPK), a key cel-lular energy sensor that is activated by bioenergetic stress(12–15).

Remarkably, the energy sensingAMPKpathway seemsdominant over the growth-promoting effects of the PI3K-AKT pathway determining cellular functional outcomes.

Authors' Affiliations: Departments of 1Systems Biology and 2GI MedicalOncology, 3Division of Cancer Medicine, and Departments of 4Genitouri-nary Medical Oncology and 5Experimental Diagnostic Imaging, TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

J.-H. Cheong and E.S. Park contributed equally to this work.

J.-H. Cheong is an Odyssey Fellow.

Current address for J.-H. Cheong: Department of Surgery, YonseiUniversity College of Medicine, 250 Seongsanno, Seodaemun-gu,Seoul 120-752, Korea.

Corresponding Author: Jae-Ho Cheong, Department of Surgery, YonseiUniversity College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul120-752, Korea. Phone: 82-2-2228-2094; Fax: 82-2-313-8289; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0497

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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Published OnlineFirst October 12, 2011; DOI: 10.1158/1535-7163.MCT-11-0497

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This ensures that proliferation does not occur in theabsence of adequate nutrients, energy and cellular build-ing blocks, providing a novel "bioenergetics checkpoint."Therefore,manipulation of theAMPK signaling pathway,thus mimicking energy stress, could represent a thera-peutic target potentially overriding the oncogenic effectsof the PI3K-AKT-mTORC1 pathway.Herein, we describe the functional outcomes of glycol-

ysis inhibition by 2-deoxyglucose (2DG) in combinationwith AMPK agonists, AICAR and metformin. Unexpect-edly, we found that combined use of 2DG andmetformin,but not 2DG and AICAR, exhibits significant antitumoreffects in vitro and in vivo. Gene expression analysisand subsequent functional assays suggest that AMPKactivation protects tumor cells in energetically stressedconditions by augmenting mitochondrial respiration.Metformin, unlike AMP-mimetic AICAR, inhibits mito-chondrial energy generation thereby explaining theobserved antitumor effect in combination with 2DG.Finally, the in vivo efficacy in mouse xenograft modelsprovides the rationale for the clinical evaluation of thisnovel strategy for the treatment of cancer patients.

Materials and Methods

Cell cultureHuman gastric and esophageal cancer cell lines paren-

tal (p)-SK4 and OE33 were kindly provided in June 2006by Dr. Julie Izzo (The University of Texas MD AndersonCancer Center, Houston, TX) and cultured in Dulbecco’sModified Eagle’s Medium (DMEM)/Ham’s F-12 50:50supplemented with 10% FBS in a humidified incubatorcontaining 5%CO2 at 37

�C. U2OS, MCF-7, MDA-MB-468,MDA-MB-231, and MCF10A were obtained in May 2007from the American Type Culture Collection (ATCC) andgrown inmedium RPMI-1640 with 5% FBS. The identitiesof all cell lines were validated by short tandem repeat(STR) DNA fingerprinting using the AmpF_STR Identi-filer Kit according to manufacturer’s instructions(Applied Biosystems; catalogue no: 4322288) at Charac-terizedCell LineCoreFacility (All the cellswere last testedin October 2009). The STR profiles were compared withknown ATCC fingerprints (http://atcc.org) and to theCell Line Integrated Molecular Authentication database(CLIMA) version 0.1.200808 (http://bioinformatics.istge.it/clima/;NucleicAcidsResearch 37:D925–D932PMCID:PMC2686526). The STR profiles matched known DNAfingerprints or were unique.

Cell viability assayCell viability was determined by trypan blue dye exclu-

sion. For the assay, 0.3 � 106 cells were plated in 6-wellplates and treated the next day. Methyl pyruvate (MP, 10mmol/L) was added 2 hours before treatment, whereindicated. Cells were trypsinized, resuspended, andmixed with 1:1 0.4% trypan blue. Percentage cell death¼ number of stained cells/(number of stained þunstained cells) � 100.

Reverse phase protein arrayReverse phase protein array (RPPA) was processed as

previously described (16, 17). Briefly, serially dilutedlysates were spotted on FAST slides (Schleicher & SchuellBioSciences) by a robotic GeneTAC arrayer (GenomicSolutions). After printing, slideswere blotted sequentiallywith Re-Blot (Chemicon), I-Block, and biotin blockingsystem (Dako), probed with primary antibodies andincubated with biotin-conjugated secondary antibodies.The signals were then amplified by a catalyzed signalamplification kit (DakoCytomation) according to themanufacturer’s instructions. The processed slides werescanned and quantified with MicroVigene software(VigeneTech Inc.).

Measurement of intracellular ATP levels andmitochondrial transmembrane potential (Dcm)

Intracellular ATP was measured by a luciferin/lucif-erase-based assay. Cells were grown under each exper-imental condition for indicated times, harvested, andcounted. Aliquots containing equal number of cellswere processed following manufacturer’s guidelines(Roche).

Rhodamine-123, a cationic voltage-sensitive mitochon-drial probe, was used to detect changes in mitochondrialtransmembrane potential (Dym). Cells were incubated asindicated and labeled with 1 mmol/L rhodamine-123 at37�C for 30 minutes. After washing, the samples wereanalyzed by flow cytometry.

