High expression of Cullin1 indicates poor prognosis for NSCLC patients

Pathology–ResearchandPractice210(2014)397–401

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Pathology–ResearchandPractice

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OriginalArticle

HighexpressionofCullin1indicatespoorprognosisforNSCLCpatients

MingmingXua,1,XiaomingYangb,1,JinliZhaoc,JianguoZhangd,ShuZhangd,HuaHuangd,YifeiLiud, ,JunhuaLiua,

a

DepartmentofCardiothoracicSurgery,Af liatedHospitalofNantongUniversity,Nantong226001,Jiangsu,ChinaDepartmentofNeuralBiology,NantongUniversity,Nantong226001,Jiangsu,Chinac

DepartmentofRadiology,Af liatedHospitalofNantongUniversity,Nantong226001,Jiangsu,Chinad

DepartmentofPathology,Af liatedHospitalofNantongUniversity,Nantong226001,Jiangsu,China

b

article

info

abstract

Articlehistory:

Received18October2013

Receivedinrevisedform5January2014Accepted30January2014

Keywords:

Non-small-celllungcancerCullin1

ProliferationPrognosis

Background:Cullin1isascaffoldproteinoftheubiquitinE3ligaseSkp1/Cullin1/Rbx1/F-boxproteincom-plexwhichubiquitinatesabroadrangeofproteinsparticipatinginbiochemicaleventslikecell-cycleprogression,signaltransduction,andtranscription.Cullin1isinvolvedintheprogressionofseveralcancers,suchasmelanoma,breastcancer,andgastriccancer.

Methods:ToinvestigatetheroleofCullin1inthedevelopmentofnon-small-celllungcancer(NSCLC),weexaminedtheexpressionofCullin1in8-pairedfreshNSCLCtissues.Wethenconstructedimmuno-histochemistry(IHC)on114paraf n-embeddedslicesandevaluatedthecorrelationbetweenCullin1expressionandclinicopathologicvariables,aswellaspatients’overallsurvival.

Results:WefoundthatCullin1washighlyexpressedinNSCLCtissuesandsigni cantlyassociatedwithNSCLC’shistologicaldifferentiation(P=0.002),clinicalstage(P=0.010)andKi-67(P=0.021).Further-more,weshowedastrongcorrelationbetweenhighCullin1expressionandworseoverallsurvivalratesinNSCLCpatients(P<0.001).CoxregressionanalysisrevealedthatCullin1expressionwasanindependentprognosticfactortopredict5-yearpatientoutcomeinNSCLCcancer(P=0.033).

Conclusion:ThesedatasuggestedthatCullin1mightpromotetheprogressionofNSCLCandbeabiotargetforNSCLC’stherapy.

©2014PublishedbyElsevierGmbH.

Introduction

Lungcancerremainstheleadingcauseofcancer-relateddeathsintheworld,mainlynon-small-celllungcancer(NSCLC)andsmall-celllungcancer(SCLC)[1].NSCLCconsistsoflungsquamouscellcarcinoma,lungadenocarcinomaandlarge-cellcarcinoma,whichisthehotspotoflungcancerresearch.TheincidenceofNSCLCisincreasing,owingtoenvironmentalpollutionandsmokingcigarette.The5-yearsurvivalrateismerely15%,althoughvari-ousadvancingtherapiescomeintouse[2].Lungcarcinogenesisisinvolvedinnumerousgeneticandepigeneticevents,suchasvari-ousoncogenes,tumorsuppressorgenes,cellcycleregulators,and

Correspondingauthorat:Af liatedHospitalofNantongUniversity,19QixiuRoad,Nantong,JiangsuProvince226001,China.Tel.:+8651385052118;fax:+8651385052118.

Correspondingauthorat:Af liatedHospitalofNantongUniversity,19QixiuRoad,Nantong,JiangsuProvince226001,China.Tel.:+8651385050901;fax:+8651385050901.

E-mailaddresses:blue ime@(Y.Liu), ime@(J.Liu).1

MingmingXuandXiaomingYangcontributedequallytothiswork.

