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.
Highlights
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant hereditary cancer syndrome characterized by a predisposition to develop cutaneous and uterine leiomyomas and a very aggressive form of papillary kidney cancer [1,2,3,4,5,6,7]
To investigate and quantify aerobic glycolysis and mitochondrial respiration in fumarate hydratase (FH) null kidney cancer cells, measurements were made of the oxygen consumption (OCR) and extracellular acidification rates (ECAR), along with tracing of the carbon flow from glucose to lactate and from glutamine to fumarate
Fumarate hydratase enzyme activity assays demonstrated the absence of activity in UOK262 and UOK262EV, but recovery of 63.2% of the fumarate hydratase activity in UOK262WT when compared to the human renal cortical epithelial cells (HRCE) control cells (Figure S1 in File S1)
Summary
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant hereditary cancer syndrome characterized by a predisposition to develop cutaneous and uterine leiomyomas and a very aggressive form of papillary kidney cancer [1,2,3,4,5,6,7]. The primary genetic alteration associated with HLRCC is a germline mutation of the FH gene that encodes fumarate hydratase (FH), which is both a tumor suppressor gene and an enzyme of the Krebs cycle [9,10,11]. Mutations of several genes that encode enzymes of the Krebs cycle have recently been implicated in multiple aspects of cancer genetics and progression, and have highlighted the potential importance of altered metabolic states in cancer cells [15,16,17]
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