Negative feedback loop of BRCA1–BARD1 ubiquitin ligase on estrogen receptor alpha stability and activity antagonized by cancer-associated isoform of BARD1

Estrogen is involved in breast cancer risk, which is increased for BRCA1 mutation carriers, suggesting a role for BRCA1 in estrogen signaling. BRCA1 exerts its function through forming an E3 ubiquitin ligase with BARD1. We report that the estrogen receptor alpha is a target of the BRCA1–BARD1 ubiquitin ligase in vivo. BRCA1 and BARD1 are required for estrogen receptor alpha ubiquitination and degradation, and repression of either one leads to ERα accumulation, suggesting a feedback loop between BRCA1–BARD1 and estrogen receptor alpha, since BRCA1 and BARD1 are induced by estrogen receptor alpha. While the ubiquitin ligase activity maps to the N-terminal RING finger domains of BRCA1 and BARD1, we demonstrate that the BARD1 C-terminus is important for target recognition. Furthermore, a BARD1 isoform lacking the RING domain binds and stabilizes estrogen receptor alpha. Thus deficiencies of BRCA1 or BARD1 and/or upregulation of BARD1 isoforms lead to estrogen receptor alpha upregulation, providing a functional link between BRCA1 deficiency, estrogen signaling, and tumorigenesis.     

BRCA1 inhibits membrane estrogen and growth factor receptor signaling to cell proliferation in breast cancer

BRCA1 mutations and estrogen use are risk factors for the development of breast cancer. Recent work has identified estrogen receptors localized at the plasma membrane that signal to cell biology. We examined the impact of BRCA1 on membrane estrogen and growth factor receptor signaling to breast cancer cell proliferation. MCF-7 and ZR-75-1 cells showed a rapid and sustained activation of extracellular signal-related kinase (ERK) in response to estradiol (E2) that was substantially prevented by wild-type (wt) but not mutant BRCA1. The proliferation of MCF-7 cells induced by E2 was significantly inhibited by PD98059, a specific ERK inhibitor, or by dominant negative ERK2 expression and by expression of wt BRCA1 (but not mutant BRCA1). E2 induced the synthesis of cyclins D1 and B1, the activity of cyclin-dependent kinases Cdk4 and CDK1, and G(1)/S and G(2)/M cell cycle progression. The intact tumor suppressor inhibited all of these. wt BRCA1 also inhibited epidermal growth factor and insulin-like growth factor I-induced ERK and cell proliferation. The inhibition of ERK and cell proliferation by BRCA1 was prevented by phosphatase inhibitors and by interfering RNA knockdown of the ERK phosphatase, mitogen-activated kinase phosphatase 1. Our findings support a novel tumor suppressor function of BRCA1 that is relevant to breast cancer and identify a potential interactive risk factor for women with BRCA1 mutations.     

Promoter hypermethylation of BRCA1 correlates with absence of expression in hereditary breast cancer tumors

Germline mutations in BRCA1 account for a low proportion of hereditary cases in diverse populations. Several efforts have been made to find new genes involved in the inheritance of breast cancer with no success until today. The participation of BRCA1 in the development of breast cancer has been proposed in several studies where hypermethylation of its promoter and a decrease in expression has been reported for sporadic cases and one study on familial cases. To explore the participation of BRCA1 in hereditary carcinogenesis through a different mechanism than the inheritance of germline mutations, we studied the methylation status of its promoter in breast tumors, from patients previously screened for BRCA1/BRCA2 germline mutations. We also determined the presence of the BRCA1 protein in these tumors and correlated both events with tumor grade, hormone receptors and ERBB2 presence. Promoter hypermethylation of the BRCA1 gene was detected in 51% of our biopsies, among which 67% did not express the respective protein. This result leads us to suggest that hypermethylation could be considered as an inactivating mechanism for BRCA1 expression, either as a first or second hit. Moreover, a number of biopsies with absence of expression on BRCA1 showed negative detection of estrogen and progesterone receptors, a similar phenotype to BRCA1 mutated breast tumors.     

