modulation of glucose metabolism by cd44 contributes to ... · from glycolysis to mitochondrial...

Molecular and Cellular Pathobiology

Modulation of Glucose Metabolism by CD44 Contributes toAntioxidant Status and Drug Resistance in Cancer Cells

Mayumi Tamada1,2, Osamu Nagano1,3, Seiji Tateyama6, Mitsuyo Ohmura2, Toshifumi Yae1,3,5,Takatsugu Ishimoto1,3,7, Eiji Sugihara1,3, Nobuyuki Onishi1, Takehiro Yamamoto2, Hiroshi Yanagawa6,Makoto Suematsu2,4, and Hideyuki Saya1,3

AbstractAn increased glycolytic flux accompanied by activation of the pentose phosphate pathway (PPP) is implicated

in chemoresistance of cancer cells. In this study, we found that CD44, a cell surface marker for cancer stem cells,interacts with pyruvate kinase M2 (PKM2) and thereby enhances the glycolytic phenotype of cancer cells that areeither deficient in p53 or exposed to hypoxia. CD44 ablation by RNA interference increased metabolic flux tomitochondrial respiration and concomitantly inhibited entry into glycolysis and the PPP. Suchmetabolic changesinduced by CD44 ablation resulted in marked depletion of cellular reduced glutathione (GSH) and increased theintracellular level of reactive oxygen species in glycolytic cancer cells. Furthermore, CD44 ablation enhanced theeffect of chemotherapeutic drugs in p53-deficient or hypoxic cancer cells. Taken together, our findings suggestthat metabolic modulation by CD44 is a potential therapeutic target for glycolytic cancer cells that manifest drugresistance. Cancer Res; 72(6); 1438–48. �2012 AACR.

IntroductionMost cancer cells depend primarily on glycolysis for their

energy production regardless of the availability of oxygen. Thisunique metabolism is known as aerobic glycolysis called "War-burg effect" (1, 2). The glycolytic energetics undermitochondrialrespiratory suppression in cancer cells reduces production ofreactive oxygen species (ROS) and thereby confers resistance tovarious therapies. Indeed, interventions to tumors for switchingfrom glycolysis to mitochondrial respiration were found toreduce tumor mass (3), suggesting that aerobic glycolysis is animportant feature of cancer cells distinct from normal cells.However, precise mechanisms underlying the switch to use ofglycolysis for energy production in cancer cells remain unclear.

Dysfunction of p53, which frequently occurs in humancancers, promotes aerobic glycolysis, because p53 positivelyregulates mitochondrial respiration through inducing cyto-

chrome c oxidase 2 expression (4). Furthermore, p53 dysfunc-tion has recently been shown to increase metabolic flux topentose phosphate pathway (PPP; ref. 5).

CD44 is a major adhesionmolecule and has been implicatedin various biologic processes including cell migration and cellproliferation, as well as tumor growth and metastasis (6–8).CD44 is a cell surface marker for cancer stem cells, andCD44-expressing cancer cells are able to initiate tumors insome types of cancer (9). We recently showed that splicevariant forms of CD44 (CD44v) inhibit ROS accumulation incancer cells, thereby promoting tumor growth (10). Recentfindings suggest that p53 not only regulates glucose metabo-lism but induces CD44 expression (6). The fact raises a pos-sibility that CD44 is involved in regulating glycolytic pathway,whereas the functional relevance of CD44 to the characteristicaerobic glycolysis of cancer cells remains unknown.

With the use of in vitro virus (IVV) selection screening, wehave shown that CD44 interacts with PKM2, which has beenrecently implicated in Warburg effect (11–14). Expression ofCD44 enhanced the glycolytic phenotype of p53-deficient orhypoxic cancer cells and promoted metabolic flux to PPP andthereby increased glutathione (GSH) levels. We thus proposethat CD44 plays a role inmetabolic shift via regulation of PKM2and ROS protection in cancer cells.

Materials and MethodsCell lines

Human colorectal cancer cell HCT116 harboring wild-typep53 (p53WT) and its isogenic derivative lacking p53 (p53KO)were kindly provided by Dr. B. Vogelstein (Johns HopkinsOncology Center, Baltimore, MD). Human glioma cell U251MGand human lung carcinoma cell A549 were obtained fromAmerican Type Culture Collection. The cell lines used were

Authors' Affiliations: 1Division of Gene Regulation, Institute for AdvancedMedical Research, 2Department of Biochemistry, School of Medicine, KeioUniversity; 3Japan Science and Technology Agency, Core Research forEvolutional Science and Technology (CREST); 4Japan Science and Tech-nology Agency, ERATO, Suematsu Gas Biology Project; 5Department ofRespiratory Medicine, Juntendo University, Tokyo, Japan; 6Department ofBiosciences and Informatics, Faculty of Science and Technology, KeioUniversity, Kanagawa, Japan; and 7Department of GastroenterologicalSurgery, Graduate School of Medical Sciences, Kumamoto University,Kumamoto, Japan

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

Corresponding Author: Hideyuki Saya, Division of Gene Regulation,Institute for Advanced Medical Research, School of Medicine, Keio Uni-versity, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone:81-3-5363-3981; Fax: 81-3-5363-3982; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-3024

�2012 American Association for Cancer Research.

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tested and authenticated on the basis of an STR Multiplexmethod that uses 9 different loci: D5S818, D13S317, D7S820,D16S539, vWA, THOI, Amelogenin, TPOX, and CSFIPO (Power-Plex 1.2 system, Promega Corporation). Cells were stored andused within 3 months after resuscitation of frozen aliquots.

