Article
theColorectalCancerStem
andIschemia
Highlights
Prox1expressionisdispensableforhomeostasisinthenormal
intestine
AsubpopulationofProx1
+
cellshasstemcellactivityinintesti-
naladenomas/CRC
tumorcell
Authors
Zolta′nWiener,JennyHo¨gstro¨m,...,Yinon
Ben-Neriah,KariAlitalo
Correspondence
kari.alitalo@helsinki.fi
Wieneretal.nowshowthattheProx1
Wieneretal.,2014,CellReports8,1943–1956
September25,2014a2014TheAuthors
http://dx.doi.org/10.1016/j.celrep.2014.08.034
AnnexinA1andfilaminAmediateProx1effectsonstemcellac-
tivityinthetumors
LossofProx1decreasesadenoma/CRCstemcells,
growth,andsurvival
CellPopulationtoFuelTumorGrowth
Resistance
GraphicalAbstract
Prox1PromotesExpansionof
transcriptionfactorfunctionsasastem
cellregulatorinintestinaladenomasand
colorectalcancer(CRC),butnotinthe
normalintestine.Prox1criticallycontrib-
utestotumorcellsurvivalinhypoxia
andtotheexpansionoftheadenoma/
CRCstemcellpopulationviainhibition
oftheWnt-targetannexinA1andinduc-
tionoftheactin-bindingproteinfilamin
A.TheProx1pathwaythusrepresents
anattractivetherapeutictargetfordrug
developmentinCRC.
AccessionNumbers
GSE47568
InBrief
CellReports
Article
Prox1PromotesExpansion
oftheColorectalCancerStemCellPopulation
toFuelTumorGrowthandIschemiaResistance
Zolta′nWiener,
1,6
JennyHo¨gstro¨m,
1,6
VilleHyvo¨nen,
1
ArjaM.Band,
1
PauliinaKallio,
1
TanjaHolopainen,
1
OlliDufva,
1
CajHaglund,
3
OlliKruuna,
3
GuillermoOliver,
4
YinonBen-Neriah,
5
andKariAlitalo
1,2,
1
TranslationalCancerBiologyProgram,UniversityofHelsinki
2
WihuriResearchInstitute
BiomedicumHelsinki,00014Helsinki,Finland
3
DepartmentofSurgery,HelsinkiUniversityCentralHospital,00029Helsinki,Finland
4
DepartmentofGenetics,StJudeChildren’sResearchHospital,Memphis,TN38105,USA
5
LautenbergCenterforImmunology,HebrewUniversity-HadassahMedicalSchool,Jerusalem91120,Israel
6
Co-firstauthor
Correspondence:kari.alitalo@helsinki.fi
http://dx.doi.org/10.1016/j.celrep.2014.08.034
ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/3.0/).
SUMMARY
Colorectalcancer(CRC)initiationandgrowthisoften
attributedtostemcells,yetlittleisknownaboutthe
regulationofthesecells.Weshowherethatasub-
populationofProx1-transcription-factor-expressing
cellshavestemcellactivityinintestinaladenomas,
butnotinthenormalintestine.Usinginvivomodels
and3Dexvivoorganoidculturesofmouseade-
nomasandhumanCRC,wefoundthatProx1dele-
tionreducedthenumberofstemcellsandcell
proliferationanddecreasedintestinaltumorgrowth
viainductionofannexinA1andreductionofthe
actin-bindingproteinfilaminA,whichhasbeenimpli-
catedasaprognosticmarkerinCRC.LossofProx1
alsodecreasedautophagyandthesurvivalof
hypoxictumorcellsintumortransplants.Thus,
Prox1isessentialfortheexpansionofthestemcell
poolinintestinaladenomasandCRCwithoutbeing
criticalforthenormalfunctionsofthegut.
INTRODUCTION
Colorectalcancer(CRC)isoneoftheleadingcausesofcancer
mortalityinWesterncountries.InmostCRCpatients,aninitial
mutationoccursintheAPCorCTNNB1gene,leadingtoactiva-
tionoftheb-catenin/TCF(canonicalWnt)pathway(Fearon,
2011;FoddeandSmits,2001).Inthecryptsofthenormalgut,
theb-catenin/TCFpathwayisactiveinPanethcells,intransit-
amplifying(TA)/progenitorcellsthathavealimitedproliferative
capacity,andinLgr5
+
intestinalstemcells(Clevers,2006).
Lgr5
+
cellsarecapableofefficientlyinitiatingadenomaformation
uponmutationoftheApcgene(Barkeretal.,2009).Inaddition,
progenitorsofintestinalepithelialcellscanconverttoastem
cell-likephenotypeandcontributetotheinitiationofCRCunder
inflammatoryconditions(Schwitallaetal.,2013).Thesestudies
indicatethattheacquisitionofastemcell-likephenotypeis
criticalforCRCtumorigenesis.
Intestinaladenomasarehighlyheterogenous,containingboth
proliferatinganddifferentiatingcells,andtheyarecontinuously
maintainedbyadedicatedcellpopulation,theso-calledcancer
stemcells(SampieriandFodde,2012;Schepersetal.,2012).
Althoughneoplasticcellsarecharacterizedbyincreasedcell
proliferationandlimitedcelldifferentiationcapacity,their
detaileddifferentiationpathwaysinCRCarepoorlyknown.
ExpressionoftheLgr5genehasbeenshowntomarkacellpop-
ulationwithstemcellpropertiesinmouseintestineandinintes-
tinaladenomas(Barkeretal.,2007;Schepersetal.,2012).
Furthermore,theintestinalstemcellsignature,includingLGR5
expression,identifiesCRCstemcellsandpredictsdisease
relapsealsoinhumanCRCpatients(Kemperetal.,2012;Mer-
los-Sua′rezetal.,2011).
AfterthegeneticlesionthatactivatestheWntsignaltransduc-
tionpathwayandabnormalcellproliferation,additionalmuta-
tionsaccumulateslowlytopromoteadenomaprogression
towardCRC,tumorinvasion,andmetastasis(Sampieriand
Fodde,2012).HighlyelevatedWntactivityafterApcdeletionin-
ducesexpressionofthehomeoboxtranscriptionfactorProx1in
intestinaltumorcells.WhenProx1wasdeletedinmicewithan
Apcmutation,adenomagrowthanddevelopmentofdysplasia
inthetumorepitheliumwasinhibited(Petrovaetal.,2008).In-
terestingly,Prox1upregulationafterlossofthetp53tumorsup-
pressorcontributestointestinaltumorprogressioninsome
modelsystems(Elyadaetal.,2011).
Here,weanalyzedthemechanismofProx1-inducedintestinal
adenomaprogressioninmicrosatellite-stabletumormodels.We
foundthatasubpopulationofProx1
+
cellsdisplaysstemcell
activityinadenomas/CRC,butnotinthenormalintestinal
epithelium.Furthermore,Prox1deletionreducedthesizeofthe
Lgr5
+
adenomaandCRCstemcellpopulations,andstemcell
activityandledtoreducedgrowthanddecreasedtumorcellsur-
vivalinanunfavorablemicroenvironment.
CellReports8,1943–1956,September25,2014a2014TheAuthors1943
RESULTS
Prox1ExpressionIsInducedinLgr5
+
CellsuponApc
GeneDeletion
TocharacterizeProx1-expressingcellsinthepathogenesisofin-
testinaladenomas,weinducedanacutedeletionoftheApc
genethroughoutthewholeintestinalepitheliuminApc
flox/flox
;
villin-Cre
ER
(VApc)micebyasingletamoxifeninjection
(VApcD/D).Asexpected,Panethcells,markingthecrypt
bottomsinthewild-type(WT)smallintestine,weredislocated
towardthelumeninmostoftheApc-deletedcrypts,andex-
pansionofthecellpopulationwithanactiveWntsignaling
pathwaywasdetectedbyEphB2immunostaining6daysafter
thetamoxifeninjection(FiguresS1AandS1B)(Batlleetal.,
2002).MostcryptscontainedscatteredProx1
+
cellclusters
intermingledwithlysozyme-positivePanethcells(FigureS1A).
