WNT10B/?-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer.

1.5 MB·English

by  Wend, Peter

OPEN TRANSPARENT ResearchArticle
ACCESS PROCESS WNT10Bisamodelfortriple-negativebreastcancer
WNT10B/b-catenin signalling induces
HMGA2 and proliferation in metastatic
triple-negative breast cancer
Peter Wend1, Stephanie Runke1, Korinna Wend2, Brenda Anchondo1, Maria Yesayan1,
Meghan Jardon1, Natalie Hardie1, Christoph Loddenkemper3, Ilya Ulasov4, Maciej S. Lesniak4,
Rebecca Wolsky5, Laurent A. Bentolila6, Stephen G. Grant7, David Elashoff8, Stephan Lehr9,
Jean J. Latimer7, Shikha Bose10,11, Husain Sattar5, Susan A. Krum2, Gustavo A. Miranda-Carboni1*
Keywords: cancer stemcells;HMGA2; Wnt/b-catenin signalling has been suggested to be active in basal-like breast
metastasis; triple negative breast cancer.However,inhighlyaggressivemetastatictriple-negativebreastcancers
cancer;wntsignalling (TNBC) the role of b-catenin and the underlying mechanism(s) for the aggres-
siveness of TNBC remain unknown. We illustrate that WNT10B induces tran-
scriptionallyactiveb-catenininhumanTNBCandpredictssurvival-outcomeof
DOI10.1002/emmm.201201320
patients with both TNBC and basal-like tumours. We provide evidence that
ReceivedFebruary22,2012 transgenic murine Wnt10b-driven tumours are devoid of ERa, PR and HER2
RevisedNovember13,2012 expression and can model human TNBC. Importantly, HMGA2 is specifically
AcceptedNovember14,2012
expressed during early stages of embryonic mammogenesis and absent when
WNT10B expression is lost, suggesting a developmentally conserved mode of
action. Mechanistically, ChIP analysis uncovered that WNT10B activates cano-
nical b-catenin signalling leading to up-regulation of HMGA2. Treatment of
mouseandhumantriple-negativetumourcellswithtwoWnt/b-cateninpathway
modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We
demonstratethatWNT10BhasepistaticactivityonHMGA2,whichisnecessary
and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression
predicts relapse-free-survival andmetastasis inTNBC patients.
INTRODUCTION cancers are divided into 6–10 subtypes that are highly
heterogeneous both histologically (Weigelt & Reis-Filho,
In2011,over230,000newcasesofinvasivebreastcancerwere 2009) and by gene expression profiling (Banerji et al, 2012;
diagnosedintheUnitedStatesandconsequentlybreastcanceris Perou et al, 2000; Sorlie et al, 2003): luminal A, luminal B,
the second leading cause of death in women. These breast ERBB2/HER2,normalbreast-like,basal-likeandtriple-negative
(1) DepartmentofObstetricsandGynecology,DavidGeffenSchoolofMedicine (7) DepartmentofPharmaceuticalSciences,NovaSoutheasternUniversity,
atUCLA,JonssonComprehensiveCancerCenter,LosAngeles,CA,USA FortLauderdale-Davie,FL,USA
(2) UCLAandOrthopaedicHospitalDepartmentofOrthopaedicSurgeryand (8) DepartmentofBiostatistics,UCLASchoolofPublicHealth,DavidGeffen
the Orthopaedic Hospital Research Center, David Geffen School of SchoolofMedicineatUCLA,LosAngeles,CA,USA
MedicineatUCLA,LosAngeles,CA,USA (9) BaxterInnovationsGmbH,Vienna,Austria
(3) Institute of Pathology, Charit´e University Medicine/UKBF, Berlin, (10) DepartmentofPathologyandLaboratoryMedicine,DavidGeffenSchool
Germany ofMedicineatUCLA,LosAngeles,CA,USA
(4) DepartmentofBrainTumorBiology,TheUniversityofChicago,Chicago, (11) Department of Pathology and Laboratory Medicine, Cedars Sinai
IL,USA MedicalCenter,LosAngeles,CA,USA
(5) DepartmentofPathology,TheUniversityofChicago,Chicago,IL,USA *Correspondingauthor:Tel:þ13107941216;Fax:þ1310-206-6531;
(6) California NanoSystems Institute and Department of Chemistry and E-mail:[email protected]
Biochemistry,UniversityofCalifornia,LosAngeles,CA,USA
(cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO.Thisisanopenaccessarticleunder
thetermsoftheCreativeCommonsAttributionLicense(CCBY3.0),whichpermitsuse,distributionandreproduction
264 inanymedium,providedtheoriginalworkisproperlycited. EMBOMolMed(2013)5,264–279
www.embomolmed.org ResearchArticle
PeterWendetal.
(TN). Targeted therapies for certain of these subtypes exist: activity leading to increased proliferation (Miranda-Carboni
luminal A breast cancers are oestrogen receptor alpha (ER) etal,2008).
positive and are treated with ER pathway inhibitors, such as InthisstudywehaveidentifiedHMGA2asthemosthighly
tamoxifen or aromatase inhibitors. ERBB2/HER2/neu (HER2) expressedgeneinWnt10b-driventumours.TheHMGAprotein
positive tumours are treated with the monoclonal antibody family includes HMGA1a and HMGA1c, which are encoded
herceptin. The triple-negative breast cancer (TNBC) subtype by the same gene, and the closely related HMGA2, which is
[i.e.lackingexpression(oroverexpression)ofERandHER2,as known to be over-expressed in breast cancer (Peluso &
well as progesterone receptor (PR)] is specifically associated Chiappetta, 2010). HMGA2 is expressed in breast tumours of
with a poor prognosis and a highly metastatic phenotype. high histological grade (3), but not in those of lower grades
Currently,treatmentoptionsforTNBCarelimitedtocytotoxic (Rogalla et al, 1997). HMGA family members play important
chemotherapy(Paletal,2011).TNBCismoreprevalentamong rolesinstemcellself-renewal,proliferationanddifferentiation
premenopausal women of African American ancestry (39%) (Fusco & Fedele, 2007). They are widely expressed during
thaninnon-AfricanAmericanwomen(16%;Careyetal,2006). early embryogenesis but are restricted as foetal development
Survival disadvantage is seen in TNBC when compared to progresses;theyarenotexpressedinnormaladulttissue(Fusco
other cancer subtypes regardless of stage at diagnosis, with a & Fedele, 2007; Zhou et al, 1995). HMGA2 associates with
5-year survival of only 77% compared with 93% for other chromatin and regulates transcription by altering chromatin
breast cancers. Much work is still needed to develop targeted structurebutitselfdoesnotbindDNA(Reeves,2000).HMGA2
neoadjuvanttherapiesfortheTNBCsubtype. directly regulates CCNA2, CCNB2 and physical phospho-RB/
TheWnt/b-cateninpathwayhasbeenshowntobeactivated E2F1interactions(reviewedinFusco&Fedele,2007).
