The DNp63 Proteins Are Key Allies of BRCA1 in the...

Tumor and Stem Cell Biology

The DNp63 Proteins Are Key Allies of BRCA1 in thePrevention of Basal-Like Breast Cancer

Niamh E. Buckley1, Susan J. Conlon2, Karin Jirstrom3, Elaine W. Kay2, Nyree T. Crawford1, Anthony O'Grady2,Katherine Sheehan2, Simon S. Mc Dade1, Ching-Wei Wang4, Dennis J. McCance1, Patrick G. Johnston1,Richard D. Kennedy1, D. Paul Harkin1, and Paul B. Mullan1

AbstractLittle is known about the origin of basal-like breast cancers, an aggressive disease that is highly similar to

BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein areknown to function in stem cell regulation and stratified epithelia development in multiple tissues, and p63expression may be a marker of basal-like breast cancers. Here we report that DNp63 isoforms of p63 aretranscriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays andBRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like orBRCA1 mutant cancers. Nevertheless, we found that BRCA1 interacts with the specific p63 isoform DNp63galong with transcription factor isoforms AP-2a and AP-2g . BRCA1 required DNp63g and AP-2g to localize to anintronic enhancer region within the p63 gene to upregulate transcription of the DNp63 isoforms. In mammarystem/progenitor cells, siRNA-mediated knockdown of DNp63 expression resulted in genomic instability,increased cell proliferation, loss of DNA damage checkpoint control, and impaired growth control. Together,our findings establish that transcriptional upregulation of DNp63 proteins is critical for BRCA1 suppressorfunction and that defects in BRCA1-DNp63 signaling are key events in the pathogenesis of basal-like breastcancer. Cancer Res; 71(5); 1933–44. �2011 AACR.

Introduction

BRCA1 was initially cloned in 1994 as one of the genespredisposing to early onset breast and ovarian cancer (1).BRCA1 germline mutations confer a cumulative lifetime riskof 50% to 85% and 12% to 60% of developing breast andovarian cancer, respectively (2). BRCA1 is a multifunctionalprotein with roles in DNA damage repair, cell-cycle control,transcriptional regulation, and ubiquitination. It partici-pates in transcriptional regulation at several levels, inter-acting with sequence-specific transcription factors, RNApolymerase II, and enzymes involved in chromatin remodel-ing (3–6). BRCA1 transcriptional targets also possess tumor

suppressor functions with roles in stress responses, cell-cycle checkpoints, apoptosis, and DNA repair (7, 8). BRCA1mutant breast cancers often occur at an early age of onsetand are referred to as "basal-like," because they often expressthe basal cytokeratins-5 and -17 (CK-5, CK-17), markersusually found in normal basal/myoepithelial breast cells(9). They are also often referred to as "triple negative"[estrogen receptor alpha (ERa)- and progesterone receptor(PR)-negative, and low HER2 expression] possessing p53mutations, and are poorly differentiated and associated withpoor prognosis. Downregulation of BRCA1 mRNA and pro-tein expression has also been reported in approximately 30%of sporadic breast cancers (10). Indeed sporadic basal-likebreast cancers share features of BRCA1 mutant breastcancers indicating a potential common defect, previouslydescribed as "BRCAness" (11). Despite showing strongexpression of basal cytokeratins, recent studies indicate thatbasal-like and BRCA1 mutant tumors are now thought toarise from aberrant luminal progenitors rather than basalstem cells because targeting BRCA1 deficiency to basal stemcells results in tumors that do not resemble BRCA1 mutantor basal-like breast cancers (12, 13).

The transcription factor p63 is a member of the p53family of transcription factors, which also includes p73 (14).p63 knockout mice exhibit a striking developmental phe-notype with neonatal lethality accompanied by lack of allstratified squamous epithelia (notably mammary, lacrimal,and salivary glands; ref. 15). It is normally expressed in the

Authors' Affiliations: 1Centre for Cancer Research and Cell Biology,Queen's University Belfast, Belfast, United Kingdom; 2Department ofSurgery, Beaumont Hospital and Royal College of Surgeons in Ireland,Dublin, Ireland; 3Center for Molecular Pathology, Department of Labora-tory Medicine, Malm€o University Hospital, Lund University, Malm€o, Swe-den; and 4National Taiwan University of Science and Technology,Graduate institute of Bioengineering, Taipei, Taiwan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Paul Mullan, Centre for Cancer Research and CellBiology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL,Northern Ireland, United Kingdom. Phone: 44-28-90972941; Fax: 44-28-90972776. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-2717

�2011 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1933

Research. on August 18, 2018. © 2011 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

basal/myoepithelial layer of epidermal tissues and isenriched in putative stem cell populations. p63 regulationis complex; it encodes at least 10 open reading frames,resulting in the expression of the "transactivating" TAp63(containing an amino-terminal transactivation domain) and"deltaN" DNp63 (lacking transactivation domain) isoforms(16). DNp63 isoforms are the predominant species in manyepithelial tissues, originally thought to act as dominantnegatives of TAp63 and p53 function, but now have beenattributed with important transcriptional activities of theirown (17). They are known to be key regulators of basalgenes often in association with activator protein-2g (AP-2g ;ref 18). Deregulation of p63 expression is observed in anumber of different cancer types (19–21). A role for p63 as amarker of basal-like breast cancer had originally beensuggested on the basis of a few limited immunohistochem-ical studies (22, 23), and also it has been proposed to be asurvival factor in the basal-like breast cancer subtype (23).

However, other studies using similar immunohistochemicaltechniques showed directly conflicting data (24, 25). Inaddition, numerous microarray studies have never shown acorrelation between p63 expression and basal-like breastcancer (26, 27).

Here we show that several DNp63 isoforms are BRCA1transcriptional targets and identify the mechanism throughwhich BRCA1 regulates DNp63 proteins by targeting anenhancer region within the p63 gene, a finding first describedin keratinocytes (28). Using breast cancer cell lines andprimary breast tumors, we show that DNp63 isoform expres-sion is a marker of normal basal cells, but not of basal-likebreast cancers, or BRCA1 mutant tumors. We show thatDNp63 proteins are key growth control genes downstreamof BRCA1 required for the functioning of normal basal breastepithelia. We propose that the transcriptional regulation ofDNp63 proteins is important for the tumor suppressor func-tion of BRCA1 and that defects in this BRCA1-DNp63 signaling

(i)

A

B

C

(ii)HCC1937 MDA468

1.75EVBR

EVBR1.50

1.25

1.00

0.75

mR

NA

exp

ress

ion

mR

NA

exp

ress

ion

0.50

0.25

0.00 012

3456789

ΔN p63

TAp6

3

pan

p63

ERalpha

p-ca

dher

in

ΔN p63

TAp6

3

pan

p63

BRCA1

ERalpha

p-ca

dher

in

(i) (ii)T47D MCF-7

ScrBRsi

ScrBRsi

mR

NA

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

mR

NA

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

ΔN p63

TAp6

3

pan

p63

BRCA1

ERalpha

p-ca

dher

in

ΔN p63

TAp6

3

pan

p63

BRCA1

ERalpha

p-ca

dher

in

BRCA1

HCC1937(i) (ii) (iii) (iv)

EV BR

MDA-MB-468

EV BR

T47D

Scr BRsiMCF-7

Scr BRsi

GAPDH

ΔN p63αΔN p63β

BRCA1

GAPDH

ΔN p63α

BRCA1

GAPDH

ΔN p63α

BRCA1

GAPDH

ΔN p63αΔN p63β

Figure 1. DNp63 isoforms arepositively regulated by BRCA1. A,immunoblots showing whole celllysates collected from (i) HCC1937BRCA1 mutant cells stablytransfected with empty vector (EV)or wild-type BRCA1 (BR)constructs, (ii) MDA-MB-468stably transfected with the sameconstructs as in (i), or (iii) T47Dor (iv) MCF-7 cells transientlytransfected with scrambledcontrol (Scr) or BRCA1 siRNA(BRsi) and lysates extracted after72 hours; blots were probed forBRCA1 and p63 expression withglyceraldehyde-3-phosphatedehydrogenase (GAPDH) used asa loading control. B, real-timequantitative PCR (RqPCR)analyses showing mRNA samplesextracted from (i) HCC1937 EVand BR cells, (ii) MDA-MB-468 EVand BR cells. C, RqPCR analysesshowing mRNA samplesextracted from (i) T47D cells and(ii) MCF-7 cells (siRNA treated asdescribed above). RqPCRanalysis was performed usingprimers specific for DNp63,TAp63, pan p63, BRCA1, estrogenreceptor alpha (ERa), andp-cadherin. GAPDH mRNAlevels were used for normalization(n ¼ 3).

