Fas, FasL, and cleaved caspases 8 and 3 in glioblastomas: A tissue microarray-based study

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Pathology

–

Research

and

Practice

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / p r p

Original

Article

Fas,

FasL,

and

cleaved

caspases

8 and

3 in

glioblastomas:

A

tissue

microarray-based

study

夽

Fabiano

P. Saggioro

a,∗

,

Luciano

Neder

a

,

João

Norberto

Stávale

b

,

Aline

Nazareth

P. Paixão-Becker

a

_,

_Suzana

_M.F.

_Malheiros

c

_,

_Fernando

_A.

_Soares

d

_,

José

Eymard

H. Pittella

a

_,

_Caio

_César

_M.S.

_Matias

e

_,

_Benedicto

_O.

_Colli

e

_,

Carlos

Gilberto

Carlotti

Jr

e

_,

_Marcello

_Franco

b

a_Department_of_Pathology,_Faculty_of_Medicine_of_Ribeirão_Preto_of_the_University_of_São_Paulo_(FMRP-USP),_Ribeirão_Preto,_SP,_Brazil b_Department_of_Pathology,_Federal_University_of_São_Paulo_{(UNIFESP/EPM),}_São_Paulo,_SP,_Brazil

c_Department_of_Neurology,_Federal_University_of_São_Paulo_{(UNIFESP/EPM),}_São_Paulo,_SP,_Brazil d_Department_of_Pathology,_Hospital_of_Cancer_A.C._Camargo,_São_Paulo,_SP,_Brazil

e_Department_of_Surgery,_Faculty_of_Medicine_of_Ribeirão_Preto_of_the_University_of_São_Paulo_(FMRP-USP),_Ribeirão_Preto,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received3July2013

Receivedinrevisedform3December2013 Accepted30December2013

Keywords: Glioblastoma Cleavedcaspase8 Apoptosis Survival Caspase3

a

b

s

t

r

a

c

t

ThisinvestigationanalyzedtheimmunoexpressionofFasL,Fas,cleavedcaspase-8,andcleavedcaspase-3 inglioblastomas.Formalin-fixedandparaffin-embeddedglioblastoma tissuesandcontrolbrain tis-suesfrom97patientswereanalyzedbytissuemicroarraysandimmunohistochemistry.Patientswith glioblastomasthatwerenegativeorweaklystained(<50%ofcellspositive)forcleavedcaspase-8had worsecancer-specificoverallsurvival(median=8.5months)thandidpatientswithtumorsthathighly expressedcleavedcaspase-8(median=11.7months;P=0.0325),independentofclinicalvariables.There wasnoassociationofothermarkerswithsurvival,treatment,sex,age,tumorsize,andprimarysite. Amongthetumors,therewerereasonabletogoodpositivecorrelationsbetweentheexpressionofFasL andFas(r=0.47)andbetweenFasandcleavedcaspase-8(r=0.41),andtherewerepoorpositive corre-lationsbetweenFasandcleavedcaspase-3(r=0.26),FasLandcleavedcaspase-8(r=0.22),andcleaved caspase-8and-3(r=0.31).OurresultssuggestthatFas-Fas-ligandsignaltransductioncouldbeinhibited, especiallyatthestageofcaspase-8activation,therebyestablishingamajormechanismforevasionof apoptosisbythesetumors.Theabsenceorlowexpressionofcleavedcaspase-8inthetumorswasa negativeprognosticindicatorforpatientsurvival.

Introduction

Glioblastoma(GBM)isthemostcommonmalignantprimary braincancer, andit hasadismal outcome.Despite advancesin diagnosisandtreatment,themediansurvivalofpatientswho suf-ferfrom GBM remains approximately 15 months, accordingto themorerecentstudieswithtemozolomide,becauseofinherent resistanceto both chemo-and radiotherapy [8,9].For decades, surgeryandradiotherapyhavebeenthetraditionalcornerstones

夽_Presented_in_part:_28th_{International}_Congress_of_the International-Academy-of-Pathology,IntAcadPathol,SãoPaulo,Brazil,10–15October2010(Histopathology 2010;57[Suppl1]:200).

∗_{Corresponding}_author_at:_University_of_São_Paulo,_School_Hospital_of_the_Faculty ofMedicineofRibeirãoPreto,AvenidaBandeirantes,3900,CEP14049-900,Ribeirão Preto,SP,Brazil.Tel.:+551636023240;fax:+551636334476.

