Abstract 3930: Ligand-independent activity of the mutant LNCaP androgen receptor drives cell proliferation in hormone-independent LNCaP tumor cells

Abstract

Abstract Androgen-dependent prostate tumor cells can adapt to androgen ablation by up-regulating expression of the androgen receptor (AR). Increased expression of AR may sensitize cells to residual androgens in the environment or to de novo-synthesized ligands. We have shown previously that hormone-independent LNCaP 104-R1 cells, which arise after long-term androgen deprivation of androgen-dependent 104-S cells, express much higher levels of AR than 104-S cells. LNCaP prostate tumor cells express a mutant T877A AR that can be activated by progesterone, raising the possibility that LNCaP cells progress to apparent hormone independence during androgen deprivation by initiating de novo synthesis of progesterone. In support of this, we have found that androgen-dependent LNCaP 104-S cells grow independently of exogenous androgen after lentivirus-mediated over-expression of T877A AR, but not wild-type (wt) AR. This difference was attributable to the T877A substitution and not to sequence differences in the N-terminus between wt and LNCaP cell-derived AR. However, this growth was insensitive to the anti-androgen bicalutamide, similar to the bicalutamide-insensitive growth of hormone-independent 104-R1 cells. Lentiviral sh-RNAi-mediated knockdown of AR resulted in growth inhibition in both 104-S and 104-R1 cells. Analysis of T877A AR transactivation in 104-S and 104-R1 cells by reporter gene assay revealed that T877A AR displayed significant constitutive and bicalutamide-insensitive activity. Wild-type AR, on the other hand, showed low basal activity in the absence of added ligand and modest induction by bicalutamide. Co-expression of CYP11A1 (cholesterol side chain cleavage enzyme) and HSD3B2 (3-beta hydroxysteroid dehydrogenase-2), which sequentially generate pregnenolone and progesterone, strongly activated T877A AR, but not wt AR in 104-S cells. This activation could be inhibited by bicalutamide or ketoconazole, an inhibitor of CYP11A1. Expression of CYP11A1 alone in 104-S cells increased cell proliferation in the absence of androgen that was bicalutamide-sensitive. This indicates that 104-S cells may express low level but functional HSD3B activity, because pregnenolone does not activate T877A AR. Secondly, the bicalutamide-sensitivity of progesterone-driven cell proliferation and bicalutamide-insensitivity of T877A AR-driven proliferation, together with the observations above, suggest that de novo synthesis of progesterone or another ligand in LNCaP cells is most likely not responsible for T877A AR-driven proliferation in cells in which T877A AR is over-expressed. Rather, the T877A mutation may confer upon the receptor constitutive ligand-independent activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3930. doi:1538-7445.AM2012-3930