Fig 2: sMICA/B correlates with poor survival in melanoma patients. Androgens trigger ADAM10 activation and AR/β1 integrin/ADAM 10 complex assembly.A, B Variables correlating with survival in 29 Pembrolizumab (anti-PD1)-treated stage IV melanoma patients. MICA serum concentration was estimated by ELISA and expressed as pg/ml. The Kaplan–Meier curves for OS (A) and PFS (B) were generated. High and low levels of MICA correspond to dotted and solid lines, respectively. The mean value of MICA serum concentration (< or > of 438 pg/ml) was calculated on the entire cohort, chosen as cut-off and reported on the right of the panels. Log-rank test was used to compare curves. p-values are reported on the top of each panel. C Correlation between PFS and serum concentration of MICB in 29 Pembrolizumab (anti-PD1)-treated stage IV melanoma patients was calculated by Spearman correlation test. p-value and r coefficient are reported on the top of the panel. In D–F, AMM16 cells were employed and the presented results are representative of three different experiments. D Cells were unstimulated or stimulated for 6 h with R1881, in the absence or presence of bicalutamide (Bic). Lysate proteins were analyzed for ADAM10 expression. The filter was re-probed with anti-GAPDH antibody, as loading control. E Cells were unchallenged or challenged for 20 min with R1881, in the absence or presence of Bic. Lysate proteins were immune-precipitated using the anti-AR (anti-AR) or control (ctrl IgG) antibodies. WB analysis using antibodies against the indicated proteins was done to reveal co-immunoprecipitated proteins. F Cells on coverslips were left untreated or treated for 20 min with R1881 and, then, stained for AR and ADAM10. Images captured by confocal microscope show the staining of AR (green) and ADAM10 (red). Merged images are presented in the right panels. Bar, 10 μm. AR/ADAM10 co-localization ratio calculated by NIH Image J was 0.6 ± 0.06 in untreated cells and 3.11 ± 0.44 in R1881-stimulated cells. p < 0.05.

Fig 3: The androgen promotes the MICA/B shedding in melanoma cells, leading to the reduction of the NKG2D expression in NK cells.A, B Frequency of MICA/B in untreated (white bars) or R1881-treated (black bars) AMM16 cells. B Data from three independent experiments are represented as mean ± SD. n = 3; **p-value < 0.01. C AMM16 were untreated or treated for 6 h with R1881, in absence or presence of bicalutamide (Bic). When indicated, cells were pretreated with MG132. WB using the indicated antibodies was done. D Quiescent AMM16 cells on coverslips were left untreated or treated for 6 h with R1881. They were stained for MICA/B. Images captured by confocal microscope show the staining of MICA/B (green) and nuclei (blu). Merged images are presented in the right panels. They are representative of three independent experiments. Bar, 10 μm. E Cells were unstimulated or stimulated with R1881, in the absence or presence of Bic for 6 h. sMICA was assayed and quantified by ELISA. Graph is representative of three independent experiments; *p-value < 0.05. F, G NK cells were untreated or treated with AMM16 cells derived CM, as indicated. F WB with the antibodies against the indicated proteins was done. G NK cells were stained as reported in Methods section. Images captured by confocal microscopy show the staining of NKG2D (red) and nuclei (blu). Merged images are presented in the right panels. They are representative of three independent experiments. Bar, 2.5 μm.

Fig 4: Androgen promotes melanoma immune-escape by NK cells and invasiveness: graphical representation.Androgen stimulation of melanoma cells triggers the assembly of the AR/ADAM10/β1 integrin complex. Such complex enables ADAM10 to act as MICA sheddase, on one hand. The MICA shedding, hence, causes the effective masking from NKG2D recognition allowing the immune-escape. On the other, the complex mediates the androgen-induced invasiveness of melanoma cells through the activation of FAK and ERK.