Investigating the Hedgehog pathway and Carbonic Anhydrase XII as prognostic and therapeutic target of melanoma dissemination.

Malignant melanoma is the deadliest form of skin cancer and its tendency to easy migrate and give metastasis makes much more difficult its management. Several therapeutic strategies have been developed but long patient survival benefits reported widely range of chemoresistance. In this scenario, the Hedgehog (Hh) pathway, an essential pathway involved in embryonic development and aberrant re-activated in melanoma, may represent a promising therapeutic target. In primary melanoma Hh has been already investigated, but the mechanism by which the primary tumor disseminates and gives metastases has not been elucidated yet. Tumor microenvironment (TME) is characterized by low O2 levels (defined as intratumoral hypoxia), which contributes to cancer progression and poor prognosis. Within the TME, Carbonic anhydrase XII (CAXII) plays pivotal roles in making the environment more acidic, allowing tumor cell adaptation and survival. Indeed, it represents a poor prognostic target for many tumors. Recent studies correlated the Hh pathway and CAXII in breast cancer, but no evidence regards melanoma. For this reason, this thesis aimed to investigate whether a correlation between the Hh pathway and CAXII exists also in melanoma and if it may be responsible for melanoma motility. At this purpose, firstly transient silencing of Smoothened (SMO), glioma-associated oncogene protein 1 (GLI1) and CAXII were achieved, to study their role in migration and invasion in two malignant melanoma cell lines (SK-MEL-28, and A375) with different invasiveness capabilities; then two new designed SMO and GLI1 inhibitors were used, to deep understand the role of the two major factors of the Hh cascade in controlling melanoma migration via CAXII inhibition. Lastly, we also tested newly designed chemical compounds targeting CAIX and CAXII to evaluate them as possible new therapeutic agents for malignant melanoma. From this manuscript emerged that both SMO and GLI1 inhibition reduced melanoma cell migration, invasion and CAXII expression, under both normoxic and hypoxic conditions. Suppression of CAXII also resulted in decreased migration and invasiveness ability. Same results were confirmed by means of chemical compounds targeting the Hh cascade. Finally, I observed a more crucial role of CAXII rather than CAIX in controlling melanoma motility, as even a direct CAXII blockade resulted in impaired both cell migration and invasion under hypoxia. To conclude, this thesis brought out that CAXII plays crucial role in melanoma migration and invasion and its inhibition may be achieved with a direct or indirect, via Hh pathway, therapeutic approach.