Dovmark T H, Saccomano M, Hulikova A, Alves F, Swietach P
Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK.
Max-Planck-Institute of Experimental Medicine, Goettingen, Germany.
Oncogene. 2017 Aug 10;36(32):4538-4550. doi: 10.1038/onc.2017.71. Epub 2017 Apr 3.
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.
糖酵解癌细胞会产生大量乳酸，在缺乏氧化磷酸化的情况下，必须将这些乳酸排出以维持新陈代谢。目前已知唯一能以足够速率进行此操作的排出机制是单羧酸转运体（MCT）上的H⁺偶联乳酸外流。然而，向外的MCT活性在热力学上会受到细胞外酸性环境的抑制，而细胞外酸性是实体瘤的一个特征。这种抑制会对新陈代谢和生长产生不利的反馈，这就增加了在灌注不足的肿瘤中其他排出机制变得重要的可能性。我们研究了连接蛋白组装的间隙连接作为胰腺导管腺癌（PDAC）细胞排出乳酸的另一条途径。在体外，Colo357细胞之间的扩散偶联（钙黄绿素传递）很强，BxPC3细胞之间较弱但缺氧诱导性较强，而MiaPaCa2细胞之间非常低。偶联与连接蛋白43（Cx43）的水平相关，Cx43是一种先前与晚期疾病相关的蛋白质。在Colo357球体中，以细胞质pH作为读数对诱发的乳酸动态进行成像，结果表明乳酸阴离子透过间隙连接的速度比高度缓冲的H⁺离子快。在稳态下，乳酸（一种化学碱）从球体核心通过间隙连接的传递对边缘有碱化作用，在其中产生有利于生长的环境。代谢物分析表明，敲低Cx43会增加Colo357球体（直径约150μm）中细胞质乳酸的保留。在单层培养中Cx43呈阴性的MiaPaCa2细胞，在原位异种移植小鼠模型的侵袭区域显示出Cx43免疫反应性明显增加。这些组织区域与慢性细胞外酸中毒有关（如质膜附近/处的标志物LAMP2所示），这可以解释间隙连接传递在体内相对于MCT的优势。我们提出，Cx43通道是从糖酵解的PDAC细胞中消散乳酸阴离子的重要途径。此外，乳酸进入灌注较好的受体细胞具有有利的碱化作用，并为氧化磷酸化提供底物。因此，Cx43是影响连接偶联肿瘤中代谢物处理的一个新靶点。