ImmunoblottingCell lysis and immunoblotting were carried out as

previously described (18). A total of 50 mg protein wasused for the immunoblotting, unless otherwise indicated.b-Actin and glyceraldehyde-3-phosphate dehydrogenasewere used as loading controls. Anti-LC3 antibody was agift from Dr. S. Kondo. All other antibodies were pur-chased from Cell Signaling.

Transmission electron microscopySamples were fixed with a solution containing 3%

glutaraldehyde plus 2% paraformaldehyde in 0.1 mol/Lcacodylate buffer, pH 7.3, for 1 hour. After fixation,samples were washed and treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid, postfixedwith 1%buffered osmium tetroxide for 30 minutes, and stained enbloc with 1% Millipore-filtered uranyl acetate. Sampleswere dehydrated in increasing concentrations of ethanol,infiltrated, and embedded in LX-112 medium. Sampleswere polymerized in a 70�C oven for 2 days. Ultrathinsections were cut in a Leica Ultracut microtome (Leica),stained with uranyl acetate and lead citrate in a LeicaEM stainer, and examined in a JEM 1010 transmissionelectron microscope (JEOL USA, Inc.) at an acceleratingvoltage of 80 kV.Digital imageswere obtained by theAMTImaging System (Advanced Microscopy TechniquesCorporation).

Anticancer Effect of Dual Inhibition of Tumor Bioenergetics

www.aacrjournals.org Mol Cancer Ther; 10(12) December 2011 2351




Animal protocolAthymic female nude mice (CD-1 nu/nu from the NIH

National Cancer Institute repository)were used for in vivotumor growth studies. All of the in vivo studies werecarried out under approved institutional experimentalanimal care and use protocols. For the breast tumororthotopic model, MDA-MD-231 cells were resuspendedat 6.5 � 106 cells per 200 mL in PBS and injected into theseventh mammary gland fat pad of 4-week-old athymicnude mice. When tumors were approximately 50 mm3

in size (14 days), the animals were randomly allocatedinto 4 groups. Following randomization, vehicle, 0.2 mLof 2DG (500 mg/kg), metformin (250 mg/kg, equivalentto a human dose of 20 mg/kg by normalization tosurface area), or both were delivered via intraperitonealadministration daily for the duration of the experiment.For the esophagogastric cancer xenograft model, nudemice received subcutaneous implantation with 200 mLof the s-SK4 (selected from p-SK4 cells) at 4 � 106 cellsper 200 mL in PBS. When tumors were approximately100 mm3 in size (one week later), the animals wererandomized. A total of 0.2 mL of PBS or 2DG (500mg/kg) þ metformin (250 mg/kg) were delivered viaintraperitoneal administration, daily for the durationof the experiment. MRI was conducted, and tumorvolumes in vivo were assessed.

Tumor size was measured with digital calipers 2 to 3times weekly. Tumor measurements were converted totumor volume using the formula as follows: L � S2/2(where L, longest diameter; S, shortest diameter). Micewere killed when either L or S exceeded 15 mm in controlgroup. At sacrifice, tumors were excised and assessedhistologically for verification of tumor growth. Statisticalsignificance was determined by the Student t test.

Microarray experiment and data analysisTotal RNA was isolated from cells harvested after

each treatment by the mirVana miRNA IsolationKit (Ambion Inc.) according to manufacturer’s protocol.Biotin-labeled cRNA samples were prepared by using theIllumina TotalPrep RNAAmplificationKit (Ambion Inc.).A total of 500 ng of RNA was used for the synthesis ofcDNA and then followed by amplification and biotinlabeling as recommended by the manufacturer. A totalof 1.5 mg of biotinylated cRNAper samplewas hybridizedto Illumina Human-6 BeadChip v.2 microarray, andsignals were developed by Amersham fluorolink strepta-vidin-Cy3 (GE Healthcare Bio-Sciences). Data wereanalyzed by using the Illumina Bead Studio v3.0 afterscanning with Illumina bead Array Reader confocal scan-ner (BeadStation 500GXDW; Illumina Inc.) All statisticalanalysis was conducted with the R 2.3.0 and BRB Array-tools Version 3.5 (http://linus.nci.nih.gov./BRB-Array-Tools.html). To minimize the effect of variation fromnonbiological factors, the values of each sample werenormalized by a quantile normalizationmethod. Randomvariance t test was applied for the calculation ofsignificance of each gene in the comparison of 2 classes,

and one-way ANOVA was applied for the evaluation ofsignificance in multigroup comparison. Cluster analysiswas conducted with Cluster and Treeview (http://rana.lbl.gov/EisenSoftware.htm). For cluster analysis, log2-transformed data were centered to mean values of eachgene expression. Gene set enrichment analysis was con-ducted against Gene Ontology (GO) of biological process,and Kolmogorov–Smirnov statistic was applied for theevaluation of statistical significance of each GO category.

Results

Inhibition of glycolysis by 2DG is not sufficient toactivate AMPK or promote cell death

To investigate the consequences of metabolic stress onkey signaling pathways, a panel of cancer cell lines ofmultiple lineages was subjected to glucose deprivationand combinations of 2DG and known AMPK activators(AICAR and metformin). The cell lysates were subject tomultiplexed RPPA (refs. 16, 17) to evaluate multipleprotein expression levels simultaneously (Fig. 1A andB, Supplementary Fig. S1).

