Reversed argininosuccinate lyase activity in fumarate hydratase-deficient cancer cells - PubMed (original) (raw)
Reversed argininosuccinate lyase activity in fumarate hydratase-deficient cancer cells
Liang Zheng et al. Cancer Metab. 2013.
Abstract
Background: Loss of function of fumarate hydratase (FH), the mitochondrial tumor suppressor and tricarboxylic acid (TCA) cycle enzyme, is associated with a highly malignant form of papillary and collecting duct renal cell cancer. The accumulation of fumarate in these cells has been linked to the tumorigenic process. However, little is known about the overall effects of the loss of FH on cellular metabolism.
Methods: We performed comprehensive metabolomic analyses of urine from Fh1-deficient mice and stable isotopologue tracing from human and mouse FH-deficient cell lines to investigate the biochemical signature of the loss of FH.
Results: The metabolomics analysis revealed that the urea cycle metabolite argininosuccinate is a common metabolic biomarker of FH deficiency. Argininosuccinate was found to be produced from arginine and fumarate by the reverse activity of the urea cycle enzyme argininosuccinate lyase (ASL), making these cells auxotrophic for arginine. Depleting arginine from the growth media by the addition of pegylated arginine deiminase (ADI-PEG 20) decreased the production of argininosuccinate in FH-deficient cells and reduced cell survival and proliferation.
Conclusions: These results unravel a previously unidentified correlation between fumarate accumulation and the urea cycle enzyme ASL in FH-deficient cells. The finding that FH-deficient cells become auxotrophic for arginine opens a new therapeutic perspective for the cure of hereditary leiomyomatosis and renal cell cancer (HLRCC).
Figures
Figure 1
Identification of urine biomarkers in AhCreFh1 fl/fl mice. (A) Representative images from the ultrasound scanning of the kidneys of AhCreFh1 fl/fl mice. Arrows indicate the cysts. (B) Quantification of cyst number in kidney sections from 1-year-old AhCreFh1 fl/fl mice performed by manual counting. Cysts with an average diameter above 50 μm were counted. (C) Principal component analysis (PCA) of the LC-MS urine metabolomic data obtained from mice with the indicated genotype. (D) Sigmoidal plot representation of the ratio of urinary metabolites based on the comparison between AhCreFh1 fl/fl and Fh1 fl/fl mice. Metabolites of interest are indicated. (E) LC-MS analyses of argininosuccinate and fumarate in kidney samples of AhCreFh1 fl/fl and Fh1 fl/fl mice sectioned as indicated in the figure. LC-MS, liquid chromatography-mass spectrometry; PCA, principal component analysis.
Figure 2
Metabolic fingerprint of FH-deficient cell lines. (A) PCA and (B) sigmoidal plot of the metabolomic data from growth media of the indicated cell lines. Results were obtained from nine independent cultures. UOK cells present a distinct metabolic signature compared to their FH-proficient counterpart, UOKpFH. (C) Extracellular and (D) intracellular fumarate and argininosuccinate levels in the indicated cell lines. Results were obtained from three independent cultures and represented as average ± SEM. PCA, principal component analysis; SEM, standard error of the mean; UOK, UOK262.
Figure 3
Argininosuccinate is produced from arginine and fumarate. (A) Isotopologue distribution analysis of intracellular metabolites after incubation with U-13C-glutamine for 24 hours. (B) In light of the isotopologues distribution (13C4) of glutamine-derived 13C-labeled argininosuccinate in FH-deficient cells (panel A), this scheme represents the most likely biochemical pathway that links TCA cycle metabolites in FH-deficient cells to the urea cycle and to argininosuccinate production. Arginine and fumarate were mixed in a physiological buffer in the presence or absence of protein lysates from (C) Fh1 −/− cells or (D) Fh1 −/− cells in which ASL expression was silenced. The formation of argininosuccinate is enzymatic and requires ASL. Where indicated, the cell extracts were heat inactivated (HI) prior to the in vitro reaction. Results were obtained from three independent experiments and expressed as average ± SEM. ASL, argininosuccinate lyase; FH, fumarate hydratase; HI, heat inactivated; SEM, standard error of the mean.
Figure 4
Argininosuccinate is produced by the reverse activity of the urea cycle enzyme ASL. (A) mRNA expression levels of ASL after acute infection of the indicated cell lines with shRNAs against human (sh_ASL_) or mouse (sh_Asl_) ASL. Values are normalized to cells infected with the control plasmid. Argininosuccinate production is decreased in _ASL_-silenced (B) human or (C) mouse cells. Cells were incubated with U-13C-arginine for 24 hours and the isotopologue distribution of argininosuccinate was analyzed by LC-MS. (D) Schematic representation of the fate of arginine in FH-deficient cells. Orange arrows indicate the major metabolic flux from arginine; light yellow arrow indicates low metabolic flux, gray arrows indicate undetectable metabolic flux; and dark gray arrow indicates the crosstalk between the TCA cycle and the urea cycle. All the results were obtained from three independent cultures and expressed as average ± SEM. ASL, argininosuccinate lyase; FH, fumarate hydratase; LC-MS, liquid chromatography-mass spectrometry; NTC, non-targeting control; SEM, standard error of the mean; shRNA, short hairpin RNA; TCA, tricarboxylic acid.
Figure 5
Arginine depletion by ADI-PEG 20 reduces argininosuccinate and inhibits cellular proliferation in FH-deficient cells. (A) Arginine levels determined in the media of the indicated cell lines after the incubation for 4 hours with 125 ng/mL ADI-PEG 20. (B) Excreted and intracellular levels of argininosuccinate after treatment with ADI-PEG 20, as in (A), were detected by LC-MS. (C) Representative images of a colony survival assay after treatment with either 125 ng/mL (mouse cells) or 50 ng/mL (human cells) ADI-PEG 20 for 8 days. The media was replaced and cells were left to grow for a further 4 days before fixation and staining. All the results were obtained from three independent cultures and expressed as average ± SEM. FH, fumarate hydratase; LC-MS, liquid chromatography-mass spectrometry; SEM, standard error of the mean.
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