Background/Objectives:Interferon gamma (IFN-γ) in the melanoma tumor microenvironment plays opposing roles, orchestrating both pro-tumorigenic activity and anticancer immune responses. Our previous studies demonstrated the role of neuronal nitric oxide synthase (nNOS) in IFN-γ-stimulated melanoma progression. However, the underlying mechanism has not been well defined. This study determined whether the nNOS/NO and COX-2/PGE2signaling pathways crosstalk and augment the pro-tumorigenic effects of IFN-γ in melanoma.Methods: Bioinformatic analysis of patient and cellular proteomic data was conducted to identify proteins of interest associated with IFN-γ treatment in melanoma. Changes in protein expression were determined using various analytical techniques including western blot, flow cytometry, and confocal microscopy. The levels of PGE2and nitric oxide (NO) were analyzed by HPLC chromatography and flow cytometry. In vivo antitumor efficacy was determined utilizing a human melanoma xenograft mouse model.Results: Our omics analyses revealed that the induction of COX-2 was significantly predictive of IFN-γ treatment in melanoma cells. In the presence of IFN-γ, PGE2further enhanced PD-L1 expression and amplified the induction of nNOS, which increased intracellular NO levels. Cotreatment with celecoxib effectively diminished these changes induced by PGE2. In addition, nNOS blockade using a selective small molecule inhibitor (HH044), efficiently inhibited IFN-γ-induced PGE2and COX-2 expression levels in melanoma cells. STAT3 inhibitor napabucasin also inhibited COX-2 expression both in the presence and absence of IFN-γ. Furthermore, celecoxib was shown to enhance HH044 cytotoxicity in vitro and effectively inhibit human melanoma tumor growth in vivo. HH044 treatment also significantly reduced tumor PGE2levels in vivo.Conclusions: Our study demonstrates the positive feedback loop linking nNOS-mediated NO signaling to the COX-2/PGE2signaling axis in melanoma, which further potentiates the pro-tumorigenic activity of IFN-γ.
背景/目的：在黑色素瘤肿瘤微环境中，γ干扰素（IFN-γ）发挥着相互矛盾的作用，既能协调促肿瘤活性，又能引发抗癌免疫反应。我们先前的研究揭示了神经元型一氧化氮合酶（nNOS）在IFN-γ刺激的黑色素瘤进展中的作用，但其潜在机制尚未明确。本研究旨在探讨nNOS/NO与COX-2/PGE2信号通路是否存在交互作用，并增强IFN-γ在黑色素瘤中的促肿瘤效应。 方法：通过对患者及细胞蛋白质组学数据进行生物信息学分析，筛选与黑色素瘤中IFN-γ处理相关的目标蛋白。采用蛋白质印迹、流式细胞术和共聚焦显微镜等多种分析技术检测蛋白表达变化。通过高效液相色谱法和流式细胞术分析PGE2和一氧化氮（NO）水平。利用人黑色素瘤异种移植小鼠模型评估体内抗肿瘤疗效。 结果：组学分析显示，COX-2的诱导表达对黑色素瘤细胞中IFN-γ处理具有显著预测价值。在IFN-γ存在条件下，PGE2进一步增强了PD-L1表达，并放大了nNOS的诱导，从而增加细胞内NO水平。塞来昔布联合处理能有效抑制PGE2诱导的这些变化。此外，使用选择性小分子抑制剂HH044阻断nNOS，可有效抑制黑色素瘤细胞中IFN-γ诱导的PGE2和COX-2表达水平。STAT3抑制剂萘帕布卡辛在IFN-γ存在或缺失条件下均能抑制COX-2表达。进一步研究发现，塞来昔布在体外能增强HH044的细胞毒性，在体内可有效抑制人黑色素瘤肿瘤生长。HH044处理也显著降低了体内肿瘤PGE2水平。 结论：本研究证实了黑色素瘤中nNOS介导的NO信号通路与COX-2/PGE2信号轴之间存在正反馈循环，该机制进一步增强了IFN-γ的促肿瘤活性。
Crosstalk Between nNOS/NO and COX-2 Enhances Interferon-Gamma-Stimulated Melanoma Progression
肿瘤(癌症)患者之家