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microenvironment interactions_Cancer Res_2004

来源：知库网

DOI:10.1158/0008-5472.CAN-03-4038

[CANCERRESEARCH64,6571–6578,September15,2004]

Tumor-MicroenvironmentInteractions:TheFucose-GeneratingFXEnzymeControlsAdhesivePropertiesofColorectalCancerCells

AdiZipin,1MiraIsraeli-Amit,1TsipiMeshel,1OritSagi-Assif,1IlanaYron,1VeronicaLifshitz,1EranBacharach,1NechamaI.Smorodinsky,1ArielMany,2PeterA.Czernilofsky,3DonaldL.Morton,4andIsaacP.Witz1DepartmentofCellResearchandImmunology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel;2LisMaternityHospital,TelAvivSouraskyMedicalCenter,TelAviv,Israel;3FacultyofMedicine,UniversityofVienna,Vienna,Austria;and4JohnWayneCancerInstitute,SantaMonica,California

1ABSTRACT

Extravasationoftumorcellsisapivotalstepinmetastasisformation.Thisstepisinitiatedbyaninteractionofextravasatingtumorcellswithendothelialcells.Amongthemoleculesmediatingtumor-endotheliumin-teractionsareselectinsandtheirfucosylatedligands.Inapreviousstudy,wedemonstratedthatthefucose-generatingFXenzymeregulatestheexpressionofselectinligandsbyBandTlymphocytesandbyheadandnecksquamouscellcarcinomacells.ItwasalsoshownthattheFXenzymeregulatedimportantinteractionparametersbetweenthesecancercellsandendothelialcells.ThepresentstudywasaimedtodeterminewhethertheFXenzymecontrolsadhesiveinteractionsbetweencolorectalcancercellsandendothelialcells.Theresultsclearlyindicatethatthisisindeedthecase.OverexpressingtheFXenzymebythetransferofFXcDNAtolowFX-expressingcolorectalcancercellsresultedinanincreasedadhe-sivecapacityofthetransfectantstoactivatedendothelialcellsandtorecombinantE-selectin.Down-regulatingFXlevelsincolorectalcancercellsexpressinghighlevelsofendogenousFXbytransfectionwithsmall-interferingRNAresultedinadown-regulatedexpressionoftheselectinligandsialylLewis-aandadecreaseintheadhesivecapacityofthetransfectantstoactivatedendothelialcellsandtorecombinantE-selectin.ThesetransfectionexperimentsalsoindicatedthatmanipulatingthelevelsoftheFXenzymeaffectedglobalcellularfucosylationandalteredtheinteractionofcolorectalcancercellswithsomeextracellularmatrixcom-ponentssuchasfibronectin.Wealsofoundthathighlymetastaticcolo-rectalcancervariantsexpresshigherlevelsofFXandofsialylLewis-athanlowmetastaticvariantsoriginatinginthesametumors.TheseresultsleadustohypothesizethattheFXenzymecontrolsthecapacityofcolorectalcancertoextravasateandformmetastasis.IfthishypothesiswillbeconfirmedtheFXenzymecouldbecomeatargetmoleculeformetastasisprevention.

INTRODUCTION

Fucoseisacomponentofmanysurface-localizedandsecretedmolecules.ItdecoratestheterminalportionsofN-,O-,orlipid-linkedglycansandmodifiesthecoreofsomeN-linkedglycans(1).Terminalfucosylatedglycansinhumansconstituteseveralbloodgroupantigensandfunctionasselectinligands(2).Fucosylationoftheseligandsdeterminestheirabilitytobindtotheselectinfamilyofcelladhesionmoleculesandthereforecontrolspivotalstepsofselectin-dependentleukocyteandtumorcelladhesionandtrafficking(1,3,4).Inadditiontoitsroleinadhesivereactions,fucosylationinfluencesNotchandCriptosignalingevents(5–9).

ThefinalstepsoffucosebiosynthesisaremediatedbytheGDP-D-mannose4,6dehydratasegeneratingGDP-mannose-4-keto-6-D-

deoxymannose.ThissugarisconvertedtoGDP-L-fucosebytheFXenzyme,functioningbothasanepimeraseandareductase(10,11).TheGDP-LfucoseisthentransportedtotheGolgi.Fucosylationofmammalianglycansiscatalyzedbydistinctfucosyltransferases,withcatalyticactivitiescharacterizedbyspecificityforspecificglycocon-jugatesubstratesandarequirementforGDP-fucose(12).

ThegenerationofFXknockoutmiceenabledustoconcludethatfucosylationeventsareessentialforfertility,earlygrowth,anddevel-opment,aswellasforintercellularadhesion(1).FXknockoutresultedinamassiveintrauterinemortality.Live-bornFX-nullmiceexhibitedavirtuallycompletedeficiencyofcellularfucosylationandapostnatalfailuretothrive.FX(Ϫ/Ϫ)adultssufferfromanextremeneutrophilia,myeloproliferation,andabsenceofleukocyteselectinligandexpres-sionreminiscentofLAD-II/CDG-IIc(1).

Allthesestudiesdemonstratethatfucosylationplaysanimportantroleindevelopmentandinadultphysiologybyinfluencingatleasttwodifferentpathways:biosynthesisofselectinligandsandsignalingthroughtheNotchandCriptopathways.

PreviousstudiesfromourlaboratorydemonstratedthattheFXenzymeisinvolvedinthebiosynthesisofsLe-xinactivatedTorBcells(13).Inheadandnecksquamouscellcarcinomas,theFXenzymeplaysakeyroleinthebiosynthesisofselectinligandssuchassialylLewis-a(sLe-a)andintheinteractionofthesecancercellswithendothelialcells(14,15).Inlymphocytes,aswellasinheadandnecksquamouscellcarcinomas,theFXenzymeisregulatedbyoutside-insignaling(13,14).

Numerousreportsindicatedacorrelationbetweenahighexpressionofselectinligandsbyepithelialcancercells,notablycolorectalcancer,andahighrateofmetastasisandpoorprognosis(16–18).Thefucose-generatingFXenzymemaythusbeapivotalelementincancer-associatedperturbationsofdifferentiation,survival,andpro-liferation,aswellasincancercellextravasation.ItisthereforeimportanttofindoutiftheFXenzymeisinvolvedincontrollingselectinligandexpressionbycolorectalcancercellsandintheirinteractionwithendothelialselectin.Thepresentstudyprovidesproofthatthisisindeedthecase.MATERIALSANDMETHODS

CellLines.Thehumancolorectalcancercelllines:474,0485,1086,1203,044,and427wereestablishedattheJohnWayneCancerInstitute(SantaMonica,CA).The474and1203celllineswerederivedfromprimarycolo-rectalcancertumors.The1086celllinewasderivedfromacolorectalcancerlivermetastasis.The0485celllinewasderivedfromalymphnodemetastasis.ThecellsweremaintainedinRPMI1640supplementedwith20%FCS,2

Received12/30/03;revised5/20/04;accepted7/20/04.

mmol/LL-glutamine,100units/mLpenicillin,0.1mg/mLstreptomycin,andGrantsupport:TheJacquelineSeroussiMemorialFoundationforCancerResearch,

12.5units/mLnystatin.AllofthemediumcomponentswereobtainedfromTheIsraelCancerAssociation,TheFainbargFamilyFund(OrangeCounty,CA),The

FredAugustandAdeleWolpersCharitableFund(Clifton,NJ),ArnoldandRuthFeuer-BiologicalIndustries(Beit-Haemek,Israel).KM12C,KM12L4,andKM12SM

stein(OrangeCounty,CA),andthePikovskyFund(Jerusalem,Israel).I.Witzisthe

werekindlyprovidedbyDr.IsaiahJ.Fidler(DepartmentofCellBiology,

incumbentoftheDavidFurmanChairinImmunobiologyofCancer.

M.D.AndersonCancerCenter,Houston,TX).TheKM12CcelllinewasThecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage

derivedfromaDuke’sB2colorectalcancerprimarytumor.TheKM12L4andcharges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordancewith

18U.S.C.Section1734solelytoindicatethisfact.KM12SMvariantsoriginatedinlivermetastasesthatdevelopedinBALB/cRequestsforreprints:IsaacP.Witz,DepartmentofCellResearchandImmunology

nudemiceinoculatedwithKM12Ccellstospleenorcecum,respectively(19).

GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,69978TelAviv,Israel.

KM12C,KM12L4,andKM12SMcellsweremaintainedinEagle’sMEMPhone:972-3-6406979;Fax:972-3-6422046;E-mailipwitz@post.tau.ac.il.

