4931

The Androgen receptor (AR) plays critical roles in both androgen-dependent and ablation-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate its roles in disease progression. We have employed a novel methodology we developed called Chromatin Immunoprecipitation (ChIP) Display (CD) to discover novel AR targets genes in the C4-2B ablation-resistant PCa cell line. Briefly, cells were subjected to ChIP with anti-AR antibodies and the precipitated DNA was subjected to digestion with AvaII, ligation-mediated PCR with sets of nested primers, and polyacrylamide gel electrophoresis. Bands enriched in replicate ChIPs as compared to mock-ChIPs were sequenced and mapped to the human genome. Of 19 novel AR-occupied regions we discovered, only five mapped to 5’ promoter proximal regions. Seven were found within the body of annotated genes and three within 4-kb 3’ of the nearest gene. We next measured, by RT-qPCR, the androgen responsiveness of genes in the vicinity of, but up to 100-kb away from, our AR-occupied regions. In C4-2B cells stimulated with androgens we found many genes, surrounding the AR binding regions, which responded significantly to AR activation. Many genes were induced: CRELD2, PRKCD, GSTT2, DDT, TRPV3, PYCR1 and PGR1; and some genes were repressed: MUC6, WBSCR28, KIAA1217 and CHRM1. Interestingly, we find that proximity of genes to AR binding regions does not necessarily dictate responsiveness to androgens. Overall, of the 15 AR-occupied regions near annotated genes, 11 regions contained androgen responsive genes. None of the tested genes near the remaining four AR-occupied regions significantly responded to androgens. To test whether AR regulated any of these genes in the absence of ligand, we performed AR siRNA experiments and discovered that the expression one gene, OAT, was decreased upon AR knockdown, indicating that although it is not DHT responsive, it is AR dependent. We also stumbled upon the discovery that PGR1, a DHT induced gene, is negatively regulated by AR in the absence of ligand, as the expression of this gene increased in siAR-treated cells. Lastly, to pursue the clinical significance of our cell culture findings, we mined a microarray study of human PCa for the expression of genes near our AR binding regions as a function of disease progression. Compared to primary untreated PCa tumors, the expression of DDT, PRKCD, GSTT2 and PYCR1, like that of known AR targets PSA and TMPRSS2, was decreased in androgen ablated PCa tumors, consistent with AR-mediated control. Furthermore, expression of many of these novel AR target genes was increased in metastatic tumors, where AR activity is known to be restored in a ligand-independent manner. As many of these novel AR target genes have functions relevant to cancer biology, they may provide opportunities for the development of novel therapeutic approaches to manage advanced PCa.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA