Abstract LB-098: ChIP-seq studies unravel novel AR and OGT co-regulated genes in prostate cancer cells

Abstract

Abstract Background and Objective: Androgen receptor (AR) is a major driver of prostate cancer initiation and progression, and is a master regulator of anabolic transcriptional network in prostate epithelial cells. AR activation leads to enhanced flux through hexosamine biosynthetic pathway (HBP) and subsequent increase in the bioavailability of UDP-GlcNAc, resulting in increased O-GlcNAcylation of wide range of intracellular proteins. O-GlcNAc transferase (OGT), the enzyme that catalyzes the covalent addition of UDP-GlcNAc to serine and threonine residues of proteins, is often up-regulated in prostate cancer with its expression correlated with high Gleason score. AR activation enhances global O-GlcNAcylation status of the prostate cancer cells which acts as a central communicator of environmental status to metabolic and transcriptional regulatory networks. Here we have performed chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) to identify binding sites common to AR and O-GlcNAc modified transcription factors/co-activators in an attempt to identify novel regulatory mechanisms, biomarkers/druggable targets. Materials and Methods: Prostate cancer cells (LNCaPs) were cultured in androgen deprivation media for 3 days prior to treatment with synthetic androgen, R1881, for 4h and 24h. Cells were fixed with 1% formaldehyde and chromatin prepared by sonication in Bioruptor plus. Fragmentation efficiency was analysed using fragment analyser and ChIP carried out using diagenode iDeal ChIP-seq kit for transcription factors. Commercially available antibodies targeted against AR and O-GlcNAc modification (RL2) were used for the IPs. Percentage recovery of immunoprecipitated DNA was measured through ChIP-qPCR of known targets. Libraries were prepared using diagenode Microplex library preparation kit V2 and sequenced. Galaxy cistrome integrative analysis platform was used to analyse the peaks. Results: AR and RL2 binding profiles were compared and the overlapping and differential binding sites identified. Comparison of early (4h) versus late (24h) androgen treatment time points demonstrated time dependent changes in AR/O-GlcNAc binding to a subset of binding sites. Analysis revealed AR- O-GlcNAc co-regulated genes which are key regulators of various intracellular processes. Few interesting candidate genes shortlisted from this study are being characterized in the lab for their ability to mediate cellular transformation in prostate cancer. Conclusion: Our study reveals a complex regulatory landscape of AR and OGT signalling in prostate cancer. Further characterization of AR-OGT axis will have significant impact on our understanding AR dependent prostate cancer and in developing novel therapeutic targets. Citation Format: Ninu Poulose, Rebecca E. Steele, Sarah Maguire, Simon McDade, Ian G. Mills. ChIP-seq studies unravel novel AR and OGT co-regulated genes in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-098.