E-mailaddresses:[email protected],[email protected] (F.P.Saggioro).

oftherapy forGBM.Severalchemotherapeuticagents, including thenitrosoureaderivativesandtemozolomide,havealsobeenused withlimitedsuccess,resultinginmediansurvivaltimesof12–15 monthsandlong-termremissionsinafewtemozolomidepatients

[9,39]. The poor efficacy of these agents is mostly attributed to the highly mutated genome of GBM, which is manifested by the deregulationof many key signalingpathways involving growth,proliferation,survival,andapoptosis[24].Moreover,O6

-methylguanine-DNAmethyltransferase(MGMT),arepairprotein thatspecificallyremovespromutagenicalkylgroupsfromtheO6

positionofguanineinDNA,protectscellsagainstalkylatingdrugs, resultinginresistanceofGBMtothesechemotherapeuticagents

[25].

Apoptosisisabasicbiologicalprocessthatpromotessurvivalof theorganismattheexpenseofindividualcells.Itiswidelyusedby multicellularorganismstoremoveundesirablecellswithout injur-ingneighboring cellsoreliciting aninflammatoryreaction[32]. Nevertheless,tumorcellscanevadeapoptosis,andthusperturb

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thebalancebetweenapoptosisandcellproliferation[14].Because cytotoxicdrugsandradiationtherapyinducetumorcellstodieby apoptosis,understandingthemechanismsinvolvedintheextrinsic apoptoticsignalingpathwayinglioblastomasmayidentifytarget moleculesformoleculartherapies.

TheactivationoftheextrinsicapoptoticpathwayfollowingFas bindinghasbeenwellcharacterized[1,40].Fasligand(FasL)isa typeIImembraneproteinwithanintracellulardomainthat con-tainsconsensussequencesforphosphorylationandanextended proline-richregionthattightlyregulatesFasLsurfaceexpression inthenervoussystem[41].Fas(APO-1/CD95)is a 48-kDatype I membrane protein with a cysteine-rich extracellular domain of155 aminoacids. The triggeringof Fas byits ligandinduces apoptosis in target cells. Although Fas is ubiquitous in human tissues, it is highly expressed in rapidlyproliferating cells and injuredtissues [29].Theoligomerizationof FasbyFasLrecruits theadaptormoleculeFas-associateddeathdomainprotein(FADD) tothe deathdomain (DD) of the Fas intracellular region [4,7]. Procaspase-8(FLICE/MACH1/Mch5),inturn,associateswithFADD toform the death-inducingsignaling complex (DISC), whereby procaspase-8convertsitselftoanactivecleavedform[4,27].Next, thecleavedcaspase-8activatesthedownstreameffector, caspase-3 [21]. Previous reports have demonstrated that the extrinsic apoptotic pathway is severely inhibited in high-grade gliomas

[2,13,14,16,19,26,33,35,44].

Severalfindingshaveindicatedthat thederegulationof apo-ptosisisinvolvedinthedevelopmentofmalignantgliomas.The upregulatedexpressionofFasLanddownregulatedexpressionof caspase-3andcaspase-8inmalignantgliomacellsareinvolvedin gliomagenesis[19,42].Forexample,FasLisimplicatedin glioblas-toma growth and invasion through the induction of apoptosis in infiltrating lymphocytes, which facilitate the evasion of the immunesystembythetumor[19].Inaddition,ithasbeenshown thatglioblastomasareresistanttoFas-relatedapoptosis,showing absentorlowlevelsofcaspases-8andcaspase-3[2,33,38,42].

Because theextrinsic apoptotic pathway in treatment-naïve humanGBMshasnotyetbeensystematicallystudied,weaimed todeterminetheexpressionofFasL,Fas,cleavedcaspase-8,and cleaved caspase-3 in not otherwise specified GBMs using tis-suemicroarraysandimmunohistochemistryandtocorrelatethe expressionofthesemoleculeswithvariousclinicalfindings.We alsoreviewedthemolecularbasisof Fas-mediatedapoptosisin malignantgliomas.

Materialsandmethods

Tissues

Glioblastomaspecimens from97patientswhohad notbeen previouslytreatedwereretrievedfromthearchivesofthe Depart-mentsofPathologyatSãoPauloFederalUniversity (n=60) and RibeirãoPretoMedicineFacultyatSãoPauloUniversity(n=37). The tumor specimens were re-examined and confirmed to be glioblastomasaccordingtothecriteriaofthemostrecentWHO ClassificationofCentral NervousSystemTumors[22].Allofthe patientshadundergonesurgeryduringthe15-yearperiodfrom 1992through2006.ThisstudywasapprovedbytheEthics Commit-teesofbothinstitutions(ResolutionNo.196ofBrazilianNational HealthCouncil).

