TRANSCRIPTIONAL REGULATION OF PROSTATE CANCER PROGNOSTIC GENES BY THE eNOS/ER? COMBINATORIAL COMPLEX: SILENCING OF GLUTATHIONE TRANSFERASE P1

Glutathione Transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents. Among them the P1 class isoenzymes have been associated with pre-neoplastic and neoplastic changes. In prostate tumors (PCa) expression of GSTP1-1 is frequently lost as a consequence of epigenetic modifications such as hypermethylation of its promoter, a common and early event in prostate carcinogenesis. We have recently identified in PCa a transcriptional prognostic signature comprising a significant number of genes upregulated in cells from patients with worse clinical outcome. The transcriptional induction was due to localized chromatin remodeling mediated by a combinatorial complex between endothelial Nitric Oxide Synthase, eNOS and Estrogen Receptor Beta, ER?. In the same cells, other prognostic genes, among which GSTP1, were downregulated. We are currently investigating whether eNOS, acting as "co-factor" of ER?, plays a role in its transcriptional silencing. First, in Tissue Micro Arrays from a retrospective cohort of patients (n.100), we were able to validate loss of GSTP1-1 in vivo and to correlate silencing of its expression with decreased disease-specific survival. Further, in our ex-vivo model of tumor cell lines established from PCa patients, we recapitulated loss of GSTP1 mRNA and protein expression and of biochemical activity, particularly in cells from patients with worse outcome. Silencing of GSTP1 did not appear to involve methylation of its promoter, unlike the case with LNCaP cells and what generally reported for PCa. Rather, ChIP and re-ChIP assays on cultured cells and more importantly on fresh surgical tissues explanted from PCa patients (ChIP in vivo) have provided evidence in favor of an active repression mechanism, apparently mediated by dynamic recruitment of the combinatorial complex eNOS/ER? along a 5-kb region of the GSTP1 regulatory sequences. In support of these observations, a synthetic antagonist of ER and/or overexpression of a dominant negative eNOS relieved the repression of GSTP1 mRNA in cells from patients with adverse prognosis. Conversely, a NO donor silenced this mRNA in cells still expressing GSTP1. Overall our findings strongly support a model where eNOS functions as a co-factor of ER? in gene transcription, and formation of eNOS/ER? complex induces local production of NO with major effects on the acquisition of a malignant phenotype by human prostate epithelial cells.