Incubation in glucose-free medium consistentlyinactivated PI3K-AKT-mTORC1 downstream signals(p-p70S6K, p-S6, and cyclin D1) and reduced expressionof cell-cycle regulators (cyclin D1, cyclin B1, and p-Rb;P�0.01, SupplementaryFig. S1). p-S6 levelsweremarked-ly reduced by glucose deprivation representing the mostsensitive biomarker (85%decrease, Fig. 1A). Interestingly,p-AMPK and p-ACC levels, markers of cellular bioener-getic stress, were essentially unchanged at 24 hoursdespite glucose deprivation or the addition of 2DG, sug-gesting that the cells were able to compensate for the lossof glucose-derived ATP production (Fig. 1B and C).

Of note, metformin, which is known to activate AMPK,didnot induceAMPKactivation or subsequent phosphor-ylation of acetyl-CoA carboxylase (ACC) and raptorwhenhigh concentrations of glucose (25 mmol/L) were present(Fig. 1C). In contrast, the combination of 2DG and met-formin markedly activated AMPK and subsequentlydeactivated mTORC1 downstream signaling. These coor-dinated alterationswere compatible with activation of thetuberous sclerosis protein 2 (TSC2) by AMPK resulting indecreased signaling through mTORC1 and downstreamtargets (19–22). The AMP-mimetic AICAR, however,markedly activatedAMPKand inhibitedmTORC1down-stream effectors even in the presence of 25 mmol/Lglucose, and these effects were not augmented whencombined with 2DG. These results suggest that the phar-macobiological outcomes betweenmetformin andAICARwould bedifferentwhenhigh level of glucose is present orglucose is deprived.

On the basis of these results, we predicted that inhibi-tion of glycolysis alonewould not be sufficient to promotebioenergetic stress. Indeed, clinically achievable concen-trations of 3 to 4 mmol/L 2DG (23) did not substantiallyactivate AMPK and affect cell signaling in the majority ofthe cell lines assessed (Fig. 1B). Furthermore, inhibition of

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Mol Cancer Ther; 10(12) December 2011 Molecular Cancer Therapeutics2352




glycolysis with 2DG at clinically achievable doseswas notsufficient to induce a significant degree of cell death, evenwith prolonged incubation (Fig. 2A and B).

Metformin but not AICAR induces cell death incombination with 2DGOn the basis of the alterations in signaling pathways

(Fig. 1C), we next compared the effects of each agent ontumor cell viability. We hypothesized that the AMP-mimetic AICAR, 2DG þ metformin, and 2DG þ AICARshould reduce cell viability based on the substantialchanges in AMPK and mTORC1 downstream signals(Fig. 1C).Surprisingly, neither AICAR nor 2DG þ AICAR

induced cell death, whereas the 2DG þ Met significantlyreduced cell viability in a time-dependent manner (Fig.2A and C). Furthermore, the combination effect of 2DGandmetformin resulted in amarked increase in cell deathcompared with either agent alone (Fig. 2A, B, and D).Unexpectedly, AICAR as a single agent or in combinationwith 2DG failed to mimic the effects of metformin incombination with 2DG on cell death (Fig. 2A–D). Thissuggests that activation of AMPK was not sufficient toinduce cancer cell death irrespective of the presence of2DG. Instead, it suggests that enhanced cell deathobservedwithmetformin in combinationwith 2DGmightbe caused by AMPK-independent mechanisms orthrough mechanisms in addition to AMPK activation.

Metformin and AICAR induce markedly differentgene sets controlling mitochondrial energytransductionAlthoughmetformin andAICARhave been reported to

activateAMPK (24–29), our data indicated that the admin-istration of each alone or in combinationwith 2DG leads to

markedly different biological outcomes. Furthermore,when glucose was sufficient, metformin did not substan-tially activate AMPK at least in the cell lines tested. Toexplore potential mechanisms underlying the ability ofmetformin, but not AICAR, to induce cell death whencombined with 2DG in cancer cells, we conductedgenome-wide transcriptional profile analysis. Geneexpression patterns of each treatment group were dra-matically different (Fig. 3A; 2,527 significant genes;P < 10�6 one-way ANOVA). Furthermore, whereas 2DGand control groups clustered closely together, metforminand AICAR showed distinct and distant clusters inde-pendent of the presence of 2DG, indicative of markedlydifferent mechanisms of action (Fig. 3A).

To estimate the functional and biological consequencesof differences in gene expression between the treatments,we conducted gene set enrichment analysis (Kolmo-gorov–Smirnov permutation test, P < 0.001) against thebiological process category of GO. Of note, metformin inthe presence or absence of 2DG significantly downregu-lated expression of 12 components of the electron trans-port chain (ETC) complex 1 in the mitochondria withoutsignificantly altering expression of other components ofthe ETC complexes (Supplementary Table S1 and Fig. 3B).Other components of the ETC or tricarboxylic acid (TCA)cycle were unaffected (Supplementary Tables S1, S2and Fig. 3C). This selective effect of metformin on ETC1could limit the extraction of electrons from NADH there-by suppressing the mitochondrial ATP-generatingcapacity.

In contrast, AICAR alone or in combination with 2DGsignificantly induced expression of multiple genes in theTCA cycle and the ETC complex I, II, III, and IV and F0F1-ATPases that could coordinately increase the efficiency ofOXPHOS (Supplementary Table S1 and Fig. 3D and E).

