Mechanisms of immune escape in melanoma progression

Background: Cutaneous melanoma is the deadliest form of skin cancer, and its incidence has been steadily increasing over the years. Melanoma progression involves complex interactions between cancer cells and immune cells within the tumor microenvironment (TME). This network also includes endothelial cells and fibroblasts, which act as a bridge between cancer and immune cells, contributing to tumor proliferation, invasion, and metastasis. Therefore, a better understanding of the mechanisms that govern TME interactions and its composition could enhance treatment strategies for this challenging disease. Research from various groups, including ours, has shown that the activation of Hedgehog-GLI (HH-GLI) signaling is crucial for melanoma growth and stemness. Moreover, it has been widely reported that one of the most common alterations during melanoma progression is the abnormal expression of sialyltransferases. Notably, a recent study from our group highlighted that the gene expression of -2,3 sialyltransferase ST3GAL1 is coregulated by the transcription factors SOX2 and GLI1, which is final mediator of the HH pathway. This work also revealed the significance of ST3GAL1 as a key driver of melanoma metastasis. However, the precise roles of GLI1 and ST3GAL1 in remodeling melanoma TME remain not fully understood. Methods: The immune-modulatory activities of GLI1 and ST3GAL1 were evaluated in a syngeneic B16F10 melanoma mouse model analyzing TME-infiltrating immune cells by flow cytometry. Primary cultures of murine polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were generated from differentiated bone marrow cells. Their immunosuppressive function was assessed by measuring the inhibition of T cell activity. Conditioned media (CM) from GLI1-overexpressing mouse melanoma cells (B16F10 and YUMM 1.7) was used to culture PMN-MDSCs, and the effects of CM were evaluated by Transwell invasion assay and T cell inhibition. Additionally, the chemoattractant effects of CM from ST3GAL1-overexpression B16F10 and YUMM 1.7 cells were assessed by Transwell invasion assay. To explore the modulation of CX3CL1 by GLI1 and CCL5 by ST3GAL1, we conducted a cytokine array analysis, adenosine and nitric oxide assays, Western blotting, and qPCR. Chromatin immunoprecipitation was finally performed to assess the transcriptional regulation of CX3CL1 expression by GLI1. Blocking antibodies anti-CX3CL1 and anti-CCL5 were used for in vitro functional assays. Results: Melanoma cell-intrinsic activation of GLI1 promotes the infiltration of immune cells, leading to the accumulation of immunosuppressive PMN-MDSCs and regulatory T (Treg) cells, and to decreased infiltration of dendric cells (DCs), CD8+ and CD4+ T cells in the TME. Likewise, ST3GAL1-overexpression in vivo increases the recruitment of PMN-MDSCs and Treg cells and decreases the infiltration of CD4+ T cells in the TME. In addition, ectopic expression of ST3GAL1 in melanoma leads to a reduced infiltration of innate cytotoxic cells such as natural killer cells (NKs). Functionally, we show that ectopic expression of GLI1 in melanoma cells promotes PMN-MDSC expansion and recruitment and increases their ability to inhibit T cells. Mechanistically, the chemokine CX3CL1, a direct transcriptional target of GLI1, is involved in PMN-MDSC expansion and recruitment. Turning to sialylation in melanoma TME, we further demonstrate that tumor-derived ST3GAL1 enables the infiltration of PMN-MDSCs and enhances their immunosuppressive functions in releasing inhibitory factors. We also show that ST3GAL1-expressing melanoma cells promote PMN-MDSC recruitment through the CCL5-CCR5 axis. Conclusions: Findings from this thesis highlight the impact of tumor-derived GLI1 and ST3GAL1 in promoting an immune-suppressive TME. Both mechanisms allow melanoma cells to evade the immune system, providing insights for designing new combination treatments targeting GLI1 or ST3GAL1 to reprogram antitumor immunity.