Role of the androgen receptor in melanoma aggressiveness

Abstract

Malignant melanoma represents the fifth most common cancer in the world and its incidence is rising. Novel therapies targeting receptor tyrosine kinases, kinases and immune checkpoints have been employed with a significant improvement of the overall survival and long-term disease containment. Nevertheless, the disease often progresses and becomes resistant to the therapies. As such, the discovery of new targets and drugs for advanced melanoma still remains a difficult task. Gender disparities, with a female advantage in melanoma incidence and outcome, have been reported. Although emerging studies support the pro-tumorigenic role of androgen/androgen receptor axis in melanoma, the molecular bases of such evidence are still under intense investigation. We now report that ligand activation of the androgen receptor drives melanoma invasiveness and its escape from natural killer-mediated cytotoxic effect. By combining different experimental approaches, we observe that melanoma escape is mediated by the androgen-triggered shedding of the surface molecule MICA. Specific blockade of ADAM10 or androgen receptor impairs the androgen-induced MICA shedding and melanoma immune-escape. Further, the increase in MICA serum levels correlates with a poor outcome in melanoma patients treated with the anti-PD-1 monoclonal antibody, pembrolizumab. At last, melanoma cells depleted of the androgen receptor become more responsive to the most commonly used immunocheckpoint inhibitors, suggesting that the receptor dampens the immunotherapy efficacy. Taken together, our findings identify the androgen receptor as a diagnostic guidance in melanoma and support the repositioning of AR blockers in clinical management of patients.

Fig. 1. AR is expressed in metastatic melanoma and its ligand activation prevents NK cell-mediated killing.

A AR gene expression levels in primary melanoma and in melanoma metastatic specimens (n = 481). AR was higher expressed in metastatic lesions (blu; n = 368; median 1.96) than in primary tumors (red; n = 103; median 1.33). One-way ANOVA with p = 0.03271 (f = 2.943). B WB analysis for AR in the indicated cell lines. Tubulin was revealed as a loading control. Representative experiment (left) and statistical analysis (right) of cytotoxicity assays done in untreated (white dots and bars) or 10 nM R1881-treated (black dots and bars) AR positive WM266-4 (C), AMM16 (D), Mel-CAL (E), LCM-Mel (F) and AR negative MEL-STA (G) cells with NK cells used at different E:T ratios. C–G Data from at least three independent experiments (n = 3) were represented as means ± SD. **p-value < 0.01; *p-value < 0.05.

Fig. 2. 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. 3. 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. 4. AR and ADAM inhibition impairs the MICA shedding and the androgen-induced melanoma escape from NK cells.

A, B Cells were unstimulated or stimulated for 6 h with the indicated compounds. A Lysate proteins were analyzed for MICA/B expression using the anti-MICA/B antibody. The filter was re-probed using anti-GAPDH antibody, as loading control. B sMICA was quantified by ELISA. Data from three (n = 3) different experiments are expressed as mean ± SD; *p < 0.05. Representative experiment (C, E, G) and statistical analysis (D, F, H) of cytotoxicity assays performed in untreated or treated AMM16 (C, D), WM266-4 (E, F) and LCM-Mel (G, H) cells co-coltured for 6 h with NK cells used at different E:T ratios (50:1; 25:1). D, F, H Data derived from different experiments (n = 3) or (n = 4) are expressed as mean ± SD; *p < 0.05, **p < 0.01.

Fig. 5. AR and ADAM inhibition impairs the androgen-induced melanoma escape from NK cells.

A, C AMM16-derived spheroids were untreated or treated as indicated in the figure and stained. Images of spheroids before (A) and after (C) the NK cells addition are presented. The green fluorescence from the acridin orange (AO) staining indicates total cells, while the red fluorescence from the propidium iodide (PI) staining indicates dead cells. C Spheroids were co-coltured for 4 h with NK cells. Images are representative of three different experiments. Bar, 100 µm. B, D The graphs represent the dead cells/total cells. Values of dead and total cells were analyzed using NIH Image J. They derive from red fluorescence mean/green fluorescence mean intensity and are expressed as mean ± SD of 3 different experiments (n = 3); *p < 0.05.

Fig. 6. AR ligand-stimulation promotes migration and invasiveness of melanoma cells.