CyclinD1markstheproliferatingcellpopulation,includingthe
progenitorcellsinWTintestine(GregorieffandClevers,2005).
Inlinewithpreviouslypublishedresults,thecyclinD1
+
cellpop-
ulationexpandedaftertheApcdeletion(Sansometal.,2005)
(FiguresS1AandS1B).CyclinD1
high
cellswerelocated
frequentlyclosetotheProx1
+
cells;however,Prox1
+
cells
werenotcyclinD1
high
inVApcD/Dmiceandintumorsfrom
Apc
min/+
mice(FiguresS1CandS1D)orfromCRCpatients(Fig-
ureS1E).Similarlytotheinvivoexperiments,whenweisolated
intestinalorganoidsfromtheVApcmiceandinducedApc
genedeletionin3DMatrigelculturebytheadditionof4-hy-
droxy-tamoxifen(4-OH-Tam),theorganoidsshowedemerging
Prox1
+
cellclusters2daysafter4-OH-Tamtreatment(Fig-
ureS1F).Atlatertimepoints,Prox1expressionwasmaintained
inisolatedcellclusters(FigureS1G).
ArecentstudyreportedthatPROX1ispartoftheWnt
high
CRC
stemcellgenesignature(deSousaEMeloetal.,2011).Wefound
thatProx1wasexpressedintheearliesthistologicaladenoma-
touslesions,theaberrantcryptfoci,oftheApc
min/+
mice,which
representawidelyusedmouseintestinaladenomamodel(Fig-
ure1A).ToexamineProx1expressionintheLgr5
+
intestinal
stemcellsoftheadenomas,weproducedApc
flox/flox
;Lgr5-
EGFP-IRES-Cre
ER
(LApc)miceandinducedApcdeletioninthe
stemcells(LApcD/D).LApcandLgr5-EGFP-IRES-Cre
ER
mouse
intestinesshowgreenEGFPsignalsintheLgr5
+
stemcellsatthe
cryptbottom(Barkeretal.,2007).Notably,thecyclinD1
high
cell
clusterswereenrichedmostlyamongtheLgr5
low
andLgr5
C0
cell
populationsintheintestinaladenomasafterApcdeletion,con-
firmingthatcyclinD1
high
cellsdonotaccumulateinthestem
cellpopulation(Figure1B).Prox1wasexpressedinsomeof
thePanethcellsintheileumandProx1
+
cellsinthenormalintes-
tinewereoftenlocatedneartheLgr5
+
cells(FigureS1H;Fig-
ure1C),buttherewasnooverlapbetweentheEGFPand
Prox1signalsintheuntreatedintestineorduringepithelialrepair
at4,6,and10daysafter6Gyirradiation(FigureS1Ianddatanot
shown).However,5daysaftertamoxifeninjection,Prox1
expressionwasobservedalsointheLgr5
+
cellsofthedevel-
opingadenomas(Figure1C).Takentogether,theseresults
indicatethatProx1isinducedduringtheearlystepsoftumori-
genesisinadenomacells,includingLgr5
+
stemcells,bothinvivo
andexvivo,butnotinthecyclinD1
high
cellsthatmayrepresent
moredifferentiatedorintestinalprogenitor-likecells.
ASubpopulationoftheProx1
+
CellsHasStemCell
ActivityinIntestinalAdenomas,butNotintheNormal
Intestine
TostudyifProx1
+
cellshavestemcellactivityinintestinalade-
nomas,weproducedProx1-Cre
ER
(Srinivasanetal.,2007);
Rosa26-tdTomato
flox/Stop/flox
;Apc
min/+
miceforlineage-tracing
experiments.Inthismodel,asingletamoxifeninjectionactivated
theCreallele,resultinginexpressionofthetdTomatoredfluo-
rescentproteinonlyintheProx1
+
tumorcells1dayafterthe
tamoxifeninjection(Figure1D),confirmingthattheactivation
oftheCreproteinisspecifictotheProx1-expressingtumorcells.
However,28daysaftertamoxifeninjection,weobservedthatthe
Prox1
+
cellshadproducedadjacenttdTomato
+
/Prox1
C0
prog-
eny,whichoccasionallystainedformucin2ofgobletcellsor
forlysozymeofPanethcells,indicatingthatProx1
+
tumorcells
cangiverisetodifferentiatedcellsintheintestinaladenomas
(Figures1Dand1E).
IntheProx1-Cre
ER
;Rosa26-tdTomato
flox/Stop/flox
;Apc
min/+
mice,Prox1
+
epithelialcellswereveryrarelylabeledoutsideof
thetumorsaftertamoxifeninjection.Inordertoactivatethe
lineagemarkermoreeffectively,weusedmiceharboringa
Prox1-Cre
ER
bacterialartificialchromosomeatanectopic
genomicsite(Bazigouetal.,2011).Inthesemice,onlysporadic
Prox1
+
intestinalepithelialcellswerepositivefortheredlineage
marker7daysaftertamoxifentreatment(Figure1F).Further-
more,Prox1deletionintheintestinalepitheliumofProx1
flox/flox
;
villin-Cre
ER
mice(VPD/D)didnotresultinanyobviousphenotype
(seeSupplementalResults).Thesedataindicatethatasubpop-
ulationoftheProx1
+
cellshasstemcellpropertiesinadenomas,
butnotinnormalintestine.
Prox1DeletionLeadstoLossofLgr5
+
StemCellsin
IntestinalAdenomas
ToanalyzethesignificanceofProx1specificallyinadenoma
stemcells,wedeletedApcandProx1intheLgr5
+
cellsof
Apc
flox/flox
;Prox1
flox/flox
;Lgr5-EGFP-IRES-Cre
ER
(LApcP)mice.
Inordertoobtainanefficientdeletion,tamoxifenwasinjected
during2consecutivedaysandthesizeoftheLgr5
+
cellpopula-
tionwasanalyzed21daysthereafter.Interestingly,themajority
oftheadenomascontainedsomeProx1
+
cells,suggestingan
incompletedeletionofProx1inthecryptstemcells(Figure2A).
However,theProx1
+
adenomascontainedfewerLgr5
+
cellsin
tamoxifen-treatedLApcP(LApcPD/D)mouseintestinesthanin
theLApcD/Dcontrols(Figure2B).Furthermore,thenumberof
stemcellswasevenlowerintheProx1
C0
crypt-likestructures
thatcontaineddislocatedLgr5
+
cellsbothinthesmallandlarge
intestine,indicatingthatsuccessfulProx1deletioninhibitsthe
expansionofLgr5
+
cellsintheadenomas(Figure2B).
Tofurthertestthishypothesisexvivo,weusedLApcD/Dand
LApcPD/Dorganoids.TheWnt-agonistR-Spondin1isrequired
forthesurvivalandgrowthoftheWT(LApcorLApcP)intestinal
organoids(Satoetal.,2009).WithoutR-Spondin1,onlyorgano-
idswithApcdeletionandresultingactiveb-catenin/TCF
pathwaysurvivebeyond4days(Wieneretal.,2014).Almostall
theProx1
+
cellsofLApcD/DorganoidswereLgr5-EGFPpositive
8daysaftertheadditionof4-OH-TamwithoutR-Spondin1(Fig-
ure2C).However,therewasamarkedreductioninthenumberof
viableorganoids8daysafterthesimultaneousdeletionofApc
1944CellReports8,1943–1956,September25,2014a2014TheAuthors
andProx1intheLgr5
+
cells(Figures2D–2F).Theviableorgano-
idscontainedsomeProx1
+
cellsatthistimepoint(Figure2C),
suggestingthatProx1deletionwasnotcompleteintheorgano-
ids.SinceProx1wasnotexpressedintheLgr5
+
stemcells
beforeApcwasdeleted,butonlyafter5daysofApcdeletion
invivo,thesedataindicatethatlossofProx1expressiondoes
Figure1.Prox1ExpressionandCellLine-
ageTracinginIntestinalAdenomasafter
ApcDeletion
(A)Prox1expressioninanaberrantcryptfocusin
theApc
min/+
intestine.
