Site-specific S-nitrosylation of integrin α6 increases the extent of prostate cancer cell migration by enhancing integrin ß1 association and weakening adherence to laminin-1.

The increased mortality in prostate cancer is usually the result of metastatic progression of the disease from the organ-confined location. Among the major events in this progression cascade are enhanced cell migration and loss of adhesion. Moreover, elevated levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) found within the tumor microenvironment are hallmarks of progression of this cancer. To understand the role of nitrosative stress in prostate cancer progression, we investigated the effects of NO and iNOS on prostate cancer cell migration and adhesion. Our results indicate that ectopic expression of iNOS in prostate cancer cells increased the extent of cell migration, which could be blocked by selective ITGα6 blocking antibody or iNOS inhibitors. Furthermore, iNOS was found to cause S-nitrosylation of ITGα6 at Cys86 in prostate cancer cells. By comparing the activities of wild-type ITGα6 and a Cys86 mutant, we showed that treatment of prostate cancer cells with NO increased the level of ITGα6 heterodimerization with ITGß1 but not with ITGß4. Finally, S-nitrosylation of ITGα6 weakened its binding to laminin-ß1 and weakened the adhesion of prostate cancer cells to laminin-1. In conclusion, S-nitrosylation of ITGα6 increased the extent of prostate cancer cell migration, which could be a potential mechanism of NO- and iNOS-induced enhancement of prostate cancer metastasis.

Comprehensive identification and modified-site mapping of S-nitrosylated targets in prostate epithelial cells.

BACKGROUND: Although overexpression of nitric oxide synthases (NOSs) has been found associated with prostate diseases, the underlying mechanisms for NOS-related prostatic diseases remain unclear. One proposed mechanism is related to the S-nitrosylation of key regulatory proteins in cell-signaling pathways due to elevated levels of NO in the prostate. Thus, our primary objective was to identify S-nitrosylated targets in an immortalized normal prostate epithelial cell line, NPrEC. METHODOLOGY/PRINCIPAL FINDINGS: We treated NPrEC with nitroso-cysteine and used the biotin switch technique followed by gel-based separation and mass spectrometry protein identification (using the LTQ-Orbitrap) to discover S-nitrosylated (SNO) proteins in the treated cells. In parallel, we adapted a peptide pull-down methodology to locate the site(s) of S-nitrosylation on the protein SNO targets identified by the first technique. This combined approach identified 116 SNO proteins and determined the sites of modification for 82 of them. Over 60% of these proteins belong to four functional groups: cell structure/cell motility/protein trafficking, protein folding/protein response/protein assembly, mRNA splicing/processing/transcriptional regulation, and metabolism. Western blot analysis validated a subset of targets related to disease development (proliferating cell nuclear antigen, maspin, integrin beta4, alpha-catenin, karyopherin [importin] beta1, and elongation factor 1A1). We analyzed the SNO sequences for their primary and secondary structures, solvent accessibility, and three-dimensional structural context. We found that about 80% of the SNO sites that can be mapped into resolved structures are buried, of which approximately half have charged amino acids in their three-dimensional neighborhood, and the other half residing within primarily hydrophobic pockets. CONCLUSIONS/SIGNIFICANCE: We here identified 116 potential SNO targets and mapped their putative SNO sites in NPrEC. Elucidation of how this post-translational modification alters the function of these proteins should shed light on the role of NO in prostate pathologies. To our knowledge, this is the first report identifying SNO targets in prostate epithelial cells.