Buckley et al.

Cancer Res; 71(5) March 1, 2011 Cancer Research1934



3.5

(i)A

B

C

D

(ii) (iii)

3.0

2.5

2.0

1.5

Luci

fera

se n

orm

aliz

edto

Ren

illa

and

EV

nor

mal

ized

toR

enill

a an

d E

V

nor

mal

ized

to R

enill

aan

d T

K-L

uc

1.0

0.5

0.0 0 0

C40AP2A

AP2B

p53

RE

TK-Luc

10

20

30

1

2

3

C40 TK-LUC C40 TK-LUC

HCC EV HCC BR

SCRBRsi

(i) (ii) (iii)

Luci

fera

se n

orm

aliz

edto

Ren

illa

and

TK

-Luc

0

1

2

3

4

5

6

7

0.00

0.25

0.50

0.75

1.00

exp

ress

ion

norm

aliz

ed to

GA

PD

H

C40 TK-LUC

SCR

ΔNp63si

AP-2γ si

SCRΔNp6

3si

AP-2γ s

i

SCR

ΔNp63s

i

AP-2γ s

i

ΔNp63α

GAPDH

AP-2γ

(i) (ii) (iii)

norm

aliz

ed to

Ren

illa

and

TK

-Luc

0.0

2.5

5.0

7.5

10.0

exp

ress

ion

norm

aliz

ed to

GA

PD

H

0

1

2

3

C40 TK-LUC

Scr

BRsi

p53si

DN p63 s

TA p63 si

SCRBRsi

p53s

i

ΔNp63s

i

TA p

63 si

SCRBRsi

p53s

i

TA p

63 si

ΔNp63

si

ΔNp63α

BRCA1

GAPDH

(i) (ii) (iii)

exp

ress

ion

norm

aliz

edto

Ren

illa

and

TK

-Luc

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

exp

ress

ion

norm

aliz

ed to

GA

PD

H

0

1

2

3

C40 TK-LUC

SCR

BRsi

ΔNp63si

p63α si

p63γ si

SCRBRsi

ΔNp63s

i

p63α si

p63γ s

i

SCRBRsi

ΔNp63s

i

p63α si

p63γ s

i

ΔNp63α

BRCA1

GAPDH

Figure 2. BRCA1 regulates p63 through an internal enhancer region. A, (i) luciferase reporter assay of MCF-7 cells transfected with scrambled (Scr) or BRCA1(BRsi) siRNA 48 hours prior to transfection or (ii) HCC EV and HCC BR cells transfected with C40 TK-Luc (C40) or empty vector control (TK-LUC) constructs;(iii) luciferase reporter assay of MCF-7 cells transfected with a wild-type (C40), point mutated C40 construct (AP2A, AP2B, or p53RE), or a controlempty vector construct (TK-luc). Values were normalized for cotransfected Renilla luciferase activity (n ¼ 3). B, (i) luciferase reporter assay of MCF-7 cellstransfected with scrambled (Scr)-, BRCA1 (BRsi)-, p53 (p53si)-, DNp63 (DNp63si)-, or TAp63 (TAp63si)-specific siRNA. Activity was normalized as outlinedin (A); (ii) immunoblots showing BRCA1 and DNp63a levels following siRNA knockdowns described in (i); GAPDH antibody was used as a loading control;(iii) RqPCR analyses showing DNp63 mRNA levels following the siRNA knockdowns described in (i) with expression of GAPDH used as a housekeeper(n ¼ 3). C, (i) luciferase reporter assay of MCF-7 cells transiently transfected with scrambled (Scr)-, BRCA1 (BRsi)-, DNp63 (DNp63si)-, p63a (p63asi)-,or p63g (p63gsi)-specific siRNAs; activity was normalized as outlined in (A); (ii) immunoblots showing BRCA1 and DNp63a levels following siRNAknockdowns described in (i), GAPDH antibody used as a loading control; (iii) RqPCR analyses showing DNp63 mRNA levels following the siRNA treatmentsdescribed in (i) with expression of GAPDH used as a housekeeper (n ¼ 3). D, (i) luciferase reporter assay of MCF-7 cells transfected withscrambled (Scr)-, DNp63 (DNp63si)-, or AP-2g (AP-2gsi)-specific siRNAs; activity was normalized as outlined in (A); (ii) immunoblots showing DNp63aand AP-2g levels following siRNA knockdowns described in (i); (iii) RqPCR analyses showing DNp63 mRNA levels following the siRNA treatments describedin (i) with expression of GAPDH used as a housekeeper (n ¼ 3).

DNp63 Proteins—Key Effectors of BRCA1 Tumor Suppression

www.aacrjournals.org Cancer Res; 71(5) March 1, 2011 1935



axis are key events in driving the proliferation of basal-likebreast cancers.

Materials and Methods

Cell linesAll cell lines were purchased from American Type Culture

Collection (ATCC). The cell lines were characterized by iso-enzyme/cytochrome c oxidase I (COI) assay and short tandemrepeat (STR) analysis by the cell bank. Complete details of theHCC-EV/BR, MCF7, and T47D cell lines are provided inreference (8). The MDA468-EV and MDA468-BR cell lineswere generated following stable transfection of MDA-MB-468 cells with the same constructs described in reference(29). MCF10A-pSuper, MCF10A-BRsh, and MCF10A-p63shcells lines were generated by stable retrovirus transfectionof the MCF10A cells with (i) pSuper scrambled (30), (ii) pSuperBRCA1 (30), or (iii) pSuper pan-p63 short hairpin RNA(shRNA) constructs. Infected cells were selected in the pre-sence of 1 mg/mL puromycin. HME-1 is a nontransformed, h-TERT immortalized cell line derived from reduction mammo-plasty and maintained as described by ATCC. Primary humanmammary epithelial cells (HMEC) were obtained from Dr.Martha Stampfer (University of California, Berkeley) andmaintained as previously described (31). All other cell lineswere maintained as previously described (32).

Short interfering RNATransfections were performed using Oligofectamine

reagent (Invitrogen), as outlined in the manufacturer'sinstructions. siRNA oligonucleotide was obtained form Dhar-macon and used at a final concentration of 100 nmol/L.

Aldehyde dehydrogenase 1 and senescence assaysAldehyde dehydrogenase 1 (ALDH1) activity was assayed by

use of Aldefluor kit technology (Stem Cell Technologies) andsenescence assayed by the Senescence Detection kit (BioVi-sion Research Products), both according to themanufacturer'sinstructions.

