BRD7) in Epithelial Ovarian Carcinoma

Published OnlineFirst November 6, 2013; DOI: 10.1158/1078-0432.CCR-13-1271

Clinical Cancer Human Cancer Biology Research

Tumor Suppressive Effects of Bromodomain-Containing Protein 7 (BRD7) in Epithelial Ovarian Carcinoma

Young-Ae Park1, Jeong-Won Lee1, Hye-Sun Kim2, Yoo-Young Lee1, Tae-Joong Kim1, Chel Hun Choi1, Jung-Joo Choi1, Hye-Kyung Jeon1, Young Jae Cho1, Ji Yoon Ryu1, Byoung-Gie Kim1, and Duk-Soo Bae1

Abstract Purpose: Bromodomain-containing protein 7 (BRD7), which is a subunit of SWI/SNF complex, has been recently suggested as a novel tumor suppressor in several cancers. In this study, we investigated the tumor suppressive effect of BRD7 in epithelial ovarian cancer. Experimental Design: We analyzed the expression of BRD7 in human ovarian tissues with real-time PCR. To investigate the functional role of BRD7, we transfected ovarian cancer cells (A2780 and SKOV3) with BRD7 plasmid and checked the cell viability, apoptosis, and invasion. The activities of BRD7 in the signaling pathways associated with carcinogenesis were also tested. In addition, we used the orthotopic mouse model for ovarian cancer to evaluate tumor growth-inhibiting effect by administration of BRD7 plasmid. Results: The BRD7 expression was downregulated in the ovarian cancer tissues compared with normal (P < 0.05), high-grade serous cancer exhibited signiﬁcantly decreased expression of BRD7 compared with low-grade (P < 0.01) serous cancer. Transfection of BRD7 plasmid to A2780 (p53-wild) or SKOV3 (p53- null) ovarian cancer cells showed the tumor suppressive effects assessed by cell viability, apoptosis, and invasion assay and especially signiﬁcantly decreased tumor weight in orthotopic mouse model (A2780). Moreover, we found that tumor suppressive effects of BRD7 are independent to the presence of p53 activity in ovarian cancer cells. BRD7 negatively regulated b-catenin pathway, resulting in decreased its accumulation in the nucleus. Conclusions: These results suggested that BRD7 acts as a tumor suppressor in epithelial ovarian cancers independently of p53 activity, via negative regulation of b-catenin pathway. Clin Cancer Res; 20(3); 565–75. 2013 AACR.

Introduction the ras-raf-MEK-ERK, b-catenin, or PI3K pathways have Ovarian carcinoma is the fifth leading cause of cancer- been suggested as potential mechanisms of ovarian car- related death among females worldwide (1, 2). Func- cinogenesis (3–5). Among these, mutation of p53 is tional inactivation of the p53 tumor suppressor protein, most frequently thought to be the primary cellular expression of the BRCAness phenotype, and upregula- event leading to the development of ovarian cancer tion of several growth-regulatory genes that activate (6–8). Bromodomain-containing protein 7 (BRD7, chromo- some 16q12) is a subunit of the SWI/SNF complex specific Authors' Affiliations: 1Department of Obstetrics & Gynecology, Samsung for polybromo BRG1-associated factor (PBAF; ref. 9). Sev- Medical Center, Sungkyunkwan University School of Medicine; and eral previous studies have described a tumor suppressor role 2 Department of Pathology, Cheil General Hospital and Women's Health- for BRD7 (10–13). BRD7 expression was shown to be care Center, Kwandong University College of Medicine, Seoul, Korea reduced in several subsets of cancer relative to that of Note: Supplementary data for this article are available at Clinical Cancer individually matched normal cells (11, 12). Moreover, loss Research Online (http://clincancerres.aacrjournals.org/). of heterozygosity (LOH) on the chromosomal locus for Y.-A. Park and J.-W. Lee contributed equally to this work. BRD7 (16: 50347398-50402485) has been revealed one of Corresponding Authors: Duk-Soo Bae, Department of Obstetrics and the most frequent genomic events in breast and nasopha- Gynecology, Samsung Medical Center, Sungkyunkwan University School ryngeal cancer (11, 12). In nasopharyngeal cancer cells, of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Korea. Phone: 82-2-3410-3519; Fax: 82-2-3410-0630; E-mail: BRD7 reduced the activity of both ras-raf-MEK-ERK and Rb/ [email protected]; and Byoung-Gie Kim, E2F signaling, and also inhibited the b-catenin pathway [email protected] (12). Our laboratory previously investigated the tumor doi: 10.1158/1078-0432.CCR-13-1271 suppressive role of BRD7 in endometrial cancer cells 0 2013 American Association for Cancer Research. (14). However, the detailed mechanism of BRD7 s tumor

