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Peptides
jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s
Gonadotropin-releasing hormone neuropeptides and receptor in human breast cancer: Correlation to poor prognosis parameters
Kalliopi Pazaitou-Panayiotou
a, Christina Chemonidou
b, Aliki Poupi
b, Maria Koureta
b,
Athina Kaprara
b, Maria Lambropoulou
c, Theodoros C. Constantinidis
d, Grammati Galaktidou
a, Maria Koffa
e, Anastasia Kiziridou
a, Stylianos Kakolyris
f, George Kolios
b, Alexandros Kortsaris
g, Ekaterini Chatzaki
b,∗aDepartmentofEndocrinology– EndocrineOncology,“Theagenio”CancerHospital,Thessaloniki,Greece
bLaboratoryofPharmacology,FacultyofMedicine,DemocritusUniversityofThrace,Alexandroupolis,Greece
cLaboratoryofHistology-Embryology,FacultyofMedicine,DemocritusUniversityofThrace,Alexandroupolis,Greece
dLaboratoryofHygieneandEnvironmentalProtection,FacultyofMedicine,DemocritusUniversityofThrace,Alexandroupolis,Greece
eLaboratoryofCellularandMolecularBiology,DepartmentofMolecularBiologyandGenetics,FacultyofMedicine,DemocritusUniversityofThrace,Alexandroupolis,Greece
fDepartmentofOncology,FacultyofMedicine,DemocritusUniversityofThrace,Alexandroupolis,Greece
gLaboratoryofBiochemistry,FacultyofMedicine,DemocritusUniversityofThrace,Alexandroupolis,Greece
a r t i c l e i n f o
Articlehistory:
Received4September2012 Receivedinrevisedform 14December2012 Accepted17December2012 Availableonline31December2012
Keywords:
Gonadotropinreleasinghormone Receptor
Neuropeptide Human Breast Cancer
a b s t r a c t
Expressionofthetwogonadotropin-releasinghormonehomologuepeptidesGnRHIandGnRHIIandtheir receptorGnRHRhasbeendemonstratedinanumberofmalignancies.Inhormone-dependentbreastcan- cer,GnRHanalogsareusedfortherapyinpremenopausalwomen.GeneexpressionofGnRHI,IIandR wasstudiedinbreastbiopsiesfromprimarybreastadenocarcinomaobtainedfromthetumorandthe adjacentbenigntissue.Levelswereevaluatedbyamultiplexreal-timeRT-PCR.GnRHItranscriptswere detectedin14.7%ofthebenignand29.4%malignantbiopsiesandGnRHIIin21.2%benignand44.1%
malignantbiopsies.GnRHRwasalsomorefrequentinthemalignant(54.2%)thaninthebenign(24.0%) biopsies,atsimilarexpressionlevels.Notranscriptsweredetectedinbiopsiesfromhealthyindividuals.
TherewasastrongcorrelationbetweenthepresenceofGnRHIandGnRHIItranscriptsandtheirreceptor inthebenignandthemalignantbiopsies.GnRHI,IIandRexpressioncorrelatedsignificantlywithpoor prognosispathologicalparameters.ImmunohistochemistryforGnRHRrevealedexpressioninmalignant cellsandinepithelialcellsofmammaryductsoftheadjacentareawithpre-cancerousfeatures.Incon- trast,GnRHIandIIpeptideswererarelyexpressedatlowlevelsinbreastcancercells.Inconclusion GnRHpeptidesandreceptorareexpressedmorefrequentlyinbreasttumorsthanintheadjacentmam- marytissue,representingamalignantfeature.Theirexpressioncorrelatedtotumorcharacteristicsof poorprognosisandwasthereforerelatedtomoreaggressivemalignancies.Concomitantexpressionof peptidesandreceptorsupportsanautocrine/paracrineregulatingrole.
©2012ElsevierInc.Allrightsreserved.
1. Introduction
Gonadotropin-releasinghormone(GnRH)isadecapeptidepro- duced in the hypothalamus [10]. It interacts with a G-protein coupledreceptor(GnRHR)intheanteriorpituitary[23],controlling thegonadalfunctioninbothsexes.Twohumanisoformshavebeen identified,namelyGnRH-IandGnRH-II.Thefirstisthehypothala- micisoformresponsibleforthesecretionofLHandFSH.Thesecond differsbythree aminoacids [6,27] andis widelydistributed in
∗Correspondingauthorat:LaboratoryofPharmacology,DUTH,Dragana,Alexan- droupolis68100,Thrace,Greece.Tel.:+302551030533;fax:+302551030533.
E-mailaddress:achatzak@med.duth.gr(E.Chatzaki).
thecentralandperipheralnervoussystem.Itisalsoexpressedat significantlyhigherthanGnRHIlevelsoutsidethebrainandithas beenshowntoactasaneuromodulatorinthebehavioralcompo- nentsofreproduction[16,27,28].
