As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues, cancer cells must adapt their metabolism to the tumour microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here we developed a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low-glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNA interference (RNAi) screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the OXPHOS upregulation that is normally caused by glucose limitation as a result of either mitochondrial DNA (mtDNA) mutations in complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, antidiabetic drugs that inhibit OXPHOS, when cancer cells are grown in low glucose or as tumour xenografts. Notably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of complex I function. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors.

Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides

Cancer cells display a high rate of glycolysis in the presence of oxygen to promote proliferation. Gluconeogenesis, the reverse pathway of glycolysis, can antagonize aerobic glycolysis in cancer via three key enzymes – phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase). Recent studies have revealed that, in addition to metabolic regulation, these enzymes also play a role in signaling, proliferation, and the cancer stem cell (CSC) tumor phenotype. Multifaceted regulation of PEPCK, FBPase, and G6Pase through transcription, epigenetics, post-translational modification, and enzymatic activity is observed in different cancers. We review here the function and regulation of key gluconeogenic enzymes and new therapeutic opportunities.

Gluconeogenesis in Cancer: Function and Regulation of PEPCK, FBPase, and G6Pase

Gluconeogenesis in cancer cells - Repurposing of a starvation-induced metabolic pathway?

Recommendations for good practice in MS-based lipidomics

Distribution and prognostic significance of gluconeogenesis and glycolysis in lung cancer.

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation.

https://www.pnas.org/content/pnas/115/24/6225.full.pdf

The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells.

The metabolic microenvironment, comprising all soluble and insoluble nutrients and co-factors in the extracellular milieu, has a major impact on cancer cell proliferation and survival A large body of evidence from recent studies suggests that tumor cells show a high degree of metabolic flexibility and adapt to variations in nutrient availability Insufficient vascular networks and an imbalance of supply and demand shape the metabolic tumor microenvironment, which typically contains a lower concentration of glucose compared to normal tissues The present review sheds light on the recent literature on adaptive responses in cancer cells to nutrient deprivation It focuses on the utilization of alternative nutrients in anabolic metabolic pathways in cancer cells, including soluble metabolites and macromolecules and outlines the role of central metabolic enzymes conferring metabolic flexibility, like gluconeogenesis enzymes Moreover, a conceptual framework for potential therapies targeting metabolically flexible cancer cells is presented

https://www.mdpi.com/1422-0067/22/3/1476/pdf

Flexibility and Adaptation of Cancer Cells in a Heterogenous Metabolic Microenvironment.

Cancer cells frequently lack nutrients like glucose, due to insufficient vascular networks. A decrease of extracellular glucose is accompanied by enhanced mitochondrial respiration in cancer cells, which promotes the formation of potentially harmful reactive oxygen species (ROS). Here we show that a gluconeogenesis enzyme, mitochondrial phosphoenolpyruvate carboxykinase, PCK2, acts as a regulator of mitochondrial respiration and maintains the redox balance in nutrient-deprived lung cancer cells. PCK2 silencing increased the abundance and interconversion of tricarboxylic acid (TCA) cycle intermediates, augmented mitochondrial respiration and enhanced glutathione oxidation under glucose and serum starvation, in a PCK2 re-expression reversible manner. Moreover, augmenting the TCA cycle by PCK2 inhibition severely reduced colony formation. As a conclusion, PCK2 contributes to maintaining a reduced glutathione pool upon starvation besides mediating the biosynthesis of gluconeogenic/glycolytic intermediates. The study sheds light on adaptive responses in cancer cells to nutrient deprivation and identifies gluconeogenesis as starvation-induced pathway that limits respiration-induced oxidative stress.

/pdf/pck2-opposes-mitochondrial-respiration-and-maintains-the-57yiuzv564.pdf

Gabriele Grasmann

Papers

Gluconeogenesis in cancer cells - Repurposing of a starvation-induced metabolic pathway?

The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells.

Flexibility and Adaptation of Cancer Cells in a Heterogenous Metabolic Microenvironment.

PCK2 opposes mitochondrial respiration and maintains the redox balance in starved lung cancer cells