The Unliganded Glucocorticoid Receptor Positively Regulates the Tumor Suppressor Gene BRCA1 Through GABP Beta

Published OnlineFirst February 10, 2012; DOI: 10.1158/1541-7786.MCR-11-0423-T

Molecular Cancer Signaling and Regulation Research

The Unliganded Glucocorticoid Receptor Positively Regulates the Tumor Suppressor Gene BRCA1 through GABP Beta

Heather D. Ritter1,3, Lilia Antonova2,3, and Christopher R. Mueller1,2,3

Abstract Loss of BRCA1 tumor suppressor function is a critical event in breast tumorigenesis. We have previously identified the stress hormone hydrocortisone as a negative regulator of BRCA1 expression in nonmalignant mammary cells. Here, we have identified a direct role for the unliganded glucocorticoid receptor (GR) in BRCA1 upregulation in the absence of hydrocortisone. The positive regulatory effect of GR is lost upon the addition of hydrocortisone. We have shown that GR interacts with the BRCA1 promoter only in the absence of hydrocortisone, and that this interaction is mediated through the b-subunit of the ets transcription factor GA-binding protein (GABP) at the RIBS promoter element. GR and GABPb interact in both coimmunoprecipitation and mammalian two-hybrid assays, and this interaction involves the N-terminal to central regions of both proteins. This work presents the first evidence of a ligand-independent role for GR as a positive regulator of gene expression, and loss of GR from the BRCA1 promoter in response to stress hormones leads to decreased BRCA1 expression. Because low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk. Mol Cancer Res; 10(4); 558–69. 2012 AACR.

Introduction levels on the order of 50% are biologically significant. The Germ line mutations in the BRCA1 tumor suppressor importance of BRCA1 in breast cancer is attributable to its contribute to familial breast tumor formation but BRCA1 involvement in a number of regulatory processes impor- mutations do not seem to occur in sporadic breast cancer tant for maintenance of genomic stability and prevention tumors (1). Instead, sporadic breast tumors exhibit of cell transformation (8). More recently, BRCA1 has decreased levels of BRCA1 expression, and the degree of been shown to be required for mammary stem/progenitor downregulation seems to be correlated with tumor grade, cell differentiation, whereby loss of BRCA1 results in – expansionofthestem/progenitorpopulation(9).Consis- rate of tumor progression, and risk of metastasis (2 6). BRCA1 This implies that loss of BRCA1 function, either through tent with this, some genes enriched in mutant decreased activity or downregulation of expression, is a one-hit cells are expressed in stem and progenitor cells critical event in the etiology of breast cancer. Phenotyp- (7, 9). Because BRCA1 also participates in maintaining ically normal breast epithelial cells from individuals har- genomic integrity, loss of BRCA1 function could lead to boring a BRCA1 germ line mutation (a so called "one-hit" cancer development through the production of genetically BRCA1 unstable stem/progenitor cells (9). mutation accompanied by a 50% decrease in BRCA1 function) express an altered mRNA profile compared with The transcriptional regulation of is modulated by BRCA1 a variety of hormones, developmental cues, and other normal cells from individuals without a mutation Eco (7). This suggests that decreases in functional BRCA1 effectors (10). We have shown that the RIBS ( RI Band Shift) element is required for maximal BRCA1 promoter activity and contains 2 binding sites for the ets transcription Authors' Affiliations: Departments of 1Biochemistry and 2Pathology and factor GA-binding protein (GABP; ref. 11). Human GABP Molecular Medicine, and 3Queen's University Cancer Research Institute, exists as a heterodimer consisting of an ets helix-loop-helix Queen's University, Kingston, Ontario, Canada DNA-binding domain (DBD) subunit (GABPa) and a Note: Supplementary data for this article are available at Molecular Cancer Notch-ankyrin repeat subunit (GABPb) which contains the Research Online (http://mcr.aacrjournals.org/). activation domain as well as a domain required for the Corresponding Author: Christopher R. Mueller, Departments of Biochem- formation of tetrameric complexes (12). GABP has been istry and Pathology and Molecular Medicine, Division of Cancer Biology and Genetics, Queen's Cancer Research Institute, Queen's University, 18 implicated in the regulation of genes in response to cell Stuart Street, Botterell Hall Room 368, Kingston, Ontario K7L 3N6, Canada. growth, activation of respiration-related genes (13), as a Phone: 613-533-6751; Fax: 613-533-6830; E-mail: [email protected] downstream mediator of ErbB3 and ErbB4 signaling doi: 10.1158/1541-7786.MCR-11-0423-T (14), and recently in connecting mitochondrial metabolism 2012 American Association for Cancer Research. and breast differentiation (15). The interaction of the a- and