in basal-like tumours (Khramtsov et al, 2010). Wnt/b-catenin Inthepresentstudy,weprovideevidencethatWnt10b-driven
signalling is activated by interaction of Wnt ligands with its tumours are devoid of ERa, PR and HER2 protein expression
receptorssubsequentlyleadingtothestabilizationofb-catenin. andcanbeusedasatranslationalmodelforhumanTNBC.We
Stabilized b-catenin translocates to the nucleus and induces showthatbothWNT10BandHMGA2arehighlyexpressedina
specifictranscriptionalprogramsinfluencingcellularresponses subset of human primary TNBC tumours correlating with
including, but not limited to, cellular proliferation, develop- predictivepoorsurvivaloutcomeinbothbasal-likeandTNBC
ment, differentiation, neoplasia and stem cell maintenance patients. From the mouse model, we are able to translate
(Moonetal,2004;Nusse,2005;Wendetal,2010). WNT10B,HMGA2andproliferationmarkers(amongstothers)
Wnt10b plays a very fundamental role during mammary to human TNBC cell lines and primary TNBC samples.
glanddevelopment,asitistheearliestdiscernibleectodermal Treatment of TNBC-derived cell lines and primary tumour
event (E11.25) defining the mammary gland ridge. It is mouse cells with known Wnt pathway inhibitors down
expressed in the mammary anlagen and is characteristic for regulatesexpressionofHMGA2andcellproliferationmarkers.
thedefinitivemammarylineage(Veltmaatetal,2004).Elevated WNT10B has an epistatic effect on HMGA2, and HMGA2 is
expression of Wnt10b results in mammary tumorigenesis in essential and necessary for proliferation in both mouse and
mice, and has been detected in human breast carcinoma cell human TN cell lines. More importantly, the regulation of
lines (Wend et al, 2011). In order to model human breast HMGA2 by WNT10B is developmentally conserved in the
tumorigenesis several Wnt/b-catenin pathway-related mouse embryonic mouse mammary gland anlagen. The translational
models have been generated, and it is believed that tumours Wnt10bmodelmayprovideanoveltherapeutictooltodevelop
originate from stem cells and/or progenitor cells (e.g. MMTV- inhibitors to control Wnt/b-catenin-mediated proliferation in
Wnt1, MMTV-Wnt10b and MMTV-DN-b-catenin amongst humanTNBC.
others;Wendetal,2011).
Althoughnuclearb-cateninisupregulatedinmorethan50%
ofbreastcancercases(Cowinetal,2005),mutationsingenes RESULTS
encodingintracellularsignallingcomponentsarerare.Thismay
suggest deregulation at the cell surface to be a possible key Humantriple-negativebreastcancers(TNBC)express
mechanismtoexplainhighlevelsofb-catenininbreastcancer. WNT10B,showactiveWntsignalling,andhavehigh
It is well established that signalling components that function proliferation,demonstratingthatWNT10Bhasclinical
transiently during embryonic development might become an relevanceandprognosticvalue
oncogene by constitutively re-activating embryonic signalling CompellingevidencesuggeststhatWNT10Bmaybeavaluable
programs in adult tissue(s) (Polyak & Weinberg, 2009). In candidate for the development of therapeutic regimens for
mammary gland development and breast tumorigenesis an certain human diseases [reviewed; (Wend et al, 2011)]. The
exclusiveWntpathwaycomponenttobeconsideredisWnt10b. Wnt10b-driven tumour mouse model has been shown to be
However,theknowledgeaboutthefunctionalandmechanistic useful to model human breast cancer (Miranda-Carboni et al,
consequences of Wnt/b-catenin signalling in mammary 2008),butstilllackingisdirectevidencethatWNT10Bcanbe
progenitor cells and breast oncogenesis is still limited. We linkedtohumanprimaryTNBCpatients.Thuswequestioned:
have recently shown that mechanistically Wnt10b-driven (i)IsWNT10BexpressiondetectableinprimaryTNBCsamples?
(Wnt10bTG) tumours degrade the tumour suppressor p27Kip1 (ii)CanWNT10BexpressionactivatecanonicalWnt-signalling-
in a SKP2-independent manner mediated by CUL4A E3-ligase mediated gene expression in primary TNBC? and (iii) Does
EMBOMolMed(2013)5,264–279 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. 265
ResearchArticle www.embomolmed.org
WNT10Bisamodelfortriple-negativebreastcancer
WNT10Bexpressionpredictsurvivaloutcome?Itisimportantto Concurrently, we employed a publicly available database
note that the molecular, clinical and pathological profiles of andKaplan–Meiersoftware(KM-plotter)thathastheabilityto
basal-like and TNBC are similar as they overlap by 60–90% assess 22,277 genes in over (cid:2)3000 breast cancer samples
(Al Tamimi et al, 2010); the terms are often interchangeable [http://kmplot.com/breast/;(Gyorffyetal,2010)].Weutilized
but they are not identical in their gene expression profiles thebasal-likedatabaseandidentified>280 patientsthatwere
leading to at least six to eight complex subtypes (Perou et al, screenedforbothWNT10BandWNT1expression.Recurrence-
2000). In our study the term basal-like will be utilized when free survival was depicted using the Kaplan–Meier survival
referencing microarray data (i.e. mRNA expression of ERa(cid:1), estimates with a medium cutoff for WNT10B and WNT1
PR(cid:1)andHER2(cid:1))andthetermTNBCwillrefertoanalysisbya expression (Fig1E–F). Remarkably,survivaloutcomediffered
pathologistwhosubsequentlyclassifiedthetumoursasERa(cid:1), betweenpatientswithlowandhighWNT10Bexpressionvalues.
PR(cid:1), HER2(cid:1) by immunohistochemistry (IHC). Triple-negative Coxproportionalhazardsregressionrevealedanincreasedrisk
(TN)willbeusedtodescribebothbasal-likeandTNBC. inpatientswithhighWNT10Bexpression(HR¼1.38,p¼0.03).