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our hospital approved this study (IRB #2011-04-008-002), Translational Relevance and informed consent was obtained from all patients Bromodomain-containing protein 7 (BRD7), which is between 2003 and 2010. a subunit of SWI/SNF complex, has been recently suggested as a novel tumor suppressor in several cancers. In RNA extraction, real-time PCR, and semiquantitative this study, we investigated the tumor suppressive effect PCR of BRD7 in epithelial ovarian cancer. Transfection of Total RNA including mRNA was extracted from the tissue BRD7 plasmid to A2780 (p53-wild) or SKOV3 (p53- using mirVana miRNA Isolation Kit (Ambion). cDNA was null) ovarian cancer cells showed the tumor suppressive synthesized from total RNA using oligo dT primer (Invitro- effects assessed by cell viabilitiy, apoptosis. and invasion gen) according to the TaqMan RNA Assay protocol (PE assay and especially significantly decreased tumor Applied Biosystems). Real-time PCR was performed using weight in orthotopic mouse model (A2780). Moreover, an Applied Biosystems 7900HT Sequence Detection system we found that tumor suppressive effects of BRD7 are (Applied Biosystems) according to the manufacturer’s pro- independent to the presence of p53 activity in ovarian tocol. Data normalizations were performed using GAPDH. cancer cells. BRD7 negatively regulated b-catenin path- To avoid amplification of genomic DNA, the primers and way resulting in decreased its accumulation in the nucle- probes for amplifying BRD7 and GAPDH were chosen to us. These results suggested that BRD7 acts as a tumor hybridize at the junction between the 2 exons as follows: suppressor in epithelial ovarian cancers independently BRD7 (#4351732; Applied Biosystems) and GAPDH of p53 activity, via negative regulation of b-catenin (#4310884E; Applied Biosystems). Relative quantification DD pathway. of BRD7 expression was calculated using the 2 CT meth- od. For semiquantitative PCR, we designed primers specific to the individual genes. The sequences of the primers were as follows: BRD7, sense 50-TGGAAGCCTCTCACAAGCT-30; suppressive effects remains unclear in the case of epithelial antisense 50-TGTGTACTAATGCCATGAT-30. p53, sense 50- ovarian cancer. CCGGAGGCCCATCCTCACCA-30; antisense 50-TGG CTG In this study, we investigated the tumor suppressive AGATGAGCC CTG CT-30. MMP2 sense 50-CACCTACAC effects of BRD7 in ovarian cancer. We found that transfec- CAAGAA CTTCC-30; antisense 50-AAC ACAGCCTTCTCCT- tion of the BRD7 plasmid into ovarian cancer cells induced CCTG-30. b-catenin sense 50-GGATCCGAGTGCGTTTATC- tumor suppressive effects in vitro and significantly decreased TGGTGGG-30; antisense 50-GTCGACACCCTTCCTCTTTA- tumor weights in the orthotopic mouse model. Moreover, GTTGC-30. b-actin sense 50-GATGCAGAAGGAGATCAC- we found that these tumor suppressive effects are indepen- TG-30; antisense 50-AGTCATAGTCCGCCTAGAAG-30. dent of p53 activity in ovarian cancer cells, and instead act by regulating the b-catenin pathway by inhibiting its accu- Cell culture mulation in the nucleus. To our knowledge, this study is the Ovarian carcinoma cell lines 2774, PA-1, OVCAR3, first to report a role for BRD7 in suppression of ovarian SKOV3, SW626 (colonic origin of ovarian cancer cell cancer. line), and Caov4 were obtained from the American Type Culture Collection (ATCC), and A2780 was obtained Materials and Methods from the European Collection of Cell Cultures (ECACC). Patients and tissue specimens HeyA8 was a gift from Dr. Anil K. Sood (Department of Study subjects were recruited from the Department of Cancer Biology, University of Texas M.D. Anderson Can- Obstetrics and Gynecology at the Samsung Medical Center, cer Center, TX). We were informed about the authentica- Sungkyunkwan University School of Medicine in Seoul, tions of whole cell lines when obtained them by the Korea, between 2003 and 2010. A total of 41 tissue speci- described providers. We checked the certificates of cell mens were collected including 11 samples of normal ovar- lines that include the results of the tests (species verifi- ian epithelium, 7 low-grade (grade 1/3), and 34 high-grade cation and short tandem repeat DNA profiling assay) (grade 3/3) serous ovarian cancer tissue samples. Tumor and procedures for authentication. More detailed infor- tissue specimens were obtained intraoperatively from wom- mation of the cell lines were stated in Supplementary en with epithelial ovarian carcinoma. Normal ovarian epi- Table S1. thelium specimens were collected from women who under- OVCAR3, 2774, and SKOV3 cells were maintained in went hysterectomy for benign disease. All specimens were RPMI and PA-1 cells were maintained in MEM.SW626 and immediately snap frozen at 80C. The specimens were Caov4 cells were maintained in Leibovitz’s L-15 Medium. stained with hematoxylin and eosin and evaluated by a Cells were grown at 37 Cin5%CO2 and supplemented single gynecologic pathologist. If the specimen had greater with 10% FBS, penicillin (100 units/mL), and streptomycin than 90% tumor cells, it was used in the analysis. Histo- (100 mg/mL). logical typing of each tumor was conducted according to the World Health Organization criteria, whereas surgical stag- Plasmid construct and siRNA ing followed the International Federation of Gynecology We tested 2 plasmid constructs, #1 and #2, that includes and Obstetrics standards. The Institutional Review Board at full length of human BRD7. #1 and its parent vector,