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b-subunits with each other and with other transcription cells were cultured as previously described (15, 19). Cell factors defines the ability of GABP to regulate the expression treatments were completed using media lacking FBS or horse of its target genes. serum and containing either 1 mg/mL hydrocortisone (Sig- BRCA1 can be considered as a central rheostat of breast ma), 1, 5, or 10 mmol/L RU-486 (Sigma), or ethanol vehicle cancer risk, with its transcriptional regulation being con- for 24 or 48 hours. trolled by a variety of factors, one of which is psychological stress. Various epidemiologic studies have indicated that DNA constructs psychological stress produces a significant increase in breast Creation of the L6-pRL, L6DR-pRL, L6mUPmE2F- cancer risk, and that the nature and magnitude of the effect pRL, L6DRmUPmE2F-pRL, RIBSn-pRL, and GF- of stress on risk is comparable with that of other well-known TATA-pRL BRCA1 promoter constructs has been described risk factors for breast cancer (16). Furthermore, specific previously (27). studies have suggested that the cancer-causing effects of The rat constructs wild-type GR (GRwt) and GRL501P stress may be fairly specific to the breast (17, 18). We (GR with a leucine to proline mutation at amino acid previously presented the first molecular evidence for a position 501, which abolishes its DNA-binding ability) were connection between stress and breast cancer with our obser- gifts of Keith Yamamoto (University of California, San vation that BRCA1 expression is repressed by the synthetic Francisco, CA), and their construction has been described stress hormone hydrocortisone in nonmalignant mouse previously (28, 29). mammary cells (19). This work was the first breast-specific Construction of the H1-2 vector has been described molecular mechanism linking stress and breast cancer previously (27). To construct the shGR vector, the oligos development. GRshRNA50 and GRshRNA30 were annealed (Supplemen- In response to a stress signal, the primary human stress tary Table S1). The product was cut with BamHI and hormone cortisol binds to its intracellular receptor, the gluco- HindIII [New England Biolabs (NEB)] and ligated into the corticoid receptor (GR), which is a member of the steroid H1-2 vector. receptor superfamily of nuclear receptors (20). While it has The constructs PRR, PRR-M1, PRR-M2, and PRR-M3 traditionally been accepted that nuclear receptors are activated were gifts of Dr. Calvin Roskelley. in response to ligand binding, several reports exist of prog- To construct pFLAG-GRa,theGRa gene was PCR esterone and estrogen receptor activation and nuclear activity amplified from MCF-7 cDNA in 2 separate reactions. The even in the absence of hormone (21). GR has been reported 50 end was amplified using primers GRa50(þ)andGRa50 to be activated by various stimuli in the absence of glucocor- (; Supplementary Table S2). The 30 end was amplified ticoid ligands (21–24), and unliganded GR has recently been using primers GRa30(þ)andGRa30(; Supplementary found to bind directly to and exert a repressive effect on the Table S2). The 50 section was cut with NotI and PstI IL-6 promoter in response to TNF-a in endocervical cells (NEB), and the 30 section was cut with PstIandBamHI (25). In addition, it has been suggested that breast cancer (NEB). The 50 and 30 sections were simultaneously ligated progression may be associated with an accumulation of GR in together and into the p3X-FLAG (pFLAG) expression the cytoplasm of tumor cells (26). vector (Sigma). In this report, we present, for the first time, evidence for a Mammalian 2 hybrid vectors pACT and pBIND, as ligand-independent role for GR in the activation of BRCA1 well as firefly luciferase reporter pG5-luc were obtained expression in breast cells. We show that this effect is from Promega. Full-length GRa,aswellasitsindividual mediated through an interaction between GR and GABPb domains, were PCR amplified from pFLAG-GRa using at the RIBS element of the BRCA1 promoter, which occurs primers listed in Supplementary Table S3. Each PCR only in the absence of hydrocortisone ligand. This interac- product was cut with NdeIandBamHI and ligated into tion and the associated positive regulatory effect are lost pACT. Similarly, full-length GABPb,aswellasitsindi- upon addition of hydrocortisone, which may represent a vidual domains, were PCR amplified from pFLAG- novel mechanism through which stress signaling increases GABPb (construction described previously; ref. 15) using breast cancer risk. The unliganded GR plays a previously primers listed in Supplementary Table S4. PCR products unknown role in gene activation with profound implications were cut with BamHI and NotI and ligated into pBIND. for our understanding of GR-mediated processes. Transient transfections and luciferase assays Transfections were carried out as described previously Materials and Methods (15, 19). For standard transfection assays, control cytomeg- Cell culture and treatments alovirus (CMV)-luc vector (Promega) was used at 25 ng The nontumorigenic murine mammary epithelial cell line per well, as were GR expression vectors and empty vector EPH-4, and the karyotypically normal, human telomerase controls. The remainder of the 250 ng per well was allott- reverse transcriptase–immortalized mammary epithelial ed to the appropriate Renilla luciferase reporter vector. cell line 184-hTERT were gifts of Dr. Calvin Roskelley For knockdown transfections, 50 ng of the short hairpin (University of British Columbia, Vancouver, Canada). The RNA (shRNA) construct or its empty vector were used. human breast carcinoma cell lines MCF-10A and MCF-7 For mammalian 2-hybrid transfections, 50 ng of each were obtained from American Type Culture Collection. All pBIND-GABPb construct was used along with 25 ng of

