Tissue-Specific Effects of Reduced β-catenin Expression on Adenomatous Polyposis Coli Mutation

30 Pages·2015·6.98 MB·English

RESEARCHARTICLE
β
Tissue-Specific Effects of Reduced -catenin
Adenomatous Polyposis Coli
Expression on
Mutation-Instigated Tumorigenesis in Mouse
Colon and Ovarian Epithelium
YingFeng1,NaoyaSakamoto1,RongWu2,Jie-yuLiu1¤a,AlexandraWiese1,Maranne
E.Green1,MeganGreen1,AytekinAkyol1¤b,BadalC.Roy1¤c,YaliZhai2,Kathleen
R.Cho1,2,EricR.Fearon1,2,3*
1 DepartmentofInternalMedicine,UniversityofMichiganMedicalSchool,AnnArbor,Michigan,United
StatesofAmerica,2 DepartmentofPathology,UniversityofMichiganMedicalSchool,AnnArbor,Michigan,
UnitedStatesofAmerica,3 DepartmentofHumanGenetics,UniversityofMichiganMedicalSchool,Ann
Arbor,Michigan,UnitedStatesofAmerica
¤a Currentaddress:GeneticsandMolecularBiologyProgram,SchoolofMedicine,UniversityofNorth
CarolinaatChapelHill,ChapelHill,NorthCarolina,UnitedStatesofAmerica
¤b Currentaddress:DepartmentofPathology,HacettepeSchoolofMedicine,Ankara,Turkey
¤c Currentaddress:DepartmentofMolecularandIntegrativePhysiology,UniversityofKansasMedical
OPENACCESS Center,KansasCity,Kansas,UnitedStatesofAmerica
*[email protected]
Citation:FengY,SakamotoN,WuR,LiuJ-y,Wiese
A,GreenME,etal.(2015)Tissue-SpecificEffectsof
Reducedβ-cateninExpressiononAdenomatous
Abstract
PolyposisColiMutation-InstigatedTumorigenesisin
MouseColonandOvarianEpithelium.PLoSGenet
11(11):e1005638.doi:10.1371/journal.pgen.1005638 Adenomatouspolyposiscoli(APC)inactivatingmutationsarepresentinmosthumancolo-
rectalcancersandsomeothercancers.TheAPCproteinregulatestheβ-cateninprotein
Editor:RiccardoFodde,ErasmusUniversityMedical
Center,NETHERLANDS poolthatfunctionsasaco-activatorofTcellfactor(TCF)-regulatedtranscriptioninWnt
pathwaysignaling.WestudiedeffectsofreduceddosageoftheCtnnb1geneencodingβ-
Received:June9,2015
catenininApc-mutation-inducedcolonandovarianmousetumorigenesisandcellculture
Accepted:October9,2015
models.ConcurrentsomaticinactivationofoneCtnnb1allele,dramaticallyinhibitedApc
Published:November3,2015
mutation-inducedcolonpolyposisandgreatlyextendedApc-mutantmousesurvival.
Copyright:©2015Fengetal.Thisisanopen Ctnnb1hemizygousdosemarkedlyinhibitedincreasesinβ-cateninlevelsinthecytoplasm
accessarticledistributedunderthetermsofthe
andnucleusfollowingApcinactivationincolonepithelium,withattenuatedexpressionof
CreativeCommonsAttributionLicense,whichpermits
keyβ-catenin/TCF-regulatedtargetgenes,includingthoseencodingtheEphB2/B3recep-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare tors,thestemcellmarkerLgr5,andMyc,leadingtomaintenanceofcryptcompartmentali-
credited. zationandrestrictionofstemandproliferatingcellstothecryptbase.Acriticalthresholdfor
DataAvailabilityStatement:Allrelevantdataare β-cateninlevelsinTCF-regulatedtranscriptionwasuncoveredforApcmutation-induced
withinthepaperanditsSupportingInformationfiles. effectsincolonepithelium,alongwithevidenceofafeed-forwardroleforβ-cateninin
Funding:Thisworkwassupportedbygrants Ctnnb1geneexpressionandCTNNB1transcription.Theactiveβ-cateninproteinpoolwas
R01CA082223(YF,MG,andERF)and highlysensitivetoCTNNB1transcriptlevelsincoloncancercells.Inmouseovarianendo-
P30CA046592fromtheNationalInstitutesofHealth.
metrioidadenocarcinomas(OEAs)arisingfromApc-andPten-inactivation,whileCtnnb1
Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,or hemizygousdoseaffectedβ-cateninlevelsandsomeβ-catenin/TCFtargetgenes,Myc
preparationofthemanuscript. inductionwasretainedandOEAsaroseinafashionakintothatseenwithintactCtnnb1
CompetingInterests:Theauthorshavedeclared genedose.OurfindingsindicateCtnnb1genedoseexertstissue-specificdifferencesin
thatnocompetinginterestsexist.
PLOSGenetics|DOI:10.1371/journal.pgen.1005638 November3,2015 1/30
β-cateninFunctioninApc-MutantTumors
Apcmutation-instigatedtumorigenesis.Differentialexpressionofselectedβ-catenin/TCF-
regulatedgenes,suchasMyc,likelyunderliescontext-dependenteffectsofCtnnb1gene
dosageintumorigenesis.
