reductase 1 mRNA levels are positively correlated with TRAMP mouse prostate most severe lesion ...

15 Pages·2017·7.27 MB·English

RESEARCHARTICLE
5α-reductase 1 mRNA levels are positively
correlated with TRAMP mouse prostate most
severe lesion scores
AlexanderB.Opoku-Acheampong1,JamieN.Henningson2,AmandaP.Beck3,Brian
L.Lindshield1*
1 DepartmentofFood,Nutrition,DieteticsandHealth,KansasStateUniversity,Manhattan,KS,UnitedStatesof
America,2 CollegeofVeterinaryMedicine,KansasStateUniversity,Manhattan,KS,UnitedStatesofAmerica,
3 DepartmentofPathology,AlbertEinsteinCollegeofMedicine,Bronx,NY,UnitedStatesofAmerica
a1111111111
*[email protected]
a1111111111
a1111111111
a1111111111
Abstract
a1111111111
Background
Thecontributionof5α-reductase1and5α-reductase2toprostatecancerdevelopmentand
progressionisnotclearlyunderstood.TRAMPmiceareacommonprostatecancermodel,
OPENACCESS
inwhich5α-reductase1and5α-reductase2expressionlevels,alongwithprostatelesions
Citation:Opoku-AcheampongAB,HenningsonJN, scores,havenotbeeninvestigatedatdifferenttimepointstofurtherunderstandprostate
BeckAP,LindshieldBL(2017)5α-reductase1
carcinogenesis.
mRNAlevelsarepositivelycorrelatedwithTRAMP
mouseprostatemostseverelesionscores.PLoS
ONE12(5):e0175874.https://doi.org/10.1371/ Method/Principalfindings
journal.pone.0175874
Tothisend,8-,12-,16-,and20-week-oldmaleC57BL/6TRAMPxFVBmiceprostatemost
Editor:MohammadSaleem,Universityof severeandmostcommonlesionscores,5α-reductase1and5α-reductase2insituhybrid-
MinnesotaHormelInstitute,UNITEDSTATES
izationexpression,andKi-67,androgenreceptor,andapoptosisimmunohistochemistry
Received:September29,2016 levelsweremeasured.Levelsofthesemarkerswerequantifiedinprostateepithelium,
Accepted:March31,2017 hyperplasia,andtumorssections.Micedevelopedlow-tohigh-gradeprostaticintraepithelial
neoplasiaat8weeksasthemostsevereandmostcommonlesions,andmoderate-and
Published:May11,2017
high-gradeprostaticintraepithelialneoplasiaat12and16weeksasthemostseverelesion
Copyright:©2017Opoku-Acheampongetal.This
inalllobes.Moderatelydifferentiatedadenocarcinomawasobservedat20weeksinall
isanopenaccessarticledistributedunderthe
termsoftheCreativeCommonsAttribution lobes.Poorlydifferentiatedcarcinomawasnotobservedinanylobeuntil12-weeks-old.5α-
License,whichpermitsunrestricteduse, reductase1and5α-reductase2werenotsignificantlydecreasedintumorscomparedto
distribution,andreproductioninanymedium,
prostateepitheliumandhyperplasiainallgroups,whileproliferation,apoptosis,andandro-
providedtheoriginalauthorandsourceare
credited. genreceptorwereeithernotablyorsignificantlydecreasedintumorscomparedwithpros-
tateepitheliumandhyperplasiainmostorallgroups.Prostate5αR1levelswerepositively
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation correlatedwithadjustedprostatemostseverelesionscores.
files.
Conclusion
Funding:ThisworkwassupportedbyNational
InstituteofHealth(https://www.nih.gov)COBRE Downregulationofandrogenreceptorand5α-reductase2,alongwithupregulationof5α-
EpithelialFunctioninHealth&Disease(NIH-Grant
reductase1intumorsmaypromoteprostaticintraepithelialneoplasiaandprostatecancer
No:P20-RR017686fromNCRR)andtheJohnson
CenterforBasicCancerResearchatKansasState developmentinTRAMPmice.
University.Thefundershadnoroleinstudy
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 1/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
design,datacollectionandanalysis,decisionto Introduction
publish,orpreparationofthemanuscript.
AmongUSmen,prostatecanceristhemostcommonlydiagnosedcancerandthethird-lead-
Competinginterests:Theauthorshavedeclared
ingcauseofcancerdeath[1].Androgensareprimarilyinvolvedinthedevelopmentandpro-
thatnocompetinginterestsexist.
gressionofprostatecancer[2].Testosteroneisconvertedbytheisoenzymes5α-reductase1
(5αR1)and5α-reductase2(5αR2)intodihydrotestosteronetoregulatetheprostategland
[3,4].Androgensbindtotheandrogenreceptor(AR),withthelatteractingasatranscription
factortoregulatetheexpressionofandrogenresponsegenesthatmodulatemanycellular
activitiessuchasproliferationandapoptosistocontributetoprostatecancerdevelopmentand
progression[5].Inthenormalprostate,5α-reductase2istheprimaryisoenzyme[6].
