Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines

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Title:
Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines
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English
Creator:
Abe, Masako
Havre, Pamela A.
Urasaki, Yasuyo
Ohnuma, Kei
Morimoto, Chiko
Dang, Long H.
Dang, Nam H.
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Bio Med Central (BMC Cancer)
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Background: CD26 (dipeptidyl peptidase IV, DPPIV) is a 110 kDa surface glycoprotein expressed in most normal tissues, and is a potential novel therapeutic target for selected cancers. Our work evaluates the mechanism involved in confluence-dependent CD26 expression in colon cancer. Methods: Colon adenocarcinoma cells were grown to confluence, and expression of CD26 and transcription factors implicated in its regulation was confirmed by immunofluorescence and Western blotting. Real-time PCR was also performed to evaluate CD26 upregulation at the transcriptional level. The influence of c-Myc on CD26 expression during different growth conditions was further evaluated following transient transfection of a c-Mycexpressing plasmid and a c-Myc specific siRNA. Results: We found that the colon cancer cell lines HCT-116 and HCT-15 exhibited a confluence-dependent increase in CD26 mRNA and protein, associated with decreased expression of c-Myc, increased USF-1 and Cdx 2 levels, and unchanged HNF-1a expression. Meanwhile, ectopic expression of c-Myc in both cell lines led to decreased CD26 expression. In contrast, transfection of a siRNA targeted to Cdx2 resulted in decreased CD26 level. Importantly, culturing of cells in serum-depleted media, but not acidic conditions, upregulated CD26. While HIF-1a level also increased when cells were cultured in serum-depleted media, its expression was required but not sufficient for CD26 upregulation. Conclusions: CD26 mRNA and protein levels increase in a confluence-dependent manner in colon carcinoma cell lines, with c-Myc acting as a repressor and Cdx2 acting as an enhancer of CD26 expression. The enhanced expression of CD26 in serum-depleted media and a requirement for HIF-1a suggest a role for nutrients or growth factors in the regulation of CD26 protein expression.
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Abe et al. BMC Cancer 2011, 11:51 http://www.biomedcentral.com/1471-2407/11/51; Pages 1-10
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doi:10.1186/1471-2407-11-51 Cite this article as: Abe et al.: Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines. BMC Cancer 2011 11:51.

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© 2011 Abe et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCHARTICLE OpenAccessMechanismsofconfluence-dependentexpression ofCD26incoloncancercelllinesMasakoAbe1,PamelaAHavre2,YasuyoUrasaki1,KeiOhnuma3,ChikaoMorimoto3,LongHDang2,NamHDang2*AbstractBackground: CD26(dipeptidylpeptidaseIV,DPPIV)isa110kDasurfaceglycoproteinexpressedinmostnormal tissues,andisapotentialnoveltherapeutictargetforselectedcancers.Ourworkevaluatesthemechanism involvedinconfluence-dependentCD26expressionincoloncancer. Methods: Colonadenocarcinomacellsweregrowntoconfluence,andexpressionofCD26andtranscription factorsimplicatedinitsregulationwasconfirmedbyimmunofluorescenceandWesternblotting.Real-timePCR wasalsoperformedtoevaluateCD26upregulationatthetranscriptionallevel.Theinfluenceofc-MyconCD26 expressionduringdifferentgrowthconditionswasfurtherevaluatedfollowingtransienttransfectionofac-Mycexpressingplasmidandac-MycspecificsiRNA. Results: WefoundthatthecoloncancercelllinesHCT-116andHCT-15exhibitedaconfluence-dependent increaseinCD26mRNAandprotein,associatedwithdecreasedexpressionofc-Myc,increasedUSF-1andCdx2 levels,andunchangedHNF-1 a expression.Meanwhile,ectopicexpressionofc-Mycinbothcelllinesledto decreasedCD26expression.Incontrast,transfectionofasiRNAtargetedtoCdx2resultedindecreasedCD26level. Importantly,culturingofcellsinserum-depletedmedia,butnotacidicconditions,upregulatedCD26.WhileHIF-1 