RNA interferenceDepletion of CD44 and xCT siRNA was conducted as pre-

viously described (10).

Measurement of cell doubling timeCells were cultured under 5% CO2 at 37�C. The number of

viable cells was determined every 24 hours by staining withtrypan blue. Doubling time was calculated by using the equa-tion shown in SupplementaryMaterials andMethods. Data aremeans � SD from 3 independent experiments.

Cell proliferation assayCell proliferation was measured at 24 and 72 hours with the

use of anXTT cell proliferation assay kit (Biological Industries).Data are means � SD from 6 independent experiments.

Measurement of ATP production, glucose consumption,lactate productionCellular ATP level was determined with the use of lumines-

cence-based assay (ATPlite; Perkin Elmer). ATP generationwas normalized by the cell number. Glucose and lactate con-centrations of the cultured medium were measured by usingglucose oxidase–based assay kit (Sigma) and F-kit L-Lactate(J.K. International), respectively. Glucose consumption andlactate production were corrected by amounts of cellular pro-tein. Data are means � SD from 6 independent experiments.

IVV screening and GST pull-down assayIVV selection and glutathione S-transferase (GST) pull-

down assay were conducted as described previously (15–17).The cDNA library for screening was obtained from U251MGcells. The intracellular domain of CD44 (CD44ICD) was pre-pared as a bait protein. Details are shown in SupplementaryMaterials and Methods.

Immunoprecipitation and immunoblot analysisImmunoprecipitation was conducted with the use of anti-

CD44 antibodies (F10-44-2 and IM7) or rabbit monoclonalantibody to PKM2, and the resulting precipitates were sub-jected to immunoblot analysis as previously described (10).The intensity of the band was measured using by Multi Gaugesoftware Ver.3.1 (FujiFilm). Data are means � SD from 3independent experiments.

Pyruvate kinase activity assayPyruvate kinase activity was determined by using a pyruvate

oxidase-based assay kit (BioVision). Data aremeans� SD from5 independent experiments.

Measurement of glucose metabolitesIntracellular metabolites of glucose were measured by cap-

illary electrophoresis combined with mass spectrometry

(CE/MS; Agilent Technology) as previously described (18,19). To measure fluxes of glucose metabolites, the cells wereincubated for 10 or 30 minutes in the presence of D-(13C6)glucose (4.5 g/L; ISOTEC) and then lysed for determination ofthe amounts of the labeled D-glucose incorporated into thecells. Data are means � SD from 3 independent experiments.Details are shown in Supplementary Materials and Methods.

Measurement ofmitochondrialmembrane potential andmitochondrial superoxide production

Mitochondrial membrane potential (Dym) and mitochon-drial superoxide production were measured in live cells by usingtetramethylrhodamine methyl ester perchlorate (TMRM) orMitoSOX Red indicator (Molecular Probes), respectively. Cellswere incubated with 200 nmol/L TMRM or 5 mmol/L MitoSOXand subjected to quantification of the mean intensity of fluo-rescence in more than 500 cells by using a Biorevo BZ-9000fluorescence microscope (Keyence) and analysis software.Nuclei were stained with Hoechst 33342 for fluorescence micro-scopy. Data are means � SD from a representative experiment.

Measurement of glucose uptake (2-NBDG uptake)Cells were subjected to staining with 2-(N-(7-nitrobenz-2-

oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Molecu-lar Probes) as described previously (20). 2-NBDG fluorescenceintensity was determined for more than 500 cells in eachexperiment. Data are means � SD from a representativeexperiment.

Quantitative and semiquantitative real-time reversetranscriptase (RT)-PCR analysis

Quantitative PCR analysis was conducted as previouslydescribed (10). Data were normalized by the amount of HPRT1mRNA. Data are means � SD of 3 independent experiments.Primer sequences are described in Supplementary Materialsand Methods.

Immunofluorescence analysisImmunofluorescence analysis of cultured cells was con-

ducted as previously described (10).

Measurement of GSH and ROSIntracellular levels of GSH and ROS were determined by

using GSH-Glo Glutathione Assay Kit (Promega) and 20,70-dichlorofluorescein-diacetate (DCF-DA), respectively, as de-scribed previously (10).

Drug treatment and cell death analysisCells were exposed to anticancer drugs for 48 hours at 37�C

under 21% O2 and 5% CO2 (normoxia) or under 1% O2 and 5%CO2 (hypoxia). Sub-G1 assessment based on cell-cycle analysiswas conducted as described previously (21). In addition, celldeath was evaluated by the trypan blue dye exclusion (22).

Statistical analysisData are presented as means � SD and were analyzed with

the unpaired Student t test by using Excel 2007 (Microsoft). A Pvalue of <0.05 was considered statistically significant.

Regulation of Glucose Metabolism by CD44 in Cancer Cells

www.aacrjournals.org Cancer Res; 72(6) March 15, 2012 1439




ResultsCD44 regulates cell proliferation and energy productionin glycolytic cancer cells

To understand roles of CD44 in cancer cell proliferation, weexamined effects of CD44 ablation by RNA interference onproliferation in 2 human cancer cells having wild-type p53(HCT116 p53WT and A549) and 2 p53-deficient cancer cells(HCT116 p53KO and U251MG; Supplementary Fig. S1A andS1B). CD44 depletion caused inhibition of proliferation ofp53-deficient cells, whereas it did not affect that of p53WTcells (Fig. 1A; Supplementary Fig. S1C and S1D). Trypan bluestaining confirmed that decreases in growth rates of p53-deficient cells resulting from CD44 ablation were not attrib-utable to the induction of apoptosis (data not shown). These

results suggest that CD44 contributes to proliferation of p53-deficient cancer cells.

