MicroRNAs as Potential Agents to Alter Resistance to Cytotox

PriorityReport

MicroRNAsasPotentialAgentstoAlterResistancetoCytotoxicAnticancerTherapy

JoanneB.Weidhaas,ImranBabar,SunithaM.Nallur,PhongTrang,SarahRoush,

212

MichelleBoehm,ErinGillespie,andFrankJ.Slack

DepartmentofTherapeuticRadiology,YaleUniversitySchoolofMedicine;and2DepartmentofMolecular,CellularandDevelopmentalBiology,YaleUniversity,NewHaven,Connecticut

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Abstract

Tumorcellsusepreexistingprosurvivalsignalingpathwaystoevadethedamagingandcytotoxiceffectsofanticanceragents.Radiationtherapyisaprimaryformofcytotoxicanticancertreatment,butagentsthatsuccessfullymodifytheradiationresponseinvivoarelacking.MicroRNAs(miRNA)areglobalgeneregulatorsthatplaycriticalrolesinoncogenesisandhavebeenfoundtoregulateprosurvivalpathways.However,thereislittleunderstandingofhowcellularmiRNAexpressionaffectstheresponseofacancertocytotoxictherapyandultimatelyoutcome.Thelet-7familyofmiRNAsregulatesexpressionofoncogenes,suchasRAS,andisspecificallydown-regulatedinmanycancersubtypes.Infact,lowlevelsoflet-7predictapooroutcomeinlungcancer.Here,wereportthatthelet-7familyofmiRNAsisoverrepresentedinaclassofmiRNAsexhibitingalteredexpressioninresponsetoradia-tion.Morestrikingly,wealsocancreatearadiosensitivestatewhentheselectlet-7familyofmiRNAsisoverexpressedinvitroinlungcancercellsandinvivoinaCaenorhabditiselegansmodelofradiation-inducedcelldeath,whereasdecreasingtheirlevelscausesradioresistance.InC.elegans,weshowthatthisispartlythroughcontroloftheproto-oncogenehomologuelet-60/RASandgenesintheDNAdamageresponsepathway.ThesefindingsarethefirstdirectevidencethatmiRNAscansuppressresistancetoanticancercytotoxictherapy,acommonfeatureofcancercells,andsuggestthatmiRNAsmaybeaviabletooltoaugmentcurrentcancertherapies.[CancerRes2007;67(23):11111–6]

Introduction

Radiationtherapyisoneofthethreeprimarymodalitiesusedincancertreatment.Althoughradiationhasbeeninpracticeforoveracentury,theglobalgeneticresponsenecessaryfortissuestosurviveradiation-inducedinjuryremainslargelyunknown.Thishaslimitedtheabilitytodevelopmeaningfulroutestominimizenormaltissuetoxicitywhileenhancingtumoreradication.Al-thoughsingle-proteintargetingstrategieshaveshownmoderatesuccessinpreclinicalmodels,fewhavebeensuccessfulinhumantrials.Afailuretoidentifyradiationmodulatorsmaybeduetothecomplexgeneticcellularresponsetoradiation,asindicatedby

Note:SupplementarydataforthisarticleareavailableatCancerResearchOnline(http://doc.guandang.net/).

I.BabarandS.M.Nallurcontributedequallytothiswork.

Requestsforreprints:JoanneB.Weidhaas,DepartmentofTherapeuticRadiology,YaleUniversitySchoolofMedicine,333CedarStreet,P.O.Box208040,NewHaven,CT06520.Phone:203-737-4267;Fax:203-785-6309;E-mail:joanne.weidhaas@yale.eduorFrankJ.Slack,DepartmentofMolecular,CellularandDevelopmentalBiology,YaleUniversity,P.O.Box208103,NewHaven,CT06520.E-mail:frank.slack@yale.edu.I2007AmericanAssociationforCancerResearch.doi:10.1158/0008-5472.CAN-07-2858

microarraystudiesshowingsignificantchangesintheexpressionofatleast855genes(>1.5-fold)within4hofradiation(1).Thissuggeststhatregulatorymoleculescapableofaffectingexpressionlevelsofalargenumberoftargetgenesinarapidmannermayberequiredtoaffecttheradiationresponse.OnesuchclassofpotentialregulatorsismicroRNAs(miRNA;ref.2).

