A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia

Abstract

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

Graphical abstract

Figure 1

FH-Deficient Cells Synthesize Argininosuccinate Directly from Fumarate (A–D) Concentrations of specific urea cycle metabolites (μM) in control and FH1KO kidneys as determined by CE-TOFMS (Soga et al., 2009). All differences between control and FH1KO mice were significant (p < 0.01, Student’s t test). For metabolomic analyses, six mice aged 15 weeks were analyzed from each group. (E) CE-TOFMS analyses of deuterium label incorporation into key Krebs cycle and urea cycle metabolites in FH1WT (blue) and KO (red) MEFs after 9 hr incubation in culture containing [D5]-glutamine. Transit of label through the canonical oxidative Krebs cycle would result in 2OG+4, succinate+4, fumarate+2, malate+2, and Asp+1, while reductive carboxylation of glutamate would result in isocitrate m+2, citrate m+2, and aspartate m+1. We did not observe label enrichment in citrate, so the reductive mechanism is not used for citrate synthesis. Argininosuccinate produced from arginine and fumarate has m+2, whereas that produced from citrulline and aspartate has m+1. We detected predominantly argininosuccinate m+2, which has a similar isotopomer distribution pattern to fumarate, suggesting it is synthesized directly from fumarate. For each graph, the concentration of metabolites (fmol/cell) is indicated on the y axis and label enrichment of [D5]-glutamine in FhWT and KO MEFs are represented on the x axis in the following order: 12C, 12C-1d, 12C-2d, 12C-3d, and 12C-4d. See also Table S1 for absolute metabolite levels. ACL, ATP citrate lyase; ACO1, -2, aconitase 1, -2; IDH1, -2, -3, isocitrate dehydrogenase 1, 2, 3; CS, citrate synthase; SUC, succinyl CoA synthetase; SDH, succinate dehydrogenase; OGDH, oxoglutarate dehydrogenase; FH, fumarate hydratase; MDH, malate dehydrogenase; PDH, pyruvate dehydrogenase; GLS, glutaminase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; OCT, ornithine carbamoyltransferase; ARG, arginase.

Figure 2

Generation and Analyses of FH-Expressing Transgenic Mice to Investigate the Role of Cytosolic FH/Fumarate in Renal Cyst Development (A) The FH and FH^cyt transgenes were cloned into the CB92 vector and targeted to the Rosa26 locus, using phage-mediated recombination (Chen et al., 2011). (B and C) Localization of FH and FH^cyt was confirmed in ES cells by immunocytochemistry. Colocalization (yellow) of FH (green) and mitochondria (red) is evident (B), whereas FH^cyt is absent from the mitochondria (C). Nuclei (blue) are visualized with DAPI. (D) Kidneys harvested from 30-week-old control, transgene-only (FH, FH^cyt), and genetically “rescued” mice (FH1KO+FH and FH1KO+FH^cyt) appeared macroscopically normal compared to FH1KO kidneys, which appeared enlarged and cystic. (E) Hematoxylin and eosin (H&E) staining of kidneys harvested from mice at 13, 20, and 30 weeks reveal that transgenic expression of either FH or FH^cyt is sufficient to ameliorate cyst development. (F–H) Numbers and frequency of macrocysts (>0.5 mm) and microcysts (>0.1 mm) were determined in each group (n = 6). Error bars represent the SEM. Scale bars, 5 μm (B and C), 10 mm (D), and 100 μm (E). Error bars indicate SEM. See also Figure S1.

Figure 3

Metabolite Analysis of Kidneys from FH1KO- and FH-Reconstituted Mice (A) Heatmap showing relative levels of specific metabolites in kidneys from control, FH1 KO, and reconstituted (FH1 KO+FH and FH1 KO+FH^cyt) mice. For each group, kidneys from six mice (aged 15 weeks) were analyzed using CE-TOFMS (Soga et al., 2009), and the data were filtered to select only metabolites where there was a 2-fold difference between any group and a p value <0.05 (one-way ANOVA). Relative metabolite levels are indicated by red (high) and green (low). (B–E) Concentrations (μM) of the specific urea cycle metabolites fumarate (B), argininosuccinate (C), aspartate (D), and citrulline (E) measured in kidneys from mice of each of the above four genotypes. (F and G) Analysis of FH mutant renal cancers (n = 3) and adjacent normal tissue (n = 2) indicates accumulation of fumarate (F) and aspartate (G). Error bars indicate SEM. See also Table S1.

