Expression Profiling in Progressive Stages of Fumarate- Hydratase Deficiency: the Contribution of Metabolic Changes to Tumorigenesis
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Published OnlineFirst October 26, 2010; DOI: 10.1158/0008-5472.CAN-10-1949 Molecular and Cellular Pathobiology Cancer Research Expression Profiling in Progressive Stages of Fumarate- Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis Houman Ashrafian1, Linda O'Flaherty2, Julie Adam2, Violetta Steeples1, Yuen-Li Chung5, Phil East6, Sakari Vanharanta13,15, Heli Lehtonen15, Emma Nye7, Emine Hatipoglu2, Melroy Miranda2, Kimberley Howarth3, Deepa Shukla9, Helen Troy5, John Griffiths12, Bradley Spencer-Dene7, Mohammed Yusuf4, Emanuela Volpi4, Patrick H. Maxwell9, Gordon Stamp7,10, Richard Poulsom8, Christopher W. Pugh2, Barbara Costa16, Chiara Bardella3, Maria Flavia Di Renzo16, Michael I. Kotlikoff14, Virpi Launonen15, Lauri Aaltonen15,MonaEl-Bahrawy11, Ian Tomlinson3,andPatrickJ.Pollard2 Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by mutations in the Krebs cycle enzyme fumarate hydratase (FH). It has been proposed that “pseudohypoxic” stabilization of hypoxia- inducible factor-α (HIF-α) by fumarate accumulation contributes to tumorigenesis in HLRCC. We hypothe- sized that an additional direct consequence of FH deficiency is the establishment of a biosynthetic milieu. To investigate this hypothesis, we isolated primary mouse embryonic fibroblast (MEF) lines from Fh1-deficient mice. As predicted, these MEFs upregulated Hif-1α and HIF target genes directly as a result of FH deficiency. In addition, detailed metabolic assessment of these MEFs confirmed their dependence on glycolysis, and an elevated rate of lactate efflux, associated with the upregulation of glycolytic enzymes known to be associated with tumorigenesis. Correspondingly, Fh1-deficient benign murine renal cysts and an advanced human HLRCC-related renal cell carcinoma manifested a prominent and progressive increase in the expression of HIF-α target genes and in genes known to be relevant to tumorigenesis and metastasis. In accord with our hypothesis, in a variety of different FH-deficient tissues, including a novel murine model of Fh1-deficient smooth muscle, we show a striking and progressive upregulation of a tumorigenic metabolic profile, as man- ifested by increased PKM2 and LDHA protein. Based on the models assessed herein, we infer that that FH deficiency compels cells to adopt an early, reversible, and progressive protumorigenic metabolic milieu that is reminiscent of that driving the Warburg effect. Targets identified in these novel and diverse FH-deficient mod- els represent excellent potential candidates for further mechanistic investigation and therapeutic metabolic manipulation in tumors. Cancer Res; 70(22); 9153–65. ©2010 AACR. Introduction ref. 1). These RCCs are relatively resistant to systemic thera- pies (2–4). Although patients with HLRCC carry heterozygous The hereditary leiomyomatosis and renal cell carcinoma germline mutations in the gene encoding the Krebs cycle (HLRCC) syndrome is an inherited condition in which affect- enzyme fumarate hydratase (FH E.C. 4.2.1.2.; refs. 3, 4), the ed individuals are at risk of developing predominantly benign mechanisms of tumorigenesis and tissue specificity remain cutaneous and uterine leiomyomas, and aggressive type II to be defined. Elucidation of these mechanisms would sup- papillary and collecting duct renal cell carcinomas (RCC; port the development of targeted therapies for HLRCC and Authors' Affiliations: 1Department of Cardiovascular Medicine, Sloan-Kettering Cancer Center, New York, New York; 14Biomedical University of Oxford, John Radcliffe Hospital, Headington, 2Henry Sciences Department, College of Veterinary Medicine, Cornell 15 Wellcome Building for Molecular Physiology, University of Oxford, and University, Ithaca, New York; Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; and 3Molecular and Population Genetics and 4Molecular Cytogenetics and 16Laboratory of Cancer Genetics, Institute for Cancer Research and Microscopy Core, Wellcome Trust Centre for Human Genetics, Treatment, Candiolo, Turin, Italy Oxford, United Kingdom; 5Cancer Research UK and Engineering and Physical Sciences Research Council Cancer Imaging Centre, The Note: Supplementary data for this article are available at Cancer Institute of Cancer Research and Royal Marsden Hospital, Sutton, Research Online (http://cancerres.aacrjournals.org/). 6 Surrey, United Kingdom; Bioinformatics and Biostatistics, H. Ashrafian and L. O'Flaherty contributed equally. 