Manystudies[7–10]althoughnotall[11–13],haveobservedincreased5α-reductase1and/
ordecreased5α-reductase2mRNAexpressionoractivityinprostatecancercomparedwith
nonmalignantprostatetissue.Additionally,5α-reductase1and5α-reductase2wereobserved
in73%and56%,respectively,ofhumanprostatecancertissues[12].Takentogether,5α-
reductasesmaybeinvolvedinprostatecarcinogenesis[14].
Acommonmodelisthetransgenicmousemodel(TRAMP),whichusestheratprostate-
specificpromoterprobasintodirecttheexpressionofthesimianvirus40(SV40)largeand
smallTantigentotheprostateepitheliumtocausecelltransformation.TRAMPmicedevelop
progressiveformsofprostatecancerwithlesionsrangingfrommildprostaticintraepithelial
neoplasia(PIN)throughwell-differentiated(WD)adenocarcinoma,toinvasivePDadenocar-
cinomawithdistantsitemetastasistopelviclymphnodesandlungs[15,16].InTRAMPmice,
thetransgeneisdetectedasearlyas3weeksofage[17],withpathologicalfeaturessimilarto
low-gradePINdevelopingasearlyas4–6weeksofage[18].C57BL/6TRAMPxFVBmice
developepithelialhyperplasiaby8weeks[19],PINandWDadenocarcinomaby12weeks,
andinvasivecarcinomaappearingbetween18–30weeksandmetastasizingintolymphnodes
andlungsandoccasionallykidney,boneandadrenalglands[18,20].By30weeksofage,
TRAMPmiceonanFVBbackground(TRAMPxFVB)display100%metastasistolungsand
lymphnodesalongwithbonemetastasis[16,21].
Inadditiontonotunderstanding5α-reductaselevelsinTRAMPmice,modifiedgrading
schemes[22]havenotbeenemployedtoassessthedevelopmentandprogressionofprostate
cancerinthesemice.Thus,thisstudywasdesignedtoquantifythecell-typespecificexpression
patternsof5αR1and5αR2mRNA,ARandKi-67protein,alongwithapoptoticDNAfragmen-
tation,inprostatetissuesectionsof8-,12-,16-,and20-week-oldC57BL/6TRAMPxFVBmice
tocharacterizehowtheirlevelsvaryatdifferentagesandstagesofprostatecarcinogenesis.Six-
teenweeksisintermediatebetween12and20weekswhenTRAMPmicewouldhavedeveloped
PDandinvasivecarcinoma[20].Thesetimepointswereusedpreviouslytostudytheincidence
anddistributionofpathologicchangeswithineachlobeofC57BL/6TRAMPxFVBmicepros-
tateasafunctionoftime[20].Tothebestofourknowledge,thisstudyisthefirsttograde
lesionsandquantify5α-reductaselevelsatdifferentagesinTRAMPmouseprostate.
Materialsandmethods
Ethicsstatement
TheInstitutionalAnimalCareandUseCommittee(IACUC)atKansasStateUniversity
approvedallanimalprocedures(protocol2969).
Studymiceanddesign
Micewerebredasdescribedpreviously[23].MaleC57BL/6TRAMPxFVBmicewereweaned
andbeganconsumingAIN-93Gdietat3weeksofagebeforebeingrandomizedto8-(n=5),
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 2/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
12-(n=8),16-(n=9),and20-(n=12)weekgroups.Micewerecohouseduntil8weeks,and
thenindividuallyhoused,monitoreddaily,weighedweekly,andprovidedAIN-93Gdietand
wateradlibitum.Micewereterminatedattheirrespectivestudytimepointsbyanesthetizing
viaCO inhalationandeuthanizedbyexsanguination.Thegenitourinary(GU)tracts,kidneys,
2
andlungsweredissected,andtheGUtractswereweighed.Iliaclymphnodeswerealsocol-
lectedwheneverpossible.Alltissueswerefixedbyimmersionin10%neutralbufferedformalin
for48hours,andthenmovedto70%alcoholuntilprocessingintheKansasStateUniversity
VeterinaryDiagnosticLaboratory.
Histopathology
Histologicalprocessingofprostatetissues,sectioning,scoringformostcommonandmost
severelesions,andadjustingfordistribution,wereperformedasdescribedpreviously[22,23].
