Ferroptosis-Death by Lipid Peroxidation.pdf

ReviewFerroptosis:Death by LipidPeroxidationWan Seok Yang1,4,*and Brent R.Stockwell1,2,3,*Ferroptosis is a regulated form of cell death driven by loss of activity of the lipidrepair enzyme glutathione peroxidase 4(GPX4)and subsequent accumulationof lipid-based reactive oxygen species(ROS),particularly lipid hydroperoxides.This form of iron-dependent cell death is genetically,biochemically,and mor-phologically distinct from other cell death modalities,including apoptosis,unregulated necrosis,and necroptosis.Ferroptosis is regulated by specificpathways and is involved in diverse biological contexts.Here we summarizethe discovery of ferroptosis,the mechanism of ferroptosis regulation,and itsincreasingly appreciated relevance to both normal and pathological physiology.Discovery of FerroptosisCell death is essential for fundamental physiological processes such as development,immunity,and tissue homeostasis;moreover,cell death is often dysregulated in degenerative andneoplastic diseases.Both apoptotic and nonapoptotic cell death modalities have increasinglybeen necessary to explain diverse biological processes involving cell loss.Two regulated formsof nonapoptotic cell death,necroptosis and ferroptosis,have been shown recently to playsignificant roles in numerous biological contexts 1,2.While the mechanisms and physiologicalrelevance of necroptosis have been reviewed recently 3,we focus here on the molecularmechanisms controlling ferroptosis and its relevance to health and disease.Early life on Earth developed in the absence of oxygen 4;approximately 2.4 billion years ago,the composition of the atmosphere changed dramatically,probably due to oxygen productionfrom emerging photosynthetic organisms 5,6.After this Great Oxygenation Event,oxygen inthe atmosphere ultimately rose from only trace abundances to its current 21%abundance 5,6.Theemergence ofcopiousamountsofoxygenintheatmospherewaschallengingfororganismswith membranes having polyunsaturated lipids that contain bis-allylic carbons,because theseare highly susceptible to lipid peroxidation in the presence of oxygen 7.This peroxidationreaction is dramatically accelerated by divalent metals,especially Fe(II)7.While life probably originated using saturated amphiphilic lipids(i.e.,charged but hydrophobicmolecules lacking carboncarbon double bonds,such as simple fatty acids)8,before long,monounsaturated and polyunsaturated lipids were integrated into lipid metabolism and mem-brane biochemistry,as they allow increased tunability of membrane fluidity 9.However,thepresence of these polyunsaturated fatty acids created a liability after the Great OxygenationEvent,especially because of the abundance of Fe(II)and Fe(II)-dependent enzymes:without ameans to prevent lipid peroxidation,these membranes became a source of damaging oxidativespecies as reactive lipid peroxides were generated.These observations on the early origin and essential functions of polyunsaturated fatty16_TD$DIFF acids,Fe(II)-dependent oxidation chemistry and abundant oxygen suggest the hypothesis that theevolution of defenses against lipid peroxidation was an early selective event in the developmentTrendsFerroptosis is a regulated,nonapopto-tic form of cell death distinct from othercell death modalities.Loss of GPX4 activity and subsequentaccumulation of lipid hydroperoxidesexecutes ferroptosis.DiffuselargeBcelllymphomasandrenalcell carcinomas are particularly suscep-tible to GPX4-regulated ferroptosis.Inhibitionofferroptosismayrepresentapromising therapeutic approach fortreating pathological conditions suchas acute kidney injury15_TD$DIFF.1Department of Biological Sciences,Howard Hughes Medical Institute,Columbia University,New York,NY,USA2Department