Metabolic traits of cancer stem cells

REVIEW SPECIAL COLLECTION: CANCER METABOLISM Me abolic ai s o cance s em cells Joana Peixo o 1,2,3,4 and Jo ge Lima 1,2,3, * ABSTRACT Cance s em cells a e a subpopula ion o cells wi hin a umou belie ed o con e esis ance o s anda d cance he apies. Al hough many s udies ha e add essed he speci ic mechanisms o umou ecu ence d i en by cance s em cells, cellula me abolism is an o en-neglec ed a ibu e. The me abolic ea u es o cance s em cells a e s ill poo ly unde s ood, and hey hus cons i u e a p omising ield in cance esea ch. The indings published so a poin o a dis inc me abolic pheno ype in cance s em cells, which migh depend on he cance ype, he model sys em used o e en he expe imen al design, and se e al con o e sies s ill need o be ackled. This Re iew desc ibes he me abolic pheno ype o cance s em cells by add essing he main me abolic ai s in di e en umou s, including glycolysis and oxida i e, glu amine, a y acid and amino acid me abolism. In he con ex o hese pa hways, we also men ion he speci ic al e a ions in me abolic enzymes and me aboli e le els ha ha e a ole in he egula ion o cance s emness. De e mining he ole o me abolism in suppo ing esis ance o he apy d i en by cance s em cells can aise he oppo uni y o no el he apeu ic a ge s, which migh no only elimina e his esis an popula ion, bu , mo e impo an ly, e adica e he whole umou in a elapse- ee scena io. KEY WORDS: Cance me abolism, Cance s em cells, The apy esis ance, Tumou he e ogenei y In oduc ion Cance is widely known o be a he e ogeneous disease, in which malignan cells communica e wi h a ious o he cell ypes, such as endo helial, haema opoie ic and s omal cells (see Glossa y, Box 1). This complex sys em wi hin a umou can in luence i s own beha iou . Al hough i is clea ha umou he e ogenei y is ela ed o p og ession, he apy esis ance and ecu ence, he mechanisms behind hese links a e s ill o be unco e ed. In his con ex , a subpopula ion o cells wi hin he umou wi h he po en ial o long- e m clonal g ow h and sel - enewal capaci ies – he so-called cance s em-like cells (CSCs) –has been desc ibed as a d i e o umou su i al and esis ance agains commonly used cance he apies. The ole o hese cells in se e al cance s has been s udied equen ly, aiming a disclosing he molecula p og ams ha go e n and main ain he s emness (Box 1) o his popula ion. One o hese molecula p og ams encompasses me abolic al e a ions, which could po en ially become impo an a ge s o he apies aimed a elimina ing his esis an cell popula ion. This Re iew ocuses on he me abolism o cance s em cells, which is cu en ly an eme ging ho opic ha esea che s need o add ess u he and in a sys ema ic way. S em cells and cance s em cells In he la e 19 h cen u y, E ns Haeckel used he e m s em cell (SC) o he i s ime o designa e he commi ed cell ha gi es ise o he ge mline o an o ganism. La e in ha cen u y, Theodo Bo e i and Valen in Häcke pu sued and amelio a ed he concep o SCs in hei emb yological s udies (Bo e i, 1892; Häcke , 1892). In pa allel, A u Pappenheim used he same e m o desc ibe he cell ha is a he basis o he e ol ing genealogy o haema opoiesis (Box 1). I was only in he 1960s ha James Till, E nes McCulloch and o he s p o ided clea e idence o he exis ence o a common haema opoie ic SC (Till and McCulloch, 1961; Till e al., 1964). These disco e ies allowed he es ablishmen o he e m SC, which is nowadays used o de ine a cell capable o p oli e a ing inde ini ely and gi e ise o specialized daugh e cells. By aising many ques ions ega ding emb yonic de elopmen , cellula di e en ia ion and o gan main enance, he ole o SCs began o be exploi ed in disease se ings, speci ically in cance (Ramalho- San os and Willenb ing, 2007). In a umou he e ogenei y – he hie a chical and s ochas ic models The concep o CSCs being iden i ied by he exp ession o a combina ion o ma ke s, and he ac ha hese dis inc popula ions a e able o de elop a seconda y umou ha ecapi ula es he p ope ies o he p ima y umou , was con i med in se e al s udies (Box 2). The e o e, he CSC model o iginally pos ula ed a unidi ec ional hie a chy, whe e asymme ic and symme ic di isions o CSCs p oduce he bulk o he umou o gene a e di e en ia ed cance cells and o sel - enew he CSC pool, espec i ely. Howe e , o he s udies ques ioned he uni e sali y o his hie a chical model, as hey showed ha cance cell plas ici y o en occu s in umou s and ha CSCs pa icipa e in his p ocess (Nassa and Blanpain, 2016; P ase yan i and Medema, 2017). Fo example, s em-like basal and luminal cell popula ions isola ed om human b eas cance cell lines could e e sibly con e in o dis inc cell s a es o p oduce he same h ee subpopula ions in simila p opo ions o he o iginal cell line hey we e isola ed om. Howe e , only he s em-like cells we e able o gene a e umou s upon xeno ansplan a ion, a ypical ea u e o CSCs (Gup a e al., 2011). In a melanoma model, he CSCs exp essing he su ace ma ke CD127 we e shown o be umou igenic, bu we e also able o a ise om CD127 − p ogeny, again indica ing he e e sible pheno ype o cance cells (Quin ana e al., 2010; Ci enni e al., 2011). Also