Enhancement of BRCA1 gene expression by the peroxisome ... - Nature

Oncogene (2003) 22, 5446–5450

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Enhancement of BRCA1 gene expression by the peroxisome proliferator-activated receptor c in the MCF-7 breast cancer cell line Miguel Pignatelli1, Claudia Cocca1,3, Angel Santos2 and Ana Perez-Castillo*,1 1 2

Instituto de Investigaciones Biome´dicas, Consejo Superior de Investigaciones Cientı´ficas-Universidad Autońoma de Madrid, Spain; Departamento de Bioquı´mica y Biologıá Molecular. Facultad de Medicina, Universidad Complutense de Madrid, Spain

BRCA1 has been linked to the genetic susceptibility of a majority of familial breast and ovarian cancers. Several lines of evidence indicate that BRCA1 is a tumor suppressor and its expression is downregulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 gene expression might lead to new insights into the pathogenesis and treatment of these tumors. Peroxisome proliferator-activated receptor gamma (PPARc) is a member of the nuclear receptor superfamily that has well-established roles in the regulation of adipocyte development and glucose homeostasis. More recently, it has been shown that ligands of PPARc have a potent antitumorigenic activity in breast cancer cells. In the present study we have found that two distinct ligands of PPARc; 15-deoxyD-12,14-prostaglandin J2 (15dPG-J2) and rosiglitazone, increase the levels of BRCA1 protein in human MCF-7 breast cancer cells. Immunofluorescence microscopy analysis showed that, after treatment with 15dPG-J2, the BRCA1 protein is mainly localized in the nucleus. Functional analysis by transient transfection of different 50 -flanking region fragments, as well as gel mobility shift assays and mutagenic analysis, suggests that the effects of 15dPG-J2 and rosiglitazone are mediated through a functional DR1 located between the nucleotides 241 and 229, which is a canonical PPARc type response element. Our data suggest that PPARc is a crucial gene regulating BRCA1 gene expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer. Oncogene (2003) 22, 5446–5450. doi:10.1038/sj.onc.1206824 Keywords: BRCA1; PPARg; regulation

Breast cancer is one of the most common neoplasms in women and is a leading cause of cancer-related deaths worldwide. Despite significant improvements in cancer diagnosis and treatment, approximately one-quarter of breast cancer patients will succumb to their disease. *Correspondence: A Perez-Castillo; Arturo Duperier, 4. 28029Madrid, E-mail: [email protected] 3 Current address: Departamento de Radioiso´topos, Facultad de Farmacia y Bioquı´ mica, Universidad de Buenos Aires, Argentina Received 12 December 2002; revised 25 April 2003; accepted 30 May 2003

Therefore, the development and application of new, molecularly based therapies is of utmost importance. The key to the development of such rational preventative and therapeutic approaches lies in the identification of genes and biochemical pathways involved in breast tumorigenesis. A multitude of molecular genetic changes have been reported in human breast cancer, however, the pathogenic significance of these events as it relates to the progression of breast cancer is poorly understood (Nathanson et al., 2001; Baer and Ludwig, 2002). Many data implicate BRCA1 as a tumor suppressor gene in breast and ovarian carcinomas. Overexpression of BRCA1 induces genes in the apoptotic pathway (Harkin et al., 1999; MacLachlan et al., 2000) and increased expression of this protein leads to repression of estrogen receptor-mediated transcription (Fan et al., 1999, 2001). Accordingly with this role, mutations in the BRCA1 gene are associated with familial breast and ovarian cancer and its expression is reduced or undetectable in sporadic breast and ovarian carcinomas and in different breast cancer cell lines (Thompson et al., 1995; Wilson et al., 1999). However, the regulation of BRCA1 expression is not well established, although several regions of its promoter (Suen and Goss, 1999; Thakur and Croce, 1999), some cellular factors (Budhram-Mahadeo et al., 1999; Wang et al., 2000), and DNA methylation (Mancini et al., 1998; Catteau et al., 1999) have been implicated. In this study, we have investigated the modulation of BRCA1 expression by 15dPG-J2 (a natural ligand of peroxisome proliferator-activated receptor g (PPARg) and rosiglitazone (a member of the thiazolidinedione family of PPARg activators) (Forman et al., 1995, 1996; Jiang et al., 1998; Lebovitz and Banerji. 2001), in MCF7 breast cancer cells. Nuclear extracts were prepared and analysed by immunoblotting using an antibody specific for human BRCA1. We show that the endogenous nuclear levels of BRCA1 protein increased significantly 16-h after treatment of MCF-7 cells with both 15dPG-J2 and rosiglitazone, compared to the untreated control (Figure 1a). Given the role of BRCA1 in critical nuclear processes, its localization within the cell is an important issue. In this regard, a series of conflicting reports regarding BRCA1 subcellular localization in different breast cancer cell lines (Chen et al., 1995; Scully et al., 1996; Thomas et al., 1996; Wilson et al., 1999) gave rise to some controversy regarding the nuclear-cytoplasmic