of Chemistry,ColumbiaUniversity,New York,NY,USA3Department of Systems Biology,Columbia University,New York,NY,USA4Current address:Department ofBiological Sciences,St.JohnsUniversity,Queens,NY,USA*Correspondence:yangwstjohns.edu(W.S.Yang)andbstockwellcolumbia.edu(B.R.Stockwell).TICB 1195 No.of Pages 12Trends in Cell Biology,Month Year,Vol.xx,No.yyhttp:/dx.doi.org/10.1016/j.tcb.2015.10.0141 2015 Elsevier Ltd.All rights reserved.TICB 1195 No.of Pages 12of life.Over time,this defense system was adapted to a more complex control mechanism thatallowed fine-tuned control over lipid peroxidation,enabling it to be harnessed to generatesignaling molecules(e.g.,in the inflammatory cascade),suppressed to preserve cell integrityunder high peroxidation stress(e.g.,in neurons or renal tubules),or unleashed to cause lethallipid peroxidation(e.g.,in17_TD$DIFF nascent neoplastic cells).Ferroptosis is this nonapoptotic,peroxidation-driven form of regulated cell death that requiresabundant and accessible cellular iron;the existence of this ancient form of cell death wasunknown and discovered only recently using a pharmacological approach 10.The firstferroptosis-inducing compounds,erastin 11 and RSL3 12,were discovered using high-throughput screening of small-molecule libraries 11,12.The mode of cell death induced bythese compounds was surprisingly found to be nonapoptotic,as cells treated with erastin andRSL3 died in the absence of apoptotic hallmarks 1113,and in cells where the core apoptosismachinery caspases,BAX,and BAK was suppressed 14.Although nonapoptotic,erastin-induced cell death proceeds normally on knockdown of RIPK1/RIPK3 or pharmacologicalinhibition of RIPK1 10,15(our unpublished data),a known component of necroptosis 16.Therefore,the cell death phenotype induced by erastin and RSL3 is distinct from other reportedmodalities,including apoptosis and necroptosis.Further studies 13,17 identified lipophilic antioxidants(/-tocopherol,butylated hydroxyto-luene,and b-carotene)as strong suppressors of erastin-induced cell death,suggesting thatROS,probably lipophilic in nature,are involved in this cell death process.Analysis withdichlorofluorescein(DCF),a ROS-detecting dye,revealed that erastin causes the generationof ROS in sensitive cell lines 10,13.Moreover,iron chelators were identified as inhibitors of celldeath induction after RSL3 treatment,revealing the requirement for cellular iron 12.Using modulatory profiling,an unbiased pharmacological and genetic profiling system in whichlethal compounds areclassified basedon theirfunctional profiles14,18,erastinand RSL3werefound to cluster together,suggesting that they share a similar cell death mechanism.ThiserastinRSL3 cluster is distinct from other lethal compounds that induce apoptosis andnecrosis.Taking these findings together,the mode of cell death induced by erastin,RSL3,and related compounds was proposed to be a previously unrecognized form of cell deathtermed ferroptosis 18_TD$DIFFferro,ferrous ion(Fe2+);ptosis,fall,suggesting a critical role for cellulariron in this regulated form of oxidative cell death 10.Mechanism of Ferroptosis Induction by Erastin and RSL3Key regulators of ferroptosis have been discovered through characterizing the mechanism ofaction of erastin and RSL3 using multipronged approaches(Figure 1,Key Figure).Ferroptosis Induction by System xc?19_TD$DIFF InhibitionGlutamate-induced toxicity can be initiated by calcium influx after glutamate receptor activation19 or by competitive inhibition of system xc?,the glutamate/cystine antiporter 20,21(Box 1).Glutamate-induced neurotoxicity is an oxidative,iron-dependent process,suggesting thatferroptosis is involved 22,23.Calcium chelators showed no effect on erastin-induced celldeath 