Hypoxia onducible factor (HIF) promotes reductive carboxylation in vivo and renders VHL-deficient cells sensitive to glutamine deprivation

Abstract

Hypoxia is a hallmark of tumor microenvironment. We showed before taht exposure of cells to hypoxia reprograms cell metabolism and directs and IDH1-mediated reductive carboxylation (RC) of glutamine-derived alpha-ketoglutarate (a-KG) for lipogenesis. In addition, Von Hippel-Lindau (VHL)-deficient renal cell carcinoma (RCC) cells use glutamine to generate citrate and lipids through RC of a-KG. 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 RCC cells. Expression of a VHL-independent HIF mutant in VHL-restored RCC cells is sufficient to promote RC. HIF expression drastically reduces intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate, or knocking-down PDK-1 and ACLY enzymes restored intracellularcitrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action, to maintain lipogenesis. Expression of HIF renders hypoxic and VHL-deficient cells addicted to glutamine in vitro. Systemic administration of glutaminase inhibitor (BPTES) suppressed the growth of VHL-deficient human RCC cell lines as xenografts in nude mice. Lastly, we investigated whether RC occurs in vivo. We metabolically labeled mice bearing VHL-deficient tumors by infusing them with 13C-1Glutamine for up to 6 hours and we detected the early formation of labeled citrate in the tumors; thus whe showed for the first time RC in vivo. Our data provide mechanistic insights into the signaling events that mediate hypoxia-induced RC, strongly suggest that RC is an in vivo phenomenon in growing tumors and highlight potential novel therapeutic approaches for treatment of hypoxic and VHL-deficient tumors based on this metabolic signature.