DNArepairgenes.Thehighproliferativecapacityoflungtumor-ouscellsappearstobestronglyrelatedtoabnormalregulationthroughcriticalcheckpointsinvolvedincellcycleprogression[3].Soitisespeciallyimportanttoidentifynovelandselectivecell-cycle-relatedtargetsfortherapeuticinterventionofNSCLC.

Cancerdevelopmentistheprocessbywhichnormalcellsaretransformedintocarcinomacellsuponaberrantcellularstimuli.Thisprocessisregulatedbytranscription,translation,post-translationalmodi cationsanddegradationofkeyregulatoryproteinswhichhasacrucialroleinmaintainingandregulatingcel-lularhomeostasis[4,5].Speci cproteolysisofcell-cycleregulatorscanregulatethecell-cycleprogression[6],whiletwomajorE3lig-asecomplexes,Anaphase-promotingcomplex/cyclosome(APC/C)andSkp1/Cullin1/F-boxprotein(SCF)complex,ubiquitinatethetargetgenes[7].AsthemajorcategoryofE3ubiquitinligase,theSCFcomplexisinvolvedintheproteolysisofmaincomponentsofthecell-cycle-relatedproteins[8–10],regulatingtheexpressionofproteinsincellcycleprogression,suchasp53,p27,CDK,andcyclins[6,11–13].Ubiquitin-mediateddegradationofregulatoryproteinsplaysimportantrolesinG1-Stransition,signaltransductionandtranscriptionalregulation[14,15].AnaberrantE3ubiquitin

/10.1016/j.prp.2014.01.015

0344-0338/©2014PublishedbyElsevierGmbH.

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M.Xuetal./Pathology–ResearchandPractice210(2014)397–401

ligasehasbeenimplicatedinthepathogenesisofnumeroushumandiseasesandcontributestodysregulatedcell-cyclecontrolanddifferentiation,whichleadstocarcinogenesis[16–18].Thusitisinvitingtoidentifyregulatorsintheseeventsinordertoresearchcancer.

Cullin1,arigidscaffoldcomponentofSCFcomplexes,isinvolvedintheproteasomaldegradationofnumerouspro-teinsrelatedtocell-cycleprogression.AbnormalexpressionofCullin1resultsinthedysfunctionofSCFE3ligases[17].Var-iousproteinswerereportedtobeinvolvedintheexpressionofCullin1.c-MycpromotedtheexpressionofCullin1;inturn,Cullin1acceleratedubiquitin-dependentproteolysis,aswellascellcycleprogression[19].Thisenzymecouldalsoplayacrit-icalroleintumorprogression,anditspresencewasassociatedwithpoorclinicaloutcomeforseveralcancers[20].AnotherstudyshowedthatCullin1mightfunctionasatumorsup-pressorbyregulatingPLK4proteinlevels[20,21].Cullin1wasalsoamatrixdegradingenzymeknowntobeinvolvedintheremodelingofextracellularmatrixproteins.Inhigh-gradeneu-roendocrinelungtumors,neddylatedformsofCullin1werespeci callyexpressedandassociatedwithahighlevelofcyclinE[10,22].GUANGDICHENandGANGLIfoundthatCullin1expres-sionwasincreasedinearlystagesofmelanomaandregulatedmelanomacellgrowththroughdegradationofp27byfunctionalSCFcomplex[17].LossofCullin1resultedinearlyembryoniclethality,andderegulationofcyclinE.Cullin1over-expressionwassigni cantlyassociatedwithhigh-gradetumorsandpre-dictedpoorprognosisininvasiveductalcarcinomaofthebreast[20].

However,lesswasknownaboutdifferentialCullin1expressionanditsfunctioninNSCLC.Inthisstudy,wedetectedtheexpressionofCullin1in8-pairedfreshlungtissues,andevaluateditsstainingin114NSCLCparaf n-embeddedslicesusingimmunohistochem-istryandanalyzedthecorrelationbetweenCullin1expressionandclinicopathologicparametersandpatients’survival.