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0.1mg/mLstreptomycin,and12.5units/mLnystatin,5mmol/Lsodiumpyruvate,nonessentialaminoacids,and2-foldvitaminsolution(19).ThecolorectalcancerSW480andSW620cellswereobtainedfromAmericanTypeCultureCollection(Manassas,VA).TheSW480celllinewasderivedfromaprimarycolorectalcancer,whereastheSW620linewasderivedfromalymphnodemetastasisofthesamepatient.SW480cellsweremaintainedinRPMI1640supplementedwith10%FCS.FX-cDNASW480-transfectedcellsweremaintainedinthesamemediumsupplementedwith800␮g/mLG418(Cal-biochem,SanDiego,CA).SW620cellsweremaintainedinLeibovitzL-15mediumsupplementedwith15%FCS,2mmol/LL-glutamine,100units/mLpenicillin,0.1mg/mLstreptomycin,12.5units/mLnystatin,10mmol/LHEPESbuffer,and0.075%sodiumbicarbonate.ThecolorectalcancerHT-29celllinewaskindlyprovidedbyDr.LiviaTheodor(GastroenterologyDepart-ment,TheChaimShebaMedicalCenter,TelHashomer,Ramat-Gan,Israel).TheHT-29cellsweremaintainedinMcCoy’smodifiedmedium(Invitrogen-LifeTechnologies,Inc.,Paisley,Scotland,UnitedKingdom)supplementedwith10%FCS,2mmol/LL-glutamine,100units/mLpenicillin,0.1mg/mLstreptomycin,and12.5units/mLnystatin.Allofthecelllineswereroutinelyculturedinhumidifiedairwith5%CO2at37°C.HumanUmbilicalVeinEndothelialCells(HUVECs).HUVECswereeitherpurchasedfromDr.NeomyLanir(DepartmentofHematologyandBoneMarrowTransplantation,RambamMedicalCenter,Haifa,Israel)orpreparedinourlaboratoryfromumbilicalcordsasfollows:endothelialcellswereharvestedby0.25mg/mLcollagenasetypeII(Sigma,Holon,Israel).Cellsweregrowntoconfluenceintissuecultureflasksprecoatedwithfibronectin(20␮g/mL;BiologicalIndustries,Beit-Haemek,Israel).Cellswereestab-lishedasprimaryculturesinM199mediumsupplementedwith20%FCS,50␮g/mLendothelialcellgrowthfactor(BiomedicalTechnologies,Inc.,Stough-ton,MA),heparin(5units/mL;LaboratoireChoay,Paris,France),andanti-biotics.Cellsfromthethirdpassageweretakenforexperiments.

Antibodies.Thefollowingantibodieswereusedinflowcytometryassays:anti-sialylLewis-a(sLe-a),anti-Lewis-a(Le-a),andanti-Lewisb(Le-b)werepurchasedfromSeikagakuAmerica(Falmouth,MA).Anti-sialylLewis-x(sLe-x)waspurchasedfromAmericanTypeCultureCollection.Anti-Lewis-x(Le-x)andanti-CD24werepurchasedfromPharMingen(SanDiego,CA).Anti-Lewis-y(Le-y)andanti-PSGL-1werepurchasedfromSerotec(Oxford,UnitedKingdom).AntihumanVIM2antibodieswereakindgiftfromDr.WalterKnapp(InstituteofImmunology,UniversityofVienna,Vienna,Aus-tria).Anti-CD62EwaspurchasedfromSouthernBiotechnologyAssociates(Birmingham,AL).FITC-conjugatedgoatantimouseIgMandIgGwerepur-chasedfromJacksonImmunosearchLaboratory,Inc.(WestGrove,PA).ThefollowingantibodieswereusedinWesternblottingassays:mousemonoclonalantihumanFXantibody(3G10/12)preparedinourlaboratory,rabbitpoly-clonalantihumanFXantibody701(13)alsopreparedinourlaboratory,anti-glyceraldehyde-3-phosphatedehydrogenaseantibody(ChemiconInterna-tional,Inc.,Temecula,CA),andhorseradishperoxidase-conjugatedsecondarygoatantibodyagainstmouseIgG(JacksonImmunosearchLaboratory,Inc.).Plasmids.TheplasmidpSUPERwaskindlyprovidedbyDr.ReuvenAgami(DivisionofTumorBiology,TheNetherlandsCancerInstitute,Ples-manlaan,Amsterdam,theNetherlands).TheplasmidpRc/CMVwaspurchasedfromInvitrogenBV(Groningen,theNetherlands).TheplasmidpEGFP-CwaspurchasedfromClontechLaboratories,Inc.(PaloAlto,CA).

ConstructionofFXSmallInterferingRNA(siRNA).FXmRNAsup-pressionwasachievedbyusingthepSUPERvector.Twogene-specificoligonucleotidesweredesignedasfollows:5Ј-GATCCCCAGACGCCG-ATCTCACGGATTTCAAGAGAATCCGTGAGATCGGCGTCTTTT-TTGGAAA-3Јand5Ј-AGCTTTTCCAAAAAAGACGCCGATCTCA-CGGATTCTCTTGAAATCCGTGAGATCGGCGTCTGGG-3Ј.RegularcharactersrepresentregionsrequiredforthegenerationofthesiRNAaspreviouslydescribed(20),andboldcharactersrepresentFX-specificcomple-mentarysequences.Botholigonucleotidesweredenaturedat95°Cfor4minutes,annealedat70°Cfor10minutes,andcooleddownslowly.Oligonu-cleotideswerethenphosphorylatedbytheuseofT4polynucleotidekinaseat37°Cfor30minutes.ThisoligonucleotidemixturewasligatedintothepSU-PERvectorpredigestedwithBglIIandHindIIIandpretreatedwithcalfintestinalphosphatase.AmutatedFXsiRNAthatservedascontrolwasgeneratedbyintroducingapointmutation(GtoC)atposition24inthefirstFX-specificcomplementarysequenceoligonucleotideshownabove.Thisoli-

gonucleotidewasclonedtothepSUPERvectoralongwiththesecondFX-specificcomplementaryoligonucleotideshownabove.

FlowCytometry.Cells(5ϫ105)wereincubatedfor45minutesat4°Cwithprimaryantibodiesdirectedagainstthetestedselectinligand.Afterawashwithcellsortermedium(RPMI1640supplementedwith5%FCSand0.01%sodiumazide),thecellswereincubatedfor45minutesat4°CwithFITC-conjugatedgoatantimouseIgGorIgM.Afteranadditionalwash,antigenexpressionon5000livecellswasdeterminedusingaBectonDickinsonFACSort(MountainView,CA)andCellQuestsoftware.Baselinestainingwasobtainedbyaddingcellsortermediumtothecellsinsteadofprimaryantibody.Flowcytometryscoresforselectinligandexpressionwerecalculatedasdescribedpreviously(13).Thescoresrepresentthemultiplicationofthemeanfluorescencebythepercentpositivecellsϫ10-4.Scoresforeachselectinligandweredividedintothreecategories:high,medium,andlow.ForsLe-a,highϭ8.2to19.7,mediumϭ5.7to7.6,andlowϭ1.8to0.2;forLe-b,highϭ8.7to11.6,mediumϭ2.2to5.6,andlowϭ0.1to1.3;forsLe-x,highϭ7andmediumϭ2.3to4.9.

RNAPreparationandNorthernBlotAnalysis.TotalRNAwasprepared,andNorthernblottingwasperformedasdescribedbyEsheletal.(14)inourlab.

WesternBlotting.ColorectalcancercellswerelysedwithLaemmlisam-plebuffer(21).Lysateswereboiledfor10minutes,centrifuged,andappliedonaminiproteanIIsystem(Bio-Rad,Hercules,CA)forSDS-PAGEusinga12%slabgelasdescribedbyLaemmli(21).Electrophoretictransferofproteinsfromthepolyacrylamidegeltonitrocellulose(Schleicher&Schull,Dassel,Germany)wasperformedbyamini-transblotelectrophoreticcell(Bio-Rad)at250mAfor2hours.Aftertransfer,thenitrocellulosemembranewasincubatedatroomtemperaturewith3%BSAinTBS-Tweenfor30minutestoblockfreebindingsitesonthemembrane.Theblockednitrocellu-losemembranewasincubatedovernightwithanti-FX3G10/12monoclonalantibody,diluted1:16in1%BSAinTBS-Tweenwith0.02%sodiumazideorwithrabbitpolyclonalantihumanFXantibody701(2),diluted1:1000in5%milkinTBS-Tweenwith0.02%sodiumazide,thenwashedthreetimesfor5minuteswithTBS-Tweenandincubatedfor50minuteswithhorseradishperoxidase-conjugatedsecondarygoatantibodyagainstmouseorrabbitIgGdiluted1:10,000with5%milkinTBS-Tweenatroomtemperature.Finally,thenitrocellulosemembranewaswashedfivetimesfor5minuteswithTBS-Tween.Thebandswerevisualizedbychemiluminescence-enhancedchemilu-minescencereaction(Amersham,Buckinghamshire,UnitedKingdom)andautoradiographybyexposuretoKodakXAR5film(EastmanKodakCo.,Rochester,NY)for1to5minutes.