非小细胞肺癌BRCA2的表达及其与ER、PR及c—erbB-2的相关性研究

目的在非小细胞肺癌中检测乳腺癌易感基因（BRCA2）的表达和ER、PR及c-erbB-2蛋白表达特点,探讨BRCA2的表达与ER、PR及c-erbB-2之间的相关性。方法免疫组织化学SP法检测了42例手术切除的非小细胞肺癌中ER、PR、c-erbB-2和BRCA2蛋白表达,根据各种临床病理因素分组进行BRCA2表达阳性率统计学分析。结果BRCA2蛋白表达阳性15例（35．7％）;BRCA2蛋白表达阳性率在大于或等于60岁与小于60岁两年龄组间、男女组间、鳞状细胞癌组与腺癌组间及其分化程度以及c-erbB-2表达阳性组与阴性组间比较有显著性差异（P〈0．01）;而在有无淋巴结转移组间BRCA2蛋白表达阳性率比较无显著性差异（P〉0．05）;ER、PR在所有非小细胞肺癌病例中均为阴性。结论非小细胞肺癌可见BRCA2和c-erbB-2的表达,其两者的表达具有相关性,可能存在协同作用。提示BRCA2和c-erbB-2可作为反映非小细胞肺癌恶性特征的一个检测指标。     

Ubiquitination and proteasome-mediated degradation of BRCA1 and BARD1 during steroidogenesis in human ovarian granulosa cells

Germ-line mutations in BRCA1 predispose women to early-onset, familial breast and ovarian cancers. However, BRCA1 expression is not restricted to breast and ovarian epithelial cells. For example, ovarian BRCA1 expression is enriched in ovarian granulosa cells, which are responsible for ovarian estrogen production in premenopausal women. Furthermore, recent tissue culture and animal studies suggest a functional role of BRCA1 in ovarian granulosa cells. Although levels of BRCA1 are known to fluctuate significantly during folliculogenesis and steroidogenesis, the mechanism by which BRCA1 expression is regulated in granulosa cells remains to be elucidated. Here we show that the ubiquitin-proteasome degradation pathway plays a significant role in the coordinated protein stability of BRCA1 and its partner BARD1 in ovarian granulosa cells. Our work identifies the amino-terminal RING domain-containing region of BRCA1 as the degron sequence that is both necessary and sufficient for polyubiquitination and proteasome-mediated protein degradation. Interestingly, mutations in the RING domain that abolish the ubiquitin E3 ligase activity of BRCA1 do not affect its own ubiquitination or degradation in ovarian granulosa cells. The proteasome-mediated degradation of BRCA1 and BARD1 also occurs during the cAMP-dependent steroidogenic process. Thus, the dynamic changes of BRCA1/BARD1 protein stability in ovarian granulosa cells provide an excellent paradigm for investigating the regulation of this protein complex under physiological conditions.     

Prophylactic mastectomy: indications, options, and reconstructive alternatives

Prophylactic mastectomy continues to be a controversial procedure as a preventive tool against breast cancer. Recent research and other scientific advances, however, have refocused attention on better risk estimation, evidence of efficacy, and improvements in reconstruction. The recently discovered genetic markers BRCA1 and BRCA2 have become increasingly important in determining risk; a BRCA1-positive patient's risk of developing breast cancer by the age of 65 is estimated at 50 percent to 80 percent. BRCA1- and BRCA2-positive breast cancers also tend to be higher grade and occur in younger women (making mammography less effective). Genetically linked breast cancers are usually estrogen receptor negative, making them less susceptible to chemoprevention. Various predictive models and recommendations by experts in the field are also available for today's clinicians to ascertain who should be genetically tested. The benefit of bilateral prophylactic mastectomy, although difficult to estimate, can be evaluated by looking at the incidence of breast cancer in studies of patients who have previously undergone prophylactic mastectomy. The estimated risk reduction from these studies is 80 percent to 95 percent. Similarly, life expectancy is believed to be increased from 2.9 to 5.3 years. The psychological benefits include a 70 percent rate of satisfaction and a decrease in emotional concern over developing breast cancer by 74 percent of women who underwent prophylactic mastectomy. Although reconstruction results may vary, most patients have been very satisfied and some may achieve cosmetic results that are better than their preoperative situation. Patient selection for specific types of reconstruction after prophylactic mastectomy and the decision to proceed should be based on surgical risk and the likelihood of a good outcome. The choice of mastectomy incision should consider the size of the breast, preexisting scars, patient risk factors, and the planned method and goal of reconstruction. The authors propose certain guidelines based on degree of ptosis and cup size when planning prophylactic mastectomies with reconstruction. In certain cases, a nipple-sparing mastectomy may provide cosmetic advantages that could outweigh the additional oncologic risk.     