Tissuemicroarrays(TMAs)

Histologicalsections(4␮m)werecutfromeachtissueblock,

stainedbyhematoxylin–eosin,andcarefullyreviewedby3 inde-pendentpathologists.Theareasmostrepresentativeofeachtumor

wereselectedforanalysis.Cylindricalcoreswereremovedandused intheconstructionoftissuemicroarray(TMA)blocks.FiveTMA blockswereconstructedusingaBeechertissuearrayinstrumentTM

(BeecherInstruments,Silver Spring,MD,USA), accordingtothe manufacturer’sinstructions,inthefollowingstages:(1)Two dif-ferentareasofthetumorweremarkedintheoriginaldonorblock forsampling(necroticzonesandperinecroticpalisadingcellswere notincludedinthesamples), (2)cylindricalholes werecreated inthereceptorblockusingtheTMAplatform.Positionswere cre-atedinthereceptorblocksandwereseparatedbyapproximately 500␮msuchthatamatrixofholesforthetissuesampleswas

cre-ated,(3)1-mmdiametercylindersoftissuewereextractedfromthe areasofinterestinthedonorblocksusinga1-mm-diameterneedle (TMArrayerPunchBeecherInstrumentsTM_),₍₄₎_the_cylindrical

tis-suesobtainedfromthedonorblocksweretransferredtotheholes inthereceptorblocks,and(5)finally,thequalityoftheblocks (rep-resentativenessofthetumorsamples)wasassessedbeforestorage. Twenty-five controlcores obtainedfrom normal brains har-vested from 25 autopsied patients (6–12h postmortem) were includedascontrols.

Immunohistochemistry

Theimmunohistochemicalprocedureswereperformedon

4-␮m-thick sectionsthatwereobtainedfromtheTMAblocksand

mountedonslidespretreatedwith3-minopropyl-triethoxysilane (Sigma).ToaidintheadhesionoftheslicesfromtheTMAblocks tothesilane-treatedslides,anadhesivetapesystem(Instrumedics Inc.,Hackensak,NJ,USA)wasalsoused.

Briefly,forimmunostaining,theslidesweredeparaffinized,and rehydratedthroughagradedethanolseries.Forantigenretrieval, slideswereplaced ina0.01Mcitratebuffer(pH6.0),heatedin a steambath for 3min,and allowedtocool at room tempera-turefor30min.Endogenousperoxidaseactivitywasblockedusing 3%hydrogenperoxidefor15min,followedbywashingin0.05M Trisbuffer(pH9.5).Theslideswerethensubjectedtomicrowave irradiationat700Wfor7min.Theslideswereagainplacedin phos-phatebufferedsaline(0.01MPBS[pH7.4])andallowedtocool atroomtemperaturefor30min.Alloftheimmunomarkersthat wereevaluatedwereexaminedonslides thatunderwent treat-ment forantigen retrieval. The endogenous biotinwasblocked using 0.02M PBS/0.3% Triton X100 (pH 7.4) and 5% skimmilk for 4h at room temperature. Incubation with anti-FasL rabbit polyclonal antibody (C-178, 1:500; Santa Cruz Biotechnology), anti-Fas rabbit polyclonal antibody (FL-335, 1:200; Santa Cruz Biotechnology), anti-cleavedcaspase-8 mouse monoclonal anti-body(AP1013,1:100;Calbiochem),anti-cleavedcaspase-3rabbit polyclonalantibody(AP1027,1:500;Calbiochem),anti-IDH1 rab-bitpolyclonalantibody(AP7454a,1:50; Abgent),or anti-MGMT mousemonoclonalantibody(SPM287,1:150;SantaCruz Biotech-nology)dilutedinPBSwith1.0%bovineserumalbumin(Sigma, USA)lastedfor12hinamoistchamberat4◦_C. _The_slides_were

thenwashed inPBS andincubatedwithsecondary biotinylated antibodyfollowedbystreptavidin–biotin-peroxidase(anti-mouse oranti-rabbitKitLSAB,DAKO)for30mineach.Finally,tovisualize thereactions,theslideswereincubatedwithlight-sensitive3,3′

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andMGMT,respectively.Negativecontrolsconsistedofslidesthat underwentthesameprocedure,excepttheincubationwith pri-maryantibodywaseliminated.

