Abstract 2201: A novel androgen receptor regulatory loop: Posttranscriptional androgen receptor regulation through Midline 1 ribonuclear protein complex

Objective: Exploration of posttranscriptional regulation of androgen receptor (AR) protein through interaction with the midline 1/protein phosphatase 2A/Alpha4 (MID1/PP2A/α4) ribonuclear protein complex. Background: Deregulation of the androgen receptor (AR) signaling pathway is a hallmark of prostate cancer progression to an advanced, castration-resistant tumor stage. Several mechanisms like amplification of the AR gene, mutations, altered nuclear receptor cofactor expression, aberrant activation and AR overexpression have been proposed. In addition to gene amplification and enhanced transcription also posttranscriptional mechanisms contribute to regulation of AR protein in prostate cancer. The MID1/PP2A/ β4 complex which is a microtubule-associated ribonucleoprotein complex was shown to regulate the stability and intracellular localization of associated mRNAs, among them AR mRNA. Materials and Methods: RNA interference (siRNA) was used to knockdown proteins in several cellular prostate cancer models. For AR activitation or inhibition synthetic androgen analog R1881 and anti-androgen bicalutamide, respectively, were used. AR DNA binding sites in the MID1 gene were identified in DuCaP cells by chromatin immunoprecipitation (ChIP) followed by DNA sequencing. Reporter gene vectors were constructed with putative wild type and mutated androgen responsive elements (AREs) and their functional activities were determined in reporter gene assays. Results: Down regulation of MID1 or β4 proteins by specific siRNAs resulted in a reduced AR protein level in the AR positive cell lines. MID1 knockdown also affected proliferation and migration in both AR positive and negative prostate cancer cell lines. Androgen treatment decreased the MID1 protein as well as MID 1 mRNA level and bicalutamide abrogated this effect. Several AR binding sites were identified in the MID1 gene using ChIP sequencing. Their functional activities as androgen responsive elements (AREs) were analyzed and confirmed in reporter gene assays using reporter vectors with wild type and mutated AREs. Immunohistochemistry revealed a specific and unique expression pattern of MID1. Whereas immunoreactivity was absent or very weak in benign epithelium, there was a very heterogeneous expression in prostate tumors with expression especially in high grade tumors displaying a cribriform growth pattern. Very high immunoreactivity was also detected in some lymph-node metastases. Conclusions: Our results support a ne