NOS1 Inhibits The Interferon Response Of Melanoma Cells By S-nitrosylation Of HDAC2

Background and ObjectivesIFNa binding to its transmembrane receptor induces the expression of interferon-stimulated genes(ISGs)to exert anti-proliferative,pro-apoptotic,anti-viral effects,which plays an crucial role in tumor immunosurveillance and tumor therapy.Deacetylases(HDACs)are usually considered to be associate with transcriptional repression.While studies have shown that type ? HDACs(HDAC1,2,3)was required for IFNa response,although the exact mechanism remains unclear.Nitric oxide,synthesized by three isoforms of NO synthase(NOS1,NOS2,and NOS3),is a key signaling molecule and involves in various biological pathway.Studies have revealed that NO synthase has a high expression level in multiple cancers and participates in various cancer processes,such as tumorigenesis,progression and metastasis.The three NOSs isoforms varied in cell biological function through the distinct modes of regulatory and protein interactions.One key mechanism which NO regulates the function of various target proteins is through the process of S-nitrosylation,that coupling a mitroso moiety and a reactive thiol group in specific cysteine residues and leading to the formation of an S-nitrosothiol(SNO).Numerous S-nitrosylated proteins,such as P53,Ras,HIF,Bcl-2,and EGFR,have been reported to be involved in various cancer-related events including apoptosis,angiogenesis,cell cycle modification,tissue invasion,metastasis and response to cancer treatments.NO/NOS1 which is highly expressed in melanoma cells,can inhibit tumor immunity and reduces the response of peripheral blood mononuclear cells to IFNa,but the specific mechanism is still unknown.HDAC2 is a target protein for NO-induced S-nitrosylation modification.Whether does NOS1 participate in the regulation of IFNa response through S-nitrosylation of HDAC2 in tumor cells are unclear.In this study,we explore the regulation of NOS1 on melanoma interferon response and clarify the relevant molecular mechanisms,which provided a new idea on the treatment of melanoma by interferon.Results1.NOS1 inhibits the expression of IFNa-stimulated genes(ISGs)in melanoma cellsThe melanoma cells were treated with NO donor and NOSs inhibitor,and the results showed that NO involved in the regulation of ISGs that is induced by IFNa.To test the role of NOS 1 in regulation of ISGs expression,we modified NOS1 expression genetically in melanoma cancer cell through transferring GV358-NOS1 lenti-virus and constructed a melanoma cell line with stable over-expression of NOS 1.The results showed that NOS 1 inhibited the transcription and expression of ISGs induced by IFNa.2.HDAC2 regulates the expression of ISGs in melanoma cells.To explore the role of HDAC2 in the regulation of ISGs expression,siRNA and exogenous plasmids transfection methods were carried out.The results of RT-PCR and pISRE luciferase reporter gene assay demonstrated that HDAC2 promoted the transcription and expression of ISGF3-dependent genes.Analysis of ChIP-qPCR showed that IFNa induced the recruitment of HDAC2 to the promoter of ISGs,accompanying with deacetylation of histone H4K16.Which promoted the recruitment of RNA polymerase ? to the promoter and initiated transcription.At the same time,HDAC2 did not affect the acetylation status of H3 and H4K5.3.NOS1 inhibits HDAC2 by S-nitrosylation at C262/C274 site.Biotin-switch assay showed that the S-nitrosylation level of HDAC2 was increased in NOS 1-overexpressed A3 75 and SKOV3 cells.Cys262 and Cys274 of HDAC2 were sensitive to S-nitrosylation modification,and these site mutations significantly reduce HDAC2-SNO level induced by NOS1 overexpression.The results of co-immunoprecipitation showed that NOS1 binding with HDAC2.Moreover,NOS 1 inhibits the ISGs expression,the deacetylation of H4K16 and recruitment of RNA polymerase II in tumor cells by S-nitrosylation of HDAC2.4.NOS1 promotes mouse melanoma lung metastasis.The mouse lung metastasis model was established by injecting mouse melanoma cells with stable over-expression of NOS 1 into tail vein.The results indicated that NOS1 increased lung metastasis in mouse melanoma model.Transfection of HDAC2-C262A/C274A plasmid to over-expressing NOS1 cells reversed the effects of NOS 1.Knockout of HDAC2 in melanoma cells decreased the lung tumorigenesis in mice model.Stable expression of wild-type HDAC2 and mutant HDAC2 in B16-HDAC2-KO cells restored the formation of melanoma lung tumors,and HDAC2 mutation partially reversed melanoma lung metastasis.ConclusionOur research demonstrated that NOS1 inhibited the transcription and expression of ISGs induced by interferon in melanoma cells.IFNa stimulated the recruitment of HDAC2 to the ISGs promoter and deacetylated histone H4K16,which enhanced the binding of RNA polymerase ? to the promoter and facilitated the transcription and expression of ISGs.NOS1 inhibited the interferon response of melanoma cells and promoted tumor progression by S-nitrosylation of HDAC2.This study provides clues and theoretical basis for targeting NO/NOS1 treatment of tumors.