RESEARCH ARTICLE PKM2 Regulates Hepatocellular Carcinoma

Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 1961

DOI:http://dx.doi.org/10.7314/APJCP.2014.15.5.1961PKM2 Regulates HCC Epithelial-mesenchymal Transition and Migration upon EGFR Activation

Asian Pac J Cancer Prev, 15 (5), 1961-1970

Introduction

Hepatocellular carcinoma (HCC) is the fifthmostfrequentlydiagnosedcancerandthesecondleadingcauseofcancer-relateddeathintheworld(Altekruseetal.,2012;Wiangnonetal.,2012;Norsa’adahetal.,2013).Surgicalresection,providingalong-termsurvivalofHCCpatients,isregardedasoneofthestandardtreatmentsofHCCifthetumorisresectable(Personenietal.,2012;Zhouetal.,2012b).However,approximately30-50%patientsdevelopHCCrecurrenceduringthefirstyearaftersurgery,andingeneral,therecurrentHCCsaremorediffuseandhardlytreatable (Li et al., 2013;Zhu et al., 2013).Therefore,preventionofHCCrecurrenceisthekeytoimprovethesurvival rate of the patients.However, unfortunately,themolecularmechanismsandthebiomarkersforHCCrecurrenceremainlargelyunknown. EMT is a process inwhich cells with epithelialphenotypes acquire mesenchymal characteristics.This transition is characterized by combined loss ofepithelialcell junctionproteinssuchasE-cadherinandby concomitant gain ofmesenchymalmarkers such as

1Department of Pharmacology, College of Pharmacy, 2Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, 3Department of Biology, Hefei Vocational and Technical College, Hefei, China, 4Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center Shreveport, Louisiana, USA &Equal contributors *For correspondence: [email protected]

Abstract

Pyruvatekinase isozyme typeM2 (PKM2)wasfirst found inhepatocellular carcinoma (HCC), and itsexpressionhasbeenthoughttocorrelatewithprognosis.Alargenumberofstudieshavedemonstratedthatepithelial-mesenchymaltransition(EMT)isacrucialeventinhepatocellularcarcinoma(HCC)andassociatedmetastasis,resultinginenhancedmalignancyofHCC.However,therolesofPKM2inHCCEMTandmetastasisremainlargelyunknown.ThepresentstudyaimedtodeterminetheeffectsofPKM2inEGF-inducedHCCEMTandelucidatethemolecularmechanismsinvitro.OurresultsshowedthatEGFpromotedEMTinHCCcelllinesasevidencedbyalteredmorphology,expressionofEMT-associatedmarkers,andenhancedinvasioncapacity.Furthermore,thepresentstudyalsorevealedthatnucleartranslocationofPKM2,whichisregulatedbytheERKpathway,regulatedβ-catenin-TCF/LEF-1transcriptionalactivityandassociatedEMTinHCCcelllines.ThesediscoveriesprovideevidenceofnovelrolesofPKM2intheprogressionofHCCandpotentialtherapeutictargetforadvancedcases. Keywords: Epithelialgrowthfactorreceptor-PKM2-β-catenin-epithelial-mesenchymaltransition-HCC

RESEARCHARTICLE

PKM2RegulatesHepatocellularCarcinomaCellEpithelial-mesenchymalTransitionandMigrationuponEGFRActivation

Fang-TianFan1,2,3&,Cun-SiShen1&,LiTao1,ChaoTian1,Zhao-GuoLiu1,Zhi-JieZhu1,Yu-PingLiu1,Chang-SongPei1,Hong-YanWu1,LeiZhang1,Ai-YunWang1,2,Shi-ZhongZheng1,2,Shi-LeHuang4,YinLu1,2*

VimentinandN-cadherin(Easthametal.,2007).Recently,it iswidely accepted that EMT is the initiate step ofintra-hepatic and extra-hepatic,Whichwere necessaryforrecurrenceofHCCafterresection(Tsujietal.,2009).However,themechanismsofEMTinHCCremainlargelyunknown. PyruvatekinaseisozymetypeM2(PKM2)isexpressedincellswithahighrateofnucleicacidsynthesis,suchasnormalproliferatingcells,embryoniccells,andespeciallytumor cells (Tamada et al., 2012). PKM2 catalyzesphosphoenolpyruvatetopyruvateandisresponsiblefornetATPproductionwithinglycolysis(Macintyreetal.,2011).PKM2containsaninduciblenucleartranslocationsignal in itsC-domain (Yanget al., 2012). It hasbeendescribed that nuclear PKM2 is pro-proliferative orpro-apoptotic, depending on environmental conditions(Spodenet al., 2009).Recently, it hasbeen found thatPKM2inhibitsorpromotesEMTingastriccancercells,dependingondifferentiationstatusofthecells(Wangetal.,2013).Furthermore,expressionofPKM2mRNAissignificantlycorrelatedwiththeprogressionofmalignantphenotypesinHCC(Tanakaetal.,2013).However,the

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Asian Pacific Journal of Cancer Prevention, Vol 15, 20141962

roleofPKM2inEMTofHCCremainstobedetermined.Dysregulationofsignaltransductionpathwaysofgrowthfactors, including transforming growth factor beta(TGF-β)/Smad,platelet-derivedgrowthfactor(PDGF)/PDGFreceptorandhepatocytegrowthfactor(HGF)/c-Met,contributestoEMTofHCC(Fischeretal.,2007;Ogunwobi et al., 2011;Xu et al., 2003).Activationofepidermal growth factor (EGF) signaling is initiatedby induction of the phosphorylation ofEGF receptor(EGFR).IthasbeenshownthatEGFRisactivatedin40-70%ofHCC,andtheactivationofEGFRcorrelateswithpathogenesisandprognosisofHCC(Buckleyetal.,2008).IthadbeenreportedthatEGFRsignalwasnecessaryforPKM2promoting tumorprogression.Here, thepresentstudywastoinvestigatetheroleofPKM2ininductionofHCCEMTanddisclosetheunderlyingmechanism.

