Studying The Mechanisms By Which Androgen Receptor Splice Variants Promote Castration-resistant Growth Of Proslate Cancer Cells

Prostate cancer (PCa) is the second most common cancer and the fifth leading cause ofdeath from cancer in men worldwide base on the statistics from WHO in2012. For nearly10years, the morbidity and mortality of PCa in China have shown a rising trend. PCa hasbecome the seventh most commonly diagnosed cancer among men in China. Therefore,studies of PCa have been included in the national "973" project in2011.Androgen receptor (AR) plays a critical role in the development and progression ofPCa. After bound by its ligand (androgen), the AR translocates to the nucleus, then binds tothe specific androgen response elements and promotes target gene expression. Prostatespecific antigen (PSA) is one of the most important target genes of AR as well as animportant biomarker for prostate cancer diagnosis and prognosis. Endocrine therapy orandrogen deprivation therapy (ADT) has been regarded as the classic therapy for advancedPCa because of the important role of the AR signaling pathway in PCa. Due to the lack ofeffective PCa screening programs in our country, most of the patients are diagnosed at anadvanced stage, and androgen deprivation therapy is the mainstay treatment.Despite the initial efficacy of ADT, relapse with incurable and lethal castration-resistantprostate cancer (CRPC) invariably occurs and remains the most critical challenge in theclinical management of prostate cancer.Resurgent androgen receptor activity is an established driver of castration-resistantprogression. Both ligand-dependent and–independent mechanisms have been proposed tounderlie reactivation of canonical AR signaling after androgen-directed therapies. Severalnew drugs targeting androgen receptor reactivation, such as the FDA-approved abirateroneand Enzalutamide, can prolong the overall survival of patients with castration-resistantprostate cancer. However, even with these new drugs, the time to disease progression andoverall survival still remain relatively short.AR-Vs are up-regulated in most CRPCs compared to hormone-na ve cancers.Differential splicing of pre-mRNA is a frequent mechanism for generation of protein variantswith oncogenic activity, and the expression of posttranscriptional AR splice variants withcapacity for constitutive AR transactivation has recently been recognized as a potential mechanism of CRPC progression. Approximately25variants have been identified in humanprostate tissues and cell lines. Strikingly, patients with high levels of AR-V7(aka AR3) andARv567es, two major AR-Vs, had significantly shorter survival than other CRPC patients,indicating an association between AR-V expression and a more lethal form of prostate cancer.Understanding the mechanisms of AR-V transactivation is vital for rational drug designto improve the efficacy of androgen-directed therapies in the battle against CRPC.AR-FL, AR-V7and ARv567es cDNA were sub-cloned into BiFc expression system.All recombined expression vectors were transfected into AR-null PCa cell line PC-3by usingthe lipofectamine transfection method. The protein expression levels were detected by thewestern blot using anti-N-terminal of AR-FL antibody; Homodimerization between AR-V orHeterdimerization between AR-V and AR-FL was detected under fluorescence microscopewhile transfected cells were alive. Flow Cytometry assay was used to quantitate the signal ofVenus fluorescence protein. The effect of AR-V homodimerization or AR-V/AR-FLheterdimerizaiton on the sub-cellular localization of themselves was determined by the laserscanning confocal microscope. The ARE report gene assay and qRT-PCR were used to detectthe influence of AR-V homodimerization or AR-V/AR-FL heterdimerization on target-geneexpression. The effect of AR-V homodimerizaiton or AR-V/AR-FL heterdimerizaiton onpromoting castration-resistant growth of prostate cancer cells was determined by the SRBmethod.Western Blot analysis showed that all BiFc-AR-FL or BiFc-AR-V were of the expectedsize. Mutating the D-box, but not the FxxLF motif, abolished the interactions, indicating thatdimerization between AR-Vs is mediated through DBD/DBD interaction. To delineate thedimerization interface, we mutated AR-Vs at the FxxLF motif (F23,27A/L26A) and/or D-box (A596T/S597T), which mediate AR-FL homo-dimerization through N/C-terminalinteraction or DBD/DBD interaction, respectively. AR-V homo-dimerization is not requiredfor its nuclear translocation, on the contrary, AR-V/AR-FL heterdimerization is required forAR-FL nuclear translocation under androgen deprivation condition. ARE-luciferase assayand qRT-PCR showed that AR-V or AR-V/AR-FL regulation of target-gene expression ishomo-or heterdimerization dependent. AR-V homo-dimerization or AR-V/AR-FLheterdimerization can promote castration-resistant growth of prostate cancer cells.Under androgen deprivation condition, in vitro, AR-V regulate the expression of itsspecific target genes through homo-dimerization, and AR-V can form a heterodimer withAR-FL to regulate the expression of classic AR target genes. These mechanisms limit theefficacy of all androgen-directed therapies and promote CRPC development and progression.