Abstract 3917: Regulatory role of ROBO-1, a novel tumor suppressor on Androgen receptor and Wnt signaling during castration-resistant prostate cancer development: A novel molecular target for gene therapy

Abstract

Abstract Introduction and Objectives: Prostatic epithelial cells while progressing from premalignant stages to malignant stages (hormone-sensitive and castration-resistance phenotypes) undergo a lot of phenotypical changes which include increased clonal expansion; and modulation in cytoskeleton and cell adhesion characteristics. During organogenesis, the Roundabout-1 (ROBO-1) regulates cell proliferation, migration and adhesion. We for the first time (1) established the mechanismαbased role of ROBO1 tumor suppressor gene in the development of castrationαresistant CaP (CRPC) and (2) determined its significance as potential target for gene therapy. Methods: Immunoblot analysis, real time PCR and immunohistochemistry was used to analyze human prostatic tissues and cell lines for ROBO1. ROBOα1 was (i) overαexpressed by transfecting ROBO1αexpressing plasmid (ii) knocked down by employing vectorαbased shRNA. ROBO1αsuppressed and ROBO1αoverexpressing cells were tested for proliferation, clonogenicity, sensitivity towards chemotherapeutic agents, androgen receptor (AR)α binding to target genes, AR and Tcfαtranscriptional activities by employing 3[H]thymidine uptake, softαagar, luciferase reporter and ChIP assays. Results: We provide evidence that mRNA and protein levels of ROBO1 are lost in (i) CRPC cells, ii) prostatic tissues of NKx3.1/Pten mouse model and (ii) human prostatic tissues during the progression of disease from early stage (low) to late stage (highαGleason score). We show that Loss of ROBO-1 was observed to increase (i) AR transcriptional activity, (ii)Tcf-transcriptional activity (marker of Wnt signaling) (iii) accumulation of β-catenin and (iv) Akt activity and (v) proliferation of CRPC cells. Importantly, CRPC cells (which survived therapy) exhibited high potential to proliferate after repopulation when ROBO-1 was silenced. We provide compelling evidence that under ablation conditions ROBO1 is lost in some malignant CaP cells which results in a subtype that is castration-resistant. We show that ROBO-1 is inactivated due to epigenetic changes in CRPC cells under castration-conditions. ROBO-1-deficient CRPC cells were more chemoresistant and exhibited increased AR-activity than ROBO-1-expressing counterparts. ROBO1αexpressing CRPC cells exhibited reduced tumorigenicity as compared to ROBO-1-silenced CRPC cells. Finally, reactivation of ROBO-1 by gene therapy resulted in decreased AR/Wnt signaling and sensitization of CRPC cells to chemotherapy. Conclusions: We suggest that inactivation of ROBO1 during androgen ablation treatment could be one of the possible mechanisms involved in the recurrence of disease in CaP patients. Therefore ROBO-1 offers a potential to be exploited as a molecular target for gene therapy to treat CRPC condition in humans. 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 3917. doi:1538-7445.AM2012-3917