p53-Dependent BRCA1 Nuclear Export Controls Cellular ... · Therapeutics, Targets, and Chemical...

Therapeutics, Targets, and Chemical Biology

p53-Dependent BRCA1 Nuclear Export Controls CellularSusceptibility to DNA Damage

Juhong Jiang1, Eddy S. Yang1,2, Guochun Jiang1, Somaira Nowsheen1,2, Hong Wang1, Tong Wang1,Yihan Wang3, Dean Billheimer4, A. Bapsi Chakravarthy1, Melissa Brown6, Bruce Haffty5, and Fen Xia1

AbstractSubcellular localization regulates BRCA1 function, and BRCA1 is exported to the cytoplasm following DNA

damage in a p53-dependent manner. Because more than 50% of solid tumors harbor p53 mutations, it is possiblethat genetically wild-type (wt) BRCA1 is functionally abnormal through compromised nuclear-cytoplasmicshuttling in sporadic breast cancer patients with dysfunctional p53. In this study, we have investigated themechanisms of p53-dependent BRCA1 subcellular distribution and DNA damage-induced nuclear export, as wellas the impact on the resulting cytotoxic response to therapy in human breast cancer. We first show that p53mediates BRCA1 nuclear export via protein–protein binding, rather than by modulation of its transcription.Furthermore, it is the C-terminal (BRCT) region of BRCA1 that is critical for its interaction with p53, and p53maypromote BRCA1 nuclear export by interrupting the association of BRCA1 with BARD1. In sporadic breast cancerspecimens, dysfunctional p53 strongly correlates with nuclear retention of sequence-verified wt BRCA1. Thisp53-dependent BRCA1 shuttling determines cellular susceptibility to DNA damage as augmentation of cytosolicBRCA1 significantly enhances cancer cell susceptibility to ionizing radiation. Taken together, our data suggestthat p53 dysfunction compromises nuclear export of wt BRCA1 as a mechanism to increase cellular resistance toDNA damage in sporadic breast cancer. We propose that targeting nuclear BRCA1 to the cytoplasm may offer aunique strategy to sensitize p53-deficient sporadic breast cancers to DNA damage–based therapy. Cancer Res;71(16); 5546–57. �2011 AACR.

Introduction

BRCA1 serves critical roles in multiple cellular processes,such as chromatin remodeling, DNA replication, DNA repair,transcriptional regulation, cell-cycle checkpoint, and apopto-sis (1, 2). These BRCA1 functions are controlled by multiplemechanisms, including phosphorylation, protein–proteininteractions, and subcellular localization.

BRCA1 is a nucleocytoplasmic shuttling protein (3). NuclearBRCA1 functions inDNArepair (4, 5) and cell-cycle checkpoints(1), whereas cytoplasmic BRCA1 regulates centrosome func-

tion (2) and p53 independent apoptosis (6). BARD1 has beenshown to promote BRCA1 nuclear import and inhibit BRCA1nuclear export (7). We have reported that p53 regulates BRCA1shuttling (8). Specifically, p53 dysfunction leads to increasednuclear retention of BRCA1 and diminished DNA damage-induced BRCA1 nuclear export (8). Furthermore, DNA damagerepair depends on nuclear BRCA1, whereas DNA damage-induced cytotoxicity depends on BRCA1 cytosolic transloca-tion (6, 9). Thus, p53 statusmay play a critical role in regulatingBRCA1 shuttling and cellular susceptibility to DNA damage.

p53 is the most commonly mutated tumor suppressor geneand is dysfunctional in greater than 50% of solid malignancies(10–12). Dysfunctional p53 correlates with poor prognosis inbreast and many other types of cancer (12–14). Given thatBRCA1 subcellular localization controls its function in DNArepair and apoptosis (6, 9), and p53 regulates BRCA1 nuclearexport (8), it is a possibility that genetically wild-type (wt)BRCA1 is rendered functionally abnormal through compro-mised nuclear-cytoplasmic shuttling in sporadic breast cancerwith dysfunctional p53. Specifically, this altered shuttling mayaffect tumor response to DNA damage-based therapies (6, 9).

In this study, we sought to determine the mechanism ofp53-dependent BRCA1 subcellular distribution and DNAdamage-induced nuclear export and the impact on cytotoxicresponse to therapy in breast cancer. Interestingly, our find-ings suggest that p53-dependent BRCA1 nuclear export isindependent of its transcriptional activity, but instead

Authors' Affiliations: 1Departments of Radiation Oncology and CancerBiology, Vanderbilt University School of Medicine, Nashville, Tennessee;2Department of Radiation Oncology, University of Alabama-BirminghamSchool ofMedicine, Birmingham, Alabama; 3Nephropathology Associates,Little Rock, Arkansas; 4Huntsman Cancer Institute, Salt Lake City, Utah;5UMDNJ-RWJMS and Cancer Institute of New Jersey, New Brunswick,NewJersey; and 6School ofMolecular andMicrobial Sciences,University ofQueensland, St. Lucia, Queensland, Australia