14,suggesting that glutamate receptor activation is not involved.In addition,themodulatory profiles of erastin and sulfasalazine(SAS),an inhibitor of system xc?,were similar,suggesting that erastin might act as a system xc?inhibitor to initiate ferroptosis 10.Erastintreatment abolished the import of radiolabeled cystine 10,a substrate for the systemxc?antiporter,confirming that erastin inhibits system xc?.One metabolic consequence of system xc?inhibition is depletion of the intracellular cysteinepool,which is a precursor for glutathione synthesis.A metabolite profiling assay revealed that2Trends in Cell Biology,Month Year,Vol.xx,No.yyTICB 1195 No.of Pages 12glutathione was the most decreased cellular metabolite during erastin-induced ferroptosis 24.This glutathione depletion is sufficient for erastin-dependent cell death,as glutathione depletionby another reagent,buthionine sulfoxamine(BSO),also initiated ferroptosis 24.BSO is aninhibitor of glutamatecysteine ligase,the rate-limiting enzyme for glutathione synthesis.Gluta-thione depletion causes loss of cellular antioxidant capacity as well as inhibition of glutathione-dependent enzymes such as glutathione peroxidases 24,25.Direct Inhibition of GPX4 by RSL3Celldeath inducedbyRSL3sharescommon featureswitherastin-mediated ferroptosis,suchasiron,MEK,and ROS dependency 12.However,RSL3 treatment does not prevent the import ofradiolabeled cystine,indicating that RSL3 does not inhibit system xc?10.Moreover,glutathi-one is not depleted during RSL3-induced cell death 24,supporting the presence of analternative ferroptosis-initiating mechanism for RSL3.Analysis of mass spectrometry-based proteomic data from an affinity pull-down experimentranked GPX4(PHGPx)as the top protein target for(1S,3R)-RSL3 24;western blotting furtherconfirmed binding between(1S,3R)-RSL3 and GPX4.Cell lysates prepared from(1S,3R)-RSL3-treated cells could not reduce phosphatidylcholine hydroperoxides into the correspondingalcohols,demonstrating that RSL3 inhibits GPX4 enzyme activity 24.Moreover,knock downofGPX4expressiongenerateslipidROSandinducesferroptosis,supportingthehypothesisthat(1S,3R)-RSL3 induces ferroptosis through inhibition of GPX4 24.Key FigureMolecular Pathways of Ferroptosis Regulation.System xcGlutamateCys?neCysteineGSHLipid ROS(1S,3R)-RSL3 DPIs Ferroptosis GlutamateEras?nSulfasalazineSorafenibGPX4SeTS pathwayMethioninesiCARSvit.EFerrosta?nsLiproxsta?n-1CoQ10HMG-CoAMevalonatepathwayFIN56 SeLipidFe2+BSODFOCPXFigure 1.Small-molecule inducers of ferroptosis are colored red;small-molecule inhibitors of ferroptosis are colored blue.2_TD$DIFFAbbreviations:TS pathway,transsulfuration pathway;Se,selenocysteine;DFO,deferoxamine;CPX,ciclopirox olamine;CoQ10,coenzyme Q10.Trends in Cell Biology,Month Year,Vol.xx,No.yy3TICB 1195 No.of Pages 12TheidentificationofGPX4asthetargetproteinforRSL3isintriguingfortworeasons.First,GPX4is the only enzyme that can reduce lipid hydroperoxides within biological membranes 25.ThisexplainswhycellstreatedwithRSL3displayelevatedlipidROSduringferroptosisintheabsenceof GSH depletion.The strong protective effect of lipophilic antioxidants such as vitamin E is likelyto be due to their ability to suppress the formation and propagation of oxidized lipids on GPX4inhibition.Second,GPX4 uses glutathione as an essential cofactor for its enzymatic activity 25.As erastin inhibits system xc?and depletes glutathione,the result is that erastin inhibits GPX4 inan indirect manner,which explains how erastin and RSL3 share a common cell death executionmechanism but have different triggering mechanisms(Figure 1).Emerging Ferroptosis-Inducing Compounds and MechanismsAdditional ferroptosis-inducing compounds and mechanisms have been identified and arebroadly categorized