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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1364
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 11 4514-4519
Copyright © 2006 by The Endocrine Society

RAPID COMMUNICATION

BRCA1 Negatively Regulates the Cancer-Associated Aromatase Promoters I.3 and II in Breast Adipose Fibroblasts and Malignant Epithelial Cells

Meiling Lu, Dong Chen, Zhihong Lin, Scott Reierstad, Amy M. Trauernicht, Thomas G. Boyer and Serdar E. Bulun

Department of Obstetrics and Gynecology (M.L., S.R., Z.L., D.C., S.E.B.), Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611; and Department of Molecular Medicine (A.M.T., T.G.B.), Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245

Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D., Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611. E-mail: s-bulun{at}northwestern.edu.

Abstract

Context: Heterozygous mutations of the breast cancer susceptibility gene 1 (BRCA1) gene that lead to haploinsufficiency increase the risk of breast cancer. The underlying mechanism is unknown.

Objective: Because excessive estrogen production increases the risk of breast cancer, we determined whether BRCA1 suppresses aromatase expression and thus local estrogen production in breast adipose fibroblasts (BAFs) and breast malignant epithelial cells by interacting with the cancer-associated promoter I.3/II region of the aromatase gene.

Results: Treatment of BAFs with prostaglandin E2 or a surrogate hormonal cocktail of dibutyryl (Bt2) cAMP plus phorbol 12, 13-diacetate (PDA) significantly reduced BRCA1 levels and induced aromatase mRNA levels. Reduction of BRCA1 in BAFs and in MCF7 and SKBR3 malignant breast epithelial cells using small interfering RNA (siRNA) or small hairpin RNA significantly increased aromatase mRNA levels and enzyme activity. This effect of BRCA1 was mediated via selective inhibition of aromatase promoters I.3 and II that are up-regulated by prostaglandin E2 or Bt2cAMP+PDA treatment. Chromatin immunoprecipitation assays revealed that BRCA1 binds directly to the aromatase promoter I.3/II region and that BRCA1 binding is abolished by treatment with Bt2cAMP+PDA.

Conclusions: Selective inhibition of aromatase expression by BRCA1 binding to the I.3/II tumorigenic promoter region may be an important protective mechanism against breast cancer development.

ESTROGEN PLAYS AN important role in the development and progression of breast cancer (1), which has led to the development of aromatase inhibitors that block estrogen biosynthesis to treat estrogen receptor (ER)-positive breast tumors (2). The main source of circulating estrogen in premenopausal women is the ovary. In postmenopausal women, the predominant site of estrogen synthesis shifts to the peripheral tissues such as fat and skin (3, 4). As such, peripheral estrogen production is believed to be a key factor in the development of estrogen-dependent diseases such as breast cancer in postmenopausal women (3).

The aromatase gene encodes the key enzyme that converts androgens to estrogens (4). It has been reported that postmenopausal breast cancer is strongly associated with elevated aromatase expression and estrogen biosynthesis in breast adipose tissue (5), and recent evidence indicates that up-regulation of local aromatase expression and activity within breast tumor tissue is critical for tumor growth (6). Aromatase is expressed in a number of human cells and tissues, including ovarian granulosa cells, skin, and adipose fibroblasts (7). Aromatase mRNA and activity in breast adipose fibroblasts (BAFs) has been implicated in the pathophysiology of breast cancer (8) such that adipose fibroblasts provide not only structural support for cancer growth but also hormonal support via up-regulation of local estrogen production. Aromatase expression in malignant epithelial cells may also be an important source of estrogen that promotes tumor growth (9). Indeed, mouse models in which aromatase is overexpressed support the role of local aromatase production in breast tissue proliferation and hyperplasia (9, 10).