Western blot analysisProtein lysates were extracted in EDTA lysis buffer (ELB;

0.25 mol/L NaCl, 0.1% IEPGAL, 0.25 mol/L HEPES, 5 mmol/LEDTA, and 0.5 mmol/L DTT), separated on a SDS PAGE gel,and transferred to a polyvinylidene difluoride membranefollowed by immunoblotting. Complete details of antibodiesused are listed in the Materials and Methods section ofSupplementary Data.

Immunofluorescence studiesCells were fixed in 4% paraformaldehyde followed by per-

meabilization with 0.1% Triton X-100. The cells were thenblocked with 2% fetal calf serum before incubation with p63antibody (4A4) for 4 hours at room temperature. Slides thenwere incubated with a fluorescently conjugated secondary(AlexaFluor 488) for 1 hour (room temperature). Vectashieldcontaining DAPI (40, 6 diamidino 2 phenylindole; VectorLabs)was used to counterstain and mount slides.

RNA extraction, reverse transcription, and real-timequantitative PCR

RNA was extracted using RNA STAT60 Total RNA extrac-tion Reagent (Tel-Test Inc.), reverse transcribed using theTranscriptor First Strand cDNA Synthesis kit (Roche), andRqPCR analysis was performed on the Opticon 2 system (Bio-Rad) using SYBR Green (Roche) according to the man-ufacturer's instructions. BRCA1 and p63 primers are as pre-viously described (33). Additional primers used are listed inthe Materials and Methods section of Supplementary Data.

Luciferase assaysDNp63 and TAp63 promoter luciferase reporter constructs

were a kind gift from Dr. Matthias Dobbelstein (University ofGottingen, Germany; ref 34). p63 C40-enhancer luciferaseconstruct was a kind gift from Dr. Caterina Missero (Napoli,Italy; ref 28). Site-directed mutagenesis was carried out usingKOD polymerase (Novagen) according to the manufacturer'sinstructions. The primers used are described elsewhere (28).Cells were cotransfected with the relevant luciferase con-structs and Renilla using GeneJuice (Novagen) according tothe manufacturer's instructions. After 24 hours, the cells werelysed with passive lysis buffer (Promega) and luciferase andRenilla activity assessed by luminescence using D-Luciferinand coelenterazine as substrates, respectively.

Tissue microarraysTissue microarrays (TMA) were generated from Formalin-

fixed paraffin-embedded primary tumor sections by ProfessorE. Kay at the Beaumont Hospital (Dublin, Ireland). Each tumorsample was represented by 4 independent cores. Immunohis-tochemistry was performed for a number of standard markergenes for ERa, PR, and HER2 with p63 staining performedusing the pan-p63 4A4 antibody.

Flow cytometryDNA content was evaluated following propidium iodide

staining as previously described (35). Phosphorylated serineHistone H2AX was detected by primary antibody (Millipore)staining followed by staining with a fluorescently taggedsecondary antibody (AlexaFluor). Staining was assessed usingthe Becton Dickinson FACSCalibur, and levels were analyzedusing WinMDI 2.9.

Chromatin immunoprecipitation assaysChromatin immunoprecipitation (ChIP) assays were per-

formed as previously described (33). PCR was performed onextracted DNA using Taq DNA polymerase (Roche) accordingto the manufacturer's instructions. Primers used for ChIP arelisted in the Materials and Methods section of the Supple-mentary Data.

Results

DNp63 isoform mRNA and protein expression arepositively regulated by BRCA1

DNp63a was identified as a BRCA1 transcriptional targetfollowing microarray comparison of BRCA1 mutant (HCC-EV)

Buckley et al.




IP-BRCA1(i)

A

B

C

D

(ii)

(i) (ii)

(i) (ii)

(i) (ii)

(iii)

IgG

Inpu

tBRCA1

p63

IgG2a

Inpu

tBRCA1

Ab1

BRCA1 D9

p63

IgG1

IgG2a

AP2γ

T47D

HCC-BR

MCF-7

ΔNp63α

ΔNp63s

i

Scr

AP-2γ s

i

Scr

AP-2γ s

i

Scr AP-2γ s

i

Scr AP-2γ s

i

Scr AP-2γ s

i

Scr AP-2γ s

i

Scr AP-2γ s

i

Scr

ΔNp63s

i

Scr ΔNp63s

i

Scr ΔNp63s

i

Scr ΔNp63s

i

Scr

ΔNp63γ

ΔNp63α

C40

C40

C40

C40

Distal C40

ΔNp63γ

BRCA1

BRCA1

BRCA1

BRCA1

IgG1

Lysa

te

BRCA1

IgG1

Lysa

te

AP-2α

BRCA1

AP-2γ

GAPDH

AP-2γ

p63 IgG2AInput

BRCA1 p63 IgG1 IgG2AAP-2γγInput

ΔNp63α

GAPDH

IP IP

293T

Figure 3. BRCA1 interacts with DNp63g and requires DNp63 and AP-2g for recruitment to the p63 enhancer region. A, (i) immunoblots showingcoimmunoprecipitation of BRCA1 with DNp63g . Nuclear extracts were immunoprecipitated (I) with either a BRCA1-specific (Ab-1) or an IgG control antibodyand immunoblotted using a pan-p63 antibody (4A4, top) or a BRCA1 antibody (D9, bottom). Lysates from 293T cells overexpressing either DNp63a or DNp63gwere run alongside pulldowns to determine identity of p63 isoforms; (ii) ChIP assay in HCC-BR cells of the C40 region or a region upstream (distal C40).One percent of total DNA was used as a loading control (input); (iii) C40 ChIP assay of MCF-7 cells with antibodies against BRCA1 (D9 and AB1), p63, AP-2g ,or isotyped matched IgG control antibodies, with input as described in (ii). B, (i) immunoblots of MCF-7 cells following treatment with either scrambled(Scr) or DNp63 (DNp63si) siRNA (72 hours) and probed for DNp63 and GAPDH antibody as loading control; (ii) C40 ChIP assay in MCF-7 cells pretreatedwith either scrambled (Scr) or DNp63 (p63si) siRNA (72 hours) followed by immunoprecipitation with BRCA1, p63, or isotyped matched IgG control antibodies,with input as described in A (ii). C, immunoblots showing coimmunoprecipitation of BRCA1 with (i) AP-2a or (ii) AP-2g ; whole cell extracts wereimmunoprecipitated with either a BRCA1-specific (Ab-1) or an IgG control antibody and immunoblotted using specific AP-2 antibodies (top) or a BRCA1antibody (D9, bottom); lysates show expression levels in 60 mg of total extract prior to immunoprecipitation. D, (i) immunoblots of MCF-7 cells followingtreatment with either scrambled (Scr) or AP-2g (AP-2gsi) siRNA (72 hours) and probed for AP-2g and GAPDH antibody as loading control; (ii) C40 ChIP assayof MCF-7 cells pretreated with either scrambled (Scr) or AP-2g siRNA (72 hours) followed by immunoprecipitation with BRCA1, p63, AP-2g , or isotypedmatched IgG control antibodies, with input as described in A (ii).





cells with wild-type BRCA1 expressing (HCC-BR) cells. Immu-noblotting confirmed p63 as a BRCA1 transcriptional target,with at least 2 p63 isoforms upregulated [Fig. 1A (i)]. Weobserved similar p63 reexpression following reconstitutionof BRCA1 into basal-like MDA-MB-468 cells [Fig. 1A (ii)].Conversely, siRNA knockdown of BRCA1 in T47D and MCF7cells resulted in reduced p63 expression [Fig. 1A (iii) and (iv),respectively]. To prove that this regulation was transcrip-tional, we performed RqPCR for BRCA1-reconstituted andsiRNA-depleted cell lines confirming our immunoblottingresults (Figs. 1B and 1C, respectively). RqPCR analysesindicated that DNp63 isoforms showed the most pro-nounced BRCA1-dependent regulation. P-cadherin andERa were used as exemplars of well-characterized basaland luminal marker genes, respectively (29, 33). Togetherthese data confirm our microarray findings that p63 iso-forms are downstream of BRCA1 and dependent on a fullyfunctional BRCA1 for expression.