Thequantificationofproteininthelaneswasdeterminedinreferencetotheamountofglyceraldehyde-3-phosphatedehydrogenaseinthelanes.Thiswasperformedbyincubatingthemembranewithanti-glyceraldehyde-3-phosphatedehydrogenasediluted1:1000with5%milkinTBS-Tweenwith0.02%sodiumazide.

LectinBlot.ThefirststepsofblottingwereperformedasdescribedaboveforWesternblotting.Aftertransferoftheproteinstonitrocellulose,themembranewasincubatedatroomtemperaturefor60minuteswith3%BSAinPBS-0.05%Tween-20toblockfreebindingsitesonthemembrane.Theblockednitrocellulosemembranewasincubatedfor2hourswithhorseradishperoxidase-conjugatedUlexeuropaeusagglutininlectin(Sigma),whichbindstoL-fucose(22),diluted1:2000inPBScontaining3%BSAand0.1%Tween-20,thenwashedfourtimesfor10minuteswithPBScontaining0.1%Tween-20.Thebandswerevisualizedbythechemiluminescence-enhancedchemilu-minescencereaction(Amersham)andautoradiographedbyexposuretoKodakX-AR5film(EastmanKodakCo.)for5seconds.

Transfection.SW620cellsweretransfectedwithpSUPER-FXsiRNA(205cells)orwithpSUPER-FX-mutatedsiRNAoligonucleotides(360controlcells)byelectroporationusingtheElectrocellmanipulator830(BTX-Genetronics,SanDiego,CA).ThirtymicrogramsofpSUPERand3␮gofthepBabe-puroplasmidwereusedfortransfection.Forty-eighthoursafterelectro-poration,thecellswereselectedwith1␮g/mLpuromycin.Individualcloneswerethenpicked,expandedandanalyzedforFXproteinlevels.Toincreasetransfectionyield,cellswerere-transfectedusingthepSUPERcoupledwithpcDNA3.1-zeovectors.Inthiscase,500␮g/mLzeocinwasusedasaselectionmarker.Thetransfectedcellsweregrowninthepresenceof25mmol/Lfucose.SW480cellswerecotransfectedeitherwithFX-cDNApRc/CMVvectoror6572

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Table1Expressionlevelsofselectinligandsbycolorectalcancercelllines

Selectinligand

CelllineHT-29044108647404854271203SW480SW620KM12CKM12L4KM12SM

sLe-a6.517.17.68.25.719.79.70.25.90.20.71.8

Le-b5.60.91.35.62.511.68.72.20.10.10.20.3

Le-a4.41.41.70.2Ͼ0.113.53.10.12.30.10.10.1

sLe-x2.3Ͼ0.1Ͼ0.14.7Ͼ0.174.9Ͼ0.1Ͼ0.1Ͼ0.10.10.9

Le-x0.6Ͼ0.10.62.21.68.216.34.37.3Ͼ0.10.3Ͼ0.1

Le-y0.40.2Ͼ0.10.71.1Ͼ0.10.22.82Ͼ0.10.10.2

CD2430.10.50.51.60.60.2Ͼ0.11.14.23.44.1

VIM20.1Ͼ0.10.4Ͼ0.1Ͼ0.1Ͼ0.12.77.50.50.320.1

PSGL-1Ͼ0.1Ͼ0.1Ͼ0.10.8Ͼ0.10.2Ͼ0.10.1Ͼ0.1Ͼ0.10.10.4

NOTE.Selectinligandexpressionwasdeterminedbyflowcytometry.Valuesrepresentexpressionscoresobtainedbythemultiplicationofmeanfluorescencevaluesbythepercentofpositivecells(ϫ10Ϫ4)asdescribedpreviously(13).

withpRc/CMVvectorwithoutinserttogetherwithpEGFP-Cvectorbyelec-troporation.

AdhesionofColorectalCancertoHUVECs.Ninety-six-wellcultureplateswerecoatedwith50␮Lof20␮g/mLfibronectinperwellfor30minutesat37°C.AfteronewashwithPBS,3ϫ104HUVECsinavolumeof100␮L/wellwereculturedontheplatefor16hourstoformaconfluentmonolayer.Endothelialcellswerestimulatedfor4hourswithhumanrecombinantIFN-␥(100units/mL)andrecombinanttumornecrosisfactor␣(100units/mL)andwashed.Atotalof2ϫ106colorectalcancercellswerewashedtwicewithPBSandsuspendedin1mLofcoldPBS,labeledwiththeCFDA-SEreagent(250nmol/L;MolecularProbes,Eugene,OR)for5minutes,andthenwashedwith2mLofFCS.AfteranadditionalwashwithPBS,thelabeledcells(1ϫ105/100␮L)wereaddedtoastimulatedHUVECmonolayerinTBScontaining2mmol/LCaCl2(forselectin-dependedadhesion)andincubatedfor30minutesat37°C.Thetotalfluorescencesignalwasthatoflabeledcellsaddedtothewellbeforeremovingthenonadherentcells,whichwereremovedbythreewasheswithPBS.TotalfluorescenceandthatofadheringcellswasmeasuredbyafluorescentELISAreader(Bio-TekFL500;Bio-TekInstruments,Inc.,Winooski,VT)atwavelengthof490/530.Theresultsarepresentedaspercentadhesion.Thenumberofadherentgreenfluorescentprotein(GFP)-transfectedcellswasdeterminedbycountingfluorescentcellsinseveralfieldswiththeaidofafluorescencemicroscope(magnificationϫ100,FITCfilter,OlympusIX70;Olympus,Hamburg,Germany).

AdhesionofColorectalCancertorE-Selectin,ExtracellularMatrix(ECM),orFibronectin.Nontissuecultureplates(Nunc-ImmunoplateNUNC;NuncInternational,Roskilde,Denmark)werecoatedfor60minutesatroomtempwith100␮L/wellof2␮g/mLrecombinantE-selectin(R&DSystems,Minneapolis,MN)dilutedinTBS-Tweencontaining2mmol/LCaCl2.Threewellswerenotcoatedfordetectionofnonspecificbinding.Supernatantswereaspiratedfromthewells,and200␮Lofblockingmedium(1%BSAinTBS-Tweencontaining2mmol/LCaCl2)wereaddedforatleast30minutesatroomtemperature.CellswerelabeledwithCFDA-SEreagentasdescribedabove.Labeledcells(1ϫ105/100␮L)suspendedin1%BSAinTBS-Tween,2mmol/LCaCl2wereaddedtorE-selectinoruncoatedwellsfor45minutesat37°C.Therestoftheassaywasperformedasdescribedabove.Asimilarprocedurewasperformedwithplatescoatedwithamixtureofmatrixproteins(E-TCMT-FNOVAmed;Jerusalem,Israel)orwithfibronectininthepresenceof5mmol/LMgCl2.

StatisticalAnalysis.Significancewascalculatedusingthetwo-wayStu-dent’sttest.

levelsofsLe-a.HighlevelsofLe-bwereexpressedbycellsoftwolines(427and1203,whichalsoexpressedhighlevelsofsLe-a).FourlinesexpressedmediumlevelsofLe-b,andsixlinesexpressedlowlevelsofthisfucosylatedglycan.Inall,itseemsthattheexpressionpatternofsLe-abythevariouscelllineswassimilartothatofLe-bandtosomeextenttoLe-a.TheexpressionlevelsofsLe-x,adomi-nantselectinligandonactivatedlymphocytes,wereratherlow.Withtheexceptionoffourcelllines,ofwhich,oneexpressedhighandthreemediumlevelsofsLe-x,allotherlinesexpressedeitherlowlevelsofthisselectinligandordidnotexpressitatall.

Takentogether,theseresultsindicatedthatsLe-aisthedominantselectinligandinallcelllinestested.Becauseofitsrelativehighexpressionbymostofthecolorectalcancercelllinesassayed,weconsidersLe-atobetherepresentativeselectinligandofcolorectalcancercells.

Inviewofthepossibilitythatincolorectalcancer,asinHNSCC(14),thefucose-generatingFXenzymefunctionsasalimitingfactorinsLe-abiosynthesis,westudied,inthefollowingseriesofexperi-ments,itscontributiontosLe-aexpression.