Materialsandmethods

Patientsandtissuesamples

The8-pairedfreshsampleswerefrozeninliquidnitrogenimmediatelyaftersurgicalremovalandmaintainedat 80 CuntiluseforWesternblotanalysis.NSCLCtissueswereobtainedfrom114patientsduring2005and2007,whounderwentlungresec-tionwithoutpreoperativesystemicchemotherapyattheSurgeryDepartmentoftheAf liatedHospitalofNantongUniversity.Forhistologicexamination,alltissueportionswere xedinformalinandembeddedinparaf n.Themainclinicalandpathologicvari-ablesofthepatientsaresummarizedinTable1.Thefollow-uptimewas5years,witharangeof1–rmedcon-sentfortissueusewasobtainedfromallpatients.AllhumantissuewascollectedusingprotocolsapprovedbytheEthicsCommitteeofNantongUniversityCancerHospital.

Westernblotanalysisandantibodies

Tissueandcellproteinswereimmediatelyhomogenizedinahomogenizationbuffer(RocheDiagnostics).Proteinconcen-trationsweremeasuredwithaBio-Radproteinassay(BioRad,Hercules,CA,USA).ProteinswereseparatedwithSDS-PAGEandthentransferredtoPVDFmembranes(Millipore,Bedford,MA).Themembraneswereblockedwith5%no-fatmilkinTBST(150mMNaCl,20mMTris,0.05%Tween-20)for2hatroomtemperaturelater.ThenthemembraneswerewashedwithTBSTforthreetimes.Themembraneswereincubatedovernightwithrabbit

Table1

Cullin1andKi-67expressionandclinicopathologicparametersin114NSCLCspecimens.

Parameters

Total

Cullinexpression

P-value

Low

HighAge(year)<604718290.089

≥6067

37

30

GenderMale6530350.706

Female

49

25

24

Tumorsize(cm)<37841370.227

≥3

36

14

22

SmokingstatusYes4720270.345

No

67

35

32

HistologicaltypeAdenocarcinoma

4827210.328

Squamouscellcarcinoma532330Adenosquamouscarcinoma13

5

8

ClinicalstageI40*

27130.010II421626III

32

12

20

HistologicaldifferentiationWell4028120.002*

Mod401723Poor

34

10

24

Lymphnodestatus04926230.450

>0

65

29

36

Ki-67expressionLow452817*

0.021High

69

27

42

Note:StatisticalanalyseswereperformedbythePearson 2test.*

P<0.05wasconsideredsigni cant.

anti-humanGAPDHpolyclonalantibody(diluted1:1000)andmouseanti-humanCullin1monoclonalantibody(diluted1:500)fromSantaCruzBiotechnology,USA,andlaterhorseradishperoxidase-linkedIgGasthesecondaryantibodies.Thebanddensitywasmeasuredwithacomputer-assistedimage-analysissystem(ImagingTechnology,Ontario,Canada),andnormalizedagainstGAPDHlevels.Valueswereresponsibleforatleastthreeindependentreactions.

Immunohistochemistry(IHC)stainingandevaluation

Immunostainingwasperformedusingtheavidinbiotinperox-idasecomplex.Thesectionsweredeparaf nizedusingagradedethanolseries,andendogenousperoxidaseactivitywasblockedbysoakingin3%hydrogenperoxidefor10min.Andthen,thesectionswereprocessedin10mmol/Lcitratebuffer(PH=6.0)andheatedto121 Cinanautoclavefor20mintoretrievetheantigen.AfterrinsinginPBST(PH=7.2),thesectionswerethenincubatedwithanti-Cullin1antibody(diluted1:400;SantaCruzBiotechnology,USA)andanti-Ki-67antibody(diluted1:400;SantaCruzBiotech-nology,USA)for2hatroomtemperature.Negativecontrolslideswereprocessedinparallelusinganonspeci cimmunoglobulinIgG(SigmaChemicalCo.,St.Louis,MO)atthesameconcentrationastheprimaryantibody.Allslideswereprocessedusingtheperoxidase-anti-peroxidasemethod(DAKO,Hamburg,Germany).AfterbeingrinsedinPBST,theperoxidasereactionwasvisualizedbyincu-batingthesectionswithDAB(DAKO).Afterrinsinginwater,thesectionswerecounterstainedwithhematoxylin,dehydrated,andcoverslipped.

