CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia

Abstract

Tumor hypoxia represents a severe microenvironmental stress that is frequently associated with acidosis. Cancer cells respond to these stresses with changes in gene expression that promote survival at least in part through pH regulation and metabolic reprogramming. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a critical adaptive role in response to hypoxic and acidic environments by catalytically hydrating extracellular CO₂ to produce bicarbonate for buffering intracellular pH (pHi). We used proteome-wide profiling to study the cellular response to transient CA IX knockdown in hypoxia and found a decrease in the levels of key glycolytic enzymes and lactate dehydrogenase A (LDHA). Interestingly, the activity of LDH was also decreased as demonstrated by native in-gel activity assay. These changes led to a significant reduction in glycolytic flux and extracellular lactate levels in cancer cells in vitro, contributing to a decrease in proliferation. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate restored LDHA activity, extracellular acidification, pHi, and cellular proliferation. These results indicate that in the absence of CA IX, reduction of pHi disrupts LDHA activity and hinders the cellular capacity to regenerate NAD⁺ and secrete protons to the extracellular space. Hypoxia-induced CA IX therefore mediates adaptation to microenvironmental hypoxia and acidosis directly, by enzymatically converting extracellular CO₂ to bicarbonate, and indirectly, by maintaining glycolysis-permissive intracellular milieu.

Keywords: carbonic anhydrase IX; glycolysis; hypoxia; metabolism; pH regulation.

Figure 1

CA IX knockdown decreases the abundance of key glycolytic enzymes. (A) Average fold change of proteins affected by doxycycline (DOX)-induced CA IX knockdown in hypoxic HeLa DOX cells incubated in 0.5% serum for 48 h, determined by 2D-DIGE analysis (n = 3). (B) KEGG pathway analysis of under-represented proteins in response to knockdown of CA IX. P-value is computed from the Fisher exact test for probability of the set of genes belonging to a specific pathway. Odds ratio and Combined score are additional biostatistical means representing the probability that the selected gene set is component of the respective pathway. All computations were provided by the Enrichr data analysis platform (15, 16). (C) Representative immunoblots showing the effect of CA IX knockdown in HeLa DOX (left) and HeLa KD (right) cells incubated in 2% O₂ for 48 h (n = 3). (D) Impact of CA IX knockdown (siCA9) on LDH and PGK1 levels normalized to β-actin in HeLa KD cells incubated in 2% O₂ for 48 h (n = 5). (E) Dose-response relationship between CA IX knockdown and reduction of LDH levels in hypoxic HeLa KD cells. (F) Representative immunoblot showing increased ALDOA and LDH in CA IX-transfected C-33 A cells (C33 A-FL) that lack endogenous CA IX expression incubated in 2% O₂ for 48h (n = 3). (G) LDH5 activity of HT-1080 CA IX knockout (HT-1080 KO) in normoxia and 48h 1% hypoxia (n = 3). (H) Representative LDH activity assay showing separated LDH5-LDH3 isoforms stained for LDH activity (n = 3). Error bars are ±SD. P-values were calculated against control by t-test. *P < 0.05.

Figure 2

Catalytic activity of CA IX maintains glycolytic flux. (A) Maximum glycolytic flux rate of HT-1080 KD cells incubated for 24 h in 1% O₂, treated with oligomycin A after establishing baseline ECAR (extracellular acidification rate). (B) Representative western blot showing decreased levels of glycolytic enzymes ENO1, ALDOA and LDHA in HeLA dCA cells that overexpress CA IX lacking the catalytic domain (dCA, right lane). HeLa dCA cells were incubated in 2% O₂ for 48 h (n = 3), the full length CA IX (FL CA IX) is shown in the top of the blot (C) Normalized extracellular lactate concentration of HeLa DOX, HeLa dCA, and C-33A-FL cells incubated in 2% O₂ for 48 h. Mean baseline lactate concentrations are: Hela DMSO 0.11 mM, HeLa DOX 0.08 mM, HeLa Control 0.09 mM, HeLa dCA 0.04 mM, C-33A-Neo 0.16 mM, C-33A-FL 0.26 mM. (D) Seahorse analysis of oxygen consumption rate (OCR) and proton production rate (PPR) in posthypoxic parental HT-1080 cells in low glucose (1mM) or with CA IX inhibitor 4-aminometylbenzensulfonamid (HSFA). (E) Normalized ECAR in HT-1080 cells pre-incubated in 1% O₂ for 24 h in 10 and 0.5% serum. After establishing the baseline, the cells were treated with HSFA for 30 min (right). Error bars are ±SD. P-values were calculated against Control by t-test. *P < 0.05; **P < 0.01.

Figure 3

Alternative LDH substrate alpha-ketobutyrate restores pHi, proliferation and LDH activity in CA IX deficient cells. (A) Intracellular pH (pHi) of HT-1080 KO cells incubated for 48 h in 1% O₂ with 1 mM alpha-ketobutyrate (α-KB). (B) Seahorse analysis of baseline ECAR (black) and the maximal glycolytic capacity (white) of HT-1080 KO cells. (C) Normalized cell number of HT-1080 KO cells after 72 h in 1% O₂ in the presence or absence of α-KB. Error bars are ±SEM. P-values were calculated against control by t-test. *P < 0.05; **P < 0.01 ***P < 0.001.

Figure 4

Intracellular acidosis decreases LDHA level and activity. (A) Representative immunoblot (n = 3) showing LDHA levels in control and α-KB-treated A-549 KD cells incubated for 48 h in 1% O₂, numbers represent relative LDHA levels quantified against β-actin in the presented blot. (B) Representative immunoblot (n = 3) showing the effect of sodium-hydrogen antiporter 1 (NHE-1) inhibition by cariporide on LDHA levels in normoxic parental HT-1080 cells, numbers represent relative LDHA levels quantified against β-actin in the presented blot. (C) Representative immunoblot (n = 3) showing the effect of 48h cariporide and α-KB treatment on the levels of activating LDHA tyrosine 10 phosphorylation (pLDHA Tyr10) in normoxic HT-1080 cells, numbers represent pTyr10/LDHA ratio in the presented blot. (D) LDH5 activity of normoxic HT-1080 cells in response to α-KB (n = 3). (E) Representative LDH activity assay showing LDH5 in the enhanced contrast field (bright, top) and resolved LDH5-LDH3 isoforms (bottom) in response to α-KB and 20 μM cariporide. (F) Schematic indicating the interaction between CAIX, pHi, and the regulation of glycolysis and lactate production. Error bars are ±SEM. P-values were calculated against Control by t-test. *P < 0.05; **P < 0.01 ***P < 0.001.