The Unliganded Glucocorticoid Receptor Positively Regulates the Tumor Suppressor GeneBRCA1through GABP Beta

Abstract

Loss of BRCA1 tumor suppressor function is a critical event in breast tumorigenesis. We have previously identified the stress hormone hydrocortisone as a negative regulator of BRCA1 expression in nonmalignant mammary cells. Here, we have identified a direct role for the unliganded glucocorticoid receptor (GR) in BRCA1 upregulation in the absence of hydrocortisone. The positive regulatory effect of GR is lost upon the addition of hydrocortisone. We have shown that GR interacts with the BRCA1 promoter only in the absence of hydrocortisone, and that this interaction is mediated through the b-subunit of the ets transcription factor GA-binding protein (GABP) at the RIBS promoter element. GR and GABPb interact in both coimmunoprecipitation and mammalian two-hybrid assays, and this interaction involves the N-terminal to central regions of both proteins. This work presents the first evidence of a ligand-independent role for GR as a positive regulator of gene expression, and loss of GR from the BRCA1 promoter in response to stress hormones leads to decreased BRCA1 expression. Because low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk. Mol Cancer Res; 10(4); 558-69. Ó2012 AACR. IntroductionGerm line mutations in the BRCA1 tumor suppressor contribute to familial breast tumor formation but BRCA1 mutations do not seem to occur in sporadic breast cancer tumors (1). Instead, sporadic breast tumors exhibit decreased levels of BRCA1 expression, and the degree of downregulation seems to be correlated with tumor grade, rate of tumor progression, and risk of metastasis (2-6). This implies that loss of BRCA1 function, either through decreased activity or downregulation of expression, is a critical event in the etiology of breast cancer. Phenotypically normal breast epithelial cells from individuals harboring a BRCA1 germ line mutation (a so called "one-hit" mutation accompanied by a 50% decrease in BRCA1 function) express an altered mRNA profile compared with normal cells from individuals without a BRCA1 mutation (7). This suggests that decreases in functional BRCA1

Expression microarray identifies the unliganded glucocorticoid receptor as a regulator of gene expression in mammary epithelial cells

“…EPH-4 cells were cultured as previously described [13,19]. EPH-4 cells stably transfected with H1-2 empty vector or shGR (see below) were maintained in serum-free media with 2 μg/mL puromycin (Sigma).…”

“…The addition of hydrocortisone (HC) abolishes this effect and results in decreased BRCA1 expression [13]. The positive regulatory effect of unliganded GR appeared to be constitutive, involving basal GR levels within breast cells, since no stimulus or secondary messenger was required for its activation, unlike other reports of ligand-independent activation by other steroid hormone receptors which have typically been in response to other stimuli [14].…”

“…We found that in cells depleted of GR, 260 genes were significantly upregulated, while 343 genes were significantly downregulated. Since the downregulated genes represented those which are positively regulated by unliganded GR, potentially through a mechanism similar to that reported for BRCA1 [13], we examined the most significant networks comprised of this gene set via pathway analyses, and determined that several of these genes were involved in pro-apoptotic networks. Validation and further analysis of five candidates of positive regulation by unliganded GR indicated that two of these, Hsd11b1 and Ch25h , were also downregulated following HC treatment, in a manner similar to Brca1 .…”

Glucocorticoid receptors in lung cancer: new perspectives

“…In breast cancer, unliganded GR interacts with BRCA1. Thus, loss of GR from the BRCA1 promoter as a result of Gc stimulation leads to reduced BRCA1 expression, which may contribute to the development of sporadic breast tumours which do not show mutations in BRCA1 (Ritter et al 2012). Decreased GR expression, therefore, appears to be a ubiquitous and important event, leading to the suggestion that the GR is a TSG in the breast as high GR expression is correlated with anti-proliferative signalling.…”

“…Decreased GR expression, therefore, appears to be a ubiquitous and important event, leading to the suggestion that the GR is a TSG in the breast as high GR expression is correlated with anti-proliferative signalling. Importantly, genetic heterogeneity influences this effect as loss of GR has a better outcome in ER − breast cancer but a poorer outcome in ER+ breast cancer, indicating that GR effects in breast cancer may be tumour sub-type dependent (Ritter et al 2012). The GR has also been postulated to act as a tumour suppressor in skin and prostate cancers .…”