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Therapeutics, Targets, and Chemical Biology

Blocking Lactate Export by Inhibiting the Myc Target MCT1Disables Glycolysis and Glutathione Synthesis

Joanne R. Doherty1, Chunying Yang1, Kristen E.N. Scott1, Michael D. Cameron4, Mohammad Fallahi1,Weimin Li1, Mark A. Hall1,†, Antonio L. Amelio1, Jitendra K. Mishra2, Fangzheng Li2, Mariola Tortosa2,Heide Marika Genau1,5, Robert J. Rounbehler1, Yunqi Lu6, Chi V. Dang6,7, K. Ganesh Kumar3,Andrew A. Butler3, Thomas D. Bannister2, Andrea T. Hooper8, Keziban Unsal-Kacmaz8,William R. Roush2, and John L. Cleveland1

AbstractMyc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that

generates lactate. Here, we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1and that elevated MCT1 levels are manifest in premalignant and neoplastic Em-Myc transgenic B cells and inhuman malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to anaccumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provokingmarked alterations in glycolytic intermediates, reductions in glucose transport, and in levels of ATP,NADPH, and ultimately, glutathione (GSH). Reductions in GSH then lead to increases in hydrogen peroxide,mitochondrial damage, and ultimately, cell death. Finally, forcing glycolysis by metformin treatmentaugments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapyfor MYC/MCT1-expressing malignancies. Cancer Res; 74(3); 908–20. �2013 AACR.

IntroductionMyc oncogenic transcription factors are activated in a large

cast of human malignancies, and it has been estimated that100,000 deaths per year are associated with deregulated MYCexpression (1). Myc drives continuous cell growth and division,which triggers DNA damage and apoptotic checkpoints that arethen bypassed by mutations that lead to frank malignancy.Accordingly, forced expression of Myc is sufficient to provoketumor formation in mouse models of human cancer (1, 2).Furthermore, Myc is required to sustain the malignant state, asMyc inactivation usually provokes rapid tumor regression (3, 4).

Myc oncoproteins are basic-helix-loop-helix-leucine zipper(bHLH-Zip) transcription factors that regulate a large cast oftargets to coordinate cell growth, metabolism, and division (5).Myc functions require dimerization with the related bHLH-Zippartner Max, and Myc:Max dimers activate target genes bybinding to E-box elements (CACGTG; ref. 6). In addition, Mycrepresses transcription via inhibitory interactions with thetranscriptional activatorMiz-1 (7, 8). Finally,Myc oncoproteinshave recently been suggested to function as universal ampli-fiers of active genes (9, 10), which may occur through theirability to recruit histone modifying enzymes (11) and/or byoccupying preexisting open chromatin and promoting tran-scription or pause release at promoters loaded with RNApolymerase II (9, 10).

Prominent targets induced by Myc include a cast of meta-bolic enzymes, some of which drive aerobic glycolysis, ahallmark of cancer cells (12, 13). Indeed, in cell culture models,Myc oncoproteins induce many aspects of cancer cell metab-olism, where Myc targets include glucose and glutaminetransporters, glutaminase and glycolytic enzymes, includinglactate dehydrogenase-A (LDH-A; refs. 14–17).

Increased glycolytic flux in cancer cells leads to high levels oflactate that is exported by proton-dependent twelve-mem-brane pass monocarboxylic acid solute transporters coinedMCT1-4 (18). Cell surface expression of MCT1 and MCT4requires coexpression of the immunoglobulin-like moleculeCD147 (19). Although MCT4 transcription is regulated byhypoxia-inducible factor-1a (HIF-1a; ref. 20) and in renal clearcell cancer by promoter methylation (21), much less is knownabout the control of MCT1 transcription, other than MCT1expression is elevated in Myc-expressing MCF10 breast epi-thelial cells and in some tumors (22).

Authors' Affiliations: Departments of 1Cancer Biology, 2Chemistry, and3Metabolism and Aging; 4The Translational Research Institute, The ScrippsResearch Institute, Scripps Florida, Jupiter, Florida; 5Institute of Biochem-istry II, Goethe University Medical School, Frankfurt am Main, Germany;6Division of Hematology, Department of Medicine, The Johns HopkinsUniversity School of Medicine, Baltimore, Maryland; 7The Abramson Can-cer Center, University of Pennsylvania, Philadelphia, Pennsylvania; and8Pfizer Oncology, Pearl River, New Jersey

†Deceased.

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

J. R. Doherty and C. Yang contributed equally to this work.

This study is dedicated to thememory of our beloved colleague Dr. Mark A.Hall.

CorrespondingAuthor: JohnL.Cleveland,Department ofCancerBiology,The Scripps Research Institute, Scripps Florida, 130 Scripps Way, #2C1,Jupiter, FL 33458. Phone: 561-228-3220; Fax: 561-228-3072; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-2034

�2013 American Association for Cancer Research.