Thestainingpatternswereanalyzedaccordingtotheir distri-butionand intensity, and thepathologists were blindedtothe clinicopathologicaldataoftheGBMpatients.Anumericalscoring systemconsisting of 2 categories wasused toassess FasL, Fas, cleavedcaspase-8andcleavedcaspase-3expression.CategoryA documentedthenumberofimmunoreactivecells(onlyoneswith theirrespectivenucleiinsidewerecounted)asfollows:0or neg-ative(noimmunoreactivecellsor<10%immunoreactivecells),1 (≥10%and<50%immunoreactivecells),or2(≥50% immunoreac-tivecells).

CategoryBdocumentedtheintensityoftheimmunostaining asfollows:0or1(noimmunostainingorweakstaining, respec-tively)or2(moderateorstrong).ThevaluesforcategoriesAand Bweresummedtoprovidean“immunoreactivityscore”, which couldrangefrom0to4.Scoresof0,1,and2wereconsideredto benegativetoweakimmunoreactivityandcalled“low immunoex-pression”.Scoresof3(moderate)and4(strong)wereconsidered tobe“highimmunoexpression”.

IDH1immunostainingwasscoredin thenucleiand/or cyto-plasm,and MGMT were scoredin the nucleiof tumor cells as negative(nostainorlimitedto<10%positivetumorcells)or posi-tive(≥10%tumorcells).

Statisticalanalysis

The immunohistochemistry scoresdetermined for FasL, Fas, cleaved caspase-8, and cleaved caspase-3 expression of the TMAs and control nervous tissues were compared using the Mann–Whitneytest,andcorrelationsineachgroupwere deter-minedusingthenonparametricSpearmantest.

To construct thesurvival curves illustrating overall survival betweenthepatientgroupswith“lowexpression”(scores0,1,and 2)vs.“highexpression”(scores3and4)immunohistochemistry scoresforFasL,Fas,cleavedcasp-8and-3,IDH1,andMGMT,we usedtheKaplan–Meiermethod.Tocomparetheoverallsurvival curves,weusedthelog-ranktest.

Tosimultaneously analyzethe prognosticeffect of the vari-ousfactors(treatment, age,gender,tumorsize,tumorlocation, andtheimmunoexpressionscoresoflowandhighexpressionof FasL,Fas,cleavedcaspase-8,and-3)onthetimeofsurvival,we useda multivariate analysiswiththeCox proportional-hazards regressionmodelusingacovariateofprimaryinterestand adjust-mentcovariates.Allstatisticalanalysesandgraphconstructions wereperformedusingGraphPadPrismversion4.00forWindows (GraphPadSoftwareInc.,SanDiego,CA,USA),andSASversion9 forWindows(SASInstitute,Inc.;Cary,NC,USA).Thelevelof signif-icancewas0.05(P<0.05).Unlessspecified,dataarepresentedas themean±SDormedian.

Results

Ageandsexdistribution

Themeanageofpatientsatdiagnosiswas55.5±14years(range, 18–78years;median=56years),with64.9%ofpatients≥50years ofage,and35.1%<50years.Theagedistributionofpatientswasas follows:<39years,12.4%;40–49years,22.7%;50–59years,23.7%; 60–69years,26.8%;and≥70years,14.4%.Thefemale/maleratio was0.8:1(Table1).Therewerenodifferencesinthesurvivalamong theagegroups(P=0.78)orthegenders(P=0.24)asdeterminedby bothunivariateandmultivariateanalyses(Table3).

Table1

Clinicaldataofpatientswithglioblastomas.

Characteristics No.(%)

Gender Male 53(54.6)

Female 44(45.4)

Age ≥50years 63(64.9)

<50years 34(35.1)

Tumorsizea

Supratentorial ≤5cm 5(9.1)

>5cm 7(12.7)

>5cmwithventricularinvasionor compression

33(60)

>5cmwithmiddle-linecrossoveror infratentorialinvasion

6(10.9)

Infratentorial >5cm 4(7.3)

Primarysite

Frontallobe 25(25.8)

Parietallobe 6(6.2)

Temporallobe 12(12.4)

Occipitallobe 1(1.0)

Supratentorialsitesb ₄₆_(47.4)

Infratentorial 4(4.1)

Notavailable 3(3.1)

Follow-upc

Aliveat1year 29(38.7)

Aliveat2years 8(10.7)

Aliveat3years 3(4)

Aliveat>3to5years 1(1.3)

Lost 10(10.3)

a_Tumor_sizes_available_in₅₅_patients. b_Two_or_more_{supratentorial}_sites. c _Follow-up_of₈₇_patients_(89.7%).