TodeterminetheexpressionofWNT10Binhumanprimary In contrast, WNT1 expression was unable to predict survival
breast cancer, we analysed commercially available tumour outcome (HR¼1.13, p¼0.42). Additionally, WNT10B expres-
microarrays (TMA) consisting of 125 samples of all different sioncouldnotpredictsurvivaloutcomeofpatientsofanyother
subtypesbyIHC(TNBC(cid:2)15%oftotal:OhioSt.Univ.Human breastcancersubtypetestedwiththeKM-plotter.
Tissue Bank). These 18 TNBC tumours on the TMA from the In summary, these results illustrate for the first time that
OhioTissueBankCohortwereselectedtobebetweentheagesof WNT10B expression has significant clinical relevance and
33 and 45 years old at the time of diagnosis. Expression of probability for predicting recurrence-free survival in breast
WNT10B is absent or low in ERþ, PRþ and HER2þ tumours cancerforbothbasal-likeandTNBCtumours.Ourhypothesis
(Fig 1A). In contrast, most of the TNBC samples found in the that WNT10B would activate canonical Wnt-signalling and
TMA were very high for WNT10B expression. Additionally, activategeneexpressionwasvalidatedwithAXIN2expression
we collected 59 TNBC from patients and our pathologist inourTNBCpatientsamples.
quantifiedandscoredoursamplesaseitherpositiveornegative
for WNT10B expression for this analysis (Fig 1B). Most TheWnt10bLacZmammarytumoursaretriple-negative
of the samples in the TMA were negative (>75%) and a few To determine how well our novel MMTV-Wnt10b-IRES-LacZ
were positive (<10%) for the presence of WNT10B protein. (Wnt10bLacZ) mouse model resembles human TNBC, we
Conversely,mostofthesamplesinourcollectionof59TNBC conductedIHCforestrogenreceptor-alpha(ERa),progesterone
scorepositive(>80%)forWNT10B. receptor (PR) and HER2 protein expression levels (Fig 2A).
We next analysed primary TNBC samples from a German Wnt10b-driven tumours are phenotypically like human
cohort (n¼14) for the presence of both WNT10B and TNBC—devoid of ERa, PR and HER2. In contrast, ERa and
b-CATENINtoverifyiftheyoverlapped(Fig1C).Insequential PRproteinexpression,intheuterusofwild-typefemalemice,is
tumour sections the expression of WNT10B and b-CATENIN present in various subpopulations of myoepithelial, stromal
correlateinsimilarregions(i.e.arrowandarrow-heads).More cells and both luminal and glandular epithelial cells (Fig 2A).
importantly, the same areas also show high expression of Interestingly, the Wnt10b tumorigenic mouse model contrasts
AXIN2,awell-knowncanonicalWntdirect-target(Lustigetal, theMMTV-Wnt1model,inpart,becauseWnt1-driventumours
2002) demonstrated by in situ hybridization (ISH). The same have been reported to express hormone receptors (Teissedre
tumours also had high expression of Ki67 (Fig 1C). We have et al, 2009). HER2 protein expression is very high in mouse
validated the specificity of the WNT10B antibody utilizing MMTV-neu2/ErbB2 (ErbB2TG) tumours but not expressed at
transgenic MMTV-Wnt10b-IRES-LacZ-derived tumours and all in the Wnt10bLacZ tumours. Furthermore, Wnt10b-driven
cell lines, human triple negative breast cancer cell lines and tumoursexpressthebasal-epithelialmarkersCK5andCK6,in
Wnt10b-knockout embryos (E.14.5; Supporting Information contrasttoErbB2TGtumours(SupportingInformationFigS2A–
Fig S1A and B). We also contrasted the expression of both C).WealsoshowthatWnt10bLacZ-driventumoursexpresshigh
b-CATENIN [i.e. non-phospho (active) b-catenin (SER33/37/ levelsofb-galactosidase activity,havetranscriptionally active
Thr41)vs.apan-b-catenin-antibody]andAXIN2inTNBCwith nuclear b-catenin and correlating with high levels of AXIN2
other breast cancer subtypes (ERþ, PRþ, HER2þ and TPþ; proteinexpression(SupportingInformationFigS2D–F).
SupportingInformationFigS8A–C).
The hallmark of clinical relevance in cancer biology is HMGA2ishighlyexpressedinWnt10bTGmammary
to evaluate the association of a given gene expression with tumoursandlackinginembryomammaryplacodesof
survival outcome. To this end we utilized the available Wnt10b-knockoutmice
clinical data from our collection of 59 TNBC samples, and Insearchfornovelgenenetworksthatareactivatedduringearly
identified two clinical parameters that exhibit significant stagesofdevelopment,repressedinadulttissueandreactivated
correlations with high WNT10B expression (Fig 1D): (i) big during tumorigenesis, we identified a substantial number of
tumoursize>1.5cm(n¼45,t¼0.28,p¼0.021),and(ii)high candidates within our tumour microarray data (Miranda-
nuclear grade status 3 (n¼26, t¼0.420, p¼0.025). Patho- Carbonietal,2008).Ofthosecandidates,highmobilitygroup
logistsassociatethesetwocharacteristicsforagiventumourto A family member 2 (Hmga2) was the highest regulated gene
haveanoverallpooreroutcome. [125-foldincreasedexpressionoverwild-typecells(p<10(cid:1)9);
266 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,264–279
www.embomolmed.org ResearchArticle
PeterWendetal.
Figure1. Humantriple-negativebreastcancers(TNBC)expressWNT10B,showactiveWntsignallingandhavehighproliferation,andWNT10Bhasclinical
relevanceandprognosticvalue.
A,B. Immunohistochemical(IHC)analysisoftissuemicroarraysofdifferentbreastcancersubtypesshowspredominantWNT10BexpressioninTNBC
(quantificationsareshowninB).
C. TNBCtumourswereanalysedbyIHCforWNT10B,b-cateninandKi-67expression.AXIN2(aWnt/b-catenintargetgene)mRNAlevelsweredeterminedby
insituhybridization(ISH).ArrowsindicateasimilarlocalizationforIHCandISH.Tentimesmagnificationsareshown.Insertsshow40(cid:3)magnifications.
D. HighWNT10BexpressioninTNBCissignificantlycorrelatedwithlargetumoursizeandhighnucleargrade(WNT10BexpressionwasdeterminedbyIHCin
TNBCpatientsamplesandresultswerecorrelatedwithpatientdata).Associationsbetweenmarkersandclinicalparametersthatweremeasuredona
continuousorordinalscalewereevaluatedusingKendall’sTaucoefficient.Associationsbetweenmarkersandbinaryclinicalparametersweretestedusing
anexacttrendtest,associationswithnominalparametersweretestedusingFisher’sexacttest.pValuesandpatientnumbers(n)areindicatedinthetable.
