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Nucleotide pools need to be constantly replenished in cancer cells to support cell proliferation. The synthesis of nucleotides requires glutamine and 5-phosphoribosyl-1-pyrophosphate produced from ribose-5-phosphate via the oxidative branch of the pentose phosphate pathway (ox-PPP). Both PPP and glutamine also play a key role in maintaining the redox status of cancer cells. Enhanced glutamine metabolism and increased glucose 6-phosphate dehydrogenase (G6PD) expression have been related to a malignant phenotype in tumors. However, the association between G6PD overexpression and glutamine consumption in cancer cell proliferation is still incompletely understood. In this study, we demonstrated that both inhibition of G6PD and glutamine deprivation decrease the proliferation of colon cancer cells and induce cell cycle arrest and apoptosis. Moreover, we unveiled that glutamine deprivation induce an increase of G6PD expression that is mediated through the activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2). This crosstalk between G6PD and glutamine points out the potential of combined therapies targeting oxidative PPP enzymes and glutamine catabolism to combat colon cancer.

The pentose phosphate pathway (PPP) plays an essential role in the metabolism of breast cancer cells for the management of oxidative stress and the synthesis of nucleotides. 6-phosphogluconate dehydrogenase (6PGD) is one of the key enzymes of the oxidative branch of PPP and is involved in nucleotide biosynthesis and redox maintenance status. Here, we aimed to analyze the functional importance of 6PGD in a breast cancer cell model. Inhibition of 6PGD in MCF7 reduced cell proliferation and showed a significant decrease in glucose consumption and an increase in glutamine consumption, resulting in an important alteration in the metabolism of these cells. No difference in reactive oxygen species (ROS) production levels was observed after 6PGD inhibition, indicating that 6PGD, in contrast to glucose 6-phosphate dehydrogenase, is not involved in redox balance. We found that 6PGD inhibition also altered the stem cell characteristics and mammosphere formation capabilities of MCF7 cells, opening new avenues to prevent cancer recurrance after surgery or chemotherapy. Moreover, inhibition of 6PGD via chemical inhibitor S3 resulted in an induction of senescence, which, together with the cell cycle arrest and apoptosis induction, might be orchestrated by p53 activation. Therefore, we postulate 6PGD as a novel therapeutic target to treat breast cancer.

Metabolic regulation of immune cells has arisen as a critical set of processes required for appropriate response to immunological signals. While our knowledge in this area has rapidly expanded in leukocytes, much less is known about the metabolic regulation of brain-resident microglia. In particular, the role of alternative nutrients to glucose remains poorly understood. Here, we use stable-isotope (13C) tracing strategies and metabolomics to characterize the oxidative metabolism of β-hydroxybutyrate (BHB) in human (HMC3) and murine (BV2) microglia cells and the interplay with glucose in resting and LPS-activated BV2 cells. We found that BHB is imported and oxidised in the TCA cycle in both cell lines with a subsequent increase in the cytosolic NADH:NAD+ ratio. In BV2 cells, stimulation with LPS upregulated the glycolytic flux, increased the cytosolic NADH:NAD+ ratio and promoted the accumulation of the glycolytic intermediate dihydroxyacetone phosphate (DHAP). The addition of BHB enhanced LPS-induced accumulation of DHAP and promoted glucose-derived lactate export. BHB also synergistically increased LPS-induced accumulation of succinate and other key immunometabolites, such as α-ketoglutarate and fumarate generated by the TCA cycle. Finally, BHB upregulated the expression of a key pro-inflammatory (M1 polarisation) marker gene, NOS2, in BV2 cells activated with LPS. In conclusion, we identify BHB as a potentially immunomodulatory metabolic substrate for microglia that promotes metabolic reprogramming during pro-inflammatory response.

The monocarboxylate transporter 1 (MCT1) is a key element in tumor cell metabolism and inhibition of MCT1 with AZD3965 is undergoing clinical trials. We aimed to investigate nutrient fluxes associated with MCT1 inhibition by AZD3965 to identify possible biomarkers of drug action. We synthesized an 18F-labeled lactate analogue, [18F]-S-fluorolactate ([18F]-S-FL), that was used alongside [18F]fluorodeoxyglucose ([18F]FDG), and 13C-labeled glucose and lactate, to investigate the modulation of metabolism with AZD3965 in diffuse large B-cell lymphoma models in NOD/SCID mice. Comparative analysis of glucose and lactate-based probes showed a preference for glycolytic metabolism in vitro, whereas in vivo, both glucose and lactate were used as metabolic fuel. While intratumoral L-[1-13C]lactate and [18F]-S-FL were unchanged or lower at early (5 or 30 min) timepoints, these variables were higher compared to vehicle controls at 4 h following treatment with AZD3965, which indicates that inhibition of MCT1-mediated lactate import is reversed over time. Nonetheless, AZD3965 treatment impaired DLBCL tumor growth in mice. This was hypothesized to be a consequence of metabolic strain, as AZD3965 treatment showed a reduction in glycolytic intermediates and inhibition of the TCA cycle likely due to downregulated PDH activity. Glucose ([18F]FDG and D-[13C6]glucose) and lactate-based probes ([18F]-S-FL and L-[1-13C]lactate) can be successfully used as biomarkers for AZD3965 treatment.