AuthorSummary
EnhancedWntsignalingcontributestocolorectalandothercancers.β-cateninfunctions
inWntsignalingasaTcellfactor(TCF)transcriptionalco-activator.Previousstudies
showedspecificβ-catenindosagefavorsWntsignaling-dependenttumorigenesisforsome
tumortypes.However,earlierstudiesemphasizedtheroleofconstitutionalCtnnb1and
Apcgenevariations,ratherthansomaticgenetargeting,andtheworkfocusedonsmall
intestinetumorsandnoeffectsoncolontumorphenotypesweredescribed.Furthermore,
definitiveinsightswerelackingintohowreducedCtnnb1genedosageaffectedApcmuta-
tion-dependenttumorigenesis.Here,weshowsomaticinactivationofoneCtnnb1allele
dramaticallyinhibitsmousecolonadenomatouspolyposisinducedbysomaticbi-allelic
Apcinactivation.Incontrast,Ctnnb1hemizygousinactivationdoesnotaffectmouseovar-
ianendometrioidadenocarcinomadevelopmentarisingfromApc-andPten-inactivation.
Ctnnb1hemizygousgenedosedramaticallyreducestheactivepoolofβ-catenin,leadingto
thesignificantinhibitionofβ-catenin/TCF-regulatedtargetgeneexpression,including
thoseencodingkeystemcellregulatoryandcryptcompartmentalizationfactorsincolon
epithelium.Tissue-specificdifferencesforexpressionofselectedβ-catenin/TCF-regulated
genes,suchasMyc,maycontributetothecontext-dependenteffectsofCtnnb1genedos-
ageinApcmutation-drivencolonandovariantumors.
Introduction
Colorectalcancers(CRCs)harboraccumulatedmutationsintumorsuppressorgenesand
oncogenesalongwithepigeneticalterations.ManyCRCsarisefromprecursorlesions,suchas
adenomatouspolypsorserratedepitheliallesionswithdysplasia.Inactivatingmutationsinthe
APC(adenomatouspolyposiscoli)andTP53tumorsuppressorgenesarefoundinroughly
80%and60%ofCRCs,respectively[1].Oncogenicmutationsactivatingthefunctionsofthe
KRASandPI3KCA(phosphoinositide-3-kinase,catalytic,alphapolypeptide)proteinsare
foundinabout40%and20%ofCRCs,respectively[1].Constitutionalmutationsinactivating
oneAPCalleleunderliethefamilialadenomatouspolyposis(FAP)syndrome,whereaffected
individualsoftendevelophundredstothousandsofcolonadenomasduringtheirsecondor
thirddecadesoflife.ThewildtypeAPCalleleissomaticallyinactivatedinadenomasarisingin
thosewithFAP[1,2].Micecarryingcertainheterozygous,constitutionalmutationsinactivat-
ingApc,suchastheApcMinmutation,maydevelop50–100smallintestinaltumorsandocca-
sionalcolontumorsby140daysofageandnearlyallofthetumorsareadenomas.Similarto
thesituationinFAPtumors,intestinaltumorsinApcMinmiceshowsomaticinactivationofthe
wildtypeApcallele[3].
Thebestunderstoodfunctionoftheroughly300kDAPCproteinisregulationofthepool
ofβ-cateninproteinthatfunctionsinthecanonical(β-catenin-dependent)Wntsignalingpath-
way[4–6].IntheabsenceofanactivatingWntligandsignal,theβ-catenindestructioncom-
plex—comprisedbytheAPC,AXIN,caseinkinaseI,andglycogensynthasekinase-3βfactors
andotherproteins—promotesphosphorylationofconservedserine/threonineresiduesinthe
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β-cateninFunctioninApc-MutantTumors
β-cateninamino(N)-terminalregion.TheN-terminallyphosphorylatedβ-catenincanthenbe
βubiquitinatedanddegradedbytheproteasome.ActivatingWntligandsinhibitdegradation
ofthe“free”orWntsignalingpoolofβ-cateninviabindingatthecellsurfacetothefrizzled
andLRP5/6(lowdensitylipoprotein-relatedproteins5and6)cognatereceptorcomplex,
resultingininhibitionofβ-cateninphosphorylationand/orubiquitinationbythedestruction
complex[4,6].IncolonadenomasandCRCswherebothAPCalíelesaredefective,destruction
ofthefreepoolofβ-cateninisimpairedandactiveβ-cateninaccumulatesinthecytoplasmand
nucleus,whereitcancomplexwithDNAbindingproteinsoftheTCF(T-cellfactorfamily)/
Lef(lymphoidenhancerfamily)family.β-cateninfunctionsasatranscriptionalco-activator
forTCFs[7].Normally,β-catenin/TCFtranscriptionalactivationisrestrictedtothecryptbase,
especiallyintheso-calledcryptbasecolumnarstemcellscharacterizedbyexpressionofthe
Wnt-regulatedLgr5presumptivestemcellmarkerprotein[8].Constitutiveactivationofβ-
catenin/TCFtranscriptioninWntpathway-defectiveadenomasandCRCsmaypromotea
stemorprogenitorcellphenotypeinepithelialcellsindependentofcellpositioninthecrypt[9,
10].Activationofβ-catenin/TCF-dependenttranscriptionalsoalterscryptcompartmentaliza-
tionandcoordinatedmigrationofcells,apparentlythroughincreasedexpressionoftheEphB2
andEphB3receptorsandviainhibitionoftheexpressionoftheirligandsephrinB1andB2[11,
12].TheMYCgenehasbeenhighlightedasapotentiallykeytargetgeneregulatedbyβ-cate-
nin/TCFinCRCs.Genesencodingnegative-feedbackinhibitorproteinsfunctioninginthe
Wnt/β-catenin/TCFpathway,suchasAXIN2,DKK1,andNKD1,arealsoactivatedbyβ-cate-
nin/TCF(seehttp://www.stanford.edu/~rnusse/pathways/targets.htmlforalistofcandidates).
