Unveiling the role of Carbonic Anhydrase XII in human melanoma and dendritic cells in a hypoxic microenvironment

The tumor microenvironment (TME) is characterized by low oxygen levels, a condition known as hypoxia which plays a critical role in cancer progression, immune evasion and resistance to therapy. Along with cancer cells, the TME is populated by stromal cells, which include infiltrating immune cells. The aim of the thesis was to study the relevance of Carbonic Anhydrase XII (CAXII) in the migration of Dendritic Cells (DCs) and the interplay between CAXII and Hedgehog (Hh) pathway in the migration of melanoma cells, through new small molecules, especially under hypoxia. At first, we decided to study the relationship between the Hh pathway and CAXII, underlining their involvement in melanoma cell migration under hypoxia. Indeed, such signaling is well-known for its role in various physiological processes but aberrantly re-activated in cancer cells and carbonic anhydrases (CAs), key enzymes in pH regulation, have been linked to cell migration. In this study, we targeted components of the Hh pathway using Glabrescione B and C22, as well as Cyclopamine and SAG, to assess their impact on CAXII expression, particularly under hypoxic conditions. Migration and invasion assays were conducted on two melanoma cell lines, SK-MEL-28 and A375, where Smoothened, a pivotal upstream regulator of the Hh pathway, and GLI1, its primary transcription factor, were chemically inhibited. The findings revealed a connection between CAXII and the Hh pathway, demonstrating that their inhibition significantly impaired melanoma migration and invasion, especially under hypoxia. Then, due to the relevance of DCs in the TME and DCs-based immunotherapy in melanoma, we focused our attention on DCs with the aim to study CAXII expression and role. For the first time, we observed an increased expression of CAXII in mature DCs (stimulated with lipopolysaccharide, LPS) either under normoxia or hypoxia. To understand the involvement of CAXII in DC migration we first inhibited CAs with the chemical compound acetazolamide (AAZ), observing a downregulation of CAXII expression. We performed a migration assay in the presence of AAZ, observing a reduction in DC migration. In addition, to define if CAXII itself was crucial in DC migration, we knocked down the protein with a siRNA approach under normoxia and hypoxia, either in the presence or not of LPS. Finally, we performed a migration assay, demonstrating that CAXII siRNA significantly reduced DC migration under all experimental conditions. Overall, these data suggest a similar role of CAXII in two different cellular models highlighting its involvement in cell migration, specifically related to Hh pathway in melanoma cells, especially under hypoxic conditions. These findings acquire a major impact on the development of new therapeutical strategies targeting CAXII in a hypoxic microenvironment, a typical feature of physiological and pathological conditions.