ExpressionLevelsoftheFXEnzymeCorrelatewithThoseofsLe-a

Fig.1showsaregressioncurvecorrelatingtheexpressionofFXproteintothatofsLe-ainseveralcolorectalcancercelllines.A

RESULTS

AnExpressionProfileofFucosylatedGlycansbyColorectalCancerCellLines

Table1showstheexpressionprofileofseveralfucosylatedglycansby12colorectalcancercelllines.Thisprofilewasderivedfromseveralrepetitionsofflowcytometryexperiments.AlllinesexpressedsLe-aaswellasLe-b.HighlevelsofsLe-awereexpressedbyfourcelllinesandmediumlevelsbyfourlines.Fourcelllinesexpressedlow

Fig.1.CorrelationbetweentheexpressionoftheFXproteinandsLe-abycolorectalcancercells.LysatesofcolorectalcancercellswereassayedforFXproteinbyWesternblotanalysisusingantiFXantibodiespreparedinourlab.Anti-glyceraldehyde-3-phosphatedehydrogenase(GAPDH)antibodieswereusedascontrol.ColorectalcancercellswereassayedbyflowcytometryforsLe-aexpression.ThevaluesofsLe-aexpression

͉,1086F,474Œ,0485ϩ,427»,1203Ϫ,werecalculatedasforTable1.HT-29:,044ϫ

SW480s,andSW620E.

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Fig.2.ExpressionofFXproteinandofsLe-abySW480andSW620cells.A,expressionofFXprotein.LysatesofSW480andSW620cellswereassayedforFXexpressionbyWesternblotanalysisusingantiFXantibodies.Anti-glyceraldehyde-3-phosphatedehydrogenase(GAPDH)antibodieswereusedascontrol.ValuesrepresenttheratiobetweenthesignalofFXproteinineachcellandthesignalofGAPDHinthesamecell.B,expressionofsLe-a.SW480andSW620cellswereassayedbyflowcytometryforsLe-aexpression.(Mϭmeanfluorescence,%posϭpercentofpositivecells).Arepresentativeexperiment(ofthreeperformed)ispresented.

significantcorrelationbetweenthesetwoparameterswasseen.AsimilarcorrelationbetweenFXexpressionlevelsandotherselectinligandswasnotfound(resultsnotshown).

MetastaticColorectalCancerCellLineVariantsExpressHigherLevelsoftheFXEnzymeandofsLe-aThantheCorrespondingNonmetastaticVariants

ToassessthecontributionoftheFXenzymetovariousmalig-nancyassociatedcharacteristics,wecomparedcelllineshavinganidenticalgeneticbackgroundbutthatdifferintheirmetastaticphenotype.Highandlowmetastaticvariantsoriginatingfromtwopatientswereused.SW480andSW620cellsoriginatedinonepatient,andKM12C,KM12SM,andKM12L4originatedfromanotherpatient.

SW480andSW620arecolorectalcancercelllinesderived,corre-spondingly,fromtheprimarytumorandfromalymphnodemetas-tasisofasinglepatient.Thiscellpairwasrecentlyvalidatedasanappropriatemodeltostudydifferencesbetweenprimaryandsecond-arytumors(23).

Fig.2AdemonstratesthatthemetastaticSW620cellsexpresshigherlevelsoftheFXenzymethanthenonmetastaticSW480cells.ThesecellsalsoexpresshigherlevelsofsLe-a(Fig.2B).

TheKM12Ccelllinewasestablishedfromaprimarycolorectalcancer,andtheKM12SMandKM12L4linesaremetastaticvariantsofKM12Cderivedfromnudemousexenotransplants.Thesethreecelllinesthushavethesamegeneticbackground(19).TheKM12SMcellsaremoremetastaticthantheKM12L4cells(19).Fig.3Ademon-stratesthatthemetastaticKM12SMvariantexpresseshigherlevelsoftheFXenzymethantheprimaryKM12Ccells.ThemoremetastaticKM12SMvariantexpressestwiceasmuchFXenzymethanthelessmetastaticKM12L4variantinwhichFXexpressionwasonlymar-ginallyhigherthanthatbytheprimaryKM12Ccells.Fig.3BshowsthattheexpressionofsLe-ainthethreecelllinescorrelatedwiththeFXexpressionbythesecells.

Fig.3.ExpressionofFXproteinandofsLe-abyKM12C,KM12L4,andKM12SMcells.PleaseseelegendtoFig.2above.Arepresentativeexperiment(ofthreeperformed)ispresented.

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Fig.4.AdhesionofSW480andSW620cells.A,adhesiontoHUVECs.SW480andSW620cellswerelabeledwithCFDA-SEreagentandincubatedonactivatedHUVECsfor30minutesasdescribedinMaterialsandMethods.Nonadherentcellswereremoved,andthenumberofadherentcellswasdeterminedbyafluorescenceELISAreaderinreferencetoastandardcurve.ThebarsrepresentmeanϮSDofvaluesobtainedinfourindependentexperiments.ء,PϽ0.03.B,adhesiontorE-selectin.CFDA-SE–labeledSW480andSW620cellswereincubatedonrE-selectin–coatedplatesfor45minutesasdescribedinMaterialsandMethods.Percentageofadheringcellswascalculatedastheratiobetweenfluorescencesignalofadheringcellsandthatobtainedfromthetotalnumberofplatedcells.Fluorescenceofthecellswasdeterminedasdescribedabove.ThebarsrepresentmeanϮSDofvaluesobtainedinthreeindependentexperiments.ء,PϽ0.02.

Theresultspresentedaboveestablished,thus,acorrelationbetweenthemetastaticphenotypeofcolorectalcancerandexpressionlevelsoftheFXenzymeandofsLe-a.

TheAdhesiveCapacityofColorectalCancerCellstoActivatedEndothelialCellsandtoE-SelectinIsLinkedtotheExpressionLevelsofEndogenousFXandsLe-a

Fig.4AdemonstratesthatthemetastaticSW620cellsexpressinghigherlevelsofFXandsLe-athanSW480cells(Fig.2),adherebettertoactivatedHUVEC(PϽ0.03).

TotestwhetherthedifferentialadhesionofSW620andSW480cellstoactivatedHUVECsreflectstheiradhesioncapacitytoE-selectin,wemeasuredtheadhesionofthesecellstorecombinantE-selectin.TheresultspresentedinFig.4BshowthatthemetastaticSW620cellsadheresignificantlybettertorE-selectinthanSW480cells(PϽ0.02),suggestingthattheadhesionofthecellsismediatedbyaselectin-selectinligandinteraction.

Similarresultswereobtainedwhencomparingtheadhesivecapac-ityofprimaryKM12CcellswiththatofthemetastaticKM12SMcellvariant.ThehighlymetastaticKM12SMcells,expressinghighlevelsoftheFXenzymeandofsLe-a(Fig.3),adheredbettertoactivatedHUVECs(PϽ0.03;Fig.5A)andtorE-selectin(PϽ0.02;Fig.5B)thantheKM12Ccells,whichexpressalowmetastaticbehaviorandlowlevelsofFXandsLe-a.

TheFXEnzymeDirectlyRegulatestheExpressionofsLe-aandtheSelectin-MediatedAdhesionofColorectalCancerCellsFXcDNATransfection.Theresultspresentedintheprevioussectionsshowapositivecorrelationbetweenexpressionlevelsofthefucose-generatingFXenzyme,expressionlevelsoftheselectinligandsLe-a,andadhesionofcolorectalcancercellstoactivatedHUVECsandrE-selectin.ThenextsetofexperimentswasaimedtodetermineiftheFXenzymedirectlyregulatestheexpressionofsLe-aoncolorectalcancercellsandtheiradhesiontoactivatedHUVECsandrE-selectin.ItwasassumedthatiffucoseisindeedalimitingfactorinselectinligandbiosynthesisincolorectalcancercellsandthusoftheirE-selectin–dependentadhesiontoactivatedHUVECs,thenoverex-Fig.5.AdhesionofKM12CandKM12SMcelllines.A,adhesiontoHUVECs.KM12CandKM12SMcellswerelabeledwithCFDA-SEandincubatedonactivatedHUVECsfor30minutesasdescribedinMaterialsandMethods.ThepercentageofadherentKM12Cwasgivenavalueof1.0.NormalizedadherencevalueswereobtainedbydividingpercentageofadhesionofKM12SMcellsbypercentageofadhesionofKM12Ccells.ThebarsrepresentmeanϮSDofvaluesobtainedinfiveindependentexperiments.ء,PϽ0.03.B,adhesiontorE-selectin.PleaseseelegendtoFig.4Babove.ThebarsrepresentmeanϮSDofvaluesobtainedinthreeindependentexperiments.ء,PϽ0.02.(CϭKM12C,SMϭKM12SM).

pressingtheFXenzymeinlowFX-expressingcellswouldresultinhigherlevelsofselectinligandexpressionandinanup-regulatedadhesivecapacityofFXcDNA-transfectedcells.Bythesametoken,down-regulatingthelevelsofFXbysiRNAshoulddecreasetheexpressionlevelsofselectinligands.TheFXsiRNA-transfectedcellsshould,asaresult,expressadown-regulatedadhesivecapacity.Theexperimentsdescribedbelowdemonstratethatthisisindeedthecase.WefirstoverexpressedtheFXenzymeinSW480cells,whichexpressverylowlevelsofendogenousFXandsLe-a(Fig.2).ThesecellsweretransientlycotransfectedwithFXandGFPcDNA(ina10:1ratio).ThesecellsexpressedhigherlevelsofFXproteinthancontrolcellstransfectedwithGFPalone(Fig.6A).Fig.6BshowsthatthesetransfectantsadheredbettertoactivatedHUVECsthancellstransfectedwithGFPcDNAalone(PϽ0.001)andshowsthattheadhesionoftheFX-transfectantswasreducedtobackgroundlevels(i.e.,adhesiontononactivatedHUVECs)byantibodiesdirectedagainstE-selectin(PϽ0.001).