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pcDNA3.0 were kindly gifted from Fr. Giannino Del Sal followed by addition of propidium iodide (Sigma; 0.5 (Laboratorio Nazionale CIB, Trieste, Italy) and #2 was mg/mL) for 30 minutes. Cells were analyzed with a obtained from ATCC (#10436864), and its parental vector FACSort flow cytometer (BD Bioscience). Data were pro- that was used as mock control was made by recirculating the cessed using CellQuest (BD Bioscience) and ModFit BRD7 construct. p53 siRNA 50-CACUACAACUACAUGU- (www.Vsh.com) and presented by using FCSPress 1.4 GUGUA-30, b-catenin siRNA 50-CGUUCUCCUCAGAUG- (http://www.fcspress.com/). GUGU-30, and AccuTarget Negative control siRNA were obtained from Bioneer (Bioneer). Cells were transfected Preparation of cellular extracts and Western blot with the described combination of the corresponding Cells were lysed using a lysis buffer (INTRON BIO Tech- expression plasmid and/or siRNA in each experiment. After nology). To avoid degradation and dephosphorylation of 36 hours, cell lysates were subjected to Western blot analysis proteins, a protease and phosphatase inhibitor cocktail to determine the protein levels for each gene. The Western (Sigma-Aldrich) was added. Extraction of cytoplasm and blot results demonstrated that the corresponding plasmid nuclear lysates from whole cells was performed using NE- and/or siRNA efficiently regulated the expression of each PER Nuclear and Cytoplasm Extraction Reagent Kit (Pierce) gene. according to the manufacturer’s protocol. The following antibodies were subsequently used: BRD7 (Lifespan Bio- Transfection technology), b-catenin (R&D), MMP-2, a-catenin, lamin b Cells were transfected using the described combination of receptor (LBR), cyclin B1 and MEK1 (Epitomics), MMP-9 the corresponding expression plasmid and/or siRNA using (Thermo), b-actin, cyclin D1, and p53 (Santa Cruz Biotech- lipofectamine (Invitrogen) according to the manufacturer’s nology), and cyclin E2, ERK, and p-ERK(1/2) (Thr202/ protocol. For upregulation of BRD7 in vivo, we injected the Tyr204; Cell Signaling). Protein bands were then labeled BRD7 plasmid or mock vector into the xenografts using with horseradish peroxidase-conjugated anti-mouse or GenJet Plus transfection reagent (SignaGen Laboratories) anti-rabbit antibody (Amersham). according to the manufacturer’s protocol. We used high- quality plasmid DNA (A260/A280 ratio of 1.8 or higher). We Wound healing assay transfected 10 mg of plasmid via intraperitoneal injection For the wound healing assay, confluent and quiescent once weekly for 1 month beginning 7 days after cancer cell monolayers of cells that were transfected with correspond- injection. ing expression plasmid and siRNA as indicated were wounded using the end of a sterilized tip. After 24 hours, MTT assay to observe changes involved with healing, photographs Cells were seeded in 96-well plates at a density of 5,000 were taken 9 and 12 hours after wounding of each subset cells/well for 24 hours. Cells were then transfected with the of SKOV3 and A2780 cells, respectively, based on the corresponding expression plasmid and/or siRNA as indi- migratory ability of each cell line. All photographs were cated for 36 hours. MTT assays were performed as previously taken at 100 magnification. described (15). Values are expressed as means of three independent experiments SD. Cell invasion assay For measuring the ability of cells to invade, we used a Apoptosis assay transwell system (8-mm pore size; Corning). Cells were Cells were transfected with the corresponding expres- transfected with the corresponding expression plasmid sion plasmid and siRNA as indicated for 12 hours and and/or siRNA for 12 hours and trypsinized for dissocia- then treated with etoposide (Sigma-Aldrich) for 24 hours tion into single cells in a serum-free medium. A total of for the induction of apoptosis. Because SKOV3 is p53 null 100 mL of the cell suspension (1 105 cells) was then cell line, we estimated apoptosis by measuring activated added to the upper chamber of the insert that was coated caspase-3onanELISAassay(Invitrogen),whichisa with martrigel (50 mg/mL; BD Biosciences). Cells were common final mediator of apoptosis in both p53-depen- allowed to invade for 12 hours in response to FBS as a dent and independent pathways. The apoptosis for each chemoattractant in the lower compartment. After incu- subset were determined as previously described (16). bation, cells on the upper surface of the inner membrane Values are expressed as means of 3 independent experi- were removed with the cotton swab, and the final number ments SD. of invaded cells was determined using crystal violet staining. Flow cytometry (FACS) and sorting For the FACS assay, cells were seeded in 175 T flasks for 24 ELISA assay hours and then starved in a serum-free medium for 24 For quantitative measurement of MMP2 and MMP9 hours. Cells were trypsinized for dissociation into a single secretion, we used ELISA assays (R&D and Abnova, Taoyuan cell in potassium-buffered saline (PBS), pH 7.5. Cell sus- Country, Taiwan, respectively). Cells were transfected with pensions were rinsed twice with ice-cold PBS and added the corresponding expression plasmid and siRNA as indi- dropwise ice-cold 70% ethanol for 12 hours at 4C. After cated for 36 hours. Protein from suppurates of each subset removing ethanol, cells were treated with RNase A (1 mg/mL) were then purified and concentrated using Amicon Ultra

