Glioma Cells with the IDH1 Mutation Modulate Metabolic Fractional Flux through Pyruvate Carboxylase

Abstract

BackgroundOver 70% of low-grade gliomas carry a heterozygous R132H mutation in the gene coding for isocitrate dehydrogenase 1 (IDH1). This confers the enzyme with the novel ability to convert α-ketoglutarate to 2-hydroxyglutarate, ultimately leading to tumorigenesis. The major source of 2-hydroxyglutarate production is glutamine, which, in cancer, is also a source for tricarboxylic acid cycle (TCA) anaplerosis. An alternate source of anaplerosis is pyruvate flux via pyruvate carboxylase (PC), which is a common pathway in normal astrocytes. The goal of this study was to determine whether PC serves as a source of TCA anaplerosis in IDH1 mutant cells wherein glutamine is used for 2-hydroxyglutarate production.MethodsImmortalized normal human astrocytes engineered to express heterozygous mutant IDH1 or wild-type IDH1 were investigated. Flux of pyruvate via PC and via pyruvate dehydrogenase (PDH) was determined by using magnetic resonance spectroscopy to probe the labeling of [2-13C]glucose-derived 13C-labeled glutamate and glutamine. Activity assays, RT-PCR and western blotting were used to probe the expression and activity of relevant enzymes. The Cancer Genome Atlas (TCGA) data was analyzed to assess the expression of enzymes in human glioma samples.ResultsCompared to wild-type cells, mutant IDH1 cells significantly increased fractional flux through PC. This was associated with a significant increase in PC activity and expression. Concurrently, PDH activity significantly decreased, likely mediated by significantly increased inhibitory PDH phosphorylation by PDH kinase 3. Consistent with the observation in cells, analysis of TCGA data indicated a significant increase in PC expression in mutant IDH-expressing human glioma samples compared to wild-type IDH.ConclusionsOur findings suggest that changes in PC and PDH may be an important part of cellular adaptation to the IDH1 mutation and may serve as potential therapeutic targets.

Exploring cancer metabolism using stable isotope-resolved metabolomics (SIRM)

“…Tracer studies show that SDH-null cells produce citrate predominantly through reductive carboxylation of Gln and accumulate Gln-derived succinate, a product inhibitor of the ␣-KG-dependent dioxygenases, while relying on PC for anaplerosis and synthesis of aspartate to support nucleotide biosynthesis required for proliferation (59,86,87). Likewise, IDH1 mutant cells increased conversion of [ 13 C 1 -2]Glc to PC-derived [ 13 C 1 -3]Glu, which implicates the importance of PC anaplerosis for proliferation of these cells (88). More intriguingly, PC expression and in vivo activity were enhanced in human NSCLC tumors relative to adjacent non-cancerous lung tissues, and silencing PC expression in lung cancer lines slowed tumor growth in mouse xenografts (8,53 (89).…”

The BRAF Inhibitor Vemurafenib Activates Mitochondrial Metabolism and Inhibits Hyperpolarized Pyruvate–Lactate Exchange in BRAF-Mutant Human Melanoma Cells

“…Cells (2 Â 10 6 ) were lyzed in Ripa buffer (Cell Signaling Technology) containing PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche) and cOmplete ULTRA Tablets (Roche), and processed as previously described (24). A spectrophotometric reading in kinetic mode at 412 nm was taken for 10 minutes at 30 C (Ultrospec 2100pro, GE Healthcare Life Sciences) and data normalized for protein content.…”

Metabolic consequences of oncogenic IDH mutations

“…Furthermore, in vivo tracing studies in an orthotopic model of human glioblastoma using 13C-labeled glucose have indicated that pyruvate carboxylase and PDH are highly active in GBM (Marin-Valencia et al, 2012). One recent study demonstrated that IDH mutant overexpression in astrocytes results in an increased fractional flux through pyruvate carboxylase and an increase in PC expression, suggesting this pathway is critical for IDH mutant cells to maintain TCA activity (Izquierdo-Garcia et al, 2014). Consistent with this observation, we observed increased pyruvate cycling through malic enzymes and PC in HCT116 cells harboring heterozygous IDH1 mutations (Grassian et al, 2014).…”