AdditionalinformationcanbefoundinSupportingInformationFigS7andtheMaterialsandMethodsSection.
E,F. Kaplan–Meiersurvivalanalysisshowingsignificantlyimprovedrecurrence-freesurvivalofbasal-likebreastcancerpatientswithtumoursthatexpresslower
levelsofWNT10B(E)butnotWNT1(F).Patientswithhigher(red)andlowerexpression(black)areindicatedaswellasnumbersofpatientsatriskatspecific
timepoints(beloweachdiagram).Hazardratios(HR)andpvalues(logrankp)aredepictedforeachsurvivalanalysis.Kaplan–Meiersurvivaldatawere
generatedusingthepubliclyaccessibleonlinetoolKM-plotter.pValuesof<0.05wereconsideredtobestatisticallysignificant(D–F).
Fig 2B]. Furthermore, the related family member Hmga1 was driventumours(Fig2D).ThereisstrongexpressionofHMGA2
foundtobeup-regulatedby10-fold.WenextvalidatedHmga2 proteinlevelsinmostoftheWnt10bLacZ-driventumourscells.
and Hmga1 expression by qt-PCR (Fig 2C). There is twice as In contrast, both virgin wild-type mammary gland tissue and
much Hmga2 expression in both primary tumour cells and ErbB2TGtumoursamplesdidnotexpressanydetectableHMGA2
(Lin(cid:1)) LacZþ FACS-sorted tumour cells, than the Hmga1 protein,aswouldbeexpected.
expressionprofileinthesamecells.NeitherHmga1norHmga2 We next determined if the Wnt10b-induced expression of
werefoundtobeexpressedinvirginwild-typemammarytissue Hmga2isembryologicallyconservedduringearlymammogen-
or ErbB2TG tumours analysed by qt-PCR (Fig 2B–C). We next esis. To this end we analysed embryonic tissues (E14.5) from
validated the expression of HMGA2 by IHC in Wnt10bLacZ- bothwild-typeandWnt10b-knockoutmice(Wnt10b(cid:1)/(cid:1))forthe
EMBOMolMed(2013)5,264–279 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. 267
ResearchArticle www.embomolmed.org
WNT10Bisamodelfortriple-negativebreastcancer
Figure2.Theself-renewalmarkerHMGA2ishighlyexpressedinWnt10bTGmammarytumoursandmammaryplacodes.
A. IHCforoestrogenreceptora(ERa),progesteronereceptor(PR)andHer2tovalidatethetriple-negativephenotypeofWnt10bLacZtumours.Wild-type(WT)
uterusandHer2tumoursampleswereusedaspositivecontrols.Highlightedaremyoepithelial(myo),glandularepithelial(ge),luminalepithelial(le)and
stromalcells(s).
B. HierarchicalclusteringofmicroarraydataofHmga2andHmga1expressioninWnt10b-driventumours.
C. ValidationofHmga2andHmga1expressionintumourcells(Tu),Lin(cid:1)LacZþ(LacZþ)tumourcellscomparedtowild-typevirgin(Virg)andErbB2TGtumour
cells,asdeterminedbyqt-PCR.Errorbarsrepresentthemeansandthestandarddeviationsfromthreeindependentexperiments;(cid:4)(cid:4)p-value¼0.007versus
VirgorErbB2TGTusamples(Student’st-test).
D. IHCofHMGA2inWTmammaryglands,andErbB2TGandWnt10bLacZtumours.RedarrowheadshighlightcellspositiveforHMGA2andblackarrowheads
negativecells,respectively.
E. IHCofHMGA2inWTandWnt10bknockout(Wnt10b(cid:1)/(cid:1))mammaryplacodes(mp)atembryonicDay14.5revealslossofHMGA2expressionin
Wnt10b(cid:1)/(cid:1)mp.DashedblacklinesindicateWnt10b(cid:1)/(cid:1)mp.Mesenchymal(M:redarrowhead),epidermislayer(EL:shortblackarrow)andmammary
anlagen(MA:longblackarrow).
F,G. Mammosphereassays(MSA)ofWTandWnt10b(cid:1)/(cid:1)mammaryglandcellsandMMTV-Wnt10bLacZprimarytumourcellsthatwereseriallypassaged(atDay10
and20)andanalysedatDay30.Secondarysphereswerecountedinsequentialdilutionsandimagedusingaphasecontrastmicroscope.Biologicalreplicates
(n¼3)andsixtechnicalreplicatesareshown.Wnt10bLacZ-derivedMSAsanalysedbyIHCforHMGA2isshown(10(cid:3))inlowerrightpanelofG(bar,50mm).
InFerrorbarsrepresentthemeansandthestandarddeviationsfromthreeindependentexperiments;pvalues:a¼0.04,b¼0.03versuswild-typeor
Wnt10b(cid:1)/(cid:1)samples(Student’st-test).pValuesof<0.05wereconsideredtobestatisticallysignificant(C,F).
268 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,264–279
www.embomolmed.org ResearchArticle
PeterWendetal.
presenceofHMGA2byIHC(Fig2E).Thereisspecificexpression duetoHMGA2expression.Weobserveddecreasedproliferation
ofHMGA2inmammaryanlagenepithelialcells(MA),aswellas (>35%) after Hmga2 silencing, but not restored to NMG
the epidermis layer (EL) and mesenchymal (M) cells of wild- parentalproliferationlevels(SupportingInformationFigS3C).
type mice. In contrast, HMGA2 levels are strongly decreased To determine if the regulation of Hmga2 expression by
throughout the three subtypes of tissues, particularly, in the Wnt10b was indirect or mediated directly by b-CATENIN
mammary anlagen of the Wnt10b(cid:1)/(cid:1) mice. Furthermore, this signallingloci,weutilizedinsilicoanalysistoidentifypotential
timepointistheidenticalstageinwhichendogenousWnt10b TCF/LEF1-binding sites that could represent Wnt response
ishighduringearlymammogenesis(Veltmaatetal,2004). elements (WRE) within the mouse Hmga2 loci (http://
Basedontheabove,wehypothesizedthatthelossoftheself- genome.ucsc.edu/: for the genomic upstream sequences and
renewalmarkerHMGA2wouldhaveafunctionalconsequence http://www.gene-regulation.com/cgi-bin/pub/programs/patch/
inadultWnt10b(cid:1)/(cid:1)mammarycellstogeneratemammospheres. bin/patch.cgi:PATCHanalysisfortranscriptionbindingsites).