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Centrifugal Filter (Millipore). To measure the quantity of type T-cell factor (Tcf) (OT) with the corresponding expres- MMP-2 in tumor tissues from the xenografts, tumor samples sion plasmid (5 mg). Renilla luciferase reporter (100 ng) was were placed into 200 mL of protein lysis buffer (INTRON cotransfected in individual cases to normalize transfection BIO Technology) and homogenized. The quantities of efficiency. All reporter plasmids were kindly gifted from Dr. MMP2 or MMP9 were assessed using ELISA assay according Sunjoo Jeong (Dankook University, Yongin Si, Korea). After to the manufacturer’s protocol. 24 hours, cells were harvested and lysed in passive lysis buffer from the Luciferase Assay Kit (Promega). All readings Animal care and orthotopic implantation of tumor cells were made using a Wallac Victor2 1420 Microplate Reader Female BALB/c nude mice were purchased from ORI- PerkinElmer Life Sciences Inc.). Relative firefly luciferase ENT BIO. This study was approved by the Institutional activities were calculated by normalization with Renilla Animal Care and Use Committee (IACUC) of the Sam- luciferase activity. sung Biomedical Research Institute (SBRI). SBRI is an Association for Assessment and Accreditation of Labora- Data analysis tory Animal Care International (AAALAC International, Mann–Whitney U tests were used to evaluate the signif- protocol No. H-A9-003) accredited facility and abides by icance between gene expression in tumors and normal the Institute of Laboratory Animal Resources (ILAR) ovarian tissues as assessed by real-time PCR, and to compare guide. To establish the orthotopic model, A2780 cells differences among the groups for both in vitro and in vivo (1 106 cells/0.2 mL HBSS) were injected into the assays. All statistical tests were two-sided, and P-values < peritoneal cavity (17). The mice were 6 to 8 weeks of 0.05 were considered to be statistically significant. All age. Mice (n ¼ 6 per group) were monitored daily for statistical analyses were performed using statistical SPSS tumor development and were sacrificed on day 35 after software package (SPSS Inc.). the cancer cell injection. Body weight, tumor weight, and the number of tumor nodules were recorded. Tumors Results were fixed in formalin and embedded in paraffin or snap The expression of BRD7 is decreased in high-grade frozen in OCT compound (Sakura Finetek Japan). serous ovarian cancer relative to normal tissue or low- grade serous ovarian cancer Immunohistochemistry We estimated the relative expression pattern of BRD7 Immunohistochemical procedures for Ki67 and BRD7 using human ovarian tissue from 11 normal ovarian epi- were performed as previously described (14, 16). To quan- thelium and 41 serous cancer tissue specimens, which tify Ki67 expression, the number of Ki67-positive cells and included 7 low-grade and 34 high-grade cancers. Actually, the total number of tumor cells were counted in 5 random we did tried estimation of protein level of BRD7 in the fields at 100 magnification, followed by calculation of the ovarian cancer tissues through immunohistochemistry percentage of positive cells. (IHC), but the qualities of antibodies were not sufficient for sensitive analysis for the expression pattern of BRD7 Luciferase assay (data not shown). We found that BRD7 mRNA expression For the luciferase assay, cells were transiently transfected was significantly lower in the serous ovarian cancer with Firefly luciferase reporter (100 ng) containing wild- tissues relative to the normal tissues (P ¼ 0.02; Fig. 1A).