MaterialsandMethods

Reagents and antibodies Recombinant human EGF was purchased fromPeprotech (RockyHill, New Jersey, USA) andwasdissolvedin0.1%bovineserumalbumin(BSA;Shanghai,China).ChemicalsofLY294002,rapamycin,U0126,andSKI-1werepurchasedfromCellSignalingTechnology(Danvers,MA,USA). Primary antibodies used forWestern blotting analyseswere against p-β-catenin(Ser552), ERK1/2, p-ERK1/2 (Thr202/Tyr204), LRP,p-LRP(Ser1490),Axin1,SRC,p-SRC(Tyr416),Snail,Twist,Slug,EGFR,vimentin,E-cadherin(CellSignalingTechnologyInc,Danvers,MA,USA),β-catenin,GSK3β,p-GSK3β (Ser9),EGFR,p-EGFR (Thr 669), p-mTOR(Ser2448), laminA/C (Epitomics, SanFrancisco,CA,USA),mTOR, p-β-catenin (Tyr333),AKT, p-AKT(Ser473),PKM2(SignalwayAntibody,Baltimore,USA),c-Myc, cyclinD1 (GeneTex, Irvine, CA,USA), andGAPDH(Sigma,StLouis,MO,USA).Allhorseradishperoxidase-conjugated secondary antibodies and thefluoresceinisothiocyanate(FITC)-conjugatedsecondaryantibodies were commercially obtained from CellSignalingTechnologyInc.(Danvers,MA,USA). Cell lines and culture HepG2,SMMC7721andMHCC-97-HcelllineswereobtainedfromtheCellBankofTypeCultureCollectionofChineseAcademyofSciences(Shanghai,China),andwere cultured inDulbecco’smodified eaglemedium(DMEM) orRPMI-1640medium (Invitrogen,GrandIsland,NY,USA)supplementedwith10%fetalbovineserum(FBS,Gibco,GrandIsland,NY,USA),100U/mlpenicillinand100μg/mlstreptomycin,andgrownina95%airand5%CO2humidifiedatmosphereat37°C.

Cell proliferation assays TheeffectofEGFoncellproliferationwasdeterminedusing CellTiter 96®AQueous One Solution CellProliferationAssay (MTS) (Promega,Madison,WI,USA).Briefly,HCCcells,seededin96-wellplates(3×104 cells/well),wereserum-starvedinDMEMfor24h,andthentreatedwithEGF(0-100ng/ml)for24h,followedbyincubationwiththeMTSreagent(20μl/well)for4h.

Finally,theopticaldensity(OD)wasmeasuredat490nmusingaSynergyTM2Multi-functionMicroplateReader(Bio-TekInstruments,Winooski,Vermont,USA).

Wound healing assays Cellswere seeded in 6-well plates andwere thensubjected to various treatments as indicated.Afterreaching 90% confluence, the cellmonolayerswerewoundedwithasterileplastic tipandwereculturedinserum-freeDMEMin thepresenceofEGF (0-100ng/ml).Migrationofcellsintowoundedareaswasevaluatedandphotographedunderaninvertedmicroscopeequippedwithadigitalcamera.

Transwell cell motility assays Cellmotilitywasalsoassessedusingan8-μmporesizeTranswellsystem(Corning,NY,USA).Briefly,serum-starvedcellswere trypsinized, resuspended inDMEM,andaddedtotheupperchamberin24-wellplates(1×105 cells/well)intheabsenceorpresenceofEGFand/ortheinhibitors at indicated concentrations,while the lowerchamber contained 600μl ofDMEMwith 10%FBS.Afterincubationfor24h,cellsthatmigratedtothelowersurfaceofthefiltermembranewerefixedinethanolandstainedwithcrystalviolet.Migratedcellswerecountedandphotographedunderaninvertedmicroscopeequippedwithadigitalcamera.

RNA isolation and quantitative real-time PCR (qRT-PCR) Total RNA was isolated using TRIzol reagent(Invitrogen,Carlsbad,CA,USA). First-strand cDNAwas synthesized with 1 μg of total RNA using aPrimeScriptRT reagentkit (TakaraBio,Tokyo, Japan).The qRT-PCRwas performed using IQTM SYBRGreen supermix and the iQ5 real-time detectionsystem (Bio-RadLaboratories,Hercules, CA,USA).Primers used for qRT-PCRwere as described in thefollowing (Zhang et al., 2013):E-cadherin: (forward)5’-TCCCATCAGCTGCCCAGAAA-3’, (reverse)3’-TGACTCCTGTGTTCCTGTTA-5’; Vimentin:(forward)5’-ATGAGTACCGGAGACAGGTGCAGA-3’;(reverse)5’-ATAGCAGCTTCAACGGCAAAGTTCA-3’;Snail:(forward)5’-TTCTTCTGCGCTACTGCTGCG-3’;(reverse) 3’-GGGCAGGTATGGAGAGGAAGA-5’;GAPDH:(forward)5’-TGGTATCGTGGAAGGACTCATGAC-3’;(reverse)3’-ATGCCAGTGAGCTTCCCGTTCAGC-5’. Reactionmixturescontained7.5μlofSYBRGreenIdyemastermix(Quanta),2pMofforwardandreverseprimers. Thermocycle conditions included initialdenaturationat50°Cand95°C(10mineach),followedby40cyclesat95°C(15s)and60°C(1min).mRNAabundancewasdeterminedby2-ΔΔCTmethod.Inthemeantime,thePCRproductsweresize-fractionatedon1.5%agarosegelsandvisualizedonaUVtransilluminator.