Mammaryglandcellswereisolated fromwild-typevirginand TotestthefunctionalityoftheseputativeLef1/Tcf4sites,and
Wnt10b(cid:1)/(cid:1)miceaswellasfromprimaryWnt10bLacZtumours. to examine whether Hmga2 represents a previously unrecog-
Cellsweregrownundermammosphereassay(MSA)culturing nizedWnttargetgene,chromatinimmunoprecipitation(ChIP)
conditionsandsequentiallypassagedat10daysandat20days analysiswasperformedwithanantibodytob-catenin.NMGand
generating tertiary-mammospheres, in a series of dilutions, NMG-Wnt10bcellsweresynchronizedandreleasedfor8h.We
followedbytheassessmentofMSAnumbersat30days(Fig2F). chose8hafterreleasefromconfluencybecausemaximalHmga2
Wnt10bLacZ tumours have approximately one self-renewing mRNA expression levels were obtained at that time point
stem cell per 200 cells. In contrast, wild-type mammary cells (Fig3A).Athistimepoint,b-cateninisenrichedbyeightfoldat
wereunabletogenerateMSAatthesamefrequencyandthecells theHmga2proximalpromoter inWnt10b-expressing cellsbut
fromWnt10b(cid:1)/(cid:1)micedonothavethecapabilityofself-renewal notintheparentalcellline(Fig3B).b-CATENINisalsoenriched
(Fig2F).Finally,mammospheresfromtime-point30daysshow 2.5-fold at the promoter of a well-known Wnt-direct target
highexpressionofHMGA2(Fig2G). gene,c-Myc,overtheparentalline(Heetal,1998).Incontrast,
In summary, Wnt10bLacZ-driven tumours are devoid of b-CATENIN is not enriched at an upstream (cid:1)6Kb element of
hormonereceptorsandHER2expression,withspecificexpres- theHmga2chromosomalORF-locidevoidofWREsitesnorto
sion of HMGA2 in tumour tissue only. More importantly, we thepromoterofanegativecontrolgene(GADPH)ineitherNMG
also provide evidence that HMGA2 is regulated by Wnt10b- orNMG-10bcelllines.
expression in a developing embryo (E.14.5 early mammogen- TofurthercharacterizeiftheexpressionofHmga2depends
esis)andintheabsenceofWNT10BexpressionHMGA2protein onactiveb-cateninsignallingwetreatedourcelllineswiththe
levelsarediminishedorcompletelylostinmultipletissues. Wnt/b-catenin signalling inhibitor ICG-001 (10mM; Fig 3C).
The inhibitor selectively inhibits TCF/b-catenin transcription
Wnt10b/b-cateninsignallingdirectlyregulatesHMGA2 inaCBP-dependentmannerwithoutinterferingwithb-catenin/
expressioninmousemammarycelllinesandregulates p300 interactions (Emami et al, 2004). The data show that
proliferationinmammarytumourcelllines NMG-10b cells respond to ICG-001 treatment with decreased
Basedontheprecedingexperiments,wehypothesizedthatone Hmga2 expression close to the levels of the parental NMG
potential function of HMGA2 in Wnt10b-driven tumours is cells.TogethertheresultssuggestthatexpressionofHmga2is
the control of proliferation. HMGA2 is known to directly dependentonb-catenin/CBPinteractionsmediatedbyWnt10b
regulatethecellcycleproteinsCCNA2andCCNB2,andRb/E2F1 signalling.
protein–proteininteractions(amongstothertargets;DeMartino Based on the above experiments we hypothesized that
et al, 2009; Fedele et al, 2006; Tessari et al, 2003). These cell if we silence Hmga2 in a Wnt10bLacZ tumour-derived cell
proliferationmarkers(amongstothers)arealsoupregulatedin line (WZALacZ) we would block proliferation. To this end we
Wnt10b-driventumours,asshownbymicroarraygeneexpres- generated stable clonal lines with lentivirally expressed short
sion analysis and verifying qt-PCR (Supporting Information hairpintoHmga2orthecontrolGFP(Fig3D).Twoindependent
Fig S3A and B). Wnt10b-driven tumours, when compared to shHmga2-clones decreased proliferation by 38% (pool) and
ErbB2TG-driventumours,haveanincreasedexpressionpattern the other by 72% (#1) relative to control shGFP. Silencing of
forG1-,S-andG2-phaseCyclins. Hmga2 and concurrent downregulation of Ccna2 expression
To gain mechanistic insights on how Wnt10b regulates wasverifiedbyqt-PCR(SupportingInformationFigS3DandF).
HMGA2 expression we turned to our previously published The results suggest that HMGA2 expression is necessary for
mouse cell line system NMuMG (NMG) and NMuMG-Wnt10b maximalproliferationofWZALacZcells.
(NMG-10b;Miranda-Carbonietal,2008).NMGandNMG-10b We next wanted to verify if these tumour-derived cells,
cells were synchronized in early G by maintenance at 100% WZALacZ,wouldrespondtotheWnt-inhibitorICG-001.Tothis
1
confluencyfor2–3days.Cellswerethenreleasedandharvested end, we used non-synchronized randomly cycling cells in the
at various times for analysis by qt-PCR. NMG-10b cell lines presence or absence of ICG-001 (10mM) and quantitated the
inducefive-toeightfoldgreaterHmga2expression,atall-time effectsonproliferation(Fig3E).Consistentwithourmodelling,
points,thanthecontrolparentalcelllineNMG(Fig3A).Wealso growth-inhibition was observed (>60%) after ICG-001 treat-
silencedHmga2intheNMG-10bcelllineutilizingaLentiviral- ment. Furthermore, a second Wnt10bLacZ cell line responded
shHmga2systemtoverifythattheincreasedproliferationwas as well (Supporting Information Fig S3E). To confirm if the
EMBOMolMed(2013)5,264–279 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. 269
ResearchArticle www.embomolmed.org
WNT10Bisamodelfortriple-negativebreastcancer
Figure3. Wnt10binducesHmga2-dependentproliferationviab-catenininmousemammaryepithelialandtumourcells.
A. TimecourseexperimenttoanalyseHmga2expressioninparentalNMuMG(NMG)cellsandNMuMGcellsoverexpressingWnt10b(NMG-10b)afterrelease
fromgrowtharrest(determinedbyqt-PCR).Errorbarsrepresentthemeansandthestandarddeviationsfromthreeindependentexperiments.
B. Assessmentofenrichmentofb-cateninattheHmga2,c-MycandGapdhpromotersbychromatinimmunoprecipitation(ChIP)8hafterreleasefromgrowth
arrest.Anupstream(cid:1)6KbelementoftheHmga2chromosomalORF-locidevoidofWREsiteswasusedasnegativecontrol.
