Crosstalk Between nNOS/NO and COX-2 Enhances Interferon-Gamma-Stimulated Melanoma Progression.

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/PGE(2) signaling 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 PGE(2) and 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, PGE(2) further enhanced PD-L1 expression and amplified the induction of nNOS, which increased intracellular NO levels. Cotreatment with celecoxib effectively diminished these changes induced by PGE(2). In addition, nNOS blockade using a selective small molecule inhibitor (HH044), efficiently inhibited IFN-γ-induced PGE(2) and 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 PGE(2) levels in vivo. Conclusions: Our study demonstrates the positive feedback loop linking nNOS-mediated NO signaling to the COX-2/PGE(2) signaling axis in melanoma, which further potentiates the pro-tumorigenic activity of IFN-γ.