Western blotting analyses Total lysates from treated cellswere preparedwithRIPAbuffer[50mMTris,pH7.2;150mMNaCl;0.5%sodiumdeoxycholate;0.1%sodiumdodecylsulfate;1%Nonidet P-40; 10mMNaF; 1mMNa3VO4; protease



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Figure1.ActivationofEGFSignalingStimulatestheExpression ofEMTMarkers andCellMotility ofHCCCells.Serum-starvedHCCcellsweretreatedwithEGFatindicatedconcentrationsfor24h.(A)ExpressionofEGFRwasevaluatedinHCCcells linesofBel-7402,MHCC-97-H,SMMC-7721andHepG2byWesternblottinganalyses;(B)Cellmorphologywas assessed inHepG2andSMMC-7721 cells;ExpressionofEMTmarkersinSMMC-7721werereceptivelyanalyzedbyWesternblottinganalyses (C)andbyq-RT-PCR(D);HCCcellinvasionwasassessedinSMMC-7721bywoundhealing (E1 andE2) andby transwell invasion (F1 andF2);(G)EGF inhibitedHCCcells proliferation significantly in adosedependentmannerin24hours.(H)flowcytometriccellcycleanalysisshowedEGFtreatedfor24hoursdramaticallydecreasedtheproportionofcellsintheSorG2phase.GAPDHwasusedinWesternblottinganalysesasaninvariantcontrolforequalloading.Representativeswerefromthreeindependentexperiments.Valuesinassayswereexpressedasmean±SD.*P ≤0.05,**P≤0.01.NS=non-significant

inhibitor cocktail (1:1000, Sigma, St. Louis,MO)].Lysateswere sonicated for10 s andcentrifugedat14,000rpmfor10minat4°C.NucleiwereisolatedusingNE-PERnuclear and cytoplasmic extraction reagents(Pierce,Rockford,IL,USA)followingthemanufacturer’sprotocol. Protein concentration was determined bybicinchoninic acid assay with BSA as a standard(Pierce,Rockford, IL,USA). Equivalent amounts ofprotein(50μg/lane)wereseparatedon7.5%-12%SDS-polyacrylamide gel and transferred to polyvinylidenedifluoridemembranes(Millipore,Bedford,MA,USA).MembraneswereincubatedwithPBScontaining0.05%

Tween20and5%nonfatdrymilktoblocknonspecificbinding andwere incubatedwith primary antibodies,thenwith appropriate secondary antibodies conjugatedtohorseradishperoxidase. Immunoreactivebandswerevisualized by usingRenaissance chemiluminescencereagent(Perkin-ElmerLifeScience,Boston,MA,USA).

Knock-down protein expression by small interfering RNA (siRNA) Cellswere transfectedwithβ-cateninsiRNA,SnailsiRNA, PKM2 siRNAor non-specific control siRNA(SantaCruzBiotechnology,SantaCruz,CA,USA)usingthe opti-MEMplusX-tremeGENE siRNA transfectionreagent(Roche,Basel,Switzerland)preparedinantibiotic-freecompleteculturemedium.After24h,thecellswereused for experiments.The effect of each siRNAwasconfirmedbyWesternblotanalysesusingcorrespondingantibodies.

Dual luciferase reporter gene assays Cells in 96-well plateswere transiently transfectedwith0.1μg/wellof theTCF/LEF-responsiveluciferaseconstruct (Luc2P/TCF-LEF/Hygro,Promega,Madison,WI, USA) for 24 h. Transfection efficiencies werenormalizedwith renilla luciferase reporter plasmid.Relative promoter activity wasmeasured by dual-luciferase reporter assay systemusing theGlomax96MicroplateLuminometer(Promega,Madison,WI,USA)

Immunofluorescence (IF) staining Cells plated on glass coverslips in 6-well plateswereincubatedwith100ng/mlEGFand/orinhibitorsatindicatedconcentrationsfor24handwerethenfixedin4%paraformaldehyde for15min.Afterbeingblockedfor1hatroomtemperaturewith1%BSA,thecellswereincubated overnight at 4°Cwithmousemonoclonalantibodytoβ-catenin(1:500).FollowingwashingwithPBS for 3 times at room temperature, the cellswerefurther incubated with FITC-conjugated goat anti-mouseIgGandHoechst33342(Sigma)for1hatroomtemperature.Thecoverslipsweremountedtoglassslides,and the immunofluorescence stainingwas visualizedand photographed using aZeiss invertedfluorescencemicroscope.Negativecontrolstainingwasperformedbyomittingtheprimaryantibody.

Flow cytometry assay Cells treatedwith orwithout EGF for 24 hweredetachedwith trypsin andwashed in cold phosphatebufferedsaline(PBS).Forcellcycleanalysis,precipitatedcellswerefixedbycold70%ethanolof500μlovernightat-20°CandwashedincoldPBS.FixedcellswerethenincubatedwithRNase at 37°C for 30min and stainedwithpropidiumiodidefor15minatroomtemperatureindarkandimmediatelyanalyzedbyflowcytometry(FACSCalibur,BDBiosciences,SanJose,CA).

Statistical analysis Datawerepresentedasmean±SD,andresultswereanalyzedusingSPSS16.0software.Thesignificanceofdifferencewas determined by one-wayANOVAwith

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the post-hocDunnett’s test.Values ofP < 0.05wereconsideredtobestatisticallysignificant.