in melanoma, he exp ession o he his one deme hylase (Box 1) JARID1B (also known as KDM5B) de ines a popula ion o slow-cycling cells wi h umou igenic po en ial, i.e. CSCs. In e es ingly, JARID1B − cells we e shown o e-exp ess he 1 Cance Signalling and Me abolism G oup, Ins i u o de In es igaçao e Ino açao em Saude (I3S), Uni e sidade do Po o, 4200-135 Po o, Po ugal. 2 Cance Signalling and Me abolism G oup, Ins i u e o Molecula Pa hology and Immunology o he Uni e si y o Po o (Ipa imup), 4200-465 Po o, Po ugal. 3 Medical Facul y o he Uni e si y o Po o, 4200-319 Po o, Po ugal. 4 Depa men o Biochemis y and Molecula Biology, Theodo -Bo e i-Ins i u e, Biocen e , 97074 Wu  zbu g, Ge many. *Au ho o co espondence ([email p o ec ed]) J.L., 0000-0001-7780-0901 This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/3.0), which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium p o ided ha he o iginal wo k is p ope ly a ibu ed. 1 © 2018. Published by The Company o Biologis s L d | Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms ma ke upon ansplan a ion, meaning ha non-CSCs can ewi e o a CSC s a e (Roesch e al., 2010). CSCs om mouse skin squamous cell ca cinoma exp ess CD34 and he ansc ip ion ac o Sox2 and show plas ici y. In his mu ine model, CD34 − and Sox2 − cells we e bo h able o o m umou s a e ansplan a ion, p oducing CD34- and Sox2-posi i e and -nega i e popula ions in simila p opo ions o he pa en al umou (Schobe and Fuchs, 2011; Boumahdi e al., 2014). In con as , abla ion o s em-like cells in a mouse model o glioblas oma was appa en ly su icien o umou g ow h a es , as non-CSCs did no eplenish a CSC popula ion, which sugges s a unidi ec ional hie a chy in his umou (Chen e al., 2012). Mo e ecen s udies, howe e , sugges ed ha plas ici y migh occu in glioblas oma unde speci ic condi ions and lead o cell ep og amming (Su à e al., 2014). In ac , non-CSCs can dedi e en ia e and acqui e he exp ession o CSC ma ke s in glioblas oma unde hypoxic condi ions (Wang e al., 2017a) o a e ea men wi h emozolomide (Box 1) (Au inge e al., 2014) o ionizing adia ion (Dahan e al., 2014). Thus, in con as o he unidi ec ional hie a chic umou model, a s ochas ic umou model wi h a mo e luid hie a chy is now accep ed, whe e cance cells ha e he plas ici y o dynamically con e om a non-CSC o a CSC pheno ype and ice e sa in esponse o app op ia e s imuli (Meacham and Mo ison, 2013; Medema, 2013). Ne e heless, u he s udies a e equi ed o explo e he mechanisms ha egula e he e e sibili y o he CSC pheno ype, because his plas ici y implies majo challenges in he e adica ion o he apy- esis an cells in cance , such as CSCs. Speci ically, ex insic ac o s can in luence and al e umou me abolism, a e y impo an ea u e o cance cells ha can egula e and main ain cellula i ness in ha sh condi ions. Al e ed me abolism as a hallma k o cance The ep og amming o cellula ene gy me abolism is a classical ea u e o cance , mainly used by cance cells o sus ain hei highly p oli e a i e s a us (Hanahan and Weinbe g, 2011). Unde ae obic condi ions, no mal nonp oli e a ing cells use glycolysis in he cy oplasm o o m py u a e, which is hen oxidized in mi ochond ial oxida i e phospho yla ion (OXPHOS) o gene a e ene gy in he o m o adenosine iphospha e (ATP). Unde anae obic condi ions, glycolysis-de i ed py u a e is mainly di ec ed o lac a e p oduc ion. In con as , cance cells ely mo e on glycolysis o ene gy p oduc ion e en in he p esence o oxygen, a phenomenon i s obse ed by O o Wa bu g and e med ‘ae obic glycolysis’o ‘ he Wa bu g e ec ’(Wa bu g, 1956a,b). This me abolic adap a ion, al hough gene a ing ATP mo e apidly, is a less e icien han OXPHOS, esul ing in abno mally high glucose up ake o sus ain ATP p oduc ion. The a idi y o cance cells o glucose, mainly media ed by he up egula ion o glucose anspo e 1 (GLUT1; also known as SLC2A1), con ibu ed o he de elopmen o luo odeoxyglucose-posi on emission omog aphy (FDG-PET; Box 1) echniques o cance de ec ion and moni o ing (Vande Heiden e al., 2009). As demons a ed in se e al s udies discussed below, inc eased glycolysis allows he p oduc ion o se e al me abolic in e media es ha can eed al e na i e biosyn he ic pa hways o gene a e mac omolecules, such as nucleosides, amino acids and lipids, which can hen be used as building blocks o suppo he high p oli e a ion and di ision a es o cance cells, con e ing a selec i e ad an age. The ac o s unde lying he me abolic al e a ions o cance cells a e he subjec o in ense s udy. Oncogenes o umou supp esso s, bu also he umou mic oen i onmen (TME), can ep og am Box 1. Glossa y Anaple o ic lux: he ac o eplenishing TCA cycle in e media es, as opposed o he use o hese molecules as subs a es o biosyn he ic eac ions (ca aple osis). Cispla in: a pla inum-based chemo he apeu ic d ug used o ea se e al cance s, including sa comas, lung cance , o a ian cance , lymphomas and ge m cell umou s. Endo helial cells: a g oup o cells ha o m he su ace o blood and lympha ic essels and unc ion as a ba ie be ween he essel lumen and he su ounding issue, being in ol ed in he o ma ion o new blood essels (angiogenesis). Fluo