InAPC-mutantneoplasticcells,theabilityoftheseinducedregulatorproteinstoinhibitthe
Wntsignalingpathwayisabrogatedbecausethefactorsfunctionupstreamoforatthelevelof
theAPCproteininthepathway[13].
Besidesthesefindings,otherevidenceindicatesthatAPCinactivationmaypromotecancer
developmentthroughβ-catenindysregulation.Forinstance,whilemostCRCsharborAPC
mutations,asubsetofCRCsandothercancerslackingAPCmutationshaveCTNNB1gene
mutationsresultinginproductionofoncogenicβ-cateninproteinsthatareresistanttoregula-
tionbythedestructioncomplexandthatactivateβ-catenin/TCFtranscription[6,13].Also,
somepriorstudieshaveusedgeneticapproachestostudyeffectsofCtnnb1genedosageon
liver,smallintestine,andmammaryglandtumorphenotypesinmousemodelsaswellaseffects
ofCtnnb1hemizygousinactivationstate(Ctnnb1+/-)inApc-mutationinducedmouseembry-
onicdevelopmentphenotypes[14,15].ThepriorstudiesindicatedtheCtnnb1+/-constitutional
statecaninhibitintestinalandlivertumorigenesisinmicecarryingmutationsintheApcgene
(Apc1638N,ApcMin,orApcfl)[14,15].Incontrast,mammaryglandtumorigenesiswasenhanced
inApc1638NCtnnb1+/-mice,perhapsbecauseCtnnb1functionsasatumorsuppressorgenein
themammaryglandtumorsviaβ-catenin’sroleinE-cadherin-dependenttumorsuppression
[14].Nonetheless,whilethepriorstudiesyieldedevidencethatβ-cateninsignalingdosage
impactsApcmutation-inducedtumorigenesisinsometissues,thepriorworkdidnotassessthe
roleofCtnnb1dosageinApcmutation-inducedcolontumorigenesis,thechiefsiteofAPC
mutation-dependenttumorigenesisinhumans.Moreover,theworkusedmiceconstitutionally
deficientinβ-catenin,notjustinApc-mutantepithelialcells,andthefindingsdidnothighlight
specificfactorsandmechanismsthatmightaccountforeffectsofCtnnb1dosageinApcmuta-
tion-instigatedtumorigenesisindifferentcontexts.Wereporthereonstudiesoftheeffectsof
Ctnnb1genedosageonβ-cateninproteinexpressionandβ-catenin/TCFtranscriptioninApc
mutation-inducedcolonandovarianmousetumorsandcellculturemodels.Weprovideevi-
dencethatApcmutation-inducedtumorigenesisinthecolonisinhibitedbyCtnnb1hemizy-
gousgenestatusthroughmarkedeffectsonthefreepoolofβ-catenininthecytoplasmand
nucleusanditsabilitytoactivatekeyβ-catenin/TCF-regulatedtargetgenes,includingthose
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β-cateninFunctioninApc-MutantTumors
encodingkeystemfactors,suchasLgr5,andregulatorsofcryptcompartmentalization,suchas
theEphB2/B3receptors.Wealsouncoveredanovelfeed-forwardmechanismwhereβ-catenin
proteinstabilizationandβ-catenin/TCFtranscriptionappearcriticalinregulatingCtnnb1/
CTNNB1transcriptioninthesettingofApcinactivationinmousecolonandhumancoloncan-
cercells.Moreover,wefoundthatdifferencesintheabilitytoactivateMycexpressionmay
underliecolonversusovarytissue-specificdifferencesinApcmutation-instigatedtumorigene-
sisinthesettingofCtnnb1hemizygousgenedosage.