BRCA-1 in a 15dPG-J2 target gene M Pignatelli et al

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Figure 1 Detection of BRCA1 expression in MCF-7 cells by Western and Immunofluorescence analysis. (a), Representative Western blot analysis of BRCA1 carried out in basal conditions or after a 16 h incubation with different concentrations of 15dPG-J2 or rosiglitazone (Calbiochem, San Diego, CA, USA). MCF-7 cells were collected in PBS, pelleted, and resuspended in ice-cold hypotonic lysis buffer. After a 10 min incubation on ice, samples were centrifuged for 10 s and the nuclei were collected by centrifugation at 15 000 g for 1 min. The nuclear pellet was resuspended in high-salt extraction buffer containing 25% glycerol and 0.4 m NaCl, incubated on ice for 20 min, and the cellular debris removed by centrifugation for 2 min. Nuclear extracts containing an equal amount of nuclear protein (20 mg) were separated by SDS– PAGE. After transfer to nylon membranes, immunoblotting with a polyclonal antibody to human BRCA1 (Santa Cruz Biotechnology, Sc642) was carried out as previously described (Pignatelli et al., 2001). For protein loading control, the blot was reprobed with a histone H1-specific antibody (Santa Cruz Biotechnology, sc10806). This experiment has been performed at least four times with essentially the same results. (b), MCF-7 cells were plated on glass coverslips in 24-well cell culture plates and grown in regular medium for 24 h. The cells were then treated or not with 15dPG-J2 for 12 h, fixed for 10 min with methanol at 201C, and permeabilized with 0.1% Triton X-100 for 30 min at 371C. After a 1 h incubation with the primary antibody, cells were washed with phosphate-buffered saline and incubated with a fluorescein-labeled secondary antibody for 45 min at 371C. Subcellular localization was determined using a TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems). Bar scale, 8 mm

distribution of BRCA1. Therefore, in order to further establish the intracellular localization of BRCA1 protein after 15dPG-J2 induction, we next performed immunocytochemistry analysis. The confocal images shown in Figure 1b show that, in control cells, there was a light cytoplasmic signal. In contrast, after 15dPG-J2 treatment, BRCA1 protein was localized in the nucleus and was distributed in multiple bright foci reminiscent of active transcription sites. We and others have obtained evidence that activation of PPARg by 15dPG-J2 results in a more differentiated, less malignant state, a reduction in growth rate, and an

enhancement of apoptosis (Mueller et al., 1998; Pignatelli et al., 2001) of several breast cancer cell lines. Specifically, we have shown that 15dPG-J2 almost completely blocks the phosphorylation/activation of the ErbB receptor tyrosine kinase family and therefore plays a suppressive regulatory role in the tumor growth of human breast carcinoma cells that express these receptors. However, although the growth-inhibitory and apoptotic effects of 15dPG-J2 on breast cancer cells have been well established, the effects of 15dPG-J2 on gene expression are less well documented. Here, we show that the two PPARg agonists, 15dPG-J2 and rosiglitazone, clearly induce the expression of BRCA1, a critical gene involved in breast tumorigenesis. Although 15dPG-J2 is a high-affinity ligand for the transcription factor PPARg (Forman et al., 1995; Jiang et al., 1998), the effects of this prostaglandin can also be mediated by PPARg-independent mechanisms such as the regulation of NF-kB activation (Castrillo et al., 2000; Straus et al., 2000). Therefore, to elucidate whether the regulation of BRCA1 expression by 15dPG-J2 was the consequence of a direct effect of PPARg on BRCA1 gene expression, we next performed transient transfection experiments into MCF-7 cells with a reporter construct containing the BRCA1 promoter (pBRC805). Treatment of MCF-7 cells with 15dPG-J2 or rosiglitazone resulted in a nine- and eight-fold increase, respectively, in promoter activity (Figure 2). It has been shown that the PPARg receptor activates transcription acting as a permissive PPARg/RXR heterodimer (reviewed in Aranda and Pascual, 2001). This kind of nuclear receptor heterodimers can be activated by ligands of either RXR or its partner receptor and are synergistically activated in the presence of both ligands. Therefore, we next analysed the response of the BRCA1 promoter to the RXR ligand (9-cis retinoic acid) alone or in combination with 15dPG-J2 or rosiglitazone. Figure 2 shows that treatment of MCF-7 cells with 9-cis retinoic acid resulted in a significant increase (three-fold) in promoter activity. Moreover, when the cells were incubated with 9-cis retinoic acid and 15dPG-J2 or rosiglitazone, a synergistic activation of the BRCA1 promoter was observed (18and 15-fold, respectively). These results further suggest that 15dPG-J2 regulates BRCA1 gene expression through a PPARg-dependent mechanism. Sequence analysis of the promoter region located between nucleotides 805 and þ 74 revealed the presence of one potential PPARg binding site between nucleotides 241 and 229 that resembles the PPARg type response element (PPRE) referred as direct repeat with one nucleotide of separation between the two hexamers (DR1). In order to know whether this element could be responsible for the effect of 15dPG-J2 and rosiglitazone on BRCA1 promoter activity, we first generated a deletion construct lacking the putative PPRE element (pBRCD204). As shown in Figure 2, the increase in luciferase activity observed in response to the ligands of PPARg receptor was completely abolished when MCF-7 cells were transfected with this construct, suggesting an implication of this element in the Oncogene