Figure 1. AICAR and thecombination of 2DG and metformin(Met) activate AMPK and reducephosphorylation of mTORC1-regulated proteins. A and B, proteinlevels of lysates from multiple celllines were quantified by RPPA aftertreatment with the indicatedcompounds for 24 hours.Phosphorylations of S6 and ACCwere quantified to evaluatesignaling changes downstream ofmTORC1 and AMPK, respectively.Values were compared with theuntreated controls (broken lines)using a 2-tailed t test. �, P < 0.05;��, P < 0.01; ��, P < 0.001.C, Western blot analysis of p-SK4cells lysate.






Furthermore, expression of a set of genes involved ingluconeogenesis and vacuolar type ATPases that con-sume ATP was concomitantly decreased (Fig. 3D and E,Supplementary Table S2). This suggests that activation of

AMPK by anAMP-mimetic drugmay increasemitochon-drial ATP production by augmenting the efficiency of theTCA cycle, ETC, and OXPHOS, enabling cells to maintainbioenergetic homeostasis.

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Figure 2. 2DG combined with metformin selectively promotes death of cancer cells. Cell viability was assessed by trypan blue exclusion. Data are the mean� SD (n ¼ 3) of at least 100 cells from one representative experiment of at least 4 independent experiments. A, p-SK4 cells were treated with indicatedagents and viability was assessed. B, viability of breast (MDA-231 andMCF-7) and osteosarcoma (U2OS) cell lines was assessed at 72 hours. C, p-SK4 cellswere treated with 2DG þ Met or 2DG þ AICAR for the indicated times and representative photomicrographs taken. Scale bar, 100 mm. D, GFP-LC3–transfected U2OS cells were incubated for 72 hours with the indicated compounds (top, fluorescence analysis; bottom, bright fields; scale bars, 100 mm).

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Metformin and AICAR exert opposite effects onmitochondrial energy generationThe disparate effects of AICAR and metformin on

expression of components of the mitochondrial respira-tory chain suggested that they would have differentialeffects on mitochondrial energy generation capabilitysuch as transmembrane potential (Dym), the protonmotive force that drives mitochondrial ATP production(30, 31). Consistent with the mRNAmicroarray data (Fig.3D and E), AICAR alone or combinedwith 2DG increasedmitochondrial function as shown by a significant increasein Dym (Fig. 3G). In contrast, metformin in the presenceor absence of 2DG induced a time-dependent decreasein Dym (Fig. 3G) andmitochondrial shrinking with acqui-sition of an electron dense matrix (Fig. 3F). Similar to theeffects of metformin, accumulation of mitochondrial elec-tron dense deposits has been previously reported after adecrease in ETC complex I and III functions (32).Consistent with the alterations in mitochondrial Dym,

we found that 2DG induced a modest time-dependentdecrease in cellular ATP levels (Fig. 3H). As predicted,metformin induced a decrease in cellular ATP levelswhereas AICAR did not significantly alter cellular ATPlevels (Fig. 3H). The combination of 2DG andmetforminreduced cellular ATP by 70%, suggesting that decreas-ing ATP levels below a critical threshold could result inthe cell death associated with 2DG and metformin (Fig.2A and B). Strikingly, AICAR significantly reversedthe effects of 2DG on ATP levels at delayed time points(48 hours), compatible with the changes in gene expres-sion and augmented mitochondrial function describedearlier.

MP rescues energetic stress-induced cell death bymetformin plus 2DGBecause energetic stress activates AMPK and sup-

presses mTORC1 thereby inducing autophagy (19), weexamined cellular ATP levels, AMPK and mTORC1 sig-nals, and autophagy over time of treatment with metfor-min and 2DG. The combination of metformin and 2DGinduced progressive depletion of ATP (Fig. 4A) andincreased LC3-II levels indicative of sustained autophagy(Fig. 4B) compatible with the prolonged activation ofAMPK and suppression of mTORC1 signals (Fig. 4C).These changes were reflected by a time-dependentdecrease in cell size, an accumulation of autophagocyticvesicles and electron dense bodies, and a lack of char-acteristics typical of apoptosis or necrosis (Fig. 4D and E).Metformin compromises but does not completely block

mitochondrial ATP production, because it selectivelyinhibits ETC1 while leaving ETC complex II intact (33,34). Thus, administering high levels of a substrate that canprovide electrons to the ETC complex II should reverse, atleast in part, the effects of metformin. We exploited MP, agrowth factor independent exogenous cell permeableenergy substrate that bypasses glycolysis and directlyenters the mitochondrial TCA cycle (35–37). In theory,MP should lead to the production of FADH2 in the TCA

cyclewith an electron being extracted fromcomplex II andtransferred in the ETC, thus producing ATP even whenETC1 is compromised by metformin. Indeed, MPincreased intracellular ATP levels in cells treated with2DG and metformin (Fig. 5A), indicating a partial bypassof the effects of metformin. Compatible with the increasein ATP levels, MP markedly suppressed the increase inphosphorylation of AMPK andACC induced by 2DG andmetformin (Fig. 5B), indicating that AMPK activation by2DG and metformin is likely a consequence of a compro-mise in energyproduction rather thandue to adirect effectof either compound onAMPK activity. In agreement withthe ability of MP to restore cellular ATP levels and atten-uate AMPK activity, MP reduced the formation of GFP-LC3 punctate vesicles as well as the number of autopha-gosomes (Fig. 5C and D). Consistent with the effects onGFP-LC3, there was a reduced accumulation of LC3-II onWestern blotting in cells treatedwithMP (Fig. 5E). Impor-tantly, MP decreased cell death induced by 2DG plusmetformin (Fig. 5F). The ability of MP to attenuate theeffects of 2DG plus metformin were confirmed in U2OScells in terms of autophagy, cellular morphology, celldeath, and ATP levels (Supplementary Fig. S2). Thus, theability of the combination of 2DG and metformin toinduce autophagy, AMPK activation, and cell death canbe attributed to decreased energy production due to apartial but coordinated compromise of glycolysis andmitochondrial function.

