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2002Integrin expression in colon cancer cells is regulated by the cytoplasmic domain of the 6 integrin subunit

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INTEGRINEXPRESSIONINCOLONCANCERCELLSISREGULATEDBYTHE

CYTOPLASMICDOMAINOFTHE6INTEGRINSUBUNIT

JunNIU1,DouglasJ.DORAHY1,XinhuaGU1,R.J.SCOTT1,BrianDRAGANIC1,NuzhatAHMED2andMichaelV.AGREZ1*

1

NewcastleBowelCancerResearchCollaborative,HunterMedicalResearchInstitute,JohnHunterHospitalandFacultyof

MedicineandHealthSciences,TheUniversityofNewcastle,Callaghan,NewSouthWales,Australia

2

GynaecologicalCancerResearchCentre,TheRoyalWomen’sHospital,MelbourneandDepartmentofObstetricsand

Gynaecology,UniversityofMelbourne,Melbourne,Australia

Wehavepreviouslyreportedthatthe

v

6integrinup-

regulatesitsownexpressioninaproteinkinaseC-dependent

mannerwithincreasingcelldensity.Thewild-type

6inte-

grinsubunithasalsobeenshowntopromotetumourgrowth

invivo

anditsgrowth-enhancingeffectisregulatedbybotha

MAPkinasebindingmotifon

6andthe11aminoacid

C-terminalcytoplasmicextensionuniquetothe

6subunit.

Herein,weshowthatthe11aminoacidcytoplasmicexten-

sionisessentialforthecelldensity-dependentincreasein

6

expressionandthatthe11aminoacidtailexertsadominant

negativeeffectoncelldensity-andPKC-mediated

5expres-

sionin

v

6-expressingcoloncancercells.Cellsthatexpress

6lackingthe11aminoacidtailrespondtoPKCsimulation

withincreasedexpressionofonlythe

5subunitasseenfor

cellsthatlackconstitutive

v

6expression.Incontrast,loss

oftheERKbindingsiteon

6markedlyimpairscelldensity-

andPKC-dependentexpressionofeither

6or

5inthe

presenceorabsenceofthe11aminoacidtail,respectively.

Ourfindingssuggestthatin

v

6-expressingcells,ahierar-

chyofkinasesignallingcascadesexistsandthatthe

6-ERK2

interactiondominatesoverPKC-mediatedsignallingpath-

waysresponsibleforintegrinupregulationwithcellconflu-

ence.Giventhedominanceofthe

6-ERK2interactionover

PKC-mediatedexpressionofboth

5and

6integrinsub-

units,targetingthe

6-ERK2interactionmayproveusefulas

ananticancerstrategyincoloncancer.

©

2002Wiley-Liss,Inc.

Keywords:

integrins;celldensity;kinasesignalling;coloncancer

Amongstthevariousfamiliesofcelladhesionmolecules,inte-

grinexpressionpatternsappeartobedirectlyimplicatedinthe

progressionofmalignantdisease.1Integrinsaretransmembrane

glycoproteinreceptorseachcomprisinganalpha()andabeta()

subunitinnoncovalentassociationthatmediatedynamiclinkages

betweentheactincytoskeletonandtheextracellularmatrixaswell

astransducingsignalstoandfromthecellinterior.2–4Withinthe

vsubfamily,thev6integriniseithernotexpressedorex-

pressedatlowlevelsinnormaladultepithelia;however,itbe-

comeshighlyexpressedduringtumourigenesis.5,6Forexample,

inductionofv6expressioninoralleukoplakiaappearstobea

necessaryprerequisiteforprogressiontosquamouscellcancer7

and

denovo

expressionofv6hasbeenobservedinoralsqua-

mousandcoloncancers.8,9Inlungcancer,approximately50%of

tumoursexhibitupregulationofthev6subunit,10andinbreast

cancerv6expressionhasrecentlybeenlinkedtomoreadvanced

tumours.11

Wehavereportedthathighcelldensityina2-dimensional

monolayerculturemodelselectivelyupregulates6integrinsub-

unitexpressionincoloncancercellsinaproteinkinaseC(PKC)-

dependentmannerinpreferencetoothersubunits.12Moreover,

PKCactivityhasbeenshowntoincreasewithcellconfluencein

coloncancercells,andtheriseinPKCactivityismuchgreaterfor

v6-expressingcellsthanforcoloncancercellsthatlackv6.