BRCA1 regulates p63 through an internal enhancerregion

To define BRCA1-responsive element(s) regulating DNp63transcription, we obtained DNp63 promoter luciferase repor-ter constructs but failed to observe BRCA1-dependent regula-tion (data not shown). However, p63 had previously beenshown to be regulated through an enhancer region within thep63 gene in keratinocytes (28). This small 260-bp region (C40)located 160 kb and 42 kb downstream from the TA- and DN-transcriptional start sites, respectively, contains few notabletranscription factor consensus binding sites, except for a p53/p63 response element (p53RE) and 2 AP-2 binding sites. C40reporter activity in keratinocytes was shown to require thep53RE and AP-2 sites, and a p63 autoregulatory loop wasidentified (28). Using a minimal C40 enhancer-thymidinekinase promoter luciferase construct, we observed a dramaticdecrease in luciferase activity in MCF7 cells following BRCA1siRNA knockdown [Fig. 2A (i)]. Conversely, reconstitution ofBRCA1 into HCC1937 cells increased luciferase activity of thesame construct [Fig. 2A (ii)]. Following site-directed mutationof the p53RE and both AP-2 sites, we observed using MCF7cells that the p53RE and an overlapping AP-2B site wererequired for luciferase activity, while mutation of the AP-2Asite had no effect on BRCA1-dependent regulation [Fig. 2A(iii)]/ In siRNA-mediated knockdown experiments in MCF7cells, we observed significant decreases in luciferase activity ofC40 following knockdown of BRCA1 and DNp63 but not TAp63or p53 [Fig. 2B (i)]/ This coincided with a correspondingdownregulation of DNp63a protein and mRNA levels [Fig.2B (ii) and (iii), respectively]. To identify which p63 isoformwas responsible for this autoregulatory effect, we used DN-specific, a/b-, or g-specific siRNA in MCF7 cells. We observedthat DN-specific siRNA was as potent as BRCA1 siRNA inreducing reporter activity and that g-specific rather thana/b-specific siRNA was most effective, suggesting thatDNp63g is the p63 species most likely to be involved[Fig. 2C (i)]. However, all 3 p63 siRNAs had dramatic inhibitoryeffects on DNp63a protein levels and mRNA [Fig. 2C (ii) and(iii), respectively]. As AP-2g had been shown to co-activate p63

genes, we performed AP-2g siRNA knockdown that alsoresulted in a reduction in C40-luciferase activity, almostcomparable to DNp63 siRNA [Fig. 2D (i)], accompanied bydownregulation of p63 protein expression and mRNA [Fig. 2D(ii) and (iii), respectively]. Together these data show thatBRCA1 regulates DNp63 proteins by targeting a p53RE andan overlapping AP-2 site in the enhancer region of the p63gene, an autoregulatory process that appears to requireDNp63g .

BRCA1 interacts with DNp63g and requires DNp63 andAP-2g for recruitment to the p63 enhancer region

To prove that BRCA1 facilitated DNp63 autoregulation, weneeded to demonstrate the interaction of BRCA1 with DNp63proteins. Interestingly, in coimmunoprecipitation (co-IP)experiments, we found that it was not the predominantDNp63a isoform but the smaller DNp63g isoform thatinteracted with BRCA1 [Fig. 3A (i)]. ChIP assays of MCF7and HCC-BR cells showed that BRCA1, DNp63, and AP-2gproteins all localized on the p63 C40 enhancer and not on adistal promoter region [Fig. 3A (ii) and (iii)]. Because BRCA1regulates transcription as a coactivator/corepressor andcannot bind DNA in a sequence-specific manner, we wantedto demonstrate the requirement for DNp63 for BRCA1recruitment to C40. Following DNp63 siRNA knockdownin MCF7 cells [Fig. 3B (i)], we observed complete loss ofBRCA1 recruitment to this region [Fig. 3B (ii)]. Because AP-2g knockdown reduced C40-luciferase reporter activity(Fig. 2), we suspected that AP-2 proteins may also localizewith BRCA1 on this region. In fact, we show by co-IP thatboth AP-2a and AP-2g are novel BRCA1 interacting proteins[Fig. 3C (i) and (ii), respectively]. We could also demonstratethat knockdown of AP-2g [Fig. 3D (i)] resulted in reducedBRCA1 recruitment to the C40 region [without affectingDNp63 recruitment; Fig. 3D (ii)]. Together these data showthat BRCA1 interacts specifically with DNp63g and requires

Table 1. Correlation of p63 expression with arange of biological parameters following immu-nohistochemical staining of a primary breastcancer TMA

p63 positive p63 negative

Triple negative and BRCA1 statusER/PR/HER� 4/15 (26.7%) 11/15 (73.3%)BRCA1 mutant 1/20 (5%) 19/20 (95%)

Estrogen receptor-a and HER2 statusER�/HER2þ 6/26 (23%) 20/26 (76.9%)ERþ/HER2� 15/104 (14.4%) 89/104 (85.6%)ER/PR/HERþ 4/20 (20%) 16/20 (80%)

Tumor gradeGrade 1 14/118 (11.9%) 3/26 (11.5%)Grade 2 54/118 (45.8%) 9/26 (34.5%)Grade 3/4 51/118 (43.2%) 13/26 (50%)Undetermined 1/26 (3.8%)

Buckley et al.




Basal-like(i)A

B

C

D

(ii) (iii)

(i) (ii)

(i) (ii)

(iii)