AdhesionexperimentsinwhichrE-selectinwasusedinsteadofactivatedHUVECs(Fig.6C)yieldedsimilarresults(PϽ0.001).AntibodiesagainstE-selectinreducedtheadhesionoftheFX-transfectantstorE-selectinalsointhiscase(PϽ0.001).

FXsiRNATransfection.Thenextstepwastodown-regulatetheexpressionoftheFXenzymeinSW620cellsbystablytransfectingthemwithFXsiRNA(205cells;ref.20).SW620cellstransfectedwithamutatedFXsiRNAsequenceinthepSupervector(360cells)servedascontrols.NorthernblottingindicatedthattheFXsiRNA-transfected205cellsexpressedlowerlevelsofFXmRNAthanthecontrol-transfected360cells(Fig.7A).TheFXsiRNA-transfected205cellsexpressedalsolowerlevelsoftheFXproteinthanthecontroltransfected360cells(Fig.7B).Itisinterestingtonotethatthedown-regulationofFXproteinwasmoreremarkableattheproteinlevelthanatthemRNAlevel.Noexplanationforthisobservationisavailableatthisstage.Fig.7CdemonstratesthatthesiRNA-trans-fected205cellsexpresslesssLe-athancontroltransfectants.

Fig.8showsthattheFXsiRNA-transfected205cellsadheredlesswelltorE-selectinthancontrol360cells(PϽ0.01).InconformitywiththeresultsreportedinFig.6C,antibodiesdirectedagainstE-

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Fig.6.AdhesivepropertiesofSW480cellstransientlyoverexpressingFXcDNA.A,expressionofFXprotein.SW480cellswerecotransfectedwithFXcDNAandGFP.ControlsweretransfectedwithGFPalone.LysatesweresubjectedtoWesternblotanalysisasdescribedinthelegendforFig.2A.B,adhesiontoHUVECsanditsblockingbyantibodiesagainstE-selectin.ThetransfectedcellswereincubatedonactivatedHUVECs,asdescribedinthelegendforFig.4A.ThebarsrepresentmeanϮSDofvaluesobtainedfromfiveindependentexperiments.PϽ0.001forthedifferencebetweenFXcDNAandcontroltransfectants.SimilarexperimentswereperformedinthepresenceofantibodiesagainstE-selectin.Twoindependentexperimentswereperformed.PϽ0.001forthedifferencebetweentheadhesionofFXcDNAtransfectantsinthepresenceorabsenceoftheantibody.C,adhesiontorE-selectinanditsblockingbyantibodiesagainstE-selectin.ThetransfectedcellswereincubatedonrE-selectin–coatedplates,asdescribedinthelegendforFig.4B.ThebarsrepresentmeanϮSDofvaluesobtainedbycountingseveralfieldsintwoindependentexperiments.Adhesionwasblocked,intwoindependentexperiments,byanti-E-selectinantibodies(␣E-selectin;PϽ0.001).TheadhesionpresentedinBandCwasdeterminedbycountingadherent,GFP-expressingcellsunderafluorescencemicroscope(theaveragenumberofcellsperfieldwasobtainedbycountingseveralfields).ControlϭSW480cellscotransfectedwithcontrolpRc/CMVvectorandGFP;FXcDNAϭSW480cellscotransfectedwithFXcDNAandGFP;seeMaterialsandMethods.

selectinblockedtobackgroundlevelstheadhesionofthecontrol360cellstorE-selectin(PϽ0.001;Fig.8).ThelowadhesionoftheFXsiRNA-transfected205cellstorE-selectinwasalsoblockedtoback-groundlevelsbytheseantibodies(PϽ0.001;Fig.8).Anisotypecontrolantibodyusedintheseexperimentshadnoblockingactivitywhatsoever.

ThetransfectionexperimentsdescribedaboveprovideconclusiveevidencethataFX3selectinligand3adhesionpathwayoperatesincolorectalcancer.However,theinvolvementofotherfucosylatedadhesionmoleculescontrolledbythefucose-generatingFXenzymeintheadhesionofcolorectalcancercannotberuledout.

ThesiRNAtransfectantsadheredlesswellalsotoECM(PϽ0.002;Fig.9A).ToidentifytheECMproteintowhichadhesionwasreducedbydown-regulatingthefucose-generatingFXenzyme,wecomparedtheadhesionofFXsiRNAtransfectants(205cells)andofcontroltransfectants(360cells)tocollagen,laminin,andfibronectin.Whereastheadhesionofbothtypesoftransfectantstocollagenandlamininwassimilar,theFXsiRNA-transfectedcellsadheredsignif-icantlylesswelltofibronectinthanthecontrols(PϽ0.04;Fig.9B).Thisfindingledustotentativelyconcludethatcellularadhesiontofibronectinmaybefucosedependent.

WealsotestedifknockingdownFXlevelsbyFXsiRNAtrans-fectionwouldaffectadhesiontouncoatedtissue-cultureplasticves-selsundernormalcultureconditions.ThreeindependentexperimentsshowedareducedsurvivalofSW620cellsinwhichlevelsofFXwereknockeddownbyFXsiRNAtransfection,ascomparedwithcontrolcellstransfectedwithmutatedFXsiRNA.Atthetimepointof96hoursaftertheinitiationofculture,theaveragenumberofviable-adherentcontrolcellswas1.96-foldhigherthanthatoftheFXsiRNAtransfectants(PϽ0.02).

TheaboveresultssuggestthatFXknockdownmayinfluenceglobalfucosylationofcolorectalcancerglycomolecules.Totestthispossi-bility,weevaluatedlevelsoffucoconjugatesinFXsiRNAtransfec-tants(205cells)andincontrol360cells.Fig.10showsalectinblot

Fig.7.ExpressionofFXmRNA,FXproteinandofsLe-abySW620cellsstablytransfectedwithFXsiRNA.A,FXmRNA.ExpressionwasdeterminedbyNorthernblotanalysis.Valuesrep-resenttheratiobetweenthesignalofFXmRNAinthecellsandthesignalof18SrRNAinthesamecellsample.B,FXprotein.Expressionwasdeter-minedasinFig.2A.C,sLe-a.ExpressionwasdeterminedasinFig.2B.Arepresentativeexperi-ment(ofthreeperformed)ispresented(360cellsϭcontrolcells;205cellsϭFXsiRNAtrans-fectants).Mϭmeanfluorescence;%posϭ%positivecells.

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Fig.8.AdhesionofSW620cellstransfectedwithFXsiRNAtorE-selectin.205and360cellswerelabeledwithCFDA-SEreagentandincubatedonrE-selectininthepresenceofantibodiesagainstE-selectin(␣E-selectin)orofirrelevantantibodiesofthesameisotype(iso.con).Fordetails,pleaseseelegendtoFig.4B.ThebarsrepresentmeanϮSDvaluesobtainedinfourindependentreplicates(ء,PϽ0.01).360cellsϭcon-trolcells;205cellsϭFXsiRNAtransfectants.

ofextractsderivedfromcontrolorFXsiRNA-transfectedcellsde-velopedwiththeUlexeuropaeusagglutinin1agglutinin,afucose-specificlectin(22).Aclearreductioninglobalfucosylationisindi-catedintheFXsiRNAtransfectants.DISCUSSION

Theattachmentofselectinligand-expressingtumorcellstoselec-tin-expressingendothelialcellsiscrucialeventintheinitialstepoftumorcell-endotheliuminteractions,includingextravasation(24–29).Therefore,understandingtheregulationofselectinligandsynthesisintumorcellsiscrucialforthedevelopmentofmetastasis-targetingmanipulations.

Theproperfunctioningofselectinligandsrequirestheirfucosyla-tion(1,30–34),mainlybythefucose-generatingFXenzyme(1,13,14,33,34).ThepresentstudydocumentsafunctionalrelationshipbetweenexpressionlevelsoftheFXenzymeandthoseoftheselectinligandsLe-abycolorectalcancercells.ThisconclusionwasbasedonadirectandpositivecorrelationbetweenexpressionlevelsofFXandthoseofsLe-abyseveralcolorectalcancercelllines.Furthermoredown-regulatingFXexpressionbyFXsiRNAtransfectiondecreasedsLe-aexpression.

