Characterization of Androgen Action in Breast Cancer

Breast cancer (BCa) is the most common malignancy in Canadian women. Male breast cancer accounts for only 1% of all BCa, yet it is potentially fatal. Regardless of gender, normal breast and BCa cells have receptors for female hormones, estrogen and prolactin, and male hormone, androgen. We have identified an enzyme, called CPD, whose activity increases BCa cell survival. CPD is found on the cell surface and it releases amino-acid arginine (Arg) from proteins found outside the cell. The released Arg enters the cell and is converted to a molecule called nitric oxide (NO). NO can stimulate BCa cell growth and, hence, some preclinical studies aim at modulating NO levels. We reported that the events governed by the CPD-Arg-NO pathway are stimulated by estrogen and prolactin. Our preliminary data show that it is also stimulated by androgen. This fortuitous convergence of hormone action makes this pathway useful for characterizing androgen action, as compared to the female hormones, in BCa cells. In this proposal, we will investigate the effects of androgen on the CPD protein and NO in BCa cells. Secondly, we will study the CPD gene (i.e., the DNA) to determine how androgen, as compared to estrogen/prolactin, activates it to make the CPD protein. The CPD-Arg-NO pathway is a potential new therapeutic target for modulating NO levels. Elucidation of androgen activation of this pathway in BCa may reveal new strategies for the treatment and/or management of this disease in both gender. To characterize androgen regulation of carboxypeptidase-D (CPD) expression and nitric oxide (NO) production in breast cancer cells.We will fully characterize androgen (e.g., T, dihydrotestosterone) regulation of CPD mRNA/protein expression and activity in human MCF-7 and T47D BCa cells. Androgenic effects on NO production and NOS expression will be determined, and correlated with BCa cell viability, using fluorescent DAF-2T assays, Western analysis and colorimetric MTS assays, respectively. We will delineate the effects of androgens versus estrogen, using synthetic androgen R1881 or aromatase inhibitors (inhibits T aromatization to estrogen).To characterize hormonal activation of CPD gene transcription.T versus PRL regulation of CPD promoter activity will be determined using CPD promoter constructs containing the GAS and the putative AREs in luciferase reporter assays. GAS and ARE activities will be verified using EMSA and ChIP assays. EMSA will help to determine the binding of i) Stat5a/b to GAS or ii) AR to the putative AREs in vitro. ChIP will show recruitment of Stat5a/b to GAS or the liganded AR to AREs in vivo.