Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer

Abstract

Androgen receptor (AR) is overexpressed in the majority of castration-resistant prostate cancers (CRPCs). Our goal was to study the effect of AR overexpression on the chromatin binding of the receptor and to identify AR target genes that may be important in the emergence of CRPC. We have established two sublines of LNCaP prostate cancer (PC) cell line, one overexpressing AR 2-3-fold and the other 4-5-fold compared with the control cells. We used chromatin immunoprecipitation (ChIP) and deep-sequencing (seq) to identify AR-binding sites (ARBSs). We found that the number of ARBSs and the AR-binding strength were positively associated with the level of AR when cells were stimulated with low concentrations of androgens. In cells overexpressing AR, the chromatin binding of the receptor took place in 100-fold lower concentration of the ligand than in control cells. We confirmed the association of AR level and chromatin binding in two PC xenografts, one containing AR gene amplification with high AR expression, and the other with low expression. By combining the ChIP-seq and expression profiling, we identified AR target genes that are upregulated in PC. Of them, the expression of ZWINT, SKP2 (S-phase kinase-associated protein 2 (p45)) and FEN1 (flap structure-specific endonuclease 1) was demonstrated to be increased in CRPC, while the expression of SNAI2 was decreased in both PC and CRPC. FEN1 protein expression was also associated with poor prognosis in prostatectomy-treated patients. Finally, the knock-down of FEN1 with small interfering RNA inhibited the growth of LNCaP cells. Our data demonstrate that the overexpression of AR sensitizes the receptor binding to chromatin, thus, explaining how AR signaling pathway is reactivated in CRPC cells.