Abstract
Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.
Highlights
Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer
The biallelic inactivation of FH leads to hereditary leiomyomatosis and renal cell cancer (HLRCC), a hereditary cancer syndrome characterized by the presence of benign tumours of the skin and uterus, and a highly malignant form of renal cell cancer[1]
We demonstrated that the oxidative stress induced by GSH succination is necessary and sufficient to elicit cellular senescence in non-transformed cells and that the ablation of senescence can initiate a tumorigenic programme in the kidneys of Fh1-deficient mice
Summary
We generated in silico genome-scale metabolic models of the FH-deficient and wt cells by incorporating the gene expression and metabolite exchange rates measured in these cell lines within a generic metabolic model[13] (see Methods) Of note, these implemented models explore essential reactions for biomass production, as previously done[8], and allowed us to predict changes in metabolic fluxes on loss of FH. Among the most significant metabolites accumulated in FH-deficient cells a putative GSH adduct was detected (annotated by Metlin database as the formate adduct of pyruvilGSH; Fig. 2a) Since this metabolite is specific to FHdeficient cells and is poorly characterized, we decided to elucidate its structure by LC-MS/MS and NMR analyses. MRNA levels (normalized to Fh1fl/fl) Nqo[1] Ptgs[1] Hmox[1] Pycr[1] Ddit[3] Gclc Lonp[1] Cat Pla2g4a Map2k1 Fancc Txn[2] Slc23a2 Ercc[3] Adam[9] Jak[2]
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