CancerResearch

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Blocking lactate transport impairs tumor cell growth throughseveral mechanisms. First, blocking lactate export leads to anaccumulation of lactic acid and decreases intracellular pH (23).This response seems to contribute to growth arrest of Ras-transformedfibroblasts triggered byMCT1 inhibitors and to theeffects of CD147 knockdown on tumor xenografts (24). Second,some tumor cells rely on lactate as a substrate for oxidativephosphorylation, and in this scenario blocking lactate importinhibits tumor cell growth (25, 26). However, this is the excep-tion as most tumor cells express high levels of LDH-A, whichdrives the production of lactate from pyruvate (27). Finally,lactate uptake in vascular endothelial cells via MCT1 seems topromote tumor angiogenesis; thus blocking this responseimpairs tumorigenesis (28, 29). Given these effects, a recentAstraZeneca patent application claims the use of MCT1 inhi-bitors for the treatment of certain cancers (30).Given their effects on aerobic glycolysis, we reasoned that

Myc oncoproteins would control lactate transport. Here, wereport that Myc directly and selectively activates MCT1 tran-scription and that elevated MCT1 levels are a hallmark ofhumanmalignancies withMYC orMYCN involvement. Notably,we show that blocking MCT1 function rapidly disables glycol-ysis, leading to reductions in ATP and glutathione (GSH) levels,and that cotreatment with metformin, which forces the gly-colytic phenotype, augments the in vivo efficacy of MCT1inhibitors against MYC-expressing malignances.

Materials and MethodsRNA analysesTotal RNAwas prepared fromB220þ B cells of the spleens or

bone marrow of 4- to 6-week-old wild-type (WT) and prema-lignant Em-Myc littermates. Lymphomas were from individualanimals. B cells were purified using magnetic-activated cellsorting and beads conjugated with antibodies to B220 (Milte-nyi). RNA was isolated from purified B220þ B cells or tumorsusing RNA shredder and RNA easy kits (Qiagen) or theNucleoSpin RNA II kit (Macherey-Nagel). For quantitativereal-time PCR (qRT-PCR) analyses, cDNA was synthesizedusing iScript reverse transcriptase (Bio-Rad), amplified withPerfecta Sybr Green (Quanta), and measured using the iCyclerreal-time PCRmachine (Bio-Rad). Experimental Ct values werenormalized with ubiquitin Ct values and were expressed rela-tive to WT Ct values. Sequences of primers are provided uponrequest.

Proliferation, lactate, and ATP assaysProliferation was measured by counting cells daily or by

MTT assay (Millipore). Lactate was measured using lactateassay kits [A-108, University of Buffalo (Buffalo, NY) and K607-100, BioVision] according to the manufacturer's instructions.Cells cultured at 5 � 105 cells/mL � MCT1 inhibitors (100nmol/L) and/or metformin (1 mmol/L) for 24 hours werewashed twice in cold PBS, lysed in 100 mL cold water, andlactate concentrations measured from the lysate. ATP wasmeasured using an ATP detection kit (Perkin Elmer) accordingto the manufacturer's instructions. Raji cells (5� 104/well in a96-well plate) were treated with inhibitors (100 nmol/L) forindicated times before assay.

siRNA knockdownMCF7 cells (2.5� 105 cells/well) were transfected with 50 to

100 nmol/L siRNAOn-TARGETplus SMARTpool siRNA againsthSLC16a1 (L-007402-00), c-Myc (L-003282-00-0005), or controlsiRNA (D-001810-01-05) from Thermo Scientific together with3 mL Lipofectamine-2000 (Invitrogen 11668-027) into a 6-welldish. Cells were incubated for 24 hours before plating forgrowth assays, metabolic assays, and protein and RNA isola-tion. For growth assays, cells were plated at 10,000 cells/wellinto 24-well dishes and three wells/day were counted.

Metabolic analysisExtracellular acidification rate (ECAR) and oxygen con-

sumption rate (OCR) were measured using the SeahorseBioscience XF96 Analyzer. Before each measurement, wellswere mixed for 2 minutes followed by analyte measurementsevery 22 seconds for 5minutes. Each data point is the change inanalyte concentration over 5 minutes and is reported as ECAR(mpH/minute) and OCR (pmol oxygen/minute). Agents wereinjected into wells through preloaded reagent delivery cham-bers in the sensor probe. Final concentration of agents usedwas MCT1 inhibitors 10 nmol/L to 1 mmol/L, oligomycin 1mmol/L (Calbiochem), carbonyl cyanide 4-(trifluoromethoxy)phenylhyrazone (FCCP) 300 nmol/L (Sigma), and rotenone 100nmol/L (Sigma). Raji, Daudi, and Ramos Burkitt lymphomacells (60,000 cells/well) were adhered to poly-D-lysine–coated96-well plates by centrifuging. MCF7 cells were plated at 15,000cells/well the day before analysis. Protein concentration inwells was measured to normalize ECAR and OCR data.

Glutathione measurements and glutathione rescueTotal glutathione [GSH; GSH plus oxidized GSH (GSSG)]

was measured according the manufacturer's protocol (Cay-man's Glutathione Assay Kit) fromRaji cells treatedwith vehicleor SR13800 (1 mmol/L) for 8 hours. Raji cells (2� 105 cells/mL),cultured in RPMI with FBS, were pretreated with GSH-reducedethyl ester (3–5 mmol/L; Sigma) for 1 hour followed by treat-ment with SR13800 (1 mmol/L) and metformin (1 mmol/L).Hydrogen peroxide (H2O2) was measured by H2DCFDA. Viabil-ity was measured by Trypan blue dye exclusion. Proliferationwas measured by MTT assay 3 days after treatment.

ResultsMyc coordinates the control of lactate homeostasis

To assess whether Myc controls lactate production andexport in vivo, we used the Em-Myc transgenic mouse modelof human B lymphoma (1), where a protracted premalignantstate allows one to identify targets that play roles inMyc-driventumorigenesis (31, 32). Expression analyses of B220þ splenicand bone marrow B cells from WT and premalignant Em-Myclittermates established that Em-Myc B cells express elevatedlevels of several glycolytic enzymes, including Ldh-A, andthat this response was augmented in neoplastic Em-Myc Bcells (Fig. 1A and Supplementary Fig. S1A and S1B).