Results

EGF promotes EMT and cell migration in HCC cell lines EMTdescribesthededifferentiationswitchbetweenpolarizedepithelialcancercellsandcontractileandmotilemesenchymal(invasive)cellsduringcancerprogressionandmetastasis(Guarino,2007).WefirstexaminedEGFRexpression infourHCCcell lines, includingBel-7402,MHC-97-H,SMMC-7721andHepG2.ItappearedthatHepG2 andSMMC-7721 cellswere expressingmuchmoreEGFRthanMHCC-97andBel-7402cells(Figure1A).Of interest, treatmentwithEGF (25 and100ng/ml)for24hinducedaremarkablemorphologicalchange(fromcobble-stone-likeappearancetoelongatedleading-trailingmesenchymalshape,indicatingEMT)inHepG2and SMMC-7721 cells, and a weakmorphologicalchangeinMHCC-97cells(Figure1B).ThisobservationsuggestedthathigherexpressionofEGFRinHCCcellsmightbebettercorrelatedwiththedevelopmentofEMT.To further explore the role of the activation of EGFsignalinginthedevelopmentofEMT,theexpressionofagroupofmarkersforEMTwereexaminedinSMMC-7721 cells after treatmentwithEGF for 24 h.ResultsfromWesternblottinganalysesshowedthattheepithelialmarkerE-cadherinwasmarkedlydown-regulated,whilethemesenchymalmarkers vimentin and Snail weredramatically up-regulated after treatmentwithEGF inthe cells in a concentration-dependentmanner (Figure1C), though the expressionofTwist andSlugwasnotobviouslyaltered.Similarly,EGFalsoup-regulatedthemRNAlevelsofvimentinandSnail,anddown-regulatedthemRNAlevelofE-cadherin,asdetectedbyqRT-PCR(Figure1D). Additionalexperimentswereconducted toevaluatetheroleofEGFinregulatingcellmotility,anothermajorphenotype ofEMT. Itwas observed that bothwoundhealingassays(Figure1E1andE2)andTranswellassays(Figure 1F1 and F2) consistently demonstrated thattreatmentwithEGFfor24hstimulatedcellmotilityinaconcentration-dependentmanner inSMMC-7721cells.Takentogether,theseresultsindicatedthatactivationofEGFsignalingstimulatedtheexpressionofthemarkersofEMTandcellmotilityofHCCcells. ItisknownthatEGFcanstimulatecellproliferation.However, our current data showed that EGF did notincreaseHCCcellproliferationsignificantly(Figure1G).FurtherflowcytometryanalysesshowedthatEGFinducedcellcyclearrestatSphaseinHCCcelllines(Figure1H).Takentogether,theseresultsindicatedthatEGFinducedEMTandenhancedcellmotilitywithin24hinHCCcelllines,whileitcannotpromoteHCCcellproliferationinthesamestimulatingtime.

β-Catenin mediates EGF-induced EMT and cell motility in SMMC-7721 β-Catenin has been found to be co-activatedwithEGFRinHCC(Llovetetal.,2008).Todeterminewhetherβ-cateninmediatesEMTand cellmotility, expression

Figure2.β-CateninMediatesEGF-inducedEMTandCellMotility inHCCCells.For inhibition ofβ-cateninexpressionbysiRNA,cellswerefirsttransfectedwithβ-cateninsiRNAor nc-siRNA.After 24h recovery, cellswere treatedwithEGFat indicatedconcentrations for anadditional24h.GAPDHorLamA/CwasusedinWesternblottinganalysesasaninvariantcontrolforequalloading.Representativeswerefromthreeindependentexperiments.Valuesinassayswereexpressedasmean±SD.*P≤0.05,**P≤0.01.NS=non-significant.(A)Westernblottinganalysesofβ-cateninexpressioninHCCcellsaftertransienttransfectionwithβ-cateninsiRNAornc-siRNA;(B)EvaluationofeffectsofEGFoncellmorphology(×400)aftertransienttransfectionwithβ-cateninsiRNAornc-siRNA;DeterminationoftheroleofEGFinregulationofexpressionof proteinmarkers ofEMTafter transient transfectionwithβ-cateninsiRNAbyWesternblottinganalyses(C)andq-RT-PCR assays (D); (E) Evaluation of effects of EGF on cellinvasionofSMMC-7721cellsbytranswellinvasionassaysaftertransienttransfectionwithβ-cateninsiRNA;(F)SMMC-7721cellswerefirsttransfectedwithβ-cateninsiRNAornc-siRNA,pGL4.49[luc2p /TCF -LEFRE/Hygro] and internal controlplasmidfor24h,thencellsweretreatedwithEGFatindicatedconcentrationsforadditional24h,dualluciferasereportergenewasusedtoaddresstheeffectofEGFoninductingofβ-catenintranscriptionalactivityinHCCcells.SMMC-7721cellsweretreatedwith EGF at indicated concentrations for indicatedtime,(G)Westernblotand(H)IFwereusedtoexploretheeffectofEGFsignalingoninducingβ-cateninnucleartranslocationinHCCcelllines.Representativeswerefromthreeindependentexperiments.NE:nuclearextracts,WCL:whole-celllysates



of β-cateninwas silenced by siRNA in SMMC-7721cells.ItwasobservedthatsiRNAsignificantlyanddose-dependently inhibited the expression of β-catenin inSMMC-7721cells(Figure2A).Thedown-regulationofβ-cateninbysiRNAeffectivelyabrogatedEGF-inducedEMTmorphological change in the cells (Figure 2B).Further experiments observed thatEGF failed to altertheexpressionoftheEMTmarkersatproteinandmRNAlevelsinβ-catenin-silencedHCCcellsdemonstratedbyWestern blotting analyses (Figure 2C) and qRT-PCRanalyses(Figure2D).Consistently,thedown-regulationof β-catenin by siRNAdramatically suppressedEGF-stimulatedcellmotility(Figure2E).Theseobservationscollectivelyindicatedthatβ-cateninplayedacriticalroleinmediatingEGF-inducedEMTandmotilityinHCCcell.As β-catenin regulates EMT and tumormetastasisusually by enhancing transcriptional activity of thetranscriptionfactorTCF-LEF(Fangetal.,2007;Jietal.,2009;Luet al., 2003),we investigated it is, therefore,plausible to elucidatewhetherEGFup-regulatesTCF-

LEFtranscriptionalactivityinHCCcells.SMMC-7721cellswere transfectedwithaβ-catenin trans-activationactivity luciferase reporter plasmidLuc2P/TCF-LEF/Hygro.After recovery, cellswere treatedwithEGFatvariousconcentrationsfor24h.Asshownin(Figure2F)by luciferase assays,EGFdose-dependently increasedβ-catenin-TCF-LEF trans-activation demonstrated byelevating luciferase activities in SMMC-7721 cells.Further experiments confirmed that EGF induced theaccumulation of nuclear β-catenin in the cells aftertreatmentwithEGFatindicatedconcentrations,indicatedbyWesternblottinganalyses(Figure2G)andbyimmuno-fluorescent staining (Figure 2H).Taken together, ourresults indicatedthatβ-CateninmediatedEGF-inducedEMTandcellmotilityinHCCcells.