Theanterior,dorsal,lateralandventrallobesofC57BL/6TRAMP×FVBmicewereassigned
twogradeseachbetween0–7.Thefirstgradeisthemostseverelesionwithinthelobe[normal
prostateasleastsevere(grade0),andPDcarcinomaasmostsevere(grade7)].Thesecond
gradeisthemostcommonlesionwithinthelobe[normalprostateasleastcommon(grade0),
andPDcarcinomaasmostcommon(grade7)].Themostsevereandmostcommonlesions
withinalobewereadjustedfordistribution,eitherasfocal,multifocal,ordiffuse.Withfocal,
therearefewerthanthreefociwithinthelobe.Multifocalindicatestherearethreeormorefoci
withinthelobe,withlessthan50%ofthelobecontainingthelesionofinterest.Diffuseindi-
catesgreaterthan50%ofthelobeisaffected[23].Prostatelesionswerescoredblindlytwiceby
aboard-certifiedpathologist(JNH).Selecttissueswerealsoscoredblindlytwicebyaboard-
certifiedsecondveterinarypathologist(APB)toensurescoringconsistency.Iliaclymphnodes
examinationformetastaseswasdoneasdescribedpreviously[23].Formalin-fixed,paraffin-
embeddedprostatetissuesections(4μm)of8-,12-,16-,and20-week-old(n=5)AIN-93G-
fedmaleC57BL/6TRAMPxFVBmicewerepreparedforbiomarkerdetection.Mostprostate
samplesatthetimeofeuthanasiawerecompletelyobliteratedbytumorandthereforeinmost
samples,prostatelobescouldnotbeidentified.Insomeinstances,allfourlobeswerereplaced
bytumor.
5α-reductase1and5α-reductase2insituhybridization
5αR1and5αR2accessionsNM175283.3andNM053188.2,respectively,obtainedfrom
NationalCenterforBiotechnologyInformation(NCBI)websiteweresubmittedtoAdvanced
CellDiagnostics,Inc.,[(ACD),Hayward,CA)]forcustomprobedesignandsynthesisforuse
intheirRNAscopeassay.ProstatesectionswerebakedinaFisherScientific(model77)slide
warmerpreheatedto55˚Cfor25minutes.Sectionsweredeparaffinizedinxylene,rehydrated
in100%ethanol,andair-driedfor5minutesatroomtemperature.Sectionswerethentreated
seriallywith:Pre-Treatment1solution(endogenoushydrogenperoxidaseblockwithPretreat
1solutionfor10minutesatroomtemperature);Pre-Treatment2(100–104˚C,25minutes
immersioninPretreat2solution);and,Pre-Treatment3(proteasedigestion,40˚Cfor28min-
utes);rinseswithdistilledwater(2X)wereperformedaftereachPre-Treatmentstep.Sections
werethenhybridizedin5αR1or5αR2probes,withoutacoverslip,at40˚Cfor2hoursina
HybEZOven(ACD).Afterwashbuffersteps,signalamplificationfromthehybridizedprobes
wereperformedbytheserialapplicationofAmp1,40˚Cfor30minutes(PreAmplifierstep);
Amp2,40˚Cfor15minutes(signalenhancerstep);Amp3,40˚Cfor30minutes(amplifier
step);Amp4,40˚Cfor15minutes(LabelProbestep);Amp5,ambienttemperaturefor30min-
utes,andAmp6,ambienttemperaturefor15minutes(signalamplificationssteps).Wash
bufferstepswithWashBuffer(ACD,proprietary)wereperformedaftereachAmpstep.
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 3/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
Horseradishperoxidase(HRP)activitywasthenobservedbytheapplicationof3,30-diamino-
benzidine(DAB)for10minutesatambienttemperature.Sectionswerethencounterstained
withGill’shematoxylin,dehydratedthroughgradedethanol[95%(1X)and100%(2X)]and
xyleneandthenmounted.SectionswerequalitycontrolledforRNAintegritywithanRNA-
scopeprobeforcyclophilinB(Mm-Ppib)RNA(positivecontrol)andfornonspecificback-
groundwithaprobeforbacterialdapBRNA(negativecontrol).SpecificRNAstainingsignal
wasidentifiedasbrown,punctatedots.
Immunohistochemistry
Androgenreceptor. ProstatesectionswerebakedinaFisherScientificIsotemp(model
281A)vacuumovenpreheatedto60˚Cfor30minutes.Afterbaking,sectionsweredeparaffi-
nizedinxyleneandrehydratedusingadecreasingethanolgradient[100%(2X),95%and80%
(1X)].Endogenousalkalinephosphatasewasblockedusing3%hydrogenperoxideinmetha-
nol.Antigenretrievalwascarriedoutbymicrowavingat540Win10mMTris/1mMEDTA
buffer,pH9,fourtimesfor5minutes.Sectionswereblockedwith2.5%normalhorseserum
(VectorLaboratories,Inc.,Burlingame,CA)beforeincubationfor1hourat37˚Cwitharabbit
polyclonalantibodytoAR(1:50dilution;N-20,sc-816;SantaCruzBiotechnology,SantaCruz,
CA).Afterwashing,sectionswereincubatedwithImmPRESS-APanti-rabbitIgG(alkaline
phosphatase)polymerdetectionreagent(MP-5401;VectorLaboratories,Inc.,)for30minutes
atroomtemperature.ColorsweredevelopedwithaVectorRedalkalinephosphatasesubstrate
kit(SK-5100;VectorLaboratories,Inc.,).Slidesweresubsequentlycounterstainedwithhema-
toxylin(VectorLaboratories,Inc.,),andprocessedbacktoxylenethroughanincreasingetha-
nolgradient[80%and95%(1X),and100%(2X)]andthenmounted.NormalrabbitIgG
(1:100;sc-2027:SantaCruzBiotechnology)wasusedasanegativecontrol.