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

J. Jiang and E.S. Yang contributed equally to this work.

Corresponding Author: Fen Xia, Department of Radiation Oncology,Vanderbilt University Medical Center, Nashville, TN 37232-5671. Phone:615-322-2555; Fax: 615-343-6589; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-3423

�2011 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst July 8, 2011; DOI: 10.1158/0008-5472.CAN-10-3423

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depends on the binding of the 2 proteins. Furthermore, we findthat dysfunctional p53 strongly correlates with nuclear reten-tion of sequence-verified wt BRCA1 in sporadic breast cancerspecimens. Furthermore, interrupting BRCA1–BARD1 bindingmay be one of the mechanisms of p53-mediated BRCA1nuclear export. Re-expression of wt-p53, but not the mutantdeficient in binding to BRCA1, restored BRCA1 cytoplasmicshuttling and enhanced ionizing radiation (IR)-induced cyto-toxicity. Finally, augmenting cytosolic BRCA1 significantlyenhances p53-deficient cancer cell susceptibility to IR. Theseresults suggest that regulation of subcellular localization andnuclear export of genetically wt-BRCA1 in sporadic breastcancer by other regulatory proteins, such as p53, may be animportant determinant of tumor response to DNA-damagingagents.

Materials and Methods

Cell cultures and treatmentHCC1937 cells were purchased from American Type Culture

Collection. The genetic background, including expression andfunction of BRCA1, BARD1, p53, p21, and caspase 9, as well asthe growth characteristics and their response to genotoxicagents, was tested most recently in May 2010 using Westernblot analysis, immunohistochemistry, and colony formationassays. HCC1937 cells were maintained in Iscove's modifiedDulbecco's medium with 10% FBS, 100 U of penicillin (pen),and 100 mg/mL of streptomycin (strep). MCF7/E6 cells werepreviously described (8) and were maintained in Dulbecco'smodified Eagle medium (DMEM) supplemented with 10%FBS, 100 U pen, and 100 mg/mL strep. EJ-p53 cells were a giftfrom Dr. Stuart A. Aaronson, Mount Sinai School of Medicine,New York. Cells were cultured in complete DMEM, containing10% FBS, 100 U pen, 100 mg/mL strep, 1 mg/mL tetracycline(Tet), 75 mg/mL hygromycin B (Sigma-Aldrich), and 500 mg/mL G418 (Cellgro). Removal of Tet from the medium results inp53 induction (15). For irradiation of cells, the Mark I 137Csirradiator (J.L. Shepherd and Associates) was used, delivering1.84 Gy/min. A turntable ensured that the radiation wasequally distributed. As a control, mock irradiation consistedof placing plates containing cells in the irradiator for thedesignated times without turning on the machine.

Plasmids and adenovirusPlasmids and adenovirus are discussed in Supplementary

data.

ImmunofluorescenceImmunofluorescence is discussed in Supplementary data.

Subcellular fractionation, immunoprecipitation, andWestern blot analysisSubcellular fractionation, immunoprecipitation, and Wes-

tern blot analysis are discussed in Supplementary data.

Colony formation assaysColony formation assays are discussed in Supplementary

data.

Analysis of apoptosisAnalysis of apoptosis is discussed in Supplementary data.

Flow cytometryFlow cytometry is discussed in Supplementary data.

Statistical analysisStatistical analysis is discussed in Supplementary data.

Results

Regulation of BRCA1 shuttling is dissociated from p53transcriptional activity

Transcriptional regulation is one of the commonlyemployed mechanisms of p53 function in controlling geno-toxic stress-induced cell-cycle arrest and apoptosis. Wehypothesize that p53 controls BRCA1 nuclear export throughtranscriptional regulation. The effect of p53 transcriptionalactivity on the subcellular distribution of BRCA1 was deter-mined in HCC1937 human breast cancer cells that are func-tionally null in both p53 and BRCA1 (16, 17) followingco-expression of HA-tagged wt-BRCA1 with wt-p53 or 2tumor-associated p53 mutants (p53-179Q and p53-273H).The transcriptional activation deficiency of both p53 mutants(p53-179Q and p53-273H) was confirmed in HCC1937 cells(Supplementary Fig. S1a). The location of BRCA1 in nuclearor/and cytoplasmic compartments was assessed by subcel-lular fractionation. As shown in Figure 1A, the presence of wt-p53 resulted in BRCA1 cytoplasmic translocation following IR.Nuclear BRCA1 levels decreased by 2-fold (P < 0.05), whereascytoplasmic levels increased by more than 2-fold (P < 0.05).Interestingly, expression of the p53-179Q mutant resulted in asimilar level of IR-induced BRCA1 nuclear export as in cellswith wt-p53 expression. This effect, however, was significantlyreduced in cells expressing the p53-273H mutant, suggestingthat the p53-273H mutant, but not p53-179Q, is deficient inregulating BRCA1 shuttling. To our surprise, these results didnot support our initial hypothesis and indicate that p53transcriptional activity is not required to regulate BRCA1nuclear export following IR. To confirm the results obtainedby cell fractionation, immunohistochemistry was conductedon these cells to assess BRCA1 localization as a function of p53by co-immunostaining of exogenously expressed BRCA1 andp53 using anti-HA and anti-p53 antibodies (Fig. 1B). BRCA1subcellular distribution was scored as nuclear (N), nuclear/cytoplasmic (N/C), or cytoplasmic (C). Consistent with ournuclear-cytoplasmic fractionation results, an increased per-centage of cells with cytoplasmic BRCA1 was seen in cellsexpressing wt-p53 or p53-179Q following DNA damage, butnot in cells expressing p53-273H. Together, these data implythat p53-mediated regulation of BRCA1 nuclear export is notdependent on the transcriptional activation of p53.