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Figure 3 Mutation analysis of human BRCA1 promoter. (a) Point mutations of the DR1 site in the 805/ þ 74 fragment were created by PCR using the DNA polymerase PfuTurbo following the instructions of the QuickChange site-directed mutagenesis method (Stratagene, CA, USA). The sequences of the oligonucleotides (coding strand) used for mutagenesis are as follows (mutated nucleotides are in bold): MUT1: Figure 2 Activation of human BRCA1 promoter by 15dPG-J2. Human BRCA1 promoter was PCR-amplified from human genomic DNA using the high fidelity Platinum s Pfx DNA polymerase (Invitrogen, Karlsruhe, Germany). The primers used were: 50 -TGG AAC TAC GAG TGC GCA GAC A-30 (forward sequence) and 50 -TTA TCT GAG AAA CCC CAC AGC C-30 (reverse sequence). They were designed according to the published sequence of human BRCA1 promoter (GenBank accession number U37574). PCR reactions were performed according to the manufacturer’s recommendations and the amplification products were cloned and sequenced in both directions. The entire promoter fragment (805/ þ 74, pBRC805) and a 50 -deletion 0 mutant (204/ þ 74, pBRCD204) were subcloned in the promoterless luciferase reporter vector pXP1. MCF-7 cells were transiently transfected with these two constructs using Transfast (Promega Corporation, Madison,WI, USA) according to the manufacturer’s guidelines. Typically, cells received 2 mg of luciferase reporter plasmid and were harvested 24 h after treatment with 10 mm 15dPG-J2, 30 mm rosiglitazone and/or 1 mm 9-cis retinoid acid (9cis-RA), for determination of luciferase activity. Data are expressed relative to the basal values and represent the mean7s.d. luciferase activity determined in triplicate in at least three independent experiments. *** , Pp0.001

induction of BRCA1 promoter expression by 15dPG-J2 and rosiglitazone. Moreover, point mutations in either of the half sites of the sequence of the DR1 (MUT1 and MUT2 constructs) abolished transactivation, indicating that these two modules play, in fact, a primary role in the regulation of BRCA1 by 15dPG-J2 (Figure 3a). On the other hand, transfection of MCF-7 cells with a Oncogene

MUT2: MUT3:

50 -AGT AGG ATC CGG AGG GCA GGC ACT TTA TGG-30 50 -AGT AGA GGC TAG GAT CCG GGC ACT TTA TGG-30 50 -AGT AGA GGT CAG AGG TCA GGC ACT TTA TGG-30

The PCR product was digested with DpnI and the DpnI-treated DNA was transformed into DH5a competent cells. Plasmids carrying inserts of the appropriate size were sequenced in both directions. Wild-type and mutant promoter constructs were transfected into MCF-7 cells. Transfection experiments were performed as described in Figure 2. (b) A double-stranded DNA oligonucleotide (nt 254/216), containing the DR1 sequence of the BRCA1 promoter, was subcloned in the pT109-luc plasmid upstream of the minimal thymidine kinase promoter (pBRCDR1) and MCF-7 cells were transfected as indicated in Figure 2. Data are expressed relative to the basal values and represent the mean7s.d. luciferase activity determined in triplicate in at least three independent experiments. ***, Pp0.001