Expression of aromatase is controlled by several distinct and somewhat tissue-specific promoters (11). In disease-free breast adipose tissue, aromatase is usually expressed at low levels, primarily under direction of the distal promoter I.4, whereas in adipose tissue of tumor-bearing breasts, aromatase mRNA levels are increased via stimulation of three promoters, I.7, I.3, and II (12). The proximal promoters I.3 and II are located close to each other and are coordinately up-regulated by prostaglandin E2 (PGE2) or cAMP plus phorbol esters (13, 14). That activation of promoters I.3 and II is a critical event responsible for aberrant aromatase expression and local estrogen biosynthesis in tumor-bearing breast tissues has led to the possibility of promoter-specific aromatase inhibitors for the treatment of breast cancer (3).

The breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene identified over 12 yr ago (15), and germline mutations in BRCA1 predominantly lead to breast and ovarian cancers. A significant proportion of sporadic breast cancers have low levels of BRCA1 expression, suggesting that even partial loss of BRCA1 function may facilitate tumorigenesis (16). BRCA1 is ubiquitously expressed and is involved in several key cellular processes, including DNA repair, DNA damage control, chromatin remodeling, and mitotic spindle formation (17). BRCA1 is also involved in transcriptional regulation through interaction with a number of transcription factors, including p53, ER{alpha}, ATF1, and Jun (18, 19, 20, 21). Yet, the general cellular functions of BRCA1 fail to explain the tissue-specific nature of cancers caused by mutations in the BRCA1 gene.

Considering that germline mutation of BRCA1 predominantly causes breast and ovarian cancers, two major estrogen-responsive tissues, we speculated that BRCA1 tumor suppressor function might be related to modulation of local estrogen production or action. Animal studies have shown that BRCA1 expression is inversely correlated with aromatase expression in the ovary, such that levels are high in the granulosa cells of developing follicles, and low in large antral/preovulatory follicles, when aromatase expression is at its peak (22). In addition, Hu et al. (23) recently reported that BRCA1 modulates aromatase expression in an ovarian granulosa cell line and in stromal cells obtained from abdominal adipose tissue.

Thus, we decided to investigate the role of BRCA1 in the regulation of aromatase expression in primary human BAFs and breast cancer epithelial cell lines. Here, we demonstrate that knockdown of BRCA1 expression increased aromatase expression and enzyme activity. In addition, the negative regulation of aromatase transcripts by BRCA1 was promoter-specific in that BRCA1 knockdown led to increases in I.3 and II, but not I.4, activity and BRCA1 directly interacted with a sequence within the aromatase proximal promoters I.3 and II. These data suggest that the tumor suppressive effect of BRCA1 may be partially due to its repression of local estrogen synthesis in breast tissue and may reveal opportunities for the development of therapeutics targeted to estrogen-responsive tumors.

Materials and Methods

Cell culture and reagents

Human breast adipose tissues were obtained at the time of surgery from women undergoing reduction mammoplasty following a protocol approved by the Institutional Review Board for Human Research of Northwestern University. BAFs were isolated as described previously (24, 25). MCF-7 and SKBR3 mammary carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). MCF7 cells were grown as described previously (25). SKBR3 cells were grown in RPMI 1640 with 4 mM L-glutamine and 10% fetal bovine serum. At approximately 80% confluence, BAFs were placed in serum-free DMEM/F-12 for 16 h and treated with vehicle, dibutyryl (Bt2) cAMP (0.5 mM) plus phorbol 12, 13-diacetate (PDA, 100 nM), or PGE2 (1 µM). Cells were collected for RNA or protein isolation. Bt2cAMP, PDA, and PGE2 were purchased from Sigma-Aldrich (St. Louis, MO).

Immunoblotting

Cell lysates were prepared in modified RIPA buffer and analyzed by immunoblotting as described previously (24) using mouse monoclonal anti-BRCA1 (Ab-1) (Calbiochem, La Jolla, CA) and monoclonal anti-ß-Actin (Sigma-Aldrich).