GnRH peptides and GnRH R have been found in extra- pituitarytissuesandtumorsofthereproductiveandothersystems [4,20,29,34,2].ExtrapituitaryGnRH bindingsitesareoftenasso- ciated with many novel cellular responses [7]. Furthermore, expressionofGnRHRseemstoberelatedwithadvancedcancer stageinovariancarcinomas[8].
The GnRH system has been reported to play an autocrine/paracrineroleintheinhibitionofcellulargrowthand metastaticpotentialinbreastcancercelllines[24,35],andbreast tumorregressioninnudemouse[14,26].However,itsexpression 0196-9781/$–seefrontmatter©2012ElsevierInc.Allrightsreserved.
http://dx.doi.org/10.1016/j.peptides.2012.12.016
wasassociatedwithaprotectiveeffectonthechemotherapeutic drug-producedapoptosis[30].AsGnRHagonists(orantagonists) showclinicalbenefitwhenusedasadjuvantpharmacotherapyin premenopausalbreastcancerpatients[13],thestudyoftheGnRH systemofneuropeptidesandreceptorinbreasttumors remains emerging.
Inthepresentstudy,theexpressionofthetwoGnRHneuro- peptidegenes (GnRHI, II)and theirreceptor wasevaluated in a seriesof biopsies fromprimary breast cancers in a quantita- tivemannerbymultiplexreal-timeRT-PCR.Transcriptlevelsfrom themalignanttissueswerecomparedtothesefromtheadjacent non-neoplastictissueandtissueswithoutmalignancy,andwere correlatedtomultipleclinicopathologicalanddemographicparam- etersand clinicaloutputin ordertorevealpotentialprognostic ordiagnosticvalue.Finally,histologicalmappingofpeptideand receptorexpressioninbreastcancerbiopsieswasdonebyimmuno- histochemistry,torevealspecifictargetcellulartypes.
2. Materialsandmethods 2.1. Tissues
Patientsnewlydiagnosedwithprimarybreastadenocarcinoma in the “Theagenio” Cancer Hospital, Thessaloniki, Greece were enrolledinthestudy.Biopsieswereobtainedfromthetumorand theadjacentnon-neoplastictissue.Diagnosis wasconfirmedby thehistologicalexaminationinallpatients.Fullmedicalhistory, follow-upandhistopathologicaldatawereavailable.Patientshave notbeenreceivinganyhormonaltreatmentchemotherapyorradi- ation.Patientswithpreviousorpresentneoplasticdiseaseatany othersitewereexcludedfromthestudy.Biopsieswithoutsignsof malignancyorotherpathologyobtainedfordiagnosticusewere alsoused.HumantermplacentawasobtainedbytheObstetrics andGynecology Department oftheGeneralUniversity Hospital inAlexandroupolis.TheprojectwasapprovedbythelocalEthical Committee.Consenthasbeenobtainedfromeachpatientafterfull explanationofthepurposeandnatureofalltheproceduresused, inaccordance tothe HelsinkiDeclaration. Tissuesampleswere storedinRNAlater(Invitrogen,Carlsbad,CA)at−80◦Cuntilusedfor RT-PCR.Breastcancertissuesectionswerealsotakenfromparaffin- embeddedarchivalfilesandusedforimmunohistochemistry.
2.2. Cellculture
Thehuman breast cancercell linesMDA MB231,MCF7 and T47wereculturedinDulbecco’sModifiedEagleMedium(DMEM) supplemented with 10% fetal calf serum (FCS) and 1% peni- cillin/streptomycin(allpurchasedfromInvitrogen,UK),at37◦Cin a5%CO2humidifiedatmosphere.Cellswereplatedataconcentra- tionof2×105cells/mlandwereharvestedfortotalRNAextraction whentheyhadreachedapproximately80%confluence.
2.3. Multiplexreal-timequantitativeRT-PCR
TotalRNAwasextractedfrombiopsiesusingTrizolReagent, accordingtothemanufacturer’sinstructions.Reversetranscrip- tion(RT)wasperformedusingtheSuperScriptPreamplification System(Invitrogen)and randomhexamersina total volumeof 20l.TwomicroliterofthesameRTproductwasusedasatem- plateforeachgene,amplifiedbyPCRusing2mMMgCl2,PCRbuffer, 0.2mMofsenseandantisenseprimers,0.2mMdNTPsand2.5U TaqPolymerase (Invitrogen)ina finalreactionvolumeof50l.
