Abstract A12: Identification of Nrdp1 as a novel androgen receptor transcription target differentially regulated in androgen-dependent and independent prostate cancer

Abstract

Abstract Background: The ErbB receptor tyrosine kinase family regulates proliferation and survival in prostate cancer (PCa). We recently showed that ErbB3 plays a significant role in increasing androgen receptor (AR) transcriptional activity and in causing castration-resistant PCa (CRPC). Reciprocally, AR maintained castration sensitivity by suppressing ErbB3 levels through transcriptional regulation of E3 ubiquitin ligase Nrdp1, while loss of AR regulation of Nrdp1 resulted in an unrestricted surge in ErbB3 levels, and cell growth. Here, we investigate whether Nrdp1 is a transcriptional target of the AR in PCa cells. Results: The promoter region of Nrdp1 contains three androgen response elements (AREs) - one located 215 aa upstream of the transcriptional start site (ARE.03), and two within an internal promoter (ARE.01 and ARE.02). Chromatin immunoprecipitation (ChIP) studies revealed AR binding in PCa cells to ARE.03. AR binding to ARE.03 was found to be androgen regulated in androgen-dependent LNCaP cells, whereas no AR binding to ARE.01 or ARE.02 has been detected in this cell line. Luciferase assay to determine AR transcriptional activity on ARE.03 showed significant response to androgens, whereas full mutation of ARE.03 abolished AR transcriptional activity. However, the AR failed to bind to ARE.03 in C4-2 and LNCaP-AI cell lines, CRPC sublines of LNCaP cells, although no mutations in these regions were identified, while AR continued to bind the AR to PSA ARE in these cells, indicating that the AR was still active. Thus the AR binds to PSA ARE in both androgen-dependent and –independent cells, but to ARE.03 only in androgen-dependent but not –independent cells. Luciferase assay in LNCaP-AI cells showed decreased AR transcriptional activity on ARE.03 when compared to LNCaP, supporting decreased AR binding to this region. Hence we investigated the cause for the differential binding of the AR in these two areas. The structural protein Filamin A (FlnA) was earlier shown to cleave to a 90 kDa fragment (FlnA16-24) whose nuclear localization maintained androgen dependence. We investigated whether this protein played a role in regulation of AR binding to ARE.03 in PCa cells. Transfection of FlnA16-24 in C4-2 cells resulted in localization of FlnA 16-24 to the nucleus, and restored AR binding to ARE.03. FlnA 16-24 significantly increased luciferase activity on ARE.03, while suppressing AR activity on PSA indicating that this AR binding protein is required for AR-mediated transcription of Nrdp1. Conclusions: Our results indicated that Nrdp1 is regulated in androgen sensitive cells, but not in CRPC cells, by AR binding to ARE.03, whereas no such difference in AR binding to PSA ARE exists. We show that AR binding to ARE.03 is abolished in CRPC cells because of a decreased expression of nuclear FlnA, and that restoration of nuclear FlnA will restore androgen-sensitive cell growth. Our data indicate that the AR-coregulator FlnA is required for AR binding to the Nrdp1 promoter and that loss of this protein results in the failure of AR to regulate Nrdp1 transcription, resulting in unrestricted increase in ErbB3. These data identified Nrdp1 as a novel target of AR transcriptional activity in androgen-dependent but not in CRPC cells.