p53-BRCA1 interaction is critical for p53-dependentBRCA1 nuclear export

It has been reported that BRCA1 and p53 physically interact(18–20). Because our results revealed that regulation of BRCA1nuclear export is not dependent on p53 transcriptional activ-

p53 Regulates BRCA1 Shuttling and Cytotoxicity

www.aacrjournals.org Cancer Res; 71(16) August 15, 2011 5547




ity, we proposed the alternative hypothesis that a protein–protein interaction may be the mechanism by which p53regulates DNA damage-induced BRCA1 nuclear export.

To determine the association of p53 and BRCA1, we con-ducted co-immunoprecipitation experiments in HCC1937cells described above. As shown in Figure 2A, similar amountsof wt-BRCA1 were present in the p53-immunocomplexespulled down in cells expressing either wt-p53 or p53-179Q.In contrast, a significantly decreased amount of BRCA1 waspresent in the p53-immunocomplex in cells expressing thep53-273H mutant. Quantitative analysis revealed a 2-fold

decrease in BRCA1 co-precipitated with p53-273H comparedwith that with wt-p53 (P < 0.05, Fig. 2A bottom). Reciprocalimmunoprecipitation experiments using HA antibody yieldedsimilar results (P < 0.05, Fig. 2B). These findings suggest that aprotein–protein interaction, and not transcriptional activity,may be important for p53 to regulate BRCA1 nuclear export,and the deficiency of the p53-273H mutant in promoting DNAdamage-induced BRCA1 nuclear export may be due, at least inpart, to its compromised ability to interact with BRCA1.

In contrast to p53, BARD1 inhibits BRCA1 export by bindingand masking the BRCA1 nuclear export sequence (NES; ref. 7).

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Figure 1. BRCA1 nuclear exportfollowing DNA damage does notrequire p53 transcriptional activity.A, top, BRCA1 subcellularlocalization at 24 hours followingmock or 5 Gy IR was determinedas a function of p53 status bysubcellular fractionation (8, 9). Thefold change relative to BRCA1expression without treatment ineach respective fraction isshown (*, P < 0.05; bottom).B, BRCA1 subcellular localizationwas confirmed byimmunofluorescence usinganti-HA antibody (9). Top,representative images of p53 (thinarrows), BRCA1 (thick arrows),DAPI, and merged staining.Bottom, BRCA1 staining wasscored as nuclear only (N),cytoplasmic only (C), or mixednuclear and cytoplasmic(N/C). Shown is the average fromat least 3 independentexperiments. Error bars representthe SEM (*, P < 0.001; **, P < 0.01).

Jiang et al.

Cancer Res; 71(16) August 15, 2011 Cancer Research5548




To further dissect the mechanisms by which p53 promotesBRCA1 cytosol translocation, we examined whether binding ofp53 to BRCA1 affects the interaction between BRCA1 andBARD1. As shown in Figure 2B, when BRCA1 was immuno-precipitated using HA antibody, similar amounts of BARD1were present in the BRCA1-immunocomplexes pulled down incells expressing either wt-p53 or p53-179Q. In contrast, asignificantly 1.9-fold increased amount of BARD1 was presentin the BRCA1-immunocomplex pulled down in cells expressingthe p53-273H mutant compared with that in cells expressingBRCA1-binding proficient wt-p53 or p53-179Q (P < 0.05, Fig. 2Bbottom). These findings support the model that binding of p53to BRCA1 disrupts the interaction of BRCA1 with BARD1. Thedissociation of BRCA1 from BARD1 uncovers the BRCA1 NESand thereby promotes BRCA1 nuclear export.

p53–BRCA1 binding confers cellular sensitivity toradiation-induced cytotoxicity

We next determined whether the p53–BRCA1 interactionwhich promotes BRCA1 nuclear export would also enhanceDNA damage-induced cell killing. To address this, we assessedthe cytotoxic response of HCC1937 cells co-transfected withwt-BRCA1 and wt-p53 or p53 mutants. IR-induced cytotoxicitywas determined by colony formation capacity. As shown inFigure 3A, increased radiosensitivity was observed in cellsexpressing wt-p53 or p53-179Q mutant compared with that incells expressing BRCA1-binding–deficient p53-273H mutant(Fig. 3A, P < 0.05).