Figure 1. Real-time PCR analysis of BRD7 expression in human ovarian tissue. A, the expression levels of BRD7 mRNA were signiﬁcantly reduced in serous ovarian cancers relative to normal ovarian tissue (, P ¼ 0.02). B, expression was signiﬁcantly reduced in high-grade serous cancer relative to low-grade serous cancer (P ¼ 0.01) or normal tissue (, P < 0.01).

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Interestingly, although there was no statistical difference in BRD7 was variably expressed among the cell lines (Fig. 2A), BRD7 expression between normal tissues and low-grade so we chose to study A2780 and SKOV3 cells because of carcinomas (P ¼ 0.49), high-grade carcinomas showed their relatively regular and minor expression, respectively. significantly decreased expression of BRD7 compared with Transfection of BRD7 plasmid construct either #1 or #2 normal (P ¼ 0.01) or low-grade cancer tissues (P ¼ 0.01; increased expression of BRD7 in a dose-dependent manner Fig. 1B). in both cells with similar transfection efficiently (approx- imately up to 3-fold upregulation of BRD7 in both 2 cell Upregulation of BRD7 affects cell survival and lines; Fig. 2B). Cells were transiently transfected with BRD7 apoptosis of ovarian cancer cells plasmid for 36 hours, and cell survival was estimated using We evaluated endogenous expression of BRD7 in epithe- MTT assay. The cell survival was significantly decreased by lial ovarian cancer cell lines using Western blot analysis. BRD7 plasmid construct #1 or #2 in a dose-dependent

Figure 2. BRD7 overexpression affects cell survival and apoptosis in ovarian cancer cells. A, Western blot analysis exhibited variable expression of BRD7 in ovarian cancer cell lines. B, A2780 and SKOV3 cells were transfected with BRD7 plasmid construct #1 or #2 and Western blot analysis subsequently showed upregulation of BRD7 in a dose- dependent manner in both cell lines. C, upregulation of BRD7 decreased cell survival in both cell lines compared with controls as assessed by MTT assays after 36 hours of transfection with corresponding BRD7 plasmid construct (, P < 0.05 and , P < 0.01). D, apoptosis was estimated by detection of activated caspase- 3 using ELISA assay after 36 hours of transfection with BRD7 plasmid (, P < 0.05 and , P < 0.01). Values are expressed as means of 3 independent experiments SD.

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manner in both cells (Fig. 2C; P < 0.05). Comparison Upregulation of BRD7 decreases cell migration/ showed that plasmid constructs induced upregulation of invasion and MMP2 activity in ovarian cancer cells BRD7 expression was similar, but the reduction of cell BRD7 inhibited cell migration in both cells as assessed by viability by construct #1 was relatively more effective than the wound healing assay (Fig. 3A). Using transwell-invasion by #2 in both 2 cell lines. So we selected construct #1 for the assay, we identified that cell invasion was more obviously rest tests for the estimation of the cellular effects by BRD7 inhibited by BRD7 than cell viability in both cells (Fig. 3B). (marked as BRD7 plasmid). In addition, MMP2 expression was significantly decreased BRD7 also significantly increased apoptosis in both cells by BRD7 both cells (Fig. 3C), although there were no compared with mock control (Fig. 2D; P < 0.05). changes in MMP9 expression in either cell (data not We also assessed the effect of BRD7 to the progress of shown). Using ELISA assay, we subsequently found that cell cycle in ovarian cancer cells. First, we sorted the cells secretion of MMP2 was significantly reduced by BRD7 (Fig. in the individual phase of cell cycle, G0–G1,S,andG2–M 3D; P < 0.01). The secretion of MMP9 was not changed by by using FACS assay and then isolated whole mRNA and BRD7 in either cell (data not shown). analyzed the expression of BRD7 mRNA. Compared with the G0–G1 phase, the expression of BRD7 in either S or BRD7 overexpression inhibits tumor growth and G2–M phase did not show significant differences in both metastatic capability in orthotopic model the cell lines (Supplementary Fig. 1A). Subsequently, we Orthotopic model of ovarian cancer was established in estimated the regulation of cyclin D1 and E2 expression nude mice by intraperitoneal injection of A2780 cells. We followed by upregulation of BRD7. And we found that injected the BRD7 plasmid or the mock vector into the mice both cyclins were not regulated by BRD7 in either cell line once weekly starting 7 days after cancer cell injection. (Supplementary Fig. 1B). These results imply that BRD7 Plasmid DNA injections resulted in significantly decreased might not be associated with the process of cell cycle in tumor weight and the number of tumor nodules (Fig. 4A ovarian cancer cells. and B; P < 0.05). In addition, when we examined tumor cell

