Evaluation of carbonic anhydrase IX as a potential therapeutic target in urothelial carcinoma

Carbonic anhydrase IX (CA9) is important in the regulation of intra- and extracellular pH in solid tumors, contributing to cell growth and invasion. In urothelial carcinoma (UC), CA9 has been identified as a urinary marker for disease detection, but its biologic role is unknown. To date, differential gene expression patterns of CA9 in various molecular subtypes and potential effects of CA9 inhibition in UC cells are unknown. We aimed to investigate the function of CA9 and the effects of CA9 inhibition in invasive UC.

Immunohistochemistry was used to assess CA9 expression in a cohort of 153 patients undergoing radical cystectomy. CA9 expression was correlated with molecular subtype by analysis of the TCGA data and of our own cohort of 223 patients with invasive UC receiving neoadjuvant chemotherapy. CA9 expression was assessed in a panel of 12 UC cell lines by Western Blot and qPCR, and multiple siRNAs were used to silence CA9 in 2 cell lines. Effects of CA9 silencing on cell growth, migration, and invasion were assessed. We also used the small molecule inhibitor U-104 to inhibit CA9 in vitro and in an orthotopic xenograft model.

CA9 expression was higher in cancer tissue compared to benign urothelium and was particularly highly expressed in luminal papillary and basal squamous tumors. CA9 expression did not correlate with outcome after neoadjuvant chemotherapy and/or radical cystectomy. Silencing of CA9 by siRNA diminished invasion but did not induce a consistent change of cell growth and migration. Treatment with U-104 led to cell growth reduction only at high concentrations in vitro and failed to have a significant effect on tumor growth in vivo.

The present study confirms over-expression of CA9 in UC and for the first time shows a correlation with molecular subtypes. However, CA9 expression showed no association with the outcome of patients with muscle invasive bladder cancer and inhibition of CA9 did not lead to a consistent inhibition of tumor growth. Based on these data, CA9 exhibits a role neither as a predictive or prognostic marker nor as a therapeutic target in invasive UC.