QuantitativePCRwasperformedusingtheLightCyclerMX3005P (Stratagene,LaJolla,CA)withthefollowingcyclingparameters:
a pre-amplification cycle(denaturation for 10min at95◦C), 40 cyclesofamplification(denaturationfor30sat95◦C, annealing
for40secat53◦C,54◦C,50◦C forGnRHI,GnRHIIandGnRHR respectively,andextensionfor50sat72◦C),andafinaldissocia- tioncycle(1minat95◦C,40sat57◦Cand30secat95◦C).Primers weredesignedaccordingtotheGenBankpublishedsequencesas follows:forhumanGnRHRsense5-CCTTGTCTGGAAAGATCCGA- 3 andantisense 5-GGAGCGGTCCAGGCTGAT-3 [33],for human GnRHIsense5-CTACTGACTTGGTGCGTGGA-3 andantisense5- CTGCCCAGTTTCCTCTTCAA-3andforhumanGnRHIIsense5-TCC- TGCTGCTGCTGACTG-3 and antisense 5- CTAAGGGCATTCTGGG- GAT-3 [25]. Productsizeswere319, 240and 119bpfor GnRH R,GnRHIandGnRHIIrespectively.Reactionsinduplicatewere carriedoutusingtheSYBERGreenMMQPCRBrilliantmix(Strata- gene),0.4Mofeachprimer,2mMMgCl2and0.5LofcDNAina finalvolumeof20L.ResultswerecalculatedusingMaxProQPCR SoftwareVersion4.0(Stratagene)usingthecomparativethreshold cyclemethod.Analysisofrelativegeneexpressiondatawasper- formedaccordingtothe2−CT method[21]using-actinasa referencegeneandRNAfromhumanplacentaasapositivecontrol.
Resultsareexpressedasthemeanfromduplicatevaluesofgene expressioninrelationto-actininthesameRNApreparation.Sam- pleswithpoor-actingeneamplificationwereexcludedfromthe study.Negativecontrolsamples,wherenoRTenzymewasadded (noRT)orwithoutDNAtemplate(noDNA),wereincludedinevery assayinordertoexcludethepossibilityofgenomicorotherDNA contamination.
2.4. Immunohistochemistry
Immunohistochemistrywasconductedaspreviouslydescribed [32].Briefly,tissuespecimenswerefixedinformalinandembed- dedinparaffin.Sections(4m)weredeparaffinized,rehydrated, and treatedwith0.3%H2O2 for 5mininmethanol. Slideswere incubatedfor75minwithprimarymousemonoclonalantibodies forhumanGnRHR(ab22168,Abcam,UK),GnRHI(HU11B,San- taCruzBiotechnologyInc.,CA,USA)andGnRHII(D-9,SantaCruz BiotechnologyInc.)diluted1:250, 1:100and 1:100respectively in10%normalmouseseruminPBS.Negativecontrolslideswere incubated for the same period withnormal mouse serum IgG.
Immunostaining was detectedby the Kwik Kit (Thermo Shan- don,Pittsbutgh,PA,USA).Finally,boundantibodycomplexeswere stainedfor10minwith0.05%diaminobenzidine,counterstained withMayer’shematoxylin,mountedandexaminedunderanOlym- pusBX40microscope.
2.5. Statisticalanalysis
Allmeasurementsweredonein duplicate. Statisticalsignifi- cancewasassessedbyMann–WhitneyU–WilcoxonRankSumW Test,usingtheSPSS17.0statisticalsoftware(SPSSInc.,Chicago, IL,USA).Groupdifferenceswereassessedbychisquaretest.Sig- nificancewassetatapvalue<0.05.Analysisofthedatainpairs ofbenignandmalignantbiopsiesfromthesamepatientwasper- formedbytheMcNemartest.KaplanMeiersurvivalanalysiswas alsoperformed.
3. Results
3.1. Patientandtumorinformation
Thirty-fivewomenwithprimarybreastcancerwereenrolledin thestudy,withmeanage61±13years,meanBMI28.9±5.4kg/m2 andmeanageofmenarche13±1.3years.Atthetimeofdiagno- sis,27/35(71.4%)weremenopausalwithmeanageofmenopause 48.5±3.9 years. Two of them did not report any history of pregnancy,whereasfortherestthemeannumberoffull-termpreg- nancieswas1.9±0.8,withmeanageoffirstpregnancy25.1±4.3
Table1
Patient’shistopathologicalfindings.
Characteristic No.ofpatients Percentage
Tumorsize
<2cm2 14/35 40.0
>2cm2 21/35 60.0
Type
Ductandother 30/35 85.7
Lobular 5/35 14.3
Grade
I 6/35 17.1
II 18/35 51.4
III 8/35 22.9
IV 3/35 8.6
Infiltration
No 19/35 54.3
Yes 16/35 45.8
Lymphnodes
0 15/35 42.9
1 6/35 17.1
>2 14/35 40.0
Metastasis
no 32/35 91.4
yes 3/35 8.6
Estrogenreceptors
(+) 25/35 71.4
(−) 10/35 28.6
Progesteronereceptors
(+) 22/35 62.8
(−) 10/35 27.2
c-erbB2
(+) 12/35 34.2
(−) 23/35 65.8
yearsandmeandurationoflactationforallchildren11.2±12.8 months.Meanvalueforpregnancyterminationswas0.8±1.0.One of them(2.85%) reported alcohol consumption (≤250ml/day of drinkwith5–10%alcohol)and 4/35(11.4%) had beenadminis- teredcontraceptivepills(0.25–2years).Theireducationlevelwas at21/35(60.0%)basic,10/35(28.5%)high-schooland4/35(11.4%) university.17/35(48.5%)hadfamilyhistoryofmalignancyand5/35 (14.2%)inbreast.3/35(8.5%)hadbeenpreviouslydiagnosedwith benignbreastdisease.Noneofthepatientshadreceivedradiation foranyreasonorhadhistoryofothermalignancyorsyndrome.