WealsodocumentedafunctionalaxislinkingFXandsLe-aex-pressiontothecapacityofcolorectalcancercellstoadheretoE-selectin.ItwasthusfoundthattheFXenzymeisalimitingfactorforthecapacityofatleastthosecolorectalcancercellsusedinthisstudytoadheretoendothelium.Thisconclusionissupportedbothbycorrelativeevidence,aswellasbydirectevidenceprovidedbytrans-fectionexperiments.

Itisassumedthathighlymetastatictumorvariantsextravasatemoreefficientlythancellswithalowmetastaticphenotype(18,26,29).Thisimpliesthattheformervariantswouldadherebettertoendothe-

lialcellsthanthelatterones.Indeed,thehighlymetastaticvariantsSW620(23)andKM12SM(19)adheredbettertoendothelialcellsthanthecorrespondinglowmetastaticvariantsSW480andKM12C.Eachofthesevariantpairsoriginatedfromasinglepatient.ThehighlymetastaticvariantsexpressedalsohigherlevelsoftheFXenzymeandofsLe-athanthevariantsexpressingalowmetastaticphenotype.ItwasinterestingtonotethatKM12SM,themosthighlymetastaticvariantoftheKM12Cprimarytumor(19),expressedhigherlevelsoftheFXenzymeandofsLe-athanKM12L4,thelessmetastaticvariantfromthesametumor.Itseems,therefore,thatthedegreeofmalig-nancycorrelatespositivelywithexpressionofthefucose-generatingFXenzymeandofitsselectinligandproductsLea.

Takentogether,theseresultsleadustohypothesizethattheFXenzymecontrols,byregulatingselectinligandbiosynthesis,theinter-actionofatleastcertaincolorectalcancercellswithendotheliumandthusthecapacitytoextravasateandformmetastasis.Ifthishypothesiswillbeconfirmedbytestingadditionalhighandlowmetastaticcellpairs,theFXenzymecouldbecomeatargetmoleculeforpreventionofmetastasis.

ItwasalsodemonstratedthattheFXsiRNA-mediateddecreaseofFXexpressionbycolorectalcancercellsdown-regulatedtheabilityofthesiRNA-transfectedcellstobindtotheECMproteinfibronectin.Thisraisesthepossibilitythatfucoseisalsoinvolvedintheinterac-

Fig.10.ExpressionoffucosylatedproteinsbySW620cellsstablytransfectedwithFXsiRNA.Lysatesofcontrol(360)andFXsiRNA(205)transfectedcellswereassayedfortheexpressionoffucosylatedproteinsbyWesternblotanalysisusinghorseradishperox-idase-conjugatedUlexeuropaeusagglutinin1.ExpressionofFXproteininthetwocellpopulationswasdeterminedasinFig.2A.Glyceraldehyde-3-phosphatedehydrogenase(GAPDH)servedasloadingcontrol.

Fig.9.AdhesionofSW620cellstransfectedwithFXsiRNAtoECM.205andcontrol360cellswerelabeledwithCFDA-SEreagentandincubatedonECM(A)oronfibronectin(B).Fordetails,pleaseseelegendtoFig.8.ThebarsrepresentmeanϮSDofvaluesobtainedinthreeindependentexperiments.ء,PϽ0.04;ءء,PϽ0.002.360cellsϭcontrolcells;205cellsϭFXsiRNAtransfectants.

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tionbetweentumorcellsandECM.Indeeditwasdemonstratedthatfucosylationofintegrins,e.g.,␣3␤1,isessentialforacorrectassemblyoftheintegrin␣and␤subunitsandforthebindingtofibronectin(35).Fucoseparticipatesnotonlyincontrollingadhesivepropertiesbutalsoinotherfunctionssuchasmodifyingsignaltransductionevents.Forexample,Fringe,afucose-specificglycosyltransferaseinitiateselongationofO-linkedfucoseresiduesattachedtoepidermalgrowthfactor-likesequencerepeatsofNotch(6).Thisglycosylationmodu-latesNotch-mediatingsignaling(5).AnotherexampleistheO-fucosemodificationofCripto.ThismodificationisessentialforNodal-dependentsignaling(7).BecausebothNotchaswellasNodal-medi-atedsignalingbearimportancewithrespecttotumorprogression(9,36–39),itwouldbeofinteresttocomparethesesignalingpathwaysincolorectalcancervariantsexpressinghighorlowlevelsofFX.Takentogether,theresultsofthepresentstudy,aswellasthoseofotherstudiescitedabove,suggestthatalteringexpressionlevelsoftheFXenzyme,therebyalteringglobalfucosylationofcolorectalcancercells,couldhavefarreachingeffectsonthesurvivalandphenotypeofthesecells.

TheFXenzymesuppliesϳ90%ofthecellularfucosewhiletherestissuppliedbythesalvagepathway(1,40).Inthispathway,extracel-lularfucoseistakenupbythecell,phosphorylatedbyafucose-kinase,andsubsequentlyconvertedtoGDPL-fucosebyGDP-fucose-pyro-phosphorylase.TheGDPL-fucosegeneratedbythesalvagepathwayundergoesanidenticalbiosyntheticrouteastheGDPL-fucosegen-eratedbytheFXenzyme(34,40).TofullyassesstheroleoftheFXenzymeinregulatingvariousfucose-dependentreactionsincolorectalcancercellssuchasadhesion,Notch,orCripto-mediatedsignaling,itwouldbeimportanttodeterminetherelativecontributionofthesalvagepathwaytothecellularfucosesupply.REFERENCES

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Corrections

FXEnzymeControlstheAdhesivePropertiesofCRC

InthearticleonhowFXenzymecontrolstheadhesivepropertiesofCRCintheSeptember15,2004issueofCancerResearch(1),someofthelabelinginFigures7and10wasincorrect.Thecorrectedfiguresarebelow.

1.ZipinA,Israeli-AmitM,MeshelT,Sagi-AssifO,YronI,LifshitzV,BacharachE,SmorodinskyNI,ManyA,CzernilofskyPA,MortonDL,WitzIP:Tumor-microenviron-mentinteractions:thefucose-generatingFXenzymecontrolsadhesivepropertiesofcolo-rectalcancercells.CancerRes2004;64:6571–8.

Chromosome11qLOHinHumanBreastCancer

Inthearticleonchromosome11qLOHinhumanbreastcancerintheSeptember1,1994issueofCancerResearch(1),thenameofoneofthecontributingauthorswasmisspelled.ThecorrectspellingisRobertWinqvist.

1.HamptonGM,MannermaaA,WinqvistR,AlavaikkoM,BlancoG,TaskinenPJ,KiviniemiH,NewshamI,CaveneeWK,EvansGA:Lossofheterozygosityinsporadichumanbreastcarcinoma:acommonregionbetween11q22and11q23.3CancerRes1994;54:4586–9.

p110␦IsoformofPI3KinaseinTumorEndothelium

Inthearticleonp110␦IsoformofPI3KinaseinTumorEndothe-liumintheJuly15,2004issueofCancerResearch(1),thenameofoneofthecontributingauthors,JeffreyBrousal,wasmissing.Thecorrectlistofauthorsshouldread:LingGeng,JiahuaiTan,EricHimmelfarb,AaronSchueneman,KenNiermann,JeffreyBrousal,AllieFu,KyleCuneo,EdwardA.Kesicki,JenniferTreiberg,JoelS.Hayflick,andDennisE.Hallahan.Dr.Brousal’saffiliationistheDepartmentofRadiationOncology,VanderbiltUniversitySchoolofMedicine,Nashville,Tennessee.

1.GengL,TanJ,HimmelfarbE,SchuenemanA,NiermannK,BrousalJ,FuA,CuneoK,KesickiEA,TreibergJ,HayflickJS,HallahanDE.Aspecificantagonistofthep110␦catalyticcomponentofphosphatidylinositol3Ј-kinase,IC486068,enhancesradiation-inducedtumorvasculardestruction.CancerRes2004;64:4893–9.

Glioblastoma-FoundingHumanNeuralPrecursors

Fig.7.ExpressionofFXmRNA,FXproteinandofsLe-abySW620cellsstablytransfectedwithFXsiRNA.A,FXmRNA.ExpressionwasdeterminedbyNorthernblotanalysis.ValuesrepresenttheratiobetweenthesignalofFXmRNAinthecellsandthesignalof18SrRNAinthesamecellsample.B,FXprotein.ExpressionwasdeterminedasinFig.2A.C,sLe-a.ExpressionwasdeterminedasinFig.2B.Arepresentativeexperiment(ofthreeperformed)ispresented(360cellsϭcontrolcells;205cellsϭFXsiRNAtransfectants).Mϭmeanfluorescence;%posϭ%positivecells.