a levelalsoincreasedwhencellswereculturedinserum-depletedmedia,itsexpressionwasrequiredbutnot sufficientforCD26upregulation. Conclusions: CD26mRNAandproteinlevelsincreaseinaconfluence-dependentmannerincoloncarcinomacell lines,withc-MycactingasarepressorandCdx2actingasanenhancerofCD26expression.Theenhanced expressionofCD26inserum-depletedmediaandarequirementforHIF-1 a suggestarolefornutrientsorgrowth factorsintheregulationofCD26proteinexpression.BackgroundCD26/dipeptidylpeptidaseIVisa110kDasurfaceglycoproteinwithdiversefunctionalpropertieswhich includepeptidaseactivity andfunctionalandphysical associationwithkeymoleculesinvarioussignaltransductionpathways[1,2].Duringthepastdecades,CD26 hasbeenshowntoparticipateinT-cellbiologyasa costimulatorymoleculeabletoregulatesignalingtransductionpathways[3-5].InadditiontoitsroleinT-cell biology,recentstudieshaveshownthatCD26alsoplays animportantroleintumorbiology. Furthermore,CD26itselfmaybeanoveltherapeutic target.Anti-CD26monoclonalantibody(mAb)treatment resultsinboth invitro and invivo antitumoractivity againstseveraltumortypes,includinglymphoma, mesotheliomaandrenalcellcarcinoma[6-8]. However,CD26expressionvariesamongdifferentcell linesandtumorhistologies,resultinginconflicting reportsregardingtheroleofCD26inthedevelopment ofdifferentneoplasms,includingcoloncancer[1].For example,CD26isupregulatedduringenterocyticdifferentiationofthecolonadenocarcinomacelllinesCaco-2 andHT-29[9].Interestingl y,trypsinizingandseeding latepost-confluentCaco-2cellsledtoreducedCD26 expressionanddipeptidylpeptidaseIVactivity,aswell asthedisappearanceofotherdifferentiationmarkers suchassucraseisomaltaseandGLUT5[10].However, theprecisemechanismresponsibleforthiscelldensitydependentregulationofCD26expressionanddipeptidyl peptidaseIVactivityhasnotbeenfullyelucidated. *Correspondence:nam.dang@medicine.ufl.edu2DivisionofHematology/Oncology,UniversityofFlorida,Gainesville,FL 32610,USA FulllistofauthorinformationisavailableattheendofthearticleAbe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 2011Abeetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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Comparedwithmostnormaltissues,solidtumorstend toexhibithighcelldensityduetorapidgrowth,and highcelldensityhasbeenlinkedtoanti-cancerdrug resistance[11,12].InviewoftheemergingroleofCD26 asanoveltherapeutictarget,weinvestigatedthe mechanismofconfluence-dependentexpressionof CD26tofurtherunderstandCD26biology. Inthispaper,weinvestigatedthemechanisminvolved intheconfluence-dependentincreaseinCD26expressioninthecoloncancercelllinesHCT-116and HCT-15.Wedemonstratedthatc-Mycisinvolvedin theregulationofCD26expressionasarepressorof transcriptioninbothcelllines,andthatCdx2significantlycontributestotheconfluence-dependentincrease inCD26expressioninHCT-15cells.Furthermore,we foundthatserum-depletedcultureconditions,butnot acidicconditions,significantlyupregulatedCD26levels, henceimplicatingapotentialrolefornutrientsorserum growthfactorsintheregulationofCD26protein expression.MethodsCellcultureThehumancolorectalcarcinomacelllinesHCT-116 andHCT-15werepurchasedfromtheAmericanType CultureCollection(ATCC,Manassas,VA).HCT15, HCT-116,andHCT-116HIF1 a -/-cells[13]weremaintainedinMcCoy s5Amedium(ATCC)supplemented with10%fetalbovineserum(FBS)(Hyclone,South Logan,Utah),100units/mlofpenicillinand100 g/ml ofstreptomycin(Invitrogen)at37Cand5%CO2.For confluence-dependentexperiments,HCT-116cellsor HCT-15cellswereseededat7.5105cells/6-cmdish and1.0106cells/6-cmdish(day-0),respectively,and weregrownseveraldaystoobtainapost-confluent state.Mediumwaschangeddaily.Forimmunofluorescencestaining,cellsweregrownonaglassslidein RPMI-1640(Hyclone)withoutphenolredandsupplementedwith10%FBS,100units/mlofpenicillin,and 100 g/mlofstreptomycinat37C.Cellswerecultured atsub-confluenceorpost-confluencebychangingmediumdaily.Fornormoxiccultureconditions,cellswere