Effect of enzalutamide (EZ) exposure time on androgen receptor (AR) splice variant (SV) expression and gene transcription correlates in androgen-dependent (AD) prostate cancer (PC) cells.

e22066 Background: The emergence of castration-resistant (CR) PC limits the benefits of androgen deprivation therapy (ADT) in controlling recurrent AD PC. ARSVs lacking the ligand-binding domain are frequent in CRPC tissue/cells where they mediate resistance to anti-androgens and androgen synthesis inhibitors by regulation of a mitosis-dependent, ARSV-driven transcriptome program (TP) that differs from the differentiation-driving program of full-length (fl) AR. We postulated that ARSVs emerge as a stress reaction to AR suppression and only later induce the TP changes of CRPC. Methods: We assessed the effects of treatment with the anti-androgen EZ (EZt) over 19 culture passages (cp) on AD LNCaP cells (AD-cells), isogenic androgen-independent (AI) cells (AI-cells) and AI CW22RV1 (RV1) cells: growth in androgen-replete or –deprived medium (RM, DM) (MTT assay, cell counts); flAR and ARSV expression (Q-PCR) and the genome-wide TP (Affymetrix Human Exon microarray using 1.3X differential expression and t-tests for significance). Results: In EZt AD-cells, known pathogenic ARSV7 increased 5X relative to flAR by cp 3 – 11 while growth remained AD (EZ-inhibited); by cp 16, the relative increase was >300X and growth suddenly became AI (EZ-resistant). In both AI-cells and RV1 cells, ARSV7 increased after only 3 cp associated with enhanced AI/EZ-resistant growth. TP analysis demonstrated a striking difference between AD-cells and AI-cells or RV1 cells at baseline and after 3 cp on EZ. The TP of EZt AD-cells showed downregulation of AR-regulated transcripts (KLK3, TMPRSS), a biphasic response of cell cycle genes (CDK1, CCNA2, UBE2C) (cp3 downregulation, cp16 upregulation) and upregulation of neuroendocrine genes (AGR2,AURKA, SSTR2) at cp 16. The profound changes in cp16 EZt AD-cells (203 differentially-expressed genes/baseline) rendered a TP similar to CRPC-like RV1 cells with high endogenous ARSV7 levels. Conclusions: After an effective interval of EZt, a precipitous increase of ARSV7 occurred in AD LNCaP cells concurrent with the acquisition of AI/CR PC characteristics, an observation potentially relevant to clinical ADT in AD PC.