Given that energy production is necessary for proliferation,we examined whether CD44 expression affects ATP levels.CD44 ablation decreased in ATP contents of p53-deficientcells but not in those of p53WT cells (Fig. 1B), suggesting thatCD44 regulates energy production in p53-deficient cells. Thesource of ATP production in cancer cells has been found todiffer depending on p53 status (4). Consistent with previousobservations (4, 5), we found that HCT116 p53KO cells man-ifest a glycolytic phenotype, characterized by increased glucoseconsumption, lactate production, and expression of the glu-cose transporter Glut1 (Supplementary Fig. S1E and S1G).CD44 ablation resulted in a decrease in glucose consumption

Figure1. CD44ablation suppressescell proliferation and decreasesATP production in p53-deficientcancer cells. A, time courseanalysis of cell growth beginningafter transfection with control andCD44 siRNAs for 48 hours. �, P <0.005. B and C, ATP productionand glucose consumption in cellscultured for 24 hours aftertransfection with indicated siRNAsfor 48 hours. Data are expressed asa percentage of the value forcontrol siRNA cells.�, P < 0.001. NS, not significant.

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in p53-deficient cells but not in p53WT cells (Fig. 1C). Wetherefore hypothesized that CD44 increases energy productionin p53-deficient cells by promoting glycolysis.

CD44 interacts with PKM2 and inhibits its activity tomaintain the glycolytic phenotype in cancer cellsTo investigate mechanisms by which CD44 regulates glycol-

ysis, we attempted to identify CD44-interacting molecules thatparticipate in glycolysis by IVV screening (Supplementary Fig.S2A). The cDNA library for screening was obtained fromU251MG cells, which manifests the glycolytic phenotypebecause of p53 mutation. The intracellular domain of CD44(CD44ICD)was prepared as a bait protein. Among 292 candidateCD44ICD-binding proteins identified by the IVV screening, we

focused on PKM2, a molecule regulating aerobic glycolysis andtumor growth (11). Sequencing revealed that 2 selected PKM2clones, designated 61–17C, encoded the 61 NH2-terminal aminoacids of PKM2 fused with 18 amino acids translated from the 50-untranslated region of the PKM2 gene (Supplementary Fig. S2B).To determine whether CD44ICD interacts with the NH2-termi-nal region or with the protein fragment encoded by the 50-untranslated region, we prepared 4 GST fusion proteins con-taining full-length, the 61–17C fragment, the 61 NH2-terminalresidues (N61AA), and a deletionmutant lacking residues 1 to 61(DN61AA; Supplementary Fig. S2C) and subjected these fusionproteins to a pull-down assay with recombinant CD44ICD.The assay revealed that CD44ICD bound to full-length PKM2,to 61–17C, and to N61AA, but not to DN61AA (Fig. 2A),

Figure 2. CD44 contributes to theglycolytic phenotype of cancer cellsthrough interactionwithPKM2.A, theGST–PKM2 fusion proteins weresubjected to a pull-down assay withT7-tagged CD44ICD. The bead-bound proteins as well as the T7-CD44ICD input to the bindingmixtures (Input) were subjected toimmunoblot (IB) analysis withindicated antibodies. FL, full-length.B and C, the cell lysates weresubjected to immunoprecipitation(IP) with indicated antibody and theresulting precipitates as well as theoriginal cell lysates (Input) wereimmunoblotted with indicatedantibodies. D and E, enzymaticactivity of pyruvate kinase in cellstransfected for 72 hours withindicated siRNAs or with both aCD44 siRNA#2 and an expressionvector for a corresponding RNAinterference (RNAi)–resistant form ofCD44s or CD44v. �, P < 0.001. F andG, lysates of cells transfected withindicated siRNAs for 48 hours wereimmunoblotted with indicatedantibodies. The graphs indicate theratio of phosphorylation to totalPKM2.�, P < 0.05. MW, molecularweight.






indicating that CD44ICD interacts directly to the NH2-terminalregion of PKM2 encompassing residues 1 to 61. We also con-ducted immunoprecipitation analysis to verify the interactionbetween endogenous CD44 and PKM2 proteins. PKM2 was co-immunoprecipitated with CD44 (Fig. 2B), as was the cystinetransporter xCT, which was previously shown to interact withCD44 (10), and confirmed that both CD44v and CD44s were co-immunoprecipitated with PKM2 (Fig. 2C).

Given that low activity of PKM2 is thought to promoteaerobic glycolysis (11, 12), we examined that whetherCD44 expression affects PKM2 activity. CD44 ablation inglycolytic cancer cells increased the PKM2 activity (Fig. 2Dand E). In addition, the expression of siRNA-resistantform of CD44s or CD44v in CD44-depleted cells (Supple-mentary Fig. S2D) significantly inhibited the increase inPKM2 activity induced by ablation of endogenous CD44(Fig. 2D and E). Tyrosine phosphorylation (Tyr105) ofPKM2 was reported to suppress PKM2 activity (12, 13).We found that CD44 ablation reduced Tyr105 phosphor-ylation of PKM2 (Fig. 2F and G). These data suggestedthat the CD44/PKM2 interaction suppresses PKM2 activitythrough increasing its phosphorylation and thereby pro-motes the glycolytic phenotype in p53-deficient cancercells.