Both PI3K and ERK pathways are involved in EGF-induced β-catenin-TCF-LEF transactivation, EMT and cell motility Wnt-dependent and -independentmechanismshave

Figure3.BothPI3KandERKPathwaysareInvolvedinEGF-inducedβ-catenin-TCF-LEFTransactivation.ForDualluciferasereporterassay,cellswerefirsttransfectedwithpGL4.49[luc2p/TCF-LEFRE/Hygro]andinternalcontrolplasmidfor24h,thencellsweresubjectedtodifferenttreatmentsdescribedineachpanelsbelow.(A1andA2)SMMC-7721cellswastreatedwithEGFatindicatedconcentrationsfor24h,WesternblotwasusedtoinvestigatetheeffectofEGFontheexpressionofrelatedWntpathwayproteins.SMMC-7721cellswerepretreatedwithWntinhibitor(rhDKK)atindicatedconcentrationsfor30min,thentreatedwithEGFatindicatedconcentrationsfor24h,(B1)Westernblotand(B2)dualluciferasereportergeneassayeswereusedtoinvestigatetheeffectofWntinhibitoronEGF-inducedβ-catenintransactivation.(C)SMMC-7721cellsweretreatedwithEGFatindicatedconcentrationsfor9h,WesternblotwasusedtoinvestigatetheeffectofEGFonphosphorylationlevelofβ-cateninatTyr333andSer552.(D1andD2)SMMC-7721cellswerepretreatedwithspecificinhibitorsofPI3K(LY294002),mTOR(rapamycin),SRC(SKI-1),FAK(Y15)andERK(U0126)atindicatedconcentrationsfor30min,respectively,thentreatedwithEGFatindicatedconcentrationsfor24h,LuciferasereportergeneassaywasusedtoaddresstheroleofaboveinhibitorsinEGF-inducedβ-catenintransactivation.SMMC-7721cellswerepretreatedwithspecificinhibitorsofPI3K(LY294002),andERK(U0126)atindicatedconcentrationsfor30min,respectively,thentreatedwithEGFatindicatedconcentrationsfor24h,(E1,E2,E3andE4)Westernblotand(F)IFassayswereusedtoexploretheroleofPI3KandERKsignalsinEGF-inducedβ-cateninnucleartranslocation.GAPDHandLamA/Careloadingcontrols.Representativeswerefromthreeindependentexperiments.NE:nuclearextracts,WCL:whole-celllysates.Dataareexpressedasmean±SD.*P≤0.05,**P≤0.01,***P≤0.001,NS=non-significant

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beenobservedtobeinvolvedinEGF-inducedβ-catenin-TCF-LEFtransactivation(Huetal.,2010).However,the

roleofWntpathwayinHCCcellsisunclear.Toaddressthisissue,SMMC-7721cellsweretreatedwithEGFfor

Figure4.BothPI3KandERKPathwaysareInvolvedinEGF-inducedEMTandCellMotility.SMMC-7721cellswerepretreatedwithspecificinhibitorsofPI3K(LY294002),mTOR(rapamycin),SRC(SKI-1),FAK(Y15)orERK(U0126)atindicatedconcentrationsfor30min,respectively,followedbythetreatmentwithEGFatindicatedconcentrationsfor24h.(AandB)Westernblottinganalyses,(C)q-RT-PCRand(G)cellmorphology(×400)assayswereusedtoinvestigatetheeffectofaboveinhibitorsonEGF-inducedHCCEMT;(D,EandF)transwellassayswereusedtoaddresstheeffectofaboveinhibitorsonEGF-inducedHCCcellmotility.GAPDHwasusedinWesternblottinganalysesasaninvariantcontrolforequalloading.Representativeswerefromthreeindependentexperiments.Valuesinassayswereexpressedasmean±SD.*P≤0.05,**P≤0.01,***P≤0.001,NS=non-significant

Figure5.ERK1/2-dependentNuclearTranslocationofPKM2Regulatesβ-catenin-TCF-LEFTranscriptionalActivity,EMTandMotilityinHCCCells.FortransienttransfectionwithsiRNAs,cellswerefirsttransfectedwith160nMPKM2siRNAor160nMnc-siRNAfor24h,thencellsweresubjectedtodifferenttreatmentsdescribedineachpanelsbelow.(A)SMMC-7721cellsweretreatedwithEGFatindicatedconcentrationsfor9h,WesternblotassaywasusedtoaddresstheeffectsofEGFontheexpressionofPKM2.(B1)WesternblotanalysisshowedthatPKM2wasknockeddowninHCCcells.(B2)ForLuciferaseassay,SMMC-7721cellswereco-transfectedwithpGL4.49[luc2p/TCF-LEFRE/Hygro]andinternalcontrolplasmidfor24h,thencellsweretreatedwithEGFatindicatedconcentrationsforadditional24h,LuciferasereportergeneassaywasusedtoexploretheroleofPKM2inEGF-inducedTCF-LEFtranscriptionalactivityinHCCcells.(C1andC2)SMMC-7721cellswerepretreatedwithPI3K,ERKinhibitorsfor30min,respectively,thentreatedwithEGFatindicatedconcentrationsfor9h.WesternblotwasusedtoinvestigatetheroleofPI3KandERKsignalsinEGF-inducednucleartranslocationofPKM2inHCCcells.(D)Westernblot,(E)q-PCR,(F)cellmorphology(×400)assayswereusedtoinvestigatetheroleofPKM2inEGF-inducedEMTinHCCcells;(G)TranswellanalysiswasusedtoinvestigatetheroleofPKM2inEGF-inducedmotilityinHCCcells.Representativeswerefromthreeindependentexperiments.GAPDHandLamA/Careloadingcontrols.NE:nuclearextracts,WCL:whole-celllysates.Dataareexpressedasmean±SD.*P≤0.05,**P≤0.01,***P≤0.001,NS=non-significant



