Investigating pseudohypoxia in renal cells deficient in fumarate hydratase.
The discovery that inactivating mutations in various metabolic enzymes leads to tumorigenesis has provided strong support for a primary role of altered metabolism in driving cancer. One of these enzymes, fumarate hydratase (FH), is found mutated in hereditary and leiomyomatosis and renal cell cancer syndrome, whereby affected cells are characterized by high subcellular accumulation of the metabolite fumarate. Fumarate has been shown to inhibit the prolyl hydroxylase-dependent degradation of hypoxia inducible factor-α (HIFα), leading to stabilization and transcriptional activation of HIF-1 under abundant oxygen conditions. This phenomenon, known as 'pseudohypoxia', is considered an important driver of tumorigenesis. However, the role of HIF activation and pseudohypoxia in FH-deficient renal cells is unclear. This aim of this thesis was to delineate the role of hypoxia biology in the phenotypic consequences of loss of fumarate hydratase in renal cells, by taking a holistic, systems-based approach, particularly with regards to transcriptional and metabolic alterations. Using a range of biochemical, molecular, and cell biology techniques, this thesis performed a comprehensive characterisation of the pseudohypoxic signature of Fh1-deficient mouse renal cells. Initial analyses of transcriptomic data sets showed a significant enrichment of generic hallmark hypoxic and HIF-1 target gene expression in Fh1-deficient cells under normoxic conditions. These features are abolished when Fh1 expression is re-constituted. To elucidate the role of Hif in the biology of Fh1-deficient cells, Hif1b (Arnt) was stably knocked out in Fh1-deficient and Fh1-proficient renal cells via CRISPR/Cas9 technology. These cells were then used in RNA-sequencing and metabolomics experiments to systematically delineate the oxygen-dependent/-independent and Hif-dependent/-independent metabolic and transcriptional signatures of Fh1-deficient renal cells. The resulting findings from this holistic, unbiased transcriptional and metabolic profiling have challenged the notion of pseudohypoxia in Fh1-deficient cells. Importantly, a novel link between purine metabolites, hypoxia, and Hif was discovered in Fh1-deficient cells, which may have important ramifications for tumorigenesis, driven by FH loss.
Fumarate Hydratase, Hypoxia, Hypoxia-Inducible Factor, Metabolism, Pseudohypoxia
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