Consistent with the colony formation assay results, weobserved a significant 2-fold decrease in the levels of cleavedcaspase 9 following the induction of DNA damage for the p53-273H mutant compared with that for wt-p53 and p53-179Q(Fig. 3B, P < 0.05). This reduction in IR-induced apoptosis wasverified using annexin V staining. As shown in Figure 3C,apoptosis was decreased by at least 2 fold in cells expressingp53-273H compared with wt-p53 or p53-179Q (P < 0.05). Thus,our results support the hypothesis that loss of p53–BRCA1binding, which fails to promote BRCA1 export, leads to areduced cytotoxic response to DNA damage.

The capacities of BRCA1 mutants to bind p53 affecttheir nuclear export and the subsequent cytotoxicresponse following DNA damage.

Our data thus far support the importance of p53–BRCA1interaction in BRCA1 shuttling and subsequent IR-inducedcytotoxicity. We reasoned that BRCA1 mutants that cannotbind to p53 should also be aberrant in their location andcytotoxic response to DNA damage. To test this, HA-epi-tope-tagged wt-BRCA1 or the BRCA1-5382insC BRCT-trun-cated mutant, or BRCA1-S1423/1524A mutant which resiststo ATM protein kinase-mediated phosphorylation and is defi-cient in G2/M-phase checkpoint (21), was co-expressed withwt-p53 or vector control in HCC1937 cells. wt p53 function wasverified by assessing IR-induced p21 induction (SupplementaryFig. S1b). Reciprocal immunoprecipitation experiments wereconducted to assess the BRCA1–p53 interaction. As shown inFigure 4A, p53 and BRCA1 were detected in the same immu-nocomplexes in cells expressingwt-p53withwt or S1423/1524A

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Figure 2. BRCA1 interaction with p53 is attenuated in the p53-273Hmutant. Reciprocal immunoprecipitation experiments with (A)p53 or (B) HA were conducted in HCC1937 cells co-transfectedwith HA-tagged BRCA1 and wt-p53, p53-179Q, or p53-273H. Thelevels of p53, HA-BRCA1, or BARD1 in immunocomplexes werenormalized to the amount of the reciprocal protein that was pulleddown. The fold change relative to cells expressing wt-p53 isshown (*, P < 0.05).






BRCA1. In contrast, in cells expressing wt-p53 and 5382insC-BRCA1, at least a 4-fold reduction in interaction wasobserved (P < 0.05). Similar results were obtained withreciprocal co-immunoprecipitation (Fig. 4B, P < 0.05). These

results confirm the observations from a previous study (19)that the BRCT domain of BRCA1 is important for interactionwith p53 (22).

To further test the model that BRCA1-p53 interaction andthe subsequent BRCA1 nuclear export are critical in DNAdamage-induced cytotoxicity, we next determined whetherp53-binding capacity of BRCA1 mutants affects its response top53-mediated and DNA damage-induced nuclear export. Thesubcellular distribution of exogenous BRCA1 in HCC1937 cellswas analyzed in relation to p53 status by subcellular fractio-nation (Fig. 4C–4E,) and immunostaining (SupplementaryFig. S2) using anti-HA antibody following either mock or 5Gy IR. Consistent with our previous findings (8), wt-BRCA1was predominantly retained in the nucleus when p53 wasdysfunctional, and this nuclear retention was attenuated bywt-p53 co-expression (Fig. 4C, P < 0.01, and SupplementaryFig. S2a, P < 0.05). Interestingly, the subcellular distribution ofp53-binding proficient S1423/1524A mutant was similar tothat of wt-BRCA1 when co-expressed with wt-p53 or controlvector (Fig. 4D, P < 0.01, and Supplementary Fig. S2b, P < 0.05).In contrast, co-expression of p53-binding–deficient 5382insCmutant exhibited an extensive cytoplasmic distribution irre-spective of p53 status and IR treatment (Fig. 4E and Supple-mentary Fig. S2c), which is consistent with previous reports(23). These data suggested that p53-dependent BRCA1 nuclearexport following DNA damage is intact in the p53-bindingproficient BRCA1 S1423/1524A mutant, but impaired in thep53-binding–deficient BRCA1-5382insC mutant.