(B)Lgr5-EGFPandcyclinD1staininginLApc
mouseintestinebefore(LApc)and21daysafter
theadditionoftamoxifen(LApcD/D).Notesome
overlapofstainingintheLgr5
low
cellpopulation
(dottedareas).
(C)DistributionoftheLgr5-EGFPandProx1sig-
nalsintheintestinalepitheliumofLApcmiceafter
theinjectionofasingledoseoftamoxifen.Note
thelackofoverlapbetweenProx1
+
(arrowhead)
andLgr5
+
cellsatday0andtheoverlap(dotted
areas)atdays5and21.
(DandE)Immunostainingofadenomasderived
fromProx1-Cre
ER
;Rosa26-tdTomato
flox/Stop/flox
;
Apc
min/+
micefortheindicatedproteinsaftera
singletamoxifeninjection(celllineagetracing).
ThearrowsmarktdTomato
+
/Prox1
C0
/Mucin2
+
or
tdTomato
+
/Prox1
C0
/Lysozyme
+
cells.
(F)Prox1andtdTomatosignalsinProx1-Cre
ER
;
Rosa26-tdTomato
flox/Stop/flox
mice7daysafter
tamoxifeninjection.Thearrowsindicatetherare
labeledProx1
+
cellsinthenormalintestinal
epithelium.
Scalebarsrepresent20mm(BandD–F)or50mm
(AandC).SeealsoFigureS1.
notinfluencethetumorinitiationfre-
quency,butinstead,itinhibitstheex-
pansionoftheLgr5
+
adenomacell
population.
Todirectlyaddresstheconnectionbe-
tweenProx1andstemcellnumberin
anotherexvivoorganoidculturesystem,
weisolatedorganoidsfromApc
flox/flox
;
villin-Cre
ER
(VApc)andApc
flox/flox
;
Prox1
flox/flox
;villin-Cre
ER
(VApcP)mice,
whereApcand/orProx1aredeletedin
thewholeintestinalepithelium,including
theprogenitorcells.DeletionofProx1
alonefromthewholeintestinalepithelium
derivedfromtheProx1
flox/flox
;villin-Cre
ER
micedidnotinducemorphological
changesintheorganoids(FigureS2A).
However,deletionofbothApcand
Prox1(VApcPD/D)fromtheorganoids
increasedtherelativeexpressionlevelof
theprogenitormarkersNol1andWdr3
(VanderFlieretal.,2007)andtherelative
numberofcyclinD1
high
cellsincompari-
sonwithorganoidswithonlyApcdeletion
(Figures3A–3C).Asinthetamoxifen-injectedVApcD/Dmice,the
Prox1
+
cellswerecyclinD1
low
(Figure3B).Furthermore,RNAs
encodingtheWnt-targetintestinalstemcellsignaturegenes
Lgr5,Tnfrsf19,andAscl2weredecreaseduponProx1deletion,
whereasc-MycRNAwasnotchanged(Figure3Canddatanot
shown).MicroarrayanalysisofVApcD/DandVApcPD/Dcultures
CellReports8,1943–1956,September25,2014a2014TheAuthors1945
7daysaftertheadditionof4-OH-Tamindicateddecreasedin-
testinalstemcellmarkersandchangesintheexpressionlevels
ofspecificWnttargetsintheVApcPD/Dorganoids(FigureS2B).
ThesedatasuggestedthatProx1deletiondecreasesthenumber
ofadenomastemcells,resultinginaskewedenrichmentofthe
progenitorpopulationintheorganoids7daysafterApcdeletion.
Indeed,theProx1-deletedorganoidswerelessefficientinform-
ingneworganoidsubcultureswhentheyweredissociatedto
smallclusterscontainingapproximatelyfourtosevencells
(Figure3D).
PROX1SilencingDecreasesStemCellsinHumanCRC
Organoids
TomodeltheeffectofPROX1onstemcellactivityinCRCpro-
gression,wechosetousethehumanSW1222cellline(mutant
CRCgenesinthiscelllinearelistedinTableS1).Thiscellline
Figure2.Prox1DeletionfromtheLgr5
+
AdenomaCellsInhibitstheExpansionoftheStemCellPool
(A)Lgr5-EGFPandProx1signalsinuntreatedLApcmouseintestineandinLApcPD/Dintestine21daysaftertamoxifenaddition.ThearrowheadmarksaProx1
C0
cryptwithdislocatedLgr5
+
cells(implicatingApcdeletion).TheasterisksmarkProx1
+
crypts.
(B)ThenumberofLgr5
+
cellsperthevarioustypesofcryptsinthesmallintestineorcolon(Kruskal-Wallistest,n=25fromthreemice).Theboxplotindicatesthe
minimum,thefirstquartile,median,thirdquartile,andmaximum.
(C)Lgr5-EGFPandProx1signalsintheLApcD/DandLApcPD/Dorganoids.
(DandE)ViableLApcD/DandLApcPD/Dorganoids(D,arrowheads)andtheirquantification(E).
(F)Prox1RNAlevel.
For(C)–(F),thesampleswereanalyzed8daysaftertheadditionof4-OH-Tamand6daysaftertheremovalofthegrowthfactors.Scalebarsrepresent20mm(C)or
100mm(AandD).Mean+SDareshown(EandF).
1946CellReports8,1943–1956,September25,2014a2014TheAuthors
isenrichedforstemcellsthatcanself-renewanddifferentiate
intomultiplelineages(Yeungetal.,2010).Whilethe‘‘small
colonies’’inSW1222cell-derived3Dcultureshavealimited
growthpotentialandlacklumens,thelargeglandular‘‘megacol-
onies’’producecrypt-likestructuresconsistingofpolarizedcells
surroundingacentrallumen(Yeungetal.,2010).Lumensare
stemcell-dependentstructurespresentinwell-differentiated
tumorsandlikewiseinexvivoandinvitro3Dculturesfrom
humanandmouseadenomasandstemcell-containingCRC
celllines(Ashleyetal.,2013).
SincetheSW1222cellshaveheterogenousPROX1andnu-
clearb-CATENINexpressionlevels(FigureS2C),weisolated
subcloneswithalow(PROX1
low
)orahigh(PROX1
high
)percent-
ageofPROX1
+
cells(FigureS2D).WethensilencedPROX1in
thePROX1
high
clonebytwodifferentlentiviralshorthairpin
RNAs(shRNAs)(FigureS2Eanddatanotshown).Interestingly,
thePROX1-silencedSW1222-PROX1
high
cellsformedastrik-
inglyreducednumberofglandularcolonies(Figure3E),suggest-
ingthatPROX1silencinghasaprofoundeffectonCRCstemcell
activity.Furthermore,PROX1silencingdecreasedthenumber
oflumensintheglandularcoloniesderivedfromSW1222-
PROX1
high
cells(Figure3F).After12daysof3Dculture,the
LGR5andTNFRSF19RNAsweredecreased,whereasno
changeswereobservedintheexpressionofotherWNT-target
genes,suchasMYC(Figure3G).PROX1silencinginCRC
patient-derivedorganoidsalsoresultedindecreasedfrequency
ofnewlumen-containingorganoids(Figure3H).Notably,the
shPROX1organoidsformedsmallcolonieswithoutlumens(Fig-
ure3H).SimilarlytotheSW1222cellline,PROX1silencingin
CRCpatient-derivedorganoidsresultedinamarkedreduction
ofLGR5andTNFRSF19RNAlevels(Figure3I),eveninthe
presenceofaKRASmutation(seeSupplementalExperimental
Procedures).LGR5insituhybridizationfurtherindicatedthat
PROX1silencingdecreasesthenumberofCRCstemcells(Fig-
ures3Jand3K).Notably,therewasnodifferenceintheLGR5
RNAinPROX1-silencedSW1222cellsin2Dculture(datanot
shown),thusrulingoutthepossibilitythatLGR5isadirect
PROX1target.TheseresultssuggestthatlossofPROX1de-
creasestheCRCstemcellsalsoinhumanCRC,andthatthis
isindependentofthepresenceofKRASmutations.