In accordancewith these analyses,marked increases in Ldh-Aproteinweremanifest in Em-Myc versusWTB cells (Fig. 1B), andthe preponderance of Ldh-A versus Ldh-B suggested that pyru-vate would be converted to lactate (27). Indeed, intracellular

Blocking Lactate Export Disables Cancer Cell Glycolysis

www.aacrjournals.org Cancer Res; 74(3) February 1, 2014 909




lactate levels were elevated 2- to 3-fold in Em-Myc versus WT Bcells (Fig. 1C). Thus, Myc activates glycolysis and drives lactateproduction during lymphomagenesis.

We reasoned that lactate was exported from Em-Myc B cellsby SLC16A/MCT transporters. Notably, there were markedincreases in Mct1 mRNA levels, but not of Mct2, Mct3, and

Mct4 transcripts, in Em-Myc B cells (Fig. 1D and SupplementaryFig. S1C). Levels of CD147 transcripts, the obligate MCT1/MCT4 chaperone (19), were similar in WT and Em-Myc B cells(Fig. 1D). Immunoblot analyses confirmed elevated MCT1protein levels in premalignant and neoplastic Em-Myc B cells(Fig. 1E). MCT4 protein was also detected at low levels in some

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Figure 1. Myc augments lactate production and MCT1 expression. A–F, B220þ B cells isolated from bone marrow (BM) or spleen of 4- to 6-week-old WT orpremalignant Em-Myc littermates or from Em-Myc lymphoma were analyzed: A, by qRT-PCR for Ldh-A (left) or Ldh-B (right) expression (WT BM, n ¼ 2;premalignant Em-Myc, n¼ 2; lymphoma, n¼ 3; error bars, SD); B, for Ldh-A protein levels; C, intracellular lactate (lactatei; triplicate assays); D, by expressionprofiling analysis of Mct1, Mct4, and CD147 mRNA [circles, WT splenic B cells, n ¼ 5; squares, premalignant Em-Myc, n ¼ 5; triangles, lymphoma,n ¼ 13; significant differences in meanMct1 levels in premalignant (P < 0.00002) and neoplastic (P < 0.000002) Em-Myc B cells vs. WT B cells]; E, for MCT1protein levels (Mct1þ, lysate from 293T cells expressing MCT1); F, by flow cytometry for CD147 levels on B220þ bone marrow B cells from WT (orange line)and premalignant Em-Myc (blue line) littermates, and of Em-Myc lymphoma (red line). MFI, mean fluorescence intensity. G, gene expression profilingof MYC, MCT1, MCT2, MCT3, and MCT4 in human Burkitt lymphoma (n ¼ 44) and non–Burkitt lymphoma (n ¼ 129, from GSE4475; ref. 33). H, MCT1expression in primary human MYCN-amplified neuroblastoma (n ¼ 20) versus non–MYCN-amplified neuroblastoma (n ¼ 81, from GSE3960; ref. 34).

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Cancer Res; 74(3) February 1, 2014 Cancer Research910




Em-Myc lymphomas (Supplementary Fig. S1D), presumablydue to hypoxic regions in these tumors (20, 25). Cell surfaceexpression of MCT1 and CD147 are codependent (19); thus,fluorescence-activated cell sorting (FACS) analyses of CD147serves as a surrogate for cell surfaceMCT1. Notably, there weremarked increases in the cell surface levels of CD147 in pre-malignant and neoplastic Em-Myc B cells versus WT B cells(Fig. 1F). Thus, there aremarked increases inMCT1 expressionduring Myc-induced lymphomagenesis.

Elevated MCT1 expression is a hallmark of MYC-drivenmalignanciesTodeterminewhether elevatedMCT1 expression ismanifest

in human malignancies having MYC involvement, we firstqueried expression datasets of Burkitt lymphoma havingMYC/immunoglobulin chromosomal translocations and ofMYCN-amplified neuroblastoma (33, 34). Notably, there areconcordantly high levels of MCT1 and MYC mRNA in Burkittlymphoma, and of MCT1 and MYCN in MYCN-amplified neu-roblastoma (Fig. 1G and H). In contrast, MCT2, MCT3, andMCT4 are not specifically elevated in tumors withMYC/MYCNinvolvement; indeed, MCT4 transcript levels are low in MYC-expressing tumors (Fig. 1G and H). Furthermore, the high-MCT1/low-MCT4 profile is a hallmark ofMYC-expressing coloncancer (Supplementary Fig. S2A; ref. 35; though normal colonwas heterogeneous inMCT1mRNA levels) and elevatedMCT1levels are manifest in the squamous carcinoma subtype ofnon–small cell lung cancer (Supplementary Fig. S2C; ref. 36).Lung adenocarcinoma and breast adenocarcinoma thatexpress high levels ofMYC also expressed high levels ofMCT1,yet some of these also elevated levels ofMCT4, possibly due tohypoxic regions in these tumors (Supplementary Fig. S2C andS2D). Finally, a high-MYC-high-MCT1 signature connotes pooroutcome in basal-like breast cancer and all lung cancers(Supplementary Fig. S2E–S2G).