24h, followedbyWesternblottinganalysesandTCF-LEF luciferase reporter gene analyses.We found thatEGFdid not obviously alter the expression ofAxin1,frizzled,NKD2,p-GSK3β,GSK3β,p-LRPandLRPinthe cells (Figure3A1andA2).Furthermore, treatmentwithrhDKK-1(100-200ng/ml),aselectiveWntinhibitor,for24h,didnotpreventEGF-inducedβ-cateninnucleartranslocation(Figure3B1)andTCF-LEFtranscriptionalactivity (Figure 3B2).The results suggested thatEGFup-regulatedβ-catenintranscriptionalactivityviaaWnt-independentmechanism. Next,weaskedwhetherEGFaffectedphosphorylationof β-catenin,which has been shown to be positivelycorrelatedwithitsnucleartranslocationandindependentofWntsignaling(Fangetal.,2007;Yangetal.,2011).Interestingly, treatmentwith EGF for 24 h inducedphosphorylationofβ-cateninatSer552andTyr333 in

aconcentration-dependentmannerinSMMC-7721cells(Figure3C).ThisobservationsupportedthehypothesisthattheactivationofEGFRresultedinphosphorylationof β-catenin, leading toWnt-independent nucleartranslocationofβ-catenininHCCcell. Several signaling pathways, includingPI3K/AKT,mTOR,SRC,FAK,andERKpathways,havebeenfoundto be involved in β-catenin-TCF-LEF transactivation(Fang et al., 2007; Ji et al., 2009;Yang et al., 2011).To definewhich pathway contributes toEGF-inducedtranscriptional activity ofβ-catenin, specific inhibitorsofPI3K(LY294002),mTOR(rapamycin),SRC(SKI-1),FAK(Y15)andERK(U0126)wereutilized.Wefoundthat treatmentwithLY294002 (2.5-5μM) andU0126(5-2.5 μM), but not SKI-1 (20-40 nM), rapamycin(20-40nM), orY15 (5μM), for 24h, inhibitedEGF-inducedTCF-LEFluciferaseactivityinaconcentration-dependentway(Figure3D1-D2).Furthermore,treatmentwith LY294002 for 9 h concentration-dependentlyalso attenuatedEGF-inducednuclear translocation andphosphorylation of β-catenin (Ser552) (Figure 3E1).Tooursurprise, treatmentwithU0126, likeSKI-1andrapamycin,didnotobviouslyaffectEGF-inducednucleartranslocationandphosphorylationofβ-catenin(Ser552)(Figure3E2-E4).Moreover,immunofluorescencestainingshowedthatLY294002notonlypreventedEGF-inducedβ-cateninnuclear translocation,butalsoblockedEGF-inducedmorphological change in SMMC-7721 cells(Figure3F).Unexpectedly,U0126blockedEGF-inducedmorphologicalchange,butdidnotaffectEGF-inducedβ-cateninnuclear translocation (Figure3F), suggestingthatanothermoleculeregulatedbyERK,togetherwithβ-catenin,might co-regulate EGF-inducedTCF-LEFtranscriptionalactivityinHCCcells.Thefindingsrevealedthat PI3K and ERK, but not SRC,mTOR and FAKpathways,mediatedEGF-inducedβ-catenin-TCF-LEFtransactivationinHCCcells. To further demonstrate the role of PI3K andERKpathwaysinEGF-inducedEMTandcellmotility,SMMC-7721cellswerepretreatedwithLY294002andU0126for 30min, followedby stimulatingwithEGF for 24h.AsshowninFigure4,EGFup-regulatedexpressionof vimentin andSnail and down-regulated expressionofE-cadherin in the cells at both protein andmRNAlevels (Figure 4A-C). PretreatmentwithLY294002orU0126attenuatedtheeffectofEGFontheexpressionofvimentin,SnailandE-cadherin(Figure4A-C).Similarly,LY294002orU0126alsoattenuatedEGF-stimulatedcellmotility(Figure4Dand4E).Inaddition,wenoticedthatthe inhibitors ofSRC,FAK,mTORorWnt under thesame condition did not obviously affectEGF-inducedcellmorphologicalchange(Figure4G)andcellmotility(Figure4F).Collectively,ourobservationsindicatedthatEGF increased β-catenin transcriptional activity, andinducedEMTandcellmotilitybyactivatingPI3KandERKpathwaysinHCCcells.

ERK1/2-dependent nuclear translocation of PKM2 regulates β-catenin-TCF-LEF transcriptional activity, EMT and motility in SMMC-7721 To test whether ERK1/2-dependent nuclear

Figure6.SnailisCriticalforEGF-inducedEMTandMotilityinHCCCells.ForinhibitionofSnailexpressionbysiRNA,SMMC-7721cellswerefirsttransfectedwithSnailsiRNAornc-siRNA.After24hrecovery,cellsweretreatedwithEGF(100ng/ml)foranadditional24h.(A)WesternblottinganalysesshowedthatSnailwasknockeddowninHCCcells;(B)Westernblottinganalysesand(C)q-RT-PCRand(D)cellmorphology(×400)assayswereusedtoinvestigatetheroleofSnailinEGF-inducedEMTinHCCcells;(E)TranswellassaywasusedtoaddresstheroleofSnailinEGF-inducedmotilityinHCCcells.(F)Westernblotanalysesshowedthatc-mycandcyclinD1wasdecreaseddose-dependentlyafter treatingwithindicatedconcentrationsofEGFinSMMC-7721andHepG2celllines.GAPDHwasusedinWesternblottinganalysesasaninvariantcontrolforequalloading.Representativeswerefromthreeindependentexperiments.Valuesinassayswereexpressedasmean±SD.*P≤0.05,**P≤0.01.NS=non-significant