However, the cytosolic accumulation of the 5382insCmutant seems to conflict with the results shown in Figures1 and 2 and our overall model: we would have predicted thatthe defect in binding to p53 would lead to retention of the5382insCmutant in the nucleus. To further investigate this, weexamined the interaction of the 5382insCmutant with BARD1,which enhances BRCA1 nuclear import and prohibits BRCA1nuclear export. Interestingly, a 4-fold reduction in the associa-tion of 5382insC-BRCA1 with BARD1 was observed (Fig. 4B, P< 0.05), compared with wt or S1423/1524A BRCA1. The lack ofinteraction between BRCA1 and p53, as well as BRCA1 andBARD1, may be due to restriction of BRCA1 to the cytoplasmiccompartment where it is inaccessible to its nuclear interactingpartners p53 and BARD1. To rule out this possibility, weexpressed 3XNLS–5382insC-BRCA1 fusion protein, in which3X-nuclear localization sequence (NLS) was inserted in-frameat the N-terminus of 5382insC-BRCA1, in HCC1937 cells. Asshown in Supplementary Figure S3, increased nuclear NLS–5383insC-BRCA1 remains incapable of interacting with eitherp53 or BARD1 and is unable to translocate to the cytoplasmfollowing IR regardless of p53 status.

These results suggest that the BRCT domain of BRCA1 iscritical not only for its interaction with p53 but also for itsassociationwith other proteins, includingBARD1. In support ofthis interpretation, it has been previously reported that cancer-associated mutations within the BRCT domain cause signifi-cant relocalization of BRCA1 from the nucleus to the cytoplasm(23). Since BARD1 binds to the N-terminal of BRCA1 to facil-itate BRCA1 nuclear import and prevents its export to thecytosol, we thus propose that the effect of BRCA1-5382insC

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Figure 3. p53-dependent BRCA1 nuclear export enhances IR-inducedcytotoxicity. A, cytotoxicity following various doses of IR was assessed bycolony formation assays (P < 0.05 for p53-273H versus wt-p53 or versusp53-179Q). Apoptosis was measured at 48 hours following mock or5 Gy IR by (B) the levels of cleaved caspase 9 (the levels of cleavedcaspase 9 were normalized to the levels of actin and the fold changerelative to cells expressing wt-p53 is shown), and (C) annexin V staining(*, P < 0.05; **, P < 0.01).

Jiang et al.





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Figure 4.C-terminal BRCT domain is critical for BRCA1 to interact with p53 and response to p53-dependent nuclear export following DNA damage. Reciprocalimmunoprecipitation experiments with (A) p53 or (B) HA were conducted in HCC1937 cells co-transfected with wt-p53 or vector control and HA-taggedwt or mutant (5382insC or S1423/1523A) BRCA1. The fold change relative to cells expressing wt-BRCA1 is shown (*, P < 0.05). Subcellular localization of (C)wt-BRCA1, (D) S1423/1524A BRCA1, or (E) 5382insC-BRCA1 following mock or 5 Gy IR was determined as a function of p53 by subcellular fractionation. Thefold change relative to BRCA1 expression without treatment in each respective fraction is shown (bottom of B and D; *, P < 0.05; **, P < 0.01).






dissociation from BARD1 on BRCA1 shuttling overrides p530sregulation and results in its exclusively cytosolic location.

To investigate whether the capacity of BRCA1 mutant tointeract with p53 also determines its effect on DNA damage-induced cytotoxicity, IR-induced cytotoxicity was assessed bycolony formation assays, caspase 9 cleavage, and annexin Vstaining. Consistent with previous reports (9, 17, 21), HCC1937cells expressing wt-BRCA1 alone, in which BRCA1 accumu-lated in the nucleus (Fig. 4C and Supplementary Fig. S2a),increased cellular radioresistance was observed comparedwith cells coexpressing wt-BRCA1 and wt-p53, in whichBRCA1 export to cytoplasm following IR (Fig. 5A, P < 0.01).In contrast, cells expressing the 5382insC mutant which ispredominantly located in the cytoplasm were exquisitelysensitive to IR-induced toxicity regardless of whether p53was co-expressed (Fig. 5B). Interestingly, the BRCA1 S1423/1524A mutant, which is retained in the nucleus following DNA

damage without p53, rendered HCC1937 cells more radio-resistant. Co-expression of wt-p53, however, restored nuclearexport and re-sensitized cells to IR (Fig. 5C, P < 0.01). Theseresults again support the notion that p53-mediated and DNAdamage-induced BRCA1 nuclear export is critical for thecytotoxic response induced by DNA damage.

To confirm our cytotoxicity results, we next analyzed theeffect of the various BRCA1 mutants on IR-induced apoptosisby assessing cleaved caspase 9 and annexin V. Increasedcaspase cleavage was observed following IR in cells expressingwt-BRCA1 and p53 (Fig. 5D, P < 0.05). Consistent with colonyformation assays, no significant differencewas observed in cellsexpressing the 5382insC-BRCA1mutant (Fig. 5E). However, theBRCA1 S1423/1524Amutant, which binds and responds to p53-mediated shuttling, behaves similarly to wt-BRCA1 (Fig. 5F, P <0.05). As shown in Figure 5G, restoration of p53-mediatedBRCA1 shuttling similarly increased the annexin V-positive

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Figure 5. p53-mediated increasein IR-induced cytotoxicity andapoptosis requires the BRCA1C-terminal region. wt, 5382insC,or S1432/1524A BRCA1 wascoexpressed in HCC1937 cellstogether with wt-p53 or vectorcontrol. Cytotoxicity followingvarious doses of IR was assessedby (A–C) colony formation assays.D–F, apoptosis was measured at48 hours followingmock or 5 Gy IRby the levels of cleaved caspase 9,and (G) annexin V staining(*, P < 0.05; **, P < 0.01).