Metformin plus 2DG treatment inhibits tumorgrowth and metastasis in xenograft tumor models

We next examined the in vivo efficacy of 2DG andmetformin by using 2 different xenograft mouse models.First, MDA-MB-231 cells were injected into themammaryfat pad of nudemice and allowed to establish measurabletumors for 14 days. Mice were randomized to differenttreatment groups and treated intraperitoneally daily for36 days at which time the experiment was terminatedbecause of excessive tumor volume in control mice. Asindicated in Fig. 6A, 2DG or metformin, at doses deliv-ered, did not alter tumor growth. In contrast, the 2DG andmetformin combination significantly decreased tumorgrowth from day 21 to the termination of the study atday 36 (Fig. 6B). Second, using a tumorigenic line selectedfrom p-SK4 cells (s-SK4), we determined whether theeffect of 2DG and metformin on in vivo tumor growth ofMDA-MB-231 cells could be generalized to other lineages.Two weeks of treatment with metformin and 2DGdecreased subcutaneous tumor growth of s-SK4 (Fig.6C). The decrease in tumor volume was confirmed byMRI during the course of treatment (see Fig. 6D, for arepresentative pair).Aswith theMDA-MB-231model, thedecrease in tumor growth inducedby 2DGandmetforminwas confirmed by weighing the tumor at the terminationof the study (Fig. 6E). Notably, immunohistochemicalanalysis of the tumor sections revealed substantialdecreases in p-S6 and cyclin D1 expression levels,






recapitulating the effects of metformin and 2DG observedin vitro (Fig. 6F).

Discussion

The Warburg effect is an attractive therapeutic targetbecause many cancers are highly glycolytic and is asso-

ciated with tumor aggressiveness and clinical outcomes.Despite exciting preclinical studies, however, 2DG failedto fulfill its expectations in clinical trials.

Likewise in our studies, 2DG at clinically achievabledoses did not substantially activate AMPK, reflectinginsufficient suppression of tumor bioenergetics. Theseeffects were inadequate to induce death of most cancer

Figure 3. AICAR and metformin effects on gene expression are distinct and opposing for genes related to the mitochondrial energy transduction.p-SK4 cells were treated with 2DG (4 mmol/L), metformin (5 mmol/L), AICAR (2 mmol/L), 2DG þ Met, or 2DG þ AICAR for 12 hours. A, class comparisonswere conducted between the 6 treatment groups using genes filtered with one-way ANOVA. Clustering analysis of genes showed significant differencesbetween AICAR and metformin treatment (2,527 significant genes, P < 10�6). B–E, supervised clustering analyses were conducted for genes related tothe mitochondrial energy transduction, gluconeogensis, and the TCA cycle.�, P < 0.05; ��, P < 0.01; ��, P < 0.001.

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cell lines assessed or to inhibit tumor growth in vivo. Thisprompted us to explore whether the activation of AMPK,thus mimicking energy stress, would lead to increases incell death. We, therefore, used metformin and AICAR,pharmacologic mediators that have been reported toactivate AMPK (24–29).Activation of AMPK has been reported to lead to sup-

pression of mTORC1 signaling (10, 11), cell cycle exit dueto stabilization of p27KIP1 (18), and a decrease in energyconsumption through diminishing protein and lipid bio-synthesis (13–15, 38). Our study suggests an additionalrole for AMPK activation in tumor bioenergetics. Directactivation of AMPK by the AMP-mimetic AICARdecreased expression of mRNA for enzymes involved ingluconeogenesis, induced expression of mRNA for mul-tiple proteins involved in the ETC and OXPHOS, andincreased mitochondrial respiration. This suggests thatAMPK induces a switch from dependence on glycolysis

for ATP production to energy transduction throughmito-chondrial respiration. In the current study, we directlyshowed a role for AMPK in the ability to bypass effects ofinhibition of glycolysis; MEFs lacking AMPKwere exqui-sitely sensitive to the effects of glucose deprivation aswellas to 2DG (Supplementary Fig. S3A and S3B). Thus, in thecontext of energy stress, activation of AMPK could limitthe effects of glycolysis inhibitors, suggesting a noveltherapeutic combination would be required to overridethe energetic protective effect of AMPK.