odeoxyglucose-posi on emission omog aphy (FDG-PET): an imaging echnique ha uses a glucose analog (FDG) o he isualiza ion o solid cance s. This echnique elies on he ac ha cance cells ha e inc eased glucose up ake, p o iding aluable in o ma ion ega ding he localiza ion and size o he umou o diagnosis, s aging and moni o ing pu poses. Haema opoiesis: he p ocess ha yields he o ma ion o blood cells, om a s em cell in o a ully di e en ia ed blood cell. Haema opoie ic cells: s em cells gene ally ound in he bone ma ow ha p oduce all he di e en blood cell ypes, by p og essing h ough commi ed p ogeni o s ages un il hey ully di e en ia e in o ma u e cells. Hexosamine pa hway: a b anch o he glycolysis pa hway in which he building blocks o glycosyl side chains o p o eins and lipids a e p oduced. I is associa ed wi h pos - ansla ional modi ica ions, speci ically glycosyla ion. His one deme hylase: an enzyme esponsible o emo ing me hyl g oups om his ones ha egula e ch oma in a chi ec u e. Induced plu ipo en s em cells: a ype o plu ipo en s em cell de i ed om adul cells by in oducing a speci ic se o ep og amming ac o s, which induce plu ipo ency-associa ed genes. In es inal c yp s: ana omical uni s whe e in es inal s em cells a e loca ed o he ac i e sel - enewal o he in es inal epi helium. Leuko ienes: a amily o eicosanoid in lamma o y media o s p oduced by leukocy es,mas ocy oma cells, mac ophages and o he cells, in esponse o immunological s imuli. Mammosphe es: ound-shaped s uc u es o mamma y cells, o med in i o unde ce ain cul u e condi ions o en ich o s em cells. Maphosphamide: he ac i e analogue o he chemo he apeu ic d ug cyclophosphamide, which is equen ly used o in i o expe imen s. Me o min: a biguanide d ug used as a i s -line he apy o ype 2 diabe es. I is also used as an an i umou agen ha a ec s me abolism by di ec ly inhibi ing espi a o y chain complex I in he mi ochond ia. Nanog: a DNA-binding homeobox ansc ip ion ac o in ol ed in sel - enewal and undi e en ia ion o emb yonic s em cells. I is also b oadly exp essed in human cance s, hus used as a cance s em cell ma ke . Pacli axel: a chemo he apeu ic d ug ha binds o ubulin and inhibi s he disassembly o mic o ubules, ul ima ely inhibi ing cell di ision. Pane h cells: cells in he in es inal epi helium ha a e loca ed in he c yp s along wi h in es inal s em cells. Pen ose phospha e pa hway (PPP): a mul i-s ep me abolic pa hway pa allel o glycolysis o he oxida ion o glucose, which p oduces NADPH and ibose 5-phospha e ha can be used o nucleo ide syn hesis. Sa elli e muscle cells: quiescen s em cells o he skele al muscle ha unc ion as a ese e popula ion o cells and p oli e a e in esponse o inju y. Sec e ome: he collec ion o ac o s eleased by a cell, including ex acellula ma ix p o eins, ansmemb ane p o eins and esicle p o eins. S emness: he essen ial ai o s em cells: hei abili y o sel - enew and di e en ia e in o a ious commi ed cells. S omal cells: a g oup o connec i e issue cells (such as ib oblas s) ha suppo he unc ion o o he cells wi hin an o gan. Temozolomide: an alkyla ing chemo he apeu ic d ug used as ea men o b ain umou s. 13 C-glucose: a non adioac i e na u ally occu ing glucose iso opome in which all six ca bons a e 13 C labelled. 2 REVIEW Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms cance me abolism by di ec ly egula ing speci ic me abolic enzymes. Oncogenic mu a ions in phospha idylinosi ol 3-kinases (PI3K; also known as PIK3CA) p omo e me abolic ep og amming by enhancing AKT [also known as AKT1 o p o ein kinase B (PKB)] signalling, which, in u n, d i es glycoly ic me abolism by inc easing cellula glucose up ake and inducing he ac i a ion o phospho uc okinase 1 (PFK1) (Dep ez e al., 1997; Els om e al., 2004; Manning and Can ley, 2007). In addi ion, AKT s imula es he mammalian a ge o apamycin (mTOR) pa hway, which also p omo es glycolysis and he pen ose phospha e pa hway (PPP; Box 1) h ough he egula ion o hypoxia-inducible ac o s (HIFs) (Dü el e al., 2010). Simila ly, Myc dys egula ion in cance is associa ed wi h inducing he exp ession o glycoly ic genes, causing a shi o glucose consump ion, as well as biomolecule p oduc ion ia nucleo ide and lipid syn hesis (Shim e al., 1997; Os hus e al., 2000; Niki o o e al., 2002; Kim e al., 2007; Mo ish e al., 2010). Mu a ions in he small GTPase RAS sub amily ha e also been associa ed wi h me abolic ep og amming owa ds glycolysis, he hexosamine pa hway (Box 1) and he PPP, in a p ocess media ed by he PI3K/AKT/mTOR axis o by MYC (Ramana han e al., 2005; Gaglio e al., 2011; Ying e al., 2012). The umou supp esso p53 (also known as TP53) inhibi s glucose anspo e s and igge s he up egula ion o TP53-induced glycolysis egula o (TIGAR), causing a dec ease in uc ose 2,6-biphospha e le els and hus inhibi ion o PFK1 (Bensaad e al., 2006). In addi ion, p53 s imula es he exp ession o he gene encoding he syn hesis o cy och ome c oxidase p o ein (SCO2), a subuni o complex IV o he elec on anspo chain equi ed o assembly o he cy och ome c oxidase (COX) complex (Ma oba e al., 2006). Thus, loss o p53 p omo es a shi in ATP p oduc ion