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each pACT-GR construct. For all cell lines, cells were treated ChIP assay with hydrocortisone, RU-486, or ethanol vehicle (as EPH-4 cells were plated and treated as described earlier. described earlier) in serum-free medium 24 hours following Chromatin immunoprecipitation (ChIP) assays were con- transfection. Either 24 or 48 hours after treatment, cells were ducted with the ChIP-IT Express Enzymatic Kit (Active harvested for the Dual Luciferase Assay as previously Motif). Each reaction was carried out using chromatin from described (15). 2 106 cells and 2 mg per reaction of affinity-purified antibody (or water as a negative control). The following Quantitative real-time PCR antibodies were used: anti-GR (ab3579; Abcam), anti- RNA and RT products were prepared as described pre- GABPa (sc-22810; Santa Cruz Biotechnology Inc.), viously (15, 19). Quantitative real-time PCR (qRT-PCR) anti-GABPb (sc-28684; Santa Cruz Biotechnology Inc.), reactions for human BRCA1 were carried out as described anti-hemagglutinin (sc-805; Santa Cruz Biotechnology previously (15), with the exception that hypoxanthine Inc.), anti-acetylated histone H3 (06-599; Upstate Bio- phosphoribosyltransferase 1 (HPRT1) was used as an inter- technology), anti-GST (sc-459; Santa Cruz Biotechnology nal control instead of TBP (primers listed in Supplementary Inc.), anti-Fra-2 (sc-604; Santa Cruz Biotechnology Inc.), Table S5). BRCA1 expression for each cell line was calculated anti-USF-2 (sc-861; Santa Cruz Biotechnology Inc.), and relative to the results for the untreated sample for each cell anti-RYBP (ab5976; Abcam). The PCR primers ampli- line with the Pfaffl method (30). qRT-PCR reactions for fied the mouse BRCA1 promoter from position 335 to mouse BRCA1 (with TBP as an internal control) were carried þ73 [(þ)50-TCCGGGAGCGATTCCCACCC and () out as described previously (ref. 19; primers listed in Sup- 50-AAGATCCGTACTTCCAAGCG]. A water blank (no plementary Table S6). BRCA1 expression for each cell line template) and EPH-4 input chromatin were also subjected was calculated relative to the results for the untreated sample to PCR amplification as controls. ChIP DNA was quantified DD C with the t method presented by PE Applied Biosystems by quantitative PCR by the QuantiTect SYBR Green PCR (Perkin Elmer). Kit (Qiagen) using 7.5 mL of ChIP DNA and the ChIP PCR primers for mouse BRCA1 above. The PCR protocol con- Immunoprecipitation assay sisted of 1 cycle of 900 seconds at 94C followed by 40 cycles MCF-7 cells were transfected as described earlier with of (30 seconds at 94C, 30 seconds at 60C, and 30 seconds different combinations of GABPa, GABPb, and FLAG- at 72C). GRa. Cells were treated 24 hours after transfection with either ethanol vehicle or 1 mg/mL hydrocortisone in serum- Transient ChIP assay free medium. Following a 24-hour incubation, whole-cell MCF-10A cells were plated in serum-containing medi- lysates were prepared in modified RIPA buffer as described um on 12-well culture dishes at a density of 5 104 cells previously (27). Fifty micrograms of protein was incubated per mL. After 24 hours, cells were transfected in triplicate with 1 mg of either anti-GABPb (sc-28684; Santa Cruz with combinations of FLAG-GRa,GABPa,andGABPb, Biotechnology Inc.) or anti-FLAG (sc-807; Santa Cruz and a series of BRCA1 promoter constructs. Cells were Biotechnology Inc.) for 4 hours at 4C. The protein– treated 24 hours after transfection with either ethanol antibody mixture was incubated overnight with 20 mLof vehicle or 1 mg/mL hydrocortisone in serum-free medium. Protein A/G PLUS-Agarose Immunoprecipitation Reagent ChIP was carried out as described earlier. Each reaction (sc-2003; Santa Cruz Biotechnology Inc.) at 4C. Beads was carried out using chromatin from 2 106 cells and 1 were washed with PBS and incubated with 50 mLof1 SDS mg per reaction of anti-FLAG antibody (sc-807; Santa loading buffer at 98C for 15 minutes to elute bound Cruz Biotechnology Inc.) or no antibody as a negative protein. control. Primers used during PCR analysis spanned the pRL vector insert [(þ)50-GCAACGCGGCCTTTT- Western blotting TACGG and ()50-CCTTAAACCTGTCTTGTAACC]. Lysates were prepared in 1 SDS loading buffer and analyzed by standard Western blotting procedures. Poly- vinylidene fluoride membranes (Millipore) were probed Results with the appropriate primary antibody: anti-GR (1:500; GR upregulates BRCA1 promoter activity in the absence ab3579; Abcam), anti-TBP (1:2,000; ab818; Abcam), anti- of ligand GABPb (1:5,000; sc-28684; Santa Cruz Biotechnology We have previously shown that BRCA1 promoter activity Inc.), or anti-FLAG (1:1,500; sc-807; Santa Cruz Biotech- is repressed in the presence of hydrocortisone in EPH-4 nology Inc.). The secondary antibodies used included goat mouse mammary cells (19). To elucidate the mechanism anti-rabbit (1:10,000; sc-2004; Santa Cruz Biotechnology through which this repression occurs, we evaluated the key Inc.) to detect GR, goat anti-mouse (1:10,000; 115-035- stress signaling protein GR in regulating the expression of 003; Jackson ImmunoResearch) to detect TBP, and Clean- BRCA1 in breast cells. In transient cotransfection experi- Blot IP Detection Reagent (1:700; Thermo Scientific/Fish- ments in both EPH-4 mouse and 184-hTERT human er) for all immunoprecipitated lysates. Secondary antibody mammary epithelial cells, overexpression of either the GRwt detection was carried out by chemiluminescence (Thermo or a rat GR mutant that cannot bind DNA (GRL501P; Scientific/Fisher). ref. 29) resulted in a dramatic increase in human BRCA1

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promoter (L6-pRL) activity in the absence of hydrocorti- Promoter sites involved in GR-induced upregulation of sone (Fig. 1A, black bars). Treatment with hydrocortisone BRCA1 promoter activity eliminated this increase in BRCA1 promoter activity We have previously shown that the RIBS, CREB (cAMP- (Fig. 1A, white bars). In the classically accepted model responsive element binding protein), and UP (Upstream) of GR function, hydrocortisone binds to GR in the promoter sites seem to play a role in the hydrocortisone- cytoplasm, which subsequently translocates to the nucleus mediated regulation of BRCA1 expression, based on changes and regulates gene expression through speciﬁcelementsor in complex formation in untreated and hydrocortisone- via bound transcription factors. In contrast, our observa- treated nuclear extracts (19). We concentrated on the RIBS tions suggest that GR acts as a positive regulator of BRCA1 element as it is a potent site of positive regulation and it in the nucleus in the absence of hydrocortisone ligand. To contains a binding element for the heterodimeric ets tran- determine whether the endogenous GR behaves as an scription factor GABP. To characterize GR-responsive pro- activator of BRCA1 expression, we transiently transfected moter elements, and to determine whether one or more of EPH-4 cells with the BRCA1 promoter (L6-pRL) and an these sites are involved in the upregulation of BRCA1 by GR, shRNA vector directed against mouse GR (shGR), which EPH-4 cells were cotransfected with GRwt and various is able to reduce GR expression (Fig. 1B). Knockdown of BRCA1 promoter mutant constructs (Fig. 1D). The addi- GR by shGR repressed BRCA1 promoter activity in the tion of GRwt increased the activity of a construct containing absence but not in the presence of hydrocortisone only the RIBS and CREB sites (Fig. 1E, PRR) in the absence (Fig. 1C), implicating GR as a positive regulator only in of ligand. Compared with the PRR promoter construct, the absence of ligand. mutation of the RIBS element (Fig. 1E, PRR-M1)

A B 4.0 Figure 1. The unliganded GR -HC is a positive regulator of BRCA1 +HC 3.0 expression. A, EPH-4 and H1-2 shGR 184-hTERT cells were transiently 2.0 transfected with the L6 BRCA1 WB: anti-GR promoter reporter construct and 1.0 expression vectors for wild-type rat

GR (GRwt) and rat GR with a mutation 0.0 WB: anti-TBP of leucine to proline at amino acid V t EV E Relative BRCA1 promoter activity position 501 (GRL501P), which GRwt L501P GRw L501P abolishes its DNA-binding ability. GR GR Cells were treated 24 hours after EPH-4 184-hTERT transfection with either ethanol m þ vehicle ( HC) or 1 g/mL HC ( HC) C D PRR RIBS CREB R-luc and assayed for luciferase activity 1.5 H1-2 following a 48-hour incubation. B–C, shGR EPH-4 cells were transiently PRR-M1 RIBSX CREB R-luc 1.0 transfected with the L6 BRCA1 promoter reporter construct and PRR-M2 RIBS CREB R-luc either an empty vector (H1-2) or an 0.5 X shRNA vector directed against endogenous GR (shGR). Cells were PRR-M3 RIBS CREB R-luc (B) lysed after 48 hours and 0.0 X X C subjected to Western blotting (WB) to Relative BRCA1 promoter activity determine GR expression or (C) EPH-4 -HC EPH-4 +H treated and assayed as above. BRCA1 D, schematic of promoter -HC +HC fragments (PRR, wild-type RIBS and E F CREB sites; PRR-M1, mutant RIBS; 2 EV 2 EV PRR-M2, mutant CREB; and PRR- GRwt GRwt M3, RIBS and CREB double mutant). E–F, EPH-4 cells were transiently transfected with a BRCA1 promoter 1 1 fragment and an expression vector for wild-type rat GR (GRwt) in the (E) absence or the (F) presence of hydrocortisone (HC). Cells were Fold promoter activity Fold promoter activity treated and assayed as above. EV, 0 0 empty vector. PRR PRR PRR-M1 PRR-M2 PRR-M3 PRR-M1 PRR-M2 PRR-M3

