In Vivo HIF-Mediated Reductive Carboxylation Is Regulated by Citrate Levels and Sensitizes VHL-Deficient Cells to Glutamine Deprivation

Abstract

SUMMARY Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [1–13C] glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.

A Tale of Two Glutaminases: Homologous Enzymes with Distinct Roles in Tumorigenesis

“…The Cancer Genome Atlas (TCGA) pan-cancer gene expression data show high expression of GLS in acute myeloid leukemia, adrenocortical cancer, triple-negative breast cancer, colorectal cancer, kidney clear or papillary cell carcinoma, lung adenocarcinoma, melanoma, mesothelioma, pancreatic cancer, sarcoma and thyroid cancer [78,79]. Of these, GLS activity has been shown to be important for growth of acute myeloid leukemia [80][81][82], breast cancer [61,64,83,84], colorectal cancer [85], kidney cancer [86,87], lung cancer [88], melanoma [89,90] and pancreatic cancer cells [91] in studies that utilized smallmolecule inhibitors or gene-silencing approaches in cell lines. Further, glioblastoma cell lines have been found to be sensitive to glutaminase inhibitors in vitro, despite having relatively low GLS expression according to TCGA data [80,92].…”

VHL Deficiency Drives Enhancer Activation of Oncogenes in Clear Cell Renal Cell Carcinoma

“…51). The presence of enhancers around metabolic enzymes and transporters is largely consistent with the metabolic contexture of ccRCC, which involves increased glycolysis and glutaminolysis (19,(52)(53)(54)(55). Indeed, gene ontology (GO) analysis of gained enhancers strongly reflected hallmark metabolic changes associated with ccRCC, including monocarboxylic acid transmembrane transporter activity (binomial FDR q-value = 1.6 × 10 −10 ; Supplementary Fig.…”

Erratum: From Krebs to clinic: glutamine metabolism to cancer therapy

“…Decreased glucose entry into the TCA cycle can be compensated for by glutamine fueled production of the TCA cycle intermediate α-ketoglutarate 151 . However, this α-ketoglutarate is largely channeled through reductive carboxylation in certain cell types to produce citrate, acetyl-coA, and lipids [89][90][91] . By contrast, glutamine is metabolized in human B-cell lymphoma model cells cultured in hypoxia largely via forward TCA cycling, with only a minor amount undergoing reductive carboxylation 151 .…”

“…Glutamine metabolism can serve as an alternative source of carbon to the TCA cycle to fuel fatty acid synthesis, through reductive carboxylation, which is a process by which glutamine-derived α-ketoglutarate is reduced through the consumption of NADPH by isocitrate dehydrogenases (IDHs) in the non-canonical reverse reaction to form citrate 87 . Reductive carboxylation, the importance of which is still somewhat controversial 88 , seems to be a major source of carbon for lipid synthesis in cancer cells that are hypoxic, have constitutive hypoxia-inducible factor-α (HIFα) stabilization or have mitochondrial defects [89][90][91][92] . Although the contribution of reductive carboxylation to lipid formation from glutamine remains unclear due to the possibility of isotope exchange 88 , studies suggest that reductive carboxylation occurs in vivo and can support lipogenesis for tumor growth and progression 89,93,94 and can also control the levels of mitochondrial reactive oxygen species (ROS) 95 .…”