In the absence of glycolysis, mitochondrial energy pro-duction is the only potential cellular source of ATP. Asdescribed earlier, inhibition of glycolysis and subsequentactivation of AMPK results in a coordinated increase inmRNA levels of genes involved in mitochondrial energyproduction, potentially triggering a switch to dependenceon OXPHOS. Metformin, an orally available antidiabeticdrug, has been proposed to inhibit complex I of the

Figure 3. (Continued) F, TEM of p-SK4 cells after incubation with indicated treatments for 12 hours (top) and 24 hours (bottom). Normal (arrowhead)and abnormally shrunken electron dense mitochondria (arrow) are indicated. Scale bar, 500nm. G, mitochondrial transmembrane potential (Dym) ofp-SK4cells incubatedwith indicatedcompounds (�,P

Figure 4. Prolonged incubation with 2DG and metformin depletes cellular ATP, activates AMPK, suppresses mTORC1 downstream signaling, and sustainsautophagy. p-SK4 cells were incubated with 2DG and metformin for the indicated times. A, cellular ATP levels were determined. Data are the mean � SD(n¼ 3). B, Western blotting for LC3 expression. b-Actin was used as loading control. C, kinetics of signaling molecules in AMPK and mTORC1 downstream.Western blotting of samples from (4B). D, TEManalysis ofmorphologic alteration of cells treatedwith 2DG in combinationwithmetformin. Severe cytoplasmiccontraction and resultant membrane blebbing is evident (48 and 60 hours). Note most of the cytoplasm is replaced with autophagosomes (60 hours).Neither typical apoptotic nor necrotic cells are evident (N, nucleus; arrowheads, autophagosome; scale bar, 10 mm for top; 500 nm for bottom). E, autophagicvesicles were counted for 3 randomly selected cells from each electron microscopy section. Data presented mean � SD.

Cheong et al.





Figure 5. Exogenous energy substrate MP increases cellular ATP, decreases AMPK activation, and reduces autophagy and cell death. p-SK4 cells weretreated with 2DG (4 mmol/L) and metformin (5 mmol/L) with and without MP (10 mmol/L). A, intracellular ATP levels were measured at 72 hours. �, P < 0.01.B, Western blotting (lysates from A) to assess AMPK activation. b-Actin was used as loading control. C, autophagic analysis by fluorescence microscopyof GFP-LC3 punctate patterns (top) and TEM (bottom; N, nucleus; arrowheads, autophagosome; scale bar, 10 mm for top; 500 nm for bottom). D, quantitationof autophagosomes on TEM. Autophagic vesicles were counted for 3 randomly selected cells from each electron microscopy section. Data presentedmean � SD. �, P < 0.01. E, cell lysates (samples from 5A) were subject to Western blotting to assess LC3 expression and lipidation (LC3-II), anautophagy marker. F, cell death assay by trypan blue exclusion. �, P < 0.05. All assays were conducted at 72 hours except C, which was 60 hours. Dataare mean � SD (n ¼ 3).






respiratory chain in themitochondria (33, 34). Indeed, ourdata show that metformin coordinately decreases mRNAlevels for ETC1 components and mitochondrial mem-branepotential compatiblewith compromisingmitochon-drial energy transduction. While metformin has beenknown to activate AMPK in intact cells (26, 39), this couldbe a consequence of its inhibitory effects onmitochondrialATP production as shown here. Furthermore, the abilityof metformin and other biguanide analogues such asphenformin to induce lactic acidosis is compatible withinhibition of mitochondrial function and consequent pro-duction of lactate due to elevated levels of pyruvate.

In light of this, we assessed whether inhibition ofglycolysis with 2DG combined with a compromise ofmitochondrial functionwith the clinically applicablemet-formin would result in cell death. Strikingly, in 13 of 15cancer cell lines assessed regardless of mutational status,the combination of 2DG and metformin was more effica-

cious at inhibiting cell growth and inducing cell deaththan either drug alone (data not shown).

Recently, Ben Sahra and colleagues reported that thecombination of metformin and 2DG induced p53-depen-dent apoptosis in prostate cancer cells (40). In their study,apoptosis was AMPK-mediated and p53 was required. Incontrast, our results show that AMPK itself prevents celldeath during glucose deprivation or 2DG treatment (SeeSupplementary Fig. S3A and S3B). Furthermore, AICARcombined with 2DG failed to induce cell death in anumber of cancer cell lines, suggesting a critical role ofAMPK as a guardian of cellular bioenergetics. Although,the distinct results between the 2 studiesmaybedue to thedifferent tissue of origin or characteristics of cell linesused, ourdata suggest that prolonged activation ofAMPKby 2DG and metformin might reflect sustained bioener-getics stress due to failure ofmitochondrial compensationrather than direct activation of AMPK.

Figure 6. Effects of 2DG andmetformin on xenograft tumorgrowth. A, tumors were establishedin athymic nude female mice bymammary fat pad injection of6.5 � 106 MDA-MB-231 cells andtreated as described in Materialsand Methods. The mean tumorvolume � SD is presented.�, P < 0.05 compared with control,2DG alone, or metformin alone.B, ex vivo tumor weight of tumorsfrom A. �, P < 0.05; ��, P < 0.01, and��, P ¼ 0.01. C, tumors wereestablished in athymic nude femalemice by subcutaneous injection of4 � 106 s-SK4 cells and treated asdescribed in Materials andMethods.Mean tumor volume�SDis presented. �, P < 0.05.D, representative MR images.E, tumor weight by 2DG þmetformin as assessed by in vivoMRI andexcised tumors. �,P