C. Forty-eighthourstreatmentofNMGandNMG-10bcellswiththeWnt/b-catenininhibitorICG-001(10mMin1%DMSO)leadstodecreasedHmga2expression,
asdeterminedbyqt-PCR.Errorbarsrepresentthemeansandthestandarddeviationsfromthreeindependentexperiments;(cid:4)p-value¼0.04versusuntreated
sample(Student’st-test).
D. shRNA-MediatedknockdownofHmga2leadstoattenuatedgrowthofWZALacZtumourcells.ShownaretwodifferentshHmga2clonescomparedtoacontrol
shGFPclone.Errorbarsrepresentthemeansandthestandarddeviationsfromthreeindependentexperiments.
E. AttenuatedproliferationofWZALacZtumourcellsupontreatmentwithICG-001(10mMin1%DMSO).Errorbarsrepresentthemeansandthestandard
deviationsfromthreeindependentexperiments.
F. ImmunoprecipitationandWesternblotanalysisverifyingdecreasedinteractionofb-cateninandCBPafterICG-001treatmentofWZALacZtumourcells.
G. DecreasedexpressionofHMGA2andPCNAinICG-001-treatedWZALacZtumourcells,asdeterminedbyimmunoblottinganalysis.
H. QuantificationofproliferationofWZALacZcellsupontreatmentwiththeWntpathwayinhibitorsICG-001orWnt-C59comparedtovehiclecontrol(DMSO).
Errorbarsrepresentthemeansandthestandarddeviationsfromthreeindependentexperiments;(cid:4)(cid:4)(cid:4)p-value¼0.0008(ANOVA).
I. ImagesofMSAfromprimaryWnt10bLacZmammarytumourcellsaftercontrol(DMSO,upperpanel)orICG-001(lowerpanel)treatment.
J. MSAformationefficiencyofprimaryWnt10bLacZmammarytumourcellsaftercontrol(DMSO),ICG-001,andWnt-C59treatment.Errorbarsrepresentthe
meansandthestandarddeviationsfrom12independentexperiments;(cid:4)(cid:4)(cid:4)pvalue¼0.0006(ANOVA).pValuesof<0.05wereconsideredtobestatistically
significant(C,H,J).
270 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,264–279
www.embomolmed.org ResearchArticle
PeterWendetal.
mechanism of action by ICG-001 is preserved in the mouse analysed by qt-PCR for the presence of WNT10B, HMGA2,
model, we conducted immunopercipitation (IP) experiments HMGA1 and BMI-1 (as a control for self-renewal markers;
with b-CATENIN in the absence or presence of the inhibitor amongstothers)inasubsetofhumanbreastcancercelllines:
and immunoblotted for CBP (Fig 3F). ICG-001-treated cells HMEC (human mammary epithelial cells; normal control),
losetheinteractionsbetweenb-CATENINandCBP.Moreover, MCF7 (ERþ tumour-derived), MCF-10A (non-tumorigenic
we verified by immunoblotting that ICG-001 treatment mammary epithelial), SK-BR-3 (HER2þ tumour-derived),
resultedindecreasedexpression of HMGA2and theprolifera- MDA-MB-231 (mesenchymal stem-cell like (MSL)/TN), JL-
tion marker PCNA (Fig 3G). We also wanted to determine BTL10(BTL10derivedfrom TNBCstage2Bandgrade3from
if proliferation of WZALacZ cells would be dependent on a 38-year-old African-American women established by Jean
Wnt10b-secretion and therefore treated the cells with Wnt- Latimer), MA-11 (TNBC metastasising to bone), and MCF7-
C59(CellagenTechnology,Inc.),whichblockspalmitylationof Wnt10b (MCF7-10b, a stable cell line expressing mouse
Wnt-proteinsmediatedbyPorcupine,similartoWnt-inhibitors Wnt10binhumanERþtumour-derivedcells(Miranda-Carboni
IWP-2, -3 and -4 (Fig 3J). The data shows that Wnt-C59 is et al, 2008; Fig 4A). WNT10B is most highly expressed in TN
capable to significantly (p¼0.001) inhibit proliferation and cell lines when compared to non-TN cell lines. Concurrently,
comparabletotheICG-001treatedcells. both HMGA2 and HMGA1 are also most highly expressed in
Wenexthypothesizedthatprimaryderivedtumourcellsthat
haveaverypotentinvitro‘self-renewal’capacity(Fig2FandG)
wouldrespondtoICG-001treatment.Todoso,mammospheres
weregeneratedinlow-adherence96-wellplates,aspreviously
described from Lin(cid:1)LacZþ sorted cells, and the effects on
mammosphere formation were measured in the presence or
absence of ICG-0001 (10mM; Fig 3I). ICG-001 completely
blocked sphere formation. In parallel, we also wanted to
determine if mammosphere formation from primary tumour
cells would be dependent on Wnt10b-secretion and therefore
treatedthecellswithWnt-C59(Fig3J).Sphereformationwas
inhibited 10-fold by ICG-001 and threefold by Wnt-C59,
respectively.ThedatasuggestthatbothupstreamWnt-ligands,
anddownstreameffectorsofb-cateninsignalling,arerequired
formaintenanceofsphereformation.
HumanTNBCcelllinesexpressWNT10BandHMGA2andare
sensitivetob-cateninpathwayinhibition
We next wanted to determine whether our identified gene
signature is also expressed in human TNBC cell lines. We
Figure4.Triple-negativebreastcancer(TNBC)cellsinhumanexpressa
specificgenesignatureandtheirself-renewaldependsoncanonical
Wnt/b-cateninsignalling.
A. HumanTNBCcelllines(MDA-231,BTL10,MA-11)andothernon-triple
negative(TN)mammarycelllinestestedbyqt-PCRforexpressionof
WNT10Bandself-renewalmarkers.RelativemRNAlevelsarenormal-
izedtohumanMCF7cells.Errorbarsrepresentthemeansandthe
standarddeviationsfromthreeindependentexperiments.
B–E. ProliferationofhumanTNBCcells(B,D)orhumannon-TNbreastcancer
cells(C,E)upontreatmentwiththeWnt/b-catenininhibitorICG-001
(10mMin1%DMSO).Errorbarsrepresentthemeansandthe
standarddeviationsfromthreeindependentexperiments.InB,
(cid:4)(cid:4)(cid:4)pvalue¼0.0005versuscontrol-treatedcells(Student’st-test).InD,
(cid:4)(cid:4)(cid:4)pvalue¼0.0007versuscontrol-treatedcells(Student’st-test).
