Hypoxia-Inducible Carbonic Anhydrase IX and XII Promote Tumor Cell Growth by Counteracting Acidosis through the Regulation of the Intracellular pH

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-1α 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 “CO2 load,” both CAs contribute to extracellular acidification and (b) both contribute to maintain a more alkaline resting intracellular pH (pHi), 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 pHi-regulating enzymes, and their combined targeting shows that they hold potential as anticancer targets. [Cancer Res 2009;69(1):358–68]

The asparaginyl hydroxylase factor-inhibiting HIF is essential for tumor growth through suppression of the p53–p21 axis

“…Athymic mice were subcutaneously injected with Tet-inducible LS174-shFIH, A375-shFIH (Figure 3e), LS174-pFIH or A375-pFIH cells ( Figure 3f). Dox had no significant effect on tumor growth of control cells as shown previously (Chiche et al, 2009). However, Dox-induced silencing of fih showed a substantial decrease in tumor growth (60%) in both LS174-shFIH and A375-shFIH tumor xenografts ( Figure 3e).…”

“…Tumors were collected for RNA, protein and immunohistochemical analysis, as described (Chiche et al, 2009). Sections were incubated with antibodies to hypoxyprobe, HIF-1a, CAIX, BNIP3, p53 or cleavage of caspase 3 for 1.5 h, followed by incubation with anti-mouse or anti-rabbit immunoglobulin G-horseradish peroxidase antibodies.…”

“…Human colon adenocarcinoma HT29 cells were grown in three-dimensional culture in suspended droplets ( Supplementary Figures S1a and b; Chiche et al, 2009). We also obtained a stable HT29 cell population expressing shRNA-FIH (HT29-shFIH), in which the expression of FIH was repressed by more than 90% compared with the control (HT29-control; Supplementary Figure S1c).…”

“…However, the expression of CAIX cannot explain the pro-tumorigenic effect of FIH, as silencing of FIH suppressed tumor growth, whereas expression of CAIX favored tumor growth (Chiche et al, 2009). We thus looked at the expression of the mRNA of 96 genes involved in different processes engaged in tumor progression (Cancer PathwayFinder PCR array from SABioscience (Frederick, MD, USA) including HIF-dependent and HIF-independent genes): angiogenesis (angpt1, angpt2, ifna1, pdgfa, pdgfb, tgfb1 and vegfA), cell senescence and apoptosis (apaf1, bad, casp8, cflar, tert, tnfrsfA and tnfrsf25), invasion and metastasis (met, mmp1, plaur and serpinb5), signal transduction and transcription factors (erbb2, ets2, jun and sncg), cell cycle control and DNA damage repair (atm, brca1, cdkn1a, rb1 and tp53) and adhesion (itga2, itga3, itgb1 and pnn; Table 1).…”

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

“…We have identified acidosis as previously unappreciated physiologic stimulus that enhances conversion of αKG to L-2HG, resulting in robust stabilization of HIF-1α in normoxia. HIF-1α facilitates cellular adaptation to an acid load by inducing expression of carbonic anhydrases 49 , and our findings suggest that acid-enhanced L-2HG may be part of this signaling axis. Indeed, we find that ablation of LDHA and MDH2 abrogates the ability of acidotic cells to stabilize HIF-1α.…”