Real-time RT-PCR

Total RNA was extracted using TRI reagent (Sigma-Aldrich). cDNAs were made from 3 µg RNA with the Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). BRCA1, aromatase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were amplified by real-time PCR using the ABI TaqMan Gene Expression system (purchased from Applied Biosystems, Foster City, CA) and the ABI Prism 7900 Sequence Detection System. Values for each gene were normalized to the expression levels of GAPDH. Three independent experiments were performed to demonstrate reproducibility.

Exon-specific RT-PCR amplification

Amplification of the untranslated 5'-ends of promoter-specific aromatase transcripts from BAFs was accomplished with exon-specific oligonucleotide pairs as described previously (24, 25).

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed as described previously using a kit from Upstate Biotechnology (Chicago, IL) (24). The sonicated chromatin fraction was immunoprecipitated with an equal amount of either mouse IgG or an anti-BRCA1 antibody (Calbiochem). After reversal of cross-linking, chromosomal DNA was purified and analyzed by PCR for the presence of the promoter I.3/II region (–302/–38 bp). The primer sequences used for PCR were as described previously (26): forward, 5' AAC CTG CTG ATG AAG TCA CAA 3'; reverse, 5' TCA GAC ATT TAG GCA AGA CT 3'.

BRCA1 knockdown in BAFs and SKBR3 and MCF7 breast cancer cell lines

BRCA1 knockdown was accomplished using siRNA oligonucleotide transfection. Control (nontargeting) siRNA and BRCA1 siRNA were purchased from Dharmacon (Chicago, IL). BAFs were plated in 60-mm dishes and were transfected with 100 nM control siRNA or BRCA1 siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) reagent according to the manufacturer’s protocol. Forty-eight hours after transfection, cells were either collected for cell lysate preparation or serum-starved for 16 h, followed by treatment with Bt2cAMP+PDA for 24 h. Total mRNA was collected for analysis by real-time RT-PCR. For SKBR3 cells, 100 nM control siRNA or BRCA1 siRNA was transfected in six-well plates using DharmaFECT 1 reagent according to the manufacturer’s protocol (Dharmacon). Cells were collected after 48 h for mRNA or protein preparation. MCF7 cells were transfected with DNA-based siRNAs using the pSUPER.retro.puro vector (OligoEngine, Seattle, WA). pSUPER.retro.puro-BRCA1 sh 385 or pSUPER.retro.puro-BRCA1 sh 5376 targeted BRCA1 sequences corresponding to nucleotides 385–403 (GCTACAGAAACCGTGCCAA), and 5376–5394 (GAAGGAGCTTTCATCATTC), respectively, according to the manufacturer’s protocol. To generate the retrovirus, plasmid DNA (10 µg) was transfected into the Phoenix amphotropic retrovirus packaging cell line (ATCC) by calcium phosphate transfection. Medium containing virus was collected 48 h after transfection. MCF7 cells were infected with one of the retroviruses (pSUPER.retro.puro vector or pSUPER.retro.puro-BRCA1 sh 385 or pSUPER.retro.puro-BRCA1 sh 5376) and selected in 1 µg/ml puromycin for 10 d (Sigma-Aldrich). Reduction of BRCA1 expression was confirmed by real-time RT-PCR and immunoblotting.

Aromatase assay

To study the effect of BRCA1 knockdown on aromatase activity in BAFs, the cells were plated in six-well plates and transfected with 100 nM control siRNA or BRCA1 siRNA as described above. Forty-eight hours after transfection, cells were serum-starved for 24 h. The mixture of [3H]-labeled and cold androstenedione was added to each well, and cells were incubated for another 6 h. Each treatment was performed in triplicate. Aromatase activity was measured using a tritiated water-release assay described previously (24).