Basal-like

BRCA1

Normalmyoepith

BRCA1mutant Luminal

CK5

#1

#2

Scr

EV BR EV BR

p63-FITC

DAPI

merge

ΔNp63si

p63

0

ZR-75T47

D

MCF-7

BT474

MDA36

1

MDA45

3

MDA46

8

MDA23

1

HC

C19

37Su

m 1

49M

DA-

MB-

436

MC

F-7

T47D

MC

F-10

A

HM

E-1

Sum14

9

ZR-75T47

D

MCF-7

BT474

MDA36

1

MDA45

3

MDA46

8

MDA23

1

HCC1937

Sum14

9

ZR-75T47

D

MCF-7

BT474

MDA36

1

MDA45

3

MDA46

8

MDA23

1

HCC1937

Sum14

9

1

2

3

mR

NA

exp

ress

ion

norm

aliz

ed to

GA

PD

H

0 0.00.51.01.52.02.53.03.54.04.55.05.5

123456789

mR

NA

exp

ress

ion

norm

aliz

ed to

GA

PD

H

mR

NA

exp

ress

ion

norm

aliz

ed to

GA

PD

H

CD44 ΔNp63

Luminal

Luminal Basal-likeLuminal Basal-likeLuminal

293T

ΔNp63αΔNp63β

β-tubulin

ΔNp63α

β-tubulin

ΔNp63α

ΔN-αTA

-αTA

-γΔN-γ

ΔNp63β

β-tubulin

ΔNp63αTA p63α

TA p63γΔNp63γ

β-tubulin

MDA-M

B-157

MDA-M

B-453

MDA-M

B-231

MDA-M

B-468

Sum14

9

MCF10

A

MCF-7

IBEP-1

ZR-75/

1

KPL1T47

DBT47

4

HCC1954

HCC1937

Hs578

T

Figure 4. p63 expression does not correlate with basal-like breast cancer cell lines or primary basal-like breast cancers. A, immunoblots showing p63expression in (i) basal-like, (ii) luminal breast cancer cell lines, and (iii) 293 T cells transfected with different p63 constructs, using the pan-p63 antibody4A4; b-tubulin was used as a loading control. B, (i) to (iii) RqPCR analyses showing BRCA1, CD44, and DNp63 mRNA levels, respectively, in the samecell line panels. GAPDH mRNA was used for normalization (n ¼ 3). C, (i) immunoblots showing p63 expression in normal myoepithelial (myoepith), BRCA1mutant, and luminal cell lines immunoblotted with 4A4; b-tubulin was used as a loading control; (ii) immunohistochemical staining of sequential tumor sectionstaken from 2 individual tumors with antibodies specific for cytokeratin 5 (CK5) and p63. D, P63 immunofluorescence staining of HCC-EV (EV) and HCC-BR(BR) cells following treatment with scrambled (Scr) or DNp63-specific (DNp63si) siRNA. DAPI staining was used to demonstrate nuclei and a mergedimage generated to show colocalization of p63 and DAPI staining.





BRCA1

(i) (ii) (iii)

(i) (ii) (iii)

(i) (ii)

(iii)

(iv)

(iii) (iv)

Scr BRsi

GAPDH

ΔNp63α

ΔNp63α

BRCA1

BRCA1

17.5

15.0

12.5

10.0

%M

FU

7.55.0

2.50.0

SCRsh

BRshp6

3sh

SCRsh

(ii)

EV Scr

BR Scr

EV DNsi

BR DNsi

BRsh

p63s

h

GAPDH

GAPDH

0 1 2 3Days

4 5

ΔNp63α

ΔNp63α

ΔNp63γ

ΔNp63α

EV ΔNp63γ

ΔNp63αEV

ΔNp63γ

P-γH2AX

ΔNp63s

i

ScrBRsi

BRsip6

3si

ΔNp63s

i

ScrBRsip63si

Scr BRsiΔNp6

3si

Scr BRsi ΔNp63si

β-tubulin

A

B

C

(i)

MCF10A HMEI

D17.5

15.012.5

10.07.5

5.02.5

0.0

45

110100

90807060

Ab5

40nm

(%

EV

)

5040302010

0

40353025251510

05

% a

ldef

luor

pos

itive

cells

ald

eflu

or p

ositi

ve c

ells 1.1

1.00.90.80.70.60.50.40.30.20.1

1.2

0.1

1.21.11.00.90.80.70.60.50.40.30.20.10.0

mR

NA

exp

ress

ion

norm

alis

ed to

β-t

ubul

in

(v)9876543210

norm

alis

ed to

β-tu

bulin

(vi)

6

5

4

3

2

1

0

mR

NA

exp

ress

ion

norm

alis

ed to

β-t

ubul

in

CXCL1

CTPS1

p-ca

d

BRCA1p6

3SM

A

MUC1

p-ca

dhar

in

ITGA6

ITGB1

SMA

norm

alis

ed to

β-tu

bulin

BRCA1p6

3

GATA3

Muc

1

ERalpha

SCRshBRsh

p63sh

SCR

BRCA1sip63si

pSuper

BRshp63sh

pSuper

BRshp63sh

SCRBRCA1si

p63si

17.5

0

15.012.510.07.55.02.50.0

% c

ells

pos

itive

for

phos

pho-

γH2A

X

225002000017500

15000

cell

coun

ts

1250010000

750050002500

302520

% s

enes

cenc

eve

rsus

scr

con

trol

15105

0

35

Figure 5. DNp63 proteins are essential downstream effectors of BRCA1 tumor suppressor function. A, (i) immunoblots of HMECs following transfectionwith scrambled (Scr), BRCA1 (BRsi), or DNp63 (DNp63si) siRNA; blots were probed with antibodies against BRCA1 and p63 with GAPDH expressionused as loading control; (ii) senescence associated b-galactosidase (SAb-gal) staining of HMECs 7 days following siRNA treatments; representative bright fieldimages are shown; (iii) SAb-gal staining quantified and calculated as % increase over scrambled control. B, (i) immunoblots of immortalized HME-1cells following transfection with scrambled (Scr), BRCA1 (BRsi), or DNp63 (DNp63si) siRNA; blots were probed with antibodies against BRCA1, p63, orphospho-H2AX (gH2AX), with GAPDH expression used as a loading control; (ii) gH2AX staining was analyzed by flow cytometry and shown aspercentage of cells showing positive staining; (iii) 5-day growth curve of HME-1 cells following siRNA treatments outlined in B (i). C, (i) immunoblotsof MDA-MB-468 cells transfected with empty vector (EV), DNp63a, or DNp63g expression constructs (24 hours) and probed with a pan-p63 antibody(4A4) and GAPDH antibody as loading control; (ii) clonogenic assays of MDA-MB-468 cells transfected as described in (i) and stained with crystal violet

Buckley et al.




the expression of both DNp63g and AP-2g for its recruitmentto the p63 C40 enhancer region.

p63 expression does not correlate with basal-like breastcancer cell lines or primary basal-like breast cancersResults from Fig. 1 suggest that p63 is not a marker of basal-

like or BRCA1 mutant breast tumors (22). Indeed, comparingpanels of basal-like and luminal breast cancer cell lines weobserved absolutely no correlation between p63 expressionand basal/luminal status [Figs. 4A (i) and (ii), respectively].Only myoepithelial, nontumorigenic MCF10A cells werestrongly positive, with DNp63a the most abundant isoform[confirmed by running alongside lysates taken from 293T cellstransfected with specific p63 isoforms; Fig. 4A (iii)]. UsingRqPCR, we found the highest BRCA1 mRNA levels in luminalcells, basal-like cell lines expressing highest CD44 levels,whereas DNp63 mRNA levels were consistently lower inbasal-like cells [Figs. 4B (i, ii, and iii)]. Immunoblottingshowed that normal myoepithelial/basal cells had the highestDNp63 expression, followed by luminal cells, whereas BRCA1mutant cells showed no detectable expression [Fig. 4C (i)].We then investigated the correlation between p63 expressionand basal-like/triple-negative (receptor) status in a TMA of144 primary breast tumors. In agreement with previousreports, we observed p63 expression in normal myoepitheliumbut not in adjacent carcinoma from the same sections (22, 36).As Table 1 shows, p63 was expressed in 26 of 144 cases overall(18%) and in 4 of 15 triple-negative cases (26.7%). Only one of 20(5%) BRCA1 mutant tumors displayed weak to moderatestaining for p63. In fact, there appeared to be no statisticallysignificant correlation of p63 staining with any of the para-meters used including ERa or HER2 status or tumor grade(Table 1). p63 staining within tumors was generally of lowintensity, focal in appearance, and usually found in only afraction of cells within p63-positive tumors. Sampling issuesmay have contributed to the general low levels of p63 staining.However, sequential sections of basal-like tumors stained foreither CK-5 or p63 showed a complete lack of costaining[Fig. 4C (ii)]. Immunofluorescence studies in HCC-EV andHCC-BR cells using the same pan-p63 antibody (4A4) con-firmed that DNp63 proteins were the predominant p63 iso-forms since DNp63-specific siRNA completely abrogated p63staining inHCC-BRcells (Fig. 4D). BRCA1mutantHCC-EVcellswere completely devoid of p63 staining. Taken together, thesedata show that p63 expression is a reliable marker of normalmyoepithelial/basal breast cells, but not of basal-like or BRCA1mutant breast tumors.