Ki-67. ProstatesectionsweredeparaffinizedinLeicaBondDewaxSolution(LeicaMicro-
systemsInc.,BuffaloGrove,IL)at72˚C,andthenrehydratedin100%ethanol,followedby3
washesinTris-bufferedsaline.AntigenretrievalwascarriedoutwithNovocastraBondEpitope
RetrievalSolution1(citratebuffer,pH6)(AR9961;LeicaMicrosystemsInc.,)for20minutes
at100˚C.ThesectionswerestainedwithapredilutedrabbitmonoclonalantibodytoKi-67
(CPRM325AA;BiocareMedicals,Concord,CA)for15minutesatroomtemperature.Theanti-
rabbitpoly-HRP-IgGpolymerfromtheBondPolymerRefineDetectionSystem(LeicaMicro-
systemsInc.,)wasusedfortheenhancementofthesignal.Thesubstratechromogen,DAB,was
usedforthedetectionofthecomplex.SectionswerecounterstainedwithGill’shematoxylin,
andprocessedbacktoxylenethroughanincreasingethanolgradient[95%(1X)and100%
(2X)],andthenmounted.Tissuefromacaninemastcelltumorwasusedasapositivecontrol.
Apoptosis. TerminaldeoxynucleotidyltransferasedUTPnickendlabeling(TUNEL)
stainingwasperformedusingtheApoptagPeroxidaseInSituApoptosisDetectionkit(S7100;
Millipore,Temecula,CA).ProstatesectionswerebakedinaFisherScientificIsotemp(model
281A)vacuumovenpreheatedto60˚Cfor30minutes.Afterbaking,sectionsweredeparaffi-
nizedinxylene,rehydratedthroughagradedethanolseries[100%(2X),95%and70%(1X)]
andtreatedwithready-to-useproteinaseK(S302080-2;DakoNorthAmerica,Inc.,Carpin-
teria,CA)for15minutesatroomtemperature.Sectionswerewashedwith2changesofdis-
tilledwaterfor5minuteseach.Endogenousperoxidaseswereblockedwith3%hydrogen
peroxideinphosphatebufferedsaline(PBS,pH7.4),for5minutesandwashedwith2changes
ofPBS.Equilibrationbuffercontainingdigoxigenin-conjugatednucleotideswasplaceddi-
rectlyontothesectionfor10seconds.Sectionswereincubatedwithterminaldeoxynucleotide
transferase(TdT)enzyme(1:15dilution)inahumidifiedchamberat37˚Cfor1hour.Sections
werethenincubatedfor10minutesatroomtemperatureinstop-washbuffer,rinsedin3
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 4/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
changesofPBSfor1minuteeach,andthenincubatedwithanti-digoxigeninconjugatefor30
minutesatroomtemperature.Sectionswerewashedin4changesofPBSfor2minuteseach,
andthenincubatedwiththesubstratechromogen,DAB,for3minutes.Sectionswerethen
stainedwith0.5%(w/v)methylgreencounterstain,dehydratedthrough100%N-butanol(3X)
andxyleneandthenmounted.
Biomarkerquantification
Aboard-certifiedpathologistidentifiedareasofepitheliumthatwerecomposedofasingle
layerofcellsasprostateepithelium;hyperplasticregionsaswheretherewasincreaseddensity
ofcellspiledupononeanother;andtumorasdiffusesheetsofcellswithnoorganizationchar-
acterizedbyneoplasticcellularcharacteristics,usinganOlympusDP26digitalcamera(Olym-
pusAmerica,CenterValley,PA).TheseareaswerescannedusingaPannoramicMidiscanner
(3DHistech,Budapest,Hungary)witha20Xobjective,giving2-megapixelresolution.Images
werecapturedusingthe3DHistechsoftware.ScannedimageswerethenannotatedusingHalo
software(IndicaLaboratory,Coralles,NM).Intumors,areasofviableneoplasticcellsaway
fromareasofnecrosisweremeasured.Ki-67andARprotein,andapoptoticDNAfragmenta-
tionwerequantifiedusingthedoublestaincytoplasmicandnuclearimmunohistochemistry
algorithm(IndicaLaboratory),whichwassettoidentifyasinglepositivenuclearstain.5αR1
and5αR2mRNAwerequantifiedusingthechromogenicRNAinsituhybridizationalgorithm
(IndicaLaboratory),whichmeasuresRNAprobesonapercellbasis.Datawerequantifiedfor
Ki-67,AR,apoptosis,5αR1and5αR2inprostateepithelium,hyperplasia,andtumorofpros-
tatesections.Prostateepitheliumwasdefinedashistologicallynormalprostate.Representative
imagesinFig1werecapturedat2Xmagnification.