Figure 3. BRD7 overexpression reduces cell migration/invasion and MMP2 activity. A, the wound healing assay showed that upregulation of BRD7 inhibited the migratory ability of ovarian cancer cells. All photographs were taken at 100 magniﬁcation. B, the transwell-invasion assay used to estimate cell invasion showed that upregulation of BRD7 inhibited invasion of ovarian cancer cells (, P < 0.05 and , P < 0.01). C, upregulation of BRD7 reduced expression of MMP2 as measured on Western blot analysis. D, upregulation of BRD7 reduced secretion of MMP2 as measured on an ELISA assay (, P < 0.01). Values are expressed as means of 3 independent experiments SD.

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Figure 4. Upregulation of BRD7 inhibits tumor growth and metastatic capability in orthotopic mouse model. Tumor weights (A) and the number of tumor nodules (B) from the xenografts transfected with BRD7 plasmid were signiﬁcantly reduced relative to controls (, P < 0.05). Results are expressed as the mean SD (n ¼ 6 mice per group). C, tumor cell proliferation as assessed by Ki67 IHC. Original magniﬁcation was 100. Black scale bar ¼ 100 mmol/L. D, MMP2 expression in tumor tissues as assessed on ELISA assay (, P < 0.05). Results are expressed as the mean SD.

proliferation using Ki67 IHC in harvested tumor tissues, found that BRD7-mediated downregulation of MMP2 activ- positive staining was significantly decreased in the BRD7 ity did not require p53 activity in ovarian cancer cells (Fig. plasmid-treated group (Fig. 4C; P < 0.05). When we mea- 5D). sured the MMP2 levels using ELISA assay, we found that secretion of MMP2 was also significantly decreased by BRD7 BRD7 negatively regulates b-catenin pathways (Fig. 4D; P < 0.05). These results were consistent with the in Based on our previous study of endometrial cancer and vitro effects. Upregulation of BRD7 in tumor tissues was the negative regulation of the b-catenin pathway by BRD7 estimated by Western blot analysis (Supplementary Fig. S2). (14), we sought to determine whether BRD7 could upregulate a-catenin and the accumulation of b-catenin in the BRD7 regulates cell survival, apoptosis, and cell cytoplasm of ovarian cancer cells. BRD7 did not upregulate migration/invasion independent of the p53 tumor a-catenin in either cell (Supplementary Fig. S3), although it suppressor protein increased the accumulation of b-catenin in the cytoplasm in We confirmed that BRD7 mediates antitumor effects in both cells (Fig. 6A). The expression of b-catenin was not both A2780 (p53 wild) and SKOV3 (p53 null) cells, sug- modified by BRD7 in either cell (data not shown), suggest- gesting that BRD7 may act as a tumor suppressor gene in a ing that BRD7 may inhibit translocation of b-catenin to the p53-independent manner. To more precisely test this nucleus in ovarian cancer cells. In addition, reporter gene hypothesis, we evaluated whether BRD7 could mediate silencing assay using a luciferase plasmid vector showed antitumor effects when p53 is downregulated in A2780 that BRD7 decreased b-catenin–mediated Tcf-responsive cells. Cells were cotransfected with p53 siRNA and BRD7 gene transcription in both cells (Fig. 6B). Transcriptional plasmid. We then examined the protein expression levels target genes of b-catenin have been known to play roles in using Western blot analysis (Fig. 5A) and found that BRD7- various cellular events and MMP2 is also known as one of mediated changes in cell survival were not affected by them. So we next tested whether the mechanism by which downregulation of p53 (Fig. 5B, top; P < 0.01). BRD7 also BRD7 downregulate MMP2 expression in ovarian cancer induced a 1.2-fold increase in apoptosis when cells were cells could be the negative regulation of b-catenin pathway. cotransfected with p53 siRNA (P < 0.05; Fig. 5B, bottom). When b-catenin was downregulated by siRNA, BRD7 plas- Downregulation of p53 increased cell invasion in A2780 mid did not downregulate MMP2 expression (Supplemen- cells compared with negative control, and BRD7 continued tary Fig. S4) via representing BRD7-mediated downregula- to induce decreases in cell migration/invasion in the tion of MMP2 is requiring b-catenin pathway. We also absence of p53 activity (Fig. 5C; P < 0.05). In addition, we assessed MEK1 expression and phosphorylation of ERK1/2,