Inthearticleonglioblastoma-foundinghumanneuralprecursorsintheOctober1,2004issueofCancerResearch(1),thee-mailaddressofR.Gallishouldhavebeenincludedintherequestsforreprintssection.Dr.Galli’se-mailaddressisgalli.rossella@hsr.it.

1.GalliR,BindaE,OrfanelliU,CipellettiB,GrittiA,DeVitisS,FioccoR,ForoniC,DimecoF,VescoviA.Isolationandcharacterizationoftumorigenic,stem-likeneuralprecursorsfromhumanglioblastoma.CancerRes2004;64:7011–21.

NF-␬BinSquamousCellCarcinoma

InthearticleonNF-␬BinsquamouscellcarcinomaintheSep-tember15,2004issueofCancerResearch(1),theentriesinTables1and2indicatingNF-␬Bmodulatedgenesshouldhavebeenboldfaced.ThecorrectedTables1and2arereproducedbelow.

1.LoercherA,LeeTL,RickerJL,HowardA,GeoghegenJ,ChenZ,SunwooJB,SitcheranR,ChuangEY,MitchellJB,BaldwinASJr,VanWaesC:Nuclearfactor-␬Bisanimportantmodulatorofthealteredgeneexpressionprofileandmalignantphenotypeinsquamouscellcarcinoma.CancerRes2004;64:6511–23.

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Table1Selectedlistofgenesincreasedwithtumorprogression

FunctionGene

Cellcycle/growthCyclinD1*CyclinD2*

Growtharrestspecific5*

Milkfatglobule-EGFfactor8*Proteinphosphatase3*†

Proliferatingcellnuclearantigen*Apoptosis

BaculoviralIAPrepeat*

Bcl-2relatedovariankiller*†Immediateearlyresponse3*Transformationrelatedprotein*†Uchrp*

Inflammation/angiogenesis

Colonystimulatingfactor1*Complementcomponent3*†FGFreceptor*Gro1oncogene*

Histocompatibility2-L*Histocompatibility2-D*Interferonreceptor*

Lymphocyteantigencomplex*Metastasis

Integrin␣3*†Laminin␣5*†Lamininreceptor1

Plasminogenactivator,tissueProcollagentype5␣2Syndecan1*Metabolism

ATPaseH؉transport*†

Branchedchainketoaciddehyd*Cholinekinase*†Cytochromep450*

Glutathione-S-transferase*

Lowdensitylipoproteinreceptor*Mannose-6-phosphatereceptor†Potassiumintermediate*Solutecarrierfamily12*†Stressresponse

Heatshockprotein,70kDa*Heatshockprotein84kDa*Heatshockprotein86kDa*Heatshockproteincognate70*Superoxidedismutase*Signaltransduction

AXLreceptortyrosinekinase*†CD97(EGF-TM7)*†

Interleukin-1receptorassociated*Frizzled7homolog

Growtharrest&DNAdamagespecific*Growthfactorreceptorbound*N-mycdownstreamregulated*P13kinaseregulatory*

Proteintyrosinephosphatase*†Rasp21proteinactivator3*†Ras-relatedC3*

Transferrinreceptor*

Nuclearproteins/transcriptionfactorsActivatingtranscriptionfactor†Breastcancer,earlyonset†Butyrateresponsefactor*HighmobilitygroupAT*Jerky*

Myelocytomatosisoncogene*Nuclearfactor␬Bp105*Sexcombonmidleg-like1†Yes-associatedprotein65kDaRNAprocessing

DEADboxprotein3*DJ-1protein†FGFinducible14NuclearribonucleaseRNApolymerase1-1*

Proteinsynthesis/modificationERO1like*†

RibosomalproteinL27a

Foldchange

SymbolCcnd1Ccnd2Gas5Mfge8Ppp3cbPcnaBirc2Bok1Ier3Trp53UchrpCsf1C3Fgfr4Gro1H2-LH2-DIfnarLy6eItga3Lama5Lamr1PlatCol5a2Sdc1Atp6bBckdkChkCyp1b1Gstm1LdlrM6prKcnn4Slc12a2Hspa5Hsp84Hsp86Hsc70Sod1AxlCd97Il1rakFzd7Gadd45gGrb2Ndr2Pik3r1Ptpn13Rasa3Rac1TrfrAtf2Brca2Brf2Hmga1JrkMycNfkb1Scml1YapDdx3DJ-1Fin14Hnrpa1Rpo1-1Ero1lRpl27a

CloneIDH3084D05H3152D01H3113A12H3126F11H3065C08H3021F12H3074A02H3081D02H3057B07H3142D07

IMAGE:605056H3057D05H3054A08

IMAGE:406823H3051F10H3096A12H3141B11H3118F09H3027D05H3137A03H3002G01H3075G08H3080H11H3156E09H3013F05H3120H04H3136B09H3088E07J0216F07H3133A06H3014C04H3092C05H3054H04H3077B02H3032A08H3042G07H3023G01H3133H01H3130B11H3152F05H3032G06H3042E08H3031A03H3054C02H3153D02G0110H06H3067B08H3118G02H3054E01

IMAGE:477981H3059G03J0221F08H3069F08H3015E08H3029B11H3119F06H3089H11H3072E09H3113B01H3089H07H3018F11H3150D06H3018G01H3111H11H3049D09H3126B01H3009B05

NF-␬BAssociationTargetgeneTargetgeneTargetgene

InhibitorofNF-␬BTargetgeneTargetgeneTargetgeneTargetgeneTargetgeneTargetgeneTargetgeneTargetgeneTargetgeneTargetgene

InhibitorofNF-␬BInducerofNF-␬BInducerofNF-␬BTargetgeneTargetgeneTargetgeneTargetgeneInhibitorofNF-␬BInducerofNF-␬BTargetgeneTargetgeneTargetgene

InhibitorofNF-␬B

LY-2/Ker3.3512.9576.3042.5943.1175.7759.1541.9942.8652.92.18814.6395.1451.98212.3942.3422.9684.54.02915.5972.2722.1692.6675.1032.9482.2472.4556.66222.2893.0372.6896.3032.7782.55114.3033.4372.613.424.7842.4592.2831.9992.7173.1442.4832.2484.2062.8443.2232.0422.2161.6062.2162.0722.8162.8492.2242.9192.3552.0722.9763.1552.3584.9262.7032.5292.183

LY-2/IkB-aM

Ϫ2.266Ϫ2.05Ϫ7.813Ϫ3.525Ϫ2.231Ϫ2.811Ϫ6.013Ϫ3.461Ϫ3.851Ϫ4.142Ϫ4.353Ϫ5.574Ϫ8.303Ϫ1.991Ϫ4.094Ϫ3.431Ϫ3.927Ϫ3.054Ϫ2.613Ϫ3.917Ϫ2.967Ϫ1.619Ϫ2.487Ϫ2.094Ϫ2.034Ϫ2.858Ϫ3.042Ϫ3.252Ϫ4.286Ϫ3.061Ϫ3.226Ϫ3.612Ϫ2.197Ϫ2.145Ϫ6.369Ϫ7.198Ϫ2.34Ϫ10.229Ϫ2.1450.938Ϫ1.352Ϫ1.296Ϫ0.843Ϫ1.407Ϫ2.341Ϫ2.039Ϫ3.539Ϫ9.071Ϫ2.71Ϫ3.875Ϫ1.145Ϫ2.696Ϫ0.604Ϫ2.008Ϫ2.591Ϫ3.329Ϫ2.42Ϫ1.443Ϫ1.154Ϫ1.746Ϫ2.055Ϫ4.611Ϫ3.355Ϫ2.785Ϫ2.328Ϫ2.4561.369

ActivatesNF-␬BActivatesNF-␬BActivatesNF-␬BBindsNF-␬BTargetgeneInhibitorofNF-␬BActivatesNF-␬BActivatesNF-␬BActivatesNF-␬BInhibitsp50

ActivatesNF-␬BInhibitorofNF-␬BActivatesNF-␬B

TargetgeneTargetgene

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Table1Continued

FunctionGene

RibosomalproteinL8RibosomalproteinS18*

UbiquitinactivatingenzymeE1UbiquitinB

UbiquitinconjugatingenzymeE2UbiquitinconjugatingenzymeE3†

Ubiquitinspecificprotease9†StructuralproteinsAlphatropomyosin*Cadherin*

Cappingprotein␣2Dystroglycan1*

Epithelialproteinlost*†Fascinhomolog1*

FourandahalfLIMdomains*Keratincomplex1acidic*Keratincomplex2basic*PDZandLIMdomain1*Protocadherin7*†Thymopoietin*Other

Globininducingfactor†Metallothionein2NexttotheBrca1*†RANbindingprotein*Repeatfamily3gene*Ringfingerprotein19*

