AR Grassian

B De Craene

C Frezza

C Loriot

Christian Frezza

IP Tomlinson

M Sciacovelli

M Xiao

Marco Sciacovelli

PJ Pollard

SJ Song

T Laukka

PubMed

Crossref

Cell Death and Differentiation

Fumarate drives EMT in renal cancer

Sciacovelli, M

Frezza, C

Apollo (Cambridge)

OPENNews and CommentaryFumarate drives EMT in renal cancerMarco Sciacovelli1 and Christian Frezza*,1Cell Death and Differentiation (2017) 24, 1–2; doi:10.1038/cdd.2016.137; published online 25 November 2016Fumarate hydratase (FH) is a metabolic enzyme that convertsfumarate to malate. FH mutations predispose to a highlyaggressive and metastatic form of renal cancer. We haverecently shown that the accumulation of fumarate inFH-deficient tumour drives an epigenetic reprogramming thatcontributes to tumour invasiveness.Cancer is a multifactorial disease thought to be driven bygenetic alterations. Mutations in metabolic enzymes of thetricarboxylic acid (TCA) cycle, such as succinate dehydro-genase (SDH), fumarate hydratase (FH) and isocitratedehydrogenase (IDH), lead to cancer, suggesting that alteredmetabolism could play an active role in tumorigenesis.1 Ourlaboratory studies howFH loss leads to renal cancer to dissectthe link between altered metabolism and cancer.FH was originally found mutated in hereditary leiomyoma-tosis and renal cell cancer (HLRCC),2 which causes a veryaggressive and metastatic type of renal cancer. It has beenproposed that fumarate, accumulated upon FH loss, con-tributes to the molecular features of FH-deficient tumours,3acting as an oncometabolite.1 For instance, fumarateinhibits prolyl hydroxylases (PHD), α-ketoglutarate-dependentdioxygenases (aKGDD) involved in HIF degradation, leadingto HIF stabilisation and pseudohypoxia.4 Through covalentbonding with reactive thiol residues of Keap1, fumarate wasalso shown to activate a potent antioxidant responsemediatedby NFE2-related factor 2 (NRF2).1 More recently, it was foundthat fumarate could also inhibit Ten-Eleven Translocation(TET) proteins, another class of aKGDDs involved in DNAdemethylation,5,6 leading to DNA hypermethylation. The roleof this epigenetic alteration in tumorigenesis is still unclear. Inour recent work,7 we have shown that, by virtue of itsepigenetic function, fumarate contributes to the invasivefeatures of FH-deficient renal cancers.Using proteomics and transcriptomics analyses, we demon-strated that epithelial mouse and human FH-deficient renalcells exhibit overlapping mesenchymal features. Furtherfunctional characterisation revealed that the loss of FHactivates an epithelial-to-mesenchymal transition (EMT), aprocess involved in both tumour initiation and progression.8EMT is orchestrated by various transcription factors thatsuppress epithelial genes and activate mesenchymalmarkers. To investigate how the loss of FH could activateEMT, we measured the expression of several EMT-relatedtranscription factors. We showed that Fh1-deficient cellsexpress high levels of Zeb1 and Zeb2 and that miR-200, afamily of miRNAs that suppresses Zeb translation, werestrongly downregulated in these cells. It was recentlyshown9,10 that miR-200 expression is regulated by promotermethylation, which, in turn, is controlled by TET demethylatingactivity. We therefore hypothesised that the epigeneticsuppression of miRNA 200 could be caused by fumarate-driven inhibition of TET. In linewith this hypothesis, we showedthat a regulatory region of mir200ba429 cluster is hyper-methylated in Fh1-deficient cells. Importantly, the epigeneticand phenotypic changes caused by FH loss were recapitulatedby the incubation of Fh1-proficent cells with cell-permeablefumarate (see Figure 1 for a schematic of the pathway). Theseresults showed, for the first time, that fumarate triggers EMTvia the epigenetic suppression of miR-200.The link between fumarate accumulation and EMTwas alsovalidated in renal cancer samples. First, we found that HLRCCpatients exhibited a significant increase in fumarate levels,inhibition of TETactivity, and miR-200 suppression comparedto adjacent normal tissue. Then, taking advantage of the datafrom The Genome Cancer Atlas (TGCA), we found that FHloss