Oral cancer-derived exosomal NAP1 enhances cytotoxicity of natural killer cells via the IRF-3 pathway

Objective: To examine the effects of oral cancer-derived exosomes (OCEXs) on natural killer (NK) cells and to explore the underlying mechanism. Materials and Methods: OCEXs were isolated from the cell culture supernatant of oral cancer (OC) cells using ultrafiltration and affinity chromatography and were identified using electron microscopy, nanoparticle tracking analysis (NTA) and immunoblotting. The effects of OCEXs on NK cells were analyzed using laser scanning confocal microscopy and several functional assays of NK cells. To explore the mechanism of their effects, antibody array, protein mass spectrometry and RNA interference were adopted. Results: The particles isolated from the OC cells were identified as exosomes with satisfactory morphology, concentration and purity. The OCEXs were internalized by NK cells and then promoted the biological functions of NK cells, including proliferation, release of perforin and granzyme M and cytotoxicity. Furthermore, OCEXs increased the expression of interferon regulatory factor 3 (IRF-3) and its phosphorylation, which drove the expression of the type I interferon (IFN) gene and the chemokine (C-X-C motif) ligand (CXCL) genes, thereby promoting the functions of NK cells. In addition, NF-kappa B-activating kinase-associated protein 1 (NAP1), an upstream activator of IRF-3, was enriched in OCEXs, and treatment with OCEXs increased the expression of NAP1 in NK cells. Importantly, NAP1-depleted OCEXs obtained from OC cells had a dramatically weakened influence on NK cells. Conclusion: Our findings reveal a previously unrecognized function of exosomal NAP1 derived from OC cells in enhancing the cytotoxicity of NK cells via the IRF-3 pathway.