CD44 ablation induces a metabolic shift tomitochondrial respiration in glycolytic cancer cells

To examine effects of CD44 expression on glucose metab-olism, we conducted metabolomic analysis by loading the cellswith D-(13C6)glucose. CD44 ablation increased amounts ofmetabolites in tricarboxylic acid (TCA) cycle (Fig. 3A), suggest-ing that CD44 expression limits metabolic flux to the cycle.Furthermore, we measured the production of lactate, the finalproduct of glycolysis, and found that CD44 ablation reducedlactate production in p53KO cells (Fig. 3B), Collectively, ourresults suggested that CD44 ablation induces a metabolic shiftfrom aerobic glycolysis to mitochondrial respiration in cancercells.

It has been reported that the suppression of mitochon-drial respiration is characterized by high intensity stainingof Dym-sensitive dye TMRM and low mitochondrial ROS (3).To confirm the CD44-mediated metabolic shift, we con-ducted cell staining with TMRM and a fluorescent probefor mitochondrial superoxide (MitoSOX). CD44 ablationsignificantly reduced the intensity of TMRM staining andincreased the mitochondrial ROS levels (Fig. 3C and D).Effects similar to those of CD44 ablation were obtainedby treatment with dichloroacetate (DCA; SupplementaryFig. S3A and S3B), which inhibits mitochondrial pyruvate

A

C D

B

Figure 3. CD44 ablation induces ametabolic shift from glycolysis tomitochondrial respiration inglycolytic cancer cells. A,metabolomic analysis of cellsincubated for 24 hours aftertransfection with indicated siRNAsfor 48 hours and then labeled withD-(13C6) glucose. The amounts ofthe indicated metabolites of theTCA cycle were measured.�,P < 0.05. B, lactate production bycells cultured for 24 hours aftertransfection with siRNAs for 48hours. �, P < 0.001. C and D, Dymandmitochondrial ROS productionin cells that had been transfectedwith siRNAs for 48 hours. Scalebars, 20 mm. �, P < 0.001. E,doubling time of the cells that hadbeen transfected with siRNAs for48 hours was determined duringsubsequent incubation of the cellsin the absence or presence of125 nmol/L oligomycin. �, P < 0.01;��, P < 0.001. NS, not significant.

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dehydrogenase kinase and thereby shifts glucose metabo-lism from glycolysis to mitochondrial respiration (3). Thesedata therefore suggested that, like DCA treatment, CD44ablation promotes mitochondrial respiration in glycolyticcancer cells.To analyze further the metabolic shift in CD44-depleted

cancer cells, we investigated the sensitivity of the cells tooligomycin, an inhibitor of mitochondrial ATP synthesis. Oli-gomycin inhibited the proliferation of p53WT cells, whichdepend more on mitochondrial respiration, whereas it did notaffect the proliferation of p53KO cells (Fig. 3E). However,p53KO cells became sensitive to oligomycin by CD44 ablation(Fig. 3E), suggesting that loss of CD44 suppresses their glyco-lytic phenotype and renders them more dependent on mito-chondrial respiration.

CD44 ablation reduces glucose uptake and PPP fluxIn addition to the metabolic shift, we found that CD44

ablation reduced metabolic flux to the PPP. In particular,CD44 depletion resulted in a reduced amount of the PPPmetabolite 6-phosphogluconate (6-PG) in p53KO cells(Fig. 4A). However, the expression of glucose-6-phosphatedehydrogenase (G6PD), a key enzyme that catalyzes the firststep of PPP, was not affected by CD44 depletion (Supple-

mentary Fig. S4A). The data showing that CD44 ablationreduced combined amounts of G6P and fructose 6-phos-phate (F6P; Supplementary Fig. S4B) led us to speculate thatreduced PPP fluxes in CD44-deficient cells result from lackof glucose. Indeed, we found that CD44 ablation led tosuppression of glucose uptake (Fig. 4B), which may contrib-ute to reduced PPP fluxes in CD44-depleted cells.

Given that glucose uptake is mainly mediated by Glut1in cancer cells (23, 24), we examined Glut1 expression inCD44-depleted cells. Indeed, CD44 depletion resulted indownregulation of Glut1 expression (Fig. 4C and D). Phar-macologic inhibition of mitochondrial respiration was pre-viously shown to increase glucose uptake potentiallythrough upregulation of Glut1 expression (25, 26). We there-fore hypothesized that CD44 ablation suppresses Glut1expression as a result of the induced metabolic shift fromglycolysis to mitochondrial respiration. Consistent with thishypothesis, the downregulation of Glut1 expression inducedby CD44 ablation was reversed by treatment with rotenone,an inhibitor of complex I of the mitochondrial respiratorychain (Fig. 4C and D). The enhancement of mitochondrialrespiration induced by CD44 ablation may suppress Glut1expression, thereby reducing glucose uptake and PPP fluxesin cancer cells (Supplementary Fig. S4C).

Figure 4. CD44ablation suppressesglucose uptake leading to reducedflux to PPP. A, metabolomic analysisof cells incubated for 24 hours undernormoxia or hypoxia aftertransfection with indicated siRNA for48 hours. �, P < 0.05. Parts of theglycolytic pathway and PPP areindicated on the left and right,respectively. RU5P, ribulose-5-phosphate. B, glucoseuptake in cellstransfectedwith siRNAs. �,P< 0.001.C, quantitative RT-PCR analysis ofGlut1mRNA in cells transfected withsiRNAs for 48 hours and thenincubated in the absence orpresence of 5 mmol/L rotenone for2 hours. �, P < 0.001. D,immunofluorescence analysis ofGlut1 and CD44 expression in cellstransfected with siRNAs for 48 hoursand then incubated in the absence orpresence of 5 mmol/L rotenone for12 hours. Scale bars, 20 mm.