PROX1
+
TumorCellsProliferateLessThanProx1
C0
TumorCells,yetPROX1DeletionLeadstoDecreased
CellProliferationintheTumorsInVivo
Totesttheinvivosignificanceofourfindings,weimplanted
SW1222-PROX1
high
cellsandtheirPROX1-silencedcounter-
partssubcutaneouslyintoimmunocompromised(NODscid
gamma[NSG])miceandmonitoredtumorgrowth.Similarly
totheProx1-deletedintestinaladenomas(Petrovaetal.,
2008),thePROX1-silencedtumorsgrewslowerthancontrols
transducedwithscrambledshRNA(Scr)atthisectopic
implantationsite(Figure4A).Notably,wedetected60%fewer
tumornestsinthePROX1-silencedsamplesascompared
toScrcontrols(200.4±46.8and70.9±11.8tumornests,
respectively;p<0.01)whentheywereexcised21daysafter
theimplantation.UnlikethescrambledshRNA-transduced
tumors,thePROX1-silencedtumorscontainedtumornests
withonlyfewglandularstructures(Figure4B).Similarlytothe
PROX1-silencedSW1222cells,subcutaneouslyimplanted
Prox1-deletedmouseVApcPD/Dorganoidsgrewslowerand
containedfewerglandsthanVApcD/Dorganoids(Figures4C
and4D).Prox1deletiondecreasedLgr5andTnfrsf19RNAs,
butnotofMycRNAinthetumors,confirmingourexvivore-
sults(Figure4E).
Intestinalepithelialprogenitorcellsproliferaterapidly,butonly
foralimitednumberofcellcycles(GregorieffandClevers,2005).
CarefulanalysisoftheProx1
+
andProx1
C0
cellsincontrol
SW1222-PROX1
high
orinVApcD/Dorganoid-derivedtumors
indicatedthattheKi67
+
proliferatingcellswereenrichedinthe
Prox1
C0
cellpopulation(FiguresS3AandS3B),suggestingthat
mostoftheProx1
+
cellsarenonproliferativeorslowlyprolifer-
ating.Interestingly,theProx1
+
/Lgr5
+
cellpopulationhadahigher
frequencyofKi67
+
cellsthantheProx1
+
/Lgr5
C0
populationinthe
LApcD/Dintestine(Figure4F),indicatingthattherapidlyprolifer-
atingProx1
+
cellsareenrichedintheLgr5
+
population.Similarly,
bromodeoxyuridinelabelingoftwohighlyproliferatingorganoid
cultures(VApcD/DandVCKID/D)showedthatonlyasmall
proportionoftheProx1
+
cellsproliferated(FigureS3C).VCKID/D
organoidsaredeletedoftp53andCsnk1a1,encodingcasein
kinaseIa(CKIa),whichphosphorylatesb-catenin,targetingit
toubiquitin-mediateddestruction;thus,theVCKID/Dorganoids
displayactivatedWntsignalingpathway(Elyadaetal.,2011).
WhereasProx1
+
cellsappearedtoproliferateslowly,we
observedadecreasedoverallfrequencyofKi67
+
tumorcellsin
subcutaneouslygrowingPROX1-silencedSW1222-PROX1
high
tumors,inProx1-deletedVApcPD/DorganoidtumorsinNSG
mice,andintheLgr5
+
cellsofLApcPD/Dintestinaltumors(Fig-
ure4G;FigureS3D).Apossibleexplanationfortheseapparently
contradictorydataisthatProx1deletionresultsindecreased
proliferationinthestemcellpopulation,whichleadstoexhaus-
tionoftheCRCstemcellpoolandconsequentlytoadecreaseof
theoverallratioofproliferatingcells.Indeed,aslowerprolifera-
tionrateofthePROX1-silencedSW1222-PROX1
high
cell-derived
organoidswasobservedonlyaftera14-dayculturingperiod,
whenthePROX1
+
organoidsalreadyhadanextensivelumen
formingactivity,butnotat6days(FigureS3E).Thisindicatesa
delayedeffectofPROX1silencingontheoverallcellproliferation
intheorganoids,inlinewiththeideathatPROX1influences
tumorgrowthbyregulatingthesizeoftheproliferatingstem
cellpool.
ThePROX1
-
(adherent)cellsoftheSW480CRCcellline
(SW480A)areunabletoinitiatesubcutaneoustumorsinNSG
mice,whereasthePROX1
+
cells(SW480R)areround,form
cellclusters,andaretumorigenic(Petrovaetal.,2008).To
testifPROX1silencinginalreadyestablishedCRCxenografts
regulatesstemcells,weimplantedcellsfromthePROX1
+
stemcell-likeSW480Rsubclone,expressingadoxycycline-
induciblePROX1shRNAconstruct(SW480R-sh)(Petrova
etal.,2008),intoNSGmice.Doxycyclinetreatmentwas
started8daysaftersubcutaneousinjection,whenthetumors
werealreadyvisible.Weobservedreducedgrowthofthe
PROX1-silencedtumorsafterday16(Figures4Hand4I),ata
timepointwhentheLGR5RNAlevelhadalreadymarkedly
decreasedinthedoxycycline-treatedtumors(Figure4J).These
datasuggestthatPROX1regulatesstemcellsalsoinestab-
lishedtumors.
CellReports8,1943–1956,September25,2014a2014TheAuthors1947
Figure3.Prox1DeletionLeadstoReducedStemCellActivityinExVivoOrganoids
(A)Theschematicoutlineofthemouseorganoidexperiments.GF,growthfactors.
(B)Prox1andcyclinD1immunostainingofsectionsfromVApcD/DandVApcPD/Dorganoids7daysafterthedeletionand5daysafterremovingR-Spondin1from
theculturemedium.
(C)RNAlevelsofprogenitormarkers(Nol1,cyclinD1,andWdr3),stemcellmarkers(Lgr5andTnfrsf19),andMycinVApcD/DandVApcPD/Dorganoidsanalyzed
byreal-timequantitativePCR(qRT-PCR).
(D)TheorganoidinitiatingfrequencyofVApcD/DandVApcPD/Dorganoids.
(E)Theproportionofglandularcoloniesderivedfrom1,000SW1222-PROX1
high
cells(transducedwithScr,sh1,orsh2PROX1shRNAlentivirus).
(F)ThenumberoflumensintheglandularcoloniesderivedfromScrorshPROX1-transduced(sh1,sh2)SW1222-PROX1
high
cellsatday14(n=10forsh1and
n=12forsh2).Thelumens(asterisks)weredetectedbyphalloidinstainingandcountedinopticalsectionsofconfocalmicroscopicimages.Theboxplotindicates
theminimum,thefirstquartile,median,thirdquartile,andmaximum.
(G)Real-timeqPCRoftheindicatedRNAsfromSW1222-PROX1
high
cell-derivedorganoids,transducedwithshPROX1orScrlentivirusandgrowninMatrigelfor
14days.