Results
InactivationofaCtnnb1alleleextendssurvivalandinhibits
adenomatouspolyposisandepithelialabnormalitiesinducedbysomatic
bi-allelicApcinactivationinmousecolon
WepreviouslydescribedCDX2P-G22Cretransgenicmice,inwhichhumanCDX2regulatory
sequencesandanout-of-frameCretransgeneallele,carryinga22-basepairguaninenucleotide
repeattractaffectingtheCreopenreadingframe,manifestmosaicCrerecombinaseexpression
incaudalembryonictissuesandinepitheliumofthedistalileum,cecum,colon,andrectum
duringadultlife[16].WealsopreviouslydescribedCDX2P-CreERT2transgenicmicethat
expressatamoxifen(TAM)-regulatedCreprotein(CreERT2)undercontrolofhumanCDX2
regulatorysequences,allowingforTAM-inducibletargetingofloxP-containingallelesinadult
terminalileum,cecum,colon,andrectalepithelium[17].UsingtheCDX2P-G22Creor
CDX2P-CreERT2transgenicmice,wehavedescribedthephenotypicconsequencesincolon
epitheliumofsomatic,bi-allelic,inactivatingmutationsinApc[16,17].Consistentwithour
priorstudies,wefoundCDX2P-G22CreApcfl/flmicelivedonlyfor8–20daysafterbirth
(mediansurvival=13d;Fig1A).AfterthreedailydosesofTAMtoinactivatebothApcalleles
indistalintestinalepithelialtissues,CDX2P-CreERT2Apcfl/fladultmicelivedonaveragefor
22days(Fig1B).Inmarkedcontrast,concurrentsomaticinactivationofoneCtnnb1allele
alongwithbothApcalleles,usingeithertheCDX2P-G22CreorCDX2P-CreERT2transgenefor
somaticgenetargeting,ledtoadramaticallyincreasedlifespanrelativetothatseeninmice
withApcbi-allelictargeting,withmediansurvivalof168dofageinCDX2P-G22CreApcfl/fl
Ctnnb1fl/+miceandfor134dafterTAMtreatmentintheCDX2P-CreERT2Apcfl/flCtnnb1fl/+
mice(Fig1Aand1B).
Consistentwithourpriorreports[16,17],theproximalcolonandcecumofboth
CDX2P-G22CreApcfl/flmice(whenmoribundat8–20dofage)andCDX2P-CreERT2Apcfl/fl
mice(only20daysafterTAMinduction)weredramaticallythickenedandmanypolypoid
lesionswereseen(S1Fig).Histologicalanalysisofproximalcolonepithelialtissuesfromthese
miceshowedsignificanthyperplasticanddysplastic(adenomatous)changesalongwithfre-
quentcryptfissionandbranching(Fig1C).Thedramaticpolyposisincecumandcolonseen
followingbi-allelicApcinactivationwassignificantlyinhibitedatbothearlyandlatertime
pointsbyconcurrentinactivationofoneCtnnb1allele,withnogrosslydiscernableepithelial
phenotypeseenintheproximalcolonandonlytwotofourpolypsinthececumpermouseas
theCDX2P-G22CreApcfl/flCtnnb1fl/+andCDX2P-CreERT2Apcfl/flCtnnb1fl/+micewereaged
(S1Fig).ThececalpolypsarisinginCDX2P-G22CreApcfl/flCtnnb1fl/+andCDX2P-CreERT2
Apcfl/flCtnnb1fl/+micemaycontributetotheirprematuremortalityrelativetocontrolmice,as
noothergrosslydetectableintestinallesionsorpathologywerenotedinthemice.TheCre-
mediatedsomaticinactivationofbothApcallelesandoneCtnnb1alleleinproximalcolonepi-
theliumwasconfirmedbygenotyping.TherarececaladenomasarisinginCDX2P-CreERT2
Apcfl/flCtnnb1fl/+micewerefoundtohavesignificantfractionsofcellsthatescapedCre-medi-
atedCtnnb1targeting,eventhoughCre-mediatedsomaticinactivationofbothApcalleles
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β-cateninFunctioninApc-MutantTumors
Fig1.Apcmutation-inducedpolyposisinmousececumandcolonepitheliumisdramaticallyinhibitedbyconcurrentinactivationofoneCtnnb1
allele.(A)ThesurvivalofCDX2P-G22CreApcfl/flCtnnb1fl/+mice(blue;n=24)iscontrastedwiththatofCDX2P-G22CreApcfl/flmice(red;n=9).P<0.0001
bylog-rank(Mantel-Cox)test.(B)ThesurvivalofCDX2P-CreERT2Apcfl/flCtnnb1fl/+mice(blue;n=12)iscontrastedwiththatinCDX2P-CreERT2Apcfl/fl
mice(red;n=14),wheretimezerorepresentsthethirddayofthreedayswhenTAMwasadministereddaily.P<0.0001bylog-rank(Mantel-Cox)test.(C)
Hematoxylinandeosin(H&E)stainingofproximalcolontissuesfromaCDX2P-CreERT2Apcfl/flmouse20daysafterthethirdofthreedailydosesofTAM
(left,lower),andacontrol(non-gene-targeted)mouse(left,upper).RightpanelsshowH&EstainingofproximalcolontissuesfromCDX2P-CreERT2Apcfl/fl
Ctnnb1fl/+mice20days(right,upper)or2months(right,lower)afterTAMdosing.Scalebars,50μm.
doi:10.1371/journal.pgen.1005638.g001
occurredtothesameextentintherareadenomasandproximalcolonmucosa(S1Fig).Com-
paredtothesituationinCDX2P-CreERT2Apcfl/flmice,microscopicexaminationofproximal
colontissuesofCDX2P-CreERT2Apcfl/flCtnnb1fl/+micerevealedmodesthyperplasticchanges
andminimalcryptbranching(Fig1C).OureffortstoinactivatebothCtnnb1allelesincolon
epitheliumviaeitherCDX2P-G22Cre-orCDXP-CreERT2-mediatedtargetingwithorwithout
Apcinactivationindicatedthatcolonepithelialcellscompletelylackingβ-cateninexpression
andfunctioncouldnotbegenerated.Thislikelyreflectsarequiredroleforβ-cateninfunction
incolonepithelium,perhapsnotlimitedtoWntsignaling,butalsoincadherin-mediatedadhe-
sion,centrosomeassemblyorotherfunctions.