A

C D

B






Reduced PPP flux by CD44 ablation depletes GSH andincreases ROS

Considering that the PPP generates NADPH which is essen-tial for generating GSH, we measured GSH levels in CD44-depleted cells. CD44 ablation significantly reduced GSH con-

tents of p53KO cells (Fig. 5A). We have recently found thatCD44v promotes xCT-mediated cystine uptake and therebyincreases GSH synthesis (10). The depletion of GSH apparent inCD44-deficient HCT116 p53KO cells, which express bothCD44s and CD44v, might therefore have been attributable to

Figure 5. CD44 ablation reducesGSH level through suppression ofnot only cystine uptake but alsoflux to PPP, leading to ROSproduction in glycolytic cancercells. A, intracellular GSH level incells cultured for 12 hours aftertransfection with indicated siRNAsfor 48 hours. �,P < 0.001. B, cellularROS level was measured with theuse of DCFH-DA and flowcytometry in cells that had eitherbeen transfected with siRNAs for72 hours or been incubated inthe presence of 30 mmol/L DHEAor vehicle for 24 hours. The meanrelative fluorescence intensity (RFI)values are shown. C, intracellularGSH level in cells cultured for12 hours in absence or presence of30 mmol/L DHEA after transfectionwith siRNAs for 48 hours andpretreatment of the cells withDHEA for 24 hours. �, P < 0.001.DCF, dichlorofluorescin, thefluorescent product producedby ROS; NS, not significant.

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downregulation of cystine uptake. To rule out this possibility,we measured GSH levels in CD44-depleted U251MG cells,which express CD44s but not CD44v (ref. 27; SupplementaryFig. S1B). CD44 ablation in U251MG cells also resulted in asignificant decrease in GSH levels (Fig. 5A). These findingssuggested that CD44 increases cellular GSH contents not onlythroughpromotion of xCT-mediated cystine uptake but also bymaintenance of the PPP flux and consequent NADPH produc-tion in cancer cells.GSH is a major metabolite protecting against oxidative

stress. We found that intracellular ROS levels were increasedinCD44-depleted p53KO cells comparablewith those in p53KOcells treated with dehydroepiandrosterone (DHEA), a PPPinhibitor (Fig. 5B). In contrast, although CD44 ablationdecreased in GSH levels (Supplementary Fig. S5A), it did notaffect intracellular ROS accumulation in p53WT cells (Supple-mentary Fig. S5B). Given that CD44 ablation inhibits neitheruptake (data not shown) nor consumption of glucose (Fig. 1D)in p53WT cells, CD44 depletion might have a less pronouncedeffect on flux to the PPP in p53WT cells than it does in p53KOcells. This hypothesis is supported by the fact that DHEAtreatment significantly conferred an additional reduction inGSH levels in the xCT-depleted p53KO cells whereas it did notprovide the additional change in xCT-depleted p53WT cells(Fig. 5C). Together, these results suggested that CD44 limitsROS accumulation in glycolytic cancer cells, such as those withdysfunctional p53, by promoting cystine uptake andmetabolicflux to PPP, leading to increase consequent GSH synthesis.

CD44 ablation sensitizes glycolytic cancer cells toanticancer drugsGiven that aerobic glycolysis and low ROS levels are asso-

ciated with drug resistance in cancer cells (28), we examinedwhether CD44 ablation enhances the sensitivity of cancer cellsto chemotherapeutic agents. Consistent with previous obser-vations (29), cisplatin (CDDP) induced apoptotic cell death to amuch greater extent in p53WT cells (sub-G1 population,56.71%) than in p53KO cells (sub-G1 population, 15.28%;Fig. 6A). CD44 ablation was associated with enhancement ofCDDP-induced cell death in p53KO cells but not in p53WT cells(Fig. 6A, Supplementary Fig. S6A). This increased sensitivity toCDDP conferred by CD44 ablation in p53KO cells was inhibitedby pretreatment with N-acetylcysteine (NAC; Fig. 6A; Supple-mentary Fig. S6A), a precursor of GSH that functions as anantioxidant. In addition to CDDP, we obtained the similar datausing adriamycin (ADM) and 5-fluorouracil (5-FU; Supple-mentary Fig. S6A). Furthermore, similar to the effect of CD44ablation, the PPP inhibitor DHEA, which suppresses NADPHproduction and thereby increases ROS levels, also enhancedthe sensitivity of p53KO cells to CDDP (Supplementary Fig.S6B). Together, these results indicated that CD44 ablationincreased drug sensitivity, possibly by increasing ROS levels,in p53-deficient cells.Given that, like p53 deficiency, hypoxia also promotes

glycolysis and confers drug resistance in cancer cells (30,31), we investigated whether CD44 ablation affects glucosemetabolism and drug sensitivity under hypoxia. p53WTcells cultured under such conditions show more of a

glycolytic phenotype, including increased Glut1 expression(Supplementary Fig. S6C), glucose consumption (Fig. 6B),and lactate production (Fig. 6C), compared with thosecultured under normoxia. The sensitivity of these cells toanticancer drugs was also reduced on their exposure tohypoxia (Fig. 6A and D; Supplementary Fig. S6D). CD44ablation reduced both glucose consumption and lactateproduction in hypoxic p53WT cells (Fig. 6B and C) as wellas increased their sensitivity to anticancer drugs (Fig. 6D;Supplementary Fig. S6D), effects that were not observedunder the normoxia (Figs. 1D, 6A and C; Supplementary Fig.S6A). These results thus indicated that CD44 plays animportant role in resistance to chemotherapeutic drugs bymaintaining the glycolytic phenotype and thereby suppres-sing ROS production in glycolytic cancer cells, such as thosewith p53 mutation or exposed to hypoxia. Metabolic mod-ulation by CD44 ablation increases ROS production, therebysensitizing highly glycolytic cancer cells to conventionalchemotherapy.

