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Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis

Abstract

Succinate dehydrogenase (SDH) is a heterotetrameric nuclear-encoded complex responsible for the oxidation of succinate to fumarate in the tricarboxylic acid cycle. Loss-of-function mutations in any of the SDH genes are associated with cancer formation. However, the impact of SDH loss on cell metabolism and the mechanisms enabling growth of SDH-defective cells are largely unknown. Here, we generated Sdhb-ablated kidney mouse cells and used comparative metabolomics and stable-isotope-labelling approaches to identify nutritional requirements and metabolic adaptations to SDH loss. We found that lack of SDH activity commits cells to consume extracellular pyruvate, which sustains Warburg-like bioenergetic features. We further demonstrated that pyruvate carboxylation diverts glucose-derived carbons into aspartate biosynthesis, thus sustaining cell growth. By identifying pyruvate carboxylase as essential for the proliferation and tumorigenic capacity of SDH-deficient cells, this study revealed a metabolic vulnerability for potential future treatment of SDH-associated malignancies.

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Figure 1: Loss of Sdhb is sufficient to truncate the TCA cycle and disable mitochondrial respiration.
Figure 2: SDH deficiency commits cells to consume extracellular pyruvate to maintain maximal glycolytic flux and cell growth.
Figure 3: Glucose-dependent aspartate biosynthesis dictates pyruvate dependence of Sdhb-deficient cells.
Figure 4: Pyruvate carboxylase expression is induced in SDH-defective cells and SDHx-mutated human tumours.
Figure 5: Pyruvate carboxylation is a vulnerable adaptation to Sdhb loss.
Figure 6: Pyruvate carboxylase supports growth of Sdhb-null cells by sustaining aspartate biosynthesis.

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Acknowledgements

We would like to acknowledge S. Tardito and Z. T. Schug for comments and interpretation of the results, A. King for editorial work, U. Srirangalingam for help in human specimen collection and the Beatson Institute mouse facility staff for housing of mice and xenograft measurements.

Author information

Authors and Affiliations

Authors

Contributions

S.C. conceived the study, designed and carried out the experiments, interpreted the data and wrote the manuscript; L.Z. carried out the untargeted metabolomic analysis; G.M., N.J.F.v.d.B. S.T., V.B., J.J.K. and A.V. supervised the targeted LC-MS and GC-MS analyses; E.D.M. supervised the generation of kidney cells; D. Strathdee and D. Stevenson generated genetically modified Sdhbfl/fl mice; G.K. carried out the bioinformatics and statistical analyses; C.N. carried out immunohistochemistry of human SDHB-associated RCC; S.F. carried out histopathological analysis of SDHB-related RCC; F.S. collected and provided human paraganglioma and pheochromocytoma samples; K.B. supervised animal work; E.G. conceived and supervised the study, interpreted the data and revised the manuscript. All the authors discussed the results and commented on the manuscript. This work was funded by Cancer Research UK. S.C. is the recipient of a FEBS long-term fellowship.

Corresponding author

Correspondence to Eyal Gottlieb.

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Competing interests

E.G. is a shareholder and a consultant of MetaboMed Israel, Ltd.

Integrated supplementary information

Supplementary Figure 1 Generation of Sdhb-deficient cells.

(a) Schematic representation of the mouse Sdhb genomic locus and Sdhb targeted allele. Numbers specify exons, arrowheads indicate LoxP sites; Hyg, Hygromycin-resistance gene cassette. 3 LoxP U1, 3 LoxP D1 and Hyg U1 primers were used for genotyping. (b) Bright-field images of Sdhbfl/fl and SdhbΔ/Δ cells. Scale bar = 280 μm. (c) PCR analysis of gDNA of kidney epithelial cells of the indicated genotypes after infection (Cre) with Ad5-CMV-Cre-GFP; bp, base pairs. (d) Western blot analysis of SDHB levels of the indicated cells. Data in b-d derive from one experiment, performed once. (e) Determination of SDH activity of the indicated cells in the presence (+) or absence (−) of 20 mM malonate. Data are presented as mean ± s.e.m. of n = 3 wells pooled from three independent experiments. (f) % labelling of lipogenic AcCoA from glucose and glutamine. Data are presented as mean ± s.e.m. (n = 3 wells) of one experiment, performed once. Raw data of independently repeated experiments are provided in Supplementary Table 3.

Supplementary Figure 2 Sdhb loss induces Warburg-like bioenergetic features.