ThetotalpositivecellspertissueareaforKi-67,ARandapoptosiswascalculatedas:
Totalpositivecellspertissuearea(μm2)=Totalpositivecells/Tissuearea(μm2)
Thetotalprobecopiespertissueareafor5αR1and5αR2mRNAwascalculatedas:
Totalprobecopiespertissuearea(μm2)=Totalprobecopies/Totaltissuearea(μm2)
Totalpixelspertissueareafor5αR1and5αR2mRNAwasalsocalculated,butthiswasnot
usedfordataanalysisduetopoorsensitivity.Immunohistochemistryandinsituhybridization
dataarefromasingleexperimentforeachbiomarker.
Statisticalanalysis
DatawereanalyzedusingANOVAwithFisher’sLeastSignificantDifference(LSD),utilizingSAS
9.4(SASInstituteInc.,Cary,NC)withp<0.05consideredstatisticallysignificant.Celltypeswith
n<2wereexcludedfromstatisticalanalysis,andthemodifiedThompsontautechniquewas
usedtoeliminateoutliers.IliaclymphnodemetastasesincidencewasanalyzedusingtheKruskal
Wallisnon-parametricone-wayANOVA.Correlationbetweenbiomarkersandadjustedprostate
severelesionscoreswasanalyzedusingtheSpearmanrankcorrelationcoefficient(r).
Results
GUweightsandiliaclymphnodemetastases
GUweightsin8-,12-,16-,and20-week-oldC57BL/6TRAMPxFVBmicewereincreasedasa
functionofage(Table1).Twenty-week-oldmicehadsignificantlyincreasedGUweights
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 5/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
Fig1. Representativestainingfor5α-reductase1(a),5α-reductase2(d),androgenreceptor(g),Ki-67(j)and
apoptosis(m)inprostateepitheliumshowingsinglelayerofcells;5α-reductase1(b),5α-reductase2(e),
androgenreceptor(h),Ki-67(k)andapoptosis(n)inhyperplasiashowingfocalincreasedcelldensitywith
pilingupononeanother;and5α-reductase1(c),5α-reductase2(f),androgenreceptor(i),Ki-67(l)and
apoptosis(o)intumorshowingdiffusesheetsofcellswithnoorganizationandcharacterizedbyneoplastic
cellularcharacteristics.Staininginprostateandhyperplasiaareshownwithablackarrow.
https://doi.org/10.1371/journal.pone.0175874.g001
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 6/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
Table1. C57BL/6TRAMPxFVBmiceGUweights1andiliaclymphnodemetastasesincidence.
Group n GUweights(g) n Lymphnode
metastasesincidence(%)
8-weeks 5 0.30±0.01a 2 0(0)
12-weeks 8 0.38±0.02a 8 0(0)
16-weeks 9 0.62±0.07a 9 1(11)
20-weeks 12 3.88±1.09b 12 5(42)
1Dataaremean±SEM;valueswithdifferentlettersarestatisticallydifferent(p<0.05).
https://doi.org/10.1371/journal.pone.0175874.t001
versus8-,12-,and16-week-oldmice.Nosignificantdifferenceswereobservedintheincidence
ofiliaclymphnodemetastasesbetweengroups(Table1);however,anotabledifferencewas
foundinincidencebetween20-(42%)and16-(11%),12-(0%)and8-(0%)week-oldmice.
Mostsevereandmostcommonlesionscoresandhistopathological
distribution
Anterioranddorsallobesrawandadjustedmeanmostsevereandmostcommonlesionscores
weresignificantlyincreasedin20-versus8-,12-,and16-week-oldmice(Tables2and3).The
dorsallobeadjustedmeanmostcommonlesionscorewassignificantlyincreasedin16-com-
paredto12-week-oldmice.Lateralandventrallobesrawandadjustedmeanmostsevereand
mostcommonlesionscoresweresignificantlyincreasedin16-and20-versus8-and12-week-
oldmice.