DiscussionOur results indicate that the expression of CD44 affects

proliferation through regulation of energy production inp53-deficient glycolytic cancer cells. In contrast, CD44 didnot affect it in p53WT cancer cells, which obtain most oftheir energy through mitochondrial respiration. We there-fore conclude that CD44 plays an important role in theregulation of glucose metabolism in cancer cells that show aglycolytic phenotype.

IVV screening is a powerful tool for identifying biologicmacromolecules that participate in protein–protein interac-tions (15–17). By using this approach, we identified PKM2 as amediator of the promotion of the glycolytic phenotype ofcancer cells by CD44. CD44 ablation suppressed Tyr105 pho-phorylation of PKM2 and consequently increased the PKM2activity. It is therefore possible that CD44 serves as a scaffold tofacilitate the interaction between a tyrosine kinase and PKM2near the cellmembrane, thereby resulting in downregulation ofPKM2 activity (Supplementary Fig. S7).

Consistent with the observation that CD44 ablation activat-ed PKM2, the amounts of metabolites in the TCA cycle wereincreased in CD44-depleted cells. Furthermore, the increase ofnot only (13C2)malate but also (13C3)malate by CD44 ablationindicated the increment of TCA cycle flux. The findings sup-port the idea that metabolic flux to TCA cycle is promoted bylacking CD44.

We found that CD44 ablation not only induced this meta-bolic shift but also reduced glucose uptake in cancer cells. Itwas previously reported that inhibition of mitochondrial res-piration increases glucose uptake potentially through upregu-lation of Glut1 expression (25, 26), indicating that mitochon-drial respiration is a regulatory factor for Glut1 expression.Therefore, we speculated that shift from glycolysis to mito-chondrial respiration induced by CD44 ablation suppressesGlut1 expression (Supplementary Fig. S4C).

A reduction in glucose uptake has been shown to causea decrease in flux to the PPP (32, 33). Accordingly, the






downregulation of glucose uptake in CD44-depleted cellsmight lead to the reduced flux to the PPP. Because the PPPis a major source of NADPHwhich is required for regenerationof the antioxidant GSH (34), the metabolic regulation by CD44in p53 dysfunctional cancer cellswas found to affectGSH levels.We previously showed that CD44v expression promotes xCT-mediated cystineuptake and consequentGSHsynthesis (10). Inthe present study, however, we found that CD44 ablationreduced the GSH levels in U251MG cells, which express onlyCD44s, as well as in HCT116 p53KO cells, which express both

CD44s and CD44v. These results indicate that GSH synthesis isregulatednot only byCD44v but also byCD44s.We suggest thatCD44 maintains the GSH levels in glycolytic cancer cellsthrough the combination of 2 mechanisms: enhancement offlux to the PPP by both CD44v and CD44s and promotion ofxCT-mediated cystine uptake by CD44v (SupplementaryFig. S7).

In p53KO cells, CD44 ablation increased the intracellularROS levels under the basal condition. However, in p53WT cells,which are less dependent on glycolysis, CD44 ablation

Figure 6. CD44 ablation increases sensitivity to CDDP in glycolytic cancer cells. A, flow cytometric analysis of cells that had been transfected with indicatedsiRNAs for 48 hours. The transfected cells were incubated for 48 hours in the absence or presence of 50 mmol/L CDDP under normoxia (21%O2). The effect ofCDDP was also assessed after pretreatment of the cells with 1 mmol/L NAC for 1 hour. The percentage of sub-G1 cells was determined. B and C, glucoseconsumption and lactate production in cells cultured for 24 hours under normoxia and hypoxia after transfection with siRNAs for 48 hours. �, P < 0.05. D, flowcytometric analysis of cells that hadbeen transfectedwith siRNAs for 48hoursunder hypoxia. The transfectedcellswere incubated for 48hours in theabsenceor presence of 50 mmol/L CDDP under hypoxia. NS, not significant.

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increased the ROS levels only when the cells were treated withH2O2 (10). Given that CD44 ablation did not reduce glucoseuptake or glucose consumption in p53WT cells and that p53inhibits the PPP (5), the depletion of GSH induced by CD44ablation inp53WTcellsmight bemostly due to the suppressionof cystine uptake via the xCT transporter.CD44-expressing cancer cells including cancer stem cells

show chemoresistance (35, 36). Anticancer drugs, such asCDDP, adriamycin, and 5-fluorouracil, are known to induceROS generation and thereby trigger cell death (29, 37–39).Consistent with the results of previous studies (29, 31, 40–42),we found that the sensitivity of cancer cells to anticancerdrugs is markedly affected by intrinsic and extrinsic factorssuch as p53 deficiency and hypoxia. These factors rendercancer cells dependent on glycolysis. However, we furtherfound that CD44 ablation enhanced effect of the anticancerdrugs in p53KO cells and in hypoxic p53WT cells. We con-firmed that the enhancement of drug sensitivity inducedby CD44 ablation in p53KO cells was inhibited by NAC treat-ment. Furthermore, like CD44 ablation, treatment with DHEAalso increased CDDP sensitivity. Therefore, CD44 ablationtriggers a metabolic shift to mitochondrial respiration that isaccompanied by suppression of both the PPP and xCT-medi-ated cystine uptake, leading to downregulation of GSHsynthesis and a consequent increase in ROS production. Fur-thermore, these effects of CD44 ablation might function syn-ergistically with chemotherapeutic drugs. The possibility thatCD44-targeted therapy may perturb the metabolism of cancerstem–like cells and thereby impair their capacity to defendagainst ROS warrants further investigation.