incubatedat37C,in5%CO2androomair(21%O2). Forhypoxicconditions,cellswereincubatedat37Cin ahypoxiachamber(Billups-Rothenberg,DelMar,CA) flushedandequilibratedwith1%O2,94%N2and5% CO2(AirgasWest,LasVegas,NV).Foracidicculture conditions,cellswereculturedinMcCoy s5Amedium supplementedwith25mM2-N-morpholineethanesulfonicacid(MES,Sigma,StLouis,MO)and10%FBS (pH6.2)undernormoxicorhypoxicconditions.For hypoxicoracidiccultureconditions,cellswereusedat 3dayspost-confluence,andgrownonthebottomofa standingflask(25cm2)filledwith50mlofculture mediumtoavoidanychangeinpHorserumconcentration.Forserumdepletionexperiments,cellswereculturedinMcCoy s5Amediumcontaining0.3%bovine serumalbumin(BSA,Sigma).WesternblotsCellswerelysedwithRIPAbuffer[1%NP40,0.5%deoxycholate,0.1%SDS,150mMsodiumchloride,50mM Tris-HCl(pH8.0)]supplementedwithHaltprotease inhibitorcocktail(Pierce,Rockford,IL),5mMEDTA, 5mMsodiumorthovanadate,10mMsodiumfluoride and1mM b -glycerophosphate(Sigma).Forpreparation oflysatesfromcellsculturedunderhypoxicconditions, 100 MCoCl2wasaddedtothelysisbuffertoprevent degradationofHIF-1 a .ProteinconcentrationwasmeasuredbyMicroBCAProteinAssayKit(Pierce).Lysates weremixedwithLaemmlisamplebuffer(4X),boiledfor 5minutesat100C,andequalamountsofprotein (50 gperlanefora10combgel)wereseparatedby SDS-PAGE,followedbyovernightwet-transfertonitrocelluloseat4C.FordetectionofCD26,sampleswere incubatedatalowertemperature(37Cfor15min). CD26wasdetectedat220kDaasaformofhomodimer whichincreaseddetectionsensitivity[14].Weconfirmed thatthebanddetectedat220kDawasdepletedina dose-dependentmannerwithanti-CD26mousemonoclonalantibody1F7byimmunoprecipitation(datanot shown).Afterblockingwith5%nonfatmilkin0.1% Tween20-TBSfor1houratroomtemperature,membraneswereincubatedwiththeappropriateprimary antibodiesin1%blockingbufferfor2hoursatroom temperatureorovernightat4C[anti-CD26(MI1004, Calbiochem,SanDiego,CAorAF1180,R&DSystems, Minneapolis,MN),anti-Cdx2(MU392A-UC,BioGenex, SanRamon,CA),antia -tubulin(T9026,Sigma,Saint Louis,MO),anti-c-Myc(9E10),anti-USF-1(C-20),antiHNF-1 a (C-19)(sc-40,sc-229,andsc-6547,respectively, SantaCruz,SantaCruz,CA)],anti-HIF-1 a (610958,BD TransductionLaboratories,SanJose,CA)],followedby incubationwiththeappropriateHRP-conjugatedsecondaryantibodies(Pierce)for1 houratroomtemperature. MembraneswereincubatedwithSuperSignalWestDura (Pierce)for5minutes,andvisualizedusingaKODAK ImageStation2000Ror4000R(CarestreamHealth,New Haven,CT).Somemembraneswereprocessedusingthe OdysseyInfraredImagingSystembyusingtheappropriateinfrared-labeledsecondaryantibodiesinOdyssey BlockingBuffer(LI-CORBiosciences,Lincoln,NE).Real-timequantitativePCRforCD26mRNAexpressionTotalRNAwasisolatedfromcellswithRNeasyMiniKit andQIAshredder(Qiagen,Valencia,CA).Equalamountsof totalRNA(90ng)wereanalyzedbyonestepreal-timePCR usingQuantiFastSYBRGreenRT-PCRandHs_DPP4_1_SGAbe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page2of10

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QuantiTectPrimerAssay(Qiagen)withtheThermalCycler 7500FastRealTimePCRSystem(AppliedBiosystems,FosterCity,CA).Thereactionconditionswereasfollows:10 minutesat50C(RTstep),5minutesat95C,and40cycles of10secondsat95Cfollowedby30secondsat60C.RelativechangesinCD26mRNAexpressionwerecalculated basedonthe2CTmethod[15].ImmunofluorescencemicroscopyFollowingwashingwithPBS,cellswerefixedwithcold ethanolfor2min.Cellswereair-dried,rehydratedwith PBS,andblockedwith5%BSAinPBSfor30minutesat roomtemperature.AfterwashingwithPBSfor3minutes (3times),cellswereincubatedwithanti-CD26mousemAb (202.36,SantaCruz)for1houratroomtemperature.Cells werethenwashedwithPBSandincubatedwithfluorescein isothiocyanate(FITC)-conjugatedanti-mouseIgG(F8264, Sigma)for30minutesinthedarkatroomtemperature. AfterwashingwithPBS,cellsweremountedusingFluoroguard Antifade(InsitusBiotechnologies,Albuquerque, NM)andimmunofluorescencewasvisualizedusingafluorescencemicroscope(Olympus-BX51,Melville,NY).Transienttransfectionforectopicexpressionofc-MycConfluentHCT-116andHCT-15cells(95-100%confluence)in6-wellplatesweretransfectedwith5 