Tumorsizeandlocation

Thesizesof thetumorsat thefirstdiagnosiswereavailable for55patients(Table1).Mostofthem(70.9%)were supratento-rialtumors>5cmthathadinvadedorcompressedtheventricular system (60%) or had crossed over the middle line or invaded infratentorial structures (10.9%). The other21.8% of the supra-tentorialtumorsforwhichtumorlocationshadbeenrecordedin themedicalrecordsweremorecircumscribed,measuring>5cm (12.7%)or≤5cm(9.1%).Thefrontallobealoneorinassociation withtheinvolvementofothersupratentorialstructureswasthe mostaffected(49outof94cases(52.1%)).In4patients,thetumor waslocatedininfratentorialstructures,withthecerebellum, pos-teriorfossa,andpons/medullaservingastheprimarysites.Patient survivalwasnotdependentupontumorsize(P=0.22;Table3)or theprimarysitesofthetumoraccordingtobothunivariateand multivariateanalyses(P=0.08;Table3).

Treatmentandfollow-up

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Table2

FasL,Fas,cleavedcaspase-8,andcleavedcaspase-3immunoexpressionin glioblas-tomasandnormalbraintissuesaccordingtotheMann–Whitneytest.

Protein Glioblastoma Normalbraintissue P

N (%) N (%)

FasL

Positive 46 (50.5) 0 (0) <0.0001

Negative 45 (49.5) 25 (100)

Fas

Positive 62 (68.9) 4 (16) <0.0001

Negative 28 (31.1) 11 (84)

Cleavedcaspase-8

Positive 43 (45.7) 8 (32) 0.0134

Negative 51 (54.3) 17 (68)

Cleavedcaspase-3

Positive 32 (35.2) 1 (4)

Negative 59 (64.8) 24 (96) 0.0011

follow-up (10.3%). Excluding thepatients who died during the

immediatepostoperativeperiod(8postoperativeweeks)andthe 4infratentorialcases,themeanfollow-upperiodwas57.7±53.6 weeksfor 76 patients. Allofthesepatientsshowedresidual or recurrentdiseaseduringthefollow-upperiodanddiedfromcauses relatedtotheirneoplasm.Theoverall5-yearcancer-specific sur-vivalratewas1.3%(Table1).Only1patientwasaliveafter5years offollow-up, andthat patientdiedfromdisease5.6years after diagnosis.

Immunohistochemistry

FasL, Fas, and cleaved caspase 8 were positively expressed (≥10%oftumorcells)inthecytoplasmofglioblastomacellsof46 (50.5%),62(68.9%),and43patients(45.7%),respectively.Cleaved caspase-3waspositivelyexpressedintheglioblastomatissuesof 32patients(35.2%)in thefollowing patterns:cytoplasmic posi-tivitywas observedin 16 tumors, nuclearpositivity in 10, and bothcytoplasmicandnuclearpositivityin6(Table2andFig.1). In normalbrain tissues(controlgroup), theexpression of FasL, Fas,andcleavedcaspase-8andcleavedcaspase-3occurredinthe cytoplasmoftheglialcellsof0(0%),4(16%),8(32%),and1(4%) controlspecimens,respectively(Table2).TheexpressionsofFasL (P<0.0001), Fas(P<0.0001), cleaved caspase-8 (P=0.0134), and cleavedcaspase-3(P=0.0011)weresignificantlyhigherin glioblas-tomathaninnormalglialtissues.Interestingly,onlyGBMswith highorlowexpressionofcleavedcaspase-8wereassociatedwith significantdifferencesintheoverallsurvival(P=0.0325), suggest-ing that low immunoexpression (scores 0, 1, or 2) of cleaved caspase-8inglioblastomaswasindicativeofamorelocally aggres-sivetumor and was a prognostic indicatorof reduced survival (mediansurvival,8.5months;log-rank=4.57,P=0.0325,and haz-ardratio[95%confidenceinterval]=1.64[1.04–2.74])(Table3and

Fig.2).In addition,cleaved caspase-8wasdeterminedtobean independentprognosticfactoraccordingtoamultivariateanalysis (P=0.03,Table3).