AMM16 (A, F), WM266-4 (B, G), MEL-CAL (C, H), LCM-Mel (D, I) and MEL-STA (E, L) cells were left unchallenged or challenged with R1881, in the absence or presence of Bicalutamide (Bic). Cells were allowed to migrate (A–E) or invade (F–L), counted and data are expressed as fold increase. AMM16 (M), WM266-4 (N) and MEL-CAL (O) cells were transfected with siRNA Alexa Fluor 488 along with control siRNA (siRNA ctrl) or siRNA AR (siRNA AR). Lysate proteins were analyzed by WB using the indicated antibodies (left panels in M–O). Cells were left in the absence or presence of R1881 and then allowed to migrate. Migrating cells were scored and data expressed as fold increase. In A–O, data are expressed as mean ± SD of 3 different experiments (n = 3); *p < 0.05; **p < 0.01.

Fig. 7. AR and ADAM10 mediate the androgen-triggered invasiveness and growth of melanoma-derived spheroids.

A AMM16, B WM266-4 and C LCM-Mel cells were used for invasion assays. The indicated compounds were added to the upper and the lower chambers and cells were counted. Results from three different experiments were collected and expressed as fold increase. *p < 0.05; **p < 0.01. D AMM16- or F WM266-4 derived spheroids were generated by Matrigel embedding. After 4 days, spheroids were left untreated or treated with the indicated compounds for additional 15 days. Phase-contrast images captured at the 15th day are shown. They are representative of 3 different experiments. Bar, 100 μm. E, G Spheroid size was calculated using the Leica Suite software in basal conditions (4 days, not shown) or in cells unstimulated or stimulated for 15 days as indicated. Data are expressed as fold increase. In A–C, E, G, means and SD are shown. n represents the number of experiments. *p < 0.05; **p < 0,01.

Fig. 8. Signaling effectors involved in the androgen-mediated migration of melanoma cells.

AMM16 (A, B) or WM266-4 (C) cells were left unchallenged or challenged for 20 min with R1881 in the absence or presence of the indicated compounds. Lysate proteins were analyzed by WB using the antibodies against the indicated proteins. p-FAK stands for Tyr397 p-FAK; p-ERK stands for p44 and p42 ERK. The filters were re-probed using anti tubulin antibody, as a loading control. A–C Expression levels of the proteins were analyzed by densitometry analysis, using NIH Image J Software. Graphical analysis, representative of three different experiments (n = 3), is shown in the right sections. Means and SD are shown. *p < 0.05; **p < 0.01. AMM16 (D) and WM266-4 (E) cells were wounded and left untreated or treated with R1881, in the absence or presence of the indicated compounds. Phase contrast images are representative of three different experiments, each in triplicate. The wound area was calculated using Leica Suite software and data are expressed as the percentage of wound width over the control cells (analyzed at time 0).

Fig. 9. The effect of immune-checkpoint inhibitors (ICIs) on cell death of AR-depleted melanoma cells analyzed by LDH release determination.

Surface expression of PD-1 (A) and CTLA-4 (B) on NK cells, identified as CD56⁺ CD3⁻ cells. WB analysis for PD-L1 (C), PD-1 (D) and CTLA-4 (E) expression in the indicated melanoma cell lines. Tubulin or GAPDH were revealed as loading controls. AMM16 (F), WM266-4 (L) and MEL-CAL (P) cells were transfected with control siRNA (siRNA ctrl) or AR siRNA (siRNA AR). Lysate proteins were analyzed by WB using the indicated antibodies. Melanoma cells were untreated or treated with 5, 10 or 35 μg/ml of Atezolizumab (G, M, Q), Pembrolizumab (H, N, R) or Ipilimumab (I, O, S) for 26 h. LDH release was determined as indicated in Methods section. In G–I, M–O, Q–S, data are expressed as mean ± SD of 3 different experiments each in triplicate; *p < 0.05; **p < 0.01.

Fig. 10. The effect of immune-checkpoint inhibitors (ICIs) on cell death of AR-depleted melanoma cells analyzed by Immunofluorescence.

AMM16 (A, D), WM266-4 (B, E) and MelCal (C, F) cells were transfected with control siRNA (siRNA ctrl; left panels in A–C) or AR siRNA (siRNA AR; right panels in A–C) and treated with the indicated ICIs at 35 μg/ml. The red fluorescence from the propidium iodide (PI) and the green fluorescence from the acridin orange (AO) staining indicate dead and total cells, respectively. Images are representative of three different experiments. D–F The graphs represent the dead cells/total cells analyzed using NIH Image J. They derive from red fluorescence mean/green fluorescence mean intensity and are expressed as mean ± SD of 3 different experiments (n = 3); *p < 0.05.

Fig. 11. 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.