The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+-shuttle His200, functions as a “proton-collecting/distributing antenna” to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme’s intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity.

No abstract

Most carbonic anhydrases catalyse the reversible conversion of carbon dioxide to protons and bicarbonate, either as soluble cytosolic enzymes, in or at intracellular organelles, or at the extracellular face of the cell membrane as membrane-anchored proteins. Carbonic anhydrase isoform IX (CA IX), a membrane-bound enzyme with catalytic activity at the extracellular membrane surface, has come to prominence in recent years because of its association with hypoxic tissue, particularly tumours, often indicating poor prognosis. We have evaluated the catalytic activity of CA IX heterologously expressed in Xenopus laevis oocytes by measuring the amplitude and rate of cytosolic pH changes as well as pH changes at the outer membrane surface (pH s ) during addition and removal of 5% CO 2 /25 mM HCO À 3 , and by mass spectrometry. Our results indicate both extracellular and intracellular catalytic activity of CA IX. Reduced rates of CO 2 -dependent intracellular pH changes after knockdown of CA IX confirmed these findings in two breast cancer cell lines: MCF-7 and MDA-MB-231. Our results demonstrate a new function of CA IX that may be important in the search for therapeutic cancer drugs targeting CA IX.

The most aggressive and invasive tumor cells, which often reside in hypoxic environments, rely on extensive glycolysis to meet their large demand for energy and biosynthetic precursors. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms that regulate lactate transport in MCF‐7 human breast cancer cells under normoxia and hypoxia. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Measurements of intracellular pH and lactate concentration with the FRET‐based lactate nanosensor laconic show that CAIX augments lactate flux via MCT1 by a non‐catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+‐shuttle His200, functions as a “proton‐collecting/distributing antenna” to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of MCF‐7 cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.This work was supported by the Research Initiative Membrane Biology, the Stiftung Rheinland‐Pfalz für Innovation, the Graduiertenfördungskommission Rheinland‐Pfalz, and the DFG.