ROLE OF eNOS IN PROSTATE CANCER PROGRESSION

The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment, creating a need to find markers capable of anticipating clinical outcome. We have defined a molecular mechanism involving functional activation of endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factors (HIFs) in association with the ER? that characterizes the most aggressive form of PCa. Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. In vivo validation of this mechanism on TissueMicroarrays from an independent cohort of patients with very long follow up, established a hierarchical predictive power for these proteins, with expression of eNOS plus ER? and nuclear eNOS plus HIF-2? being the most relevant combinations for adverse clinical outcome. We also provided evidence for the existence and prognostic role in human PCa of functional complexes between ER? and HIF-1?/2? or eNOS. These combinatorial complexes, under specific modifications of the cell microenvironment, are capable of activating a transcriptional program associated with aggressive PCa. Genetic or pharmacological modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with opposite outcome, highlighting eNOS relevance in PCa progression. Our work has considerable clinical relevance since it may enable the earlier diagnosis of poor prognosis PCa through routine biopsy assessment of eNOS, ER?, and HIF-2? expression. The identification of genes that may be differentially targeted by nuclear eNOS will be performed by high resolution analysis with ChIP-Seq, a technique which combines standard ChIP assay with high throughput DNA Sequencing technology. Presently, the ChIP-enriched DNA of PCa samples has been converted into libraries suitable for high-throughput sequencing and we are starting now the first step of analysis, specifically the mapping of millions tags/reads to human genome. In parallel, analysis of eNOS-containing complexes bound to DNA using cutting-edge Mass Spectrometry (MS) proteomic techniques is in progress.