Glucocorticoid receptor repression mediated by BRCA1 inactivation in ovarian cancer

BMC Cancer Glucocorticoid receptor repression mediated by BRCA1 inactivation in ovarian cancer Yuan-Yuan Fang 0 Da Li 0 Chen Cao Chun-Yan Li Ting-Ting Li 0 Department of Obstetrics and Gynecology, Shengjing Hospital, China Medical University , Shenyang 110004 , China Background: BRCA mutations are the main known hereditary factor for ovarian cancer. Notably, emerging evidence indicates that the glucocorticoid receptor (GR) has drawn considerable interest in ovarian cancer development. However, dynamic cross-talk between BRCA1 and GR signaling pathways are poorly understood. Methods: The regulatory effects of BRCA on GR were assessed in 146 serous ovarian cancer patients (28 pairs of BRCA1-mutated or not, 23 pairs of BRCA2-mutated or not, and 22 pairs with hypermethylated BRCA1 promoter or not). BRCA1 promoter methylation was analyzed by bisulfite sequencing using primers flanking the core promoter region. Expression levels of BRCA1 and GR were assessed by immunohistochemistry and real-time PCR. Regression analysis was used to examine the possible relationship between BRCA1 and GR expression levels. The knockdown and overexpression of BRCA1 were achieved using a lentiviral vector in 293 T cells, SKOV3 ovarian cancer cells, and primary non-mutated and BRCA1-mutated ovarian cancer cells. Results: GR expression levels were unchanged in non-BRCA1-mutated, non-BRCA2-mutated and BRCA2-mutated ovarian cancer compared to their normal tissues; BRCA1 repression (BRCA1 mutation or BRCA1 promoter hypermethylation) ovarian cancer showed decreased GR levels compared to normal tissue; there was a positive correlation between BRCA1 and GR expression in human ovarian cancer specimens; BRCA1 knockdown was effective at inhibiting GR expression, and overexpression of BRCA1 induces an increase in GR levels in ovarian cancer cells. Conclusions: These results suggest that GR may be a potential target for BRCA1 in ovarian cancer progression. BRCA1; BRCA2; Glucocorticoid receptor; Ovarian cancer - Background Ovarian cancer is the most lethal gynecological malignancy in women worldwide [1]. To date, although the exact cause of ovarian cancer remains largely unknown, BRCA mutations are the main known hereditary factor [2], and the risk of ovarian cancer conferred by BRCA mutations can be regulated by both genetic and environmental components [3]. Glucocorticoid action in cells is mediated by the glucocorticoid receptor (GR), a member of the superfamily of ligand-inducible transcription factors that exert a variety of physiological functions, such as inflammation, autoimmune diseases, and cancer [4]. Recently, the glucocorticoid system has drawn considerable interest in the field of ovarian cancer therapy, with studies involving, for instance, glucocorticoids-induced chemotherapy resistance in ovarian cancer cells [5,6]; GR may be involved in the pathogenesis of ovarian cancer via the regulation of apoptosis and aberrant cell migration [7]. In addition, emerging evidence has suggested that: (i) both genetic and environmental factors contribute to impaired GR function [8]; (ii) GR inactivation is a hallmark for BRCA1-mutated breast cancer tissues [9]; and (iii) the BRCA1-interacting protein NELF-B participates in GRmediated gene induction [10]. However, to date, little is known about the effects of BRCA dysfunction on GR in ovarian cancer. Therefore, insights into the complex interrelationship between BRCA and GR might improve our understanding of the basic molecular mechanism of ovarian cancer. For this reason, the present study was undertaken to investigate GR expression after BRCA inactivation events (mutation, promoter methylation, or knockdown), and to provide novel insights into the regulatory mechanism of GR in ovarian cancer progression. Methods Patients and tissue collection This study was approved by the Institutional Review Board at China Medical University. Serous ovarian cancer patients were enrolled between 2010 and 2012, and all patients gave informed consent. Fresh tumor samples, adjacent normal ovarian tissues, ascites, and blood samples were obtained at the time of primary surgery before any chemotherapy or radiotherapy (28 pairs of BRCA1mutated or not, 23 pairs of BRCA2-mutated or not, and 22 pairs with hypermethylated BRCA1 promoter or not). Hematoxylin and eosin staining of the samples for histopathological diagnosis and grading were determined by three staff pathologists using the World Health Organization criteria. All patients were screened for BRCA1 and BRCA2 mutations by multiplex polymerase chain reaction (PCR) with complete sequence analysis as previously described [11]; their characteristics are given in Additional file 1. Cell culture and lentiviral transfection Primary ovarian cancer cells were obtained from the ascites of patients undergoing surgery for ovarian cancer and cultured in RPMI 1640 with 10% fetal bovine serum (Invitrogen, CA USA), using methods reported by Szlosarek [12]. Human 293 T cells and wild-type SKOV3 ovarian carcinoma cells were maintained in DMEM with 10% fetal bovine serum (Invitrogen). Lentiviral vectors expressing short hairpin RNAs (shRNAs) against BRCA1 (NM_007299) were obtained from GeneChem Co., Ltd (Shanghai, China), and synthesized as follows: Forward: 5-CCGGAACCTGTCTCCACAAAGTGTGCTCGAGC ACACTTTGT GGAGACAGGTTTTTTTG-3, and Reverse: 5-AATTCAAAAAAACCTGT CTCCACAAAGT GTGCTCGAGCACACTTTGTGGAGACAGGTT-3. The non-silencing shRNA sequence was used as a negative control and synthesized as follows: forward, 5-ccggTT CTCCGAACGTGTCACGTctcgagACGTGACACGTTCG GAGAAtttttg-3, and reverse, 5-aattcaaaaaTTCTCCGA ACGTGTCACGTctcgagACGTGACACGTTCGGAGAA3. For overexpression of BRCA1, the open reading frame of BRCA1 (NM_007299) was cloned into the lentiviral vector GV287 (Ubi-MCS-3FLAG-SV40-EGFP; GeneChem Co., Ltd). Transfections were performed using polybrene and enhanced infection solution (GeneChem Co., Ltd) according to the manufacturers recommended protocol. The efficiency of BRCA1 knockdown and overexpression was shown in Additional file 2 (supplementary methods are shown in Additional file 3). Real-time quantitative PCR Total RNA was extracted using Trizol reagents (Invitrogen) according to the manufacturers protocol. DNA contamination was removed by adding DNase I (Invitrogen) according to the manufacturers protocol. Total RNA was then reverse-transcribed from 2 g of RNA using the PrimeScript RT Master Mix kit (TaKaRa, Dalian, China) and amplified by SYBR Premix Ex TaqTM II (TaKaRa) in a Roche LightCycler 2.0 instrument (Roche Diagnostics, Mannheim, Germany). The specific primer sequences were as follows: GR: 5- TGTTTTGCTCCTGATCTGA -3 (F) and 5- TCGGGGAATTCAATACTCA-3 (R); BRCA1: 5-GGCTATCCTCTCAGAGTGACATTT-3 (F) and 5GCTTTATCAGGTTATGTTGCATGG-3 (R); GAPDH: 5-AGGTGAAGGTCGGAGTCA-3 (F) and 5-GGTCAT TGATGGCAACAA-3(R). GAPDH mRNA was amplified as an internal control for normalization of each sample. All samples were analyzed in triplicate using the 2CT meth (...truncated)