g/wellof pcDNA3-c-Myc(providedb yDr.WafikEl-Deiry,UniversityofPennsylvania,th roughAddgeneasadistributor,Cambridge,MA)oremptyvectorusing10 l/well ofLipofectamine2000.Cellsweregrownforseveral days,andmediumwaschangeddaily.TransienttransfectionofCdx-2siRNAHCT-15cellsweregrownto80-90%confluencein6-well platesandweretransfectedwith200pmol/wellofCdx-2 siRNA(sc-43680,SantaCruz)orcontrolsiRNA (sc-37007,SantaCruz)using7 lperwellofLipofectamine2000(Invitrogen).Mediumwaschangeddaily.ResultsConfluence-dependentupregulationofCD26expression inHCT-116andHCT-15cellsFollowingincubationofth ecoloncancercelllines HCT-116andHCT-15toapost-confluentstate,we detectedaconfluence-dependentincreaseinCD26 expression(Figure1Aand1B),whichwasregulatedat themRNAlevelasshownbyreal-timePCR(Figure1C). ThemembranelocalizationofCD26inbothcelllinesat post-confluencewasconfirmedbyimmunofluorescence staining(Figure1D).Thisconfluence-dependentupregulationofCD26expressionwasalsoobservedinthree othersolidtumorcelllines:malignantmesothelioma celllineJMN,colonadenocarcinomacelllineLS174T andgastriccancercelllineMKN45(datanotshown).Confluence-dependentexpressionoftranscriptionfactors c-Myc,USF-1,HNF-1 a ,andCdx2inHCT-116andHCT-15 cellsToinvestigatethemolecularmechanisminvolvedinthe confluence-dependentexpressionofCD26,weexamined theexpressionoftranscriptionfactorsknowntobe involvedinregulatingCD26expression.Inpreviousstudies,theubiquitouslyexpressedcellularupstreamstimulatoryfactor1(USF-1)andthehepatocytenuclearfactor 1 a (HNF-1 a )havebeenreportedtoexhibitpromoter activityforCD26expressionthroughbindingtotheir consensusmotifsintheCD26promoterregioninCaco2cells[16,17].USF-1isamemberoftheeukaryotic evolutionarilyconservedbas ichelix-loop-helixleucine zippertranscriptionfactorfamily[18].HNF-1 a was originallycharacterizedasaliver-specifictranscription factorbutisalsofoundinothertissuessuchaskidney, smallintestineandthymus[19].OurdatafromWestern blotanalysisshowthatUS F-1proteinexpressionis increasedinaconfluence-dependentmanner,while HNF-1 a expressionisunchangedforbothcelllines, suggestingthatonlyUSF-1iscorrelatedwiththeconfluence-dependentincreaseforCD26expression(Figure2). Wealsoexaminedtheexpre ssionoftheproto-oncogenec-Mycinthisprocess.Atranscriptionfactor belongingtothebasichelix-loop-helixleucinezipper transcriptionfactorfamily,c-Myciscloselylinkedto upregulatedproliferationandderegulatedcellcyclein tumors[20].c-MycisknowntobindtospecificDNA sequencesreferredtoasE-boxsequences.USF-1can alsobindtothesesequences,andinfact,c-Mycand USF-1havebeenreportedtoexertopposingeffectson promoteractivityforsomegenesbycompetingwith eachothertooccupyE-boxsites[21].Inaddition, c-Mychasbeenreportedtosuppressgeneexpressionof proteinsinvolvedindifferentiationandcellcyclearrest suchasp15Ink4b,p21Cip1andp27Kip1[22].Wenow demonstratethat,unlikeo urfindingswithUSF-1, c-Mycexpressiondecreasesinaconfluence-dependent mannerinbothcelllines(Figure2),suggestingthe involvementofc-MycasanegativeregulatorofCD26 expression. Cdx2isamemberofthecaudal-relatedhomeoboxgene familyandhasemergedasoneofthemajorregulatoryfactorscontrollingintestinalcelldifferentiation[23].Previous workhasindicatedthattheforcedexpressionofCdx2in HIECcells(anormalhumanintestinalepithelialcryptcell model)ledtoCD26upregulation[24].However,Cdx2can bephosphorylatedbycyclin-dependentkinase2(Cdk2), resultinginubiquitin-dependentdegradation[25].Therefore,theincreasedCD26expressioninHIECcellswas foundtobelimitedbyCdx2degradationbyproteosomes. Inourstudies,therewasadramaticincreaseinCdx2proteinexpressioninaconfluence-dependentmannerinAbe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page3of10

