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

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.

Hypoxia and Extracellular Acidification as Drivers of Melanoma Progression and Drug Resistance

“…Moreover, it has been shown that both oxygen deficiency and extracellular acidification induce the expression of genes encoding CAIX and CAXII [ 173 , 174 ]. Expression of these enzymes is associated with higher risk of metastasis and worse overall survival among melanoma patients ( Figure 2 ) [ 175 , 176 ]. Andreucci et al demonstrated that CAIX inhibitor—FC16-670A prevents upregulation of this metalloenzyme expression under acidosis, promoting apoptosis and necrosis only in acidified cancer cells [ 177 ].…”

“…This form of treatment could represent a possible therapeutic approach to reduce tumor relapse [ 177 ]. Giuntini et al indicated that CAXII inhibitor is also able to reduce invasive abilities of melanoma cells, which was associated with reduced phosphorylation of FAK kinase and the activity of MMP9 [ 176 ].…”

Hypoxia-dependent drivers of melanoma progression

“…The levels of HIF-1α and CAIX proteins under hypoxia were decreased in melanoma cells exposed to vemurafenib in a cell type-dependent manner, thus indicating hypoxia involvement in the response to drug treatment and in the induction of heterogeneity [ 97 ] . A recent paper also evidenced CAXII expression in melanoma cell lines and the role of Hedgehog pathway as mediator of melanoma cell migration and CAXII expression under normoxic, as well as hypoxic microenvironment [ 98 ] .…”

Tissue Expression of Carbonic Anhydrase IX Correlates to More Aggressive Phenotype of Basal Cell Carcinoma

“…Among the molecules most expressed in hypoxic condition, the carbonic anhydrase IX (CAIX) is considered a marker of hypoxia in vivo (17), whose overexpression has been correlated with increased tumor aggression in different types of cancer (18)(19)(20)(21)(22). To date, a series of CAIX inhibitors have been synthesized, both in the form of small inhibitory molecules and as monoclonal antibodies, used as antitumor agents in different models of neoplasms (23)(24)(25)(26)(27)(28)(29)(30)(31). In a previous work we tested the expression of CAIX in several human solid tumors, extending the CAIX expression information to the expression of the stem cells markers CD44 and nestin in solid cancers, to explore their relationship with the biological behavior of tumors.…”