The immune checkpoint CD47 as a putative target of the Hedgehog-GLI pathway in melanoma

The Hedgehog (HH) signaling is a highly conserved pathway that plays a crucial role during embryonic development and in regulating stem cell biology. Its aberrant activation has been reported to drive tumor initiation and progression in several types of cancer, including melanoma. Furthermore, HH pathway is involved in the regulation of anti-cancer immune response as its components are able to mediate a molecular crosstalk between immune cells and cancer cells, modulating immune checkpoint molecules expression. In the last few years, Smoothened (SMO), the main transducer of the HH signaling, has emerged as a promising therapeutic target for anti-cancer therapies as single agent or in combination regimen. In this study we investigated whether and how the HH pathway regulates the expression of CD47, a transmembrane protein highly expressed in cancer cells which suppresses macrophages-induced phagocytosis by binding to its ligand SIRPα expressed on the surface of macrophages. We also investigated whether inhibition of HH pathway may increase phagocytosis of melanoma cells by macrophages, decreasing the ability of melanoma cells to avoid immunosurveillance. Here we show that the HH signaling positively regulates the expression of CD47 in melanoma cells. Our results support the hypothesis of a direct transcriptional regulation of CD47 by the transcription factor GLI2, one of the main downstream effectors of the HH pathway, likely through its binding to a non-canonical GLI binding site located approximately 32Kb upstream to CD47 transcription starting site. In the second part of this work, we set up the conditions for monocyte purification and macrophage differentiation, and co-cultures of macrophages and patient-derived melanoma cells. Our preliminary data indicate that pharmacological inhibition of SMO reduces CD47 expression in melanoma cell lines, and its combination with anti-CD47 antibody slightly increase phagocytosis of melanoma cells. We also report the use of a novel GLI inhibitor which effectively inhibits CD47 expression, to be tested in combination with anti-CD47 antibody in phagocytosis assays. Altogether, these data shed light on the mechanism of regulation of CD47 expression by the HH pathway, establishing the basis for designing future combinatorial treatments targeting HH pathway and CD47 in melanoma.