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CD26 Day: 12345671234567 A. B. HCT-116HCT-15 0 2 4 6 8 Relative protein level 0 2 4 6 8 10 HCT-116 HCT-15 0 1 2 3 4 Relative mRNA level 0 2 4 6 8 10 12 C. HCT-116 HCT-15 a-Tubulin D. H C T-11 6 H C T-1 5 Figure1 Confluence-dependentupregulationofCD26expressioninHCT-116andHCT-15 .A)Confluence-dependentchangesinCD26 proteinexpressionwereanalyzedbyWesternblots.Cells(1106)wereplatedina6-cmdish(day0)andgrowntopost-confluence,withcells beingconfluentonday3.Eachlanewasloadedwith50 gprotein.B)DensitometricanalysisoftheresultsfromWesternblotsisshown.C) CD26mRNAlevelwasmeasuredbyreal-timequantitativePCRasdescribedinMaterialsandMethods.D)ImmunofluorescencestainingofCD26 inHCT-116andHCT-15cellsonday7wasperformedasdescribedinMaterialsandMethods.Whilefluorescencestainingwasnotdetectableat sub-confluence(datanotshown),membranelocalizationofCD26inbothcelllineswasdemonstratedatpost-confluence.Alldatashownare representativeofthreeindependentexperiments. Abe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page4of10

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HCT-15cells.HoweverCdx2wasundetectableinHCT116cellsatallstatesofconfluence(Figure2).Involvementofc-MycasarepressorofCD26expression inHCT-116andHCT-15cellsTofurtherinvestigatec-Mycinvolvementinregulating CD26expression,wetransientlytransfectedc-Mycinto bothcelllinesatpost-confluence.Figure3showsthat theover-expressionofc-Mycresultedinasignificant decreaseinCD26proteinexpression.Althoughc-Myc over-expressiondoesnotco mpletelyabrogatetheconfluence-dependentincreaseinCD26expression,these resultssupportaroleforc-MycasarepressorofCD26 expression.InvolvementofCdx2intheconfluence-dependent increaseinCD26expressioninHCT-15cellsSincewedetectedadramaticincreaseinCdx2protein expressionalongwithenhancedCD26levelinaconfluence-dependentmanner,wenextexaminedwhether Cdx2isinvolvedintheregulationofCD26expression. AsshowninFigure4,transienttransfectionofpostconfluentHCT-15cellsusingCdx2siRNAresultedin decreasedCD26expressioncomparedtothatseenusing thecontrolsiRNA.Theseresultsindicateapotential roleforCdx2inregulatingtheconfluence-dependent increaseinCD26expressioninthiscellline. ToevaluatetheeffectofectopicexpressionofCdx2 onCD26level,wetransientlytransfectedCdx2into HCT-116andHCT-15cellsatvariousstatesofconfluence.However,wefoundnosignificanteffectonCD26 expressionineithercellline(datanotshown).One potentialexplanationforthesefindingsisthatCdx2 mayexertitseffectontheCD26promoterbycombiningwithanunknownfactorX.Therefore,decreasing Cdx2levelbysiRNAwouldreducetheputativeCdx2factorXcomplex,leadingtoaloweredCD26promoter activity.Thisscenariomayexplainthelackofeffecton CD26transcriptionfollowingtransienttransfectionof Cdx2alone(withoutincreasingfactorX).Effectofhypoxic,acidicandserum-depletedculture conditionsonCD26expressioninHCT-116cellsTodeterminecellcultureconditionsthatmighthavean effectontheregulationo fCD26expression,wefirst evaluatedtheeffectofhypoxia.Theupregulationof CD26expressionunderhyp oxicconditionshasbeen reportedpreviouslybyourselvesandothers[26-28],as wellastheexistenceofpericellularhypoxiaindense cultures[12].Sub-confluentHCT116cellswereincubatedeitherundernormoxicorhypoxicconditions(see MaterialsandMethods)for1,4,or24hours.Aftera24 hourincubation,cellswerestillsub-confluent(~90%), andtherewasnodifferenceinpHbetweennormoxic andhypoxicmedia.Cellswereharvestedatthespecified time,lysed,runongels,and analyzedbyWesternblots (Figure5A).CD26expressiondidnotchangeunder hypoxicconditionsevenwhenexpressionofHIF-1 a was H C T-11 6 H C T-1 5 N/D CD26 c-Myc USF-1 HNF-1 -Tubulin Day:12345671234567 Cdx2 Figure2 Confluence-dependentupregulationoftranscriptionfactorsinHCT-116andHCT-15cells .Cultureconditionswerethesameas forFigure1.ProteinexpressionwasanalyzedbyWesternblottingasdescribedinMaterialsandMethods.Datashownarerepresentativeof threeindependentexperiments.Alltranscriptionfactorsexhibitedalteredproteinexpressionlevelsinaconfluence-dependentmannerexcept forHNF-1 a ,whichwasunchanged.* N/D ,Cdx2wasnotdetectedinHCT-116. Abe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page5of10