F. QuantificationofproliferationofhumanMDA-MB231andMCF7cells
upontreatmentwiththeWntpathwayinhibitorsICG-001orWnt-C59
comparedtovehiclecontrol(DMSO).Errorbarsrepresentthemeansand
thestandarddeviationsfromthreeindependentexperiments;
(cid:4)(cid:4)p-value¼0.004(ANOVA).
G. PhasecontrastimagesofBTL10cellsinadherent(upperpanel)and
mammospherecellculture(lowerpanel)upontreatmentwiththe
Wnt/b-catenininhibitorICG-001(10mMin1%DMSO,n¼3).pValues
of<0.05wereconsideredtobestatisticallysignificant(B,D,F).
EMBOMolMed(2013)5,264–279 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. 271
ResearchArticle www.embomolmed.org
WNT10Bisamodelfortriple-negativebreastcancer
the TNBC cell lines and MCF7-10b cells have a high level of Inhibitionofb-cateninsignallinginhumanTNBCcelllines
induction for HMGA2 (>30-fold) and BMI-1 (>5-fold) but disruptsLEF-1,TCF4andCBPprotein–proteininteraction
not HMGA1. This may suggest that HMGA2 and BMI-1 are thatleadstodirectrepressionofHMGA2expression
direct downstream targets of the WNT10B ligand. Moreover, Wenextrevisitedourpreviouslydefined‘intrinsicproliferation
HMGA2 is not responsive to 17b-estradiol (E2) treatment of signature’ from our Wnt10b-driven tumours (Supporting
parentalMCF7 cells orinMCF7-10b cells;MCF7-10b cells are Information Fig S3A and B) and hypothesized that a transla-
still responsive to E2 treatment by upregulation of XBP1 and tional TNBC model should preserve this signature in human
pS2 (Krum et al, 2008b), and express known canonical Wnt- TNBC cells. Moreover, these proliferation genes might be the
signalling target genes, such as c-myc, CCND1 and DKK1 target(s) for ICG-001-mediated growth arrest. To test this, we
(Supporting Information Fig S4A; The Wnt homepage: Wnt synchronizedbothMCF7andMDA-MB-231cellsatG -phaseby
1
targets,http://www.stanford.edu/group/nusselab/cgi-bin/wnt/). contact inhibition (for 2–3 days) and then harvested and
Importantly,treatmentofMCF7-10bcellswithICG-001blocks released the cells for 16h [S-phase; (Miranda-Carboni et al,
proliferationanddownregulatesHMGA2expression(Support- 2008)]. We treated the released cells with ICG-001 for an
ing Information Fig S4B and C). MA-11 cells were originally additional48h.Wholecellextractswereanalysedbyimmuno-
isolatedfromamalignantbonemarrowmetastasisofabreast blottingforHMGA2andproliferationmarkersinthepresenceor
cancer patient and express high levels of both WNT10B and absenceofICG-001(Fig5A).HMGA2,CyclinA2,CDK2,CDK4,
HMGA2(Miccietal,2001).BTL10cellsareanovelTNBCcell E2F1 and PCNA protein expression were down-regulated in
linefromanAfrican-Americanpatientthathasbeenengineered MDA-MB-231 cells treated with ICG-001. These results are
toretainanundifferentiatedstatewhileinculture(Latimeretal, consistentwithacellcyclearrestphenotype.Incontrast,MCF7
2010). BTL10 cells show a high level of HMGA2 induction cellshadnosignificantexpressionchangesoftheseproliferation
(>400-fold), express WNT10B (threefold vs. HMEC control) markers.CyclinE1andB1wereunchangedinbothcelllines.
andareWnt/b-cateninresponsive(SupportingInformationFig HCT-116 colon cancer cells have constitutively active Wnt/b-
S4D). cateninsignallingbutdonotexpressashighlevelsofHMGA2as
We next hypothesized that inhibition of canonical Wnt- inTNBCcelllines(Fig5A).
signalling would block proliferation in TN cell lines but We next wanted to determine if cell cycle arrest protein
not in cell lines derived from other breast cancer subtypes. activity is restored upon ICG-001 treatment and hypothesized
For this purpose, non-synchronized randomly cycling cells thatRBwouldbehypo-phosphorylatedduetothelossofCDK2
were treated with the Wnt/b-catenin signalling inhibitor and4.Totestthisweprobedthesameproteinextractswithboth
ICG-001 (10mM) and the effects on proliferation were a phospho-specific antibody (pRB {Ser807/811}) that can
measured (Fig 4B–E). Both of the human TNBC cell lines measure Cyclin D1/CDK4 and/or Cyclin A2, E/CDK2 activity
(MDA-MB-231 and BTL10) had a >60% reduction in and an antibody detecting total RB (Fig 5A). In MDA-MB-231
proliferation in the presence of ICG-001 (p¼0.001, Fig 4B cells,48hafterrelease,RBishyper-phosphorylated(pRB)and
and D). In contrast, proliferation of both MCF7 (ERþ) and in the presence of ICG-001 this hyper-phosphorylation is lost,
SKBR3 (HER2þ) cells was not affected by ICG-001 treatment consistent with the loss of both CDK4 and CDK2 enzymatic
(Fig4CandE).HMECcellsalsodidnotrespondtotreatment activity.
(Supporting Information Fig S4E). We next wanted to Toensure that the observedresults weredue tothe lossof
determine if the inhibition of proliferation by ICG-001 on transcriptional activation by b-catenin and not mediated by
MDA-MB-231 was also dependent on Wnt-ligand secretion. protein turnover we concurrently conducted qt-PCR on MDA-
To this end we treated the cells with Wnt-C59 inhibitor and MB-231 cells for HMGA2, proliferation markers and BIRC5
measured the effects on proliferation (Fig 4F). Inhibition [Survivin, a well-known target of ICG-001 in colon cancer
of Porcupine with Wnt-C59 blocked proliferation by >50% (Emami et al, 2004; Supporting Information Fig S5A)]. We
(p¼0.01). In contrast, in non-TN MCF7 control cells furthervalidatedtheeffectsofICG-001onvarioussubtypesof
proliferation was not affected by either ICG-001 or Wnt-C59 TNBC cells lines and purchased commercially available cell
treatment. To test the effects of ICG-001 on the capacity to lines from ATCC to ensure authenticity. ICG-001 treatment
block ‘self-renewal’, we generated mammospheres from down regulated HMGA2 and two of its downstream targets
BTL10 TNBC cell lines. We chose BTL10 because they have (CCNA2 and CCNB2) and BIRC5 a known direct target of
strong Wnt-reporter activity (Supporting Information Fig ICG-001inasubsetofMDA-MBderivedcelllines(Supporting
S4D) and retain an undifferentiated state while in culture InformationFigS5B).HMGA2mRNAlevelisdown-regulatedin
resemblingprimarybreastcancerexplants.ICG-001treatment thepresenceofICG-001,suggestingthatitisdirectlyregulated
of BTL10 cells robustly blocked mammosphere self-renewal by b-catenin/CBP interactions. We also silenced b-catenin
in 3D-cultured cells and effectively stopped growth in with siRNA (80% reduction) and verified the loss of HMGA2
2D-culturedcells (Fig4G). (30%reduction),whichwasaccompaniedbyreducedWNT10B
In summary, we have illustrated that our mouse gene expression(80%reduction)amongstothertargets(Supporting
signaturehasbeentranslatedspecificallytohumanTNBCcell Information Fig S5C). Corroborating our hypothesis, ICG-001
linesandnottoothersubtypesofbreastcancercelllines.We treatment of BTL10 cells also led to down-regulated mRNA
alsodemonstratedthatblockingofproliferationisdependenton levels of several Cyclins, HMGA2, and BIRC5 (Supporting
bothWNT-ligandsecretionandb-cateninsignalling. InformationFigS5D).Takentogether,theseresultsarguethat
272 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,264–279
www.embomolmed.org ResearchArticle
PeterWendetal.
