Modeling tumor predisposingFHmutations in yeast: Effects on fumarase activity, growth phenotype and gene expression profile

Abstract

Heterozygous mutations in the fumarase (FH) gene cause the tumor predisposition syndrome hereditary leiomyomatosis and renal cell cancer (MIM 605839). While most families segregate a benign phenotype of multiple leiomyomas, others display a phenotype with early-onset renal cancer and leiomyosarcoma. Modifier genes may play a role in this, but an alternative explanation is simple genotype-phenotype association. FH mutations predisposing to cancer appear to be truncating or in fully conserved amino acids, suggesting that mutations severely affecting FH activity might predispose to malignancy. In the present study, we analyzed 2 conserved fumarase mutations in yeast. H153R has been described in 3 cancer predisposition families; whereas all 3 reported K187R families have displayed the benign phenotype. Examining H153R and K187R should clarify whether cancerrelated FH mutations differ from their benign phenotype-associated counterparts. Yeast strains containing the 2 mutations, and knockout and wild type (WT) references, were created and the growth phenotypes studied on selected carbon sources to assess mitochondrial function. Additionally, Fum1 protein production and activity were measured, and the strains were subjected to transcriptional profiling. On nonfermentable lactate medium, the fumarase knockout strains did not grow, whereas the mutants showed no differences, as compared to WT yeast. Although both mutant strains produced fumarase, a considerable decrease in enzyme activity was seen in mutants with respect to WT. Transcription of the majority of Krebs cycle enzymes was downregulated in response to mutations in fumarase. In conclusion, both mutants displayed some, albeit greatly reduced, fumarase activity. This activity was sufficient to support normal growth on nonfermentable carbon source, unlike the deletion phenotype, demonstrating the significance of the residual activity. The findings support the hypothesis that modifier gene(s), rather than phenotype-genotype effects, display a major role in determining tumor phenotypes in families segregating FH mutations. ' 2005 Wiley-Liss, Inc.

Fumarate Hydratase Deficiency in Renal Cancer Induces Glycolytic Addiction and Hypoxia-Inducible Transcription Factor 1α Stabilization by Glucose-Dependent Generation of Reactive Oxygen Species

“…The non-fermentable sugar galactose cannot substitute for glucose in supporting UOK262 cell growth (Figure 1A), suggesting that these cells lack mitochondrial respiration and are addicted to glycolysis. This phenotype is identical to that of yeast lacking fumarase activity(22). In support of this hypothesis, UOK262 cells produce significantly more extracellular lactate as compared with HK-2 and HEK293 renal epithelial cells (Figure 1B).…”

“…Fum1 is the sole fumarase gene in yeast and it is not essential for viability, but its loss confers an obligate fermentor phenotype(22). The yeast mutants Δfum1 (fumarase knockout mutant), H153R, and K187R (both of which express different human FH mutant proteins as their sole source of fumarase) were previously reported to have depressed fumarase activity in comparison to wild-type fumarase expressing yeast strains(22). The H153R and K187R mutants represent germline mutations found in HLRCC kindreds.…”

“…However, like UOK262, yeast lacking fumarase activity cannot use mitochondrial respiration to grow on non-fermentable carbon sources(22), suggesting that addiction to glycolysis in the absence of FH activity is a highly conserved property of eukaryotic cells. Indeed, the enforced glycolysis and resultant rapid generation and secretion of lactic acid characteristic of FH-deficient cells may underlie the high metastatic potential of HLRCC, as lactic acid is reported to stimulate endothelial cell migration, which is a crucial component of a tumor's metastatic phenotype(1, 24).…”

Flux Balance Analysis of Barley Seeds: A Computational Approach to Study Systemic Properties of Central Metabolism

“…Unlike the situation in microbial systems, such as yeast, for which a variety of knockout studies exist to date (Przybyla-Zawislak et al, 1999;McCammon et al, 2003;Kokko et al, 2006), only a single transgenic study, analyzing the effect of fumarase inhibition, has been carried out in plants ( Nunes-Nesi et al, 2007). That study revealed that antisense inhibition of mitochondrial fumarase activity in tomato (Solanum lycopersicum) plants has a negative impact on photosynthesis and leads to a consequent reduction in total plant biomass.…”

“…Thus, knockout studies are needed to support further evidence for the model prediction. Nevertheless, knockout analysis in yeast suggested an important role of fumarase in central metabolism, with cells lacking fumarase being essentially unable to grow on any nonfermentable carbon source (Przybyla-Zawislak et al, 1999;McCammon et al, 2003;Kokko et al, 2006).…”