(legendcontinuedonnextpage)
1948CellReports8,1943–1956,September25,2014a2014TheAuthors
PROX1SilencingIncreasesAnnexinA1Expressionin
MultipleCRCModels
Todeterminewhichgenesareresponsiblefortheeffectof
PROX1ontheadenoma/CRCstemcells,wetestedPROX1-
(H)Imagesandtherelativelumen-containingorganoidinitiatingfrequencyofScrandshPROX1lentivirus-transducedhumanorganoidsderivedfromCRC
patients.TheGFPsignalshowsthelentiviraltransductionefficiency.
(I)RNAlevelsoftheindicatedgenesinhumanCRCpatient-derivedorganoids,transducedwithScrorshPROX1lentivirus.Notethatwhilesample1containsthe
G12DclinicallyrelevantmutationintheKRASgene,samples2and3arewild-type.
(JandK)LGR5insituhybridizationandquantificationofLGR5
+
cellsintheScrandshPROX1-transducedSW1222-PROX1
high
clone.
Scalebarsrepresent100mm(B)or50mm(E,F,H,andK).Mean+SD(C–EandG–I)ormean+SEM(J)areshown.SeealsoFigureS2.
Figure4.PROX1RegulatestheNumberof
StemCellsviaCellProliferation
(A)Growthcurveofsubcutaneoustumorsderived
fromScrorshPROX1-transducedSW1222-
PROX1
high
cellsinNSGmice(n=10).
(B)PROX1andE-CADHERINimmunostainingand
HEstainingoftumorsections21daysaftersub-
cutaneousinjectionofSW1222-PROX1
high
cells
intoNSGmice.Thedashedlineindicatesthetu-
morborder,thearrowheadspointtodegenerating
glandularstructuresandtheasteriskmarks
necroticarea.
(C–E)Immunostaining,tumorvolume(CandD)
andRNAquantification(E)ofVApcD/D(black
columns)andVApcPD/D(redcolumns)organoid-
derivedtumors12and28daysaftertheirsubcu-
taneousinjectionintoNSGmice(confocal3D
reconstruction).Notethemorecomplexglandular
structureoftheVApcD/Dtumors(asterisks).
(FandG)Thepercentageofproliferating(Ki67
+
)
cellsamongtheProx1
+
cellsintheLgr5
+
and
Lgr5
C0
populationsintheintestinalepitheliumof
LApcD/Dmice21daysafterApcdeletion(n=10–
12)(F).ThepercentageofKi67
+
cellsintheLgr5
+
cellpopulationintheintestinalepitheliumof
LApcD/DandLApcPD/Dmice(n=9–11)(G).The
boxplotsindicatetheminimum,thefirstquartile,
median,thirdquartile,andmaximum(FandG).
(H–J)GrowthofsubcutaneousSW480R-shtu-
morsinNSGmice(H)withorwithoutdoxycycline
(n=10).Doxycylinetreatmentfromday8(blue
arrow)today16(redarrow)aftercellimplantation
wasusedforthesilencingofPROX1.Immuno-
staining(I)andquantitativeRT-PCR(qRT-PCR)(J)
analysesatday16.
Scalebarsrepresent50mm(B)and100mm(B,D,
andI).Mean+SD(C,E,andJ)ormean+SEM
(AandH)areshown.SeealsoFigureS3.
regulatedcandidategenesbasedon
themicroarraydataderivedfromthe
SW480Rsubclone(Petrovaetal.,2008).
Basedonourinitialresults,wefocused
furtheronthecalcium-dependentphos-
pholipidbindingproteinAnnexinA1
(ANXA1),whichhasbeenshowntoinhibit
breastcancermetastasis(Maschleretal.,
2010).PROX1suppressedANXA1RNA
(4.21±0.08-fold,mean±SD)andprotein
expressioninSW480R-shcells(Fig-
ureS4A).Anxa1wasincreasedafter4-OH-Tamadditionto
VApcorganoidsandevenfurtherelevatedwhenalsoProx1
wasdeleted(Figure5A;FiguresS4BandS4C),suggestingthat
Prox1suppressesAnxa1expression.Furthermore,theProx1
C0
CellReports8,1943–1956,September25,2014a2014TheAuthors1949
(legendonnextpage)
1950CellReports8,1943–1956,September25,2014a2014TheAuthors
cellsshowedmoreintenseAnxa1stainingthanProx1
+
cellsin
tumorsectionsfromApc
min/+
mice(FigureS4D).
WeobservedamutuallyexclusivestainingforAnxa1and
Prox1intumorsfromVCKID/DmiceandinhumanCRCsamples
(Figures5Band5C).Furthermore,theLgr5
+
intestinalstemcells
intheLApcmiceshowedverylittleornoepithelialAnxa1expres-
sionbeforeApcdeletion(FigureS4E).Theycontainedalowlevel
ofAnxa1aftertamoxifeninjection,whereastheAnxa1
high
cells
werenegativeforLgr5intheresultingadenomas(FigureS4F).
Importantly,transfectionofadominant-negativeTCF4(tran-
scriptionfactor7-like2[TCF7L2])construct,aWntpathwaysup-
pressortothePROX1
C0
subcloneoftheSW480CRCcellline
(SW480A)(Petrovaetal.,2008),ledtosuppressionofANXA1
expression(Figure5D).ThesedataindicatethatAnxa1ismini-
mallyexpressedinthenormalintestinalepithelium,wherethe
activationoftheWntpathwayafterApcdeletionincreases
Anxa1expression,whichisthensuppressedbytheinduction
ofProx1inthetumorcells.
ANXA1SilencingMimicstheEffectsofPROX1inCRC
WenexttestedthepossibilitythatthestrongANXA1suppression
mediatestheeffectsofPROX1,suchastheassociatedrear-
rangementoftheactincytoskeleton,changesincellshape,
andadherencetothecultureplates(Petrovaetal.,2008).Indeed,
ANXA1overexpressionintheSW480Rsubcloneinducedan
elongatedcellshapeandincreasedthenumberoftightly
adherentcells,whereasANXA1suppressionresultedinrear-
rangementoftheactincytoskeleton,roundedcellshape,and
decreasedadherence(FiguresS5A–S5C).Interestingly,Anxa1
silencinginVApcD/DorganoidsincreasedtheLgr5andTnfrsf19
stemcellmarkerRNAs,withoutaffectingMyc(Figure5E).
AlthoughANXA1silencinghadnoeffectonPROX1expression
inSW1222-PROX1
low
orSW480cellsin2Dculture(datanot
shown),itresultedinincreasedlumenformation(Figure5F),
upregulationofTNFRSF19andLGR5RNAs,andincreaseof
LGR5
+
cellsin3DorganoidsderivedfromshANXA1-transduced
cells(Figures5Gand5H).Incontrast,ANXA1overexpression
inSW1222-PROX1
high
cellsdecreasedtheproportionofglan-
dularcolonies,thenumberoforganoidlumens,andLGR5
andTNFRSF19RNAlevelswhencomparedtothecontrols(Fig-
uresS5D–S5F).Furthermore,theANXA1-silencedSW1222-
PROX1
low
cell-derivedtumorsgrewfasterthancontroltumors,
containedmoreKi67
+
cells,andwerecomposedoflarger
tumornestswithmultiplelumensorganizedintolabyrinth-like
structures(Figure5I;FigureS5G).Similartotheeffectof
PROX1,ANXA1silencinginSW1222cell-derivedorganoids
wasassociatedwithenhancedcellproliferationonlyafter
16days,whentheorganoidsshowedextensiveoutpocketing
andlumenformation,butnotatday6(FigureS5H).Thesedata
indicatethatANXA1silencingmimicstheeffectsofPROX1
expression,leadingtoanexpansionofthestemcellpoolvia
increasedproliferation.