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

Authors' ContributionsConception and design: M. Tamada, O. Nagano, H. SayaDevelopment of methodology: M. Tamada, S. Tateyama, T. YaeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Tamada, S. Tateyama, M. Ohmura, T. Yae, T.Ishimoto, H, Yanagawa, M. SuematsuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Tamada, M. Ohmura, T. YaeWriting, review, and/or revision of the manuscript: M. Tamada, S.Tateyama, H. Yanagawa, M. Suematsu, H. SayaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Tamada, T. Yae, E. Sugihara, N.Onishi, T. Yamamoto, H. Yanagawa, M. SuematsuStudy supervision: H. Saya

AcknowledgmentsThe authors thank K. Arai for help with preparation of the manuscript and K.

Dan, M. Fujiwara, and K. Matsuo (Core Instrumentation Facility), S. Suzuki andY. Matsuzaki (Department of Physiology), T. Matsu-ura, Y. Nagahata, T. Hishiki, A.Kubo, Y.A Minamishima, andM. Kajimura (Department of Biochemistry, School ofMedicine, Keio University, Tokyo, Japan) for discussion and technical assistance.

Grant SupportThis work was supported in part by Grant-in-Aid for Scientific Research from

the Ministry of Education, Culture, Sports, Science and Technology (MEXT),Japan (O. Nagano and H. Saya); the Global COE Program, MEXT, Japan (M.Tamada, M. Ohmura, and M. Suematsu); the Grant-in-Aid for JSPS Fellows DC2(M. Tamada); and "Next Generation Integrated Simulation of Living Matter"project, part of the Development andUse of theNext-Generation SupercomputerProject of MEXT (M. Ohmura).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 8, 2011; revised January 12, 2012; accepted January 26,2012; published OnlineFirst January 31, 2012.

References1. WarburgO,Wind F, Negelein E. Themetabolism of tumors in the body.

J Gen Physiol 1927;8:519–30.2. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis?

Nat Rev Cancer 2004;4:891–9.3. Bonnet S, Archer SL, Allalunis-Turner J, Haromy A, Beaulieu C,

Thompson R, et al.A mitochondria-Kþ channel axis is suppressed incancer and its normalization promotes apoptosis and inhibits cancergrowth. Cancer Cell 2007;11:37–51.

4. MatobaS, Kang JG, PatinoWD,Wragg A, BoehmM,GavrilovaO, et al.p53 regulates mitochondrial respiration. Science 2006;312:1650–3.

5. Jiang P, DuW,Wang X, Mancuso A, Gao X, WuM, et al. p53 regulatesbiosynthesis through direct inactivation of glucose-6-phosphatedehydrogenase. Nat Cell Biol 2011;13:310–6.

6. Godar S, Ince TA, Bell GW, Feldser D, Donaher JL, Bergh J, et al.Growth-inhibitory and tumor- suppressive functions of p53 depend onits repression of CD44 expression. Cell 2008;134:62–73.

7. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. CD44 is theprincipal cell surface receptor for hyaluronate. Cell 1990;61:1303–13.

8. Ponta H, Sherman L, Herrlich PA. CD44: from adhesion molecules tosignalling regulators. Nat Rev Mol Cell Biol 2003;4:33–45.

9. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accu-mulating evidence and unresolved questions. Nat Rev Cancer2008;8:755–68.

10. Ishimoto T, Nagano O, Yae T, TamadaM,Motohara T, Oshima H, et al.CD44 variant regulates redox status in cancer cells by stabilizing thexCT subunit of system xc(-) and thereby promotes tumor growth.Cancer Cell 2011;19:387–400.

11. Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A,Gerszten RE, Wei R, et al. The M2 splice isoform of pyruvate kinase

is important for cancer metabolism and tumour growth. Nature2008;452:230–3.

12. Hitosugi T, Kang S, Vander Heiden MG, Chung TW, Elf S, Lythgoe K,et al. Tyrosine phosphorylation inhibits PKM2 to promote theWarburgeffect and tumor growth. Sci Signal 2009;2:ra73.

13. Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC.Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature2008;452:181–6.

14. Vander Heiden MG, Locasale JW, Swanson KD, Sharfi H, Heffron GJ,Amador-Noguez D, et al. Evidence for an alternative glycolytic path-way in rapidly proliferating cells. Science 2010;329:1492–9.

15. Tateyama S, Horisawa K, Takashima H, Miyamoto-Sato E, Doi N,Yanagawa H. Affinity selection of DNA-binding protein complexesusing mRNA display. Nucleic Acids Res 2006;34:e27.

16. Horisawa K, Tateyama S, Ishizaka M, Matsumura N, Takashima H,Miyamoto-Sato E, et al. In vitro selection of Jun-associated proteinsusing mRNA display. Nucleic Acids Res 2004;32:e169.

17. Miyamoto-Sato E, IshizakaM, Horisawa K, Tateyama S, Takashima H,FuseS, et al. Cell-free cotranslation and selection using in vitro virus forhigh-throughput analysis of protein-protein interactions and com-plexes. Genome Res 2005;15:710–7.