BRCA1 promoter construct bearing a 100% consensus PPARg binding site (MUT3) also conferred a high responsiveness to 15dPG-J2 (eight-fold the basal value) (Figure 3a). Direct repeats of an AGGNCA half-site separated by a one-base pair spacer (DR1) have been identified in the promoters of several genes whose protein products are implicated in several cellular functions known to be regulated by the PPARg receptor (Rosen and Spiegelman, 2001; Picard and Auwerx, 2002). Further confirmation that 15dPG-J2 is acting through the DR1 response element described above was


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Figure 4 Gel retardation analysis of PPARg and RXRa binding to the putative DR1 element of the BRCA1 promoter. Human PPARg and RXRa proteins were generated by expressing pETPPARg and pGEX-RXRa in NM522 E. coli strain and, after induction with 0.1 mm IPTG, purified using nickel chelating affinity chromatography or Glutathione Sepharose-4B (Amersham Pharmacia Biotech, Europe, GmbH), respectively. Binding reactions were performed with a 32P-end labeled oligonucleotide probe, DR1wt, spanning nucleotides 251/219 in the BRCA1 promoter. Gel retardation assays were carried out according to the method previously described (Menendez-Hurtado et al., 2000). To perform supershift analysis, anti-PPARg antibody (Biomol Research Laboratories, Inc., Plymouth, PA, SA 206) was added to the reaction mixture 20 min before the addition of the labeled oligonucleotide. Double-stranded oligonucleotides composed of the following sequences were used for binding and competition analysis: BRCA1 DR1wt, 50 -GGA AGA GTA GAG GCT AGA GGG CAG GCA CTT TAT-30 ; BRCA1 DR1mut, 50 -GGA AGA GTA GGA TCC GCA GGG CAG GCA CTT TAT GG-30 . DR1 sequence is underlined. The mutated bases are shown in bold and correspond to the same bases mutated in the MUT1 construct used for transfection experiments. The mobility of the PPARg/RXRa heterodimer is indicated by an arrowhead. An arrow marks the position of the complex after supershifting. Competition experiments were carried out in the presence of a 100-fold excess of the indicated oligonucleotides

obtained by subcloning an oligonucleotide sequence (nt 254/216) containing the DR1 in front of a heterologous thymidine kinase promoter (construct

pBRCDR1). As shown in Figure 3b, the PPARg ligand 15dPG-J2 was also able to elicit a significant increase (4.8-fold) in the expression of this construct. To better characterize the binding site responsible for the induction of BRCA1 promoter expression by 15dPG-J2, we radiolabeled an oligonucleotide containing the wild-type DR1 sequence and used this oligonucleotide as a probe to perform gel mobility shift assays. Since PPARg’s partner for transcriptional activation is RXRa, the 32P-radiolabeled oligonucleotides were incubated with purified PPARg and RXRa proteins. As shown in Figure 4, this sequence bound with high affinity the PPARg/RXRa heterodimers. The addition of excessive unlabeled wild-type oligonucleotides reduced the signal of the 32P-radiolabeled wild-type oligonucleotides’s binding to PPARg/RXRa complex. On the other hand, addition of oligonucleotides containing the same mutation present in the MUT1 construct did not reduce the specific signal. The retarded band was supershifted by a PPARg-specific antibody, confirming the presence of this receptor in the retarded complex. Taking together, these results indicate that the DR1 element found in BRCA1 promoter is a functional PPARg-responsive element, which could mediate the increased expression of the endogenous BRCA1 gene observed after treatment of MCF-7 cells with 15dPG-J2 and rosiglitazone. Therefore, PPARg could represent an important new pathway in the regulation of BRCA1 gene expression. Previous reports have also implicated the Rb-E2F pathway, the brn-3b POU transcription factor, and CpG methylation of the promoter as potential regulators of the expression of this tumor suppressor gene (Xu et al., 1997; Budhram-Mahadeo et al., 1999; Suen and Goss, 1999; Thakur and Croce, 1999; Wang et al., 2000). In summary, the present study provides novel insights into the mechanisms through which BRCA1 expression is regulated. These findings offer a molecular basis for the regulation of the BRCA1 gene as well as the role of PPARg as a protective factor in breast cancer tumors. Acknowledgements This work has been supported by grants from the Direccioń General de Ensenãnza Superior e Investigacioń Cientı´ fica, Grants BMC2001-2342 (APC) and PM99-0057 (AS) and by the Comunidad de Madrid, Grant 08.5/0078/2000 (AS). MP is a fellow of the Fondo de Investigaciones Sanitarias de la Seguridad Social.

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