AnteriorlobeLG-PINasthemostseverelesionwassignificantlyincreasedin8-versus
12-and20-week-oldmice(Table4).LG-PINasthemostseverelesionwassignificantlyin-
creasedintheventrallobeof8-comparedto16-and20-week-oldmice.Laterallobemoder-
ate-grade(MG)PINasthemostseverelesionwassignificantlyincreasedin8-versus12-week-
old,andin8-and12-comparedto16-and20-week-oldmice.MG-PINasthemostsevere
lesionwassignificantlyincreasedintheventrallobeof8-versus12-,16-,and20-week-old
mice.MG-PINasthemostseverelesionwassignificantlyincreasedintheventrallobeof12-
and16-comparedto20-week-oldmice.LaterallobeHG-PINasthemostseverelesionwas
significantlyincreasedin12-and16-versus8-and20-week-oldmice.HG-PINasthemost
severelesionwassignificantlyincreasedintheventrallobeof12-week-oldmicecomparedto
8-and20-week-oldmice.DorsallobeHG-PINasthemostseverelesionwassignificantly
increasedin12-and16-versus20-week-oldmice.PDcarcinomaandprostatecancerasthe
mostseverelesionsweresignificantlyincreasedintheanteriorlobeof20-comparedto8-,12-,
and16-week-oldmice.DorsallobePDcarcinomaandprostatecancerasthemostsevere
lesionsweresignificantlyincreasedin20-versus8-,12-,and16-week-oldmice.PDcarcinoma
Table2. Rawandadjustedmeanmostseverelesionscoresfortheanterior,dorsal,lateral,andventralprostatelobes1.
Anteriorprostate Dorsalprostate Lateralprostate Ventralprostate
Group n Raw Adjusted Raw Adjusted Raw Adjusted Raw Adjusted
8-weeks 5 2.10±0.28a 4.10±0.86a 2.50±0.17a 6.10±0.38a 1.90±0.10a 4.80±0.47a 1.60±0.16a 4.30±0.63a
12-weeks 8 2.56±0.13a 6.13±0.30a 3.00±0.29a 7.38±0.81a 3.00±0.41a 7.50±1.15a 1.56±0.27a 3.56±0.67a
16-weeks 9 2.39±0.31a 5.78±0.95a 3.78±0.42a 10.17±1.39a 5.17±0.50b 14.44±1.62b 4.83±0.59b 13.66±1.87b
20-weeks 12 4.92±0.51b 14.21±1.65b 5.63±0.43b 16.33±1.38b 5.42±0.51b 15.88±1.62b 4.86±0.61b 14.18±1.93b
1Dataaremean±SEM;valueswithdifferentlettersarestatisticallydifferentfromoneanother(p<0.05).
https://doi.org/10.1371/journal.pone.0175874.t002
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 7/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
Table3. Rawandadjustedmeanmostcommonlesionscoresfortheanterior,dorsal,lateral,andventralprostatelobes1.
Anteriorprostate Dorsalprostate Lateralprostate Ventralprostate
Group n Raw Adjusted Raw Adjusted Raw Adjusted Raw Adjusted
8-weeks 5 1.30±0.21a 2.70±0.58a 2.0a 5.20±0.13a,b 1.50±0.17a 3.70±0.65a 1.60±0.16a 4.30±0.63a
12-weeks 8 1.81±0.14a 4.44±0.41a 2.0a 5.50±0.26a 1.63±0.13a 4.25±0.46a 1.25±0.21a 3.13±0.62a
16-weeks 9 1.83±0.34a 4.56±1.01a 3.22±0.50a 9.39±1.49b 4.11±0.63b 11.94±1.93b 4.22±0.60b 12.28±1.87b
20-weeks 12 4.83±0.53b 14.04±1.69b 4.75±0.50b 14.00±1.55c 5.21±0.52b 15.25±1.67b 4.77±0.63b 14.09±1.96b
1Dataaremean±SEM;valueswithdifferentlettersarestatisticallydifferentfromoneanother(p<0.05).
https://doi.org/10.1371/journal.pone.0175874.t003
andprostatecancerasthemostseverelesionsweresignificantlyincreasedinthedorsallobeof
16-comparedto8-and12-week-oldmice.LateralandventrallobesPDcarcinomaandpros-
tatecancerasthemostseverelesionsweresignificantlyincreasedin16-and20-versus8-and
12-week-oldmice.Prostatecancerasthemostseverelesionwassignificantlyincreasedinthe
lateralandventrallobesof20-comparedto16-week-oldmice.
AnteriorlobeLG-PINasthemostcommonlesionwassignificantlyincreasedin8-versus
12-and20-week-oldmice(Table5).MG-PINasthemostcommonlesionwassignificantly
increasedintheanteriorlobeof12-comparedto8-,16-,and20-week-oldmice.Dorsallobe
MG-PINasthemostcommonlesionwassignificantlyincreasedin8-and12-versus16-and
Table4. Histopathologicalanalysis(mostseverelesion)ofindividualprostatelobesin8-,12-,16-,and20-week-oldC57BL/6TRAMPxFVBmice1,2.