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β-cateninFunctioninApc-MutantTumors
ImmunohistologicalanalysisofcolonsectionsfromCDX2P-CreERT2Apcfl/flmiceshowed
strongcytoplasmicandnuclearβ-cateninexpressioninmanyepithelialcells,comparedtothe
nearlyuniformmembraneβ-cateninstainingincolonepitheliumofwild-typemice(Fig2A).
InCDX2P-CreERT2Apcfl/flCtnnb1fl/+mice,weobservedinfrequentcellswithelevatedcyto-
plasmicand/ornuclearβ-cateninexpression(Fig2A).Panethcells,aspecializedsecretorycell
linagethatexpresseslysozymeandothermarkers,arefoundatthecryptbaseinnormalmouse
smallintestinalepithelium,butareabsentinnormalmousecolonepithelium.Panethcells
havebeenproposedtohaveakeyroleingenerationand/ormaintenanceoftheintestinalcrypt
stemcellniche[18].Bi-allelicApcinactivationhasbeenassociatedwiththegenerationof
manyectopiclysozyme-expressingPaneth-likecellsthroughoutthecryptsofsmallintestine
andcolon[12,17,19,20].WeconfirmedthisfindinginCDX2P-CreERT2Apcfl/flmice(Fig2A).
WhereasnoPaneth-likecellswereseeninnormalmousecolon,modestnumbersoflysozyme-
expressingcellswereseeninthecolonsofCDX2P-CreERT2Apcfl/flCtnnb1fl/+mice(Fig2A).
WealsousedatransgenicmouselinecarryingaCre-activatedenhancedyellowfluorescence
protein(EYFP)reportergeneattheubiquitouslyexpressedRosa26locustomonitorcolonepi-
thelialcellsandglandswhereCre-mediatedtargetinghadoccurred.Ectopiclysozyme-express-
ingcellswerefoundinnearlyalloftheEYFP-positivecryptsinCDX2P-G22CreApcfl/fl
Ctnnb1fl/+andCDX2P-CreERT2Apcfl/flCtnnb1fl/+mice(Fig2Band2C).Therareoccurrence
ofPaneth-likecellsincryptswithoutEYFPexpressionlikelyreflectsthepossibilitythatCre
maymoreefficientlytargettheloxPsitesattheApclocusthanattheRosa26locus.
PriorstudiesfromothergroupsandourshaveshownthatApcbi-allelicinactivation
increasesbothcellproliferationandapoptosisinintestineandcolonepithelium[12,17,19,
21].FollowingTAM-inducedApcbi-allelicinactivationinproximalcolonepithelium,wecon-
firmedsignificantlyelongatedcryptsandincreasedcellproliferationandapoptosisrelativeto
controlepithelialtissues(Fig2Dand2E).Incontrast,onlymodestlyincreasedcryptheight,
cellproliferationandapoptosisrelativetocontrolepitheliumwereseeninepitheliumof
CDX2P-CreERT2Apcfl/flCtnnb1fl/+micefollowingcombinedApcandCtnnb1geneinactivation
(Fig2Dand2E).Furthermore,althoughbi-allelicApcinactivationinducedextensivecellpro-
liferationintheupperhalfoftargetedcoloncrypts,cellproliferationfollowinggenetargeting
inCDX2P-CreERT2Apcfl/flCtnnb1fl/+micewaslargelyrestrictedtothebottomhalfofeach
crypt,withonlyaslightincreaseincellproliferationcomparedtocontrolcolonepithelium
(Fig2Dand2E).Thecellproliferationandapoptosisresultswerewellcorrelatedwiththe
immunohistochemicalstudiesofβ-cateninlevelsandlocalization(Fig2A),suggestingdiffer-
encesinthestrengthofβ-catenin-dependentWntsignalingincellswithbi-allelicApcdefects
underlietheobservedeffectsoncolonepithelialmorphology,cellfateanddifferentiation,and
cellproliferationandapoptosis.