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Figure 5. BRD7 mediates tumor suppressive effects independent of p53 activity. Cotransfection with BRD7 plasmid and p53 siRNA of A2780 (p53-wild) cells as assessed on Western blot analysis (A). BRD7 regulated cell viability (B) and apoptosis (C) of ovarian cancer cells independent of p53 activity in A2780 cells (, P < 0.05 and , P < 0.01). Values are expressed as means of 3 independent experiments SD. BRD7 inhibited invasion of ovarian cancer cells as assessed by the transwell-invasion assay (D; , P < 0.05 and , P < 0.01) and wound healing assay (E). BRD7-mediated downregulation of MMP2 expression was independent of p53 activity in A2780 cells, as assessed by semiquantitative RT-PCR (F) and ELISA (G; , P < 0.05 and , P < 0.01). Values are expressed as means of 3 independent experiments SD.

which is the final activating event of the ras-raf-MEK-ERK Recent studies have attempted to develop a more precise pathway, using Western blot analysis, but there was no model of carcinogenesis with an emphasis on differences in influence on the transduction of the pathway by BRD7 cellular origins and molecular aberrations in serous ovarian (Supplementary Fig. S5). cancer (18–20). These studies have suggested that tumor grade, a pathological index of an individual cellular aber- Discussion ration, could help to stratify serous ovarian carcinomas. The key finding of this study is that BRD7 is a novel tumor Low-grade carcinomas tend to be slow growing, indolent, suppressor in serous ovarian cancer independent of p53 and typically arise from invasive foci of tumors with low activity. The expression of BRD7 was significantly lower in malignant potential, whereas high-grade carcinomas are serous cancer tissues than in normal tissues, with high-grade rapidly growing, aggressive, and usually lack a definitive serous cancers exhibiting markedly decreased expression of precursor region. In this study, we found that the expression BRD7 compared with low-grade cancers. Although BRD7 of BRD7 mRNA in high-grade tumors was significantly has modest effects on cell viability and apoptosis, it could lower than that of low-grade tumors. These findings suggest more effectively inhibit cell migration/invasion and the that a decrease in BRD7 could play an important role in the reduction of MMP2 expression and secretion in ovarian aggressive behavior of high-grade tumors and may poten- cancer cells. Moreover, we confirmed these BRD7-induced tially be used as a prognostic marker for this disease. tumor suppressor effects in orthotopic mouse model of As a member of the bromodomain-containing protein ovarian cancer, and found that not only cell proliferation family, BRD7 is known to interact with other proteins but also secretion of MMP2 were significantly decreased by resulting in modification of their activity (21–23). In addi- BRD7. These results are consistent with the observed in vitro tion, its role as a subunit of the SWI/SNF complex specific results. for PBAF that regulates chromatin remodeling has also been

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Tumor Suppressive Effect of BRD7 in Ovarian Cancer

Figure 6. BRD7 inhibits translocation of b-catenin to the nucleus via transcriptional activity of Tcf. A, upregulation of BRD7 decreased translocation of b-catenin to the nucleus in A2780 and SKOV3 cells. To conﬁrm each fraction, a-tubulin (cytoplasm) or lamin B receptor (LBR; nucleus) were used as normalization markers. B, BRD7 reduced transcriptional activity of Tcf in A2780 and SKOV3 cells (, P < 0.01). Values are expressed as means of 3 independent experiments SD.