7Experimental Pathology, and 8Histopathology Laboratory, Cancer Research UK London Research Institute, 9The Rayne Institute, Corresponding Author: Patrick J. Pollard, University of Oxford, Roose- University College London Division of Medicine, 10Royal Marsden velt Drive, Oxford OX3 7BN, United Kingdom. Phone: 44-1865287780; Hospital, and 11Department of Histopathology, Imperial College, Fax: 44-1865287787; E-mail: [email protected]. Hammersmith Hospital, London, United Kingdom; 12Cancer Research doi: 10.1158/0008-5472.CAN-10-1949 UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom; 13Cancer Biology and Genetics Program, Memorial ©2010 American Association for Cancer Research. www.aacrjournals.org 9153 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/0008-5472.CAN-10-1949 Ashrafian et al. might inform the biology of a broadening array of tumor Ksp-Cre conditional knockout mice have been previously de- syndromes and somatic (acquired) tumors associated with scribed (19). Mice expressing Cre recombinase under the mutations in Krebs cycle enzymes (5). These include muta- smooth muscle myosin heavy chain (smMHC) promoter tions in succinate dehydrogenase (SDH) subunits B, C, and were a kind gift from Dr. Michael Kotlikoff (20). All mice were D that cause paragangliomas and pheochromocytomas (6); backcrossed onto a C57BL/6 genetic background for at least mutations in the SDH-interacting protein SDH5 (SDHAF2, re- five generations. quired for flavination of SDHA; ref. 7) that cause paraganglio- mas; and mutations affecting isocitrate dehydrogenase 1 and Gene expression arrays 2 (8) converting the Krebs cycle intermediate isocitrate to mRNA was extracted from each sample with the GenElute α-ketoglutarate that have been described in brain tumors. mammalian total RNA miniprep kit (Sigma, RTN70) All sub- Several possible mechanisms have been proposed to con- sequent steps in the microarray expression analysis were car- tribute to the tumor formation that results from FH mutations ried out by the Cancer Research UK GeneChip Microarray (5). An important observation is that elevated fumarate levels Service. Briefly, after quality control, the RNAs were reverse have been noted in HLRCC tumors (9). Because fumarate transcribed to double-stranded cDNAs and biotin-labeled inhibits 2-oxoglutarate–dependent enzymes, one potential complementary RNA (cRNA) was amplified by in vitro tran- pathogenic pathway is the inhibition of hypoxia-inducible fac- scription with T7 RNA polymerase. cRNAs were then hybrid- – μ tor (HIF) hydroxylases (Ki values of 50 80 mol/L; ref. 10), ized to either HGU133A (Human RCC) or MOE430_2 (mouse resulting in “pseudohypoxic” HIF stabilization and HIF target kidney tissue) Affymetrix Chips. Full protocols are available gene upregulation (9, 11, 12). HIF stabilization induces expres- at The Paterson Institute Microarray Service Web site sion of a broad range of target genes, including those that stim- (http://bioinformatics.picr.man.ac.uk/mbcf/protocols.jsp). ulate growth and angiogenesis that may contribute to tumorigenesis (13). Furthermore, it has been observed that Data analysis HIF stabilization in HLRCC may also result from glucose- Chips were quantile normalized and quantified using ro- dependent reactive oxygen species generation (12, 13). In a bust multiarray averaging. A linear model was fitted to the similar manner, fumarate may also inhibit other 2-oxoglutarate– knockout and wild-type groups and differential genes selected dependent dioxygenases, including JMJD6, which catalyzes by applying a 0.001 false discovery rate threshold to P values lysyl hydroxylation of U2AF65, an RNA splicing protein (14); corrected using the Benjamini and Hochberg method. T statis- the JmjC family of histone demethylases (15); and extracellular tics were moderated before transcript selection by empirical matrix processing enzymes (collagen prolyl and lysyl hydroxy- Bayes shrinkage of the standard errors. The analysis was car- lases). Recently, evidence of the contribution of bioenergetics, ried out using the Limma and Affy packages from Bioconduc- as exemplified by the Warburg effect, to tumorigenesis in tor 2.2 within R 2.7.0. FH-deficient cells has been adduced from cell lines including an immortalized line from a patient with aggressive HLRCC- Magnetic resonance spectroscopy metabolite analysis associated recurring kidney cancer (16, 17). Magnetic resonance spectroscopy (MRS) analysis was done We hypothesized that as well as activating a pseudohypoxic as previously described (9). In brief, metabolites were extracted response (18), FH deficiency activates a biosynthetic milieu, from cells and tissue using perchloric acid and freeze dried. The 1 i.e., an environment that is both permissive of and contributo- samples were resuspended in D2O and analyzed using HMR. ry to tumorigenesis. To define the progressive consequences of Media from