Metabolic Reprogramming for Producing Energy and Reducing Power in Fumarate Hydratase Null Cells from Hereditary Leiomyomatosis Renal Cell Carcinoma

Abstract

Fumarate hydratase (FH)-deficient kidney cancer undergoes metabolic remodeling, with changes in mitochondrial respiration, glucose, and glutamine metabolism. These changes represent multiple biochemical adaptations in glucose and fatty acid metabolism that supports malignant proliferation. However, the metabolic linkages between altered mitochondrial function, nucleotide biosynthesis and NADPH production required for proliferation and survival have not been elucidated. To characterize the alterations in glycolysis, the Krebs cycle and the pentose phosphate pathways (PPP) that either generate NADPH (oxidative) or do not (non-oxidative), we utilized [U-13C]-glucose, [U-13C,15N]-glutamine, and [1,2- 13C2]-glucose tracers with mass spectrometry and NMR detection to track these pathways, and measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of growing cell lines. This metabolic reprogramming in the FH null cells was compared to cells in which FH has been restored. The FH null cells showed a substantial metabolic reorganization of their intracellular metabolic fluxes to fulfill their high ATP demand, as observed by a high rate of glucose uptake, increased glucose turnover via glycolysis, high production of glucose-derived lactate, and low entry of glucose carbon into the Krebs cycle. Despite the truncation of the Krebs cycle associated with inactivation of fumarate hydratase, there was a small but persistent level of mitochondrial respiration, which was coupled to ATP production from oxidation of glutamine-derived α–ketoglutarate through to fumarate. [1,2- 13C2]-glucose tracer experiments demonstrated that the oxidative branch of PPP initiated by glucose-6-phosphate dehydrogenase activity is preferentially utilized for ribose production (56-66%) that produces increased amounts of ribose necessary for growth and NADPH. Increased NADPH is required to drive reductive carboxylation of α-ketoglutarate and fatty acid synthesis for rapid proliferation and is essential for defense against increased oxidative stress. This increased NADPH producing PPP activity was shown to be a strong consistent feature in both fumarate hydratase deficient tumors and cell line models.

Regulation of mammalian nucleotide metabolism and biosynthesis

“…The ATP yields for oxidation of different substrates assuming perfectly coupled mitochondria are given in Supplementary Table S2. Different cell types use different strategies that are also dependent on the tissue environment including the nutrient supply, which is a point of focus for understanding metabolic reprogramming in cancers (25,44,47,101–107). Although the oxygen concentration in tissues (e.g.…”

“…1 pmol ATP, it would need about 3 h to produce this amount of ATP from oxidative phosphorylation to support nucleotide synthesis alone. However, hypoxic cells also ferment glucose to lactate, producing 2 ATP/mol glucose and in cancer cells glycolysis is typically accelerated many fold (42,121–122), which may produce ATP at a rate comparable to the more efficient mitochondrial oxidation of fuels including fatty acids (104,123), Gln (25,44–45,81,106,119,124–127) and ketone bodies (46) (Supplementary Table S2). It is notable that in the interstitial fluid of solid tumors, the glucose levels are very low with depletion of lipids compared with the blood supply in the tumor (128).…”

“…Our group has developed the stable isotope resolved metabolomics or SIRM approach (173–175) for this purpose, which has been applied to mapping nucleotide biosynthesis in cultured cells or tissues in situ (19,92,101,168,173,176–177). Figure 5 tracks the expected incorporation of labeled atoms from 13 C 6 -glucose (, for 13 C) and 13 C 5 , 15 N 2 -glutamine ( for 13 C and for 15 N) into nucleotide products via purine ( PUR ) and pyrimidine ( PYR ) synthesis as well as feeder pathways including glycolysis, the Krebs cycle in the absence or presence of anaplerotic pyruvate carboxylation or glutaminolysis, PPP and one-carbon pathway.…”

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase

“…Mutations in fumarase have been implicated in a variety of human diseases, including progressive encephalopathy, fumaric aciduria, hereditary leiomyomatosis and renal cell cancer (Kerrigan et al, 2000;Yang et al, 2013;Whelan et al, 1983). Despite its remarkable importance, the only structural study presently available regarding the clinical relevance of human fumarase (HsFH) was performed on the Escherichia coli FumC homologue, which shares 60% identity with the human enzyme (Esté vez et al, 2002).…”

NADPH Oxidase 4 at the Nexus of Diabetes, Reactive Oxygen Species, and Renal Metabolism

“…A dramatic increase in glycolytic flux, despite aerobic conditions, is a hallmark of proliferating cancer cells, including FH-deficient cells, 18 and it is possible that the mitogenic effects of NOX ROS production could participate in this shift (e.g., through PI3-Akt signaling). Alternatively, because increased NOX ROS production triggers an antioxidant response through NRF2 activation (perhaps in part because of fumarate accumulation), secondary effects of NOX4 on metabolism must also be considered.…”