Figure 4

Argininosuccinate Is Produced from Arginine in FH-Deficient Cells (A) Sections of HSQC spectra for the C₄ (top panel) and C₅ (bottom panel) resonances of arginine and argininosuccinate detected in FH1WT (red) and FH1KO (blue) MEFs. Argininosuccinate was detected in FH1KO MEFs only, which also correlates with the appearance of the fumarate resonance at 6.5/138 ppm. (B and C) Cells of the indicated genotype were cultured in media containing ¹⁵N₄,¹³C₆-arginine for 12 hr prior to metabolite extraction and analyses of fumarate (B) and argininosuccinate (C) levels by CE-TOFMS (Soga et al., 2009). The m+10 isotopomer comprises >90% of total argininosuccinate detected in FH1KO MEFs and UOK262 cells, suggesting direct conversion of argininosuccinate from arginine and fumarate. Three independent cultures were used for each condition, and data shown here are representative of two independent experiments. (D–G) Concentrations (fmol/cell) of fumarate (B), argininosuccinate (C), citrulline (D), and aspartate (E) were determined in FH1WT and KO MEFs by CE-TOFMS, confirming the metabolic profile observed in equivalent mouse kidneys of significantly elevated fumarate and argininosuccinate and significantly depleted aspartate. (H) The manganese-containing enzyme arginase catalyzes the conversion of arginine and water to ornithine and urea. Levels of arginine and ornithine (μM) measured in medium by HPLC-MSMS following treatment with bovine arginase show that arginine levels are significantly reduced with time with a concomitant increase in ornithine. (I–L) WT and KO FH1 MEFs were cultured in medium treated with arginase for 1, 2, 4, and 6 hr compared to no treatment (0 hr). Cells were then returned to standard DMEM (containing arginine) and assayed separately for proliferative capacity (I and J) and colony-forming capacity (K and L). (I and J) Reduced proliferative capacity of FH1KO MEFs compared to WT controls is observed following culture in arginine-depleted medium for as little as 2 hr. 1.5 × 10⁴ cells/well of 96-well plate were plated for each experimental group and measurements made using CyQuant. Fluorescence units were measured and normalized to those of cells cultured in standard untreated medium. (K) Representative photographs of colony assays for WT and KO FH1 MEFs 10³ cells/10 cm plate were cultured for 10 days for each experimental group. Three separate dilutions were plated for each experimental group, and the experiments were repeated in triplicate. (L) The number of colonies formed by FH1WT and KO MEFs was counted and expressed relative to the colony count of cells cultured in standard untreated medium (0 hr). The graph of plating efficiency versus time cultured in arginine-depleted medium shows that FH1KO MEFs are acutely sensitive to reduced arginine even for a very short time period (2 hr). See also Figure S2.

Figure S1

Generation and Analyses of FH (FL) (FH) and FH(-MLS) (FH^cyt)-Expressing Transgenic Mice, Related to Figure 2 (A) Restriction digest profile of CB92-CAGGS-FH(FL) (FH) and CB92-CAGGS-FH(-MLS) (FH^cyt), showing the predicted fragments. (MLS = mitochondrial leader sequence). (B and C) show the plasmid maps of the two exchange vectors, including the sequenced regions and the locations of the restriction sites used for the restriction analysis. (D) The exchanged Rosa26 loci (Chen et al., 2011) are shown with the positions of the genotyping primers. (E) PCR genotyping for exchange at the 3′ end using primer combination AttL-PolyF1 and Rosa3HR-R (Clones ending in 1, 2 or 3 contain the full length construct and clones ended in 4, 5 or 6 contain the MLS deleted construct). (F) PCR genotyping for exchange at the 5′ end using primer combination ExPGK3 and ExNeo2 (top panel) and PCR genotyping for the presence of the transgenic construct allowing the two variant (full length and MLS deleted) to be distinguished on the basis of size (bottom panel). (G) Verification by immunoblot of the presence of FH-V5 in tissues of transgenic mice. Such verification was confirmed in tissues from three mice for both FH(FL) (FH) and FH(-MLS) (FH^cyt). (H) Confirmation by immunofluorescence of the localization of FH-V5 and FH^cyt-V5 in renal tubular cells. Scale bar = 5 μm.

Figure S2

Characterization of UOK262 Cells Complemented with FH and FH^cyt, Related to Figure 4 (A-C) Complementation with, and localization of, FH and FH^cyt in UOK262 cells (Yang et al., 2010b) was confirmed by immunofluorescence. The FH and FH^cyt constructs carry a C-terminal V5 tag (green); mitochondria are identified with Mitotracker red (red) and nuclei by Dapi (blue). (A) Parental UOK262 cells show no V5 immunofluorescence. Four panels are shown; three for each fluorophore separately and a merged image. (B) UOK262 cells transfected with FH show clear overlap with Mitotracker red confirming the mitochondrial localization. (C) Cytoplasmic localization of FH^cyt was confirmed since there is no overlap with Mitotracker red. The large vacuoles that are present in these cells can also be seen clearly in this set of images. (D) Complementation of UOK262 cells with FH and FH^cyt was confirmed further by Western blot analysis for FH and the C-terminal V5 tag. ACTB was used as a loading control. Note that complementation with either transgene did not ameliorate HIF-1α levels.