p63 expression does not correlate with other knownmarkers of basal-like breast cancer

We also wanted to investigate the correlation between p63and the basal-like/triple negative phenotype using geneexpression profiles of breast cancers. We mined publiclyavailable microarray data sets for expression of p63, basal-like markers (CD44, p-cadherin, CK-5, FOXC1, and c-kit),luminal markers (Gata-3, cytokeratin 8), and triple-negativestatus using the online database ONCOMINE (www.onco-mine.com). While significant correlations were observedbetween CD44, p-cadherin, CK-5, and FOXC1 with triple-negative status and basal-like tumors, no correlation wasapparent for p63 (Supplementary Table S2), in agreementwith our TMA and cell line. More limited information wasavailable on the relationship between p63 and the breastcancer subtypes as defined by the intrinsic gene signaturesidentified by Sorlie and colleagues (27). In studies where p63mRNA expression data were available, there was no significantdifference in p63 expression between subtypes (32, 37, 38). Asimilar number of studies had data available on BRCA1expression and/or mutation status (38–40). Increased p63expression was not observed in BRCA1 mutant tumors incontrast to previously published data (22). In fact, p63 mRNAexpression appeared to be reduced in BRCA1 mutant tumors(40) in keeping with our findings that p63 is not a marker ofbasal-like or BRCA1 mutant breast tumors.

DNp63 proteins are essential downstream effectors ofBRCA1 tumor suppressor function

We next wanted to assess the consequences of p63 lossfollowing BRCA1 mutation, in particular growth control andDNA damage responses. We used untransformed HMECs thatare thought to be derived of a mixture of cell lineagesincluding luminal, myoepithelial, and probably basal pluripo-tential cells (41). We were particularly interested in the factthat HMECs retain the ability to senesce, including followinggenotoxic stress. We found that knockdown of either BRCA1or DNp63 [Fig. 5A (i)], resulted in increases in senescence-associated b-galactosidase staining of HMECs [Fig. 5A (ii)],quantified at approximately 30% over scrambled control forp63 knockdown [Fig. 5A (iii)]. However, in cells that have by-passed senescence such as the HME-1, we observed increasedphosphorylation of the DNA damage marker gH2AX followingloss of BRCA1 and DNp63 [Fig. 5B (i)], which we quantified byflow cytometric analysis [Fig. 5B (ii)]. In contrast to HMECs,HME-1 cells do not senesce in response to oncogenic stimuli,although they still retain cell-cycle checkpoint controls. Inter-

after 5 days; crystal violet dye was reabsorbed, absorbance values quantified (OD540 nm) and normalized to EV control (as % EV); (iii) immunoblots ofMCF10-A cells stably transfected with shRNA targeting scrambled control (SCRsh), BRCA1 (BRsh), or all p63 isoforms (p63sh); blots were probedfor BRCA1 and p63 (DNp63a indicated), with expression of b-tubulin used as a loading control; (iv) quantification of 3-dimensional mammospherecultures of MCF10-A cells following stable transfection of the shRNA constructs outlined in (iii); mammospheres were counted and expressed aspercentage mammary forming units (% MFU). D, (i) ALDH1 activity assay of MCF10-A and HME-1 cells following the treatments outlined in C (iii) and C(iv) with % Aldefluor-positive cells calculated and shown; (ii) ALDH1 activities of HCC-EV (EV) and HCC-BR (BR) following siRNA treatment withscrambled control (Scr) or DNp63-specific (DNsi) siRNA; (iii) RqPCR analyses of MCF7 cells following treatment with BRCA1 (BRCAsi) or DNp63 siRNA(p63si) showing the mRNA levels of luminal marker genes or (iv) basal-like marker genes with b-tubulin mRNA used as loading control; (v) RqPCRanalysis of MCF10A shRNA cell lines showing mRNA levels of BRCA1, p63, and markers of basal differentiation or (vi) basal-like breast cancer withb-tubulin mRNA used as loading control.





estingly, we observed enhanced proliferation following BRCA1or DNp63 knockdown in HME-1 cells [Fig. 5B (iii)]. Conversely,re-expression of either DNp63a or DNp63g [Fig. 5C (i)] inMDA-MB-468 basal-like breast cancer cells resulted in amarked decrease in cell proliferation [Fig. 5C (ii)] of up to70% compared with control cells [Fig. 5C (iii)]. We alsoinvestigated potential roles of both BRCA1 and DNp63 asregulators of breast stem/progenitor cells, since BRCA1 hasbeen implicated in breast stem cell proliferation and differ-entiation (42) and p63 in epithelial and keratinocyte stem cellregulation (43, 44). Stable shRNA knockdown of BRCA1 andp63 was performed in nontumorigenic MCF10-A cells [Fig. 5C(iii)] and resulted in an increase in mammosphere numbers ofover 4-fold [Fig. 5C (iv)]. Although mammosphere growth isnot conclusive proof of these cells are behaving as "stem cells"per se, it is known to enrich for subpopulations which possessstem or progenitor characteristics. BRCA1 and p63 shRNAknockdowns also resulted in increased activity of the stem cellmarker enzyme ALDH1 (45) in bothMCF10-A and HME-1 cells[Fig. 5D (i)]. However, ALDH1 activity was reduced followingreconstitution of wild-type BRCA1 into mutant HCC1937(HCC-BR) cells, an effect which could be reversed followingtreatment with DNp63 siRNA [Fig. 5D (ii)]. Further evidencethat both genes promote normal breast differentiation isshown by the fact that following BRCA1 and DNp63 siRNAknockdown in MCF7 cells we observe a pronounced loss ofluminal marker expression coincident with an increase in theexpression of well-characterized basal-like markers [Fig. 5E (i)and (ii), respectively]. Similar results are also seen in theMCF10-A shRNA cell lines where loss of BRCA1 or DNp63results in loss of markers of normal basal differentiation with again in basal-like markers [Fig. 5E (iii) and (iv), respectively].Together these data show that BRCA1 and DNp63 cooperateto regulate growth control and maintain genomic stability inbreast cells and possibly to suppress the expansion of basal-like progenitor cells.

Discussion

In this study we have for the first time identified DNp63proteins as major transcriptional targets of BRCA1. P63 hadbeen proposed to be a marker of basal-like breast cancer andpossibly a surrogate marker of BRCA1 dysfunction. This hasnever been conclusively proven and from numerous micro-array studies p63 has never been associated with basal-likebreast cancer (9, 26, 27). We show by using multiple in vitromodels that DNp63 proteins are positively regulated byBRCA1 (Fig. 1) and by using primary breast tumors thatp63 is not a marker of basal-like breast cancers (Fig. 4). Wedemonstrate that p63 regulation in breast tissue is similar tothat observed in keratinocytes where p63 autoregulates itselfthrough binding to an enhancer region in association withAP-2g (Fig. 2). Using co-IP, we show that DNp63g , AP-2a, andAP-2g are novel BRCA1 interacting proteins (Fig. 3). ChIPassays show that BRCA1, DNp63, and AP-2g all localize onthe p63 enhancer and that BRCA1 requires DNp63 and AP-2gfor recruitment to this region. Finally we show that theBRCA1 transcriptional upregulation of DNp63 proteins has

important consequences for growth control in normal basalbreast epithelia including the protection from endogenousDNA damage and the suppression of basal-like progenitorproliferation (Fig. 5).