(a) Maximal and ATP-coupled respiration. Dashed line indicates the relative basal mitochondrial OCR. Data are presented as mean ± s.e.m. of three reading cycles of n = 27 wells pooled from four independent experiments. (b) Mitochondrial mass; a.u., arbitrary units. Data are presented as mean ± s.e.m. of n = 3 wells pooled from three independent experiments. (c) Representative Western blot analysis (out of 2 independent experiments) of the levels of two complex I subunits (red) and several mitochondrial proteins (green) assessed in the indicated mitochondrial extracts. (CI, Complex I; CVa, ComplexVa, CIII Core1, Complex III Core 1, CypD, Cyclophilin D, Cyt c, Cytochrome c, COX IV, Cytochrome c Oxidase). (d) Mitochondrial Complex I activity. Data are presented as mean ± s.e.m. (n = 4 wells) of one experiment, performed once. (e,f) AMP/ATP ratio (expressed as fold change compared to untreated Sdhfl/fl cells), AMP and ATP levels (expressed as % of untreated Sdhfl/fl cells) upon treatment of the indicated cells with 0.5 μM oligomycin for 8 h. (g) ECAR of the indicated cells. Data are presented as mean ± s.e.m. of n = 21 (Sdhbfl/fl) and n = 23 (SdhbΔ/Δ) wells pooled from three independent experiments. (h) Lactate secretion by Sdhbfl/fl and SdhbΔ/Δ cells cultured for 48 h with U-13C-glucose in presence/absence of 5 mM oxamate. The sum of all isotopologues is reported for clarity. Data are presented as mean ± s.e.m. (n = 6 wells) of one experiment, performed once. (i,j) AMP/ATP ratio (expressed as fold change compared to untreated Sdhfl/fl cells), AMP and ATP levels (expressed as % of untreated Sdhfl/fl cells) upon treatment with 5 mM oxamate for 24 h. (k) NAD+ and NADH levels (expressed as % of Pyruvate-fed Sdhfl/fl cells) in cells incubated for 24 h in the presence/absence of pyruvate. Data in e, f, k are presented as mean ± s.e.m. (n = 3 wells) of one representative experiment, independently replicated twice. (l,m) NAD+/NADH ratio (expressed as fold change compared to untreated Sdhfl/flcells), NAD+ and NADH levels (expressed as % of untreated Sdhfl/fl cells) in cells incubated for 24 h in the presence/absence of 5 mM oxamate. Data in i, j, l, m are presented as mean ± s.e.m. (n = 3 wells) of one experiment, performed once. Raw data of independently repeated experiments are provided in Supplementary Table 3.

Supplementary Figure 3 Pyruvate-dependency of Sdhb-deficient cells.

(a) Determination of secretion (positive bars) and consumption (negative bars) rates of the indicated metabolites of cells cultured for 48 h with U-13C-glucose. The sum of all isotopologues is reported for clarity. Data are presented as mean ± s.e.m. of n = 18 wells pooled from three independent experiments. (b) Isotopologues labeling profile of intracellular pyruvate in cells incubated for 24 h with U-13C-pyruvate. (c) Labeling of pyruvate after incubation of cells for 24 h with with U-13C-Glucose in presence (+) or absence (−) of pyruvate. Data in b, c are presented as mean ± s.e.m. (n = 3 wells) of one representative experiment, independently replicated twice. Raw data of independently repeated experiments are provided in Supplementary Table 3.

Supplementary Figure 4 Essentiality of pyruvate carboxylase for growth of Sdhb-deficient cells.

(a) Immuhistochemical assessment of PC levels in two human SDHB-related RCC samples. N.A.T., normal adjacent tissue; T., tumour. Scale bars = 100 μM. Numbers on the left indicates the Tumour ID described in Supplementary Table 2. (b) qPCR analysis of Pcx expression in cells infected with lentiviruses expressing either a non-targeting shRNAsequence (shNTC) or an shRNA targeting Pcx. (c) Measurement of malate levels in cells treated as in (b) cultured for 24 h in presence of U-13C-glucose. Data in b, c are presented as mean ± s.e.m.of n = 9 wells pooled from three independent experiments. (d) Abundance of 13C1-malate in cells treated as in (b) incubated for 10 min in presence of13C-bicarbonate. Data are presented as mean ± s.e.m. (n = 3 wells) of one representative experiment, independently replicated twice. (e) Measurement of Aspartate levels in cells treated as in (b) cultured for 24 h in presence of U-13C-glutamine. (f) PCX expression inH-RasV 12-transformed cells infected with lentiviruses expressing either a shNTC or shPcx-5 sequence before injection into nude mice. Data in e, f are presented as mean ± s.e.m. (n = 3 wells) of one representative experiment, performed once. (gi) In vivo growth of cells described in (f) xenografted in athymic nude mice. The % of tumour-free mice over time (g) and the tumour volumes of each xenografted mouse (n = 10 mice per group) (h,i) are presented. The Log-rank (Mantel–Cox) test was used to calculate the statistical significance between curves in (g). A statistical permutation test was used to compare the statistical significance between curves of the selected genotypes in (h) and (i), as described in Methods. Data derive fromone experiment, performed once. Raw data of independently repeated experiments are provided in Supplementary Table 3.

Supplementary Figure 5 Pyruvate carboxylation sustains aspartate biosynthesis.

(a) Measurement of citrate levels in cells cultured for 24 h in presence of U-13C-glucose. (b) Number of control and PCX-silenced cells supplemented with 2.5 mM aspartate measured after 96 h of culture. Data are presented as mean ± s.e.m. (n = 4 wells) of one representative experiment, performed once. Data related to shNTC control cells are shared with Fig. 6c (c) Determination of total intracellular palmitate levels in PCX-silenced cells supplemented with 50 μM palmitate for 24 h. Data in a, c are presented as mean ± s.e.m. (n = 3 wells) of one representative experiment, performed once. Raw data of independently repeated experiments are provided in Supplementary Table 3.

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Cardaci, S., Zheng, L., MacKay, G. et al. Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis. Nat Cell Biol 17, 1317–1326 (2015). https://doi.org/10.1038/ncb3233

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