Results

PGE2 and Bt2cAMP+PDA regulates BRCA1 and aromatase expression in BAFs

It has been reported that tumor-derived factors such as PGE2 or a cocktail of Bt2cAMP+PDA can trigger aberrant activation of the aromatase I.3 and II promoters in primary human BAFs (13, 14). We postulated that PGE2 or Bt2cAMP+PDA may counter the suppressive effect of BRCA1 on the aromatase I.3/II promoter by down-regulating BRCA1 expression, thus contributing to elevated aromatase expression. To test this possibility, we treated BAFs with PGE2 and analyzed BRCA1 and aromatase mRNA levels after 24 h. Aromatase mRNA levels were increased up to 2.5-fold, whereas BRCA1 mRNA levels were decreased to half (Fig. 1AGo). BAFs treated with Bt2cAMP+PDA showed a similar expression pattern, with aromatase mRNA levels increasing 128-fold and 237-fold, and BRCA1 mRNA levels decreasing 2.7-fold and 4.6-fold at 8 h and 24 h after treatment, respectively (Fig. 1Go, B and C). BRCA1 protein levels were also decreased after 24 h of Bt2cAMP+PDA treatment (Fig. 1DGo).


Figure 1
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  		FIG. 1. PGE2 and Bt2cAMP+PDA regulate BRCA1 and aromatase mRNA levels in a reciprocal fashion in BAFs. A, BAFs were treated with PGE2 for 24 h. BRCA1 and aromatase mRNA was analyzed by real-time RT-PCR. B and C, BAFs were treated with Bt2cAMP+PDA for 8 or 24 h. BRCA1 and aromatase mRNA levels were analyzed by real-time RT-PCR. D, BAFs were treated as in panels B and C, and cell lysates were prepared for immunoblotting with anti-BRCA1 and ß-actin (loading control) antibodies. Data are representative of three experiments. *, P < 0.01; and **, P < 0.01 vs. vehicle-treated cells.

 
To determine whether estradiol itself affects BRCA1 or aromatase gene expression, we treated MCF7 cells with estradiol, the antiestrogen ICI 182,780, or in combination of both. We found that none of the treatment affects BRCA1 or aromatase mRNA levels (data not shown).

Knockdown of BRCA1 increases endogenous levels of aromatase mRNA and enzyme activity in BAFs and breast malignant epithelial cells

To explore a direct functional relationship between aromatase and BRCA1 in BAFs, we reduced endogenous BRCA1 levels using BRCA1-specific siRNA. As shown in Fig. 2AGo, BRCA1 protein levels were reduced by half in BRCA1 siRNA-transfected cells compared with cells transfected with control siRNA. Reduction of BRCA1 led to an increase in aromatase mRNA levels up to 8-fold (Fig. 2BGo). This effect was further enhanced in cells treated with Bt2cAMP+PDA (Fig. 2BGo). In addition, we examined the effect of BRCA1 knockdown on aromatase enzyme activity in BAFs. As shown in Fig. 2CGo, knockdown of BRCA1 led to a significant increase in aromatase enzyme activity by 2-fold.


Figure 2
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  		FIG. 2. Reduction of BRCA1 levels leads to an elevation of endogenous aromatase expression and enzyme activity in BAFs and breast cancer epithelial cell lines. A, BAFs were transfected with 100 nM control nontargeting siRNA or BRCA1 siRNA. Lysates were collected from control siRNA and BRCA1 siRNA-transfected cells for immunoblotting with anti-BRCA1 or ß-actin (loading control) antibody 48 h after transfection. B, At 48 h after transfection, BAFs were serum-starved for 16 h, followed by treatment with vehicle or Bt2cAMP+PDA for 24 h. BRCA1 and aromatase mRNA levels were analyzed by real-time RT-PCR. C, BRCA1 knockdown in BAFs significantly increase aromatase enzyme activity measured by the rate of conversion of androstenedione to estrone. D, SKBR3 cells were transfected with 100 nM control nontargeting siRNA or BRCA1 siRNA. BRCA1 and aromatase mRNAs levels were analyzed by real-time RT-PCR 48 h after transfection. E, MCF7 cells were infected with retroviral constructs, and stable pools of BRCA1 knockdowns were selected with puromycin. BRCA1 and aromatase mRNA levels were analyzed by real-time RT-PCR. Data are representative of three experiments. *, P < 0.01; **, P < 0.01; {dagger}, P < 0.01; and {ddagger}, P < 0.01 vs. control nontargeting siRNA.