Intheglioblastomas,therewerereasonabletogood positive correlations between the expressions of FasL vs. Fas (r=0.47,

P<0.0001) and between Fas vs. cleaved caspase-8 (r=0.41,

P<0.0001)andpoorpositivecorrelationsbetweenFasvs.cleaved caspase-3(r=0.26,P=0.014),FasLvs.cleavedcaspase-8(r=0.22,

P=0.0388),andcleavedcaspase-8and-3(r=0.31,P=0.0026).No correlationswerefoundamongFasL,Fas,andcleavedcaspase-8 andcleavedcaspase-3innormalnervoustissue.

BothIDH1andMGMTwerenegativelyexpressedinall97GBMs despitethepositivecontrolsusedforimmunohistochemistry.

Table3

MultivariateanalysisoftheCoxproportional-hazardsregressionmodelforsurvival inpatientswithGBM.

Variable HR 95%CI P

Age 1.18 0.38–3.67 0.78

Gender 2.07 0.61–7.01 0.24

Tumorsize 4.37 0.42–45.89 0.22

Primarysite 4.83 0.83–28.14 0.08

Treatment

Surgery 1

Surg.+Radiotherapy 1 0.16

Surg.+Rad.+Chemo 1.76 0.80–3.89

FasL

Highexpression 1 0.85

Lowexpression 1.13 0.33–3.93

Fas

Cleavedcaspase-8

Cleavedcaspase-3

Discussion

Deregulationof thenormalmechanism forprogrammedcell

deathplaysanimportantrole inthepathogenesisand progres-sionofgliomas[14,16,20,33].Althoughevidencehasaccumulated thatgene mutations[22],microRNAs[11,36,47],growthfactors

[17,18,37],RNA-bindingproteins[45],DNA-bindingtranscription factors[23],Ca2+_binding_proteins_[31]_,_signal_transduction_proteins

[5,31],andDNAmethylation[15]havecriticalrolesinregulating cellapoptosis,thesignificanceoftheextrinsicapoptoticsignaling pathwayforglioblastomasremainsunclear[19,26].Inthisstudy, weusedTMAtechnologyandimmunohistochemistrytoassessthe expressionofproteinsinvolvedintheextrinsicpathway.Welooked atFasL,Fas,cleavedcaspase-8,andcleavedcaspase-3in treatment-naïvehuman glioblastomas and normal glial cells fromcontrol brainsandexaminedtheseimmunohistochemistryfindingsinthe contextoftheclinicopathologicaldataofthestudypatients.

Death receptors of the tumor necrosis factor (TNF) family, includingTNFR1,Fas(CD95/Apo-1), DR4/DR5,Apo-3(DR3),and theirrespectivecognateligandsTNF-␣,FasL(CD95L/Apo-1L),

TNF-relatedapoptosis-inducingligand(TRAIL/Apo-2L),andApo-3Lcan inducetheextrinsicapoptoticpathwayinthecytoplasmoftumor andnormal glialcells [1].Molecularassays oftheFassignaling pathwayusingyeastandeukaryoticcellshaveshownthatafter thebindingofFasLtotheFasreceptor,Fasbindsdirectlytothe adapterproteinFADD(Mort1)andleadstoapoptoticsignal trans-duction.Inturn,FADDinteractswithcaspase-8throughitsdeath effectordomain(DED),leadingtoDISCassemblyand caspase-8 oligomerization,whichdrivesitsownactivationinthecytoplasm throughself-cleavage.Subsequently,cleavedcaspase-8molecules intheDISCactivatedownstreameffectorcaspases,leadingtothe cleavageofcaspase-3andapoptosis[4,7,21,27].

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Fig.1.ImmunoexpressionofFasL,Fas,cleavedcaspase-8andcleavedcaspase-3inglioblastomas.(A–C)CytoplasmicexpressionofFasLisobservedintumorcells;(A)shows atumorwithhighFasLexpression(scoreof3);(B)(scoreof1)and(C)(scoreof1)showtumorswithlowFasLexpression.(D–F)CytoplasmicexpressionofFasisobserved intumorcells;(D)showsatumorwithhighFasexpression(score3);andE(score1)andF(score0)showtumorswithlow-to-negativeFasexpression.(G–I)Cytoplasmic expressionofcleavedcaspase-8isobservedintumorcells;(G)showsatumorwithhighcleavedcaspase-8expression(score4);andH(score1)andI(score0)showtumors withlowandnegativecleavedcaspase-8expression,respectively.(J–L)Expressionofcleavedcaspase-3incytoplasmand/orinnucleiisobservedintumorcells.(J)shows highexpression(score4)ofcleavedcaspase-3intumorcells,whereasK(score2)andL(score0)showtumorcellswithlowandnegativeexpressions,respectively(original magnifications:×200).