Fang-Tian Fan et al


translocation of PKM2 could regulate EGF-inducedβ-catenintransactivationinHCCcells,SMMC-7721cellsweretreatedwithEGF(5-100ng/ml)for9h,followedbynuclearextraction.WefoundthatEGFelevatednuclearPKM2 level in a concentration-dependentmanner, butdidnotobviouslyaffectthetotalcellularlevelofPKM2(Figure 5A).At 25 ng/ml, EGFwas able to inducesignificantexpressionofPKM2innuclei. To validatewhetherPKM2was really involved inEGF-inducedTCF-LEF transactivation, PKM2wassilencedbyusingsiRNAintheHCCcells.Wefoundthatdown-regulationofPKM2bysiRNAalmostcompletelyabolishedEGF-inducedTCF-LEFtranscriptionalactivity(Figure5B1-B2).Interestingly,U0126,butnotLY294002,preventedEGF-inducednucleartranslocationofPKM2(Figure5C1-C2).TheresultssupportedtheconclusionthatERK,butnotPI3K,regulatedEGF-inducedPKM2nucleartranslocationandβ-catenin-TCF-LEFtransactivationinHCCcells. In addition,we found that the down-regulation ofPKM2bysiRNApotentlyinhibitedEGF-inducedchangesofEMTmarkers,asdetectedbyWesternblottinganalyses(Figure5D)andqRT-PCR(Figure5E), aswell as cellmorphology inSMMC-7721 cells (Figure 5F). In linewith the abovefindings, silencingPKM2alsoblockedEGF-stimulatedcellmotility(Figure5G).Therefore,theresultscollectivelyindicatedthatPKM2wasessentialforEGF-inducedEMTandcellmotilityinHCCcells.

Snail is critical for EGF-induced EMT and motility in SMMC-7721 Snail isa transcriptionfactor thatcontrolsEMTbyinhibitingE-cadherinexpression(Canoetal.,2000).ToexploretheroleofSnailinEGF-inducedEMTandmotilityinHCCcells,SnailwassilencedbysiRNAinSMMC-7721cells.Wefoundthatthedown-regulationofSnailbysiRNAalmostcompletelyabrogatedEGF-inducedup-regulationofvimentinanddown-regulationofE-cadherinat both protein (Figure 6A andB) andmRNA levels(Figure6C).Furthermore,silencingSnailalsopreventedEGF-inducedmorphologicalchange(Figure6D),aswellasEGF-stimulatedmotilityinthecells(Figure6E).TheresultssupportedthenotionthatSnailplayedacrucialroleforEGF-inducedEMTandcellmotilityinHCCcells.

Discussion

AdvancedandmetastaticHCCisahighlydevastatingdiseasewith limited and largely ineffective treatmentoptions (Eggert et al., 2013;Mittal et al., 2013).Consequently, there is still a great need for a betterunderstanding of the underlying biology of advancedHCCandtheeffortstowardsfindingeffectivemolecular-targetedtherapyshouldbeintensified.HCCisassociatedwithahighpotentialforvascularinvasion,metastasisandrecurrenceevenaftersurgicalresection,leadingtopoorprognosis(Altekruseetal.,2012).Animportantbiologicalprocessthathasbeenshowntounderlietumorprogressionandmetastasis isEMT(Guarino,2007).Recently, it iswidely accepted thatEMT is the initiate step of intra-

hepatic and extra-hepatic (Maheswaran et al., 2012).Dysregulationofgrowthfactorpathwayshasbeenthoughtto be correlatedwith thepoor prognosis ofHCC.ThegrowthfactorsreportedtopromoteEMTinHCCincludeTGF-β,PDGFandHGF(Xuetal.,2003;Fischeretal.,2007;Ogunwobi et al., 2011).Recent evidence showsthatEGFRisoverexpressedin40-70%ofHCC,anditsactivationisinvolvedinHCCpathogenesisandprognosis(Buckleyetal.,2008),butfewreportsareseentoaddresstheroleofEGFRsignalingininductionofEMTinHCCcells.Thus,ourpresentinvestigationsweredesignedtoexplorewhetherEGFRactivationcouldinduceEMTinHCCcelllinesandtouncovertheunderlyingmechanism.

EGFRisaRTKthatisoverexpressedinawidevarietyof human carcinomas, including non-small cell lung,breast,headandneck,bladder,ovarian,prostatecancer,andHCC,andithasbeenassociatedwithanumberofadvanceddiseasesandpoorprognosis(Liuetal.,2002).In addition, it iswell-known that EGFR is not onlyimportantforcellproliferation,butalsoforanumberofvariedprocesseslikelytobesignificantforcarcinomasprogressionsuchascelladhesion,migrationandinvasion,whicharemajorstepsintheEMTevent(AlMoustafaetal.,2012).OurpresentdatashowedthatEGFandresultantEGFR activation could promoteEMT and associatedmigrationinHCCcelllines.Theseresultswereconsistentwith theprevious reports thatEGFcould induceEMTandmigrationinsomeothercancercells.Interestingly,theproliferativepropertyofEGFwasnot observed inourexperiments.Incontrast,wefoundthatEGFcausedcellcyclearrestintestedHCCcelllines.Actually,theseobservationshavebeenrecapturedin theEMTprocessinducedbyhypoxiaandTGF-βinHCC.However, thedifferences are that hypoxia andTGF-β induced cellcyclearrestatG1phaseinHCC,whileEGFcausedcellcyclearrestatSphase.ThisimpliedthatEGFwaslikelytohavemechanismsdifferentfromhypoxiaandTGF-βininductionofcellcyclearrestinHCCcelllines,whichisawaitedtoberevealedfurther.