resulted in hypermethylation and suppression of miR-200in a larger cohort of FH-deficient renal cancers. Finally, wefound that FH is downregulated in a set of clear cell renal cellcarcinoma, and FH expression is positively correlated to EMTmarkers, confirming the link between FH and EMT in vivo inhereditary and sporadic tumours.Although fumarate accumulation activates a plethora ofsignalling cascades, our findings suggest that the EMT is a keyphenotype of FH-deficient cancer driven caused by fumarate-driven TET-mediated suppression of miR-200. This unex-pected property of fumarate is likely shared by otheroncometabolites. Indeed, both succinate and 2-hydroxygluta-rate, the metabolites accumulated in SDH and IDH mutanttumours, respectively, are also well-established TET inhibi-tors. Interestingly, SDH-deficient pheochromocytomas andparagangliomas,11 Sdhb-deficient cells,7 and IDH-mutantcells also exhibit EMT.12 Our results indicate that, at least inSdhb-deficient cells, EMT induction is mediated by epigeneticsuppression of miR-200, similar to that observed in FH-deficient cells. These results have important implications.First, they indicate that TET inhibition and EMT induction couldbe a convergent mechanism of action of these metabolites.Second, they suggest that EMT could be a common response1MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK*Corresponding author: Christian Frezza, MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Box 197, Cambridge Biomedical Campus,Cambridge CB2 0XZ, UK. Tel: +44 1223 330608; Fax: +44 1223 763241; E-mail: cf366@MRC-CU.cam.ac.ukCell Death and Differentiation (2017) 24, 1–2Ofﬁcial journal of the Cell Death Differentiation Associationwww.nature.com/cddto loss of mitochondrial enzymes and could link mitochondrialdysfunction to tumorigenesis. It is tempting to speculate thatenvironmental conditions that affect mitochondrial functionand, as a consequence, the abundance of TCA cyclemetabolites, such as ischaemia or hypoxia, could also leadto dysregulation of TET activity, promoting tumorigenesis. Inconclusion, our findings go beyond the understanding of howFH mutations lead to HLRCC and pave the way for theunderstanding of how mitochondrial metabolites couldpromote tumorigenesis.Conflict of InterestThe authors declare no conflict of interest.1. Sciacovelli M, Frezza C. Free Radic Biol Med 2016 (this issue).2. Tomlinson IP et al. Nat Genet 2002; 30: 406–410.3. Frezza C et al. Nature 2011; 477: 225–228.4. Pollard PJ et al. Hum Mol Genet 2005; 14: 2231–2239.5. Xiao M et al. Genes Dev 2012; 26: 1326–1338.6. Laukka T et al. J Biol Chem 2015; 291: 4256–4265.7. Sciacovelli M et al. Nature 2016; 537: 544–547.8. De Craene B, Berx G. Nat Rev Cancer 2013; 13: 97–110.9. Song SJ et al. Cell Stem Cell 2013; 13: 87–101.10. Song SJ et al. Cell 2013; 154: 311–324.11. Loriot C et al. J Clin Endocrinol Metab 2012; 97: E954–E962.12. Grassian AR et al. J Biol Chem 2012; 287: 42180–42194.This work is licensed under a Creative CommonsAttribution 4.0 International License. The images orother third party material in this article are included in the article’sCreative Commons license, unless indicated otherwise in the credit line;if the material is not included under the Creative Commons license,users will need to obtain permission from the license holder toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/r The Author(s) 2017Figure 1 Schematic representation of the proposed link between FH loss and EMTactivation. Left panel: In epithelial Fh1-proficient cells, TETs bind the mir-200 regulatoryregion, favouring DNA demethylation and miR-200 expression. miR-200 can then bind its mRNA targets such as Zeb1, efficiently reducing Zeb1 translation. Right panel: After FHloss, fumarate is accumulated and inhibits TETs activity, leading to mir-200 DNA hypermethylation. The reduced expression of miR-200 favours Zeb1 translation and subsequentEMT reprogramming. Cells switch their phenotype, moving from an epithelial to a mesenchymal configurationNews and Commentary2Cell Death and Differentiation

Fumarate drives EMT in renal cancer

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