 
It has been previously reported that several breast cancer cell lines, including the ER{alpha}-positive MCF7 and ER{alpha}-negative SKBR3 lines, also express aromatase, suggesting that breast tumor cells may affect estrogen-dependent tumor growth in an intracrine manner (27). Thus, we tested whether BRCA1 knockdown also led to an up-regulation of aromatase mRNA levels in these cell lines. Indeed, reduction of BRCA1 levels in MCF7 and SKBR3 malignant breast epithelial cells significantly increased aromatase mRNA levels (Fig. 2Go, D and E).

BRCA1 regulates aromatase expression via promoters I.3 and II in BAFs

To determine the promoter involved in BRCA1-dependent regulation of aromatase expression in BAFs, we determined the levels of promoter-specific aromatase mRNA species by exon-specific semiquantitative RT-PCR. As shown in Fig. 3Go, BRCA1 knockdown led to a significant increase in aromatase promoter I.3 and II-specific transcripts, whereas BRCA1 knockdown did not have any effect on promoter I.4-specific transcript levels.


Figure 3
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  		FIG. 3. BRCA1 knockdown leads to aromatase promoter-specific activation in BAFs. RNA was isolated from control siRNA and BRCA1 siRNA-transfected cells 48 h after transfection. Semiquantitative RT-PCR was performed to measure the levels of the first exons specific for each aromatase promoter. GAPDH mRNA levels served as the control. Data are representative of three experiments.

 
BRCA1 interacts with the promoter I.3 and II region of the aromatase gene

We performed ChIP assays to determine whether BRCA1 binds directly to the aromatase promoter I.3/II region. An initial ChIP performed on BAFs grown to near confluence in the presence of 10% fetal bovine serum revealed that BRCA1 bound the aromatase I.3/II region under basal conditions (Fig. 4AGo). Whereas BRCA1 remained bound to the aromatase I.3/II regulatory region under serum-starved conditions, treatment of cells with Bt2cAMP+PDA abolished BRCA1 binding (Fig. 4BGo).


Figure 4
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  		FIG. 4. BRCA1 is associated with the promoter I.3/II region of the aromatase gene. BAFs were grown either (A) in the presence of serum or (B) under serum-starvation for 16 h, followed by treatment with vehicle or Bt2cAMP+PDA for 24 h. ChIP was performed using equal amounts of either nonspecific mouse IgG or an anti-BRCA1 antibody. A 265-bp sequence in the common regulatory region of the aromatase promoters I.3 and II was amplified by PCR. A, Under basal conditions, BRCA1 bound to the promoter I.3/II region. B, Under serum-starved conditions, BRCA-1 bound to the promoter I.3/II region (first 3 lanes), whereas treatment with Bt2cAMP+PDA abolished binding of BRCA1. Input, PCR amplification of DNA without immunoprecipitation; IgG, PCR after immunoprecipitation with nonspecific mouse IgG; BRCA1, PCR after immunoprecipitation with an anti-BRCA1 antibody.

 
Discussion

Estrogen biosynthesis in extraovarian tissues is extraordinarily important in the pathophysiology of postmenopausal breast cancer, as evidenced by the efficacy of aromatase inhibitors in the treatment of postmenopausal breast cancer (2, 3). Elevated aromatase expression in BAFs is a critical source of local estrogen that signals through ER to transactivate a number of genes that promote breast carcinoma growth (3). Thus, local up-regulation of aromatase activity and estrogen production in the adipose tissue of cancer-free breasts may contribute to the risk of developing breast carcinomas (28). This has led to a number of ongoing or planned studies for the prophylactic use of aromatase inhibitors to prevent breast cancer in high-risk groups (3).

