STAT3 Regulation By S-Nitrosylation: Implication In Cancer.

In this study, we assessed S-nitrosylation-based regulation of Janus-activated kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. Our studies show that STAT3 in stimulated microglia underwent two distinct redox-dependent modifications, S-nitrosylation and S-glutathionylation. STAT3 S-nitrosylation was associated with inducible nitric oxide synthase (iNOS)-produced nitric oxide (NO) and S-nitrosoglutathione (GSNO), whereas S-glutathionylation of STAT3 was associated with cellular oxidative stress. NO produced by iNOS or treatment of microglia with exogenous GSNO inhibited STAT3 activation via inhibiting STAT3 phosphorylation (Tyr705). Consequently, the interleukin-6 (IL-6)-induced microglial proliferation and associated gene expressions were also reduced. In cell-free kinase assay using purified JAK2 and STAT3, STAT3 phosphorylation was inhibited by its selective preincubation with GSNO, but not by preincubation of JAK2 with GSNO, indicating that GSNO-mediated mechanisms inhibit STAT3 phosphorylation through S-nitrosylation of STAT3 rather than JAK2. In this study, we identified that Cys259 was the target Cys residue of GSNO-mediated S-nitrosylation of STAT3. The replacement of Cys259 residue with Ala abolished the inhibitory role of GSNO in IL-6-induced STAT3 phosphorylation and transactivation, suggesting the role of Cys259S-nitrosylation in STAT3phosphorylation. Since STAT3 activation is involved in tumor progression and metastasis, we investigated the effect of GSNO in cell culture and mouse xenograft model of head and neck squamous cell carcinoma (HNSCC). GSNO treatment of HNSCCN cell lines reversibly decreases the activation (phosphorylation) of STAT3 in a concentration dependent manner. The reduced STAT3/NF-kB activity by GSNO correlated with decreased cell proliferation and increased apoptosis of HNSCC cells. In HNSCC mouse xenograft model, the tumor growth was reduced by systemic treatment with GSNO and was further reduced when the treatment combined with radiation and cisplatin. Accordingly, GSNO treatment also resulted in decreased levels of pSTAT3 and tumor growth regulators (ie. cyclin D2, VEGF and Bcl-2) in tumor tissue. In summary, these findings have implications for the development of new therapeutics targeting of STAT3 for treating diseases associated with inflammatory/immune responses and abnormal cell proliferation, including cancer.