Connexin-43 channels are a pathway for discharging lactate from glycolytic pancreatic ductal adenocarcinoma cells.

Glycolytic cancer cells produce large quantities of lactate that must be removed to sustain metabolism in the absence of oxidative phosphorylation. The only venting mechanism described to do this at an adequate rate is H+-coupled lactate efflux on monocarboxylate transporters (MCTs). Outward MCT activity is, however, thermodynamically inhibited by extracellular acidity, a hallmark of solid tumours. This inhibition would feedback unfavourably on metabolism and growth, raising the possibility that other venting mechanisms become important in under-perfused tumours. We investigated connexin-assembled gap junctions as an alternative route for discharging lactate from pancreatic ductal adenocarcinoma (PDAC) cells. Diffusive coupling (calcein transmission) in vitro was strong between Colo357 cells, weaker yet hypoxia-inducible between BxPC3 cells, and very low between MiaPaCa2 cells. Coupling correlated with levels of connexin-43 (Cx43), a protein previously linked to late-stage disease. Evoked lactate dynamics, imaged in Colo357 spheroids using cytoplasmic pH as a read-out, indicated that lactate anions permeate gap junctions faster than highly-buffered H+ ions. At steady-state, junctional transmission of lactate (a chemical base) from the spheroid core had an alkalinizing effect on the rim, producing therein a milieu conducive for growth. Metabolite assays demonstrated that Cx43 knockdown increased cytoplasmic lactate retention in Colo357 spheroids (diameter ~150 µm). MiaPaCa2 cells, which are Cx43 negative in monolayer culture, showed markedly increased Cx43 immunoreactivity at areas of invasion in orthotopic xenograft mouse models. These tissue areas were associated with chronic extracellular acidosis (as indicated by the marker LAMP2 near/at the plasmalemma), which can explain the advantage of junctional transmission over MCT in vivo. We propose that Cx43 channels are important conduits for dissipating lactate anions from glycolytic PDAC cells. Furthermore, lactate entry into the better-perfused recipient cells has a favourable alkalinizing effect and supplies substrate for oxidative phosphorylation. Cx43 is thus a novel target for influencing metabolite handling in junctionally-coupled tumours.

New insights into the physiological role of carbonic anhydrase IX in tumour pH regulation.

In this review, we discuss the role of the tumour-associated carbonic anhydrase isoform IX (CAIX) in the context of pH regulation. We summarise recent experimental findings on the effect of CAIX on cell growth and survival, and present a diffusion-reaction model to help in the assessment of CAIX function under physiological conditions. CAIX emerges as an important facilitator of acid diffusion and acid transport, helping to overcome large cell-to-capillary distances that are characteristic of solid tumours. The source of substrate for CAIX catalysis is likely to be CO2, generated by adequately oxygenated mitochondria or from the titration of metabolic acids with HCO3⁻ taken up from the extracellular milieu. The relative importance of these pathways will depend on oxygen and metabolite availability, the spatiotemporal patterns of the cell's exposure to hypoxia and on the regulation of metabolism by genes. This is now an important avenue for further investigation. The importance of CAIX in regulating tumour pH highlights the protein as a potential target for cancer therapy.

Carbonic anhydrase IX as an anticancer therapy target: preclinical evaluation of internalizing monoclonal antibody directed to catalytic domain.

Carbonic anhydrase IX (CA IX) is a suitable target for various anticancer strategies. It is a cell surface protein that is present in human tumors, but not in the corresponding normal tissues. Expression of CA IX is induced by hypoxia and correlates with cancer prognosis in many tumor types. Moreover, CA IX is functionally implicated in cancer progression as a pro-survival factor protecting cancer cells against hypoxia and acidosis via its capability to regulate pH and cell adhesion. Cancer-related distribution of CA IX allows for targeting cancer cells by antibodies binding to its extracellular domain, whereas functional involvement of CA IX opens the possibility to hit cancer cells by blocking their adaptation to physiologic stresses via inhibition of CA IX enzyme activity. The latter strategy is recently receiving considerable attention and great efforts are made to produce CA IX-selective inhibitor derivatives with anticancer effects. On the other hand, targeting CA IX-expressing cells by immunotherapy has reached clinical trials and is close to application in treatment of renal cell carcinoma patients. Nevertheless, development and characterization of new CA IX-specific antibodies is still ongoing. Here we describe a mouse monoclonal antibody VII/20 directed to catalytic domain of CA IX. We show that upon binding to CA IX, the VII/20 MAb undergoes efficient receptor-mediated internalization, which is a process regulating abundance and signaling of cell surface proteins and has considerable impact on immunotherapy. We evaluated biological properties of the MAb and demonstrated its capacity to elicit anti-cancer effect in mouse xenograft model of colorectal carcinoma. Thus, the VII/20 MAb might serve as a tool for preclinical studies of immunotherapeutic strategies against non-RCC tumors. These have not been explored so far and include broad spectrum of cancer types, treatment of which might benefit from CA IX-mediated targeting.

The importance of carbonic anhydrase II in red blood cells during exposure of chicken embryos to CO2.

The importance of carbonic anhydrase (CA) during exposure of chicken embryos to CO(2) during the second half of incubation was investigated. The protein abundance and activity of CAII in erythrocytes was significantly higher in CO(2)-exposed embryos compared to normal conditions. Daily injections of acetazolamide (ATZ), an inhibitor of CA, increased blood P(CO2) and decreased blood pH in both control and CO(2)-incubated embryos. ATZ increased blood bicarbonate concentration in embryos exposed to normal atmosphere and in day-12 embryos exposed to high CO(2). The tendency of an increased blood potassium concentration in ATZ-injected embryos under standard atmospheric conditions might indicate that protons were exchanged with intracellular potassium. However, there was no evidence for such an exchange in CO(2)-incubated ATZ-treated embryos. This study shows for the first time that chicken embryos adapt to CO(2) during the second half of incubation by increasing CAII protein expression and function in red blood cells. This response may serve to "buffer" elevated CO(2) levels.

Alternative splicing variant of the hypoxia marker carbonic anhydrase IX expressed independently of hypoxia and tumour phenotype.

CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8-9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia- and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control.