Jiang et al.





cells, indicative of enhanced apoptosis (P< 0.01). Thus, our datasupport our hypothesis that p53–BRCA1 interaction and p53-mediated BRCA1 nuclear export are important determinants ofcancer cell sensitivity to DNA damage–based therapy.

The subcellular distribution of genetically wt-BRCA1strongly correlates with p53 status in human breastcancer specimensAlthough BRCA1 mutations are rarely found in sporadic

breast cancers, aberrant localization of BRCA1 in patientspecimens has been reported (24). We next determinedwhether p53 functional status affects the subcellular distribu-tion of wt-BRCA1 in sporadic breast cancer patients.To address this question, we assessed BRCA1 subcellular

distribution in 31 specimens from breast cancer patientsconfirmed to have wt-BRCA1 by direct sequencing underan IRB-approved protocol. p53 functional status and BRCA1subcellular distribution were evaluated by co-immunofluor-escence staining using p53 and BRCA1 antibodies. Increasedprotein stability and nuclear accumulation of p53 have been

well documented and are routinely used to represent dysfunc-tional p53 (12). The intensity of nuclear p53 immunostainingwas graded as: 0 for negative, 1 for weak, 2 for moderate, and 3for strongly positive staining. Samples with intensity of grades1 to 3 for positive nuclear p53 staining were scored as havingdysfunctional p53, whereas samples with negative nuclearstaining of grade zero intensity were scored as having func-tional p53. The BRCA1 subcellular distribution was scored asnuclear (N), nuclear/cytoplasmic (N/C), or cytoplasmic (C).Examples of each of these BRCA1 distribution patterns inspecimens with dysfunctional or functional p53 are shown inFigure 6A, 6B, and Supplementary Figure S4. As expected (10,12), p53 functional status was heterogeneous within eachpatient specimen. Cells were divided into functional anddysfunctional p53 groups, and BRCA1 subcellular distributionwas scored with respect to p53 status. Consistent with ourprevious findings, the combined data from the 31 patientsshowed that themedian distribution of BRCA1 with functionalp53 was 36% N, 41% N/C, and 23% C (Fig. 6C). However, whenp53 was dysfunctional, increased nuclear BRCA1 was

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Figure 6. Loss of p53 function increases nuclear BRCA1 in breast cancer patients. Tissue sections from breast cancer patients genotyped as wt for BRCA1were costained for BRCA1 and p53. A, representative staining of N, N/C, and C subcellular distributions of BRCA1 in cells with dysfunctional p53. B,representative staining of N, N/C, and C distributions of BRCA1 in cells with functional p53. C, Box-whisker plots of BRCA1 subcellular distributionwith respect to p53 functional status in all 31 patients. D, Ternary diagram of BRCA1 subcellular distribution with respect to p53 functional status for eachindividual patient (P < 0.0001; detailed explanation of the Ternary diagram is in Supplementary data, Materials and Methods). The filled circles indicatelocalization of BRCA1 in cells that retain p53 function. The open circles indicate BRCA1 localization when p53 function has been lost. The gray linesjoin data points for each of the individual 31 patients.






observed, and the fraction of cells exhibiting nuclear BRCA1was increased (67% N, 25% N/C, and 8% C; Fig. 6C).

A ternary diagram (Fig. 6D) depicts BRCA1 subcellulardistribution with respect to p53 status for each individualpatient. Strikingly, in every patient, dysfunctional p53 wasassociated with a dramatic shift of wt-BRCA1 to the nucleus(P < 0.0001). The substantial increase in nuclear localization ofBRCA1 when p53 is dysfunctional was consistent across all 31patients, which shows that loss of functional p53 stronglycorrelates with the nuclear accumulation of wt-BRCA1 inbreast cancer and suggests that localization of wt-BRCA1plays a pivotal role in sporadic breast cancer.

Targeted cytosolic localization of BRCA1 enhances thecytotoxic response to DNA damage

Loss of p53 is associated with a worse prognosis and, as wehave shown here, results in nuclear retention of wt-BRCA1 insporadic breast cancer patients and increased resistance toIR-induced cytotoxicity in breast cancer cells. We furtherhypothesized that enhancing cytosolic accumulation of wt-BRCA1 can be used as a means of sensitizing cancer cells toDNA-damaging agents in a p53-dysfunction background.