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A β O H 2 1:10 InputNo Ab GST HA H3 GABP Fra-2 USF-2 RYBP GR - HC

+ HC

B C L6-pRL + GABP L6-pRL -GABP RIBS CREB UP E2F R-luc L6-pRL

L6-pRL L6ΔR-pRL CREB UP E2F R-luc L6ΔR-pRL

RIBS CREB XUP XE2F R-luc L6mUPmE2F-pRL L6ΔRmUPmE2F-pRL L6mUPmE2F-pRL + GABP CREB XXUP E2F R-luc RIBSn-GF-TATA-pRL GF-TATA-pRL L6ΔRmUPmE2F-pRL

RIBS x 8 R-luc O

RIBSn-GF-TATA-pRL 2 + + + + FLAG-GR H No Ab + + + + HC R-luc GF-TATA-pRL

Figure 2. GR interacts directly with the BRCA1 promoter via the RIBS element only in the absence of hydrocortisone. A, chromatin was prepared from EPH-4 cells 24 hours after treatment with either ethanol vehicle (HC) or 1 mg/mL hydrocortisone (þHC) in serum-free medium. ChIP analysis was carried out with the indicated antibodies and primers spanning the BRCA1 promoter. B, schematic of BRCA1 promoter constructs. C, MCF-10A cells were transiently transfected with each of the BRCA1 promoter constructs and different combinations of FLAG-tagged GR (FLAG-GRa), GABPa,and GABPb. Cells were treated 24 hours after transfection with either ethanol vehicle or 1 mg/mL hydrocortisone in serum-free medium, and chromatin was prepared following a 24-hour incubation. The interaction of FLAG-GRa with elements of the BRCA1 promoter was examined through ChIP using FLAG antibody and subsequent PCR analysis of the immunoprecipitated DNA using primers spanning each promoter construct. Ab, antibody; GST, glutathione S-transferase; HA, hemagglutinin; H3, acetylated histone H3.

eliminated activation whereas mutation of the CREB site transcription factor), which are factors known to bind to the (Fig. 1E, PRR-M2) did not. Mutation of both sites (Fig. 1E, BRCA1 promoter through various elements, interacted with PRR-M3) reduced BRCA1 upregulation to a level similar to the BRCA1 promoter in the absence of hydrocortisone that obtained with the PRR-M1 RIBS mutation construct whereas the polycomb repressor RYBP did not (Fig. 2A, alone. Therefore, RIBS is a key element in controlling top). In the presence of hydrocortisone, GR dissociated from BRCA1 expression in the presence of GR and the absence the promoter (Fig. 2A, bottom), as did GABPb and Fra-2, of ligand. In comparison, none of the BRCA1 mutant which may be a result of loss of GR binding. USF-2 and reporters were activated by GR in the presence of hydro- RYBP bound BRCA1 in the presence of hydrocortisone (Fig. cortisone ligand (Fig. 1F), suggesting that liganded GR is 2A, bottom). The ChIP DNA samples were analyzed via recruited away from the BRCA1 promoter. quantitative PCR and values obtained reflect the pattern of band intensities on the ChIP gel (Table 1). In general, there GR interacts directly with the BRCA1 promoter seems to be less transcription factor binding to BRCA1 in BRCA1 regulation by GR is not dependent on the DNA- the presence of hydrocortisone. This and the fact that the binding ability of GR, as a GR mutant deficient in DNA repressor RYBP binds BRCA1 in this case may reflect the binding (GRL501P) still activates BRCA1 expression in reduced activity of the BRCA1 promoter in the presence of transient transfection assays (Fig. 1A, black bars). Further- hydrocortisone. more, there are no defined GRE sequences within the To further characterize the promoter elements involved in BRCA1 promoter. Therefore, it is likely that GR exerts its the interaction between GR and the BRCA1 promoter, we effect on BRCA1 transcriptional activity by interacting with conducted transient ChIP assays in nonmalignant MCF- proteins present at the BRCA1 promoter, particularly at the 10A human mammary epithelial cells. Cells were transiently RIBS element. ChIP analysis of endogenous GR in EPH-4 transfected with a FLAG-tagged human GR construct, cells revealed that GR was present on the BRCA1 promoter human GABPa and GABPb, and various BRCA1 promoter in the absence of hydrocortisone (Fig. 2A, top). Transcrip- mutant constructs (Fig. 2B). GR was immunoprecipitated tion factors, GABPb, Fra-2 (Fos-related AP1 transcription with an anti-FLAG antibody to pull down GR, and BRCA1 factor), and USF-2 (basic helix-loop-helix leucine zipper constructs were detected via PCR using primers flanking the