SilencingtheActin-BindingProteinFILAMINA
DecreasesStemCellsinCRC
ReanalysisofthemicroarraydataderivedfromtheSW480R
subcloneshowedthatfocaladhesionandregulationofactin
cytoskeletonwerethetopKyotoEncyclopediaofGenesand
GenomesgenepathwaycategoriesaffectedwhenPROX1was
silenced(datanotshown).Thus,wesearchedforPROX1-regu-
latedgenesdirectlyaffectingthecytoskeletonamongpublished
CRCstemcellgenesets(deSousaEMeloetal.,2011)andprog-
nosismarkersinCRCsubgroups(Sadanandametal.,2013).We
foundthegeneencodingtheactin-bindingproteinfilaminA
(FLNA)inbothgenesets.FLNAconnectsactinfilamentsto
transmembranereceptors,includingb1integrin,modulatescell
migration,andfunctionsasacentralmechanotransduction
elementofthecytoskeleton(Ehrlicheretal.,2011;Zhouetal.,
2010).Interestingly,wefoundhighlyincreasedFlnaexpression
intheProx1
+
andLgr5
+
tumorcells(Figure6Aanddatanot
shown)andacorrelationbetweenPROX1andFLNAlevelsin
theSW1222-PROX1
high
cell-derivedorganoids(FigureS5I).
FLNAwasalsoupregulatedintheANXA1-silencedorganoids
(Figures6Band6C).Furthermore,PROX1silencinginthe
SW1222-PROX1
high
andCRCpatient-derivedorganoidsre-
sultedinmarkedlydecreasedFLNARNA(Figure6D)andprotein
(Figure6E).
BecausebothPROX1andANXA1regulatedFLNAexpression
inintestinaladenomasandinCRC3Dorganoids,westudied
FLNAfunctionbysilencingitsexpressioninVApcD/Dorganoids
andinSW1222-PROX1
high
cells.ThisledtoreducedLgr5and
Tnfrsf19RNAs(Figure6F),toadecreaseofSW1222cell-
derivedglandularcoloniesandlumens(Figure6G),andtoa
decreasednumberofKi67
+
cellsintheorganoids(Figure6H).
Surprisingly,FLNAsilencingdidnotaffectthegrowthofthecells
in2Dcultureconditions(datanotshown).Overall,theseresults
suggestamodelwherethelossofProx1leadstoanelevated
Anxa1levelandareductioninFlna,whichlimittheexpansion
Figure5.ANXA1SilencinginCRCLeadstoElevatedStemCellActivityandTumorGrowth
(A)ImmunostainingofVApcD/DandVApcPD/Dorganoidsfortheindicatedproteins.
(BandC)Immunoperoxidasestainingofsectionsfrom(B)VCKID/Dtumorsand(C)fromatumorofaCRCpatient(n=5).
(D)ANXA1expressioninSW480cellstransfectedwithadominant-negativednTCF4-FLAGoranunrelatedcontrolconstruct(TIE1-FLAG).Thearrowheads
indicatetransfectedcells.
(E)RNAanalysisofScrandshAnxa1-transducedVApcD/Dorganoids(blackandgreencolumns,respectively).
(F)NumberoflumensinSW1222-PROX1
low
subclone-derivedorganoids,transducedwitheitherScrorshANXA1-expressinglentivirus,stainedwithfluorescent
phalloidinandcountedfromopticalsectionsofconfocalimages.Thewhiteasterisksmarkthelumens(n=11forsh(1)andn=25forsh(2)).Theboxplotindicates
theminimum,thefirstquartile,median,thirdquartileandmaximum.
(GandH)RNAlevelsoftheindicatedtranscripts(G)andLGR5insituhybridization(H)(redsignal)inSW1222-PROX1
low
-derivedorganoids,growninMatrigel
for16days.
(I)Growthcurves(n=6),histologicalanalysisandimmunostainingofSW1222-PROX1
low
tumorsgrowingsubcutaneouslyinNSGmice.
Forthestaining,thetumorswereisolatedonday11aftertheimplantation.Scalebarsrepresent100mm(A–D)or50mm(F,H,andI).Mean+SD(EandG)or
mean+SEM(HandI)areshown.SeealsoFiguresS4andS5.
CellReports8,1943–1956,September25,2014a2014TheAuthors1951
oftheadenoma/CRCstemcellpopulationviadecreasingcell
proliferation.
PROX1SilencingReducesCellSurvivalinHypoxic
TumorXenografts
Interestingly,whengrowntoalargesizeinMatrigelculture
(>150mmdiameter,>5daysaftersubculture)orinjectedsubcu-
taneouslyintoNSGmice,Prox1-deletedVApcPD/Dorganoids
containedmoreapoptoticcellsthanVApcD/Dorganoids(Fig-
ure7A).Also,wedetectedanintensestainingfortheapoptosis
markeractiveCASPASE-3insidethePROX1-silencedSW1222-
PROX1
high
cell-derivedsubcutaneouslygrowingtumorsthat
werepositiveforthehypoxiamarkercarbonicanhydraseIX,
butnotatthetumormargin(Figure7Banddatanotshown),sug-
gestingreducedtumorcellsurvivalintheischemic/hypoxic
tumorinterior.Interestingly,wefoundnodifferenceintheendo-
mucin
+
bloodvesseldensitybetweenScrandshPROX1tumors,
evenwhenvascularendothelialgrowthfactor(VEGF)wasover-
expressedinthetumorcells(FiguresS6AandS6B).However,
theshPROX1tumorsweresmallerthantheScrtumors,
evenwhenVEGFwasoverexpressedinthetumorcellsand
VEGFfailedtorescuethelargenecroticareasinsidethe
Figure6.SilencingtheActin-BindingProteinFilaminAInhibitsStemCellActivity
(A)ImmunostainingforfilaminA(Flna)andProx1inanintestinaladenomaofApc
min/+
mice.
(BandC)FLNAstaining(B)andqRT-PCR(C)fromscrambledandshANXA1-transducedSW1222-PROX1
low
cell-derivedorganoids14daysafter3Dseeding.
(D)FLNARNAinCRCpatient-derivedandSW1222-PROX1
high
cell-derivedorganoids,transducedwithScrorshPROX1lentivirus(qRT-PCR).
(E)ImmunostainingforPROX1andFLNAinScrorshPROX1-transduced,SW1222-PROX1
high
cell-derivedorganoids,culturedfor7daysinMatrigel(confocal3D
reconstructions).
(F)ThelevelsoftheindicatedRNAsinVApcD/Dorganoids10daysaftertransductionwithScrorshFlnalentivirus(qRT-PCR).
(G)Theproportionofglandularcoloniesandthenumberoflumens(n=8forsh(1)andn=14forsh(2))inSW1222cell-derivedorganoids,transducedwiththe
indicatedlentivirus.
(H)ThepercentageofKi67
+
cellsinScr(blackbox)orshFLNA-transduced(graybox)SW1222cell-derivedorganoidsafter9daysof3Dculturing,countedfrom
confocalopticalsections(n=12).
Theboxplotsin(G)and(H)indicatetheminimum,thefirstquartile,median,thirdquartile,andmaximum.Mean+SDareshown(C,D,F,andG).Scalebars
represent50mm(AandB)or25mm(E).SeealsoFigureS5.
1952CellReports8,1943–1956,September25,2014a2014TheAuthors
PROX1-silencedtumors(FiguresS6AandS6B).Thus,thepres-
enceofthelargenecroticareasintheshPROX1tumorscould
notbeexplainedbythelackofangiogenicfactors.
