BACKGROUND The dysfunction of type I interferon (IFN) signaling is an important mechanism of immune escape and metastasis in tumors. Increased NOS1 expression has been detected in melanoma, which correlated with dysfunctional IFN signaling and poor response to immunotherapy, but the specific mechanism has not been determined. In this study, we investigated the regulation of NOS1 on the interferon response and clarified the relevant molecular mechanisms. METHODS After stable transfection of A375 cells with NOS1 expression plasmids, the transcription and expression of IFNα-stimulated genes (ISGs) were assessed using pISRE luciferase reporter gene analysis, RT-PCR, and western blotting, respectively. The effect of NOS1 on lung metastasis was assessed in melanoma mouse models. A biotin-switch assay was performed to detect the S-nitrosylation of HDAC2 by NOS1. ChIP-qPCR was conducted to measure the binding of HDAC2, H4K16ac, H4K5ac, H3ac, and RNA polymerase II in the promoters of ISGs after IFNα stimulation. This effect was further evaluated by altering the expression level of HDAC2 or by transfecting the HDAC2-C262A/C274A site mutant plasmids into cells. The coimmunoprecipitation assay was performed to detect the interaction of HDAC2 with STAT1 and STAT2. Loss-of-function and gain-of-function approaches were used to examine the effect of HDAC2-C262A/C274A on lung metastasis. Tumor infiltrating lymphocytes were analyzed by flow cytometry. RESULTS HDAC2 is recruited to the promoter of ISGs and deacetylates H4K16 for the optimal expression of ISGs in response to IFNα treatment. Overexpression of NOS1 in melanoma cells decreases IFNα-responsiveness and induces the S-nitrosylation of HDAC2-C262/C274. This modification decreases the binding of HDAC2 with STAT1, thereby reducing the recruitment of HDAC2 to the ISG promoter and the deacetylation of H4K16. Moreover, expression of a mutant form of HDAC2, which cannot be nitrosylated, reverses the inhibition of ISG expression by NOS1 in vitro and decreases NOS1-induced lung metastasis and inhibition of tumor infiltrating lymphocytes in a melanoma mouse model. CONCLUSIONS This study provides evidence that NOS1 induces dysfunctional IFN signaling to promote lung metastasis in melanoma, highlighting NOS1-induced S-nitrosylation of HDAC2 in the regulation of IFN signaling via histone modification.

中文翻译：

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NOS1通过HDAC2的S-亚硝基化抑制癌细胞的干扰素反应。

背景技术I型干扰素（IFN）信号传导功能障碍是肿瘤中免疫逃逸和转移的重要机制。在黑色素瘤中已检测到NOS1表达增加，这与功能障碍的IFN信号传导和对免疫疗法的不良反应有关，但具体机制尚未确定。在这项研究中，我们研究了NOS1对干扰素反应的调控，并阐明了相关的分子机制。方法用NOS1表达质粒稳定转染A375细胞后，分别通过pISRE荧光素酶报告基因分析，RT-PCR和Western blotting评估IFNα刺激基因（ISG）的转录和表达。在黑色素瘤小鼠模型中评估了NOS1对肺转移的影响。进行了生物素转换试验，以检测NOS1对HDAC2的S-亚硝基化作用。进行了ChIP-qPCR以测量在IFNα刺激后ISGs启动子中HDAC2，H4K16ac，H4K5ac，H3ac和RNA聚合酶II的结合。通过改变HDAC2的表达水平或将HDAC2-C262A / C274A位点突变质粒转染到细胞中，可以进一步评估这种效果。进行共免疫沉淀测定以检测HDAC2与STAT1和STAT2的相互作用。使用功能丧失和功能获得方法来检查HDAC2-C262A / C274A对肺转移的影响。通过流式细胞术分析肿瘤浸润的淋巴细胞。结果HDAC2被募集到ISGs的启动子，并使H4K16脱乙酰基化，以响应于IFNα处理而使ISGs最佳表达。黑色素瘤细胞中NOS1的过表达降低了IFNα反应性，并诱导HDAC2-C262 / C274的S-亚硝基化。这种修饰减少了HDAC2与STAT1的结合，从而减少了HDAC2向ISG启动子的募集和H4K16的脱乙酰作用。此外，无法亚硝化的HDAC2突变体形式的表达逆转了NOS1在体外对ISG表达的抑制，并减少了黑色素瘤小鼠模型中NOS1诱导的肺转移和对肿瘤浸润淋巴细胞的抑制。结论这项研究提供了证据，即NOS1诱导功能异常的IFN信号传导，促进黑色素瘤的肺转移，突显了NOS1诱导的HDAC2的S-亚硝基化通过组蛋白修饰对IFN信号的调节。这种修饰减少了HDAC2与STAT1的结合，从而减少了HDAC2向ISG启动子的募集和H4K16的脱乙酰作用。此外，不能亚硝化的HDAC2突变形式的表达逆转了NOS1在体外对ISG表达的抑制作用，并减少了黑色素瘤小鼠模型中NOS1诱导的肺转移和对肿瘤浸润淋巴细胞的抑制作用。结论这项研究提供了证据，即NOS1诱导功能异常的IFN信号传导促进黑色素瘤的肺转移，突显了NOS1诱导的HDAC2的S-亚硝基化通过组蛋白修饰对IFN信号的调节。这种修饰减少了HDAC2与STAT1的结合，从而减少了HDAC2向ISG启动子的募集和H4K16的脱乙酰作用。此外，不能亚硝化的HDAC2突变形式的表达逆转了NOS1在体外对ISG表达的抑制作用，并减少了黑色素瘤小鼠模型中NOS1诱导的肺转移和对肿瘤浸润淋巴细胞的抑制作用。结论这项研究提供了证据，即NOS1诱导功能异常的IFN信号传导促进黑色素瘤的肺转移，突显了NOS1诱导的HDAC2的S-亚硝基化通过组蛋白修饰对IFN信号的调节。它不能被亚硝化，在黑色素瘤小鼠模型中可以逆转NOS1在体外对ISG表达的抑制作用，并减少NOS1诱导的肺转移和对肿瘤浸润淋巴细胞的抑制作用。结论这项研究提供了证据，即NOS1诱导功能异常的IFN信号传导促进黑色素瘤的肺转移，突显了NOS1诱导的HDAC2的S-亚硝基化通过组蛋白修饰对IFN信号的调节。在黑素瘤小鼠模型中，其不能被亚硝化，可逆转NOS1在体外对ISG表达的抑制作用，并减少NOS1诱导的肺转移和对肿瘤浸润淋巴细胞的抑制作用。结论这项研究提供了证据，证明NOS1诱导功能障碍的IFN信号传导促进黑色素瘤的肺转移，突显了NOS1诱导的HDAC2的S-亚硝基化通过组蛋白修饰对IFN信号的调节。