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a b BRCA1 Table 1. ChIP DNA products were analyzed by functional GABP / , its regulatory effect on may be via a protein–protein interaction with this multisubunit quantitative PCR and expressed as relative C t transcription factor. To determine whether GR interacts values with GABP, we conducted coimmunoprecipitation experiments using lysates from human MCF-7 cells cotransfected EPH-4 -HC EPH-4 þHC with a FLAG-tagged GR construct along with untagged a b Input 34.41 35.58 vectors for the - and -subunits of GABP. These experi- b No Ab >40 >40 ments revealed that GABP , which contains the activation GST >40 >40 domain of the GABP protein, interacts with GR in the HA >40 >40 absence of hydrocortisone, both on its own and in combi- a H3 34.78 35.63 nation with the DNA-binding -subunit (Fig. 3A). More- b GABPb 36.05 >40 over, GR and GABP interact in the presence of hydrocor- Fra-2 35.15 37.4 tisone (Fig. 3B), suggesting that this interaction may be USF-2 34.52 36.02 maintained during ligand binding to GR. This possibility is fl RYBP >40 36.64 re ected by the ChIP results, which indicate that like GR, b BRCA1 GR 35.7 >40 GABP interacts with only in the absence of hydrocortisone (Fig. 2A). In contrast, GR was not shown to interact with GABPa, either in the absence or presence of hydrocortisone. promoter inserts. FLAG-tagged GR coimmunoprecipitated We further investigated the interaction between GR the L6 BRCA1 construct only when exogenous GABPa and GABP through the use of a mammalian 2-hybrid and GABPb were added (Fig. 2C, L6-pRL), indicating the system for which we created a mammalian expression requirement for this transcription factor in the interaction vector consisting of the GAL4 DBD fused to GABPa or between GR and BRCA1, and showing the limiting quan- GABPb, and used vectors for either GRwt or human GR tities present in transient transfections. A RIBS multimeric (GRa) to complete the system. Cotransfection of the construct (Fig. 2C, RIBSn-GF-TATA-pRL) and a BRCA1 GAL4-GABPb expression vector along with expression promoter construct in which a number of important sites, vectors for either GRwt or GRa in EPH-4 mouse (Fig. but not the RIBS element, had been mutated (Fig. 2C, 3C) or MCF-7 human (Fig. 3D) mammary cells resulted L6mUPmE2F-pRL) were also coimmunoprecipitated by in a dramatic increase in luciferase activity from a GAL4- GR in the presence of GABPa and GABPb. In contrast, responsive reporter in the absence of hydrocortisone, various RIBS deletion mutants were not immunoprecipi- indicating an interaction between GABPb and unliganded tated by GR (Fig. 2C, L6DR-pRL, GF-TATA-pRL, and GR. The increase in activity of the GAL4-responsive L6DRmUPmE2F-pRL). GR was found to interact with the promoter was presumed to be the result of the endogenous RIBS promoter constructs only in the absence of hydrocor- transactivation domain of GR, as the wild-type protein tisone (Fig. 2C). When cells were treated with hydrocorti- was used in place of a VP16 activation domain fusion. sone, the interaction was abolished (Fig. 2C). In this context, Treatment with hydrocortisone abolished the increase in these experiments show that the interaction between GR and luciferase activity, indicating that the transactivation by the BRCA1 promoter is dependent on GABP and the RIBS GR and GABPb had been disrupted. No increase in element. Knockdown of GABP and subsequent transient luciferase activity was observed using GAL4-GABPa as ChIP would be ideal to establish the requirement for this a target (Fig. 3C and D), indicating that the a-subunit of transcription factor in the GR–BRCA1 interaction; however, GABP alone does not interact with GR. When a VP16 knockdown of the GABP subunits results in significant cell activation domain was attached to the GRa construct death, limiting the ability to assess BRCA1 expression. [pACT-GR full-length (FL)], a similar increase in reporter Furthermore, BRCA1 expression is otherwise dependent on activity was observed in EPH-4 mouse (Fig. 3E) or MCF- GABP binding outside of the GR context (11, 15). Because 7 human (Fig. 3F) mammary cells in only the presence of knockdown of GABP dramatically decreases BRCA1 expres- the GAL4-GABPb fusion, and this activation was lost sion, the assessment of GABP dependence in this context is with hydrocortisone addition. not feasible. We have thus developed a novel model of To further characterize the interaction between GR BRCA1 regulation in which GR binds to the BRCA1 and GABPb, we created a domain-specific deletion series promoter via the RIBS site in the absence of hydrocortisone of both GR and GABPb based on the previously char- to activate expression, and in the presence of hydrocortisone, acterized domain structure of both proteins. The GR BRCA1 levels decrease due to the dissociation of GR from deletion mutants, including the full-length protein, and the promoter. different combinations of the transcriptional activation domain (TAD), DBD, hinge region (HR), and ligand- The interaction between GR and the BRCA1 promoter is binding domain (LBD), were cloned into a VP16 activa- mediated by GABPb tion domain containing vector (pACT) to create pACT- Because GR interacts with the BRCA1 promoter through GR fusions. Similarly, the GABPb deletion mutants, the GABP-binding RIBS element, and seems to require including the full-length protein, the ankyrin repeat region

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A -HC B +HC

+ + + + GABPα + + + + GABPα

+ + + + GABPβ + + + + GABPβ Figure 3. GR interacts directly with the b-subunit of the ets + + + + FLAG-GRα + + + + FLAG-GRα transcription factor GABP. MCF-7 WB: anti-FLAG WB: anti-FLAG cells were transiently transfected with GABPa, GABPb, and/or WB: anti-GABPβ WB: anti-GABPβ FLAG-GRa. Cells were treated 24 hours after transfection with either IP: anti-FLAG IP: anti-FLAG (A) ethanol vehicle (HC) or (B) 1 β β WB: anti-GABP WB: anti-GABP mg/mL hydrocortisone (þHC) in serum-free medium, and whole- IP: anti-GABPβ IP: anti-GABPβ WB: anti-FLAG WB: anti-FLAG cell lysates were prepared following a 24-hour incubation. Lysates were immunoprecipitated (IP) with 1 mg of either anti-GABPb C 250 D 250 EPH-4 -HC MCF-7 -HC or anti-FLAG antibody, and the 200 EPH-4 +HC 200 MCF-7 +HC presence of either FLAG (GR) or b 150 150 GABP in the eluted fractions was examined by Western blotting 100 100 (WB). C and D, EPH-4 and MCF-7 50 50 cells were transiently transfected with GAL4 expression vectors for Relative promoter activity 0 activity promoter Relative 0 a b a α α α α GABP or GABP (GAL4-GABP EV EV GR GR GR GR GRwt GRwt GRwt b + GRwt and GAL4-GABP , respectively) + + β + β + β and expression vectors for both rat GAL4-GABPα GAL4-GABPβ GAL4-GABPα GAL4-GABPβ and human GR (GRwt and FLAG- GAL4-GABPα GAL4-GABPβ + GAL4-GABPα + GAL4-GABP GAL4-GABPα + GAL4-GABP GAL4-GABPα GAL4-GABP GRa, respectively). Cells were treated 24 hours after transfection E F with either ethanol vehicle or 1 mg/ 10 20 EPH-4 -HC MCF-7 -HC mL hydrocortisone in serum-free 8 EPH-4 +HC MCF-7 +HC 15 medium and assayed for luciferase 6 activity following a 24-hour 10 incubation. E and F, EPH-4 and 4 MCF-7 cells were transiently 5 2 transfected with a GAL4 expression vector for GABPb Relative promoter activity promoter Relative 0 activity promoter Relative 0 b T L (pBIND-GABP FL) and a VP16 pACT expression vector for human GR + + pACT FL FL (pACT-GR FL). Cells were treated pBIND + pACT pBIND + pAC and assayed as above. FL + pACT-GR FL FL + pACT-GR F β pBIND + pACT-GR FL pBIND + pACT-GR FL pBIND-GABPβ pBIND-GABPβ