Semadomain,immunoglobin*†SuppressorofLec15†

TGF␤inducibletranscript*

Foldchange

SymbolRpl8Rps18Ube1xUbbUbe2hUbe3aUsp9xTpm1Cdh3Cappa2Dag1EplinFscn1Fhl2Krt1-18Krt2-8Pdlim1Pcdh7TmpoGbifMt2Nbr1Ranbp9Llrep3Rnf19Sema3fSupl15hTgfb1i1

CloneIDH3141F09H3006C11H3022E03H3138A08H3057B09H3102B01H3139F12H3120G06H3018F05H3085F12H3008B05H3153C05H3006D08H3033C07H3021B02H3031C01J0824B03H3067F12H3096B08H3053F12H3013D11H3061D04H3013A10H3107F07H3153A08H3134D09H3090D12H3122H01

Bindsp65InflammatoryActivatesNF-␬BActivatesNF-␬BTargetgeneInflammatoryNF-␬BAssociation

LY-2/Ker2.7782.4875.3138.1443.3334.8783.7692.4212.32814.2932.5932.0662.3152.1693.1052.0945.76.5569.9392.0032.1452.3672.873.5562.1482.1912.0013.068

LY-2/IkB-aM1.005Ϫ2.424Ϫ3.139Ϫ1.171Ϫ1.199Ϫ5.096Ϫ3.175Ϫ4.36Ϫ2.706Ϫ7.458Ϫ4.738Ϫ2.971Ϫ2.581Ϫ1.189Ϫ5.517Ϫ5.568Ϫ3.775Ϫ1.026Ϫ8.1531.002Ϫ3.029Ϫ1.888Ϫ3.189Ϫ7.704Ϫ4.402Ϫ1.667Ϫ1.383Ϫ2.974

Phosphorylates␬BLabels␬B

Phosphorylates␬BPhosphorylates␬B

InhibitsI␬BdegradationAccumulatesI␬B␣ActivatesNF-␬B

NOTE.TotalnumberofgenesregulatedbyNF-␬Bϭ105/167.TotalnumberofgenespreviouslyassociatedwithNF-␬Bϭ67/167.*Genescontaining␬Bsiteinpromoterregion.

†GenescontainingACTACAGmotifincodingsequence.

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Table2Selectedlistofgenesdecreasedwithtumorprogression

FunctionGene

Cellcycle/growth

C-srctyrosinekinase*Calmodulin

Celldivisioncyclehomolog25aCelldivisioncyclehomolog45CyclinCCyclinE2*

Cyclindependentkinase4

Cyclindependentkinaseinhibit*Plateletderivedgrowthfactor*Apoptosis

ATPbindingcassette*Bcl2/adenovirusE1B

Fasassociatingw/deathdomainInflammation/angiogenesisCoagulationfactorIII*†Interleukin17receptorInterleukin2receptor

Lymphocyteantigen6complexProthymosin␤Metastasis

Adisintegrin/MMPCadherin1*Kangai1†Lipocalin2

Procollagentype1␣ProcollagentypeII␣ProcollagentypeIII␣SecretedacidicC-richTissueinhibitorofMMPsMetabolism

ATPase,type11A†

ATPsynthaseH؉transportGlutathioneperoxidase*

Lipopolysaccharidebinding*†Phosphoproteinenriched†Pyruvatedehydrogenase*†Sterolcarrierprotein2*†StressresponseCrystallin␣2SignaltransductionAdenylatekinase†

Maxdimerizationprotein4*†MADhomolog4

NF-␬Benhancerinhibitor*NIK-relatedkinase

Phosphoglyceratekinase*Proteintyrosinephosphatase4Rho-associatedcoiled-coilTNFreceptorassociatedfactorNuclearproteins/transcriptionfactorsCbp/p300interactingtransactivationHighmobilitygroupbox1JunoncogeneRas-relatedC3RNAprocessing

Nuclearprotein220†RNApolymeraseII*

Proteinsynthesis/modificationEukaryotictranslation4g2Nedd4WW-bindingprotein4*Structuralproteins

Alpha2glycoprotein1*†Betaspectrin2*†Cateninbeta*Fibronectin†Catenin␣1*†TenascinCOther

Deletedinpolyposis

Insulin-likegrowthfactorr2Ninjurin1*Rabaptin5*†

TopoisomeraseII␣*†

Tumordifferentiallyexpressed†WWdomainbinding5*Zincfingerprotein68*†

Foldchange

SymbolCskCalmCdc25aCdc45lCcncCcne2Cdk4Cdkn1cPdgfaAbcd3Bnip3FaddF3Il17rIl2raLy6Ptmb4Adamts1Cdh1Kai1Lcn2Col1a2Col2a1Col3a1SparcTimp3Atp11aAtp5j2Gpx3LbpPea15Pdha1Scp2Crya2Ak2Mad4Madh4NfkbiaNrkPgk1Ptp4a2Rock1Traf1Cited4Hmgb1JunRac1Np220Rpo2-3Eif4g2N4wbp4Azgp1Spnb2CatnbFn1Catna1TncDp1Igfr2Ninj1Rab5epTop2aTde11Wbp5Zfp68

CloneIDL0237H04H3006H05H3050E04H3003E07C0117F09C0186A01H3147D06H3097D03H3146C02H3143E03H3103B07H3095D08H3014G02H3008A03J0052C08H3115A08H3143A02H3034B07H3076B06H3154D02H3083G02H3125D01H3026G09H3005D11H3026D08H3031E01H3097B05H3118C01J0088G08H3086G08H3014G07H3068G07H3122F12H3143B04H3052D11H3131B07H3128C04H3026A08H3008B02H3023D06H3088F03H3069C09H3015E06H3076H08H3126A05H3058C09H3018C09H3029A07H3055H08H3113E10H3062G06IMAGE:521249H3010G09H3031E05H3116A10H3018E08L0062E01J0420H06H3148G08H3072B10H3002C01H3139A05H3014H10H3127H02H3058F07

NF-␬Bassociation

LY2/KerϪ2.693Ϫ2.554Ϫ2.341Ϫ2.032Ϫ2.739Ϫ2.309Ϫ2.734Ϫ5.208Ϫ2.15Ϫ2.89Ϫ2.364Ϫ3.597Ϫ4.672Ϫ3.021Ϫ2.262Ϫ5.617Ϫ21.739Ϫ2.695Ϫ2.597Ϫ2.816Ϫ2.506Ϫ4.901Ϫ6.896Ϫ3.205Ϫ8.928Ϫ3.3Ϫ2.888Ϫ2.191Ϫ2.604Ϫ2.977Ϫ2.424Ϫ3.021Ϫ2.412Ϫ3.921Ϫ3.755Ϫ2.008Ϫ2.765Ϫ1.433Ϫ2.244Ϫ2.659Ϫ2.118Ϫ2.808Ϫ2.011Ϫ2.178Ϫ3.104Ϫ2.906Ϫ2.004Ϫ2.259Ϫ2.639Ϫ4.698Ϫ6.966Ϫ2.013Ϫ3.781Ϫ3.998Ϫ6.201Ϫ1.996Ϫ2.557Ϫ2.473Ϫ2.765Ϫ2.579Ϫ2.739Ϫ2.427Ϫ2.593Ϫ2.087Ϫ4.122

LY2/IkB-aM

2.3582.422.2692.0292.4182.1552.052.9132.9062.5352.4362.9753.8583.0482.2073.3118.8582.7362.3132.13911.2352.8862.1394.2682.8723.2892.7342.1114.0652.1031.1591.6431.4883.4793.5462.1312.1144.6114.8592.0611.6153.1831.2432.7451.7392.0573.3291.8274.5012.2222.8141.3981.0153.0614.7231.7231.9562.1372.4944.8781.3332.8931.183.3135.885

ActivatesNF-␬BviaIKK

TargetgeneofNF-␬B

NF-␬Bsiteinpromoter

TransientinhibitorofNF-␬BInducerofNF-␬B

ActivatesNF-␬BviaIKKActivatesNF-␬BviaMAPKNF-␬BsiteinpromoterInducerofNF-␬B

AssociatedwithinflammationTargetgeneofNF-␬B

AssociatedwithinflammationInducerofNF-␬B

TwoNF-␬Bsitesinpromoter

InhibitorofNF-␬B

InhibitorofNF-␬BupregulatesIkBanormalhalf-lifeActivatesNF-␬BviaMAPKInducerofNF-␬B

NF-␬BsiteinpromoterInhibitorNF-␬B

ActivatesNF-␬B

NF-␬BdependentNF-␬Bco-activatorBindsp50subunitNF-␬Bco-activatorInducerofNF-␬BCoactivatorofp65

RegulatedbyIKK

NF-␬BsiteinpromoterNF-␬Bsiteinpromoter

InducerofNF-␬B

NOTE.NumberofgenesregulatedbyNF-␬Bϭ47/141.NumberofgenespreviouslyassociatedwithNF-␬Bϭ39/141.*GenescontainingkBsiteinpromoterregion.

†GenescontainingACTACAGmotifincodingsequence.

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