Theorientationofthemitoticspindleaxismayimpactoncellfatedecisionsinintestinal
epithelium.Atcytokinesis,theorientationofthespindleaxisinaplanarfashion(i.e.,parallel
tothecryptaxis)isthoughttogeneratetwodaughtercellswithequivalentluminal(apical)and
basement(extracellularmatrix)surfaces.Ifthespindleaxisisnotorientedparalleltothecrypt
axis,cytokinesisgeneratesdaughtercellswithdifferencesinluminalandbasementmembrane
surfacesandthepotentialforresultantdifferencesinthefatesadoptedbythetwodaughter
cells.Wepreviouslyreportedsignificantincreasesinthepercentageofepithelialcellswherethe
mitoticspindleaxiswasorientedorthogonaltotheplanaraxisinApc-mutantmousecolon
cryptsrelativetowildtypecrypts[17].Consistentwithourpriorresults,incolonepitheliumof
CDX2P-CreERT2Apcfl/flmicetreatedwithTAMtoinactivatebothApcalleles,roughly50%of
thecellsinmitosishadmitoticspindleaxes(cid:1)30°degreesoutoftheplanaraxis,withnearly
20%showingspindleaxesbetween60°and90°outofplanaralignment.Incontrast,inepithe-
liumofCDX2P-CreERT2Apcfl/flCtnnb1fl/+miceandcontrolmice,>75%ofmitoticcolon
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β-cateninFunctioninApc-MutantTumors
Fig2.InactivationofaCtnnb1alleleinmousecolonepitheliumreducesapoptosisandhyperproliferationinducedbybi-allelicApcinactivation,
andalsoreduces,butdoesnotabrogate,theaberrantcelldifferentiationassociatedwithApcinactivation.(A)Representativeimmunohistochemical
stainingforβ-catenin(leftpanels)andlysozyme(rightpanels)inaCDX2P-CreERT2Apcfl/flCtnnb1fl/+mouse(middlerow)oraCdx2P-CreERT2Apcfl/fl
mouse(bottomrow)20daysfollowingthreedailydosesofTAM.ImmunohistochemicalstainingisalsoshownfornormalcontrolcolonmucosafromaCre
negativecontrollittermatemouse(toprow).Scalebars,20μm.(BandC)EYFPexpressionactivatedintargetedcoloncryptsbyCre-mediatedrecombination
wasimagedalongwithImmunofluorescencestainingforlysozyme(lyso;red)inproximalcolontissuesthatwerecounter-stainedwithHoechst33342for
detectionofnuclei.A6-montholdG22CreApcfl/flCtnnb1fl/+EYFPfl/+mouseandaCrenegativecontrollittermateareshowninpanelB.Colontissuesfroma
CDX2P-CreERT2Apcfl/flCtnnb1fl/+EYFPfl/+mouse(panelC,middle)oraCDX2P-CreERT2Apcfl/flEYFPfl/+mouse(panelC,bottom)wereanalyzed38days
afterthethirdofthreedailydosesofTAM.ColontissuesfromaCDX2P-CreERT2EYFPfl/+mouse(panelC,topleft)andaCre-negativeEYFPreportermouse
(panelC,topright)servedascontrols.ArrowheadsindicatetheEYFP-negativecryptsthatexpressedthePanethcellmaker,lysozyme.Anarrowinthe
middlepanelindicatesacryptshowingEYFPexpressionbutnolysozymestaining.Scalebars,50μm.(D)TUNELassay(middlecolumnpanels)and
immunohistochemicalstainingforlysozyme(leftcolumnpanels)andBrdU(rightcolumnpanels)inproximalcolontissuesofaCDX2P-CreERT2Apcfl/fl
Ctnnb1fl/+mouse(middlerow)oraCdx2P-CreERT2Apcfl/flmouse(bottomrow)20daysafterthreedailydosesofTAM.Thenormalcolonmucosafromacre
negativemouseservedascontrol(toprow).ArrowsindicateapoptoticcellsintissuesfromaCDX2P-CreERT2Apcfl/flCtnnb1fl/+mouseandthecontrol
mouse;arrowheadsindicatethelysozyme-positivecellsintissuesfromaCDX2P-CreERT2Apcfl/flCtnnb1fl/+mouse.Scalebars,25μm.(E)Apoptoticcells
percryptinproximalcolon,basedontheTUNELassay,werequantified(panelE,top;n=3and>100cryptscounted;*P<.001comparedtothecontrol
mouse).Proliferatingcellspercrypt,asassessedbyBrdUincorporation,wasquantified(panelE,bottom;n=3and>110cryptscounted;*P<.001
comparedtothecontrolmouse).
doi:10.1371/journal.pgen.1005638.g002
epithelialcellshadtheirmitoticspindlesalignedwithin30°oftheplanar(crypt)axis(S2Fig).
Thefindingsindicateβ-cateninlevelshaveakeyroleinthealteredmitoticspindleaxispheno-
typeofApc-mutantcolonepithelium.