pBIND-GABP pBIND-GABPβ

(ANK), the region C-terminal to the ankyrin repeats deletion mutant plus full-length pBIND-GABPb versus (CT region), the activation domain (AD), and the leucine each mutant alone. zipper region (LZ), were cloned into a GAL4 DBD con- To test whether GR lacking the entire ligand-binding taining vector (pBIND) to create pBIND-GABPb fusions. region can activate the BRCA1 promoter, the pACT-GR Transient transfections involving each set of deletion fusion series, plus deletion mutants containing the DBD mutants with the complementary full-length protein and cloned into the pFLAG vector, were transiently transfected a GAL4-responsive reporter revealed that the N-terminal to into EPH-4 cells along with the BRCA1 promoter. GR central region of GABPb (including the activation domain) domain mutants, either VP16 (pACT) or FLAG (pFLAG) was involved in the interaction with the full-length GR tagged, containing the N-terminal to central region of GR (Fig. 4A–C, ANK-CT-AD). The increase in luciferase protein, speciﬁcally either the full-length (FL) or TAD- activity from the GAL4-responsive reporter is given as the DBD-HR, activated BRCA1 in the absence of ligand (Fig. ratio of each GABPb deletion mutant plus full-length 5A). In a transient transfection of MCF-7 cells, both VP16 pACT-GR versus each mutant alone, to control for the and FLAG-tagged versions of a GR mutant lacking the entire basal activity of each fragment. Likewise, the N-terminal ligand-binding region (TAD-DBD-HR) activated BRCA1 to central region (DBD and hinge region) of GR was in both the absence and presence of hydrocortisone, empha- involved in the interaction with GABPb (Fig. 4D–F, TAD- sizing that in contrast to the wild-type protein, it is now DBD-HR). The increase in luciferase activity from the immune to the effects of hydrocortisone (Fig. 5B, GR TAD- GAL4-responsive reporter is given as the ratio of each DBD-HR black and white bars). This mutant is unable to

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Unliganded Glucocorticoid Receptor Regulates BRCA1 Expression

A Domain constructs of GABPβ D Domain constructs of GR ANK CT AD LZ TAD DBD HR LBD Figure 4. The interaction between + + b the GR and GABP requires the Full-length GR Full-length GABPβ amino terminal to central regions of both proteins. EPH-4 and MCF-7 B EPH-4 E EPH-4 cells were transiently transfected pBIND-GABPβ domain constructs ± pACT-GR FL pACT-GR domain constructs ± pBIND-GABPβ FL with (A–C) full-length pACT-GR and 5 25 various domain constructs of pBIND- 4 20 GABPb and (D–F) full-length pBIND- 3 15 GABPb and various domain constructs of pACT-GR. Cells were 2 10 Relative ratio Relative treated 24 hours after transfection 1 ratio Relative 5 with ethanol vehicle in serum-free 0 0 K Z Z R D D medium and assayed for luciferase FL CT AD L FL HR AN TAD DB LB CT-AD AD-LZ activity following a 24-hour ANK-CT DBD-HR HR-LBD pBIND EV CT-AD-L pACT EV TAD-DBD ANK-CT-AD AD-DBD-H incubation. GR domain T DBD-HR-LBD abbreviations: TAD, transcriptional C F activation domain; DBD, DNA- MCF-7 MCF-7 pBIND-GABPβ domain constructs ± pACT-GR FL pACT-GR domain constructs ± pBIND-GABPβ FL binding domain; HR, hinge region; 6 15 and LBD, ligand-binding domain. b GABP domain abbreviations: ANK, 4 10 ankyrin repeat region; CT, region C-terminal to the ankyrin repeats; 2 5 Relative ratio ratio Relative AD, activation domain; and LZ, ratio Relative leucine zipper region. 0 0 D R R FL CT A LZ FL HR ANK TAD DBD LBD CT-AD AD-LZ ANK-CT pACT EV DBD-H HR-LBD pBIND EV CT-AD-LZ TAD-DBD ANK-CT-AD AD-DBD-H T DBD-HR-LBD

bind ligand, but can still interact with GABPb via its breast specific molecular mechanism to explain the increased N-terminal to central hinge region, and activate BRCA1 ex- risk associated with psychological stress (16). In this article, pression via its N-terminal transactivation domain. This we have continued to explore the molecular mechanisms mutant also activated BRCA1 expression in the presence of involved and have determined that, contrary to expectations, titrated concentrations of the GR antagonist mifepristone GR is actually a positive regulator of the BRCA1 promoter in (RU-486; Fig. 6A, GR TAD-DBD-HR) whereas the full- the absence of ligand [Fig. 7 (1)]. This represents a novel length GR did not (Fig. 6A, GR FL). RU-486 is a hydro- mechanism of GR function and suggests that it plays a role cortisone analogue that is able to bind to GR but inhibits the in the basal transcription of the BRCA1 gene. Our original transcription of GR target genes (31). As with hydrocorti- model suggested that long-term stress would result in the sone, treatment of mouse and human mammary cells with a protracted elevation of cortisol levels and the consequent comparable concentration of RU-486 [3 mmol/L hydro- ongoing repression of BRCA1 [Fig. 7 (2)]. Although this is cortisone (1 mg/mL) compared with 5 mmol/L RU-486] still a feasible model, these new results also imply that the resulted in decreased expression of endogenous BRCA1 basal levels of GR may play a role in determining BRCA1 (Fig. 6B), indicating that ligand binding, even of a non- levels. That is, reduced GR levels would lead to reduced signaling ligand, to GR is the key physiologic stimulus transactivation of the BRCA1 promoter, which may be for decreased BRCA1 expression. The repressive ability of associated with an increased risk of breast cancer devel- RU-486 may be partly due to its capacity to disrupt unli- opment. It has been reported that GR is differentially ganded GR binding at the promoters of its target genes. expressed in a variety of breast tissues, with strong expression in normal myoepithelial cells, and rare expression in nonmetaplastic carcinomas (33). Similarly, GR status has Discussion been negatively correlated with histologic tumor grade in BRCA1 has been associated with a wide variety of cellular estrogen receptor (ER)-negative/progesterone receptor functions, most recently the ability to regulate luminal breast (PR)-negative ductal intraepithelial neoplasia and invasive cell differentiation and satellite DNA expression (9, 32). breast carcinoma tumors (34). Nuclear immunoreactivity Regardless of the means by which BRCA1 blocks tumor to GR was found to decrease significantly with both tumor formation, it is clear that its functional loss through muta- development and histologic grade (26, 34), suggesting a tional inactivation or by transcriptional downregulation connection between functional GR levels and tumorigen- leads to breast tumorigenesis. We have previously demon- esis. However, it has also been reported that GR expres- strated that BRCA1 expression is negatively regulated by sion is elevated in stage III infiltrating ductal carcinomas hydrocortisone (19) and suggested that this could provide a (35), and GR expression has also been correlated with