Consistent with the ability of the combination of 2DGand metformin to induce cell death in vitro, the combi-nation significantly suppressed tumor growth in 2 xeno-graft models in vivo. These results suggest that thetumor cell bioenergetics can be targeted, and the com-bination of 2DG andmetformin warrants further clinicalevaluation. Recently, metformin has gained much atten-tion due to its antitumor activity in some cancer types inpreclinical assays (40, 41), with AMPK activation beingsuggested as a major mechanism of action. As our dataindicate, however, AMPK activation can promote can-cer cell survival in energetically stressed conditions. Arecent study showing that metformin can exert itsinhibitory action directly on mTORC1 (42), furtherreinforces the notion that the effects of metformin canbe mediated independently of AMPK.In summary, realizing the clinical benefit of blockade

of the Warburg effect may require concomitant inhibi-tion of multiple components of cellular energy path-ways. A preemptive blockade of the Warburg effect andcompensatory mechanisms may prove to be dominantover the survival and growth-promoting effects ofgrowth factors or activated oncogenes. Because 2DGand metformin are already widely used in PET scan orseizure disorders and in type II diabetes, respectively,the expedited assessment of clinical effect of depriva-tion of cancer bioenergetics could be immediately testedin cancer patients.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Dr. Seiji Kondo and Keishi Fujiwara for criticaldiscussion, Doris Siwak and Qinghua Yu for assistance with RPPA,Dr. Peng Huang and Zhao Chen for the mitochondrial transmem-brane potential analysis, Kenneth Dunner Jr. for conducting electronmicroscopy, and Maurice J. Dufilho IV for assistance with mousenecropsy.

Grant Support

This work was supported by NIH SPORE (P50-CA83639), PO1CA64602, PO1 CA099031, CCSG grant P30 CA16672, and DAMD (17-02-01-0694) to G.B. Mills and Institutional Core grant #CA16672 HighResolution Electron Microscopy Facility, UTMDACC. STR DNA finger-printing was done by the Cancer Center Support grant funded Charac-terized Cell Line core, NCI # CA16672. J-H.Cheong is supported by theOdyssey Program of the Theodore N. Law Endowment for ScientificAchievement at the MD Anderson Cancer Center. J.B. Dennison is sup-ported by a TRIUMPHGlaxoSmithKline and theAmerican Cancer SocietyJoe and Jessie Crump Biomedical Research postdoctoral fellowships at theMD Anderson Cancer Center.

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 July 7, 2011; revised September 21, 2011; accepted September28, 2011; published OnlineFirst October 12, 2011.

References1. Warburg O. On the origin of cancer cells. Science 1956;123:

309–14.2. Downey RJ, Akhurst T Gonen M Vincent A Bains MS Larson S , et al.

Preoperative F-18 fluorodeoxyglucose-positron emission tomogra-phy maximal standardized uptake value predicts survival after lungcancer resection. J Clin Oncol 2004;22:3255–60.

3. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis?Nat Rev Cancer 2004;4:891–9.

4. Kumar R, Mavi A, Bural G, Alavi A. Fluorodeoxyglucose-PET in themanagement of malignant melanoma. Radiol Clin North Am 2005;43:23–33.

5. DeBerardinis RJ, LumJJ,HatzivassiliouG, ThompsonCB. The biologyof cancer: metabolic reprogramming fuels cell growth and prolifera-tion. Cell Metab 2008;7:11–20.

6. Brugge J, HungMC,Mills GB. A newmutational AKTivation in the PI3Kpathway. Cancer Cell 2007;12:104–7.

7. Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, et al. Highfrequency ofmutations of thePIK3CAgene in humancancers. Science2004;304:554.

8. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol3-kinases as regulators of growth and metabolism. Nat Rev Genet2006;7:606–19.

9. Shamji AF, Nghiem P, Schreiber SL. Integration of growth factor andnutrient signaling: implications for cancer biology. Mol Cell 2003;12:271–80.

10. Brenman JE. AMPK/LKB1 signaling in epithelial cell polarity and celldivision. Cell Cycle 2007;6:2755–9.

11. Buttgereit F, Brand MD. A hierarchy of ATP-consuming processes inmammalian cells. Biochem J 1995;312;163–7.

12. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activatedprotein kinase suppresses protein synthesis in rat skeletal muscle

through down-regulated mammalian target of rapamycin (mTOR)signaling. J Biol Chem 2002;277:23977–80.

13. GwinnDM,ShackelfordDB,EganDF,MihaylovaMM,MeryA,VasquezDS, et al. AMPK phosphorylation of raptor mediates a metaboliccheckpoint. Mol Cell 2008;30:214–26.

14. Hardie DG. AMP-activated/SNF1 protein kinases: conservedguardians of cellular energy. Nat Rev Mol Cell Biol 2007;8:774–85.

15. Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated proteinkinase: ancient energy gauge provides clues tomodern understandingof metabolism. Cell Metab 2005;1:15–25.

16. SheehanKM,Calvert VS, KayEW, LuY, FishmanD, Espina V, et al. Useof reverse phase protein microarrays and reference standard devel-opment for molecular network analysis of metastatic ovarian carcino-ma. Mol Cell Proteomics 2005;4:346–55.

17. Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, et al. Reversephase protein array: validation of a novel proteomic technology andutility for analysis of primary leukemia specimens and hematopoieticstem cells. Mol Cancer Ther 2006;5:2512–21.

18. Liang J, Shao SH, Xu ZX, Hennessy B, Ding Z, Larrea M, et al. Theenergy sensing LKB1-AMPK pathway regulates p27(kip1) phosphor-ylation mediating the decision to enter autophagy or apoptosis. NatCell Biol 2007;9:218–24.

19. Lum JJ, DeBerardinis RJ, Thompson CB. Autophagy in metazoans:cell survival in the land of plenty. Nat Rev Mol Cell Biol 2005;6:439–48.