The clinicopathological findings of the tumors by patholog- ical examination based on the TNM system of the American Joint Committee on Cancer, used in the study are shown in Table1.
Follow-upfor 24–68monthsshoweddiseaserelapsein4/32 (12.5%)cases.In3patientsfollow-upwasnotpossible.Onepatient developed endometrial adenocarcinoma36 months after initial diagnosis,andexacerbationwasreportedinthepatientwithliver metastasis.Atthecompletionofthestudy,26/32(81%)patients weredisease-free,4/32(13%)stillhaddiseaseand2/32(6%)died.
3.2. LevelsofgeneexpressionofGnRHneuropeptidesand receptorinbreastcancerbiopsies
GnRHI,IIandRgeneexpressionwasexaminedbycomparative real-timeRT-PCRinhumanbreastcancerbiopsiesandintheadja- centnon-malignanttissue.PCRproductsweredenaturatingatthe sametemperatureastheproductfromhumantermplacentaused aspositivecontrol.
GnRHIgenetranscriptswerefoundtwiceasfrequentlyinthe malignant(10/34,29.4%)thanthebenignbiopsies(5/34,14.7%) examined.Transcript levelsdid not differ between benign and malignant biopsies in a statistically significant manner, being 92×10−3±60×10−3 and 129×10−3±95×10−3, respectively.
Whenanalysiswasperformedinpairsofbenign andmalignant biopsiesfromthesamepatient,itwasfoundthatin18/26(69.2%)
casesbothbiopsieswerenegative,in4/26(15.4%)onlythemalig- nantbiopsywaspositive,in2/26(7.7%)onlythebenignbiopsywas positiveandin2/26(7.7%)bothbiopsieswerepositive(Fig.1).
GnRHIItranscriptswerefoundin7/33(21.2%)ofthebenign biopsiesexaminedandin15/34(44.1%)ofthemalignantbiopsies andthisdifferencewasstatisticallysignificant(p=0.04).Transcript levels,ascomparedtotheexpressionlevelsfoundinhumanterm placenta,didnotdifferbetweenbenignandmalignantbiopsiesin astatisticallysignificantmanner,being17×10−3±11×10−3and 55×10−3±30×10−3respectively.Whenanalysiswasperformed inpairsofbenignandmalignantbiopsiesfromthesamepatient, itwasfoundthatin12/24(50.0%)casesbothbiopsieswerenega- tive,in7/24(29.2%)onlythemalignantbiopsywaspositive,in3/24 (12.5%)onlythebenignbiopsywaspositiveandin2/24(8.3%)both biopsieswerepositive(Fig.2).
GnRHRgeneexpressionwasfoundtobemorefrequentinthe malignantbiopsies(13/24,54.2%)comparedtothebenignbiopsies (6/25,(24.0%),inastatisticalsignificantmanner(p=0.05).GnRH Rtranscriptlevels,expressedinrelationtohumantermplacenta, werealsohigherinthemalignanttissues(430×10−3±365×10−3) incomparisontothebenign(91×10−3±54×10−3)althoughthis differencewasnotstatisticallysignificant.Whenanalysiswasper- formedinpairsofmalignantandbenignbiopsiesfromthesame patient, the following pattern was revealed regarding GnRH R expression;in8/16(50.0)patientsGnRHRtranscriptswereabsent inbothbiopsies,in4/16(25.0%)werepresentinbothbiopsies,in 4/16(25.0%)GnRHRwasexpressedonlyinthemalignanttissues, whereasinnone(0.0%)ofthepatientstranscriptswerefoundin thebenignbutnotinthemalignantbiopsy(Fig.3).
Interestingly,statisticalanalysisrevealedacorrelationbetween GnRHRgeneexpressioninthetumorbiopsyandinthecorrespond- ingadjacentarea(p=0.021).Nosuchsignificantcorrelationwas foundforthetwoneuropeptidegenes.However,receptorexpres- sioninthebenigntissuecorrelatedpositivelywiththepresenceof GnRHI(p=0.019)butnotGnRHIItranscripts(p=0.062)inthesame tissues.Inaddition,receptorexpressioninthemalignantbiopsies correlatedpositivelywiththepresenceofGnRHI(p=0.008)and alsoGnRHIItranscripts(p=0.001).Finally,therewasastrongsta- tisticallysignificantcorrelation(p<0.0001)regardingthepresence ofGnRHIandGnRHIItranscriptsinboththebenignandthemalig- nantbiopsies.Breastbiopsiesfrom5patientsthatwerediagnosed tohavenomalignancywherefoundtoexpressnoneofthethree genes.