Ctnnb1inactivationinhibitsApcmutation-inducedcolontumorigenesis
viamaintenanceofEphB/ephrinBsignalingandrestrictionof
presumptivestemcellstothecryptbase
Asdescribedabove,bi-allelicApcinactivationacutelyinduceshyperproliferationanddysplas-
ticalterationsinmouseproximalcolonepithelium,withthealteredepitheliumarisinginpart
fromexpansionofthecryptprogenitorcompartmentattheexpenseofthedifferentiatedcom-
partment,alongwithfrequentcryptfission/branching[12,17,21].TheEphB/ephrinBsignal-
ingaxishasbeenimplicatedincontrolofintestinalepithelialcellcompartmentalizationalong
thecryptaxisandincellmigration[11,22].TheEphB2andEphB3receptorsaretwokeyeffec-
torsofcompartmentalizationandcellmigrationinthecrypt,andEphB2andEphB3areeach
encodedbyageneactivatedinintestinaltissuesbyβ-catenin/Tcftranscription.TheEphB2/B3
receptorligands,ephrinB1andephrinB2,showhighestexpressionlevelsindifferentiatedcells
atthecryptsurface,andexpressionofephrinsB1andB2isnegativelyregulatedbyβ-catenin/
Tcfactivity[11,23].Ofnote,EphB-ephrinBinteractionsgeneraterepulsiveforcesthatseparate
andcompartmentalizetheEphB-andephrinB-expressingcellstomaintaincryptarchitecture
[11,23].Innormalmousecolonepithelium,theEphB2andEphB3receptorswereexpressed
onlyinprogenitorcellsatthecryptbase(Fig3Aand3B).Wefoundbi-allelicApcinactivation
incolonepitheliumnotonlyincreasedEphB2andEphB3expression,butalsoperturbedthe
gradientofEphB2andB3receptorexpressionalongthecryptaxis,withEphB2/B3expression
seenevenatthecryptsurfaceinApc-mutantcrypts(Fig3Aand3B).Inthecaseofephrin
ligandexpression,ourstudiesdemonstratedstrongexpressionofephrinB1andB2innormal
colonsurfaceepithelialcellsandnormalcoloncryptcellsotherthanthecryptbase.Thenormal
patternofephrinB1/B2expressionremainedlargelyunaffectedinApc-mutantcryptswithone
Ctnnb1alleleinactivated(Fig3C).Incontrast,inApc-mutantcryptswhereCtnnb1dosagewas
intact,ephrinB1/B2expressionwasmarkedlydown-regulatedincolonsurfaceepithelialcells
andthroughoutthecrypt(Fig3C).Ourfindingsareconsistentwiththoseinapriorstudythat
showedincreasedexpressionofEphB2/B3andlossofephrinB1/B2expressionincolonadeno-
masofApcmin/+mice[23].AlthoughexpressionofEphB2/B3wasmoderatelyelevatedinsome
colonepithelialcellsofCDX2P-CreERT2Apcfl/flCtnnb1fl/+micecomparedtocontrolmice,
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β-cateninFunctioninApc-MutantTumors
Fig3.InactivationofoneCtnnb1alleleinApc-mutantmousecolonepitheliumpreservesthecompartmentalizationoftheEphB2/B3-and
ephrinB1/B2-expressingcellstomaintaincryptarchitecture.Mouseproximalcolontissuesectionswerestainedforlysozyme(red)andEphB2(panelA,
green)orEphB3(panelB,green)receptors,andcounter-stainedwithHoechst33342(Blue)toidentifynuclei.ThetissuesfromaCDX2P-CreERT2Apcfl/fl
Ctnnb1fl/+mouse(top,right)andaCDX2P-CreERT2Apcfl/flmouse(bottom)wereanalyzed20daysafterthethirdofthreedailydosesofTAM.Thenormal
colonmucosafromaCre-negativelittermatewasusedasacontrol(Cntrl,topleft).Scalebars,20μm.(C)Proximalcolontissuesectionsfromthesamemice
wereco-stainedforephrinB1(left,green)orephrinB2(right,green)andlysozyme(red),andcounter-stainedwithHoechst33342(Blue).Scalebars,20μm.
doi:10.1371/journal.pgen.1005638.g003
elevatedEphB2/B3expressionremainedrestrictedtothecryptbaseregion,ratherthanspread-
ingthroughoutthecryptaswasseeninApc-mutantcryptswithintactCtnnb1genedosage
(Fig3Aand3B).Thisobservationsuggeststhereducedβ-cateninlevelsinCDX2P-CreERT2
Apcfl/flCtnnb1fl/+miceleadstoafailuretoinduceenoughβ-catenin/TCF-regulatedEphB2/B3
expressiontoovercometherepulsiveeffectsoftheretainedexpressionofephrinB1/B2ligands
inApc-mutantcryptswithreducedCtnnb1dosage.
AsimilarexpressionpatterntothatseenforEphB2andEphB3wasalsofoundforSox9,a
transcriptionfactorencodedbyaβ-catenin/Tcftargetgene.Sox9expressionisrestrictedto
stem/progenitorcellsatthenormalcoloncryptbase(S3Fig).Sox9expressionwasonlymod-
estlyincreasedandexpandedfollowinggenetargetingincryptsofCDX2P-CreERT2Apcfl/fl
Ctnnb1fl/+micerelativetothemarkedchangesinSox9levelsandthenumberofSox9-expres-
singcellsincryptsfromCDX2P-CreERT2Apcfl/flmice(S3Fig).Inspiteofthereducedincrease
inβ-cateninlevelsincoloncryptsofCDX2P-CreERT2Apcfl/flCtnnb1fl/+micerelativeto
CDX2P-CreERT2Apcfl/flmice,theresultantsignalingwasstillsufficienttogeneratesome
ectopicPaneth-likecells(Fig3Aand3B).Inaddition,themodestincreaseinβ-cateninlevels
intargetedcryptsofCDX2P-CreERT2Apcfl/flCtnnb1fl/+micewassufficienttoinduceexpres-
sionintargetedcryptsofaβ-galactosidasereportergeneintegratedintotheβ-catenin/TCF-
regulatedAxin2locus.However,β-galactosidaseexpressionwasreducedincryptsandfewif
anycolonsurfaceepithelialcellsexpressedβ-galactosidaseinApc-mutantepitheliumwith
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β-cateninFunctioninApc-MutantTumors
hemizgyousCtnnb1dosage(S3Fig).Incontrast,uniformlystrongβ-galactosidaseexpression
wasseenthroughoutApc-mutantcoloncryptsandsurfaceepithelialcellswithintactCtnnb1
dosage(S3Fig).Takentogether,thefindingsindicatedistinctβ-catenin/Tcftargetgenesin
colonepitheliumdisplaydifferingtranscriptionalresponsestoβ-cateninlevels,withAxin2per-
hapsrepresentingatargetgenecapableofbeingactivatedbymodesttomoderatelevelsofβ-
cateninincolonepithelium.TheSox9,EphB2,andEphB3genesappeardependentonhigher
levelsofβ-cateninfortranscriptionalactivationincolonepithelium.