M.Xuetal./Pathology–ResearchandPractice210(2014)397–401

399

Fig.1.Expressionpro lesofCullin1inNSCLCandnon-tumorousadjacenttissues.

Immunohistochemicalevaluation

Theintensityanddistributionpatternsofthestainingreactionwereevaluatedby3blinded,independentpathologists.Forassess-mentofCullin1andKi-67, vehigh-power eldsineachspecimenwereselectedrandomly,cytoplasmandnuclearstainingwereexamined.Morethan1000cellswerecountedtodeterminethemeanpercentage,whichrepresentedthepercentageofimmunos-tainedcellsrelativetothetotalnumberofcells.Inmorethanonehalfofthesamples,stainingwasrepeatedthreetimestoavoidtechnicalerrors,andaconsensuswasachieved.ToevaluatetheCullin1proteinimmunoreaction,stainingintensewasclassi edtobe0(negatively,poorlyormoderatelystaining),1(stronglystain-ing).WhenevaluatingtheKi-67proteinimmunoreaction,stainingwasscoredinasemi-quantitativefashion.Acut-offvalueof50%ormorepositivelystainednucleiwasusedtode neKi-67staining:lowexpressiongroup(<50%)andhighexpressiongroup(≥50%).

mainlyinthenuclei(Fig.2).Tumorigenesiswasassociatedwithmultiplefactors,andCullin1overexpressionmightbeanimpor-tantfactorinNSCLC.Thus,wehypothesizedthatoverexpressionofCullin1mightcontributetotheproliferationofNSCLC.

CorrelationofCullin1expressionwithclinicopathologicfeaturesinNSCLC

Statisticalanalysis

TheclinicopathologicdataofthepatientsaresummarizedinTable1.WeevaluatedtheassociationofCullin1andKi-67expres-sionwithclinicopathologicvariables.ForstatisticalanalysisoftheexpressionofCullin1andKi-67,thecarcinomaspecimensweredividedinto2groups,aspreviouslydescribed.ExpressionofCullin1wassigni cantlyassociatedwithhistologicaldifferen-tiation(P=0.002)andclinicalstage(P=0.010),buttherewasnorelationbetweenCullin1expressionandotherprognosticfactors,likeage,gender,tumorsize,histologicaltype,andlymphnodesta-tus(Table1).Furthermore,inmostspecimens,thehighexpressionofCullin1wassimilartothehighexpressionofKi-67(P=0.021).

StatisticalanalysiswasperformedusingtheSPSS13.0statisticalprogram.TheassociationbetweenCullin1andKi-67expressionandtheclinicopathologicfeatureswasanalyzedusingthe 2test.Foranalysisofsurvivaldata,Kaplan–Meiercurveswereconstructed,andthelog-ranktestwasperformed.MultivariateanalysiswasperformedusingCoxproportionalhazardsmodel.P<0.05wasconsideredstatisticallysigni cant.

Cullin1wassigni cantlyassociatedwiththesurvivalofNSCLCpatients

Results

CullinwashighlyexpressedinNSCLCtissues

TheexpressionofCullin1wasassessedineightpairedNSCLCandadjacentnon-tumoroustissuesbyWesternblotassay.WefoundthatCullin1proteinwashighlyexpressedinNSCLCtis-suescomparedwiththeadjacentnormaltissue(Fig.1AandB).Tocon rmtheexpressionofCullin1inNSCLC,weinvestigatedtheexpressionofCullin1byusingIHCon114samplesfrompatientswithNSCLC.Expectedly,Cullin1washighlyexpressedinpoordif-ferentiatedspecimenscomparedwithwelldifferentiatedones,whichhadthesametendencywithKi-67.RepresentativeexamplesofreactivityforCullin1andKi-67areshowninFig.2.Cullin1wasexpressedmainlyinthecytoplasmwhereasKi-67wasexpressed

