Carbonic anhydrase-IX inhibition enhances the efficacy of hexokinase II inhibitor for hepatocellular carcinoma in a murine model

Hypoxic conditions, which large or infiltrative hypovascular tumors may encounter, also produce acidic environments. Carbonic anhydrase-IX (CA-IX), an enzyme involved in lowering pH, is overexpressed in hepatocellular carcinoma (HCC). In the present study, whether inhibition of CA-IX enhances the efficacy of a hexokinase II inhibitor in an in vivo murine model was examined and its prognostic implication in HCC patients was investigated. CA-IX expression was evaluated using quantitative real-time PCR and western blot analysis using human HCC cell lines. 3-bromopyruvate (3-BP), a hexokinase II inhibitor, and acetazolamide, a carbonic anhydrase inhibitor, were used to target hexokinase II and CA-IX in vitro and in vivo, respectively. A human HCC cell line (Huh-7) was tested as a subcutaneous tumor model in BALB/c nu/nu mice. The prognostic role of CA-IX was evaluated in the TCGA database. Quantitative real-time PCR and western blot analysis revealed that CA-IX expression was activated in the presence of 3-BP. Further analysis showed that introducing an additional stress by treating the orally active CA-IX inhibitor (acetazolamide) can synergistically increase the efficacy of 3-BP in vivo, which was confirmed using a mouse model. We also found that HCC patients with high CA-IX expression show poor overall survival in TCGA database. These results indicate CA-IX is a promising therapeutic target for enhancing the efficacy of 3-BP and can be a prognostic factor for HCC.