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This raises the possibility that events in utero, perinatally, A and/or during puberty in humans could result in the long- term reduction in GR levels either via systemic methylation 3 of the GR promoter, or via any other mechanism which would bring about a decrease in GR levels, including long- 2 term glucocorticoid exposure or increased rate of protein promoter activity turnover [Fig. 7 (4)]. Reduction in GR levels would subse- 1 quently result in decreased BRCA1 levels due to the loss of BRCA1 positive regulation by GR (Fig. 7). These reduced BRCA1 0 levels would then be reﬂected in increased breast cancer risk. D D V D Relative Relative TA LB Elevated progesterone levels have been suggested to lead to pACT EV HR-LBD TAD-DBD pFLAG E TAD-DBD an increase in the breast stem cell population (41, 42); altered pACT-GR FL TAD-DBD-HRDBD-HR-LBD TAD-DBD-HRDBD-HR-LB FLAG-GRa FL BRCA1 levels as a consequence of altered GR levels may BRCA1 TCAp GALFp produce the same effect through decreased blocking differentiation. B

3 -HC +HC A

2 UT

promoter activity promoter 2.0 + 1 μmol/L RU-486 + 10 μmol/L RU-486 1 1.5 BRCA1

0 1.0 Relative Relative

GR FL AG EV GR FL 0.5 pACT EV pFL

GR TAD-DBD-HR GR TAD-DBD-HR 0.0

pACT pFLAG BRCA1 promoterRelative activity EV GR FL

Figure 5. GR domains activate the BRCA1 promoter in the absence of GR TAD-DBD-HR ligand binding. A, EPH-4 cells were transiently transfected with the L6 B BRCA1 promoter reporter construct and expression vectors for full- 1.5 length FLAG-GR, full-length pACT-GR, and GR domain constructs either UT fused to VP16 (pACT) or without the VP16 fusion (pFLAG). Cells were + RU-486 treated 24 hours after transfection with ethanol vehicle in serum-free medium and assayed for luciferase activity following a 24-hour 1.0 incubation. GR domain abbreviations are as in Fig. 4. B, EPH-4 cells were expression transiently transfected with the L6 BRCA1 promoter reporter construct and both pFLAG and pACT expression vectors for each the full-length GR 0.5 (GR FL) and GR lacking the LBD (GR TAD-DBD-HR) as well as empty vectors (pFLAG EV and pACT EV). Cells were treated 24 hours after transfection with either ethanol vehicle (HC) or 1 mg/mL hydrocortisone 0.0 (þHC) and assayed for luciferase activity following a 24-hour incubation. BRCA1 Relative

EPH-4 MCF-10A 184-hTERT

ﬁ patient age, with its expression being signi cantly higher Figure 6. GR antagonist RU-486 decreases BRCA1 expression. A, EPH-4 in tumors from patients older than 50 years (36). cells were transiently transfected with the L6 BRCA1 promoter reporter Our hypothesis that reduced GR levels may result in construct and expression vectors for full-length GR (GR FL) and GR reduced BRCA1 expression is of particular interest given lacking the LBD (GR TAD-DBD-HR). Cells were treated 24 hours after recent work suggesting that GR levels may be under long- transfection with either ethanol vehicle (UT) or 1 or 10 mmol/L RU-486 and assayed for luciferase activity following a 24-hour incubation. B, 184- term epigenetic regulation (37) and that repression of GR hTERT, MCF-10A, and EPH-4 cells were treated 24 hours after plating expression may be associated with speciﬁc stressful events with either ethanol vehicle (UT) or 5 mmol/L RU-486, and RNA was occurring at various times during development (38). Both harvested following a 48-hour incubation. qRT-PCR analysis of human and rat experiments have found that perinatal stress endogenous BRCA1 expression was conducted using primers for either BRCA1 BRCA1 C can result in methylation of the GR promoter in the brain human or mouse . t values were normalized to either HPRT1 (for 184-hTERT and MCF-10A) or TBP (EPH-4) internal control Ct [refs. 38, 39; Fig. 7 (3)], though it also appears that this may values for triplicate samples and are presented as the level of expression occur systemically, being detectable in peripheral blood (40). in relation to untreated cells for each cell line.

566 Mol Cancer Res; 10(4) April 2012 Molecular Cancer Research

Unliganded Glucocorticoid Receptor Regulates BRCA1 Expression

HPA axis

Anterior Adrenal Hypothalamus pituitary cortex Breast 4 Methyl or other factors GABP

2 Stress CRH ACTH Cortisol GRnuc BRCA1 1 +GR Target GRcyto GRcortisol 3 Methyl GRcortisol −GR Target

Figure 7. Model of GR regulation. 1, in the absence of stress (black), the unliganded nuclear GR (GRnuc) interacts with and positively regulates BRCA1 via GABP. 2, stress (red) increases production of corticotrophin-releasing hormone (CRH), which induces release of adrenocorticotropic hormone (ACTH), which results in cortisol secretion. The binding of cortisol to both nuclear and cytoplasmic GR (GRcyto) causes them (GRcortisol) to relocate to regular GR target genes. This results in reduced BRCA1 expression. The presence of cortisol negatively feeds back (blue) to both the hypothalamus and the pituitary, reducing the secretion of both CRH and ACTH. 3, as a result of prolonged stress exposure, methylation of the GR promoter in the brain decreases GR expression, resulting in loss of negative feedback at the hypothalamic-pituitary-adrenal (HPA) axis. Sustained production of cortisol results in loss of BRCA1-positive regulation by unliganded GR. 4, in breast tissue, systemic methylation of the GR promoter, or other factors, such as long-term glucocorticoid exposure and/or increased rate of protein turnover, leads to decreased GR expression, and subsequently to decreased BRCA1 activation.