The biology of p63 in different tissue types is complex butappears to be critical for the development of stratified epithelialtissues. Debate exists over whether p63 acts as an oncogene or atumor suppressor. It is known tobeamplifiedandoverexpressedin head and neck cancer (46) but its loss is also associated withoncogenesis in the same disease (20). In breast cancers, p63 hasrecently been shown to play a tumor suppressor role, withabrogation of its function through interaction with mutantp53-SMAD complexes leading to enhanced metastasis (47).Our data suggest that the transcriptional control of DNp63expression by BRCA1 is important for the maintenance ofgenomic stability and for the normal differentiation of mam-mary tissue. In agreement with this, p63 and p73 have recentlybeen shown to upregulate a number of key DNA damage repairproteins (BRCA2,RAD51, andmre11) and to suppressmammarytumor formation in mice (48). In addition, DN isoforms of bothp63 and p73 were much more important for tumorigenesissuppression than TAp63/p73 isoforms (48). This may be afeature of the enhanced DNA damage we observe in our breastcell lines following DNp63 knockdown. We observe that loss ofeither BRCA1 or DNp63 in nonimmortalized HMECs results inthe induction of senescence (Fig. 5A), which is probably theprimary default growth control mechanism. Increased DNAdamage could select formutations of key senescence-associatedgenes (including p53 and p16Ink4a), allowing affected cells agrowth advantage and ultimately malignancy. BRCA1 mutantand basal-like breast cancers exhibit a high rate of p53mutationcompared with luminal tumors, suggesting abrogation of p53function is a prerequisite for tumorigenesis in this geneticbackground. The enhanced genomic instability observed follow-ing BRCA1 or DNp63 knockdown may induce a p53-dependentsenescence checkpoint which is bypassed in basal-like breastcancers through p53 mutation.

In addition to maintenance of genomic stability, p63 andBRCA1 have also been implicated as stemcell regulators in skinand breast tissue, respectively (49). BRCA1 shRNA knockdownin primary breast cells has been shown to result in increasedALDH1 activity and loss of differentiation markers (42). Inter-estingly, BRCA1 mutation carriers have been shown to harborexpanded luminal progenitors whichmay be the cell typemostsusceptible to BRCA1 dysfunction (and possibly loss of DNp63expression) rather than basal stem cells (12, 13). In this study,we observe enhanced proliferation and self-renewal capacity ofmammosphere cultures and increased ALDH1 activity follow-ing BRCA1 and DNp63 knockdowns, which although notdefinitive of a stem cell population per se indicates that bothgenes may be important for breast stem/progenitor cell reg-ulation. ALDH1 activity has been recently used with success toisolate stem and progenitor cells from mammary tissues butmay not be the most reliable method for the isolation oftumorigenic populations and is probably best used in combi-nation with defined cell surface markers [for review see (50)].We believe that the mutation or downregulation of BRCA1accompanied by the loss of DNp63 protein expression may

Buckley et al.




explainmany of the features of basal-like breast cancer. Indeed,knockdown of either gene results in loss of normal differentia-tion markers accompanied by an increase in markers asso-ciated with basal-like breast cancers (Fig. 5D).In conclusion, we show that DNp63 proteins are not mar-

kers of basal-like breast cancer. Conversely, they may actuallybe crucial downstream effectors of BRCA1 function. Wepredict that the identification of transcriptional targets down-stream of BRCA1-DNp63 will prove invaluable in the devel-opment of novel therapeutics for the treatment of basal-likebreast cancers.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Dr. Caterina Missero (Telethon Institute of Geneticsand Medicine, Naples, Italy) for her kind gift of the C40 enhancer-TK promoterluciferase reporter construct and Dr. Martha Stampfer, University of Californiafor her kind gift of HMECs.

Grant Support

This work was supported by Research and Development Northern Ireland(RRG/3264/05) and the Breast Cancer Campaign (2008NOVPR20).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 26, 2010; revised December 21, 2010; accepted December 24,2010; published OnlineFirst March 1, 2011.

References1. Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K,

Tavtigian S, et al. A strong candidate for the breast and ovariancancer susceptibility gene BRCA1. Science 1994;266:66–71.

2. King MC, Marks JH, Mandell JB. Breast and ovarian cancer risksdue to inherited mutations in BRCA1 and BRCA2. Science 2003;302:643–6.

3. Anderson SF, Schlegel BP, Nakajima T, Wolpin ES, Parvin JD. BRCA1protein is linked to the RNA polymerase II holoenzyme complex viaRNA helicase A. Nat Genet 1998;19:254–6.

4. Bochar DA, Wang L, Beniya H, Kinev A, Xue Y, LaneWS, et al. BRCA1is associated with a human SWI/SNF-related complex: linking chro-matin remodeling to breast cancer. Cell 2000;102:257–65.

5. Zhang H, Somasundaram K, Peng Y, Tian H, Bi D, Weber BL, et al.BRCA1 physically associates with p53 and stimulates its transcrip-tional activity. Oncogene 1998;16:1713–21.

6. Wang Q, Zhang H, Kajino K, Greene MI. BRCA1 binds c-Myc andinhibits its transcriptional and transforming activity in cells. Oncogene1998;17:1939–48.

7. Andrews HN, Mullan PB, McWilliams S, Sebelova S, Quinn JE,Gilmore PM, et al. BRCA1 regulates the interferon gamma-mediatedapoptotic response. J Biol Chem 2002;277:26225–32.

8. Mullan PB, Hosey AM, Buckley NE, Quinn JE, Kennedy RD, JohnstonPG, et al. The 2,5 oligoadenylate synthetase/RNaseL pathway is anovel effector of BRCA1- and interferon-gamma-mediated apoptosis.Oncogene 2005;24:5492-501.

9. Perou CM, Jeffrey SS, van de Rijn M, Rees CA, Eisen MB, Ross DT,et al. Distinctive gene expression patterns in human mammaryepithelial cells and breast cancers. Proc Natl Acad Sci U S A1999;96:9212–7.

10. Yang Q, Sakurai T, Mori I, Yoshimura G, Nakamura M, Nakamura Y,et al. Prognostic significance of BRCA1 expression in Japanesesporadic breast carcinomas. Cancer 2001;92:54–60.

11. Turner N, Tutt A, Ashworth A. Hallmarks of ‘BRCAness’ in sporadiccancers. Nat Rev Cancer 2004;4:814–9.

12. Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, et al. Aberrantluminal progenitors as the candidate target population for basaltumor development in BRCA1 mutation carriers. Nat Med 2009;15:907–13.

13. Molyneux G, Geyer FC, Magnay FA,McCarthy A, Kendrick H, NatrajanR, et al. BRCA1 basal-like breast cancers originate from luminalepithelial progenitors and not from basal stem cells. Cell Stem Cell7:403–17.

14. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, et al.p63, a p53 homolog at 3q27–29, encodes multiple products withtransactivating, death-inducing, and dominant-negative activities.Mol Cell 1998;2:305–16.

15. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, et al.p63 is essential for regenerative proliferation in limb, craniofacial andepithelial development. Nature 1999;398:714–8.

16. Murray-Zmijewski F, Lane DP, Bourdon JC. p53/p63/p73 isoforms: anorchestra of isoforms to harmonise cell differentiation and response tostress. Cell Death Differ 2006;13:962–72.

17. McKeon F. p63 and the epithelial stem cell: more than status quo?Genes Dev 2004;18:465–9.

18. Romano RA, Birkaya B, Sinha S. A functional enhancer of keratin14 isa direct transcriptional target of deltaNp63. J Invest Dermatol 2007;127:1175–86.

19. Urist MJ, Di Como CJ, Lu ML, Charytonowicz E, Verbel D, Crum CP,et al. Loss of p63 expression is associated with tumor progression inbladder cancer. Am J Pathol 2002;161:1199–1206.

20. Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N.Snail-induced down-regulation of DeltaNp63alpha acquires invasivephenotype of human squamous cell carcinoma. Cancer Res2007;67:9207–13.

21. Sniezek JC, Matheny KE, Westfall MD, Pietenpol JA. Dominantnegative p63 isoform expression in head and neck squamous cellcarcinoma. Laryngoscope 2004;114:2063–72.

22. Ribeiro-Silva A, Ramalho LN, Garcia SB, Brandao DF, Chahud F,Zucoloto S. p63 correlates with both BRCA1 and cytokeratin 5 ininvasive breast carcinomas: further evidence for the pathogenesis ofthe basal phenotype of breast cancer. Histopathology 2005;47:458–66.

23. LeongCO, VidnovicN, DeYoungMP, Sgroi D, Ellisen LW. The p63/p73networkmediates chemosensitivity to cisplatin in a biologically definedsubset of primary breast cancers. J Clin Invest 2007;117:1370–80.

24. Laakso M, Loman N, Borg A, Isola J. Cytokeratin 5/14-positive breastcancer: true basal phenotype confined to BRCA1 tumors. Mod Pathol2005;18:1321–8.

25. Burga LN, Tung NM, Troyan SL, Bostina M, Konstantinopoulos PA,Fountzilas H, et al. Altered proliferation and differentiation propertiesof primary mammary epithelial cells from BRCA1 mutation carriers.Cancer Res 2009;69:1273–8.

26. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, et al.Repeated observation of breast tumor subtypes in independent geneexpression data sets. Proc Natl Acad Sci U S A 2003;100:8418–23.

27. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al.Gene expression patterns of breast carcinomas distinguish tumorsubclasses with clinical implications. Proc Natl Acad Sci U S A2001;98:10869–74.

28. Antonini D, Rossi B, Han R, Minichiello A, Di Palma T, CorradoM, et al.An autoregulatory loop directs the tissue-specific expression of p63through a long-range evolutionarily conserved enhancer. Mol Cell Biol2006;26:3308–18.

29. Gorski JJ, James CR, Quinn JE, Stewart GE, Staunton KC, BuckleyNE, et al. BRCA1 transcriptionally regulates genes associated with thebasal-like phenotype in breast cancer. Breast Cancer Res Treat 2009;122:721–31.

30. Fabbro M, Savage K, Hobson K, Deans AJ, Powell SN, McArthur GA,et al. BRCA1-BARD1 complexes are required for p53Ser-15 phos-





phorylation and a G1/S arrest following ionizing radiation-inducedDNA damage. J Biol Chem 2004;279:31251–8.

31. Garbe JC, Bhattacharya S, Merchant B, Bassett E, Swisshelm K,Feiler HS, et al. Molecular distinctions between stasis and telomereattrition senescence barriers shown by long-term culture of normalhuman mammary epithelial cells. Cancer Res 2009;69:7557–68.

32. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. Acollection of breast cancer cell lines for the study of functionallydistinct cancer subtypes. Cancer Cell 2006;10:515–27.

33. Hosey AM, Gorski JJ, Murray MM, Quinn JE, Chung WY, Stewart GE,et al. Molecular basis for estrogen receptor alpha deficiency inBRCA1-linked breast cancer. J Natl Cancer Inst 2007;99:1683–94.

34. Waltermann A, Kartasheva NN, Dobbelstein M. Differential regulationof p63 and p73 expression. Oncogene 2003;22:5686–93.

35. Mullan PB, Quinn JE, Gilmore PM, McWilliams S, Andrews H,Gervin C, et al. BRCA1 and GADD45 mediated G2/M cell cyclearrest in response to antimicrotubule agents. Oncogene2001;20:6123–31.

36. Matos I, Dufloth R, Alvarenga M, Zeferino LC, Schmitt F. p63,cytokeratin 5, and P-cadherin: three molecular markers to distinguishbasal phenotype in breast carcinomas. Virchows Arch 2005;447:688–94.

37. Julka PK, Chacko RT, Nag S, Parshad R, Nair A, Oh DS, et al. A phaseII study of sequential neoadjuvant gemcitabine plus doxorubicinfollowed by gemcitabine plus cisplatin in patients with operable breastcancer: prediction of response using molecular profiling. Br J Cancer2008;98:1327–35.

38. Richardson AL,Wang ZC, DeNicolo A, Lu X, BrownM,Miron A, et al. Xchromosomal abnormalities in basal-like human breast cancer. Can-cer Cell 2006;9:121–132.

39. Pawitan Y, Bjohle J, Amler L, Borg AL, Egyhazi S, Hall P, et al. Geneexpression profiling spares early breast cancer patients from adjuvanttherapy: derived and validated in two population-based cohorts.Breast Cancer Res 2005;7:R953–64.

40. van't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, et al.Gene expression profiling predicts clinical outcome of breast cancer.Nature 2002;415:530–6.

41. Stampfer MR, Yaswen P. Culture systems for study of human mam-mary epithelial cell proliferation, differentiation and transformation.Cancer Surv 1993;18:7–34.

42. Liu S, Ginestier C, Charafe-Jauffret E, Foco H, Kleer CG, Merajver SD,et al. BRCA1 regulates human mammary stem/progenitor cell fate.Proc Natl Acad Sci U S A 2008;105:1680–5.

43. Senoo M, Pinto F, Crum CP, McKeon F. p63 Is essential for theproliferative potential of stem cells in stratified epithelia. Cell2007;129:523–36.

44. Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bon-danza S, et al. p63 identifies keratinocyte stem cells. Proc Natl AcadSci U S A 2001;98:3156–61.

45. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, BrownM, et al. ALDH1 is a marker of normal and malignant human mammarystem cells and a predictor of poor clinical outcome. Cell Stem Cell2007;1:555–67.

46. Rocco JW, Leong CO, Kuperwasser N, DeYoung MP, Ellisen LW. p63mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 2006;9:45–56.

47. Adorno M, Cordenonsi M, Montagner M, Dupont S, Wong C, Hann B,et al. A Mutant-p53/Smad complex opposes p63 to empowerTGFbeta-induced metastasis. Cell 2009;137:87–98.

48. Helton ES, Zhu J, Chen X. The unique NH2-terminally deleted (DeltaN)residues, the PXXP motif, and the PPXY motif are required for thetranscriptional activity of the DeltaN variant of p63. J Biol Chem2006;281:2533–42.

49. Koster MI, Kim S, Mills AA, DeMayo FJ, Roop DR. p63 is themolecularswitch for initiation of an epithelial stratification program. Genes Dev2004;18:126–131.

50. Charafe-Jauffret E, Ginestier C, BirnbaumD. Breast cancer stem cells:tools and models to rely on. BMC Cancer 2009;9:202.

Buckley et al.




2011;71:1933-1944. Cancer Res Niamh E. Buckley, Susan J. Conlon, Karin Jirstrom, et al. Prevention of Basal-Like Breast Cancer

Np63 Proteins Are Key Allies of BRCA1 in the∆The

Updated version

http://cancerres.aacrjournals.org/content/71/5/1933

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2011/02/28/71.5.1933.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/71/5/1933.full#ref-list-1

This article cites 49 articles, 16 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/71/5/1933.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/71/5/1933To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/content/suppl/2011/02/28/71.5.1933.DC1

http://cancerres.aacrjournals.org/content/71/5/1933.full#ref-list-1

http://cancerres.aacrjournals.org/content/71/5/1933.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



The DNp63 Proteins Are Key Allies of BRCA1 in the...

Documents

Transcript of The DNp63 Proteins Are Key Allies of BRCA1 in the...