Understanding the mechanisms underlying local aromatase up-regulation in the breast is critical to the development of more effective, targeted treatments. Here, we provide evidence that BRCA1 plays a pivotal role in repressing aromatase expression in the breast tumor microenvironment. Knockdown of BRCA1 led to an elevation of endogenous aromatase expression via the specific activation of the tumor-associated promoters I.3 and II in BAFs. Moreover, BRCA1 directly bound to the aromatase I.3/II promoter region, and hormonal stimulation of aromatase promoter activity decreased both BRCA1 levels and promoter binding. In malignant breast tumors, aromatase is expressed in both fibroblasts and malignant epithelial cells (29, 30). Our data indicate that BRCA1 regulates aromatase expression in MCF7 and SKBR3 breast cancer cell lines, suggesting that BRCA1 deficiency in malignant breast tumors may lead to further aromatase overexpression in breast cancer.

A number of transcription factors bind to and regulate the aromatase promoters I.3 and II, including C/EBPß (CCAAT/enhancer binding protein-ß), ATF2 (activating transcription factor 2), Jun, SF-1 (steroidogenic factor-1), LRH-1 (liver receptor homolog-1), CREB (cAMP response element binding protein), and several orphan nuclear receptors (24, 25, 26, 30). Some of these transcription factors are known to interact with BRCA1 (18, 20). Current studies are investigating the possibility that BRCA1 may interact with one or more of the transcription factors that occupy the aromatase promoter I.3/II region to suppress their enhancer function.

In this study, we used both PGE2 and a combination of Bt2cAMP+PDA to stimulate aromatase expression. It has been previously shown that both treatments induce aromatase via the promoter I.3/II region through similar signaling pathways (13, 14). We chose to use Bt2cAMP+PDA in most experiments because it provides a more robust induction of aromatase.

Although the mechanisms underlying BRCA1 tumor suppressor properties are still largely unknown, our studies suggest that suppression of aromatase activity by BRCA1 in the breast may contribute to the prevention of breast cancer development by modulating local estrogen production. If one consequence of BRCA1 haploinsufficiency is aromatase overexpression, prophylactic use of aromatase inhibitors may decrease breast cancer risk in this particular patient group.

Acknowledgments

We thank Meagan Porter and Stacey Tobin for editorial assistance.

Footnotes

This work was supported by National Cancer Institute Grant CA67167 and grants from the Northwestern Memorial Foundation, AVON Foundation, Lynn Sage Foundation, and Friends of Prentice.

Disclosure statement: The authors have nothing to disclose.

First Published Online August 29, 2006

Abbreviations: BAFs, Breast adipose fibroblasts; Bt2, dibutyryl; ChIP, chromatin immunoprecipitation; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDA, phorbol 12, 13-diacetate; PGE2, prostaglandin E2; siRNA, small interfering RNA.

Received June 26, 2006.

Accepted August 21, 2006.

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CCAAT/Enhancer Binding Protein {beta} Regulates Aromatase Expression via Multiple and Novel Cis-Regulatory Sequences in Uterine Leiomyoma
J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 981 - 991.
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 J. Biol. Chem.Home page
K. Subbaramaiah, C. Hudis, S.-H. Chang, T. Hla, and A. J. Dannenberg
EP2 and EP4 Receptors Regulate Aromatase Expression in Human Adipocytes and Breast Cancer Cells: EVIDENCE OF A BRCA1 AND p300 EXCHANGE
J. Biol. Chem., February 8, 2008; 283(6): 3433 - 3444.
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 Mol. Endocrinol.Home page
Y. Lu, A. Amleh, J. Sun, X. Jin, S. D. McCullough, R. Baer, D. Ren, R. Li, and Y. Hu
Ubiquitination and Proteasome-Mediated Degradation of BRCA1 and BARD1 during Steroidogenesis in Human Ovarian Granulosa Cells
Mol. Endocrinol., March 1, 2007; 21(3): 651 - 663.
[Abstract] [Full Text] [PDF]

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