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atitshighest(following4hrhypoxia).Inaddition,there wasnodifferencebetweennormoxicandhypoxiccells withrespecttoCD26at3dayspost-confluence(Figure 5B).Althoughhypoxiaalonedidnotleadtothe enhancementofCD26(Figure5Aand5B),HIF-1 a level wasincreasedascellsbecamemoreconfluent(Figure 5C).Itwasnotdetectedonday1(sub-confluent),but rosesteadilyuntilitpeakedatday4(1daypost-confluent),thendeclinedgraduallythroughday8(5days post-confluent).ComparisonofCD26expressionin confluentandsub-confluentHCT-116andHCT-116HIF1 a -/-cellsrevealedsignificantlyhigherCD26expressionforcellsthatareHIF-1 a positive(Figure5D). TheseresultssuggestthatHIF-1 a isrequired,butnot sufficient,forCD26upregulation. WenextexaminedwhetherlowextracellularpH affectedCD26proteinexpression.HCT-116cellsat post-confluenceorsub-confluencewereculturedin 123456123456 Control Days after transfection Ectopic c-Myc CD26 c-Myc a-Tubulin H C T-116 A. HCT-15 123456123456 ControlEctopic c-Myc Days after transfection CD26 c-Myc a-Tubulin B. Da y s after transfectionRelative CD26 expression level Control Ectopic c-Myc 0 1 2 3 123456 Da y s after transfection HCT-15 HCT-116 C. 0 4 8 12 123456 Control Ectopic c-Myc Figure3 Effectofc-Myconconfluence-dependentCD26expressioninHCT-116andHCT-15cells .TheeffectofectopicexpressionofcMycontheconfluence-dependentincreaseinCD26proteinexpressionwasexaminedinHCT-116andHCT-15cellsbytransienttransfection usingeitherpcDNA3/c-Mycortheemptyvector(control)asdescribedinMaterialsandMethods.ProteinexpressionwasanalyzedbyWestern blotting(A,B)andquantifiedbydensitometricanalysis( C ).Datashownarerepresentativeofthreeindependentexperiments. Abe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page6of10

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regular10%FBSmediumatpH7.3oracidic10%FBS mediumsupplementedwith25mMMESatpH6.2.As showninFigure5Eand5F,thepHofthemediumdid notaffectCD26expressioninsub-confluentorpostconfluentcultures. Sincenutrientsandserumgrowthfactorsmaybe depletedwhencellsreachahighcelldensity,wealso testedtheeffectofculturingcellsinserum-depleted media.Sincethesupplyofavailablenutrientsand growthfactorsmayvaryinpost-confluentcultures,the useofserum-depletedmediamayofferamoreconsistentcultureenvironmenttoevaluatetheroleofnutrientsandserumgrowthfactorsinCD26expression. WhenHCT-116cellsatsub-confluencewerecultured in0.3%BSAmediumwithoutserumfor24hours, CD26proteinlevelwassignificantlyincreasedcompared tocellsculturedin10%FBSmedium(Figure5Fand 5G).HIF-1 a levelwaslikewiseincreasedwhencells wereculturedintheabsenceofserum(Figure5G).On theotherhand,c-Mycexpressionwasdecreasedin serum-depletedmedia.Interestingly,CD26expressionin cellsculturedfor24hourswith10%FBS(following 24-hourserumdepletion)returnedtobasallevelasdid HIF1a andc-Myc(Figure5G),demonstratingthe reversibilityofthisphenomena.DiscussionInthepresentstudy,wedemonstratethatCD26expressionisincreasedincoloncarcinomalinesgrowntoconfluence,associatedwithchangesinselectedtranscription factors.Inparticular,wef oundthatc-Mycexpression decreaseswithconfluence,andthatitcanactasa repressorofCD26expression.Incontrast,Cdx2expressionparallelsthatofCD26,increasingwithconfluence. ApotentialexplanationfortheobservedconfluencedependentincreaseinCdx2proteinexpressionmaybe duetoconfluence-mediatedcellcyclearrest,resultingin decreasedexpressionofCdk2anddecreasedphosphorylationofCdx2byCdk2,leadingtoincreasedCdx2proteinexpression[25].Ourfindingsalsosuggestthe existenceofaco-factorXthathasaroleinregulating CD26expressionincombin ationwithCdx2,although 23456 Control siRNACdx2 siRNA CD26 Cdx2 a-Tubulin 23456 Days after transfection 0 1 2 3 4 5 6 7 8 Relative CD26 protein level Control siRNA Cdx2 siRNA Figure4 EffectofCdx2siRNAonconfluence-dependentCD26expressioninHCT-15cells .TheeffectofCdx2siRNAontheconfluencedependentCD26proteinexpressioninHCT-15wasevaluatedfollowingtransienttransfection(whencellswereat90%confluence)ofcontrol siRNA( left )orCdx2siRNA( right )asdescribedinMaterialsandMethods.CD26andCdx2proteinexpressionwereanalyzedbyWesternblotting andquantifiedbydensitometricanalysis.Datashownarerepresentativeofthreeindependentexperiments. Abe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page7of10