Inaddition,weobservedasignificantdifferenceinFas expres-sionbetweenglioblastomas(68.9%)andnormalglialtissue(16%) andreasonabletogoodpositivecorrelations betweenboth FasL and Fasand Fas and cleaved caspase-8in glioblastomas. Taken together,ourfindingssuggestthatneoplasticallytransformedglial cells increase theexpression ofFasL, Fas, and cleaved caspase-8, indicating the initiation of the extrinsic apoptotic pathway.

MolecularstudieshavedemonstratedthehighexpressionofFas andFasLinmalignantgliomacells,andthesefindingssupportthe conclusionthat theFasL-Fas-dependentapoptoticmechanismis intactandfunctional[14,33].

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Fig.2.Survivalcurvesaccordingtolow(scores0–2)andhigh(scores3and4) immunoexpressionofcleavedcaspase-8inglioblastomas.Patientswithtumorsthat expressedlowlevelsoftheproteinshowedlowermediansurvival(8.5months) comparedtopatientswithtumorsthatexpressedhighlevelsoftheprotein(median survival=11.7months)(Log-rank=4.57,P=0.0325,andhazardratio[95% confi-denceinterval]=1.64[1.04–2.74]).

normalglialtissues.Cleavedcaspase-3wasexpressedin35.2%of theglioblastomasandinonly4%ofthenormalglialtissues.In addi-tion,wefoundthatthelowlevelofexpressionofcleavedcaspase-8 in glioblastomas was associated with a median survival of 8.5 months,whichrepresentsasignificantdecreaseinoverallsurvival comparedtopatientswithglioblastomasexpressinghighlevelsof cleavedcaspase-8(mediansurvivalof11.7months).Thiseffecton survivalwasindependentoftreatment,gender,age,tumorsize, andtumorlocation.Usingaquantitativeimmunoblottingmethod, Ashleyetal.[2]alsofoundthatthecaspase-8proteinlevelsinex vivomalignantgliomasvariedsubstantially.Takentogether,our findingssuggestthathigh-orlow-levelsofexpressionofcleaved caspase-8andcleavedcaspase-3areindependentof clinicopatho-logicalfeaturesandarelikelyimplicatedintumorprogression.

We observed poor correlations between Fas and cleaved caspase-3, between FasL and cleaved caspase-8, and between cleaved caspase-8 and cleaved caspase-3 in thetumors. These results suggest that Fas-induced apoptosis is activated by the extrinsicpathwaybutisinhibiteddownstream. Infact,the Fas-mediatedapoptoticpathwaycanbeinhibitedinglioblastomasat severalstagesbyRIP(receptor-interactingprotein)[3],byc-FLIP (cellular Fas-associated death domain-like interleukin-1beta-convertingenzyme-inhibitoryprotein)[13],byPEA-15/PED (phos-phoproteinenrichedastrocytes-15kDa/phosphoproteinenriched indiabetes)[14,37],byBcl-2[10,12,42]orbythecytokineresponse modifierA(CrmA)[28].Inaddition,theactivationofcaspase-3by caspase-9canbeblockedbythehighexpressionof inhibitorof apoptosisproteins(IAPs)inglioblastomas[28,35,44].

Accordingtotheliterature,afterFasLisboundtotheFas recep-tor,apoptoticsignaltransductionviatheextrinsicpathwayresults intheinteractionofFADDwithcaspase-8throughtheirDED; how-ever,inourstudy,wefoundpoorcorrelationsofcleavedcaspase-8 expressionwithFasL,Fas,andcleavedcaspase-3levels.This find-ingmightbeexplainedbythehighexpressioninglioblastomasof c-FLIPandPED/PEA-15,whichareproteininhibitorsofcaspase-8 activationandcontainDEDdomainsandcanmodifyDISCsinthe non-raftfractionsoftheplasmamembrane[3,13,43].Infact,Bellail etal.[3]showedthatRIP,c-FLIP,andPED/PEA-15canmodifythe DR5-mediatedDISCinTRAIL-sensitiveandresistantglioblastoma cells,leadingtotheinhibition ofcaspase-8cleavageand NF-␬B

activation.