ToaddressmechanismsunderlyingsuppressionofcryptfissionandbranchinginApc-defi-
cientcolonepitheliumwhenoneCtnnb1allelewasinactivated,wecomparedexpressionofpre-
sumptivestemcellmarkersinApc-deficientcoloncryptswherebothCtnnb1alleleswereintact
orwhereonlyoneCtnnb1allelewasactive.Consistentwithourpriorwork[17],20daysafter
TAM-inducedbi-allelicApcinactivation,wedetectedstronginductionofenhancedgreenfluo-
rescentprotein(EGFP)expressedfromtheLgr5locus(Lgr5-EGFP)(Fig4A)inApc-deficient
colonepitheliumgeneratedbyCDX2P-CreERT2targeting.Lgr5isaβ-catenin/TCF-regulated
geneandamarkerofpresumptivecryptbasecolumnarstemcellsinnormalcolon,andthe
Lgr5allelethatweusedhasaEGFPopenreadingframeintegratedinthelocustoallowfor
monitoringofendogenousLgr5expression[8].InApc-mutantepithelium,wealsoconfirmed
stronginductionoftheMsi1RNA-bindingprotein(Fig4B),anotherpresumptiveintestinal
stemcellmarker[24,25].IncontrasttoapriorstudywhereitwasreportedthatLgr5-expres-
singcellswereonlyexpandedatthelowerpartofthecryptsincolonepitheliumfollowing
mutantβ-catenininduction[21],wedetectedEYFP-positiveandMsi-positivecellsessentially
throughouttheApc-mutantdysplasticcoloncryptswhenbothCtnnb1alleleswereactive,
thoughexpressionofEYFPwasmoreprominentnearthecryptbaseregion,includinginthede
novocrypts.WhilethenetnumberofEYFP-andMsi1-expressingcellspercryptwereslightly
increased(e.g.from3–4to5–8Lgr5-positivecellspercrypt)incolonepitheliumofTAM-
treatedCDX2P-CreERT2Apcfl/flCtnnb1fl/+micecomparedtothecontrolmice,parallelingthe
subtleincreaseincryptfission/buddingseen,theexpandedpopulationofEYFP-positivecells
remainedrestrictedtothecryptbaseregion(Fig4A),consistentwiththeEphB2andEphB3
datadescribedabove.EYFPexpressionpatternsincolonsimilartothoseseeninTAM-treated
CDX2P-CreERT2Apcfl/flCtnnb1fl/+micewerealsoobtainedwhenweusedTAM-treatmentto
activatetheLgr5-drivenCreERT2transgenetotargetApcandCtnnb1allelesandEYFPexpres-
sionwasusedtomarkLgr5-expressingcells(Fig4A).
ConsistentwithourstudiesofLgr5andMsiexpressionpatternsincolonepithelium,the
levelsoftranscriptsencodingpresumptivestemcellmarkers,includingLgr5,CD44,Msi1,and
Hopx,werealsofoundtoincreasedramaticallyinthecolontissuesofCDX2P-CreERT2Apcfl/fl
mice(S4Fig).Theinductionofgenesencodingstemcellmarkersandotherselectiveβ-cate-
nin/Tcftargetgenes(suchasAxin2,Nkd1,Ccnd1andIrs1)observedinApc-deficientcolon
epitheliumwassignificantlysuppressedincolonepitheliumfromCDX2P-CreERT2Apcfl/fl
Ctnnb1fl/+mice(S4Fig).Takentogether,ourdataindicatethattherobustinductionofmany
β-catenin/Tcf-regulatedgenesthatisseenresponsetoApcinactivationwasvariablyinhibited
inreducedCtnnb1genedosageandβ-cateninproteinlevelsinmousecolonepithelium.Inthe
settingofinactivationofoneCtnnb1allele,theinabilityofApcinactivationtosubstantially
activatecertainkeyβ-catenin/TCF-regulatedgeneswithfunctionsincoloncryptcompartmen-
talizationandcellmigration(e.g.,EphB2andEphB3)orstemcellfate(e.g.,Lgr5andMsi)is
likelytounderliethedramaticabrogationofadenomaformationinCDX2P-CreERT2Apcfl/fl
Ctnnb1fl/+mice.
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