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A CD26 HIF-1a a-Tubulin 121212 1 hr4 hrs24 hrs 1: Hypoxia 2: Normoxia B. CD26 a-Tubulin HIF-1a 12 1: Hypoxia, 24 hrs 2: Normoxia, 24 hrs C. a-Tubulin 12345678 HIF-1a 1: Day 1 (Subconfluent) 2: Day 2 (Subconfluent) 3: Day 3 (Confluent) 4: Day 4 (1 day postconfluent) 5: Day 5 (2 days postconfluent) 6: Day 6 (3 days postconfluent) 7: Day 7 (4 days postconfluent) 8: Day 8 (5 days postconfluent) D. 3 21 3 21 a-Tubulin CD26 HCT-116 (parent)HCT-116 HIF1a-/E. CD26 a-Tubulin 12 1: 10% FBS, pH 7.3, 24 hrs 2: 10% FBS, pH 6.2, 24 hrs F. 12341234 Hypoxia 24 hrs Normoxia 24 hrs 1: 10% FBS, pH 7.3 2: 10% FBS, pH 6.2 3: No serum, pH 7.3 4: No serum, pH 6.2 CD26 HIF-1a a-Tubulin G. CD26 c-Myc a-Tubulin 1234 USF-1 1: 0 hr (10% FBS) 2: 24 hrs with 10% FBS 3: 24 hrs with no serum 4: Another 24 hrs with 10% FBS from #3 HIF-1a Figure5 Effectofhypoxic,acidicandserum-depletedcultureconditionsonCD26expressioninHCT-116cells .A)Effectofhypoxia(1% O2)onCD26proteinexpressionincellsatsub-confluence.Cellsat~50%confluencewereincubatedunderhypoxicornormoxicconditions. CellswereharvestedatdifferenttimepointsandproteinexpressionwasanalyzedbyWesternblottingasdescribedinMaterialsandMethods. B)EffectofhypoxiaonCD26proteinexpressionat3dayspost-confluence.Cellsweregrownto3dayspost-confluence,thenincubatedunder hypoxicornormoxicconditionsfor24hours.C)ExpressionofHIF-1 a atsub-andpostconfluence.Cellsweregrownfor8days,fromsubconfluencethrough5dayspost-confluenceandproteinexpressionwasanalyzedbyWesternblotting.D)EnhancedCD26expressioninparental HCT-116cellscomparedtoHCT-116HIF1a -/-cells.Wholecelllysates(24 g)wererunongelsandanalyzedasdescribedpreviously.Cellswere heldatconfluencefor3days(lane1),1day(lane2),orweresub-confluent(lane3).E)TheeffectofacidicconditionsonCD26expression.Cells weregrownto3dayspost-confluenceasdescribedabove,andthenincubatedinregular10%FBSmedium(pH7.3)orinacidicmedium supplementedwith25mMMES(pH6.2)for24hours.F)Theeffectofhypoxia,acidicmedium,andserumdepletionwasexaminedincellsat sub-confluence. G )Theeffectofswitchingcellsfromserum-depletedcultureconditionsto10%FBSmediumwasevaluated.Cellsweregrownto ~25%confluence(lane#1)andthenculturedinregular10%FBSmedium(lane#2)orserum-depletedmedium(lane#3)for24hours.Cells culturedinserum-freemediumfor24hourswerere-culturedinregular10%FBSmediumforanother24hours(lane#4).Cellconfluenceatthis pointwasstill~90%.Alldatadiscussedabovearerepresentativeofatleastthreeindependentexperiments. Abe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page8of10

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additionalstudieswillneedtobedonetotestthis hypothesis.Inaddition,wedemonstratedthata hypoxia-inducedincreaseinHIF-1 a wasnotassociated withanenhancedCD26level,butthatHIF-1 a expressingcellsdisplayedahigherlevelofHIF-1 a alongwith ahigherlevelofCD26whengrowntoconfluence.In comparison,alackofenhancementofHIF-1 a and CD26levelsinHCT-116HIF1 a -/-cells,suggestthatHIF1 a expressionisrequiredbutnotsufficientforincreased expressionofCD26. Thegrowthoftumorsiskno wntobeassociatedwith hypoxiaandlowextracellularpHinthetumormicroenvironment[29].Inourexperimentalsystem,lowextracellularpHhadnoeffectonCD26proteinexpression, regardlessoftheextentofc ellconfluence.Ontheother hand,wefoundthatdepletionofserumfactorswasassociatedwitharobustupregulationofCD26.Furthermore, theeffectofserumdepletiononCD26proteinexpression wasreversible,aswasalsothecasewithc-MycandHIF1 a expression.Allthreeproteinsreturnedtobasallevel ofexpressionin24hoursfollowingculturinginregular 10%FBSmedium.Theseresultshencesuggestthatthere isaserumfactor(s)-dependentregulatorymechanismfor CD26proteinexpression,whichaffectscellsmaintained withoutserumatsub-confluence.Sincethehalflifeof CD26proteinincellsatpost -confluenceisreportedto bemuchlongerthan48hoursasassayedby[35S]methioninelabelpulse-chaseexperiments[9],ourresults suggesttheexistenceofanunidentifiedserumfactordependentdegradativemechanismintheregulationof