BARD1 prevents BRCA1 nuclear export by interacting withRING domain and thereby masks the BRCA1 NES. Expressionof a truncated BRCA1 RING domain has been shown toenhance wt-BRCA1 nuclear export by removing this compet-ing interaction (6, 7). We created a BRCA1 RING domain-yellow fluorescent fusion protein (YFP-RING; SupplementaryFig. S5a) to increase cytosolic BRCA1 in wt-BRCA1-expressingMCF7/E6 cells, in which p53 function is inactivated by expres-sion of the HPV16-E6 protein (8). Cells were sorted by flowcytometry for YFP expression, 24 hours following YFP-RING orcontrol vector transfection. The BRCA1 distribution wasdetermined by immunocytochemistry using the BRCA1 anti-body Ab-1, which recognizes the N-terminus of BRCA1. Asshown in Figure 7A, expression of the YFP-RING fusionprotein efficiently augmented cytosolic wt-BRCA1, resultingin an approximately 40% decrease in nuclear BRCA1 (P <0.001) and a concomitant 22% increase in nuclear/cytoplasmicBRCA1 (P < 0.001) and 18% increase in cytoplasmic BRCA1 (P< 0.01) compared with vector control. Similar results wereobtained using the BRCA1 antibody Ab-4 (SupplementaryFig. S5b), which recognizes exon 11 of BRCA1 and does notrecognize YFP-RING.

We next determined whether YFP-RING–induced cytosolicBRCA1 enhances the cytotoxic response to IR. The expressionof YFP-RING resulted in radiosensitization of MCF7/E6 cellsmeasured by colony formation ability (Fig. 7B, P < 0.05), andsignificant enhancement of IR-induced apoptosis measured byAnnexin V (Fig. 7C, P < 0.05) and caspase 9 cleavage (Fig. 7D, P< 0.05). These findings confirmed that BRCA1 subcellularlocation is an important determinant of the cytotoxicresponse to DNA damage.

Discussion

While the importance of BRCA1 mutations in the develop-ment of familial breast cancers is well established, sporadic

BRCA1 mutations are rare. Because of this, the significance ofBRCA1 function in sporadic breast cancers is unclear. We havepreviously reported p53-mediated BRCA1 cytoplasmic trans-location, which may play a novel role not only in inhibitingDNA repair functions but also in activating cell death pro-cesses following DNA damage (9). In this study, we provideevidence that the p53–BRCA1 interaction—and not p53 tran-scriptional activity—is one of the mechanisms for p53-mediated regulation of BRCA1 nuclear export followingDNA damage. Our results were validated in breast cancerpatient specimens with dysfunctional p53 that showedincreased nuclear accumulation of wt-BRCA1. We furtherdetermined that p53 regulates BRCA1 subcellular localizationthrough interaction with the BRCA1 C-terminal region, dis-rupting BRCA1 association with BARD1, and this regulation ofBRCA1 export determines DNA damage-induced cytotoxicity.Finally, augmenting cytosolic wt-BRCA1 by expressing a trun-cated BRCA1 RING domain enhanced cancer cell sensitivity toIR in p53-deficient cancer cells. Our findings therefore indicatethat the location of BRCA1 can serve as a therapeutic targetfor p53-mutated tumors.

Complex signaling networks contribute to cell sensitivity toDNA damage, making it difficult to pinpoint the key determi-nants of sensitivity. It has been shown that BRCA1 functions inmultiple DNA damage response and repair pathways, andBRCA1-deficient cells are sensitive to various types of DNAdamage, including oxidative stress-induced lesions (17, 21, 25,26), but it is not clear which function of BRCA1 is responsiblefor this sensitivity. Work by others has shown that loss of theG2/M checkpoint function of BRCA1 does not render cellsmore sensitive to DNA damage (21). Results from this studywith the BRCA1 S1423/1524A mutant are in agreement withthese findings. Interestingly, we have recently reported thatsubcellular localization of BRCA1 is the predominant deter-minant of its ability to regulate cellular response to differenttypes of DNA damage, in particular the increased persistentg-H2AX foci following IR due to decreasing overall DSB repairefficiency and the increasing apoptotic response when BRCA1is cytosolic (9). The complex role of cytosolic BRCA1 inconferring cytotoxicity is further supported by the enhancedcellular sensitivity not only to IR but also to UV exposure (9).Our results indicate that cytosolic BRCA1 may compromise itsnuclear functions in processing oxidative DNA lesions and invarious DNA repair pathways including homologous recom-bination, nonhomologous end-joining, and nucleotide exci-sion repair. As BRCA1 has been shown to be required for DNAdamage–induced apoptosis (27), it is intriguing to speculatethat, besides inhibition of overall DNA repair, the effect ofBRCA1 cytosolic localization on sensitivity to IR can also bepartially attributed to activation of BRCA1 cell death func-tions. Alternatively, sequestration of BRCA1 away from criticalbinding partners may block multiple nuclear processes,including DNA repair, required for cell survival after DNAdamage.