Functional characterization of a novel BRCA1-null ovarian cancer cell line in response to ionizing radiation

The breast and ovarian cancer susceptibility gene BRCA1 plays a major role in the DNA damage response pathway. The lack of well-characterized human BRCA1-null cell lines has limited the investigation of BRCA1 function, particularly with regard to its role in ovarian cancer. We propagated a novel BRCA1-null human ovarian cancer cell line UWB1.289 from a tumor of papillary serous histology, the most common form of ovarian carcinoma. UWB1.289 carries a germline BRCA1 mutation within exon 11 and has a deletion of the wild-type allele. UWB1.289 is estrogen and progesterone receptor negative and has an acquired somatic mutation in p53, similar to the commonly used BRCA1-null breast cancer cell line HCC1937. We used ionizing radiation to induce DNA damage in both UWB1.289 and in a stable UWB1.289 line in which wild-type BRCA1 was restored. We examined several responses to DNA damage in these cell lines, including sensitivity to radiation, cell cycle checkpoint function, and changes in gene expression using microarray analysis. We observed that UWB1.289 is sensitive to ionizing radiation and lacks cell cycle checkpoint functions that are a normal part of the DNA damage response. Restoration of wild-type BRCA1 function in these cells partially restores DNA damage responses. Expression array analysis not only supports this partial functional correction but also reveals interesting new information regarding BRCA1-positive regulation of the expression of claudin 6 and other metastasis-associated genes and negative regulation of multiple IFN-inducible genes.     

Clinical implications for BRCA gene mutation in breast cancer

To investigate the mutations of BRCA1 and BRCA2 and determine whether clinic-pathological factors related to BRCA gene mutation. Mastectomy specimens from 360 breast cancers were enrolled and examined in the study. The relationship between BRCA gene mutation and clinic-pathological factors was evaluated. Overall, 280 patients were BRCA negative and 80 got BRCA gene mutation. Triple-negative breast cancers—i.e., breast cancers that do not express estrogen receptors (ER), progesterone receptors (PR) or human epidermal growth factor receptor 2 (HER2/neu)—was observed in 53.85% of the BRCA1 mutation patients, in 28.57% of the BRCA2 mutation cases, while 14.29% of BRCA negative patients. BRCA1 mutation patients got a heavy lymph node metastasis and higher nuclear grade tumors than the others (P = 0.004, 0.007). Furthermore, BRCA mutation was also found to be significantly related to ER, PR and HER2/neu status (P < 0.05). BRCA1 expression was not associated with breast cancer-specific survival in the triple-negative breast cancers (P = 0.742). After Cox regression, BRCA1 mutation was not shown to be an independent prognostic factor for breast cancer. These findings substantiated the possibility of tumors associated with BRCA1 mutations divided into two distinct groups, triple-negative and non-triple-negative groups requires further investigation.     

TGF-��1 and BRCA2 Expression are Associated with Clinical Factors in Breast Cancer

The objective of this study was to investigate the possible association between the expression of transforming growth factor beta-1 (TGF-β1) and breast cancer type2 susceptibility protein (BRCA2) with clinical factors in breast cancer. TGF-β1, BRCA2, human epidermal growth factor receptor2 (HER2), estrogen receptor, and progesterone receptor protein levels were measured in 67 samples from breast cancer patients by immunohistochemistry. The expression of these proteins was correlated with various clinical factors including age, pathohistological grade and status of axillary lymph node implication. TGF-β1 and BRCA2 were expressed in breast cancer tissues and expression of HER2 and TGF-β1 was significantly correlated with BRCA2. The authors conclude that elevated expression of BRCA2 correlates with TGF-β1 and HER2 in breast cancer and these three factors act in synergy to promote cancer. Thus, detection of both TGF-β1 and BRCA2 may therefore assist in the prognosis and treatment of breast cancer.