Abstract
BackgroundThe loss-of-function mutation of fumarate hydratase (FH) is a driver of hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Fumarate accumulation results in activation of stress-related mechanisms leading to upregulation of cell survival-related genes. To better understand how cells compensate for the loss of FH in HLRCC, we determined the amino acid nutrient requirements of the FH-deficient UOK262 cell line (UOK262) and its FH-repleted control (UOK262WT).MethodsWe determined growth rates and survival of cell lines in response to amino acid depletion and supplementation. RNAseq was used to determine the transcription changes contingent on Asn and Gln supplementation, which was further followed with stable isotope resolved metabolomics (SIRM) using both [U- 13C,15N] Gln and Asn.ResultsWe found that Asn increased the growth rate of both cell lines in vitro. Gln, but not Asn, increased oxygen consumption rates and glycolytic reserve of both cell lines. Although Asn was taken up by the cells, there was little evidence of Asn-derived label in cellular metabolites, indicating that Asn was not catabolized. However, Asn strongly stimulated Gln labeling of uracil and precursors, uridine phosphates and hexosamine metabolites in the UOK262 cells and to a much lesser extent in the UOK262WT cells, indicating an activation of the hexosamine biosynthetic pathway (HBP) by Asn. Asn in combination with Gln, but not Asn or Gln alone, stimulated expression of genes associated with the endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in UOK262 to a greater extent than in FH-restored cells. The changes in expression of these genes were confirmed by RT-PCR, and the stimulation of the UPR was confirmed orthogonally by demonstration of an increase in spliced XBP1 (sXBP1) in UOK262 cells under these conditions. Asn exposure also increased both the RNA and protein expression of the HBP regulator GFPT2, which is a transcriptional target of sXBP1.ConclusionsAsn in the presence of Gln induces an ER stress response in FH-deficient UOK262 cells and stimulates increased synthesis of UDP-acetyl glycans indicative of HBP activity. These data demonstrate a novel effect of asparagine on cellular metabolism in FH-deficient cells that could be exploited therapeutically.
Highlights
The loss-of-function mutation of fumarate hydratase (FH) is a driver of hereditary leiomyomatosis and renal cell carcinoma (HLRCC)
Growth curves over a longer duration revealed that both Gln and Asn significantly contribute to cell growth for both cell lines, but only after the first 96 h of incubation (Fig. 1c and d) indicating that a period of adaptation was required for Asn availability to show an effect on cell growth
These results suggest the important role Gln and Asn play in sustaining cell growth of both FH replete and FH depleted cell lines
Summary
The loss-of-function mutation of fumarate hydratase (FH) is a driver of hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive form of RCC characterized by germline inactivating mutation of fumarate hydratase (FH), followed by somatic loss of the remaining wild-type allele. FH is a key metabolic enzyme of the mitochondrial tricarboxylic acid (TCA) or Krebs cycle, and as a result of FH loss-of-function, the metabolite fumarate accumulates This lesion has two major oncogenic effects: it directly stimulates a metabolic transition to Warburg metabolism, through the disruption of the Krebs cycle, leading the cells to shift to alternative pathways in order to sustain a balance of energy required for survival and proliferation; and the excess fumarate results in accumulation of members of the hypoxia inducible factor family of transcription factors [1, 2].
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