HLA-DR signalling and extracellular vesicles release contribute to immune escape and cancer progression of MHC class II expressing melanoma cells

Melanoma is the deadliest form of skin cancer in Western population whose incidence rate is increasing rapidly worldwide. The metastatic progression of melanoma is associated, in almost 50% of melanoma, to the constitutive expression of Major Histocompatibility Complex (MHC) class II molecules, whose engagement leads to the activation of several signalling pathways. Melanoma cells secrete extracellular vesicles (EVs), that regulating immune cell functions and interacting with different cell types, modify the tumour microenvironment, playing a main role in the metastatic progression of melanoma as mediators of metastasis. The aim of our work was to study in melanoma cells the expression and lipid raft localisation and activation of several adhesion receptors and signalling proteins, as well as the role of EVs in melanoma metastatic progression as a consequence of HLA-DR mediated signalling. In particular, we demonstrated that HLA-DR mediated signalling increases the expression and lipid raft localisation of HLA-DR, PD-L1, Integrin and CAM adhesion receptors, FAK, AKT and STAT3 signalling proteins and their activation. In EVs secreted by melanoma cell lines constitutively expressing MHC class II molecules we showed the increased localization of HLA-DR, CAM adhesion receptors, PD-L1 and STAT3 signalling proteins mediated by the signalling activated by MHC class II molecules. Furthermore, through PBMCs and EVs co-culture experiments, we demonstrated that HLA-DR mediated signalling enhances the cytotoxic effects of melanoma-derived EVs on human PBMCs. Finally, we showed the autocrine and paracrine function of melanoma-derived EVs that, in response to HLA-DR mediated signalling, induces increased migration of fibroblasts and melanoma cells. Conclusions: The results reported suggest a new model in which HLA-DR stimulation activates a signalling in melanoma cells that provides a platform useful to frustrate an effective anti-tumour response and to increase melanoma migration and metastatic dissemination regulating cell adhesion, motility and immune escape. Furthermore, our results suggest that HLA-DR mediated signalling promotes melanoma progression enhancing, through the extracellular vesicles secreted, the metastatic dissemination of melanoma cells as well as inhibiting the immune response in tumour microenvironment. In particular, our results showed a complex interaction between tumour cells and fibroblasts involving EVs secreted by tumour cells that, as important mediators of cell communication, establish a paracrine and autocrine dynamic signalling circuit that plays a crucial role in the regulation of tumour cell and fibroblast migration and cancer progression.