Abstract #234: The HIF-induced carbonic anhydrases CAIX and XII regulate intracellular pH. Validation of CAIX and XII as anticancer targets Free

Introduction: Under hypoxic conditions increased lactic acid secretion together with carbonic acid production contribute to a high acid load in the tumor. All cells express multiple isoforms of carbonic anhydrase (CA), zinc-dependent enzymes catalyzing the reversible hydration of carbon dioxide into bicarbonate and protons. The expression of membrane-bound CAIX and CAXII is tightly controlled by oxygen levels via the Hypoxia-Inducible Factor (HIF) in many cancers. Despite the recognition that tumor expression of HIF-1\#945; and CAIX correlates with poor patient survival, the physiological role of CAIX and XII in tumor growth is not fully resolved. What is the contribution of CAIX and XII in intracellular pH (pHi) homeostasis for hypoxic tumor cells evolving in an acidic environment? Experimental Procedures: To answer these questions: i) we forced the expression of human CAIX or CAXII in fibroblasts that do not express these isoforms and ii) we silenced CAIX expression combined with or without CAXII silencing in human colon adenocarcinoma cells, LS174Tr. We analyzed downstream effects of CAIX and/or CAXII on: i) extracellular and intracellular pH regulation, ii) cell survival in response to an acidic stress and iii) tumor growth in nude mice. Results: 60% of the human tumor cell lines we tested express both CAIX and CAXII in hypoxia. We demonstrated that CAIX-expressing cells strongly acidify the extracellular milieu in response to a \#8216;CO₂-load\#8217;. Expression of CAXII showed a slighly lower external acidification when compared to CAIX-expressing fibroblasts. In hypoxic tumor cells, which express both CA, double silencing is necessary to abolish extracellular acidification in response to a \#8216;CO₂-load\#8217;. Interestingly, in spite of their capacity to acidify the milieu, CAIX- or CAXII-expressing cells do maintain a more alkaline resting pHi_i than control cells, an action that preserves ATP levels and cell survival, in a range of acidic external pH (6.0 to 6.8) and low bicarbonate medium. Our results suggest that CAIX is more potent than CAXII in protecting the cells against an extracellular imposed acidic stress. However CAXII activity is nonetheless significant. In vivo experiments conducted with LS174Tr cells indicate that ca9 silencing alone leads to a 40% reduction in xenograft tumor volume. Surprisingly, a 95% decrease in ca9 leads to ca12 gene up-regulation. Combined knockdown of both ca9 and ca12 gives an impressive 85% decrease in the tumor growth rate. Finally, we are now examining the contribution of CAIX and XII to pHi regulation and to in vivo growth with several other carcinoma cell lines (melanoma A375Tr, renal Caki-1, lung A549 cells). Conclusion: Hypoxia-induced CAIX and CAXII are major tumor pro-survival pHi-regulating enzymes and their combined targeting demonstrates that they hold potential as anticancer targets.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 234.