om OXPHOS o glycolysis, bu ende s cance cells mo e sensi i e o me abolic s ess (Ma oba e al., 2006). The in ol emen o he TME in me abolic ep og amming is mainly media ed by HIF-1αand HIF-2α. The up egula ion o HIF-1αand HIF-2αunde hypoxic condi ions is one o he mechanisms by which umou cells can igge he swi ch om OXPHOS o glycolysis. Speci ically, HIF-1αinduces he exp ession o GLUT1, and up egula es glycoly ic enzymes and lac a e dehyd ogenase A (LDHA), wi h concomi an ac i a ion o Box 2. Cance s em cells Tumou he e ogenei y is o en e lec ed in he exp ession o many di e en his ological ma ke s, despi e he ac ha umou s a e belie ed o a ise om a single mu a ed cell (Nassa and Blanpain, 2016). Ad anced echnologies, such as luo escence-ac i a ed cell so ing and mouse xenog a s assays, allowed s udies in haema opoie ic s em cells (SCs) ha deli e ed se s o cell-su ace ma ke s, which we e c ucial o de ining umou he e ogenei y in acu e myeloid leukaemias. Lapido and colleagues ound ha only he popula ion o leukaemic cells ha we e posi i e o CD34 and nega i e o CD38 (CD34 + CD38 − )could ini ia e leukaemic eng a men in immune-de icien mice, and ha he equency o hese umou -ini ia ing cells was abou one pe million cance cells (Lapido e al., 1994; Bonne and Dick, 1997). The e m cance s em-like cells (CSCs) was hen coined o de ine a small popula ion o cells wi hin he cance ha exp esses speci ic ma ke s, and ha , when ansplan ed in immune-de icien mice, is able o ecapi ula e some o he he e ogenei y o he o iginal malignancy (Cle e s, 2011; Ba lle and Cle e s, 2017). These indings we e subsequen ly ecapi ula ed in s udies o solid umou s. B eas cance was he i s human umou demons a ed o consis o he e ogeneous popula ions o cells, speci ically a subpopula ion capable o ini ia ing umou g ow h in immune-de icien mice. These cells we e pheno ypically CD44 + CD24 −/low and as ew as 100 cells we e capable o o ming umou s, in con as o he ens o housands o cells wi h o he pheno ypes ha could no e o m umou s in mice (Al-Hajj e al., 2003). Mo eo e , using he same expe imen al app oaches, he speci ic ma ke s ha pinpoin ed hose cells o ini ia e umou s in immune-de icien mice we e iden i ied in se e al o he malignancies, such as panc ea ic (He mann e al., 2007; Li e al., 2007) and colon cance (O’B ien e al., 2007; Ricci-Vi iani e al., 2007), melanoma (Scha on e al., 2008; Boiko e al., 2010), o a ian (Zhang e al., 2008) and lung cance (E amo e al., 2008), and b ain umou s (Singh e al., 2004; Chen e al., 2010). Howe e , i became clea ha he cell ansplan a ion assay was mainly app op ia e o haema ological malignancies, in which a de ined s em-p ogeni o hie a chy, wi h speci ic ma ke s, was alida ed in no mal haema opoie ic SCs and used o he de ini ion o leukaemia SCs (Lapido e al., 1994; Bonne and Dick, 1997). In mos solid cance s, his cellula hie a chy and he speci ic ma ke s o he issue o o igin a e s ill unknown. Commonly used ma ke s, such as CD133, ha e been ex ensi ely used in b ain (Singh e al., 2004; Bao e al., 2006) and colon umou s (O’B ien e al., 2007; Ricci-Vi iani e al., 2007) o he cha ac e iza ion o CSCs, bu he ep oducibili y o he indings using his ma ke has been ques ioned (Shmelko e al., 2008; Wang e al., 2008; Chen e al., 2010). Mo eo e , when so ed o a speci ic ma ke , bo h ma ke -posi i e and -nega i e popula ions we e capable o egene a ing he o iginal ma ke exp ession o he umou (Shackle on e al., 2009; Quin ana e al., 2010). The ac ha in a umou he e ogenei y is mo e p onounced in solid umou s han in leukaemias, oge he wi h he lack o speci ic CSC ma ke s, also limi s he uni e sali y o his app oach o he s udy o CSCs (Ba lle and Cle e s, 2017). I should also be no ed ha ansplan a ion s udies a e limi ed o demons a ing ha a speci ic cell popula ion adap s o pa icula assay condi ions, and hus canno disclose he a e o hese cells in hei o iginal mic oen i onmen (Cle e s, 2011; Nassa and Blanpain, 2016). In an a emp o o e come he a o emen ioned limi a ions, esea che s de eloped gene ic lineage- acing app oaches o s em cell s udies. The ad an age o lineage acing, as opposed o isola ion and ansplan a ion s udies, is ha knowing which ma ke s a e exp essed by he cell o in e es is no equi ed. When acing a pa icula cell ype by pe manen ly labelling i s p ogeny wi h a epo e gene, i is possible o iden i y cells wi h SC po en ial and p o ide insigh s in o he dynamics o s em and p ogeni o cells du ing de elopmen , issue main enance and epai and, ul ima ely, hei dys egula ion in cance (K e zschma and Wa , 2012; Blanpain and Simons, 2013). Lineage acing in umou s p o ided he e idence o a hie a chical o ganiza ion in solid umou s and he e o e consis en ly p o ed he exis ence o CSCs. Lineage acing o he basal-cell-speci ic ke a in 14 in papillomas demons a ed he long- e m su i al o a popula ion o cells, which ga e ise o la ge clonal popula ions wi hin he umou (D iessens e al., 2012). Ano he s udy in a b eas cance mouse model demons a ed ha some clones apidly g ew, becoming dominan . CSCs we e he o igin o hese dominan clones and we e able o di ide in o