ChIP-Sequencing for studying estrogen-dependent eNOS-DNA associations in aggressive prostate cancer

Previously we identified endothelial NOS (eNOS) as transcriptional co-factor of Estrogen Receptor Beta and Hypoxia Inducible Factors in transcriptional regulation of genes associated with adverse prognosis in Prostate Cancer (PCa). To explore the role of nuclear eNOS in aggressive PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from PCa-cells derived from primary tumor with poor outcome and from metastatic LNCaP cells. We documented the genome-wide existence of a considerable number of eNOS-DNA associations that define transcriptional active regions modulated by estrogen. In summary: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. Estrogen increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. 55% of peaks were in extragenic DNA regions with intriguing involvement of the 5' domain of several miRs deregulated in PCa. The large number of novel eNOS-targeted identified genes suggests that eNOS participates in regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. In particular, upon estrogen treatment the eNOS/SIRT1 complex mediates a transcriptional downregulation of pri-miR34a which consequently leads to up-regulation of its-target SIRT1 that actives eNOS itself. These findings reveal unprecedented functions of eNOS and of eNOS/SIRT1 interplay, fine-tuned by estrogen, and attributed to eNOS a critical role in aggressive PCa.