Background: Malignant melanoma is the leading cause of death among skin cancer patients due to its tendency to metastasize. Alterations at the molecular level are often evident, which is why melanoma biology has garnered increasing interest. The hedgehog (Hh) pathway, which is essential for embryonic development, is aberrantly re-activated in melanoma and may represent a promising therapeutic target. In addition, carbonic anhydrase XII (CAXII) represents a poor prognostic target for hypoxic tumors, such as melanoma, and is involved in cell migration. Thus, we decided to investigate whether and how the Hh pathway and CAXII may control melanoma cell migration and invasiveness. Methods: The migratory and invasive capabilities of SK-MEL-28 and A375 cell lines, either un-transfected or transiently transfected with Smoothened (SMO), GLI1, or CAXII siRNA, were studied under normoxic or hypoxic conditions. Results: For the first time, we showed that SMO and GLI1 silencing resulted in the downregulation of CAXII expression in both moderately and highly invasive melanoma cells under hypoxia. The Hh pathway as well as CAXII inhibition by siRNA resulted in impaired malignant melanoma migration and invasion. Conclusion: Our results suggest that CAXII and the Hh pathway are relevant in melanoma invasion and may be novel and promising therapeutical targets for melanoma clinical management.

Cutaneous melanoma is a highly aggressive skin cancer, with poor prognosis. The tumor microenvironment is characterized by areas of hypoxia. Carbonic anhydrase IX (CA-IX) is a marker of tumor hypoxia and its expression is regulated by hypoxia-inducible factor-1 (HIF-1). CA-IX has been found to be highly expressed in invasive melanomas. In this study, we investigated the effects of hypoxia on the release of small extracellular vesicles (sEVs) in two melanoma in vitro models. We demonstrated that melanoma cells release sEVs under both normoxic and hypoxic conditions, but only hypoxia-induced sEVs express CA-IX mRNA and protein. Moreover, we optimized an ELISA assay to provide evidence for CA-IX protein expression on the membranes of the sEVs. These CA-IX-positive sEVs may be exploited as potential biomarkers for liquid biopsy.