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 (2429).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 (1315, 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(n 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.

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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 3).

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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 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 volumeSDis presented. , P < 0.05.D, representative MR images.E, tumor weight by 2DG metformin 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.

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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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