CD26proteinexpression.Thesedataindicatethata post-confluentstatecanbemimickedbycellsatsub-confluencebydepletingrequiredserumfactors.Whilewe didnotdetectdifferencesinCD26expressionwhencells wereincubatedinserum-freeconditionswithlowlevels ofBSA(datanotshown),itisstilltheoreticallypossible thatthepresenceofBSAintheculturemediaofcells culturedinserum-freeconditionscaninterferewith CD26metabolismandexpression.Wewouldalsoliketo notethatourobservationsarerestrictedtothespecific cellsandproteinsevaluated inourexperimentalconditions,andarenotnecessarilyapplicabletoothercell typesorproteins.Whencellsareatpost-confluence, depletionofnutrientsislikelytolimitcellgrowth.In addition,tightjunctionsbetweencellsgrowingatpostconfluencemayinhibitbindingofserumfactorstothe cellsurface,leadingtotheupregulationofCD26.However,thepotentialcontributionoffactorssecretedfrom cellsatpost-confluencecannotbeexcluded.Infact, othershavereportedincreasedHIF-1 a expressionwhen sub-confluentbreastcarcinomacellswereincubatedwith conditionedmediafromconfluentcultures[12]. Ourpreviousstudiesdemonstratedthepotentialoftargetedtherapyusinganti-CD26mAbtreatmentinseveral tumormodels,includingrenalclearcellcarcinomaand malignantmesothelioma[7,8].Inthesecelllines,upregulationofp27Kip1wasobservedfollowingtreatmentwith anti-CD26mAb.Weobservedthatp27Kip1expression waselevatedincellstreatedwithanti-CD26mAbcomparedtoisotype-matchedcontrolmAbwhentreatment wasdoneinserum-depletedcultureconditions(datanot shown).However,thisanti-CD26mAb-mediated enhancementinp27Kip1levelwasnotobservedifcells wereincubatedinserum-containingmedia,indicating thatserumdepletioncanenhancetheeffectofanti-CD26 mAbtreatmentincellsnormallyexpressinglowCD26 level(presumablybyupregulatingtargetexpression). Futurestudieswillexamineindetailthemechanism associatedwithCD26enhancementmediatedbyserumdepletedculturecondition s.Thisknowledge,combined withourpresentfindings,mayprovetohaveclinicalsignificancebyexpandingthepotentialusageofanti-CD26 mAbtherapyinselectedhumancancersthrough enhancedCD26expression.ConclusionsInsummary,wedemonstratethatCD26expressionis regulatedinaconfluence-d ependentmanner,involving potentiallytranscriptionalandposttranslationalmechanisms.Fortheconfluence-dependentincreaseinCD26 expression,decreasedexpressionofc-Mycandincreased expressionofUSF-1andCdx2maycontributetothe upregulationofCD26expressionatthetranscriptional level.Meanwhile,factorsassociatedwithserumdepletion mayalsocontributetoincreasedCD26expression,with onepotentialmechanismbeingattheposttranslational levelbyregulatinganunknownserumfactor-dependent CD26proteindegradationpathway.Importantly,ourpresentfindingsmayresultintheexpansionoftheuseof CD26-targetedtherapyintheclinicalsettingforselected tumorsbyregulatingtheexpressionofCD26itself.Authordetails1DepartmentofHematologicMalignancies,NevadaCancerInstitute,Las Vegas,Nevada,89135,USA.2DivisionofHematology/Oncology,Universityof Florida,Gainesville,FL32610,USA.3DivisionofClinicalImmunology, AdvancedClinicalResearchCenter,InstituteofMedicalScience,The UniversityofTokyo,Tokyo,108-8639,Japan. Authors contributions MAcarriedouttheexperiments.PAH,YU,KO,CM,LHD,andNHD participatedinthedesignofthestudy,itscoordination,andhelpedtodraft themanuscript.Inaddition,LHDwasresponsibleforthecreationofthe HCT-116HIF1 -/-cellline. Allauthorsreadandapprovedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:4August2010Accepted:1February2011 Published:1February2011Abe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page9of10

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JNatlCancerInst 2007, 99 :1441-1454.Pre-publicationhistory Thepre-publicationhistoryforthispapercanbeaccessedhere: http://www.biomedcentral.com/1471-2407/11/51/prepubdoi:10.1186/1471-2407-11-51 Citethisarticleas: Abe etal .: Mechanismsofconfluence-dependent expressionofCD26incoloncancercelllines. BMCCancer 2011 11 :51. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Abe etal BMCCancer 2011, 11 :51 http://www.biomedcentral.com/1471-2407/11/51 Page10of10