Inhibition of Melanoma Cell Migration and Invasion Targeting the Hypoxic Tumor Associated CAXII

SIMPLE SUMMARY: Melanoma is a potential metastatic cancer with a poor prognosis and a low free-survival rate. Thus, discovering new therapeutic strategies will be helpful to fight against it. Carbonic anhydrases IX (CAIX) and XII (CAXII) along with Hedgehog pathway aberrations are already demonstrated to be involved in melanoma progression. Here we investigated the effects of either an indirect or direct CAXII inhibition on cell migration and invasion in three melanoma cell lines. First we indirectly inhibited CAXII through the smoothened antagonist cyclopamine, which resulted in a decreased CAXII protein expression and cell migration. Thereafter, we directly blocked CAXII using new small molecules. This resulted in reducing not only cell migration, but also the invasiveness ability of highly aggressive melanoma cell lines. This evidence may contribute to further exploiting the therapeutic role of CAXII in melanoma progression and invasiveness. ABSTRACT: Background: Intratumoral hypoxia contributes to cancer progression and poor prognosis. Carbonic anhydrases IX (CAIX) and XII (CAXII) play pivotal roles in tumor cell adaptation and survival, as aberrant Hedgehog (Hh) pathway does. In malignant melanoma both features have been investigated for years, but they have not been correlated before and/or identified as a potential pharmacological target. Here, for the first time, we demonstrated that malignant melanoma cell motility was impaired by targeting CAXII via either CAs inhibitors or through the inhibition of the Hh pathway. Methods: We tested cell motility in three melanoma cell lines (WM-35, SK-MEL28, and A375), with different invasiveness capabilities. To this end we performed a scratch assay in the presence of the smoothened (SMO) antagonist cyclopamine (cyclo) or CAs inhibitors under normoxia or hypoxia. Then, we analyzed the invasiveness potential in the cell lines which were more affected by cyclo and CAs inhibitors (SK-MEL28 and A375). Western blot was employed to assess the expression of the hypoxia inducible factor 1α, CAXII, and FAK phosphorylation. Immunofluorescence staining was performed to verify the blockade of CAXII expression. Results: Hh inhibition reduced melanoma cell migration and CAXII expression under both normoxic and hypoxic conditions. Interestingly, basal CAXII expression was higher in the two more aggressive melanoma cell lines. Finally, a direct CAXII blockade impaired melanoma cell migration and invasion under hypoxia. This was associated with a decrease of FAK phosphorylation and metalloprotease activities. Conclusions: CAXII may be used as a target for melanoma treatment not only through its direct inhibition, but also through Hh blockade.