20. Inoki K, Zhu T, Guan K-L. TSC2 mediates cellular energy response tocontrol cell growth and survival. Cell 2003;115:577–90.

21. Sarbassov DD, Ali SM, Sabatini DM. Growing roles for the mTORpathway. Curr Opin Cell Biol 2005;17:596–603.

22. Wang W, Guan KL. AMP-activated protein kinase and cancer. ActaPhysiol (Oxf) 2009;196:55–63.






23. Mohanti BK, Rath GK, Anantha N, Kannan V, Das BS, ChandramouliBA, et al. Improving cancer radiotherapy with 2-deoxy-D-glucose:phase I/II clinical trials on human cerebral gliomas. Int J Radiat OncolBiol Phys 1996;35:103–11.

24. Fryer LG, Parbu-Patel A, Carling D. The Anti-diabetic drugs rosiglita-zone and metformin stimulate AMP-activated protein kinase throughdistinct signaling pathways. J Biol Chem 2002;277:25226–32.

25. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role ofAMP-activated protein kinase in mechanism of metformin action. JClin Invest 2001;108:1167–74.

26. Hawley SA, Gadalla AE, Olsen GS, Hardie DG. The antidiabetic drugmetformin activates the AMP-activated protein kinase cascade via anadenine nucleotide-independent mechanism. Diabetes 2002;51:2420–5.

27. Young ME, Radda GK, Leighton B. Activation of glycogen phosphor-ylase and glycogenolysis in rat skeletal muscle by AICAR–an activatorof AMP-activated protein kinase. FEBS Lett 1996;382:43–7

28. Corton JM, Gillespie JG, Hawley SA, Hardie DG. 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells? Eur J Biochem 1995;229:558–65.

29. Sullivan JE, Brocklehurst KJ, Marley AE, Carey F, Carling D, Beri RK.Inhibition of lipolysis and lipogenesis in isolated rat adipocytes withAICAR, a cell-permeable activator of AMP-activated protein kinase.FEBS Lett 1994;353:33–6.

30. Labajova A, Vojtiskova A, Krivakova P, Kofranek J, Drahota Z, HoustekJ. Evaluation of mitochondrial membrane potential using a comput-erized device with a tetraphenylphosphonium-selective electrode.Anal Biochem 2006;353:37–42.

31. Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, NewmeyerDD,GreenDR.Cytochromecmaintainsmitochondrial transmembranepotential and ATP generation after outer mitochondrial membranepermeabilization during the apoptotic process. J Cell Biol 2001;153:319–28.

32. Lamperth L, Dalakas MC, Dagani F, Anderson J, Ferrari R. Abnormalskeletal and cardiacmusclemitochondria induced by zidovudine (AZT)

in human muscle in vitro and in an animal model. Lab Invest 1991;65:742–51.

33. Owen MR, Doran E, Halestrap AP. Evidence that metformin exerts itsanti-diabetic effects through inhibition of complex 1 of the mitochon-drial respiratory chain. Biochem J 2000;348:607–14.

34. El-Mir MY, Nogueira V, Fontaine E, Averet N, Rigoulet M, Leverve X.Dimethylbiguanide inhibits cell respiration via an indirect effect tar-geted on the respiratory chain complex I. JBiol Chem2000;275:223–8.

35. Mertz RJ, Worley JF, Spencer B, Johnson JH, Dukes ID.Activation ofstimulus-secretion coupling in pancreatic beta-cells by specific pro-ducts of glucose metabolism. Evidence for privileged signaling byglycolysis. J Biol Chem 1996;271;4838–45.

36. Sener A, KawazuS, Hutton JC, Boschero AC,DevisG, SomersG, et al.The stimulus-secretion coupling of glucose-induced insulin release.Effect of exogenous pyruvate on islet function. Biochem J 1978;176:217–32.

37. Zawalich WS, Zawalich KC. Influence of pyruvic acid methyl ester onrat pancreatic islets. Effects on insulin secretion, phosphoinositidehydrolysis, and sensitization of the beta cell. J Biol Chem 1997;272:3527–31.

38. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, et al. AMP-activated protein kinase induces a p53-dependent metabolic check-point. Mol Cell 2005;18:283–93.

39. Shaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, Depinho RA,et al. The kinase LKB1 mediates glucose homeostasis in liver andtherapeutic effects of metformin. Science 2005;310:1642–6.

40. BenSahra I, LaurentK,GiulianoS, Larbret F, PonzioG,GounonP, et al.Targeting cancer cellmetabolism: the combinationofmetformin and2-deoxyglucose induces p53-dependent apoptosis in prostate cancercells. Cancer Res 2010;70:2465–75.

41. Buzzai M, Jones RG, Amaravadi RK, Lum JJ, DeBerardinis RJ, Zhao F,et al. Systemic treatmentwith theantidiabetic drugmetformin selectivelyimpairs p53-deficient tumor cell growth. Cancer Res 2007;67:6745–52.

42. Kalender A, Selvaraj A, Kim SY, Gulati P, Brûl�e S, Viollet B, et. al.Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner. Cell Metab 2010;11:390–401.

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2011;10:2350-2362. Published OnlineFirst October 12, 2011.Mol Cancer Ther Jae-Ho Cheong, Eun Sung Park, Jiyong Liang, et al. Preclinical Cancer Modelsand Metformin Is Effective against a Broad Spectrum of Dual Inhibition of Tumor Energy Pathway by 2-Deoxyglucose

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