Previous reportsdemonstrated thatEGFcould leadto disruption of cell-cell junctions and promotion ofinvasivenessviathephosphorylationoftheE-cadherin/β-catenin complex, thus resulting in dissociation of thelatter and functional loss ofE-cadherin.These eventscouldinturnreleaseβ-cateninandintothecytoplasmandthenintothenucleus,whereitactsasaco-activatorofT-cellandlymphoidenhancerfactors(TCF/LEF)inthetranscriptionalactivationoftargetgenes(suchasc-myc,snail-familymembers,cyclinD1andVimentin)involvedin proliferation, invasion and tumorprogression (Fangetal.,2007;Jietal.,2009).RecentstudiesshowedthatEGFpromotedglioblastomacancercellproliferationbyincreasingtheexpressionofc-mycandcyclinD1,Whichwereregulatedbythelevelofβ-cateninphosphorylationatTyr333residue(Yangetal.,2011).Ourcurrentresultssuggested that PI3K could phosphorylateβ-catenin atSer552residueinaGSK3β-independentwayuponEGFRactivation,which upregulated the expression of snailandVimentin,leadingtoHCCcellEMTandmigration.Interestingly,decreasedexpressionofc-mycandcyclin



D1werealsoobservedinpresentstudy(Figure6F).Thisreasonmaybethatdifferentdosagesandtreatingtimeofhavedifferent effectson theproliferationof somecelllines,

PKM2isakeyglycolyticenzymethatregulatestheWarburgeffectandisnecessaryfortumorgrowth(Wongetal.,2013).RecentstudiesindicatedthatnuclearPKM2couldactivategenetranscriptionandcellproliferationinhumanglioblastoma,prostatecancerandbreast cancercells(Gaoetal.,2012;Leeetal.,2008;Luoetal.,2011a;Luoet al., 2011b).Hereweobserved its role inEGF-inducedEMT and cellmotility inHCC cells.RecentclinicalinvestigationshavedemonstratedthatPKM2isup-regulatedincolorectalcancerandcorrelatedwithcoloncancermetastasis(Zhouetal.,2012a),butthemechanismis unknown.The findings from this study and othersstronglysuggestthatPKM2playsanimportantrolenotonlyincellproliferationbutalsoinEMT/cellmotility,contributingtotumormetastasis.

PreviousstudieshavedocumentedthatPDGFsignalingcould result in the nuclear accumulation of β-catenin,leadingtoEMTandcellinvasioninHCCcells(Fischeretal.,2007).Besides,HGF/c-metpathwaywasfoundtopromoteEMTviaupregulationofcyclooxygenase2andPI3K(Panetal.,2010).Recently,ithasbeenreportedthatWnt/β-cateninsignalingcouldenhancehypoxia-inducedEMT inHCC via crosstalk with hypoxia-induciblefactor-1α(HIF-1α)(Zhangetal.,2013).Inthepresentstudy, we demonstrated that EGF induced EMT byactivatingPI3KandERKpathways inHCCcell lines.OurresultsalsoindicatedthatPI3KandERKpathwayscouldupregulatenuclearlevelsofβ-cateninandPKM2,respectively,andthatthetwopathwayscouldco-regulateβ-catenin-TCF/LEFtranscriptionalactivityandEMTinHCCcelllines.Therefore,β-catenin,HIF-1αandPKM2could be keymolecules involved in theEMTprocessinHCC. Interestingly, there have beenmany reportsdemonstratingtheinteractionsbetweenthesemolecules,whichwereimportantfortumorproliferation,invasionandprogression(Luoetal.,2011a;Yangetal.,2011;Zhangetal.,2013).ItcouldbepostulatedthattheEMTprocessinducedbydifferentfactorsinHCCcouldbeattributedtosomecommonmechanismsinvolvingβ-catenin,HIF-1αandPKM2simultaneously. Inaddition,ourcurrentstudiesmightindicatethatβ-catenin,HIF-1αandPKM2could bemarkers of EMT and prognostic factor inHCC.InterventionoftheirinteractionsmightbeanewtherapeuticstrategyforHCC.

Clinicalevidencehas revealed thatEGFRcouldbeamolecular target for tumor therapy, and that aboutone-third of patients with advaced HCC exhibitedactivationofWntpathway,mostlyasaresultofβ-catenintransactivation(Guanetal.,2012;Nejak-Bowenetal.,2011).Inaddition,co-activationofEGFRandβ-cateniniscommonlyseeninpatientswithHCC(Llovetetal.,2008).Some reports have demonstrated thatWnt/β-cateninactivation could induce theEGFR activation inHCC(Tanetal.,2005).Interestingly,inthepresentstudy,weshowedthatEGFRactivationinducedβ-cateninactivationinHCC cell lines, highlighting the crosstalk betweenEGFRandWntpathways,whichcouldbeimportantfor

HCCtumorigenesis,progressionandmetastasis.Previousstudies indicated that there could be some potentialconvergent points betweenWnt andEGFRpathways,presumably including Frizzled, β-catenin andNKD2(Huetal.,2010).HereinweshowedthatEGFRsignalingcrosstalkedwithWntpathwayviaPI3Kphosphorylationofβ-catenininHCCcells.ItthereforecouldbepostulatedthatinhibitionoftheseconvergentpointsorcombinationdisruptionofWnt andEGFRpathwayscouldbemoreeffectivefortherapeuticoptionsforHCC.

Insummary,thepresentstudyrevealedthatnucleartranslocationofPKM2, regulatedHCCcellsEMTandmigration in vitro.These discoveries provided novelroleofPKM2intheprogressionofHCCandpotentialtherapeutictargetforadvancedHCC.

Acknowledgements

Thisworkwas supported by theNationalNaturalScience Foundation of China (No. 81173174 and81202655);theNationalKeyTechnologyR&DProgram(No. 2008BAI51B02); Ph.D. ProgramsFoundation ofMinistryofEducationofChina(No.20113237110008).

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RESEARCH ARTICLE PKM2 Regulates Hepatocellular Carcinoma