Abstract
Acidosis of the tumor microenvironment is typical of a malignant phenotype, particularly in hypoxic tumors. All cells express multiple isoforms of carbonic anhydrase (CA), enzymes catalyzing the reversible hydration of carbon dioxide into bicarbonate and protons. Tumor cells express membrane-bound CAIX and CAXII that are controlled via the hypoxia-inducible factor (HIF). Despite the recognition that tumor expression of HIF-1alpha and CAIX correlates with poor patient survival, the role of CAIX and CAXII in tumor growth is not fully resolved. To understand the advantage that tumor cells derive from expression of both CAIX and CAXII, we set up experiments to either force or invalidate the expression of these enzymes. In hypoxic LS174Tr tumor cells expressing either one or both CA isoforms, we show that (a) in response to a "CO(2) load," both CAs contribute to extracellular acidification and (b) both contribute to maintain a more alkaline resting intracellular pH (pH(i)), an action that preserves ATP levels and cell survival in a range of acidic outside pH (6.0-6.8) and low bicarbonate medium. In vivo experiments show that ca9 silencing alone leads to a 40% reduction in xenograft tumor volume with up-regulation of ca12 mRNA levels, whereas invalidation of both CAIX and CAXII gives an impressive 85% reduction. Thus, hypoxia-induced CAIX and CAXII are major tumor prosurvival pH(i)-regulating enzymes, and their combined targeting shows that they hold potential as anticancer targets.
Highlights
Adaptation of tumor cells to hypoxia and acidosis is a critical driving force in tumor progression and metastasis [1, 2]
Stable expression of human CAIX in normoxia was found to be plasma membrane located in PS120 (Fig. 1A) and CCL39 cells (Supplementary Fig. S1)
During the preparation of this manuscript, a study showed that ectopically expressed CAIX in human bladder carcinoma RT112 cells was able to spatially coordinate pHi, but only when cells are cultured as three-dimensional spheroids [32]
Summary
Adaptation of tumor cells to hypoxia and acidosis is a critical driving force in tumor progression and metastasis [1, 2]. Cancer cells produce a large amount of lactic acid [3], which is generated through glucose metabolism and inefficient vascular clearing, resulting in an acidic microenvironment within many solid tumors [4]. Because numerous intracellular processes require close regulation of pHi, most mammalian cells, hypoxic tumor cells, have developed key strategies to regulate their pHi. Activation of the hypoxia-inducible factor-1 (HIF-1) in hypoxia plays a major role in regulating pH homeostasis by enhancing expression of membrane located transporters, exchangers, pumps and ecto-enzymes [8]. To survive in an acidic environment, the pHi-regulating system of tumor cells actively extrudes acids via the growth factor–activated Na+/H+ exchanger 1 We showed previously that NHE-1 plays a key role in tumor development for cells producing large amounts of lactic acid [14]. In the opposite direction to H+ extrusion, HCOÀ3 influx through Na+-HCOÀ3 cotransporters (NBC) and ClÀ/HCOÀ3 exchangers (AE) contributes to cytoplasmic alkalinization [15,16,17]
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