Challenges in prostate cancer management stem from 1) inability to distinguish indolent prostate cancer from aggressive malignancy with metastatic potential; 2) limited treatment options for recurrent, castration-resistant prostate cancer (CRPC). Reactivated androgen receptor (AR) in CRPC results from elevated expression and activity of AR, and increased de novo androgen biosynthesis from DHEA. Anti-AR/anti-androgen biosynthesis drugs inhibit post chemotherapy cancer for a few months, but response is not universal. Finding new predictors for cancer progression and new drug targets are high-priority goals. We will examine a novel aspect of androgen-activated AR signaling that was revealed from our study. This entails i) regulation of androgen homeostasis by the prostate-expressed SULT2B, a sulfotransferase which mediates sulfation of cholesterol and DHEA, and ii) enhanced activity of methylated AR. We show 1) SULT2B mRNA and protein levels are markedly reduced in clinical specimens and IHC staining of tissue microarrays revealed highly statistically significant (p<0.001) loss of SULT2B in cancer tissue; 2) SULT2B silencing caused increased proliferation of prostate cancer cells; 3) Vitamin D receptor (VDR) and the oxysterol-inducible liver X receptor (LXR) can induce the SULT2B gene (SULT2B1); 4) AR is monomethylated at a lysine residue in its hinge domain by the SET9 lysine methyltransferase. SET9 depletion reduced wild type but not methylation-site-mutant AR activity; 5) LSD1, a histone lysine demethylase can convert methylated AR to unmethylated AR; 6) LXR-? is a potential regulator of genes encoding SET9 and LSD1, since LXR-responsive cis elements are present in these two genes based on in silico analysis. We have developed an antibody that can specifically recognize lysine- methylated AR by western blotting. We hypothesize that advanced prostate cancer associates with reduced SULT2B and elevated methyl-modified AR, LXR-? inhibits prostate cancer in part by preventing loss of SULT2B and elevation of methylated AR, and VDR synergizes LXR effects. The hypothesis is built from our own results and from reports that i) LXR- -/- mice show heightened androgen sensitivity in ventral prostates, but no change in AR levels; ii) steroid sulfatase (Sts) which counters DHEA sulfation, was induced in prostates of Lxr- -/- mice and suppressed in LXR- -activated mice; iii) LXR inhibited prostate cancer in xenograft tumors. We propose to determine:
Aim 1) significance of SULT2B loss in prostate cancer, and a role for DNA methylation in this loss. We will examine i) diverse specimens for association of low SULT2B in cancer tissue with i) DHT levels and AR target gene expression, ii) levels of the enzymes AKR1C3 & SRD5A1, and iii) methylation status of the SULT2B1 gene. Statistical association of clinical variables (including role of race) with above parameters will be explored.
Aim 2) the dynamics of LXR- signaling with SULT2B-regulated androgen homeostasis in prostate, possible synergy with VDR and their relevance in prostate cancer. We will investigate i) LXR-responsive DNA cis element(s) in SULT2B1 and its interaction with LXR- , coregulators, chromatin modifiers; ii) Synergy of LXR- with VDR in SULT2B induction and prostate cancer inhibition; iii) DHT levels in LXR- -silenced prostate cancer cells in vitro & in vivo, and in Lxr- -null mouse prostates and reversal of the effect of LXR silencing by calcitriol; iv) a link in clinical specimes between low SULT2B and attenuated expression of LXR- and LXR- -targeted lipogenic genes.
Aim 3) significance of AR methylation in prostate cancer and its interplay with LXR-?. We will examine i) methylated AR levels in LXR- -knocked down cancer cells in vitro & in vivo; ii) a role for LXR- in SET9 & LSD1 expression and in their association with AR iii) clinical specimens for elevated methylated AR levels (relative to total AR). Methods: IHC, tissue microarray, LCM, western blot, pyrosequencing, bisulfate-modified DNA, promoter assay, ChIP,, lentivirus, si RNAs, LC- MS/MS. Molecular biologists, pathologists and biostatisticians will collaborate. Multiple cores will be used. Significance: This study may reveal new predictive markers and new intervention targets to prevent CRPC.
At initial diagnosis, clinicians cannot distinguish between indolent and aggressive prostate cancer. The latter has poor prognosis. We will search for proteins that reliably predict risks for aggressive prostate cancer from biopsied material. Prostate tumor tissue produces androgen. Tumor growth is fueled by intra-tumor androgen. Our new results show that in some patients, tumors make very little of the enzyme SULT2B, which normally inhibits intra-prostate androgen production. We will assess whether SULT2B status in prostate tumor can reliably predict risks for aggressive cancer. The mechanism for SULT2B loss and ability for receptor proteins LXR and VDR to reverse loss will be explored. Our second novel result is that androgen receptor (AR), which mediates androgen action, is methylated in prostate cancer cells. Methylated AR is more potent than unmethylated AR in mediating androgen action, thus bolstering androgen's role in promoting tumor growth. We will explore why methylated AR shows enhanced activity, and whether its level increases in prostate cancer.