MCT1 is a Myc transcription targetWe evaluated the control of MCT1 expression in human

P493-6 B-lymphoma cells, which bear a tetracycline-repress-ible c-Myc transgene (37). The induction of c-Myc mRNA andprotein in P493-6 cells deprived of tetracycline was followed bymarked increases inMCT1mRNA and protein (Fig. 2A and B).These findings agree with RNA-seq and expression profilinganalyses of P493-6 cells (Supplementary Fig. S3A; ref. 9).To testwhetherMct1 is also induced by physiologic cues that

control c-Myc, we assessed whether Mct1 expression is cyto-kine dependent. Primary mouse bone marrow-derived pre-Bcells grown in interleukin (IL)-7 medium were deprived of andthen restimulatedwith ligand. Like c-Myc,Mct1 expressionwasalso IL-7 dependent and its induction followed that of c-Myc(Fig. 2C). Similarly,Mct1 expression in immortal 32D.3myeloidcells was IL-3 dependent and followed the induction of c-Myc(Fig. 2D).To test whetherMyc binds to the endogenousMCT1 gene via

identified E-boxes, we performed chromatin immunoprecip-itation (ChIP) analyses in P493-6 cells and 32D.3 myeloid cells.Therewas amarked and specific enrichment for c-Myc bindingto human MCT1 E-box2 and E-box1 in P493-6 cells and to

mouseMct1 E-box1 in IL-3–stimulated 32D.3 cells (Fig. 2E andF). In contrast, c-Myc binding to human E-box3 or E-box4, or tomouse E-box4was not detected (Fig. 2E andF). Similarfindingswere evident in querying the genome-wide ChIP-seq datasetsof Myc and Max target genes in P493-6 cells (9), which showedthat MCT1 was transcriptionally silent until bound by Myc(Supplementary Fig. S3B). In addition, Myc-directed control ofMCT1 was specific, as c-Myc knockdown in MCF7 breastcancer cells impaired proliferation and reducedMCT1 expres-sion but not levels of MCT2 or MCT4 (Fig. 2G), which areexpressed in these cells (Supplementary Fig. S3C). Finally, Mycknockdown selectively reduced the activity of a MCT1 pro-moter-reporter harboring E-box1 and E-box2 (Fig. 2H). Thus,MCT1 is a direct Myc transcription target in normal and tumorcells.

MCT1 is necessary for tumor cell proliferationMost tumor cells produce and export excess lactate.We thus

reasoned that blocking MCT1 function would impair thegrowth of MCT1-expressing tumor cells. Potent and specificinhibitors that disable MCT1- and MCT2-directed lactatetransport (but not that of MCT4) block growth of activatedT cells (38, 39). We synthesized two of these pyrole pyrimidine-based molecules [Supplementary Fig. S4A; AR-C122982 (here-after SR13800) and AR-C155858 (SR13801)] and assessed theireffects on Raji Burkitt lymphoma cells that only expressMCT1(Supplementary Fig. S3C). Both inhibitors blocked Raji cellproliferation (Fig. 3A andB), at doses at least 10-fold lower thanthose needed to impair growth of primary bone marrow-derived B cells (Fig. 3C). Growth inhibition was due to prolif-erative arrest, which was followed by protracted cell death(Supplementary Fig. S4B and S4C). These inhibitors alsoblocked MCF7 breast cancer cell growth (Fig. 3D), and thatof Raji Burkitt lymphoma and 70Z3mouse B-cell lymphoma inmethylcellulose (Supplementary Fig. S4D).

Manipulating MCT1 expression was not feasible in Raji Blymphoma. Thus, to confirm that these inhibitors were ontarget, MCT1 expression was silenced in MCF7 cells. SilencingMCT1 blocked the growth of MCF7 cells similar to treatmentwith the inhibitor (Fig. 3E). We also tested the effects of MCT1or MCT4 overexpression in MCF7 cells. As expected (19),overexpression of either transporter increased CD147 levels(Fig. 3F), and MCT4, but not MCT1, overexpression wassufficient to confer SR13800 resistance (Fig. 3G). MCT1 over-expression augmented lactate transport (Supplementary Fig.S4E) and shifted the IC50 for SR13800 in inhibiting transport of14C-lactate (Fig. 3H). However, forced expression of MCT1 (orMCT4) did not affect MCF7 cell proliferation (SupplementaryFig. S4F). Thus, in tumor cells that express MCT1, this trans-porter is necessary for proliferation but does not augment cellgrowth.

MCT1 inhibition derails lactate homeostasis, glycolysis,and glutathione synthesis

In Raji cells treated with SR13800 or SR13801, there wereimmediate increases in the levels of intracellular lactate and ablock in lactate export (Fig. 4A and B). However, levels ofintracellular lactate reached amaximumwithin approximately






4 hours (Fig. 4A), suggesting effects on metabolism. Indeed,there were rapid and marked reductions in the levels ofintracellular ATP (Fig. 4C) and in the ECAR (Fig. 4D and G).

In contrast, there were little effects of SR13800 on basal OCR(Fig. 4F). Rapid decreases in ECAR provoked by SR13800 weremanifest in other Burkitt lymphoma lines that express MCT1

