Expression of carbonic anhydrase IX is associated with poor prognosis through regulation of the epithelial-mesenchymal transition in hepatocellular carcinoma

Carbonic anhydrase 9 (CA9) is a plasma membrane-associated isoenzyme that catalyzes pH regulation under hypoxic conditions. CA9 is transcriptionally regulated by hypoxia-inducible factor 1. Recent studies reported that hypoxia also promoted the epithelial-mesenchymal transition (EMT) in various cancers. In the present study, we evaluated the relationship between CA9 expression and EMT in vitro with two hepatoma cell lines. We also examined the clinical significance of CA9 expression in 117 consecutive patients that underwent hepatectomies for hepatocellular carcinoma (HCC). We evaluated CA9 expression and EMT induction under hypoxia with quantitative RT-PCR, western blot analysis and immunofluorescence staining, in HuH7 and HepG2 cells. We knocked down CA9 expression with small interfering RNA to evaluate the relationship between CA9 and EMT. We found that hypoxia induced CA9 expression in HCC cells and promoted EMT, evidenced by a loss of E-cadherin and an increase in N-cadherin. Twist, a transcriptional regulator of EMT, was also upregulated with hypoxia. The CA9 deficiency attenuated hypoxia-induced changes in E-cadherin and N-cadherin. Immunohistochemical evaluations of patient samples showed that CA9 was expressed in 50.4% of patients (59/117). However, patients with and without CA9 expression were not significantly different in clinicopathological factors. Nevertheless, a multivariate analysis showed that CA9 expression was an independent factor for both recurrence and prognosis among patients that underwent curative surgery for HCC. In conclusion, this study revealed that CA9 expression was a pivotal predictive factor for poor prognosis after radical surgery for HCC. Moreover, the CA9 regulation of the expression of EMT-related molecules represented a mechanism that enhanced malignant potential.