Statisticalanalysiswasperformedinordertorevealsignificant correlationsofGnRH neuropeptideandreceptorexpressionand expressionlevelswithanyofthepatienthistopathologicalchar- acteristics,includingtumorsize,type, grade,infiltration,lymph nodes,metastasis,estrogenorprogesteronereceptorsandc-erbB2.
DetectionofGnRHIinthecancerbiopsiescorrelatedsignificantly withthesizeofthetumor(p=0.04),whiledetectionandincreased expression levels correlated with infiltration (p=0.02). Expres- sionlevelsofGnRHIintheadjacentbenignbiopsiescorrelated significantly withthe absenceofPR(p<0.001)and presencec- erbB2(p=0.05)inthetumor,whereasexpressionlevelsofGnRH IIcorrelated withabsenceofER.Inaddition,thelevelsofGnRH Rexpressionwerecorrelatedwithinfiltration(p<0.001),whereas thepresenceofGnRHRcorrelatedwithhigherBMI(p=0.04).
Nootherstatisticalcorrelationwasfoundbetweentheexpres- sionofthethreegenesandthehistopathologicalcharacteristicsof thetumorsaswellaswithage,weight,height,BMI,ageofmenar- che,children birth,ageof firstpregnancy,durationoflactation, ageofmenopause,abortions,alcoholuse,educationlevel,famil- ialbreasthistory,familialcancerhistoryandbenignbreastdisease ofthepatients.Finally,nocorrelationwasfoundbetweenGnRHI andIIandGnRHRexpressioninbothbenignandmalignanttissues withdiseaserelapseandpatientsurvival(KaplanMeieranalysis).
0 5 10 15 20 25 30 35
benign malignant
negative positive
14.7%
29.4%
No tissues
0 0,05 0,1 0,15 0,2 0,25
malignant benign
GnRHI ddCT/placenta
GnRHI benign
(-) 18/26 (69.2%)
(-) 4/26 (15.4%)
(+) 2/26 (7.7%)
(+) 2/26 (7.7%)
A
B
C malignant No of tumors (%)
(-) (+) (-) (+)
Fig.1.DetectionofGnRHIgenetranscriptsinmalignantandadjacentbenignbiopsiesfrombreasttumorsbyreal-timePCRfollowingreversetranscriptionoftotalRNA extracts.(A)PresenceofGnRHItranscriptsandpercentagesofpositivetissues.(B)Levelsofgeneexpression.Barsrepresentmeansbetweenallpositivetissuesanderror barsthestandarddeviationbetweenmeasurements.(C)Expressioninmalignantbiopsiesinrelationtotheiradjacentbenigntissue.
3.3. LevelsofgeneexpressionofGnRHneuropeptidesand receptorinbreastcancercelllines
Expressionofthethreegeneswasalsoexaminedinthehuman breast cancer cell lines MDA MB231, MCF7 and T47. All cell linesexpressedlowlevelsofGnRHI(0.50×10−3,0.06×10−3and 34.70×10−3unitsinrelationtohumanplacenta,respectively)and GnRHII(0.60×10−3,0.05×10−3and16.70×10−3unitsinrelation tohumanplacenta,respectively),whereasGnRHRwasfoundin MDAMB231andMCF7(3.4×10−3and0.2×10−3unitsinrelation tohumanplacenta,respectively),butnotinT47.
3.4. ExpressionofGnRHpeptidesandGnRHRproteininbreast cancerbiopsies
Immunohistochemical analysis was used in order to detect GnRHpeptidesandGnRHRproteinexpressionin16humanbreast tumorbiopsiesandtolocalizeitathistologicalandcellularlevel.
Immunoreactivity for GnRH I was foundin breast cancer cells in5outof16tissues(31.25%),localizedinperineuralinvasions, implantsandmacrophages(Fig.4).Similarly,themajorityofthe tumorswerenegativeforGnRHII.Immunoreactivitywasfoundin 5outof16tissues,beingalsopositiveforGnRHI,inbreastcancer
Fig.2.DetectionofGnRHIIgenetranscriptsinmalignantandadjacentbenignbiopsiesfrombreasttumorsbyreal-timePCRfollowingreversetranscriptionoftotalRNA extracts.(A)PresenceofGnRHIItranscriptsandpercentagesofpositivetissues.(B)Levelsofgeneexpression.Barsrepresentmeansbetweenallpositivetissuesanderror barsthestandarddeviationbetweenmeasurements.(C)Expressioninmalignantbiopsiesinrelationtotheiradjacentbenigntissue.
cells and in cancer implants, and in infiltrating macrophages (Fig. 5). Epithelial cells of cancerous implants of a mucinous carcinomastudiedwerepositiveforbothGnRHI(Fig.4C)andII (Fig.5C).Immunoreactivityinallcaseswascytoplasmicandmild, notuniforminallareasofthetissuesectionbutratheroccasional.
