Androgen signalling in normal and malignant breast epithelial cells.

Abstract

The growth and survival of normal breast epithelial cells and breast cancer cells is promoted by estrogens. In contrast, androgens inhibit the proliferation of normal and malignant breast epithelial cells. While this effect of androgens on breast cells appears to be androgen receptor (AR) dependent, the precise mechanism of inhibition and its functional significance are unknown. The aims of this thesis were to investigate the effect of androgen signalling on growth of normal and malignant breast epithelial cells, and to assess the interactions between androgen and estrogen signalling in the breast. To investigate the role of androgen signalling in the growth and development of the normal mammary gland, female mice were treated with either the native androgen 5α- dihydrotestosterone (DHT) or the antiandrogen, flutamide. Analysis of the mammary glands at the end of the treatment period demonstrated that DHT reduced ductal branching and mammary epithelial cell proliferation when treatment commenced mid-puberty. Conversely, flutamide treatment that commenced post-puberty significantly increased ductal branching and proliferation of mammary epithelial cells. This data demonstrates that androgen signalling inhibits proliferation in the normal mammary gland, and may therefore oppose to the growth stimulatory effects of estrogen signalling to regulate breast growth and development. The antiproliferative effects of androgens on breast epithelial cells may be due in part to direct AR-mediated activation of androgen regulated genes, or alternatively, androgens could act indirectly through AR to inhibit estrogen receptor alpha (ERα) activity. Expression of fulllength AR or a truncated, constitutively active AR (AR-T707) significantly inhibited the activity of ectopically expressed ERα in MDA-MB-231 breast cancer cells (ERα- and ARnegative), in a dose-dependent manner. The functional consequences of inhibition of estrogen signalling by overexpressing AR were investigated in the T-47D breast cancer cell line (ERα- and AR-positive). Expression of AR-T707 in T-47D cells resulted in inhibition of both basal and estradiol-induced cell proliferation and a marked reduction in the steady-state protein levels of the estrogen regulated gene, PR. The final chapter investigated the mechanism by which AR inhibits ERα activity. A coimmunoprecipitation assay demonstrated an interaction between ectopically expressed AR and ERα in COS-1 cells, but not endogenous AR and ERα in a breast cancer cell line. To delineate the regions of AR required for inhibition of ERα signalling, various functional domains of the AR were mutated or deleted. Reporter gene assays showed that the inhibitory effects of AR were abrogated by deletion or mutation of the DNA binding domain (DBD). Furthermore, overexpression of the AR-DBD alone was sufficient to inhibit ERα activity. Consistent with a requirement for the DBD of AR to inhibit ERα activity, mobility shift assays demonstrated binding of AR to the Xenopus vitellogenin A2 consensus estrogen response element (cERE); however AR/ERα heterodimers were not detected on a cERE. Consistent with these findings, molecular modelling demonstrated that it is feasible for the DBD of AR to bind to a cERE and that it is unlikely that AR/ERα heterodimers could bind. Chromatin immunoprecipitation demonstrated recruitment of AR to the promoters of endogenous estrogen regulated genes. The findings suggest that the inhibitory effect of AR on ERα activity may occur either via formation of non-functional AR/ERα heterodimers that are unable to bind to EREs, or AR homodimers competing effectively for binding to EREs, in ERα target genes. The results in this thesis demonstrate an inhibitory effect of androgen signalling on growth of normal and malignant breast epithelial cells. Additionally, the inhibition of breast epithelial cell proliferation by androgen signalling can be attributed, at least in part, to inhibition of ERα activity. These studies have provided insight into androgen action in the breast, and support a model whereby androgens balance the stimulatory effects of estrogen signalling in normal and malignant breast epithelial cells.