Hence,wehaveproposedasystemofintegrinautoregulation

wherebytheintegrinv6upregulatesitsownexpressionvia

PKC-mediatedsignallingastumourcellsbecomecrowded.12

Integrincytoplasmicdomainsmediateinside-outandoutside-in

signallingthroughinteractionswithcytoskeletalmoleculesand

intracellularkinases.Functionallinksbetweenboththemajor

integrinsubfamilies,1andv,andthekinasefamilies,PKCand

mitogen-activatedprotein(MAP)kinases,arenowwellrecog-

nised.Forexample,phosphorylationofvand1integrincyto-

plasmicdomainsonserinehavebeenshowntobePKC-depen-

dent13andcollagenI-stimulatedMAPkinaseactivityismediated

specificallythroughthecytoplasmictailofthe2integrinsub-

unit.14Thecytoplasmicdomainofthe6integrinsubunitcontains

an11aminoacidC-terminalextensionnotsharedbyother

integrinsubunits.5Wehavepreviouslyreportedthatheterologous

expressionofv6incoloncancercellspromotestumourcell

growth

invitro

and

invivo,

andthisgrowth-enhancingeffect

requiresthepresenceofthe11aminoacidcytoplasmicexten-

sion.15Moreover,v6expressionincoloncancercellsleadsto

increasedgelatinaseBsecretioninaPKC-dependentmanner,

whichisalsodependentuponthepresenceofthiscytoplasmic

extension.16,17The6cytoplasmicdomainhasrecentlybeen

shownbyustobinddirectlytoextracellularsignal-regulated

kinase2(ERK2),amemberoftheMAPkinasefamily,andthis

physicalinteractiondefinesanovelparadigmofintegrin-mediated

signallingincancer.18Thisbindingeventoccursthroughamotif

onthe6cytoplasmicdomainthatisupstreamofthe11amino

acidtail.Inourstudy,wesoughttoexaminetherespectiverolesof

theERK2-bindingsiteandthe11aminoacidtailinregulating

expressionofintegrinsubunitsthatassociatewithvinSW480

coloncancercells.

MATERIALANDMETHODS

Antibodiesandreagents

MonoclonalantibodiesR6G9andE7P6against6andPIF6

againstthe5subunithavebeendescribedpreviously.19Mono-

clonalantibodiesL230andAP3againstthevand3subunits,

respectively,werepreparedfromhybridomacellsobtainedfrom

theAmericanTypeCultureCollection(ATCC,Rockville,MD).

Themonoclonalantibodyagainstthe1subunit(MAb13)was

purchasedfromBectonDickinson(SanJose,CA).Phycoerythrin-

Grantsponsor:NewSouthWalesStateCancerCouncil;Grantsponsor:

NationalHealthandMedicalResearchCouncilofAustralia;Grantspon-

sor:RoyalAustralasianCollegeofSurgeons;Grantsponsor:NewSouth

WalesDepartmentofHealth;Grantsponsor:JohnHunterHospitalChar-

itableTrustFund.

Thefirsttwoauthorscontributedequallytothiswork.

*Correspondenceto:DisciplineofSurgicalScience,DivisionofSur-

gery,JohnHunterHospital,LockedBag1,HunterRegionMailCentre,

NewSouthWales2310,Australia.Fax:61-2-49-214970.

E-mail:Michael.Agrez@newcastle.edu.au

Received4July2001;Revised14January,12February2002;Accepted

15February2002

DOI10.1002/ijc.10397

Publishedonline8April2002inWileyInterScience(www.interscience.

wiley.com).

Int.J.Cancer:

99,

529–537(2002)

©2002Wiley-Liss,Inc.

PublicationoftheInternationalUnionAgainstCancer


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