We have shown that GR interacts with the b-subunit of absence of ligand (25), but this effect is dependent on the ets transcription factor GABP and that this interaction TNF-a and is thus not a constitutive change as we have requires the N-terminal to central regions of both proteins observed. Indeed, reports of ligand-independent activation (GR TAD-DBD-HR; GABPb ANK-CT-AD). Several pro- by other steroid hormone receptors have typically been in teins have been reported to interact with GABPb. The response to other stimuli (49). Our studies indicate that coactivator HCF binds the TAD of GABPb via its central unliganded GR is recruited to promoters through its inter- region to coordinate the assembly of the herpes simplex virus action with GABP. This suggests that a fourth class of GR- enhancer complex (43). The second zinc finger motif (CR2) regulated genes exist, possibly overlapping with GABP target of YEAF1 (YY1 and E4TF1/hGABP-associated factor 1) genes. ChIP-seq analysis of GR binding sites in A549 lung interacts with a small internal region of GABPb to repress cells has revealed some 2,600 genes that are bound by GABP-dependent transcription (44). The N-terminus of unliganded GR, though with a much lower signal than GABPb interacts with the C-terminus of E2F1, including its liganded GR targets (50), suggesting this may be a wide- transactivation and retinoblastoma protein (pRb)-binding spread mechanism for gene regulation. The regulation of domain (45). GR physically interacts with and negatively basal expression of target genes by unliganded GR suggests regulates the transcriptional activity of proteins such as NF- that in some contexts the endogenous levels of GR may be kB and activator protein (AP-1) via the second zinc finger of significant for gene regulation. That is, the level of unli- its DBD. In contrast, GR interacts with nuclear receptor ganded nuclear GR in a given cell will help to determine the coactivators of the p160 family as well as the CBP/p300 expression level of its target genes. This is in contrast to genes family mainly through its secondary activating function regulated by liganded GR where gene activation is depen- domain (AF2), located in the C-terminal LBD of the protein dent primarily on cortisol levels rather than GR levels. Given (46). Positive interactions with ligand-bound GR have been the wide degree of variability seen in GR protein levels across shown to occur through the LBD, and we have shown that different cell types and its complex transcriptional regulation the interaction between the unliganded GR and GABPb is (with numerous 50 exons; ref. 37), the regulation of genes by not dependent on the GR LBD. It is therefore possible that unliganded GR may be very particular to a particular tissue separate domains in GR may exist for targets of liganded and or cell. unliganded GR, whereas a region common to multiple Glucocorticoid signaling in the breast is associated with protein interactions may be involved with GABPb. growth as well as the regulation of involution after lactation Classic models of GR signaling include ligand-induced (51), where it seems to primarily regulate apoptosis (52). The binding to both positive and negative recognition elements nature of this regulation may actually change depending on (47) as well as directly binding and repressing other tran- the differentiation state, as dexamethasone induces cell-cycle scription factors such as NF-kB and AP-1 (48). The obser- inhibitors such as p21 in undifferentiated cells, whereas in vation of a constitutive positive regulatory role for GR in the differentiated cells it reduces their expression and inhibits absence of ligand is unique and adds a new dimension to apoptosis (53). The target genes of unliganded GR seem to the GR pathway. A recent publication has reported negative be downregulated in the presence of glucocorticoids, sug- regulation of the interleukin (IL)-6 gene by GR in the gesting that these genes may be opposing the function of

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liganded GR target genes. Given the known role of BRCA1 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- tational analysis): H.D. Ritter, L. Antonova, C.R. Mueller in regulating apoptosis (54), its induction by unliganded Writing, review, and/or revision of the manuscript: H.D. Ritter, L. Antonova, C.R. GR may represent a proapoptotic signal which is then lost Mueller Administrative, technical, or material support (i.e., reporting or organizing data, in response to the antiapoptotic signal from hydrocorti- constructing databases): C.R. Mueller sone treatment. It is possible that the stress-induced Study supervision: C.R. Mueller downregulation of BRCA1, as well as other proapoptotic genes also regulated by unliganded GR, results in reduced Acknowledgments monitoring and elimination of abnormal cells either dur- The authors thank Rachael Klinoski, Valerie Kelly-Turner, and Sherri Nicol for ing normal cellular turnover or during postlactational their excellent technical assistance, Dr. Calvin Roskelley for providing cell lines and the PRR constructs, as well as Dr. Keith Yamamoto for providing the GRwt and regression. This work provides new insight into the pos- GRL501P constructs. sible mechanisms responsible for the stress-induced increased risk of breast cancer as well as having wide Grant Support implications for understanding the action of the gluco- This work was funded by a grant from the Canadian Breast Cancer Foundation- corticoids and GR. Ontario Region, as well as a Doctoral Postgraduate Scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC PGS-D) to H.D. Ritter, a Student Fellowship from the Canadian Breast Cancer Foundation-Ontario Disclosure of Potential Conﬂicts of Interest Region to L. Antonova, and a Research Project Grant to C.R. Mueller from the No potential conﬂicts of interest were disclosed. Canadian Breast Cancer Foundation-Ontario Region. The costs of publication of this article were defrayed in part by the payment of page advertisement Authors' Contributions charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Conception and design: H.D. Ritter, L. Antonova, C.R. Mueller Development of methodology: H.D. Ritter, L. Antonova, C.R. Mueller Acquisition of data (provided animals, acquired and managed patients, provided Received September 7, 2011; revised January 10, 2012; accepted February 6, 2012; facilities, etc.): H.D. Ritter, L. Antonova published OnlineFirst February 10, 2012.

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www.aacrjournals.org Mol Cancer Res; 10(4) April 2012 569

The Unliganded Glucocorticoid Receptor Positively Regulates the Tumor Suppressor Gene BRCA1 through GABP Beta

Heather D. Ritter, Lilia Antonova and Christopher R. Mueller

Mol Cancer Res 2012;10:558-569. Published OnlineFirst February 10, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-11-0423-T

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