768

CA9

Extracellular acidification, a mandatory feature of several malignancies, has been mainly correlated with metabolic reprogramming of tumor cells toward Warburg metabolism, as well as to the expression of carbonic anydrases or proton pumps by malignant tumor cells. We report herein that for aggressive prostate carcinoma, acknowledged to be reprogrammed toward an anabolic phenotype and to upload lactate to drive proliferation, extracellular acidification is mainly mediated by stromal cells engaged in a molecular cross-talk circuitry with cancer cells. Indeed, cancer-associated fibroblasts, upon their activation by cancer delivered soluble factors, rapidly express carbonic anhydrase IX (CA IX). While expression of CAIX in cancer cells has already been correlated with poor prognosis in various human tumors, the novelty of our findings is the upregulation of CAIX in stromal cells upon activation. The de novo expression of CA IX, which is not addicted to hypoxic conditions, is driven by redox-based stabilization of hypoxia-inducible factor-1. Extracellular acidification due to carbonic anhydrase IX is mandatory to elicit activation of stromal fibroblasts delivered metalloprotease-2 and -9, driving in cancer cells the epithelial-mesenchymal transition epigenetic program, a key event associated with increased motility, survival and stemness. Both genetic silencing and pharmacological inhibition of CA IX (with sulfonamide/sulfamides potent inhibitors) or metalloprotease-9 are sufficient to impede epithelial-mesenchymal transition and invasiveness of prostate cancer cells induced by contact with cancer-associated fibroblasts. We also confirmed in vivo the upstream hierarchical role of stromal CA IX to drive successful metastatic spread of prostate carcinoma cells. These data include stromal cells, as cancer-associated fibroblasts as ideal targets for carbonic anhydrase IX-directed anticancer therapies.

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.