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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
baDepartmentofPathology,FacultyofMedicineofRibeirãoPretooftheUniversityofSãoPaulo(FMRP-USP),RibeirãoPreto,SP,Brazil bDepartmentofPathology,FederalUniversityofSãoPaulo(UNIFESP/EPM),SãoPaulo,SP,Brazil
cDepartmentofNeurology,FederalUniversityofSãoPaulo(UNIFESP/EPM),SãoPaulo,SP,Brazil dDepartmentofPathology,HospitalofCancerA.C.Camargo,SãoPaulo,SP,Brazil
eDepartmentofSurgery,FacultyofMedicineofRibeirãoPretooftheUniversityofSãoPaulo(FMRP-USP),RibeirãoPreto,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.
©2014ElsevierGmbH.Allrightsreserved.
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Presentedinpart:28thInternationalCongressofthe International-Academy-of-Pathology,IntAcadPathol,SãoPaulo,Brazil,10–15October2010(Histopathology 2010;57[Suppl1]:200).
∗Correspondingauthorat:UniversityofSãoPaulo,SchoolHospitaloftheFaculty ofMedicineofRibeirãoPreto,AvenidaBandeirantes,3900,CEP14049-900,Ribeirão Preto,SP,Brazil.Tel.:+551636023240;fax:+551636334476.
E-mailaddresses:fsaggioro@terra.com.br,fsaggioror@terra.com.br (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
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(4m)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 500msuchthatamatrixofholesforthetissuesampleswas
cre-ated,(3)1-mmdiametercylindersoftissuewereextractedfromthe areasofinterestinthedonorblocksusinga1-mm-diameterneedle (TMArrayerPunchBeecherInstrumentsTM),(4)thecylindrical
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. Theslideswere
thenwashed inPBS andincubatedwithsecondary biotinylated antibodyfollowedbystreptavidin–biotin-peroxidase(anti-mouse oranti-rabbitKitLSAB,DAKO)for30mineach.Finally,tovisualize thereactions,theslideswereincubatedwithlight-sensitive3,3′
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)
aTumorsizesavailablein55patients. bTwoormoresupratentorialsites. c Follow-upof87patients(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
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
Highexpression 1 0.11
Lowexpression 0.37 0.11–1.27
Cleavedcaspase-8
Highexpression 1 0.03
Lowexpression 11.5 1.25–106.95
Cleavedcaspase-3
Highexpression 1 0.08
Lowexpression 2.50 0.90–6.93
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+bindingproteins[31],signaltransductionproteins
[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].
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].
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.
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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