BASIGIN/CD147/EMMPRIN is a multifunctional transmembrane glycoprotein strongly expressed in tumours. BASIGIN controls tumour metabolism, particularly glycolysis by facilitating lactic acid export through the two monocarboxylate transporters MCT1 and hypoxia-inducible MCT4. However, before being recognized as a co-carrier of MCTs, BASIGIN was described as an inducer of extracellular matrix metalloproteases (MMPs). Early on, a model emerged in which, tumour cells use the extracellular domain of BASIGIN to recognize and stimulate neighbouring fibroblasts to produce MMPs. However, this model has remained hypothetical since a direct link between BASIGIN and MMPs production has not yet been clearly established. To validate the BASIGIN/MMP hypothesis, we developed BASIGIN knockouts in three human tumour cell lines derived from glioma, colon, and lung adenocarcinoma. By using co-culture experiments of either human or mouse fibroblasts and tumour cell lines we showed, contrary to what has been abundantly published, that the disruption of BASIGIN in tumour cells and in MEFs has no action on the production of MMPs. Our findings do not support the notion that the pro-tumoural action of BASIGIN is mediated via induction of MMPs. Therefore, we propose that to date, the strongest pro-tumoural action of BASIGIN is mediated through the control of fermentative glycolysis.

The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types.

The Na(+)/H(+)-exchanger NHE1 and the monocarboxylate transporters MCT1 and MCT4 are crucial for intracellular pH regulation, particularly under active metabolism. NHE1, a reversible antiporter, uses the energy provided by the Na(+) gradient to expel H(+) ions generated in the cytosol. The reversible H(+)/lactate(-) symporters MCT1 and 4 cotransport lactate and proton, leading to the net extrusion of lactic acid in glycolytic tumors. In the first two sections of this article we review important features and remaining questions on the structure, biochemical function and cellular roles of these transporters. We then use a fully-coupled mathematical model to simulate their relative contribution to pH regulation in response to lactate production, as it occurs in highly hypoxic and glycolytic tumor cells. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via 'Zinc-Finger Nucleases'. The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

Glutathione (GSH) and glutathione disulfide (GSSG) are commonly used to assess the oxidative status of a biological system. Various protocols are available for the analysis of GSH and GSSG in biomedical specimens. In this study, we present an optimized protocol for the in situ derivatization of GSH with N-ethylmaleimide (NEM) to prevent GSH autooxidation, and thus to preserve the GSH/GSSG ratio during sample preparation. The protocol comprises the incubation of cells in NEM containing phosphate buffered saline (PBS), followed by metabolite extraction with 80% methanol. Further, to preserve the use of QTOF-MS, which may lack the linear dynamic range required for the simultaneous quantification of GSH and GSSG in non-targeted metabolomics, we combined liquid chromatographic separation with the online monitoring of UV absorbance of GS-NEM at 210 nm and the detection of GSSG and its corresponding stable isotope-labeled internal standard by QTOF-MS operated with a 10 Da Q1 window. The limit of detection (LOD) for GS-NEM was 7.81 µM and the linear range extended from 15.63 µM to 1000 µM with a squared correlation coefficient R2 of 0.9997. The LOD for GSSG was 0.001 µM, and the lower limit of quantification (LLOQ) was 0.01 µM, with the linear (R2 = 0.9994) range extending up to 10 µM. The method showed high repeatability with intra-run and inter-run coefficients of variation of 3.48% and 2.51% for GS-NEM, and 3.11% and 3.66% for GSSG, respectively. Mean recoveries of three different spike-in levels (low, medium, high) of GSSG and GS-NEM were above 92%. Finally, the method was applied to the determination of changes in the GSH/GSSG ratio either in response to oxidative stress in cells lacking one or both monocarboxylate transporters MCT1 and MCT4, or in adaptation to the NADPH (nicotinamide adenine dinucleotide phosphate) consuming production of D-2-hydroxyglutarate in cells carrying mutations in the isocitrate dehydrogenase genes IDH1 and IDH2.

Rapidly growing glycolytic tumors require energy and intracellular pH (pHi) homeostasis through the activity of two major monocarboxylate transporters, MCT1 and the hypoxia-inducible MCT4, in intimate association with the glycoprotein CD147/BASIGIN (BSG). To further explore and validate the blockade of lactic acid export as an anticancer strategy, we disrupted, via zinc finger nucleases, MCT4 and BASIGIN genes in colon adenocarcinoma (LS174T) and glioblastoma (U87) human cell lines. First, we showed that homozygous loss of MCT4 dramatically sensitized cells to the MCT1 inhibitor AZD3965. Second, we demonstrated that knockout of BSG leads to a decrease in lactate transport activity of MCT1 and MCT4 by 10- and 6-fold, respectively. Consequently, cells accumulated an intracellular pool of lactic and pyruvic acids, magnified by the MCT1 inhibitor decreasing further pHi and glycolysis. As a result, we found that these glycolytic/MCT-deficient cells resumed growth by redirecting their metabolism toward OXPHOS. Third, we showed that in contrast with parental cells, BSG-null cells became highly sensitive to phenformin, an inhibitor of mitochondrial complex I. Phenformin addition to these MCT-disrupted cells in normoxic and hypoxic conditions induced a rapid drop in cellular ATP-inducing cell death by "metabolic catastrophe." Finally, xenograft analysis confirmed the deleterious tumor growth effect of MCT1/MCT4 ablation, an action enhanced by phenformin treatment. Collectively, these findings highlight that inhibition of the MCT/BSG complexes alone or in combination with phenformin provides an acute anticancer strategy to target highly glycolytic tumors. This genetic approach validates the anticancer potential of the MCT1 and MCT4 inhibitors in current development.

UCART19 is an "off-the-shelf" genome-edited anti-CD19 chimeric antigen receptor (CAR)-T cell product, manufactured from unrelated healthy donor cells.