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Figure 2. MCT1 is a Myc transcription target. A, human P493-6 B lymphoma cells bearing a tetracycline-repressible c-Myc transgene were cultured for72 hours in tetracycline (1 mg/mL, Myc-Off), washed and cultured inmedium lacking tetracycline (Myc-On), and assessed forMYC andMCT1mRNA levels byqRT-PCR. B, top, c-Myc protein levels at the indicated intervals following withdrawal of tetracycline from P493-6 cells. Bottom,MCT1 protein levels 24 hoursafter removal (�) of tetracycline. C and D, primary bone marrow-derived mouse pre-B cells grown in IL-7 (C) and mouse 32D.3 myeloid cells grownin IL-3 (D) were deprived of ligand for 16 hours and then restimulated with IL-7 or IL-3. Levels of c-Myc and Mct1 transcripts were determined by qRT-PCR(n ¼ 3). Expression is relative to Ub. E, top, schematic of human SLC16A1/MCT1. Vertical blue bars indicate four E-box (CACGTG) sequences and orangearrow indicates exon 1. Bottom, P493-6 cells cultured for 72 hours with tetracycline (white bars, Myc-Off), then washed and cultured for 8 hours withouttetracycline (black bars, Myc-On), were chromatin immunoprecipitated with c-Myc antibody. Designated E-boxes and controls [cyclin D2 (CCND2) andMDM2] were amplifiedby qRT-PCRandcomparedwith total input. F, top, schematic ofmouseSlc16a1/Mct1. Vertical blue bars denote four E-box sequencesin themouseMct1 gene and an intronic region (NC) used as a control for ChIP analyses. Orange arrow indicates exon 1. Bottom, 32D.3 cells cultured withoutIL-3 for 16 hours (white bars) and then stimulated with IL-3 for 2 hours (black bars) were chromatin immunoprecipitated with c-Myc antibody. Mouse Cadwas amplified as a positive control for c-Myc binding. G, MCF7 cells were transfected with control siRNA or c-MYC–specific siRNA. Top, cells were counteddaily (n ¼ 3). Bottom, 48 hours after transfection, cells were harvested for qRT-PCR analyses. H, top, mouseMct1 promoter regions used to generate fireflyluciferase reporters. Bottom left, fold activation of Mct1 promoter-reporters in HEK 293T cells transfected with 40 nmol/L scrambled siRNA (NSsi) orMYC-specific siRNA (MYCsi) relative to mock-transfected cells (n ¼ 3, in quadruplicate). Bottom right, c-MYC protein knockdown in HEK293T cells.

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(Supplementary Fig. S5B) and inMCF7 breast cancer cells (Fig.4H). Similar decreases were observed in MCF7 cells followingMCT1 knockdown (Fig. 4I). Furthermore, oligomycin treat-ment, which blocks ATP synthesis and activates glycolysis,induced ECAR in vehicle- but not SR13800-treated cells (Fig.4D). Notably, dissipating the proton gradient with FCCPrevealed thatMCT1 inhibition abolishes reservemitochondrialcapacity (Fig. 4F) without reducing levels of NADH (Fig. 4E), anelectron donor that drives oxidative phosphorylation. Rote-none treatment, which inhibits mitochondrial complex I,abolished the majority of OCR in Raji cells, and this wasunaffected by SR13800 (Fig. 4F). Thus, MCT1 inhibition rapidlydisables tumor cell metabolism.To assess the effects of MCT1 inhibition on metabolism, we

measured metabolic intermediates in Raji lymphoma cells by

mass spectrometry. Again, SR13800 treatment led to rapidincreases in the levels of intracellular lactate and correspondingdecreases in extracellular lactate (Fig. 5A and B). Furthermore,as MCT family members transport pyruvate (18), SR13800treatment led to marked reductions in extracellular pyruvate(Fig. 5B). Notably, MCT1 inhibition increased the levels ofglucose- and fructose-6-phosphate, fructose-bisphosphate, andglycerol-3-phosphate, and markedly reduced products of theATP-generating arm of glycolysis, including phosphoglycerateand pyruvate (Fig. 5A, B, and F). Furthermore, there weremarked reductions in NADPH levels and glucose transport(Fig. 5C and D). These changes were not associated withalterations in the levels of glycolytic enzymes (SupplementaryFig. S5C), and reduced glucose transportwas not due to changesin the cell surface levels of glucose transporters (not shown).

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Raji cells/mL were treated withvehicle (black line), 100 nmol/LSR13800 (red line) or SR13801(blue line) and counted at theindicated times (n ¼ 3,representative of threeexperiments). B, Raji cells weretreated with indicated doses ofSR13800 or SR13801 for 3 daysand assayed by MTT (n ¼ 3). C,MTT assay of Raji cells or primarymouse B cells treated withSR13800 for 3 days (n ¼ 3).D, 1 � 105 MCF7 cells treated withvehicle (black line) or 10 nmol/L(dashed black line) or 1 mmol/LSR13800 (gray line) and cellnumbers were counted atthe indicated times (n ¼ 3,representative of twoexperiments). E, siRNAknockdown of MCT1 mRNA (topleft) and MCT1 protein (bottom left)in MCF7 cells. þ, control lysatefrom 293T cells expressing Mct1.Right, cells transfectedwith controlsiRNA (black line) or MCT1 siRNA(dashed line) and counted(n ¼ 3, representative of threeexperiments). F, MCF7 cellsengineered to overexpress mouseMct1 or human MCT4 wereassessed for expression of MCT1,MCT4, and CD147 proteins. G,MCF7 cells overexpressing MCT1or MCT4 were treated with vehicle(black lines) or SR13800 (1 mmol/L,red lines) and cell numberwas determined daily (n ¼ 3,representative of twoexperiments). H, 14C lactatetransport in MCF7 cellsoverexpressing MCT1 and treatedwith indicated doses of SR13800(10 minutes). n ¼ 4, representativeof three experiments.






Decreases in NADPH following MCT1 inhibition were notassociated with changes in ribulose-5-phosphate or malate,two intermediates in NADPH production (Fig. 5E). NADPH isconsumed in the conversion of GSSG to reduced GSH byGSH reductase. The first step of GSH synthesis, the produc-tion of g-glutamylcysteine (g-GC), requires ATP (Supplemen-tary Fig. S5D). Thus, we reasoned that MCT1 inhibitionmight compromise GSH synthesis. Indeed, by two measures,MCT1 inhibition led to rapid reductions in g-GC and GSH,and in total GSH (GSH and GSSG; Fig. 5G). Reductions inGSH did not reflect changes in the levels of the catalytic(GCLC) and modulatory (GCLM) subunits of glutamyl cys-teine ligase (GCL) that catalyze g-GC production (Supple-mentary Fig. S5E). Thus, the reductions in ATP levels trig-gered by inhibiting lactate transport (Fig. 4C) are associatedwith reduced levels of GSH.