Theadjacenttothetumorareashowednoimmunoreactivityfor bothantibodies.Humantermplacenta,usedasapositivecontrol, showedstrongcytoplasmicimmunoreactivity(Figs.4Eand5E).No specificstainingwasfoundinanycelltypeafterreplacementof theprimaryantibodybynon-specificmouseIgG(negativecontrol inplacentaandbreastcancer,Figs.4Fand5Frespectively).
Tissuesectionswerealsostainedusingmonoclonalantibod- ies against the N-terminus of human GnRH R. Representative
areas of the sectionsare presented in Fig. 6. Positive GnRH R stainingwaslocalizedinmalignantcellsofthetumorwhichvar- ied in size and shape (A–C) and in the cancerousimplants of ductal in situ carcinomaof solid type with(B) or without (A) inflammation (aggregatesof periductal mononuclearinflamma- torycells), andin macrophages scatteredin a negativestroma.
Immunostainingwasclearlymembranicbutalsocytoplasmic(C).
In theadjacentto thetumorarea, epithelialcells of mammary ducts presenting features of atypia, lack of polarity, enlarged nuclei,prominentnucleoliandnumerousmitosis,showedsome immunoreactivity for the GnRH receptor, mainly cytoplasmic (B). Finally, cancer cells of perineural invasions were negative (D).
Fig.3. DetectionofGnRHRgenetranscriptsinmalignantandadjacentbenignbiopsiesfrombreasttumorsbyreal-timePCRfollowingreversetranscriptionoftotalRNA extracts.(A)PresenceofGnRHRtranscriptsandpercentagesofpositivetissues.(B)Levelsofgeneexpression.Barsrepresentmeansbetweenallpositivetissuesanderror barsthestandarddeviationbetweenmeasurements.(C)Expressioninmalignantbiopsiesinrelationtotheiradjacentbenigntissue.
4. Discussion
ThetwoGnRHneuropeptides(GnRHIandII)andtheirreceptor arekeyplayersof thenervoussystemcontrolonthereproduc- tive function. Their expression however has also been clearly establishedin many extra-pituitaryorgans in reproductive and non-reproductivetissuesandtumorsarisingthere,breastcancer beingone of the first reported [7]. Ectopic expression of neu- ropeptides is frequentlyfound in endocrine cancers. Their role seemstocontributetothecancerbiologyviaactivationoflocally expressedreceptorsin anautocrinemannerora paracrinedia- loginthetumormicroenvironmentbetweenthetumorcellsand the nearby located cells, such as stroma, immune cells or by
innervatingautonomicneurons.Breastcancertumorsareknown toexpressmultipleneuropeptidesandtheirreceptorssuchasCRF, GHRHandsomatostatinalongwithGnRH,firstlystudiedinbreast cancerbyimmunohistochemistry[9].Wehaverecentlyreported the expressionof both CRF receptors in breast cancerand the respectivebenignadjacenttissue,whichcouldserveastargetsof endogenousligandsregulatingcancergrowth[15].Inthepresent studywequantifygene expressionof GnRHIandIIalong with GnRHRinhumanbreasttumors.Itis importantthattwo biop- sieswerecollectedbythesamepatient,onefromthebreastcancer tissueandonefromtheadjacentbenigntissueandresultsinthe actualtumorwerecomparedwiththosefromitspre-cancerous milieu.
Fig.4. ImmunohistochemicalanalysisofGnRHIpeptideexpressioninhumanbreasttumorbiopsies.Tissuesectionswerestainedusingamonoclonalantibodyandrepre- sentativefieldsarepresented.Mildcytoplasmicimmunoreactivitywaslocalizedinbreastcancercellsofperineuralinvasionsinsometissues(A,arrows)butnotinothers (D),incancerimplants(B,arrows),inmacrophages(B,diamonds)andinmalignantepithelialcellsofcancerousimplantsinmucinousmatrixofamucinouscarcinoma studied(C,arrows).Humantermplacenta,usedasapositivecontrol,showedstrongcytoplasmicimmunoreactivity(E).Nospecificstainingwasfoundinplacentalcellsafter replacementoftheprimaryantibodybynon-specificmouseIgG(F).Originalmagnification200×(A,B,D,F,E),400×(C).