Our results suggest that these proteins mediate the early stagesoftheextrinsicapoptoticpathwayinglioblastomas.FasL binds toFas andsubsequently binds toFADD, transmittingthe signaltoactivatetheextrinsicpathway.Atthisstage,in glioblas-tomas, cleaved caspase-8 may be inhibited, and consequently

apoptosisofthesecells mayalsobeinhibited.Onecouldargue thatthesignalstrengthsdetectedbyimmunohistochemistryinour study,mainlyforcleaved caspases-8andcleaved caspase-3,did notcorrelatewiththeapoptotic morphologyintheGBMs. Two fundamentalexplanationsfortheseresultscouldbepostulated. First,perinecroticpalisadingcells,whereapoptoticfiguresaremore oftenobserved,werenotincludedintheanalyzedsamples. Sec-ond,thereisevidencethatthemolecularmodificationofthedeath receptor-mediatedDISCbyRIP,c-FLIPandPED/PEA-15maycontrol caspase-8cleavageandtheinitiationofapoptosisinglioblastoma cells[3].

Incontrasttootherstudies[6,30],wedidnotobserveany sig-nificantdifferencesinthesurvivalofourpatientcohort’spatient survivalbetweenolderandyoungergroups(<50yearsvs.≥50)or betweenthethreedifferenttreatmentregimens,evenwhenthe datawereadjustedfor theothervariablesstudied.These diver-gentresultsmaybeduetothedifferencesintheagerangesofthe cohorts.Forexample,Ohgakietal.[30]studied715GBMpatients inthefollowingageranges:6.9%were<39years,12.5%between40 and49years,21.1%between50and59,29.9%between60and69, 22.1%between70and79,and7.6%>80years.Inaddition,we ana-lyzedasmallersampleofpatients(n=97)comparedtotheOhgaki etal.’scohort(n=715).Itisimportanttohighlightthattheage dis-tributionofthepopulation-basedstudyofOhgakietal.[30]showed greaterfrequencyofyoungerandolderpatients(40.5%<50years and29.7%≥70)comparedtoourseries(35.1%<50yearsand14.4%

≥70).Thisdifferenceinthesurvival outcomesandresponsesto treatmentcouldbeattributedtothedifferentagedistributions pre-sentedinbothstudies.Similarly,BurgerandGreen[6]studied71 patientswithGBM,35(49%)ofwhomwereyoungerthan45years and36wereolderthan65years(51%);however,inourseries,35.1% ofthepatientswere≤49years,and41.2%were≥60years.

Unfortunately,we didnotobtainanyconclusivelabelingfor MGMT(instead, the controlswerepositive), thoughwe used a robustantibody(SPM287).Infact,thesmalltissuecores(1.0mm) andthewell-knownMGMTimmunolabeling heterogeneitymay havebeenlimitingfactorsinouranalysis,underscoringsomeof thedifficultiesin usingimmunohistochemistrytoassessMGMT expression in formalin-fixed paraffin-embedded GBM tissues, as previously reported in other studies [34,46]. Similarly, the immunohistochemistryfor IDH1wasnegativeinallGBMtissue cores(with positive controls).However,it is importanttonote thatthemajority(ifnotall)ofourGBMcaseswereprimaryGBMs thatdidnotcontaintheIDH1mutation.Althoughweuseda gen-eralIDH1-antibodyinsteadof thewell-establishedantibodyfor thedominantmutant variantoftheenzyme (IDH1-R132H),we do not believe that it impacted our results because no IDH1-immunopositivecellscouldbefoundintheTMAs.Furthermore, thestainingofsuchsmallareaswiththemutation-specificantibody maybeproblematic.

(7)

withtumors that expressedhigher levelsof cleaved caspase-8. Furtherstudiesexaminingmoleculartargetsintheextrinsic path-wayofapoptosisareneededandmayrevealpromisingtreatment strategiesforglioblastomas.

Conflictofinterest

Theauthorsdeclarethattherearenoconflictsofinterest.

Acknowledgments

WewouldliketothankJoaquimSoaresdeAlmeida,who pre-paredthetissuemicroarrays,andMariaJoséCarregosaPinheiro dosSantosfortheirexcellenttechnicalassistance.Thisworkwas supportedbyFundac¸ãodeAmparoaPesquisadoEstadodeSão

Paulo-FAPESP(04/09932-4).WritingassistancewasprovidedbyBioMed

Proofreading,Cleveland,USA.

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