Fig. 6

MITF-driven phenotypic plasticity modulates ADAM10 expression in melanoma. a Quantification of MITF and ADAM10 mRNA in SK-MEL-28 after 24 and 48 h of irradiation (10 Gy). Differences in MITF and ADAM10 mRNA expression at 24 and 48 h were statistically significant (*p < 0.05) with respect to the non-irradiated control (0 h). b Analysis of MITF and ADAM10 proteins in SK-MEL-28 cells after irradiation at different post-IR times. The histograms represent the relative amount of proteins with respect to actin. The IR-dependent increase in MITF and ADAM10 was only statistically significant in irradiated cells (*p < 0.05 at all assayed times with respect to non-irradiated controls). c, d Depletion of glutamine or CoCl₂ treatments, to mimic hypoxia conditions, resulted in decreased levels of MITF and ADAM10 in human and mouse melanoma cells. e Depletion of glutamine and hypoxia sensitizes B16/F10 melanoma cells to NK-driven cytotoxicity. Spleens of untreated C57BL/6 were used for NK purification. Purified NKs were activated with IL-2 during 2 days. Then, IL-2-activated NKs were co-cultured with irradiated melanoma cells previously subjected to depletion of glutamine or CoCl₂ (48 h treatments). NK cytotoxicity was evaluated by a ⁵¹Cr-release assay at two effector/target ratios (E/T). The results are representative of three independent experiments (*p < 0.05). f Schematic illustrating proposed mechanism by which MITF induces escape from innate immunity in melanoma. MITF expression in melanoma (left panels) directly upregulates ADAM10. MICA/B interacts with Erp5 and is shed by ADAM10 from the cell surface, which subsequently leads to a blocking of NKG2D. Lack of MITF expression in melanoma (right panels) downregulates ADAM10 and facilitates NKG2D-mediate immune response. Blots in this figure were cropped from different gels. Full blots are shown in Fig. S9 and quantified in Fig. S8