Figure5. InhibitionofWnt/b-cateninsignallinginhumanTNBCleads
todecreasedexpressionofHMGA2andcellcycleregulatorsbyblocking
CBP/LEF/TCF-dependenttranscriptionalactivity.
A. EffectofICG-001(10mM)-inducedinhibitionofWnt/b-catenin
signallingontheexpressionofcellcycleregulatorsandproliferation
markersinsynchronized(0h)andreleased(48h)humanMCF7
(non-triple-negative)andMDA-MB-231(triple-negative)cells,as
detectedbyWesternblotanalysis.OnepercentDMSOwasusedas
controltreatment.b-actinservedasaloadingcontrol.HCT-116
colorectalcarcinomacellsservedascontrolcells.
B. ReducedexpressionofCCNA2andCCNB2inMDA-MB231cellsafter
HMGA2knockdownorICG-001treatment,asdeterminedbyqPCR.Error
barsrepresentthemeansandthestandarddeviationsfromthree
independentexperiments.
C. QuantificationofproliferationofMDA-MB-231cellsaftershRNA-
mediatedknockdownofHMGA2orICG-001treatment(10mM).Error
barsrepresentthemeansandthestandarddeviationsfromfour
independentexperiments;(cid:4)pvalues¼0.02and(cid:4)(cid:4)p¼0.008versus
controltreatments(Student’st-test).
D. qt-PCRexpressionanalysisforCCNA2andCCNB2incontrol(pcDNA3)
andHMGA2-overexpressing(pcDNA3-HMGA2)MDA-MB-231cells
treatedwithvehiclecontrolorICG-001.Errorbarsrepresentthemeans
andthestandarddeviationsfromfiveindependentexperiments;
(cid:4)pvalues¼0.01and(cid:4)(cid:4)p¼0.004versusHMGA2-overexpressingcells
(Student’st-test).
E,F. WNT10B-inducedupregulationofCCNA2dependsonHMGA2,as
determinedinanepistasisexperimentandanalysedbyqPCR.Error
barsrepresentthemeansandthestandarddeviationsfromthree
independentexperiments.
G,H. qt-PCRanalysisofChIPexperimentswithMDA-MB-231cellsafter
releasefromgrowtharrestandICG-001treatment(48h)usingRNAPol
IIandb-cateninantibodies.Valueswerenormalizedtoboththeinput
templateandanonregulatedlocus(hHBB).
I. ImmunoprecipitationandWesternblotanalysisillustratingaCBP/LEF/
TCF-dependentinhibitionofb-cateninsignallingbyICG-001inhuman
TNBCMDA-MB-231cells.Actinservedasaloadingcontrol.pValuesof
<0.05wereconsideredtobestatisticallysignificant(C,D).
TorescuetheinhibitionphenotypewetransfectedMDA-MB-
231 cells with pcDNA3-GFP and pcDNA3-HMGA2 vectors for
24h and subsequently treated with ICG-001 for an additional
24h. The cells were harvested and qt-PCR was conducted for
thelossofproteinexpressionofourproliferationmarkersisdue CCNA2andCCNB2(Fig5D).HMGA2overexpressionwasable
to the loss of transcriptional activity mediated by b-catenin tomoderatelyrescuebothCCNA2(>30%p<0.05)andCCNB2
signallingandnotproteinturnover. (60% p¼0.01) in the presence of ICG-001. The above results
To determine if HMGA2 is necessary and sufficient for would suggest that HMGA2 is essential and necessary for
proliferationofMDA-MB-231cellsweconductedgeneticloss-of- cellular proliferation of MDA-MB-231 cells that depend on
functionandgain-of-functionassays.Tothisendwegenetically b-cateninsignalling.
silenced HMGA2 expression utilizing the human Lentiviral- To test our model for the order of action of genes in a
shHMGA2andshGFPsystem,aspreviouslymentioned.Thereis regulatory hierarchy that governs the Wnt10b/b-catenin
a60%decreaseinproliferationintheshHMGA2cells(p<0.05), signalling pathway, we designed an epistatic functional assay
which is very similar to the growth inhibition in the parental forWNT10B.WetransfectedMDA-MB-231cellswithpcDNA3-
cellswhentreatedwithICG-001(75%reduction,p¼0.01;Fig GFPandpcDNA3-WNT10Bvectors,cellswereharvestedandqt-
5C).Next,weexposedshGFPcontrolcellstoICG-001treatment PCRwasconductedforWNT10B,HMGA2andCCNA2(Fig5E).
for 48h and compared it to shHMGA2 untreated cells by Concurrently, we repeated the previously mentioned transfec-
analysing the mRNA expression levels of HMGA2, CCNA2 tionwithourMDA-MB-231-shHMGA2silencedverifiedcelllines
and CCNB2 using qt-PCR (Fig 5B). We observed a >70% (Fig 5F). WNT10B induced the expression of HMGA2 and its
downregulation for all the genes tested in the shHMGA2 knowndownstreamtargetCCNA2incontrolcells.Incontrast,
clones, similar to the ICG-001-induced reduction in shGFP celllinessilencedforHMGA2canstillexpressWNT10Bbutthe
controlcells. expressionofHMGA2anditsdownstreamtargetCCNA2islost.
EMBOMolMed(2013)5,264–279 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. 273