Abstract 1856: Transcriptional regulation of the UDP-glucuronosyltransferases (UGTs) by SAFB1 and SAFB2: Strategy to reduce DHT levels in prostate cancer cells

Backround: Androgen deprivation therapy (ADT) is a first-line option for prostate cancer (PC) treatment. While there has been some progress inhibiting AR activation by focusing on DHT synthesis, other mechanisms to reduce intracellular DHT have not been exploited for drug development. The UDP-glucuronosyltransferase (UGT) enzymes UGT2B15 and UGT2B17 inactivate DHT in the prostate by adding a bulky UDP-glucuronic acid moiety to the hormone. These reactions are irreversible and therefore represent a prominent mechanism for depletion of intratumoral androgen in vivo. Here we show evidence that the chromatin scaffolds SAFB1/2 activate expression of UGT2B15/17. This is a novel activity for these proteins, which are known primarily as inhibitors of nuclear receptors (Oesterreich, S. et al. Molecular Endocrinology, 2000 and Debril, MB. et al. Molecular Endocrinology, 2005). We previously showed that SAFB1 can act as an AR suppressor that cooperates with the MST1 kinase and the EZH2 methyltransferase to modify chromatin at AR-responsive genes (Mukhopadhyay et al. Oncogene, 2014). Methods: Enforced expression, silencing, RNA profiling, quantitative PCR, chromatin immunoprecipitation (ChIP) and ChIP-Seq, mass spectrometry and luciferase gene reporter assays. Results: SAFB1/2 is genomically inactivated in a high percentage (∼28%) of castration-resistant prostate cancer (CRPC). SAFB1/2 silencing, which emulates SAFB1/2 downregulation/gene inactivation, potently down-regulates UGT2B15/17 in LNCaP and 22RV1 cells. Conversely, enforced expression of SAFB1 upregulates UGT2B15/17. Genomic effects of SAFB1 loss on the AR cistrome in LNCaP cells by ChIP-seq are shown. Computational analysis indicates that SAFB1 regulates AR and EZH2-regulated genes, but not exclusively, indicating that gene regulation by SAFB1 independently of AR and EZH2 also occurs in prostate cells. SAFB1 silencing resulted in increases in several androgen species in LNCaP cells. Finally, SAFB1 is recruited to the UGT2B15/17 promoters and its effect on promoter luciferase constructs is analyzed. Conclusions: This is the first identification of a molecular mechanism of regulation independent of androgen of the UGT2B15/17 genes in prostate cells. Our findings indicate that SAFB1 is an essential node where several oncogenic pathways converge onto chromatin to regulate gene expression in PC in a manner that can affect androgen degradative mechanisms. Citation Format: Mirja Rotinen, Julie S. Yang, Sungyong You