Aberrant succination of proteins in fumarate hydratase-deficient mice and HLRCC patients is a robust biomarker of mutation status†
Chiara Bardella
Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford, UK
Search for more papers by this authorMona El-Bahrawy
Department of Histopathology, Imperial College, Hammersmith Hospital, London, UK
Search for more papers by this authorNorma Frizzell
Department of Exercise Science, School of Public Health, University of South Carolina, Columbia, SC, USA
Search for more papers by this authorJulie Adam
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorNicola Ternette
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorEmine Hatipoglu
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorKimberley Howarth
Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorLinda O'Flaherty
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorIan Roberts
Department of Cellular Pathology, John Radcliffe Hospital, Oxford, UK
Search for more papers by this authorGareth Turner
Department of Cellular Pathology, John Radcliffe Hospital, Oxford, UK
Search for more papers by this authorJennifer Taylor
Oxford Biomedical Research Centre, Wellcome Trust Centre for Human Genetics, Oxford, UK
Search for more papers by this authorKonstantinos Giaslakiotis
Department of Cellular Pathology, John Radcliffe Hospital, Oxford, UK
Search for more papers by this authorValentine M Macaulay
Weatherall Institute of Molecular Medicine, University of Oxford, UK
Search for more papers by this authorAdrian L Harris
Weatherall Institute of Molecular Medicine, University of Oxford, UK
Search for more papers by this authorAshish Chandra
Cellular Pathology, St Thomas' Hospital, London, UK
Search for more papers by this authorHeli J Lehtonen
Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Finland
Search for more papers by this authorVirpi Launonen
Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Finland
Search for more papers by this authorLauri A Aaltonen
Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Finland
Search for more papers by this authorChristopher W Pugh
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorRadu Mihai
Department of Endocrine Surgery, John Radcliffe Hospital, Oxford, UK
Search for more papers by this authorDavid Trudgian
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorBenedikt Kessler
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorJohn W Baynes
Department of Exercise Science, School of Public Health, University of South Carolina, Columbia, SC, USA
Search for more papers by this authorPeter J Ratcliffe
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorIan P Tomlinson
Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford, UK
Oxford Biomedical Research Centre, Wellcome Trust Centre for Human Genetics, Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorCorresponding Author
Patrick J Pollard
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, UK.Search for more papers by this authorChiara Bardella
Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford, UK
Search for more papers by this authorMona El-Bahrawy
Department of Histopathology, Imperial College, Hammersmith Hospital, London, UK
Search for more papers by this authorNorma Frizzell
Department of Exercise Science, School of Public Health, University of South Carolina, Columbia, SC, USA
Search for more papers by this authorJulie Adam
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorNicola Ternette
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorEmine Hatipoglu
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorKimberley Howarth
Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorLinda O'Flaherty
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorIan Roberts
Department of Cellular Pathology, John Radcliffe Hospital, Oxford, UK
Search for more papers by this authorGareth Turner
Department of Cellular Pathology, John Radcliffe Hospital, Oxford, UK
Search for more papers by this authorJennifer Taylor
Oxford Biomedical Research Centre, Wellcome Trust Centre for Human Genetics, Oxford, UK
Search for more papers by this authorKonstantinos Giaslakiotis
Department of Cellular Pathology, John Radcliffe Hospital, Oxford, UK
Search for more papers by this authorValentine M Macaulay
Weatherall Institute of Molecular Medicine, University of Oxford, UK
Search for more papers by this authorAdrian L Harris
Weatherall Institute of Molecular Medicine, University of Oxford, UK
Search for more papers by this authorAshish Chandra
Cellular Pathology, St Thomas' Hospital, London, UK
Search for more papers by this authorHeli J Lehtonen
Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Finland
Search for more papers by this authorVirpi Launonen
Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Finland
Search for more papers by this authorLauri A Aaltonen
Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Finland
Search for more papers by this authorChristopher W Pugh
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorRadu Mihai
Department of Endocrine Surgery, John Radcliffe Hospital, Oxford, UK
Search for more papers by this authorDavid Trudgian
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorBenedikt Kessler
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Search for more papers by this authorJohn W Baynes
Department of Exercise Science, School of Public Health, University of South Carolina, Columbia, SC, USA
Search for more papers by this authorPeter J Ratcliffe
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorIan P Tomlinson
Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford, UK
Oxford Biomedical Research Centre, Wellcome Trust Centre for Human Genetics, Oxford, UK
These authors contributed equally to this work.