Metformin augments the potency and antitumor activityof MCT1 inhibitors

Metformin disables oxidative phosphorylation (OXPHOS) byinhibiting mitochondrial complex I and forces a glycolyticphenotype that we reasonedmay augment sensitivity toMCT1inhibitors. Though metformin alone had little effect, cotreat-ment of Raji lymphoma with SR13800 and metformin led tohigher levels of intracellular lactate (Supplementary Fig. S6A),rapid growth arrest, and cell death (Supplementary Fig. S6B).Synergy of SR13800 and metformin was evident in all MCT1-expressing human andmouse tumor cell lines (SupplementaryFig. S6C and S6D), yet had, as expected, no effects on eitherMCT4-expressing human tumor cells or upon Em-Myc lym-phomas in transplant experiments (not shown), likely due tohypoxia-mediated induction of Mct4 (Supplementary Fig.S1D). Thus, metformin augments the anticancer potency of

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Doherty et al.





MCT1/MCT2 inhibitors in tumor cells expressing thesetransporters.The efficacy of SR13800�metforminwas tested in vivousing

nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice injected intravenously with Raji lymphoma, orinjected subcutaneously with MCT1-expressing humanT47D estrogen receptor-positive breast cancer cells. Immuno-histochemical analyses confirmed that high levels of MCT1,but not MCT4, were expressed in both tumor models (Sup-plementary Fig. S7A and S7B). In vivo drug metabolism andpharmacokinetic (DMPK) analyses demonstrated that a dailyintraperitoneal injection of 30 mg/kg of SR13800 was suffi-cient to maintain serum levels above the EC50 of the drug(�5 nmol/L; Fig. 3B), andmice tolerated long-termdaily dosing

of SR13800 for >130 days without toxic effects (not shown).There were also no significant effects of SR13800 treatmenton numbers of white or red blood cells that express highCD147 levels (not shown).

Notably, SR13800 treatment delayed the onset and pene-trance of disease relative to vehicle-treated Raji lymphomarecipients (Fig. 6A and Supplementary Fig. S7C). In contrast,metformin treatment (at doses not affecting blood glucose) didnot affect lymphoma onset (Fig. 6A). Recipients treated withboth SR13800 plus metformin had remarkably delayed disease(Fig. 6A); indeed, some recipients never developed tumors,even after being taken off drug for over 80 days (data notshown). In the T47D model, SR13800 and metformin hadcomparable activity in impairing tumorigenecity (Fig. 6B).

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However, the SR13800/metformin combination was againsuperior in blocking tumor growth, without affecting thehealth of transplant recipients (Fig. 6B and SupplementaryFig. S7D–S7F). Thus, MCT1 inhibitors have in vivo anticanceractivity and the MCT1 inhibitor/metformin combination is anattractive therapeutic strategy for treating MCT1-expressingmalignancies.

Metformin overcomes resistance toMCT1 inhibitors dueto shifts to OXPHOS

To assess whether, in addition to MCT4 expression, therewere other tumor cell intrinsic mechanisms of resistance toMCT1 inhibitors, Raji cells were cultured with low doses ofSR13800 and surviving cells serially passaged intomedia havingincreased levels in SR13800, to generate independent pools of

SR13800-resistant Raji (RajiR) cells (n ¼ 6; Supplementary Fig.S8A). The growth rates of RajiR cells were comparable withparental Raji cells in normal media yet they continued toproliferate inmedia containing SR13800, whereas parental Rajilymphoma cells underwent growth arrest (Fig. 6C).

The resistance of RajiR cells was not associated withMCT4 induction or changes in MCT1 levels (SupplementaryFig. S8B). However, RajiR cells had elevated levels of intra-cellular lactate and low ECAR compared with parental Rajicells growing in normal media (Fig. 6D and E). Notably, RajiR

cells had an increase in OCR compared with parental cells(Fig. 6E). Treatment of RajiR cells with metformin alone ledto a rapid collapse in metabolism, with a marked reductionin OCR (Fig. 6F). Although metformin treatment led toreductions in OCR in parental Raji lymphoma cells, it also,

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Figure 6. Metformin augmentsthe antitumor activity of MCT1inhibitors and overcomesresistance due to shifts toOXPHOS. A, survival of NOD/SCIDmice transplanted with Rajilymphoma. At day 3, mice weretreated with water or water with 5mg/kg metformin and dailyinjections of vehicle or 30 mg/kgSR13800. Median survival: vehicle(n¼ 8), 30 days;metformin (n¼ 10),31 days; SR13800 (n¼ 10), 39 days(P ¼ 0.016); SR13800 þmetformin(n ¼ 10), 64 days (P ¼ 0.005). B,tumor volumes measured withcalipers in NOD/SCID miceinjected sub-Q with T47D cells andtreated as in A. At day 32, therewere significant differences intumor volume for vehicle (n ¼ 5)versus SR13800 (n ¼ 9, P ¼ 0.01),metformin (n ¼ 8, P ¼ 0.002), andSR13800/metformin (n ¼ 10,P ¼ 0.0005) cohorts. C, parentalRaji and SR13800-resistant Raji(RajiR) cells were treated withvehicle or 100 nmol/L SR13800and cell numbers determined(n ¼ 4, representative of twoexperiments). D, levels ofintracellular lactate in Raji and RajiR

cells in normal growth medium(n ¼ 3, representative of twoexperiments). E, ECAR (left) andOCR (right) measurements of Raji(blue lines) andRajiR cells (red lines;n ¼ 6, representative of twoexperiments). F, parental Raji andRajiR cells were assessed for ECARand OCR � metformin (1 mmol/L)for 16 hours (n ¼ 6, representativeof two experiments). Cell number ofRaji or RajiR cells treated withvehicle (black bars) or metformin(1 mmol/L, gray bars) for 24 hours(representative of twoexperiments).

