53BP1 is a Positive Regulator of the BRCA1 Promoter

Germline mutations in the BRCA1 tumor suppressor gene contribute to familial breast and ovarian tumor formation. Sporadic breast and ovarian cancer, however, which accounts for more than 90% of total cases and virtually lacks BRCA1 mutations, exhibits reduced expression of the BRCA1 gene. The magnitude of this reduction correlates with disease progression. In this report we have identified an imperfect palindrome sequence for binding of the 53BP1-containing complex, -40TTCCGTGG CAACGGAA-25, within the BRCA1 minimal promoter. Overexpression of 53BP1 activates a luciferase reporter driven by the wild type BRCA1 minimal promoter, but not by the BRCA1 minimal promoter with mutated palindrome sequence. Depletion of endogenous 53BP1 by siRNA suppresses activity of the BRCA1 minimal promoter. In vitro and in vivo DNA-protein interaction studies demonstrate that this palindrome sequence binds to the 53BP1-containing complex. These findings establish a positive regulation of the BRCA1 promoter by 53BP1. Disruption of this regulation in cancer cells may provide a molecular mechanistic basis for sporadic breast and ovarian tumor formation.     

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.     

Frequent promoter hypermethylation and expression reduction of the glucocorticoid receptor gene in breast tumors

Previous studies have found that expression of the Glucocorticoid Receptor (GR) is altered or reduced in various cancers, while the GR promoter has been shown to be methylated in gastric, lung, and colorectal cancers. Examining a small cohort of matched normal and breast cancer samples we found that GR levels were dramatically reduced in almost all tumors in relation to their normal tissue. The methylation status of the GR promoter was assessed to determine if this observed decrease of expression in breast tumors could be due to epigenetic regulation. While it was not methylated in normal tissue, the GR proximal promoter was methylated in 15% of tumor samples, particularly, but not exclusively, in Estrogen Receptor positive tumors. GR expression in these tumors was particularly low and loss of GR expression was specifically correlated with methylation of the proximal promoter GR B region. Overall, these results show that hypermethylation of the promoter in tumors is a frequent event and suggests that GR may act as a tumor suppressor in breast tissue.     

Frequent promoter hypermethylation and expression reduction of the glucocorticoid receptor gene in breast tumors

Previous studies have found that expression of the Glucocorticoid Receptor (GR) is altered or reduced in various cancers, while the GR promoter has been shown to be methylated in gastric, lung, and colorectal cancers. Examining a small cohort of matched normal and breast cancer samples we found that GR levels were dramatically reduced in almost all tumors in relation to their normal tissue. The methylation status of the GR promoter was assessed to determine if this observed decrease of expression in breast tumors could be due to epigenetic regulation. While it was not methylated in normal tissue, the GR proximal promoter was methylated in 15% of tumor samples, particularly, but not exclusively, in Estrogen Receptor positive tumors. GR expression in these tumors was particularly low and loss of GR expression was specifically correlated with methylation of the proximal promoter GR B region. Overall, these results show that hypermethylation of the promoter in tumors is a frequent event and suggests that GR may act as a tumor suppressor in breast tissue.     

BRCA1 and BRCA2 bind Stat5a and suppress its transcriptional activity

Germline mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are thought to account for a large portion of familial breast cancer. The increased risk of breast cancer in women carrying such mutations suggests that these proteins play a critical role in the growth regulation of mammary epithelial cells. Another protein, Stat5a, is known to be essential for growth and terminal differentiation of breast epithelial cells. Here we show that Stat5a forms a complex with both BRCA1 and BRCA2 in breast epithelial cells upon stimulation with prolactin. In addition, we show that the activity of Stat5a on the beta-casein promoter is modulated by both BRCA1 and BRCA2. This interaction may be important during the expansion and terminal differentiation of breast epithelial cells, as happens during pregnancy and lactation.     

BRCA1 plays a role in the hypoxic response by regulating HIF-1alpha stability and by modulating vascular endothelial growth factor expression

A recent study of breast cancer patients with and without BRCA1 gene mutations found significantly lower levels of VEGF in serum from patients with BRCA1 mutations (Tarnowski, B., Chudecka-Glaz, A., Gorski, B., and Rzepka-Gorska, I. (2004) Breast Cancer Res. Treat. 88, 287-288). Here, we describe a possible mechanistic explanation for this correlation. Because hypoxia in tumors stimulates VEGF expression and secretion we hypothesized that altered BRCA1 protein levels in breast tumors could affect hypoxia-stimulated VEGF promoter activity. This possibility was tested in cells transfected with various combinations of expression plasmids for BRCA1, BRCA1 specific inhibitory RNAs (BRCA1-siRNAs), HIF-1alpha, and a VEGF promoter-reporter and then incubated in normoxia (21%, O2) or hypoxia (0.1%, O2). As predicted, increased BRCA1 levels enhanced both hypoxia-stimulated VEGF promoter activity and the amounts of VEGF mRNA, as determined by semiquantitative RT-PCR and quantitative real time PCR. Using the ChIP assay, we discovered that BRCA1 could be recruited to the endogenous human VEGF promoter along with HIF-1alpha following hypoxia. An interaction between BRCA1 and HIF-1alpha was found in human breast cancer cells. We also found that hypoxia-stimulated VEGF promoter activity and secretion was reduced in cells containing reduced amounts of endogenous BRCA1 protein (obtained by transfecting with BRCA1 siRNAs). A mechanistic explanation for these effects is provided by our finding a reduced half-life and reduced accumulation of HIF-1alpha in hypoxic cells transfected with BRCA1-siRNAs and that proteasome inhibitors blocked these effects of BRCA1-siRNAs. Thus, our results suggest that normal amounts of BRCA1 function in hypoxia to regulate HIF-1alpha stability, probably by interacting with HIF-1alpha.     

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway

The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.