Search for more papers by this authorCorresponding Author
Patrick J Pollard
Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, UK.Search for more papers by this authorConflict of interest statement. PP, NF, JWB and IPT have filed for a patent [No. USC-268-P(849)] covering immunohistochemical screening for the determination of FH mutations.
Abstract
Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Supporting Information
Filename | Description |
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path_2932_sm_supportinginformationsfs1.pdf73.7 KB | Supporting Information: Figure S1. 2SCP expression results from loss of Fh1 protein in mouse embryonic fibroblasts. |
path_2932_sm_supportinginformationsfs2.pdf138.5 KB | Supporting Information: Figure S2. Succination of murine Gapdh in FH-deficient kidneys. |
path_2932_sm_supportinginformationsts1.pdf47 KB | Supporting Information: Table S1. 2SCP immunohistochemistry and FH mutation status of PRCCs derived from Oxford Radcliffe Hospitals. |
path_2932_sm_supportinginformationsts2.pdf453.7 KB | Supporting Information: Table S2. Details of the biopsies screened for 2SCP by immunohistochemistry. |
path_2932_sm_supportinginformations.pdf16.4 KB | Supporting Information: Supplementary methods: PCR conditions and primer sequences for FH mutation screening. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1Astuti D, Latif F, Dallol A, et al. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet 2001; 69: 49–54.
- 2Baysal BE, Ferrell RE, Willett-Brozick JE, et al. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 2000; 287: 848–851.
- 3Burnichon N, Briere JJ, Libe R, et al. SDHA is a tumor suppressor gene causing paraganglioma. Hum Mol Genet 2010; 19: 3011–3020.
- 4Hao HX, Khalimonchuk O, Schraders M, et al. SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science 2009; 325: 1139–1142.
- 5Niemann S, Muller U. Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nature Genetics 2000; 26: 268–270.
- 6Tomlinson IP, Alam NA, Rowan AJ, et al. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet 2002; 30: 406–410.
- 7Warburg O. The Metabolism of Tumours. Arnold Constable: London, 1930.
- 8Warburg O. On the origin of cancer cells. Science 1956; 123: 309–314.
- 9Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009; 360: 765–773.
- 10Refae MA, Wong N, Patenaude F, et al. Hereditary leiomyomatosis and renal cell cancer: an unusual and aggressive form of hereditary renal carcinoma. Nat Clin Pract Oncol 2007; 4: 256–261.
- 11Alam NA, Rowan AJ, Wortham NC, et al. Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency. Hum Mol Genet 2003; 12: 1241–1252.
- 12Pollard PJ, Ratcliffe PJ. Cancer. Puzzling patterns of predisposition. Science 2009; 324: 192–194.
- 13Frizzell N, Rajesh M, Jepson MJ, et al. Succination of thiol groups in adipose tissue proteins in diabetes: succination inhibits polymerization and secretion of adiponectin. J Biol Chem 2009; 284: 25772–25781.
- 14Nagai R, Brock JW, Blatnik M, et al. Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress. J Biol Chem 2007; 282: 34219–34228.
- 15Frizzell N, Lima M, Baynes JW. Succination of proteins in diabetes. Free Radic Res 2011; 45: 101–109.
- 16Pollard PJ, Spencer-Dene B, Shukla D, et al. Targeted inactivation of fh1 causes proliferative renal cyst development and activation of the hypoxia pathway. Cancer Cell 2007; 11: 311–319.
- 17O'Flaherty L, Adam J, Heather LC, et al. Dysregulation of hypoxia pathways in fumarate hydratase-deficient cells is independent of defective mitochondrial metabolism. Hum Mol Genet 2010; 19: 3844–3851.
- 18Wessel D, Flugge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 1984; 138: 141–143.
- 19Trudgian DC, Thomas B, McGowan SJ, et al. CPFP: a central proteomics facilities pipeline. Bioinformatics 2010; 26: 1131–1132.
- 20Blatnik M, Frizzell N, Thorpe SR, et al. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by fumarate in diabetes: formation of S-(2-succinyl) cysteine, a novel chemical modification of protein and possible biomarker of mitochondrial stress. Diabetes 2008; 57: 41–49.
- 21Kiuru M, Lehtonen R, Arola J, et al. Few FH mutations in sporadic counterparts of tumor types observed in hereditary leiomyomatosis and renal cell cancer families. Cancer Res 2002; 62: 4554–4557.
- 22Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010; 363: 1693–1703.
- 23Dang L, White DW, Gross S, et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2009; 462: 739–744.
- 24Zhang Z, Tan M, Xie Z, et al. Identification of lysine succinylation as a new post-translational modification. Nat Chem Biol 2010; 7: 58–63.