GnRH I and GnRH II gene transcripts were found approxi- mately twiceas frequent in the malignant than in the benign biopsies,implyingthattheypresentacharacteristicaccompanying malignanttransformation.Transcriptlevelsshowednostatistically significant differences between malignant and benign biopsies for eithergene. Thisis inaccordance withpreviousfindings of semi-quantitativedeterminationsofGnRHImRNAinasmallnum- beroftissues[19].Chenetal.,2002[5]however,reportedhigher levelsofGnRHIandGnRHIIinthemalignantbiopsiesofbreast
incomparisonwiththeadjacentbenigntissue, asdetectedin6 breastcancerpatientsbysemi-quantitativeRT-PCR,attributedto theoverall highprotein expressionand enhanced transcription machinerythatexistincancercells.Thegreatvariabilityfoundin theexpressionlevelsbetweenthepositivesamplesmighthavepro- hibitedstatisticallysignificantdifferencestoemergeinourstudy, whichhoweveremploysastrictlyquantifyingmethodcorrecting forthetotalproteincontent(usingactinasahouse-keepinggene) andamuchlargernumberoftissues.
Fig.5.ImmunohistochemicalanalysisofGnRHIIpeptideexpressioninhumanbreasttumorbiopsies.Tissuesectionswerestainedusingamonoclonalantibodyand representativefieldsarepresented.Mildcytoplasmicimmunoreactivitywaslocalizedincellsofmalignantimplantsinsometissues(A,arrows)butnotinothers(D),in macrophages(B,arrows)andinmalignantepithelialcellsofcancerousimplantsinmucinousmatrixofamucinouscarcinomastudied(C,arrows).Humantermplacenta, usedasapositivecontrol,showedstrongcytoplasmicimmunoreactivity(E).Nospecificstainingwasfoundinbreastcancercellsafterreplacementoftheprimaryantibody bynon-specificmouseIgG(F).Originalmagnification200×.
Fig.6.ImmunohistochemicalanalysisofGnRHRexpressioninhumanbreasttumorbiopsies.TissuesectionswerestainedusingamonoclonalantibodyagainsttheN- terminusofthereceptor.Representativefieldsofmalignantcells(AandC),cancerousimplantswith(B)orwithout(A)inflammation,mammaryductsintheadjacentto thetumorarea(B)andperineuralinvasion(D)areshown.Positivemembranicandcytoplasmicreceptorstainingwaslocalizedincancercells,whereasstromawasmainly negative.Originalmagnification100×(B),200×(AandD),400×(C).
Atthepeptidelevel, GnRHIand IIwereexpressedrarelyby breastcancercells, asshown byimmunohistochemicalanalysis usingmonoclonalantibodies.Lowcytoplasmicimmunoreactivity wasfoundin31.25%ofthesamples,apercentage similartothe mRNApositivetissues.Antigenwaslocalizedinsomebreastcan- cercellsofperineuralinvasions,implantsandmacrophages(Fig.4).
TheseresultsareinagreementtothestudyofChenetal.,2002, showingGnRHIandIIpeptideimmunoreactivityin8outof14 breastcancerbiopsiesusingpolyclonalanti-serums.Theadjacent tothetumorareashowednoimmunoreactivityforbothantibod- ies.Itispossiblethatpost-transcriptionalregulationmechanisms preventpeptideexpressionatthebenigntissue.Infact,thiswas alsothecase inendometrial tissuesand celllines[4],in which GnRHtranscriptswereabundantbutnosecretedpeptidewasfound byRIA.Thesemechanismsmustbealteredincertainmalignant transformedcells,wherepeptidescanbetraced.
GnRHRtranscriptswerealsofoundathigher frequencythe malignant(54.2%)thanthebenignbiopsiesexamined(24.0%),and athigher levels.Interestingly,inall casesfoundpositive inthe benigntissue,thiswasaccompaniedbytranscriptpresenceinthe correspondingmalignantbiopsy,indicatingthatitcouldrepresent apre-malignantfeatureofthemammarytissue.Indeed,GnRHRin thetumorbiopsycorrelatedsignificantlytoitspresenceinthecor- respondingadjacentarea.GnRHRexpressionwasalsostudiedat theproteinlevelbyimmunohistochemistry.Receptorwaslocalized inthemembranesbutalsointhecytoplasmofmalignantcellsof thetumorandinthecancerousimplantsofductalinsitucarcinoma ofsolidtype,inaccordancewithpreviousreports[1,22].Perineural invasionswerenegative.Adjacenttothetumorarea,epithelialcells ofmammaryductspresentingpre-cancerouscharacteristicswere alsopositive,furthersupportinganearlytumorigenesisevent.Dif- ferentialexpressionofGnRHRbydifferentcelltypeswithinthe tumorcouldexplainthegreatvariabilityingeneexpressionlevels estimatedbyreal-timeRT-PCR.Mammarysamplesfrompatients withoutanymalignancywerenegativeforbothGnRHpeptideand receptorgeneexpression.
GnRHI expressioncorrelated significantlywiththesize and infiltration of the tumor in the cancer biopsies, while in the adjacent benign biopsies it correlated with absenceof proges- terone receptors and c-erbB2 positivity of the tumor. GnRH II was expressed more frequently than GnRH I in the same tis- sues,anditsexpressionlevelscorrelatedwithabsenceofER.In addition,levelsofGnRHRcorrelatedwithinfiltrationandhigher patientBMI.Alltheseparametersarenegativebiomarkersofbreast cancerandthereforecorrelateGnRHsystemexpressiontomore aggressiveandpoorer prognosisdisease.Our resultsimplythat GnRHsystemexpressioncouldholdaclinicalprognosticpotential for breast cancer. Fortunately, only 3 patients from our sam- pledeceasedand6developed metastasisina 5-yearfollowup, making survival analysis in relation to GnRH gene expression inconclusive.