shown to contribute specifications for assembly and target- b-catenin is a part of an E-cadherin/catenin adhesion com- ing (21, 24, 25). Research studies have demonstrated that plex, acting as a cell-to-cell adhesion molecule by regulating BRD7 regulates signaling pathways that regulate cellular the epithelial entities of cancer cells with characteristics of events involving growth, apoptosis, cell cycle, and mobility catenin proteins (35, 36). It is known that BRD7 upregulates (10, 12, 26, 27). In particular, BRD7 has been known to a-catenin, which holds b-catenin in the membrane and interact with p53 and p300 by affecting acetylation of cytoplasm in nasopharyngeal carcinoma cells (12). In addi- histones and recruiting them to specific promoters of target tion, in our previous study, we confirmed that BRD7 accu- genes (11, 27). Those studies have revealed that the inter- mulates b-catenin in the cytoplasm in endometrial cancer action of BRD7 with p53-p300 is required for a subset of cells (14). Interestingly, in this study, BRD7 did not alter the p53-regulated gene expressions through direction of pro- regulation of a-catenin but did decrease the accumulation moter-binding and/or bridging of p53 to a favorable chro- of b-catenin in the nuclei of ovarian cancer cells. Accord- matin environment. In another study, the interaction ingly, BRD7 negatively regulated b-catenin–mediated Tcf- between BRD7 and BRCA1 was shown to regulate tran- responsive gene transcription. In addition, we found that scriptional activity of downstream target genes of BRCA1 in the mechanism by which BRD7 reduces MMP2 expression breast cancer cells (9). In addition, other studies have found could be the negative regulation of b-catenin pathway. that BRD7 may act as a regulator of signaling pathways Taken together, our results show that BRD7 induced the including ras-raf-MEK-ERK and RB/E2F (12, 26). accumulation of b-catenin in the cytoplasm and inhibited As the p53 mutation is a definitive feature in high-grade transcription of Tcf-responsive genes, which may be the key ovarian cancer, we initially considered BRD7 as a coacti- mechanism by which BRD7 exhibits tumor suppressive vator of p53. It is well established that p53 regulates cellular effects in ovarian cancer cells. behavior of cancer cells by transcriptional regulation of This study demonstrated an association between BRD7 specific genes or signaling pathways implicated in those and high-grade serous ovarian carcinoma that is indepen- activities (28–34). However, our results showed that BRD7 dent of p53. These findings are significant with respect to could induce tumor suppressive effects in either SKOV3 targeted therapy, as most patients with serous ovarian (p53 null) or A2780 (p53 wild type) cells. In addition, we cancer exhibit poor responses to chemotherapeutic agents found that downregulation of p53 activity did not alter that target the p53 pathway. Thus, there is a new demand for BRD7-mediated tumor suppressive effects in A2780 cells. the development of novel treatments targeting p53 inde- These results indicate that BRD7-mediated effects may pendent pathways. involve a p53-independent signaling pathway in ovarian In conclusion, this study showed that BRD7 acts as a cancer cells. tumor suppressor in epithelial ovarian cancers indepen- As a key mediator of the Wnt signaling pathway, b-catenin dent of p53 activity via negative regulation of the b-cate- accumulates in the nucleus where it complexes with the nin pathway. Establishment of the mechanism by which Tcf/LEF family of transcription regulators in response to BRD7 negatively regulates the b-catenin pathway could Wnt, and directly binds the promoter of Tcf-responsive result in promising diagnostic and therapeutic strategies genes thereby activating transcription (35–37). Moreover, for the treatment of ovarian cancer. Future study about

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Park et al.

LOH and the correlation of the BRD7 protein expression Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): J.-W. Lee, Y.Y. Lee, J.-J. Choi, H.-K. and the clinicopathological factor could be helpful for the Jeon, Y.-J. Cho understanding the role of BRD7 in epithelial ovarian Study supervision: J.-W. Lee, T.-J. Kim, J.-J. Choi, B.G. Kim carcinoma. Disclosure of Potential Conﬂicts of Interest Grant Support This study was supported by a grant of the Korea Healthcare Technology No potential conﬂicts of interest were disclosed. R&D Project, Ministry for Health & Welfare Affairs, Republic of Korea (A092255), and by one from the National R&D Program for Cancer Control, Authors' Contributions Ministry for Health, Welfare and Family Affairs, Republic of Korea (0920010). Conception and design: D.-S. Bae, Y.-A. Park, J.-W. Lee, T.-J. Kim The costs of publication of this article were defrayed in part by the Development of methodology: J.-W. Lee, T.-J. Kim, J.-J. Choi, H.-K. Jeon, payment of page charges. This article must therefore be hereby marked Y.-J. Cho, J.Y. Ryu advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Acquisition of data (provided animals, acquired and managed patients, this fact. provided facilities, etc.): J.-W. Lee Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): J.-W. Lee, H.-S. Kim, C. Choi Received May 26, 2013; revised September 29, 2013; accepted October 17, Writing, review, and/or revision of the manuscript: J.-W. Lee, Y.Y. Lee 2013; published OnlineFirst November 6, 2013.

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Tumor Suppressive Effects of Bromodomain-Containing Protein 7 (BRD7) in Epithelial Ovarian Carcinoma

Young-Ae Park, Jeong-Won Lee, Hye-Sun Kim, et al.

Clin Cancer Res 2014;20:565-575. Published OnlineFirst November 6, 2013.

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