Prostaticintraepithelialneoplasia Adenocarcinoma Prostatecancer
n LG MG HG WD MD PD (WD-PD)
Anteriorprostate
8-weeks 5 30%a 30% 40% 0% 0% 0%a 0%a
12-weeks 8 0%b 44% 56% 0% 0% 0%a 0%a
16-weeks 9 17%a,b 50% 28% 0% 0% 6%a 6%a
20-weeks 12 8%b 8% 17% 0% 4% 54%b 58%b
Dorsalprostate
8-weeks 5 0% 50% 50%a,b 0% 0% 0%a 0%a
12-weeks 8 0% 25% 69%a 0% 0% 6%a 6%a
16-weeks 9 0% 11% 67%a 0% 0% 22%b 22%b
20-weeks 12 0% 17% 13%b 0% 4% 67%c 71%c
Lateralprostate
8-weeks 5 10% 90%a 0%a 0% 0% 0%a 0%a
12-weeks 8 0% 50%b 38%b 0% 0% 13%a 13%b
16-weeks 9 0% 6%c 39%b 0% 0% 56%b 56%c
20-weeks 12 17% 4%c 8%a 0% 4% 67%b 71%d
Ventralprostate
8-weeks 5 40%a 60%a 0%a 0% 0% 0%a 0%a
12-weeks 8 13%a,b 44%b 19%b 0% 0% 0%a 0%a
16-weeks 9 0%b 39%b 6%a,b 0% 0% 56%b 56%b
20-weeks 12 8%b 17%c 0%a 0% 4% 54%b 58%c
1Valueswithdifferentlettersarestatisticallydifferentfromoneanother(p<0.05).
2LG=low-grade,MG=moderate-grade,HG=high-grade,PIN=prostaticintraepithelialneoplasia,WD=well-differentiated,MD=moderately
differentiated,PD=poorlydifferentiated.
https://doi.org/10.1371/journal.pone.0175874.t004
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 8/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
Table5. Histopathologicalanalysis(mostcommonlesion)ofindividualprostatelobesin8-,12-,16-,and20-week-oldC57BL/6TRAMPxFVB
mice1,2.
Prostaticintraepithelialneoplasia Adenocarcinoma Prostatecancer
n LG MG HG WD MD PD (WD-PD)
Anteriorprostate
8-weeks 5 80%a 10%a 10% 0% 0% 0%a 0%a
12-weeks 8 25%b,c 69%b 6% 0% 0% 0%a 0%a
16-weeks 9 50%a,b 39%a 6% 0% 0% 6%a 6%a
20-weeks 12 13%c 21%a 8% 0% 4% 54%b 58%b
Dorsalprostate
8-weeks 5 0% 100%a 0%a 0% 0% 0%a 0%a
12-weeks 8 0% 100%a 0%a 0% 0% 0%a 0%a
16-weeks 9 0% 67%b 11%b 0% 0% 22%b 22%a
20-weeks 12 0% 38%b 8%c 0% 4% 50%c 54%b
Lateralprostate
8-weeks 5 50% 50% 0% 0% 0% 0%a 0%a
12-weeks 8 38% 63% 0% 0% 0% 0%a 0%a
16-weeks 9 11% 44% 0% 0% 0% 44%b 44%b
20-weeks 12 17% 8% 8% 0% 4% 63%c 67%c
Ventralprostate
8-weeks 5 40% 60%a 0% 0% 0% 0%a 0%a
12-weeks 8 25% 50%a 0% 0% 0% 0%a 0%a
16-weeks 9 0% 56%a 0% 0% 0% 44%b 44%b
20-weeks 12 17% 8%b 0% 0% 4% 54%c 58%c
1Valueswithdifferentlettersarestatisticallydifferentfromoneanother(p<0.05).
2LG=low-grade,MG=moderate-grade,HG=high-grade,PIN=prostaticintraepithelialneoplasia,WD=well-differentiated,MD=moderately
differentiated,PD=poorlydifferentiated.
https://doi.org/10.1371/journal.pone.0175874.t005
20-week-oldmice.VentrallobeMG-PINasthemostcommonlesionwassignificantlyin-
creasedin8-,12-,and16-comparedto20-week-oldmice.DorsallobeHG-PINasthemost
commonlesionwassignificantlyincreasedin16-and20-versus8-and12-week-oldmice.
HG-PINasthemostcommonlesionwassignificantlyincreasedinthedorsallobeof16-com-
paredto20-week-oldmice.AnterioranddorsallobesPDcarcinomaandprostatecanceras
themostcommonlesionsweresignificantlyincreasedin20-versus8-,12-and16-week-old
mice.PDcarcinomaasthemostcommonlesionwassignificantlyincreasedinthedorsallobe
of20-comparedto16-week-oldmice.PDcarcinomaandprostatecancerasthemostcommon
lesionsweresignificantlyincreasedinthelateralandventrallobesof20-versus16-week-old
mice.LateralandventrallobesPDcarcinomaandprostatecancerasthemostcommonlesions
weresignificantlyincreasedin16-and20-comparedto8-and12-week-oldmice.