SpecificbindingsitesforGnRHweredemonstratedinhuman breastcarcinomasbutnotinnon-neoplasticbreasttissuebyligand immunoblottingbackin1985[18].ThepercentageofGnRHRpos- itivetissueshere(54.2%)isinlinewithpreviousreportsdetecting bindingsitesbyamultipointassay[11].Ourstudyconfirmsthat theyrepresenttheproteinproductofthisgene.Similarly,thelow affinityGnRH-bindingsitesdescribedinhumanbreastcancercell lines(ZR-75-1,MDA-MB-231,SkBr3,MDA-MB-157andMCF-7) [18]obviouslyrepresenttheGnRHRgeneproductdetectedhere inMDA-MB231andMCF7butnotinT47humanbreastcancercell lines.ThehighGnRHneuropeptidelevelsdetectedinT47might down-regulatereceptorexpression.Thelevelsofgeneexpression inallthecelllineswerelowerthatthesedetectedinthebreast cancerbiopsies,but nodirectcomparisonscanbedone, dueto thegreatvariability betweentissuesand theheterogeneityand complexityofthecellulartypesintissues(i.e.epithelial,stromal, macrophagesetc.inthebiopsies)vs.anepithelialcancercellclone inthecelllines.Thishowevermightnotbereflectedatthetransla- tionallevel,asGnRHpeptideexpressionhasbeenshownbeforein MCF7cellsandextensivelycharacterizedwithmultipleapproaches (HPLC,confocalmicroscopy)[5].
Co-expressionofGnRHpeptideandreceptorinhumanbreast tissuessupportanautocrine/paracrinerole inhumanmammary gland.Thisisfurthersupportedbyouranalysisshowingthatrecep- tor expression in the benign and malignant tissues correlated positivelywiththe presenceof GnRH neuropeptide transcripts.
Inaddition,therewasastrongcorrelationbetweenthepresence of GnRH Iand GnRH IItranscripts in both thebenign and the malignantbiopsies,indicatingtheconcomitantexpressionofboth neuropeptideisoformsinbreast,confirmedalsobyimmunohisto- chemistry.Overall,itseemsthatthefullneuropeptideandreceptor systemissimultaneouslyexpressedinsometissuesandthiscould betheresultofinter-activationbetweenthegenesofthesystem, althoughthisdoesnotseemtobethecaseatleastforthepep- tides[3].Alternatively,co-activationofthethreegenescouldbea concurrentresponsetothesamestimulant.Severalstudieshave showndifferentialregulationoftheGnRH-IandGnRH-IIgenesby gonadotrophsandsteroidhormonesinextrapituitarysites.How- ever,othereffectorsandfactorsshouldbeexaminedfortheirability totranscriptionallyactivateGnRHgenes[17]andbreastcancercell linesreportedheretoexpressbothpeptidesand receptorcould serveasaconvenientinvitromodelforsuchstudies.
5. Conclusions
GrowtheffectsofGnRHanalogsonbreastcancercellsdepend ontheamountofGnRHatcellsurfacesandonreceptorfunction- ality[12,31],thereforequantificationofexpressionlevelsholdsa specificinteresttopredictcellularresponses.Furthermore,GnRH receptors may contribute to the breast tumor responsiveness topharmacotherapywithGnRHanalogs andthusestimation of expressionlevelscoulddefineamoresensitivepatientsubpopula- tion.Ourresultsaddtothepathophysiologicalsignificanceofthe GnRHsysteminbreastcancerbiologypossiblybycontributingto thedialogbetweenbenignandmalignantcelltypes,andimplythat itsquantificationcouldcomplementtheclinicopathologicalprofil- ingofbreastcancerwithpotentialclinicalexploitationinprognosis andtreatment.
Conflictofinterest
Allauthorsdeclarethattheyhavenoconflictofinterest.
Contributors
Kalliopi Pazaitou-Panayiotou, Alexandros Kortsaris and Eka- teriniChatzaki designed thestudy,ChristinaChemonidou, Aliki Poupi,MariaKouretaandMariaKoffaperformedtheexperiments, MariaLambropoulou,TheodorosC.ConstantinidisandGrammati Galaktidouanalyzedtheresults,AthinaKaprara,AnastasiaKiziri- douandStylianosKakolyriscollectedbiologicmaterialandclinical information,KalliopiPazaitou-Panayiotou,GeorgeKoliosandEka- teriniChatzakipreparedthemanuscript.
Allauthorshaveapprovedthefinalarticle.
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