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

Abstract

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.

Normoxic cells remotely regulate the acid‐base balance of cells at the hypoxic core of connexin‐coupled tumor growths

“…In pancreatic cancer, connexin43 immunoreactivity has been shown to increase with disease progression (25). Our recent study (23) demonstrated the presence of connexin43 in PDAC cell lines, such as Colo357, hypoxic BxPC3 in vitro and in invading or metastasizing MiaPaCa2 cells in vivo and also established a correlation between connexin43 levels and solute exchange between cells. Evidence of the loss of histone H3 methylation during subclonal evolution may provide a molecular mechanism for the upregulation of genes coding for connexins in PDAC metastases relative to the primary tumor (26).…”

“…A plausible route for supplying cytoplasm with solutes, including HCO 3 2 ions, may involve passive permeation through gap junctional channels found in many tissues, including some tumors (18)(19)(20)(21)(22). We have demonstrated that connexin43 channels conduct a flow of lactate anions away from the hypoxic core of PDAC tissue growths (23). In contrast, H + ions, coproduced by glycolytic metabolism, are heavily buffered and therefore unable to diffuse freely through connexin43 channels (23,24), which leaves hypoxic cells vulnerable to uncontrolled acidification.…”

“…Net flux of small solutes through connexin channels can occur only down cell-to-cell concentration gradients, which are notably absent in uniformly perfused monolayers but are highly significant in 3-dimensional tissue growths, where pO 2 nonuniformity establishes metabolite heterogeneity. Thus, to study connexin-dependent transport, spheroids were grown from Colo357 cells, a PDAC line with high levels of connexin43 (23). The spheroids were first seeded using the hanging-drop method and then transferred to culture dishes for another 1-3 d to gradually increase the degree of cell-cell packing (i.e., tortuosity, which favors steeper pO 2 gradients).…”

“…If the cell's entire output of lactate were exported by MCT, scalar a would equal 0 and secondary active transport would be required to balance J leak . In contrast, if lactate were catabolized or exported by an H + -independent mechanism [e.g., via connexin43 channels (23)], a greater secondary active transport flux would be necessary. J met was taken to be 15 mM/min, the midpoint of the range of lactate production rates measured in PDAC cells (6,7).…”

“…We have demonstrated that connexin43 channels conduct a flow of lactate anions away from the hypoxic core of PDAC tissue growths (23). In contrast, H + ions, coproduced by glycolytic metabolism, are heavily buffered and therefore unable to diffuse freely through connexin43 channels (23,24), which leaves hypoxic cells vulnerable to uncontrolled acidification. In the current study, we tested the extent to which HCO 3 2 permeation through connexin-assembled gap junctional channels are able to supply hypoxic cancer cells with sufficient chemical base for titrating metabolic H + ions.…”