miRNAsaresmallnoncodingRNAsfoundinplantsandanimalsthatcontrolgeneexpressionbybindingtocomplementarysitesontargetmRNAtranscripts.ThefoundingmembersofthemiRNAfamily,lin-4andlet-7,wereidentifiedinCaenorhabditiselegans(3,4),wheretheywerefoundtoplaycriticalrolesindevelopmentandprogenitorcelldifferentiation.Thelin-4andlet-7miRNAsareevolutionarilyconservedinhigheranimals,includinghumans,andrecentstudiesfromourlaboratoryandothershaveshownrolesforthese(andothermiRNAs)inhumancancers(seeref.2forreview).let-7isamemberofasmallfamilyofmiRNAs,includingmir-84andmir-48,inC.elegansandlet-7athroughlet-7hinhumans(5,6),whicharedown-regulatedinlungcancer(7),afindingassociatedwithapooroutcomeforthesepatients(8).Thelet-7familyofmiRNAshasbeenshowntoregulatethehumanRASoncogene(7),theoverexpressionofwhichiscommonlyfoundinhumantumors.RASoverexpressionintumorsisconsideredapoorprognosticfeatureandisbelievedtobeinvolvedintheresponsetocytotoxictherapy(9).Recently,RASsignalingwasshowntobecriticalforprotectionfromradiation-inducedreproductivecelldeath(10),theprimaryformofradiation-inducedtargetcelldeath(11).Unfortunately,strategiesdirectlytargetingRASoritsupstreamand/ordownstreameffectorshavenotsuccessfullyalteredtheradiationresponseinvivo(12).BecausemiRNAstargetRASaswellashundredsofadditionalgenes(13),wehypothesizedthattheirmanipulationmightmoresuccessfullyaltertheradiationresponse.

MaterialsandMethods

miRNAmicroarrays.TotalRNAwascollectedfromcellsusingthemirVanakitfromAmbion(permanufacturer’sinstructions).Atotalof10AgwasusedformiRNAmicroarraybyLCSciences.ToconfirmthequalityoftheRNA,aUVtestwasperformedandthesampleswereenrichedformiRNAsbyusingacutofffilter(um100fromMicrocon-modifiedprocedure).ThemicroRNAswerethenlabeledandhybridizedtoamicroarraychipwithmultiplerepeatregionsandamiRNAproberegion,whichdetectsmiRNAtranscriptslistedinSangermiRBaserelease8.2.Thisconsistsof440humanmiRNAsequences.Multiplecontrolprobeswereincludedineachchip.Thecontrolprobeswereusedforqualitycontrolsofchipproduction,samplelabeling,andassayconditions.Forthein-depthdataanalysisofourtimepointexperiments,LCSciencesperformedmultiarraynormalization,ANOVA,andclusteringanalysis.TheANOVAandclusteringanalysiswereperformedonratiodataofinpidualarrays(withthemultiarraynormalization)insteadoftheoftenusedintensitydataofinpidualsamples.TheyfoundthisnecessarytorevealtherathersmallmiRNAvariationsamongthesamplesofdifferenttime

http://doc.guandang.net11111CancerRes2007;67:(23).December1,2007

CancerResearch

Figure1.Relativelevelsofthelet-7microRNAschangesignificantlyafterirradiation.A,miRNAmicroarrayswereperformedontotalRNAcollectedfromirradiatedA549cellsbeforeand2,8,and24hafter2.5Gy.Thele …… 此处隐藏：22054字，全部文档内容请下载后查看。喜欢就下载吧 ……