A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells

Abstract

The most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+ shuttle system in CA IX to facilitate proton efflux through MCT4.

What do cellular responses to acidity tell us about cancer?

“…The effective diffusion coefficients of these acidic products will depend on the equilibration state of CO 2 /HCO 3 − , as this depends on the activity of exofacial carbonic anhydrases (CAs), such as CAIX or CAXII [139][140][141][142][143][144][145]. As described previously [146], the effect of CA activity on pHe depends on the nature of the disturbance.…”

Monocarboxylate Transporters (SLC16): Function, Regulation, and Role in Health and Disease

“…Subsequently, MCT1 and MCT4 transport activity was increased by extracellular catalytically active and inactive carbonic anhydrase IV (Klier et al, 2014). Additionally, carbonic anhydrase IX (active and inactive) augments the activity of MCT1 and MCT4 (Jamali et al, 2015;Mboge et al, 2019). It is hypothesized that the carbonic anhydrases function as a proton-collecting antenna, thereby enhancing the activity of proton-coupled MCTs (Fig.…”

Transport Metabolons and Acid/Base Balance in Tumor Cells

“…Indeed, inhibition of CA catalytic activity with EZA had no effect on the lactate transport capacity or proliferation of hypoxic MCF-7 breast cancer cells [91]. In line with this, selective inhibition of CAIX with three different ureido-substituted benzene sulfonamides (USBs) did not alter the glycolytic rate in DFO-treated UFH-001 breast cancer cells, while CRISPR/Cas9-induced deletion of CAIX resulted in a significant reduction of glycolysis in the same cell type [101,153]. Since the transport metabolons formed between MCTs and CAIX are insensitive to the inhibition of CAIX catalytic activity, they have to be targeted either via the physical interaction between CAIX and the MCT chaperon CD147 or via the CAIX antennal function.…”

“…Indeed, knockout of CAIX as well as the application of an antibody against the CAIX PG domain, but not inhibition of CA enzymatic activity, resulted in a significant reduction in the proliferation of hypoxic MCF-7 and MDA-MB-231 cells [91,95]. These data were confirmed in the triple-negative breast cancer cell line UFH-001 [101]. Knockdown of CAIX with a CRISPR/Cas9 approach decreased the glycolytic proton efflux rate (GlycoPER), as measured by Seahorse analysis, in pseudo-hypoxic UFH-001 cells treated with the HIF1α-stabilizing agent desferrioxamine (DFO).…”

“…This H + shuttle seems to be mediated by the amino acids Glu69 and Asp72, which have been suggested to form a part of the CAII proton-collecting antenna [92,113]. Based on these results, it was suggested that CAII, which is directly bound to MCT1/4, facilitates parts of its proton-collecting apparatus to quickly move H + between the transporter and the surrounding protonatable residues near the inner [88,90,92,94,95,[98][99][100][101] Even though there is accumulating evidence for the physical and functional interaction between acid/base transporters and CAs, both in vitro and in intact cells, several studies have questioned the existence of bicarbonate transport metabolons, both in respect to the physical as well as functional interaction between CAs and transport proteins [102][103][104][105]. For a comprehensive review on bicarbonate transport metabolons, including the criticism on this concept, we recommend the reviews [55,[106][107][108].…”