The results from this study support a model (Fig. 7E) inwhich physical interaction of p53 with BRCA1 at the BRCTdomain may alter BRCA1 conformation and interrupt BRCA1to interact with BARD1. In this case, the exposure of the

Jiang et al.





BRCA1 NES results in BRCA1 cytoplasmic translocation. Wepreviously have shown that p53-mediated BRCA1 nuclearexport takes places in all phases of the cell cycle, it is lesslikely that an aberrant G1 checkpoint in p53-deficient tumorcells plays an important role in regulation of BRCA1 subcel-lular distribution. Further investigation is warranted to shedlight on the molecular basis of these interactions and theresulting effect on their tumor suppressor roles.On the other hand, mutations in BRCA1 can affect its

interaction and response to p53-mediated nuclear export.ATM phosphorylation of BRCA1 at serine 1423 and 1524was not required for p53-mediated BRCA1 shuttling. However,the BRCT domain has been shown to play a crucial role forBRCA1 protein folding, conformation (22), nuclear foci for-

mation, and association with numerous other proteins (18, 23,28–30). Mutations that target the BRCT region of BRCA1 havebeen shown to be located in the cytoplasm (31). Our datarevealed that the 5382insC mutation at the BRCT domaincompromises its interaction with not only p53 but alsoBARD1, suggesting that the dissociation of 5382insC-BRCA1from BARD1 may compromise BRCA1 nuclear import andenhance BRCA1 nuclear export (18, 28, 29), and override p530sregulation on BRCA1 shuttling and lead to exclusive cytoplas-mic accumulation of 5382insC-BRCA1.

While the majority of known p53 mutations have beenshown to lead to increased protein stability (12, 32), weacknowledge that we cannot detect mutations that do notlead to nuclear accumulation in our study. However, the

Figure 7. BRCA1 RING domainexpression increases cytosolicBRCA1 and sensitizes breastcancer cells to IR. A, BRCA1subcellular distribution (N, N/C,and C) was determined byimmunofluorescence usingBRCA1 Ab-1 antibody(*,P < 0.001; **,P < 0.01). B, colonyformation ability, (C) annexin Vstaining, and (D) levels of cleavedcaspase 9 were assayed on thecollected YFP-positive cells(*, P < 0.05). E, model depictingpotential targeting of BRCA1 tothe cytoplasm to inhibit DNArepair, promote apoptosis, andsensitize cells to DNA-damagingagents. Following DNA damage,BRCA1 facilitates the repair ofDNA in the nucleus, which isdependent on association ofBRCA1 with BARD1. Whenunrepaired DNA damage ispersistent, p53 binds to BRCA1 atthe BRCT domain, which maydissociate BRCA1 from BARD1,BRCA1 cytoplasmictranslocation, and activateapoptosis. By targeting BRCA1 tocytoplasm, tumor cells aresensitized to DNA-damagingagents.

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nuclear accumulation of BRCA1 when p53 was determined tobe dysfunctional was consistent across all 31 patients. Further-more, the clinical data are supported by our previous (8) andcurrent biochemical and genetic studies. There is abundantevidence showing that p53-mutated tumors have a poor prog-nosis and increased resistance to therapies, including DNA-damaging agents. Because tumors with p53 mutation retainBRCA1 in the nucleus and are subsequently more resistant totreatment, the worse prognosis seen in patients with dysfunc-tional p53 may also be attributed, at least in part, to nuclearaccumulation of wt-BRCA1. Our findings also provide a newtarget in the RING domain. We have shown that the RINGdomain disrupts the BRCA1–BARD1 interaction, which resultsin BRCA1 nuclear export and a significantly enhanced sensi-tivity to DNA damage. It is thus a promising target andwarrants further investigation of thismechanism as a potentialtherapeutic strategy in p53-mutated tumors (Fig. 7E).

The findings in this study have these important implica-tions: (1) genotyping of BRCA1 and protein expression levelmay not be sufficient to determine BRCA1 function in human

cancer specimens; (2) BRCA1 localization may be an impor-tant prognostic factor and predictor of cancer cell response totherapies; and finally, (3) BRCA1 shuttling is a potentialmolecular target for improving treatment using DNA-dama-ging agents in sporadic breast cancer, especially when p53 isdysfunctional.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by grants from the NIH (R01 CA118158-02, to F.Xia), the Vanderbilt CTSA (UL1 RR024975, to J. Jiang), and the University ofAlabama-Birmingham School of Medicine IMPACT Award (to E.S. Yang).

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 September 21, 2010; revised May 25, 2011; accepted June 24,2011; published OnlineFirst July 8, 2011.

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2011;71:5546-5557. Published OnlineFirst July 8, 2011.Cancer Res Juhong Jiang, Eddy S. Yang, Guochun Jiang, et al. Susceptibility to DNA Damagep53-Dependent BRCA1 Nuclear Export Controls Cellular

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