Doherty et al.





as predicted, increased ECAR, thus providing energeticcompensation for inhibiting OXPHOS (Fig. 6F). Finally,metformin alone led to the rapid death of RajiR but notparental Raji cells (Fig. 6G), demonstrating RajiR cells aredependent on OXPHOS. Thus, a metabolic shift to OXPHOSis a mechanism of resistance to MCT1 inhibitors and met-formin overcomes this resistance.

Reductions of GSH pools trigger hydrogen peroxide-induced cell deathGSH is a major antioxidant in cells that neutralizes free

radicals and reactive oxygen species (ROS; Supplementary Fig.S5D). Indeed, themarked reduction in GSH in SR13800-treatedRaji cells was followed by increases of H2O2 but not of mito-chondrial or cytosolic superoxide anions (Fig. 7A and data

not shown). Notably, SR13800/metformin treatment led to aprofound increase in H2O2 levels that were associated with thedeath of Raji Burkitt lymphoma cells.

ROS compromise the function of mitochondria. AnalyseswithMitoTracker Green, whichmeasuresmitochondrial mass,indicated that treatment of Raji Burkitt lymphoma cells withSR13800, metformin, or SR13800/metformin did not changemitochondrial mass (Supplementary Fig. S8C). However, anal-yses with MitoTracker Red, which measures mitochondrialmembrane potential, demonstrated that SR13800 treatment,and especially SR13800/metformin, triggered marked reduc-tions in numbers of Raji Burkitt lymphoma cells having func-tional mitochondria (Fig. 7B).

These findings suggested that reductions in GSH andsubsequent increases in H2O2 triggered by SR13800 or

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SR13800/metformin compromise tumor cell survival. Indeed,pretreatment of Raji Burkitt lymphoma cells with the antiox-idants N-acetyl cysteine (NAC) or GSH blocked the deleteriouseffects of SR13800 and of SR13800/metformin on Raji cellgrowth and survival (Fig. 7C–E). Finally, GSH treatmentimpaired H2O2 production in SR13800- and SR13800/metfor-min-treated cells (Fig. 7F). Thus,metabolic demise triggered bythe inhibition of lactate transport leads to a collapse in ATPpools, reductions inGSH, and increases inH2O2 that contributeto tumor cell death (Supplementary Fig. S8D).

DiscussionAerobic glycolysis is a hallmark of most tumor types, and

our findings have established that Myc is sufficient to driveglycolysis in vivo, where premalignant Em-Myc B cells expresselevated levels of several glycolytic enzymes that leads toincreased lactate levels, in accordance with Ldh-A inductionby Myc in vitro (14, 40). Furthermore, this response isamplified in Em-Myc lymphoma. These findings parallel theincreased dependence on glycolysis and lactate productionmanifest during the stepwise transformation of primaryhuman fibroblasts (41). Finally, in rapidly expanding tumors,HIF-1a also contributes to glycolysis via the induction ofhypoxia (42).

In cancer cells, glucose transport and glycolytic flux is high,and this drives the production of ATP and intermediatesnecessary for anabolic pathways (43). A consequence of thisshift is the production of toxic levels of lactate. Here, we showthis is diverted by expression of high MCT1 levels in Myc-expressing malignancies and that MCT1 is necessary for theproliferation of these tumor cells. Furthermore, exportinglactate into the milieu may play roles in promoting tumorprogression, as lactate can trigger an inflammatory response(44) that drives tumor progression (45). Finally, lactate alsoplays other roles in tumorigenesis, where it can be convertedinto pyruvate that enters the tricarboxylic acid (TCA) cycle (25,26) and/or stimulate endothelial cell angiogenesis (28, 29).

Ourfindings establish thatMyc controls lactate homeostasisby inducingMCT1 transcription. Pharmacologic inhibition andknockdown of MCT1 demonstrate the essential role of thistransporter in maintaining the metabolism, growth, survival,and tumorigenecity of MCT1-expressing tumor cells. Thesefindings are consistent with studies showing the anticancereffects of nonspecific inhibitors of lactate transporters such asa-cyanocinnamate (25) and with the block in metabolism andgrowth of Ras-transformed fibroblasts ex vivo by MCT1 inhi-bitors (24). However, the mechanism by which inhibitinglactate transport compromises cancer cell growth has beenunclear and we now show is due to a collapse in glycolysis andaccompanying reductions in ATP and GSH (SupplementaryFig. S8D).