18. Kinoshita A, Tsukada K, Soga T, Hishiki T, Ueno Y, Nakayama Y, et al.Roles of hemoglobin Allostery in hypoxia-induced metabolic altera-tions in erythrocytes: simulation and its verification by metabolomeanalysis. J Biol Chem 2007;282:10731–41.

19. Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, et al.Differential metabolomics reveals ophthalmic acid as an oxidativestress biomarker indicating hepatic glutathione consumption. J BiolChem 2006;281:16768–76.






20. Kawauchi K, Araki K, Tobiume K, Tanaka N. p53 regulates glucosemetabolism through an IKK-NF-kappaB pathway and inhibits celltransformation. Nat Cell Biol 2008;10:611–8.

21. Arima Y, Nitta M, Kuninaka S, Zhang D, Fujiwara T, Taya Y, et al.Transcriptional blockade induces p53-dependent apoptosis associ-ated with translocation of p53 to mitochondria. J Biol Chem 2005;280:19166–76.

22. SudoT,NittaM, SayaH,UenoNT.Dependenceof paclitaxel sensitivityon a functional spindle assembly checkpoint. Cancer Res 2004;64:2502–8.

23. FurutaE,OkudaH,Kobayashi A,WatabeK.Metabolic genes in cancer:their roles in tumor progression and clinical implications. BiochimBiophys Acta 2010;1805:141–52.

24. Ganapathy V, Thangaraju M, Prasad PD. Nutrient transporters incancer: relevance to Warburg hypothesis and beyond. PharmacolTher 2009;121:29–40.

25. Hamrahian AH, Zhang JZ, Elkhairi FS, Prasad R, Ismail-Beigi F.Activation of Glut1 glucose transporter in response to inhibition ofoxidative phosphorylation. Arch Biochem Biophys 1999;368:375–9.

26. Ebert BL, Firth JD, Ratcliffe PJ. Hypoxia and mitochondrial inhibitorsregulate expression of glucose transporter-1 via distinct Cis-actingsequences. J Biol Chem 1995;270:29083–9.

27. Okamoto I, Tsuiki H, Kenyon LC, Godwin AK, Emlet DR, Holgado-MadrugaM, et al. Proteolytic cleavage of the CD44 adhesionmoleculein multiple human tumors. Am J Pathol 2002;160:441–7.

28. Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, et al.Inhibition of glycolysis in cancer cells: a novel strategy to overcomedrug resistance associated with mitochondrial respiratory defect andhypoxia. Cancer Res 2005;65:613–21.

29. Bragado P, Armesilla A, Silva A, Porras A. Apoptosis by cisplatinrequires p53 mediated p38alpha MAPK activation through ROS gen-eration. Apoptosis 2007;12:1733–42.

30. Teicher BA. Hypoxia and drug resistance. Cancer Metastasis Rev1994;13:139–68.

31. Song X, Liu X, Chi W, Liu Y, Wei L, Wang X, et al. Hypoxia-inducedresistance to cisplatin and doxorubicin in non-small cell lung cancer is

inhibited by silencing of HIF-1alpha gene. Cancer Chemother Phar-macol 2006;58:776–84.

32. Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY,et al. Antioxidant and oncogene rescue of metabolic defectscaused by loss of matrix attachment. Nature 2009;461:109–13.

33. Gottlieb E. Cancer: the fat and the furious. Nature 2009;461:44–5.34. Rahman I, Kode A, Biswas SK. Assay for quantitative determination of

glutathione and glutathione disulfide levels using enzymatic recyclingmethod. Nat Protoc 2006;1:3159–65.

35. Dallas NA, Xia L, Fan F, Gray MJ, Gaur P, van Buren G II, et al.Chemoresistant colorectal cancer cells, the cancer stem cell pheno-type, and increased sensitivity to insulin-like growth factor-I receptorinhibition. Cancer Res 2009;69:1951–7.

36. Phillips TM, McBride WH, Pajonk F. The response of CD24(-/low)/CD44 þbreast cancer-initiating cells to radiation. J Natl Cancer Inst2006;98:1777–85.

37. Choi KJ, Piao YJ, LimMJ, Kim JH, Ha J, ChoeW, et al. Overexpressedcyclophilin A in cancer cells renders resistance to hypoxia- andcisplatin-induced cell death. Cancer Res 2007;67:3654–62.

38. Hwang IT, Chung YM, Kim JJ, Chung JS, Kim BS, Kim HJ, et al. Drugresistance to 5-FU linked to reactive oxygen species modulator 1.Biochem Biophys Res Commun 2007;359:304–10.

39. Aluise CD, St Clair D, Vore M, Butterfield DA. In vivo ameliorationof adriamycin induced oxidative stress in plasma by gamma-glutamylcysteine ethyl ester (GCEE). Cancer Lett 2009;282:25–9.

40. Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis ofresistance. Oncogene 2003;22:7265–79.

41. Lowe SW, Ruley HE, Jacks T, Housman DE. p53-dependent apo-ptosis modulates the cytotoxicity of anticancer agents. Cell1993;74:957–67.

42. Liu L, Sun L, Zhang H, Li Z, Ning X, Shi Y, et al. Hypoxia-mediated up-regulation of MGr1-Ag/37LRP in gastric cancers occurs via hypoxia-inducible-factor 1-dependent mechanism and contributes to drugresistance. Int J Cancer 2009;124:1707–15.

Tamada et al.





2012;72:1438-1448. Published OnlineFirst January 31, 2012.Cancer Res Mayumi Tamada, Osamu Nagano, Seiji Tateyama, et al. Antioxidant Status and Drug Resistance in Cancer CellsModulation of Glucose Metabolism by CD44 Contributes to

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