di e en ia ed umou cells and in o new CSCs o sus ain umou g ow h (Zome e al., 2013). In colo ec al cance pa ien -de i ed o ganoids, he use o CRISPR-Cas9 gene edi ing o inse casse es in o he LGR5 locus –a bioma ke o adul s em cells in ce ain issues –enabled lineage acing and e ealed ha LGR5 + cells su i e o long ime pe iods, p oducing p ogenies ha a e capable o o ming umou s (Co ina e al., 2017; Shimokawa e al., 2017). The o ma ion o la ge clonal popula ions om APC-dele ed Lg 5 + cells was obse ed by lineage acing, enabling he iden i ica ion o CSCs in a mouse model o in es inal adenoma (Schepe s e al., 2012; Koza e al., 2013). Fu he mo e, Chen e al. used a gene ically enginee ed mouse glioma model wi h a Nes-ΔTK-IRES-GFP ansgene and iden i ied a popula ion o endogenous cells ha we e esponsible o umou ecu ence. This ansgene labelled no mal b ain SCs in he sub en icula zone and also a es ic ed popula ion o slow-cycling endogenous gliomacells. A e umou p oli e a ion a es , he nes in + cells e-en e ed he cell cycle and p oduced highly p oli e a i e cells ha con ibu ed o umou elapse. Ta ge ed abla ion o hese cells inhibi ed umou p og ession and ecu ence. This s udy showed ha nes in + cells had CSC-like p ope ies, because hey demons a ed he capaci y o long- e m umou g ow h and media ed umou elapse ollowing he apy (Chen e al., 2012). O e all, he exis ence o CSCs wi hin he umou is now accep ed as an impo an ea u e o cance and hus, new app oaches o s udying CSCs a e being de eloped o ind no el he apeu ic a ge s. 3 REVIEW Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms py u a e dehyd ogenase kinase 1 (PDK1), a nega i e egula o o py u a e dehyd ogenase (PDH) (Semenza e al., 1994; Kim e al., 2006; Papand eou e al., 2006). In addi ion o glucose me abolic changes, inc eased glu amine me abolism is a common ea u e o cance cells (Medina e al., 1992; Souba, 1993). Glu amine is essen ial in p oli e a ing umou cells, p o iding de ence mechanisms agains oxida i e s ess, syn hesising mac omolecules when glucose me abolism is no su icien and, ul ima ely, uelling cellula bioene ge ics (Debe a dinis e al., 2008). Fu he mo e, oncogenes such as MYC can in luence glu amine me abolism, as hey do o glucose me abolism (Wise e al., 2008). MYC s imula es he exp ession o su ace anspo e s o d i e glu amine me abolism and can also egula e glu aminase (GLS) by sup essing he mic oRNAs esponsible o p e en ing GLS ansla ion (Gao e al., 2009). Glu amine dependency can also modula e he signal ansduc ion pa hways ha con ibu e o umou g ow h. Fo example in HeLa cells, glu amine excess leads o a bidi ec ional anspo o his amino acid h ough memb ane anspo e s, accompanied by an impo o o he essen ial amino acids (Nicklin e al., 2009). This mechanism subsequen ly ac i a es mTORC1, s imula ing cell g ow h and sup essing ca abolism and au ophagy. ERK (also known as EPHB2) signalling is ano he example o glu amine-dependen ac i a ion ound in in es inal epi helial cells (Rhoads e al., 1997; La son e al., 2007), and in melanoma (Pollock e al., 2003; Oh ani e al., 2008) and glioma cells (A cella e al., 2005). A majo ou come o bo h glucose and glu amine me abolism is he p oduc ion o ci a e o suppo cellula bioene ge ics and o p oduce biomass, namely nucleic acids, p o eins and lipids, necessa y o cell p oli e a ion. The me abolic a e o ci a e p oduced by hese wo main me abolic p ocesses is de ined by i s subcellula localiza ion: mi ochond ial ci a e is shu led o he ica boxylic acid (TCA) cycle, whe eas cy oplasmic ci a e eeds a y acid syn hesis (Cu ie e al., 2013). Lipid me abolism is ano he majo sou ce o me abolic in e media es and ene gy o p ocesses in ol ed in cell ans o ma ion and umou p og ession (San os and Schulze, 2012). Cance cells can ul il hei s ong a idi y o lipids ei he by inc easing exogenous lipid up ake o endogenous p oduc ion h ough de no o syn hesis (Medes e al., 1953; Ookh ens e al., 1984). Lipid syn hesis equi es se e al s eps o con e ci a e in o bioac i e a y acids, which a e unde aken by ATP ci a e lyase (ACLY), ace yl-CoA ca boxylase (ACC), a y acid syn hase (FASN) and acyl-CoA syn he ase (ACS). Addi ionally, a y acid biosyn hesis is mainly con olled by s e ol egula o y elemen -binding p o eins (SREBPs), a amily o ansc ip ion ac o s ha bind o s e ol egula o y elemen s and some E-box sequences in he p omo e s o a ge genes (Röh ig and Schulze, 2016). SREBP1 (also known as SREBF1) can be ac i a ed h ough an AKT-mTORC1 signalling axis, hus p omo ing lipid syn hesis and cell g ow h (Po s mann e al., 2008). In glioblas omas, he p esence o a cons i u i ely ac i e mu an epi helial g ow h ac o ecep o (EGFR), he EGFR a ian III (EGFR III), unc ions as an enhance o ac i a ed SREBP1 in he nucleus, being co ela ed wi h inc eased le els o FASN and ACC (Guo e al., 2009). In glioblas omas ha do no ca y EGFR mu a ions, bu ins ead ha e AKT pa hway ac i a ion, silencing o SREBP1 o SREBP2 p e en ed xenog a g ow h, con i ming he impo ance o a y acid me abolism in umou main enance (G i i hs e al., 2013; Williams e al., 2013). Fu he mo e, s ea oyl-CoA desa u ase (SCD), an enzyme in ol ed in a y acid biosyn hesis and a a ge gene o SREBP1, is o e exp essed in se e al human cance s (Li e al., 1994; Fal ella e al., 2002; F i z e al., 2010). Silencing o SCD inhibi s lipid syn hesis and ac i a es AMP-ac i a ed p o ein kinase (AMPK; also known as PRKAA2), which in u n inc eases β-oxida ion o a y acids (Dob zyn e al., 2004), leading o umou size educ ion in xenog a models o li e (Budhu e al., 2013), lung (Scaglia and Igal, 2008) and s omach (Roong a e al., 2011) cance s, as well as o inhibi ion o p os a e cance p og ession in mice (F i z e al., 2010). As a disease ha equi es building blocks o cell p oli e a ion and su i al, cance is cha ac e ized by me abolic emodelling mean o accumula ing me abolic in e media es, which a e hen used as a sou ce o biomass. In he se ing o SCs and CSCs, such al e ed me abolism is also able o shape and egula e he a e and unc ion o hese speci ic cells. Me abolic pheno ypes o SCs and CSCs In gene al, no mal issue hie a chy holds a me abolic pheno ype whe e mul ipo en SCs p ima ily pe o m glycolysis, while di e en ia ed cells a e mo e elian on OXPHOS. The i s s udies on he me abolic pheno ype o SCs, again using haema opoie ic cells, e ealed ha SCs eside in hypoxic niches and use mainly glycolysis (Pa ma e al., 2007; Suda e al., 2011). Addi ionally, hey ha e ewe and less ma u e mi ochond ia han di e en ia ed cells, esul ing in a lowe p oduc ion o eac i e oxygen species (ROS) (Jang and Sha kis, 2007; P igione e al., 2010). In con as , he di e en ia ed p ogeny o haema opoie ic SCs shi o OXPHOS and inc eased p oduc ion o ROS. Al hough OXPHOS is c ucial o he ene gy demands o complex issues, SCs migh a oid his me abolic pheno ype because o he esul ing high le els o ROS, which in u n could lead o SC dys unc ion (Simsek e al., 2010; Suda e al., 2011; Ma yano ich e al., 2015). The e o e, he quiescen s a e o adul SCs can se e as a p o ec i e mechanism agains oxida i e s ess- ela ed damage, ensu ing he in ini e sel - enewal capaci y o hese cells (Folmes e al., 2012). Fu he mo e, cellula me abolism can ac ually con ol s emness. When ep og amming soma ic cells in o induced plu ipo en SCs (Box 1), he up egula ion o glycoly ic genes p eceded he exp ession o plu ipo ency ma ke s, e ealing ha he me abolic swi ch om OXPHOS o glycolysis is an ea ly e en du ing SC ep og amming (Folmes e al., 2011; Panopoulos e al., 2012). Howe e , his me abolic pa e n can di e be ween adul SC popula ions. Fo example, in in es inal c yp s (Box 1), Lg 5 + SCs ha e inc eased OXPHOS, whe eas Pane h cells (Box 1) p e e en ially use glycolysis. Pane h cells egula e he enewal o Lg 5 + SCs by p oducing lac a e o he oxida i e me abolism o he SCs (Rod íguez-Colman e al., 2017). Appa en ly, high ROS le els a e no ha m ul o in es inal SCs, bu a he induce hei di e en ia ion (Yilmaz e al., 2012; Rod íguez-Colman e al., 2017). Ano he example is sa elli e muscle cells (Box 1), which a e localized in ae obic niches and use mainly OXPHOS; in con as , commi ed p ogenies o hese SCs unde go epigene ic ep og amming consis en wi h a shi o glycoly ic me abolism (Ryall e al., 2015). The me abolic pheno ype o CSCs has been s udied o e he pas ew yea s and, con a ily o wha was hypo hesized, CSCs do no ecapi ula e he me abolic pa e n o adul SCs. In ac , CSCs can p ima ily ely ei he on glycolysis o on OXPHOS, mainly depending on he umou ype and TME s imuli ha igge cell plas ici y and me abolic ep og amming (Sancho e al., 2016). Glycolysis Glycolysis is an oxygen-independen me abolic pa hway ha occu s in he cy osol, gene a ing ATP om he con e sion o glucose 4 REVIEW Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms in o py u a e. I consis s o h ee main eac ions: phospho yla ion o glucose by hexokinase o o m glucose 6-phospha e and subsequen ly uc ose 1,6-biphospha e (F1,6P); clea age o F1,6P in o wo h ee-ca bon p oduc s (glyce aldehyde 3-phospha e and dihyd oxyace one phospha e); and oxida ion o hese h ee-ca bon p oduc s o py u a e, wi h ATP p oduc ion. As discussed below, CSCs usually ha e signi ican ly inc eased glucose up ake and lac a e p oduc ion, oge he wi h a dec ease in mi ochond ial espi a ion, when compa ed wi h hei ma u e non- CSC coun e pa s (Fig. 1A,B). In b eas cance cell lines, a swi ch om mi ochond ial espi a ion o glycolysis dec eases he le els o ROS, a mechanism ha is essen ial o he main enance o s emness in CD44 + CD24 − EPCAM + cells (Dong e al., 2013). Besides, key glycolysis enzymes, such as py u a e kinase M2 (PKM2; also known as PKM), LDH and glucose-6-phospha e dehyd ogenase (G6PDH) ha e inc eased ac i i y in b eas CSCs, while ea men wi h 2-deoxyglucose (2DG), a glucose analogue ha inhibi s hexokinase 2 (HK2), p e e en ially dec eases he p oli e a ion o hese cells compa ed wi h ma u e cance cells, showing ha glycolysis is essen ial o b eas CSCs (Cia a delli e al., 2014). CSCs isola ed om human glioblas oma xenog a s also ha e inc eased glycolysis and low mi ochond ial espi a ion, wi h a down egula ion o he succina e dehyd ogenase subuni B (SDHB) and subsequen mi ochond ial dys unc ion, leading