Survivalinformationwasavailableforallthe114patients.Whenallvariableswerecomparedseparatelywithsurvivalstatus,Cullin1(P=0.033),Ki-67(P<0.001),histologicaldifferentiation(P<0.001),tumorsize(P=0.002),andclinicalstage(P=0.023)signi cantlyin uencedthepatients’survival(Table2).Inunivariateanaly-sis,theKaplan–MeiersurvivalcurvesshowedthathighCullin1expressioncorrelatedwithpoorsurvivalwithstatisticalsigni -cance(P<0.001;Fig.3).Inaword,multivariateanalysisusingtheCox’sproportionalhazardsmodelprovedthatCullin1(P=0.033;Table2)wasaprognosticfactorforpatients’overallsurvival.

Discussion

Inthisstudy,weshowedthatCullin1expressioncorrelatedwithseveralclinicopathologicalfactorssuchashistologicaldifferentia-tionandtumorsizein114NSCLCpatients.Therefore,Cullin1couldbeconsideredtoplayanimportantroleinpromotingNSCLCpro-gression,andtheidenti cationofthisproteinmightbehelpfulinpredictingoutcomeandimprovingprognosticmodels.

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Fig.2.RepresentativemicrophotographsforCullin1byIHCinNSCLC.

Table2

ContributionofvariouspotentialprognosticfactorstosurvivalbyCoxregressionanalysisin114NSCLCspecimens.

Hazardratio

AgeGenderTumorsize

SmokingstatusHistologicaltypeClinicalstage

HistologicaldifferentiationLymphnodestatusCullin1expressionKi-67expression

95.0%Con denceinterval

P0.3340.1420.002*0.1900.2050.023*<0.001*0.0550.033*<0.001*

1.2400.6912.0751.4160.7651.3642.7580.6710.5772.464

0.802–1.9200.421–1.1321.305–3.3010.841–2.3850.505–1.1581.044–1.7842.020–3.7650.447–1.0080.347–0.9581.495–4.059

Note:StatisticalanalyseswereperformedbytheCoxregressionanalysis.*

P<0.05wasconsideredsigni cant.

Fig.3.CorrelationbetweenCullin1expressionandpatients’survival.

Asageneralrule,normalcellproliferationdependsonanorderlyandef cientlycellcycleprocesswhichensurestheduplicationandtransductionofgeneticinformationduringcellgeneration[23].Dysregulationofcellproliferationalwaysledtoanabnor-malcellcycle,whichwasthefundamentalofcarcinogenesis.Theubiquitin–proteasomesystemplayedacrucialroleinmain-tainingthebalancebetweennormalgrowthanduncontrolledproliferation,regulatingcellularhomeostasisandcontrolingtheabundanceofavarietyofcellularproteins,including -catenin,P27,andcyclins[24–27].TheSCFcomplex,acoreelementoftheubiquitin–proteasomesystem,playedwellestablishedrolesincellgrowth.Cullins,asscaffoldproteins,couldendowmulti-mericcomplexofE3ligaseswithsubstratespeci city[28].Cullin1,asascaffoldproteinoftheSCFcomplex,hadakeyroleintheubiquitin-dependentdegradationpathwayregulatingtheexpres-sionofcyclins(cyclinD1andcyclinG1)andCDKinhibitors(p27andp21)[29,30].Cullin1-mediatedsubstratedegradationdictatedawiderangeofcellularprocessessuchasproliferation,differ-entiation,andapoptosis[23].PreviousstudiesfoundthatCullin1regulatedcyclinEdegradation[28].LossofCullin1resultedinearlyembryoniclethalityanddysregulationofcyclinE[31].