5αR1,5αR2,AR,proliferation,andapoptosis
Prostateepithelium,hyperplasiaandtumorrepresentativestainingsfor5αR1areshowninFig
1A,1Band1C,respectively.Prostateepithelium,hyperplasiaandtumorrepresentativestain-
ingsfor5αR2areshowninFig1D,1Eand1F,respectively.Therewasnosignificantdifference
in5αR1and5αR2inthecelltypeswithinandbetweengroups(Tables6and7).5αR1waspre-
dominantlyexpressedinprostateepitheliumversusstromawhile5αR2washighlyexpressed
inbothprostateepitheliumandstroma.Prostateepithelium,hyperplasiaandtumorARrepre-
sentativestainingsareshowninFig1G,1Hand1I,respectively.Therewasasignificant
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 9/15
5α-reductase1levelsarepositivelycorrelatedwithmostseverelesionscores
Table6. 5α-reductase1expressionin8-,12-,16-,and20-week-oldC57BL/6TRAMPxFVBmice. Thevaluesarethemeantotal5α-reductase1probe
copiespertissuearea(μm2)±SEMinprostateepithelium,hyperplasiaortumor.
5α-reductase1
Group n Prostateepithelium n Hyperplasia n Tumor
8-weeks 5 5.8±2.5 5 5.4±1.1 - —
12-weeks 4 18.7±8.7 5 10.5±4.1 2 16.8±12.1
16-weeks 5 15.1±5.3 5 6.7±3.5 2 15.7±5.1
20-weeks 2 22.4±9.5 2 13.5±5.6 4 15.8±4.1
p<0.05vs.prostateepithelium,hyperplasiaandtumorwithinandbetweengroups.Dataaremultipliedby1000.
https://doi.org/10.1371/journal.pone.0175874.t006
increaseinARexpressioninhyperplasiacomparedtoprostateepitheliumandtumorof
16-week-oldmice(Table8).TherewasasignificantdecreaseinARexpressionintumorversus
prostateepitheliumandhyperplasiaof20-week-oldmice.Prostateepithelium,hyperplasiaand
tumorKi-67representativestainingsareshowninFig1J,1Kand1L,respectively.Therewasa
significantincreaseinKi-67expressioninprostateepitheliumandhyperplasiaof8-compared
to16-and20-week-oldmice(Table9).TherewasasignificantincreaseinKi-67expressionin
prostateepitheliumof12-versus16-week-oldmice.TherewasasignificantincreaseinKi-67
expressioninhyperplasiacomparedtoprostateepitheliumandtumorof20-week-oldmice.
Prostateepithelium,hyperplasiaandtumorapoptosisrepresentativestainingsareshownin
Fig1M,1Nand1O,respectively.Therewasnosignificantdifferenceinapoptosisinthecell
typeswithinandbetweengroups(Table10).
Biomarkercorrelationwithadjustedprostateseverelesionscores
Onlyonesignificantcorrelationbetweenbiomarkersandadjustedprostateseverelesionscores
wasidentified.Prostate5αR1levelswerepositivelycorrelatedwithadjustedprostatemost
severelesionscores(Fig2).Correlationbetween5αR1inprostateandadjustedprostatesevere
lesionscoreswascomparedin8-,12-,16-,and20-week-oldmiceandfoundtohaveasimilar
trendinallgroups,thusagedidnotappeartoimpactthiscorrelation.Adjustedprostatesevere
lesionscoreswasusedbecauseitcombineslesionseveritywithanindicationofitsdistribution
withinalobe[22].
Discussion
Therawandadjusted,meanmostseverelesionscoreswereslightlyincreasedin20-week-old
micecomparedto20-week-oldcontrolC57BL/6TRAMPxFVBmiceinourpreviousstudy
[23],whichmaysuggestthatinthisstudy,lesionsweremoresevereorprogressingfaster.
Giventhatthisstudywasconductedatthesametimeusinglittermatesofthatstudy,this
Table7. 5α-reductase2expressionin8-,12-,16-,and20-week-oldC57BL/6TRAMPxFVBmice. Thevaluesarethemeantotal5α-reductase2probe
copiespertissuearea(μm2)±SEMinprostateepithelium,hyperplasiaortumor.
5α-reductase2
Group n Prostateepithelium n Hyperplasia n Tumor
8-weeks 5 9.0±4.7 5 3.8±1.3 - —
12-weeks 5 12.2±5.3 5 9.0±4.6 2 12.7±10.6
16-weeks 4 7.5±2.6 4 6.9±3.6 2 0.7
20-weeks 1 17.7 1 1.8 3 4.1±1.6
p<0.05vs.prostateepithelium,hyperplasiaandtumorwithinandbetweengroups.Dataaremultipliedby1000.
https://doi.org/10.1371/journal.pone.0175874.t007
PLOSONE|https://doi.org/10.1371/journal.pone.0175874 May11,2017 10/15