Changes in metabolic intermediates suggest how inhibit-ing lactate export disables glycolysis. In particular, ourfindings are remarkably akin to the changes in glycolyticintermediates observed in the exercised muscle of LDH-A–deficient patients, where reduced levels of NADþ compro-mise glyceraldehyde-3-phosphate dehydrogenase (GAPDH)activity and favor the oxidation of NADH and the production

of glycerol-3-phospate by glycerol-3-phosphate dehydroge-nase (GPDH; ref. 46). In MCT1 inhibitor-treated cells, intra-cellular lactate levels and NADH levels rise, suggesting thatthe conversion of pyruvate to lactate and the coupledoxidation of NADH to NADþ are impeded by high intracel-lular lactate levels. High NADH levels favor GPDH conver-sion of DHAP to glycerol-3-phosphate, which diverts triose-phosphates from GAPDH and the ATP-generating arm ofglycolysis (Supplementary Fig. 8SD). As a consequence, thisleads to reductions in phosphoglycerate and pyruvate. Thischain of events, coupled with feedback inhibition by lactateon PFK1 (47), explains increased levels of more upstreamintermediates such as glucose-6-phosphate (G-6-P) and theeffects upon glucose transport. Specifically, G-6-P feedbackinhibits hexokinase, which results in increased intracellularfree glucose that then inhibits glucose transport. Collectivelythese events converge to markedly impair ATP levels and thesynthesis of the major antioxidant of the cell, GSH, which is akey target, as GSH is sufficient to override the acute effects ofMCT1 inhibitors on tumor cell survival.

Importantly, these findings suggest that any agent targetingglycolysis will compromise ATP production and GSH levels,resulting in H2O2-induced death. In accordance with thisnotion, LDH-A inhibition triggers oxidative stress and blockstumor cell proliferation (48) and the antioxidants NAC andGSH block the antitumor activity of MCT1 inhibitors. Ourfindings also suggest that pro-oxidant therapies will augmentthe potency of lactate transport inhibitors and that othermeans of disrupting GSH synthesis, for example, by inhibitingcysteine or cystine transporters (49), will be effective thera-peutic strategies for tumors with MYC involvement.

Elevated MCT1 expression is a hallmark of human malig-nancies with MYC involvement. Notably, many MYC-expres-sing tumors express reduced MCT4 levels, suggesting MCT1inhibitors will have a therapeutic benefit in such tumors.However, it is clear that tumor cells that express MCT4 willbe refractory to MCT1 inhibitors, and that patient tumorsshould be assessed for MCT1 and MCT4 expression beforeusing MCT1 inhibitors for treatment (25). A second mecha-nism of resistance identified herein is a shift of tumor cells toOXPHOS, and we show that agents such as metformin thatdisrupt OXPHOS disable this resistance, leading to a collapse intumor cell metabolism and rapid death. Notably metforminuse in type-II diabetes seems to decrease cancer risk and mayprovide benefit as a cancer therapeutic (50). Together, thesefindings support the use of MCT1 inhibitors in combinationwithmetformin, orwithmetformin derivatives, in therapies forMCT1- and MYC-expressing malignancies.

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

Authors' ContributionsConception and design: J.R. Doherty, C. Yang, K.E.N. Scott, Y. Lu, C.V. Dang,T.D. Bannister, W.R. Roush, J.L. ClevelandDevelopment of methodology: J.R. Doherty, C. Yang, K.E.N. Scott, M.D.Cameron, M.A. Hall, A.L. Amelio, J.K. Mishra, F. Li, M. Tortosa, Y. Lu, A.A. Butler,T.D. Bannister, K. Unsal-KacmazAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.R. Doherty, C. Yang, K.E.N. Scott, M.D. Cameron,

Doherty et al.





W.Li,M.A. Hall, A.L. Amelio, F. Li, H.M.Genau, R.J. Rounbehler, Y. Lu, K.G. Kumar,A.A. Butler, T.D. Bannister, A.T. Hooper, K. Unsal-Kacmaz, W.R. RoushAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.R. Doherty, C. Yang, K.E.N. Scott, M.D. Cameron,M. Fallahi, W. Li, M.A. Hall, A.L. Amelio, F. Li, H.M. Genau, Y. Lu, A.A. Butler, A.T.Hooper, J.L. ClevelandWriting, review, and/or revision of the manuscript: J.R. Doherty, C. Yang,K.E.N. Scott, C.V. Dang, T.D. Bannister, A.T. Hooper, W.R. Roush, J.L. ClevelandAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.R. Doherty, C. Yang,W. Li, J.K. Mishra,W.R. RoushStudy supervision: J.R. Doherty, C. Yang, K. Unsal-Kacmaz, W.R. Roush,J.L. Cleveland

AcknowledgmentsThe authors thank Nancy Philp (Thomas Jefferson University, Philadel-

phia, PA) for MCT antibodies, the FACS, Genomics, and ARC Cores of ScrippsFlorida, members of the Cleveland laboratory and Anke Klippel-Giese (Pfizer

Oncology) for discussions, Marika Kernick for editing, Justin Lucas (PfizerOncology) for assistance, and Dr. George Hu (Pfizer Oncology) for pathologicanalyses.

Grant SupportThis work was supported by NIH grants CA076379 and CA169142 (J.L.

Cleveland), CA057341 (C.V. Dang), GM026782, GM038436, CA169142, andU54MH074404 (W.R. Roush), F32 CA134121 (A.L. Amelio), the Jane & LeonardKorman Family Foundation Postdoctoral Fellowship (K.E.N. Scott), and bymonies from the State of Florida to Scripps Florida.

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 22, 2013; revised October 28, 2013; accepted October 28, 2013;published OnlineFirst November 27, 2013.

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Doherty et al.





2014;74:908-920. Published OnlineFirst November 27, 2013.Cancer Res Joanne R. Doherty, Chunying Yang, Kristen E.N. Scott, et al. Disables Glycolysis and Glutathione SynthesisBlocking Lactate Export by Inhibiting the Myc Target MCT1

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