ManyrecentstudieshavedemonstratedthatCullin1overex-pressionisassociatedwithvariousmalignanttumors,likegastriccancerandmalignantmelanoma[24,32].Cullin1mightintegratewithSkp2toregulateG1-StransitioningastriccancerviaSkp2-dependentp27degradation[24].Inparticular,Cullin1,themostcharacterizedmemberoftheCullinfamily,predictedpoorprog-nosisofpatientswithgastriccarcinomawhenoverexpressed[24].HighCullin1expressionwassigni cantlycorrelatedwithworseoverallsurvivalinbreastcancerpatients[33].Inaddition,Cullin1wasreportedtopromotetrophoblastinvasionandmigration,basedonseverallinesofevidence[29].Sinceuncontrolledcelldivisionwasafeatureofoncogenesis,itwastemptingforustoconjec-turetheroleofCullin1inNSCLC.Firstly,wefoundthatCullin1washighlyexpressedinNSCLCfreshtissuescomparedwithadja-centnormaltissues(Fig.1).Thiswascon rmedbyIHCon114

M.Xuetal./Pathology–ResearchandPractice210(2014)397–401

401

paraf n-embeddedslices,whichshowedthatCullin1wascorrela-tivelyexpressedwithKi-67.BothCullin1andKi-67werehighlyexpressedinpoorlydifferentiatedNSCLCslices(Fig.2).Besides,Cullin1wassigni cantlyassociatedwithhistologicaldifferenti-ation(P=0.002),clinicalstage(P=0.010),andKi-67(P=0.021;Table1).MultivariateanalysisusingtheCox’sproportionalhazardsmodelindicatedthatCullin1mightbeanindependentindicatorofNSCLCpatients’prognosis(P=0.033;Table2).SurvivalcurverevealedthathighCullin1expressioncorrelatedwithpoorsurvivalwithstatisticalsigni cance(P<0.001;Fig.3).Thus,our ndingssupportedthenotionthatCullin1couldbeapositiveregulatorandpotentiallyatherapeutictargetofNSCLC.Sofar,thereisbarelyanystudyaboutCullin1inNSCLC;inthisstudywearethe rsttostudythefunctionofCullin1onNSCLC.

Insummary,wefoundthathighCullin1expressionwassigni -cantlyassociatedwithhighhistologicalgradeandKi-67expressionaswellaspoorprognosisinpatientswithNSCLC.However,alargergroupofpatientsneedstobeinvestigatedtocon rmthisconclu-sion.Consequently,Cullin1expressioncouldaffectoverallsurvivalwithtumorprogressionandthusrepresentedapotentialtargetforthetreatmentofNSCLC.Becausecellproliferationhadessen-tialrolesincarcinogenesis,controllingofcancercellproliferationwasimportantforinhibitingcancerprogression.Ourpresentstud-iessupportedanimportantroleforCullin1inpromotingNSCLCprogression,andsuggestedapossiblepathologicalmechanismofNSCLC.AlltheseindicatedthatCullin1mightfunctionasatherapytargetforNSCLC.Thereby,inordertoclarifythemolecularmecha-nismsofCullin1inNSCLCpathogenesis,furtherstudiesareofgreatnecessity.

Paraf n-embeddedtissuesectionswerestainedwithantibodiesforCullin1andKi-67andcounterstainedwithhematoxylin(A–L).BothCullin1andKi-67werehighlyexpressedinlungsquamouscellcarcinomacells(A,B,E,F,I,J)andlungadenocarcinomacells(C,D,G,H,K,L).AccordingtotheintensityofCullin1expression,wedividedthesamplesintohistologicdifferentiationgradeI(A–D),histologicdifferentiationgradeII(E–H),andhistologicdifferentiationgradeIII(I–L).

Kaplan–MeiersurvivalcurvesforlowversushighCullin1expressionon114patientswithNSCLCshowedahighlysigni cantseparationbetweencurves(P<0.001).

Con ictofinterest

Alltheauthorsdeclarenocon ictofinterest.

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