o inc eased elec on leakage and ROS p oduc ion. Th ough an ROS-media ed mechanism, he basal le els o HIF-1αand HIF-2αinc ease, p omo ing glycolysis by up egula ing GLUT1 and HK2 in hese human glioblas oma SCs. Fu he mo e, hypoxia ende s glioblas oma SCs esis an o con en ional an icance agen s and sensi i e o glycoly ic inhibi ion, sugges ing ha hey ha e a p e e ence o hypoxic en i onmen s and a glycoly ic me abolism o main ain hei s emness (Zhou e al., 2011). Genes in ol ed in glycolysis, namely PKM2 and 6-phospho uc o-2-kinase/ uc ose-2,6-biphospha ase 4 (PFKFB4), we e also iden i ied as s emness egula o s in glioma SCs (Mo ouace e al., 2014) (Fig. 1A). On one hand, expe imen al knockdown o PFKFB4 caused a educ ion in lac a e and ATP p oduc ion, inducing apop osis o glioblas oma SCs. On he o he hand, o e exp ession o his enzyme was associa ed wi h sho e su i al o glioblas oma pa ien s (Goid s e al., 2012). In nu ien -dep i ed condi ions, glioblas oma SCs also showed an up egula ion o GLUT3 (also known as SLC2A3), a anspo e wi h highe a ini y o glucose han GLUT1, in o de o p ese e glycolysis and main ain s emness (Fla ahan e al., 2013). Mao e al. ound ha wo dis inc umou - de i ed glioblas oma SC sub ypes –p oneu al and mesenchymal glioblas oma SCs –we e p ominen ly co ela ed wi h he clinically ecognized p oneu al and mesenchymal sub ypes o glioblas oma and had dis inc dys egula ed signalling pa hways (Mao e al., 2013). Mesenchymal glioblas oma SCs highly exp essed he aldehyde dehyd ogenase (ALDH) amily, especially he enzyme ALDH1A3, which is in ol ed in glycolysis, among o he unc ions. Inhibi ion o ALDH1A3 a enua es he g ow h o his sub ype o CSCs, bu no o he p oneu al sub ype, sugges ing ha di e en CSC popula ions can ha e dis inc s emness- egula ing me abolic pa hways (Mao e al., 2013). To u he highligh hei me abolic a iabili y, glioblas oma SCs we e also epo ed o consume less glucose and p oduce less lac a e, while ha ing highe ATP le els, when compa ed wi h di e en ia ed cance cells (Vlashi e al., 2011). In his s udy, inhibi ion o ei he glycolysis o mi ochond ial espi a ion in CSCs had minimal e ec on ene gy p oduc ion and only he combined inhibi ion o bo h pa hways was able o deple e in acellula ATP le els. These CSCs e ealed me abolic plas ici y ea u es, indica ing ha a ge ing speci ic me abolic pa hways indi idually migh no be su icien o e adica e glioblas oma SCs (Vlashi e al., 2011). In a mouse model o hepa ocellula ca cinoma, CSCs exp essing Nanog (Box 1) ha e inc eased glycoly ic ac i i y and a y acid oxida ion (FAO), dec eased mi ochond ial espi a ion –owing o cy och ome C oxidase subuni 6A2 (Cox6a2) ep ession, and inhibi ion o ROS gene a ion (Fig. 1A), sugges ing ha a de ined me abolic pa e n egula es s emness in his model (Chen e al., 2016). Emmink e al. compa ed he sec e ome (Box 1) o CSCs and di e en ia ed cells om colo ec al umou s, and obse ed ha CSCs ha e en iched le els o p o eins in ol ed in glycolysis and an ioxidan pa hways. Fu he mo e, CSCs sec e ed high le els o ALDH, which is implica ed in he de oxi ica ion om an icance d ugs, such as maphosphamide (Box 1). By sec e ing his d ug- de oxi ying enzyme, CSCs could no only p omo e sel - p ese a ion, bu also p o ec he di e en ia ed ma u e cance cells in hei icini y. B ie ly, colo ec al CSCs had a su i al and an ioxidan signa u e, bo h o which con ibu ed o he apy esis ance (Emmink e al., 2013). In a me abolic s udy ha used 13 C-glucose (Box 1) in o a ian cance , CSCs showed an en ichmen in glycolysis, he PPP and de no o a y acid syn hesis, while ha ing a dec ease in mi ochond ial espi a ion and anaple o ic lux (Box 1, Fig. 1A); in con as , ma u e cance cells showed inc eased mi ochond ial espi a ion and highe anaple o ic lux (Fig. 1B). Addi ionally, cispla in ea men (Box 1) esul ed in highe su i al o o a ian CSCs in compa ison wi h ma u e cance cells. Thus, he au ho s concluded ha his me abolic pheno ype o CSCs migh con ibu e o mo e agg essi e umou s and con e inc eased he apy esis ance (Liao e al., 2014). Mi ochond ial espi a ion Mi ochond ial espi a ion comp ises a se ies o chemical eac ions ha occu in he mi ochond ia, o gene a ing ATP in he p esence o oxygen. This me abolic pa hway is a mo e e icien in ene gy p oduc ion han glycolysis, gene a ing 36 molecules o ATP pe molecule o glucose, as opposed o wo molecules o ATP p oduced in glycolysis. As b ie ly men ioned in he p e ious sec ion, he e a e epo s claiming ha CSCs consume less glucose, p oduce less lac a e and a e mainly OXPHOS dependen and less glycoly ic han hei di e en ia ed coun e pa s. Leukaemia SCs, al hough showing low le els o ROS, ha e an o e ac i e BCL-2-dependen OXPHOS; indeed, inhibi ing BCL-2 educes OXPHOS and can he e o e e adica e CSCs (Lagadinou e al., 2013). Likewise, b eas cance cell lines wi h highe mi ochond ial mass and ac i i y we e en iched in CSC ma ke s, had a highe e iciency in o ming mammosphe es (Box 1), had inc eased umou ini ia ion capaci y in mu ine xenog a s and we e esis an o pacli axel ea men . The au ho s hus conside ed mi ochond ial mass a po en ial me