AlthoughANXA1silencingmimickedseveraloftheeffectsof
PROX1intheintestinaladenomaandCRCmodels,the
apoptosisrateintheANXA1-silencedsubcutaneouslygrowing
tumorswasnotsignificantlyaffectedwhenanalyzed9or
24daysafterimplantation(datanotshown).Thisraisedthepos-
sibilitythatinunfavorableconditionsalsoalternativepathways
contributetotheapoptosisintheshPROX1tumors.Autophagy
isanessentialcellularprocessforthesurvivalofcellsunderhyp-
oxiaornutrientdeprivation(Satoetal.,2007).Interestingly,
PROX1silencingintheSW480Rcellsresultedinadecreaseof
theautophagy-associatedLC3-IIproteinbothinnormalmedium
andinstarvationconditions(Figures7Cand7D).Furthermore,
lackofPROX1preventedtheaccumulationoftheLC3-contain-
ingearlyautophagosomesinthepresenceofthelysosomalin-
hibitorbafilomycinAorchloroquine,whichinhibitthefusionof
autophagosomeswiththelysosomes(Figure7D).Importantly,
weobservedadecreasednumberofLC3-containingearlyauto-
phagosomesunderhypoxiainlentivirallyPROX1-silenced
SW480RorSW1222-PROX1
high
cellswhentheirfusionwithlyso-
someswasinhibited(Figure7E),suggestingthatPROX1en-
hancestheautophagicflux.Theadditionofalowconcentration
ofchloroquineorbafilomycinAresultedinlossoftheLgr5
+
cells
afterApcdeletion(Figure7F).Ofnote,thischloroquineconcen-
trationinhibitedtheformationofVApcD/Dorganoidcolonies
uponsubculture,butnottheirsurvivalwhenadded2daysafter
organoidplating(Figure7G).Furthermore,theadditionofchloro-
quineorbafilomycinatalowdosetotheLApcD/Dorganoidcul-
turesreducedtheproportionofviableorganoidstothesame
levelasProx1deletionintheLApcPD/Dcultures,thushigh-
lightingtheimportantroleofautophagyinpromotingtumorcell
survivalspecificallyintheProx1
+
cells(Figure7H).
DISCUSSION
Prox1iscriticalforthefateofseveraltypesofstemandprogen-
itorcells(Elsiretal.,2012).Here,weshowthatProx1isnotex-
pressedintheLgr5
+
stemcellsofnormalintestinalcrypts,but
isinducedsoonaftertheinitiatingmutationinintestinaltumori-
genesis.WeshowthattheProx1
+
cellsgiverisetomorediffer-
entiatedProx1
C0
cellsintheadenomas,butnotinthenormal
intestine,indicatingthatasubpopulationoftheProx1
+
cells
hascancerstemcellactivity.UponProx1deletion,thenumber
ofstemcellsdeclinedandthiswasreflectedlateronas
decreasedoveralltumorcellproliferationandtumorgrowth.
Stem-likecellsprovideanimportantdrugtargetincancer,as
theyareabletopersistintumorsasadistinctpopulation,self-
renew,anddifferentiate,andtheyareassociatedwithtumor
relapse(SampieriandFodde,2012).Therefore,development
ofspecifictherapiestargetedatcancerstemcellsmayimprove
survival.Inelegantstudies,Schepersetal.demonstratedthat
theLgr5
+
cellsinintestinaladenomashavestemcellproperties
(Schepersetal.,2012).Interestingly,recentstudiesindicated
thatintestinaladenomascontainfewerstemcellsthanLgr5
+
cells,suggestingthatonlyasubpopulationoftheLgr5
+
cells
functionasstemcells(Kozaretal.,2013).Furthermore,Myant
etal.haveshownthattheRac1GTPaseisanimportantregulator
oftheproliferationoftheLgr5
+
cellpopulationinintestinalade-
nomas(Myantetal.,2013).AlthoughtheLgr5
+
cellsmaybe
dispensableforhomeostasisofthenormalintestineandfor
increasedproliferationofintestinalepitheliumafterApcdeletion
(Metcalfeetal.,2014),theexpansionofcellsderivedfromthe
Lgr5
+
cellsinadenomasafterApcdeletionclearlyindicates
thattheycontributetotumorgrowth(Schepersetal.,2012).
Importantly,Prox1deletionnotonlydecreasedthenumberof
Lgr5
+
cellsinintestinaladenomasinvivo,butitalsoreducedtheir
proliferation,theorganoid-initiatingfrequencyandthenumberof
megacoloniesandlumensintheglandularCRCorganoids,
whichallrepresentindicatorsofcancerstemcells(Ashley
etal.,2013).Furthermore,PROX1silencingafterthetumor
establishmentresultedinadecreaseoftheLGR5
+
tumorcell
markerbeforeadeclineintheoveralltumorgrowthrate.
Mechanistically,weshowthatthephospholipid-bindingpro-
teinAnxa1isincreasedbytheWntpathwayactivationafter
Apcdeletion,whileProx1suppressesitsexpression.ANXA1
andPROX1showedalsomutuallyexclusiveexpressionpatterns
inhumanCRCsamples.Strikingly,thesilencingofANXA1inthe
PROX1
C0
cellsmimickedtheeffectsofPROX1intheCRCcells.
Amongitsothereffects,ANXA1inhibitstheproinflammatory
phospholipaseA2,whichhasbeenshowntostimulatetheprolif-
erationofCRCcellsbyproducingvariouslipidmediatorsand
whichregulatesintercellularjunctionsandtheactincytoskeleton
(Cristanteetal.,2013;ParenteandSolito,2004;Surreletal.,
2009).BothANXA1silencingandPROX1expressionincreased
stemcellmarkersandthenumberofLGR5
+
cellsintumororga-
noidsandenhancedlumenformation,proliferationoftumorcells,
andtumorgrowthinvivo.Furthermore,ANXA1silencingand
PROX1expressionincreasedFLNA,whichstabilizesthecortical
3Dactinnetworks,linksthemtothetransmembranereceptorb1
integrin,andfunctionsasacentralmechanotransduction
elementofthecytoskeleton(Ehrlicheretal.,2011;Zhouetal.,
2010).Theroleoftheactincytoskeletonintheexpansionofthe
adenomastemcellpopulationwassupportedbyourFLNAre-
sults,showingthatsilencingthisactin-bindingprotein,which
wasexpressedintheLgr5
+
stemcellsofintestinaladenomas,
dramaticallyinhibitedtheactivityandproliferationoftheade-
noma/CRCstemcellsin3D,butnotin2D,cultureconditions.
FLNAisoneofthemarkersofaCRCsubtypecharacterizedby
poordisease-freesurvival(Sadanandametal.,2013).FLNA
expressionisincreasedinanumberofcancers,andithas
beenrecentlyshowntoboostthehypoxiaresponseandtumor
progression(Zhengetal.,2014).Consistentwiththis,the
FLNA-expressingPROX1
+
tumorcellsweremoreresistantto
apoptosisthantheFLNA
low
PROX1
C0
tumorcellsbothinvivo
andintheorganoids.Therewasastrikingincreaseinapoptosis,
particularlyinthecentralpartsofthePROX1-silencedtumors
growingsubcutaneouslyinmice.However,thesensitivityofthe
PROX1
C0
tumorcellstoapoptosiswasnotduetoinsufficient
expressionofangiogenicgrowthfactors,asshownbythe
inabilityofVEGFoverexpressiontorescuethedifference.
Instead,theanalysisofautophagymarkersanduseoflysosomal
inhibitorsofautophagyindicatedthatPROX1expressionsus-
tainsautophagy,whichisknowntobeessentialforthesurvival
ofCRCcells(Satoetal.,2007).Thisfindingisparticularly
CellReports8,1943–1956,September25,2014a2014TheAuthors1953
Figure7.LossofPROX1ReducesCRCStemCellSurvivalunderUnfavorableConditions
(A)Percentageofcaspase-3
+
apoptoticcellsamongtheE-cadherin
+
epithelialcellsinVApcD/D(blackcolumns)andProx1-deleted(VApcPD/D;redcolumns)
organoids8daysaftertheadditionof4-OH-Tamand6daysaftertheremovalofR-Spondin1(rightpanel,n=14)orgrowingsubcutaneouslyinNSGmice
(leftpanel,n=22).
