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/PGE₂ 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 PGE₂ 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, PGE₂ 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 PGE₂. In addition, nNOS blockade using a selective small molecule inhibitor (HH044), efficiently inhibited IFN-γ-induced PGE₂ 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 PGE₂ levels in vivo. Conclusions: Our study demonstrates the positive feedback loop linking nNOS-mediated NO signaling to the COX-2/PGE₂ signaling axis in melanoma, which further potentiates the pro-tumorigenic activity of IFN-γ.

celecoxib; cyclooxygenase-2 (COX-2); interferon-gamma (IFN-γ); melanoma; nNOS inhibitors; neuronal nitric oxide synthase (nNOS); nitric oxide (NO); programmed death-ligand 1 (PD-L1); prostaglandin E2 (PGE2).