as system xc?inhibitors,glutathione depleters,or direct GPX4 inhibitors.Box 1.Cell Death Pathways in Glutamate-Induced NeurotoxicityWhen excessive glutamate is present near nerve cells,a nonapoptotic,oxidative form of cell death occurs.As excessiveamounts of glutamate inhibit system xc?,a cystine/glutamate antiporter,it is likely that glutamate-induced neurotoxicitydisplays many features of ferroptosis.Nerve cells treated with glutamate show decreased cystine import,becomedeficient in cellular glutathione,and undergo oxidative cell death that can be prevented by iron chelation 22,88,89.Theterm oxytosis has been used to describe this mode of neuronal cell death 22.Although oxytosis and ferroptosis displayrelated phenotypes,there exist several notable differences.During oxytosis,high levels of calcium ions(Ca2+)enter cellsand activate numerous destructive enzymes such as serine proteases,calpains,and phospholipases 9092.Theelevated calcium ions also damage mitochondria,causing translocation of apoptosis-inducing factor(AIF)from mito-chondria to the nucleus 93.These biochemical changes are required for the execution of oxytosis whereas they aredispensable for ferroptosis.Oxytosis may represent a combination of the more general ferroptosis and a specialized typeof damage induced by glutamate through other pathways and receptors.Normally,glutamateactsasignalingmolecule(neurotransmitter)thatmediatesmostexcitatorysynaptictransmissionsinthecentral nervous system.This glutamate-mediated signaling occurs through the interactions between glutamate andglutamate receptors in nervecells.It is possible that this glutamate-to-glutamate receptor interaction plays significant rolesin oxytosis in nerve cells,whereas inhibition of system xc?plays a dominant role in ferroptosis in cancer cells(Figure I).GluCysSystem xcNa+,Ca2+GGlu receptors&VDCCsInvolved inferroptosisInvolved in glutamate-induced toxicityExcess glutamateCa2+GSHMitochondriaGPX4SeFigure I.Cell Death Pathways in Glutamate-Induced Neurotoxicity.3_TD$DIFF Abbreviations:VDCCs,voltage-dependentcalcium channels.4Trends in Cell Biology,Month Year,Vol.xx,No.yyTICB 1195 No.of Pages 12Induction of Ferroptosis by DPI CompoundsThe screening assay in which erastin and RSL3 were identified later revealed ten additionalcompounds that induce ferroptosis 24,26.All but one compound(DPI2)inhibited GPX4directly,similar to RSL3.By contrast,DPI2 depleted GSH,like erastin 24.Overexpressionof GPX4 suppressed cell death induced by these compounds 24 but not other lethal agents,indicating that GPX4 is a specific and robust central regulator of ferroptotic cell death,akin toBCL-2 in apoptosis.System xc?Inhibition by SorafenibSorafenib,a multikinase inhibitor,is an FDA-approved drug used for treating advanced renalcellcarcinomaandadvancedhepatocellularcarcinoma(HCC).InHCC cell lines,sorafenibwasreported to induce a nonapoptotic form of cell death that was suppressed by lipophilicantioxidants 27,28,indicating that sorafenib activates ferroptosis in HCC cell lines.Sorafenibactsasaninhibitorofsystemxc?,similartoerastin29.Theadverseeventprofileofsorafenibinpatients was found to be consistent with the notion that sorafenib exerts additional activity invivo compared with other,similar kinase-inhibiting drugs that do not inhibit system xc?29.Recently,retinoblastoma protein(Rb)was found to suppress ferroptosis in response tosorafenib 30,suggesting that Rb status may aid in selecting ferroptosis-sensitive tumors,at least for sorafenib.Ferroptosis Induction by FIN56CIL56 and FIN56 are small molecules discovered from a systematic survey of nonapoptotic celldeath mechanisms 31.CIL56-induced and FIN56-induced cell death is accompanied by lipidROS generation;vitamin E and iron chelators su