(B)Activecaspase-3stainingofsubcutaneouslygrowingScrandshPROX1-transducedSW1222-PROX1
high
tumorsinNSGmice.NotethattheshPROX1-
transducedtumorinteriorishighlyapoptotic(asterisk).
(CandD)ImmunoblottingofSW480R-shcellsfortheindicatedproteins.Cellswereeitherculturedincompletemedium(C)orinstarvationmediumlackingamino
acids8hrbeforeproteinisolation(D)intheabsenceorpresenceof100nMbafilomycinAor30mMchloroquine.
(E)ThenumberofLC3
+
granulesintheindicatedcelllinestransducedwithScrorshPROX1lentivirusandculturedinstarvationmediumcontaining100nM
bafilomycininhypoxiafor8hr(n=50).
(F)FlowcytometricanalysisoftheLgr5-EGFP
+
cellsinLApcD/Dorganoids,9daysaftertheApcdeletionand3daysaftertheadditionof15mMchloroquineor
0.2mMbafilomycinA.
(G)TheplatingefficiencyandsurvivalpercentageofVApcD/DorganoidsinthepresenceorabsenceofchloroquineorbafilomycinA.Notethatfororganoid
survivalchloroquineorbafilomycinwereadded2daysafterorganoidsplittingfor3days.
(legendcontinuedonnextpage)
1954CellReports8,1943–1956,September25,2014a2014TheAuthors
interestingconsideringthathypoxia-inducedautophagycanpro-
motetumorcellsurvivalandadaptationtoantiangiogenictreat-
ment,whichisusedinCRCtherapy(Huetal.,2012).Ourresults
areinagreementwiththefindingsofRagusaetal.,whoshowthat
PROX1promotesthemetabolicadaptationofCRCcellsinunfa-
vorablemicroenvironments,andthuscriticallycontributestothe
metastaticoutgrowthofCRCs(Ragusaetal.,2014).
Insummary,weshowherethatPROX1isinducedinintestinal
stemcellsinadenoma/CRCsoonaftertheactivationoftheWnt
pathway.WhiletheAPCmutationinducesbothPROX1and
ANXA1expressionintheepithelium,PROX1restrictsANXA1
levelsandinducesFLNA,whichstimulatescellproliferation
andpromotesstemcellactivityintheadenomas,andmaycoun-
teractstemcellexhaustionduringtumorgrowth.Theneteffectis
theexpansionoftheadenoma/CRCstemcellpopulationand
increasedtumorgrowth.Inthehypoxicpartsoftumortrans-
plants,PROX1promotestumorcellsurvivalbyincreasingauto-
phagy.Basedonthisstudy,PROX1regulatesthenumberof
adenoma/CRCstemcellswithoutaffectingthehomeostasisof
thenormalintestine,thusprovidinganattractivetherapeutic
targetpathwayfordrugdevelopmentinCRC.
EXPERIMENTALPROCEDURES
AdetaileddescriptionoftheexperimentalproceduresisprovidedinSupple-
mentalExperimentalProcedures.
IntestinalCrypt/OrganoidCultures
TheNationalBoardforAnimalExperimentsattheProvincialStateOfficeof
SouthernFinlandapprovedallexperimentsperformedwithmice.Intestinal
cryptsfromApc
flox/flox
;villin-Cre
ER
,Apc
flox/flox
;Prox1
flox/flox
(Harveyetal.,
2005);villin-Cre
ER
,Csnk1a1
flox/flox
;tp53
flox/flox
;villin-Cre
ER
(Elyadaetal.,
2011),Apc
flox/flox
;Lgr5-EGFP-IRES-Cre
ER
(Barkeretal.,2007)andApc
flox/flox
;
Prox1
flox/flox
;Lgr5-EGFP-IRES-Cre
ER
micewereisolatedandculturedas
describedpreviously(Satoetal.,2009,2011).Toactivatetheendogenous
b-catenin/TCFpathwayinmouseorganoids,culturesweretreatedwith
300nM4-hydroxy-tamoxifen(4-OH-Tam)for48hr.Organoidswiththeendog-
enouslyactiveb-catenin/TCFpathwaywerethenselectedandculturedin
growthfactor-deficientmedium.
HumanOrganoidCultures
For3Dculture,SW1222orpatient-derivedCRCcellswereextensivelytrypsi-
nized,embeddedintoMatrigel(500–2,000cells/50mlMatrigel/well),and
grownfor3–16days.TheethicscommitteeoftheDepartmentofSurgeryat
HelsinkiUniversityHospitalapprovedallexperimentsinvolvingpatientsam-
ples,andinformedconsentwasobtainedfromthepatients.Tissuesamples
isolatedfromCRCpatientswereprocessedaccordingtoapreviouslypub-
lishedmethod(Satoetal.,2011).
InVivoExperiments
Micewereinjectedwith2mgtamoxifen(Sigma)dissolvedin200mlsunflower
oil(Sigma)attheageof8–9weeks.Themicewereeuthanizedattheindicated
timepoints.ThemicewereontheC57Bl/6background.Inallexperiments
littermatecontrolswereused.
StatisticalAnalysis
Statisticalcomparisonoftwogroupswasdonebytwo-tailedunpairedor
pairedttestusingtheSPSSsoftwareunlessotherwiseindicated.Fornonpara-
metrictests,theMann-WhitneyUtestwasused,andthedataarepresentedas
boxplots,showingthefivestatistics(minimum,firstquartile,median,third
quartile,maximum).Thestatisticalsignificanceismarkedbyp<0.05,p<
0.01,andp<0.005.
ACCESSIONNUMBERS
TheGeneExpressionOmnibusaccessionnumberforthemicroarraydatare-
portedinthispaperisGSE47568.
SUPPLEMENTALINFORMATION
SupplementalInformationincludesSupplementalResults,Supplemental
ExperimentalProcedures,sixfigures,andonetableandcanbefoundwith
thisarticleonlineathttp://dx.doi.org/10.1016/j.celrep.2014.08.034.
AUTHORCONTRIBUTIONS
Z.W.,J.H.,V.H.,A.M.B.andK.A.designedresearch;Z.W.,J.H.,V.H.,A.M.B.,
P.K.,T.H.,andO.D.performedresearch;C.H.,O.K.,G.O.,andY.B.-N.contrib-
utednewreagents/analytictools;Z.W.,J.H.,V.H.,andK.A.analyzeddata;and
Z.W.,J.H.,andK.A.wrotethepaper.
ACKNOWLEDGMENTS
WethankDr.LeifAnderssonfortheconsultationsonhistopathology,Dr.Taija
Ma¨kinenfortheProx1-Cre
ER
mice,Dr.DarrenTysonfortheANXA1-modu-
latingconstructs,Dr.DavidCalderwoodfortheFLNAantibody,Dr.Meenhard
Herlyn(WistarInstitute)fortheSW1222cellline,Dr.TatianaPetrovafordiscus-
sionsofPROX1functionsinCRC,Dr.PekkaKatajisto,Dr.TuomasTammela,
andDr.TimoOtonkoskiforcommentsonthemanuscript,andLariPyo¨ria¨,Kirsi
Lintula,KatjaSalo,LauraRaitanen,andTapioTainolafortheirhelpwiththeex-
periments.TheBiomedicumImagingUnitisacknowledgedformicroscopy
services.ThisworkwasfundedbytheSigridJuseliusFoundation,theFinnish
CancerOrganizations,andtheAcademyofFinland(262976).Z.W.wassup-
portedbytheSigridJuseliusFoundationandbytheMarie-CurieIntra-Euro-
peanFellowship(PIEF-GA-2009-236695).
Received:October23,2013
Revised:January14,2014
Accepted:August17,2014
Published:September18,2014
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