abolic bioma ke o CSCs (Fa nie e al., 2015; Lamb e al., 2015). In o a ian cance , CSCs also had an OXPHOS-domina ed me abolic p o ile wi h high ROS p oduc ion and inc eased mi ochond ial memb ane po en ial (Pas ò e al., 2014). Panc ea ic duc al adenoca cinoma (PDAC) is ano he example o OXPHOS-dependen me abolism in CD133 (also known as PROM1)-exp essing CSCs, when compa ing wi h ma u e cance cells. In his model o pa ien -de i ed xenog a s, he ansc ip ion ac o pe oxisome p oli e a o -ac i a ed ecep o - gamma coac i a o (PGC-1α), a egula o o mi ochond ial biogenesis, was essen ial o he OXPHOS pheno ype o CSCs, and also o hei sel - enewal and in i o umo igenic capaci ies. In con as , di e en ia ed PDAC cells ha e a MYC-d i en glycoly ic pheno ype, in which MYC o e exp ession nega i ely 5 REVIEW Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms Glucose A Cance s em cell Glu Glu Glu Glu Glu Glu Glu amine Glu amine ASCT2 Lac a e Py u a e Phosphoenolpy u a e 3-phosphoglyce a e Glucose 6-phospha e Glucose HK2 PFKFB PKM2 LDH PPP Nucleo ide syn hesis Amino acid syn hesis Aspa a e Oxaloace a e Fa y acids CPT1 FAO TCA cycle Ace yl-CoA III III IV V ATP Lac a e Py u a e Phosphoenolpy u a e 3-phosphoglyce a e Glucose 6-phospha e Glucose Ace yl-CoA III III IV V TCA cycle ATP ROS B Ma u e cance cell (non-CSC) Glu Glu GOT ASCT2 ASCT2 G6PD Fig. 1. Gene al me abolic ea u es o cance s em cells and ma u e cance cells (non- CSCs). (A) Cance s em cells end o ely mo e on glycolysis o ATP syn hesis, wi h o e exp ession o he glucose anspo e s GLUT1 and GLUT3, and inc eased exp ession o hexokinase 2 (HK2), 6-phospho uc o-2- kinase/ uc ose-2,6-biphospha ase (PFKFB), py u a e kinase isozyme M2 (PKM2) and lac a e dehyd ogenase (LDH). Nucleo ide biosyn hesis is o en inc eased in cance s em cells owing o o e exp ession o glucose-6- phospha e dehyd ogenase (G6PD) and amino acid syn hesis. Glu amine up ake and me aboliza ion o oxaloace a e, oge he wi h a y acid oxida ion, also appea o be impo an mechanisms in cance s em cells. (B) In con as , ma u e cance cells end o ely mo e on OXPHOS o adenosine iphospha e (ATP) p oduc ion, leading o inc eased le els o eac i e oxygen species (ROS); hese cells show low le els o glycolysis and nucleo ide syn hesis, al hough his can a y. ASCT2, alanine, se ine, cys eine-p e e ing anspo e 2; CPT1, ca ni ine-dependen anspo e 1; FAO, a y acid oxida ion; Glu , glucose anspo e (GLUT1 o GLUT3); GOT, glu ama e-oxaloace a e ansaminase; PPP, pen ose phospha e pa hway; TCA, ica boxylic acid. 6 REVIEW Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms con ols PGC-1αexp ession and inhibi s s emness (Sancho e al., 2015). Glycolysis and OXPHOS a e no necessa ily mu ually exclusi e. Indeed, in a s udy using b eas CSCs, Vlashi e al. epo ed highe glucose consump ion, concomi an wi h lowe lac a e and highe ATP p oduc ion, and wi h a consis en inc ease in mi ochond ial capaci y and ac i i y, compa ed wi h he di e en ia ed p ogeny (Vlashi e al., 2014). Glu amine me abolism Glu amine me abolism is an anabolic p ocess ha p oduces mac omolecules wi h lowe ene ge ic po en ial (Zheng, 2012). Glu amine can en e cells h ough he alanine, se ine, cys eine- p e e ing anspo e 2 (ASCT2; also known as SLC1A5) and hen be hyd olyzed o glu ama e and ammonia h ough he ac ion o GLS. Glu ama e, on one hand, can be combined wi h cys eine and glycine o o m educed glu a hione (GSH), which is a majo an ioxidan ha egula es oxida i e s ess (Es ela e al., 2006); on he o he hand, glu ama e can be con e ed in o α-ke oglu a a e (αKG) o p o ide TCA cycle in e media es and, ul ima ely, ene gy p oduc ion. This p ocess is ele an o cells ha lack ci a e p oduc ion owing o ine icien usage o glucose, cons i u ing an al e na i e pa hway o a unca ed TCA cycle (DeBe a dinis and Cheng, 2010). In human haema opoie ic SCs, he di e en ia ion o he e y h oid lineage comple ely depends on glu amine me abolism. E en in he p esence o e y h opoie in, glu amine-deple ed haema opoie ic SCs we e di e ed owa ds a myelomonocy ic di e en ia ion. Knockdown o ASCT2 dec eased glu amine up ake and inhibi ed e y h oid di e en ia ion. Addi ionally, he au ho s demons a ed ha he commi men o an e y h oid s a e was no es o ed by solely eeding he TCA cycle wi h cell-pe meable αKG, bu a he i depended on nucleo ides p oduced by glu amine me abolism (Obu oglu e al., 2014). In colo ec al cance cell lines, glu amine me abolism was ound o egula e he sensi i i y o CSCs o me o min (Box 1) h ough he AMPK-mTOR pa hway. In he p esence o glu amine, CSCs om he SW620 cell line showed esis ance o me o min, while in he absence o glu amine, hese CSCs showed ac i a ion o AMPK, supp ession o mTOR and became sensi i e o me o min ea men . In con as , CSCs om he HT29 cell line we e sensi i e o me o min, because hey ha e an ac i a ed AMPK pa hway. Ne e heless, inhibi ion o glu amine me abolism in hese cells inc eased he CSC-supp essi e e ec o me o min. CSCs om bo h cell lines showed highe exp ession o ASCT2 in compa ison wi h ma u e cance cells, and knockdown o ASCT2 signi ican ly dec eased he p opo ion o CSCs (CD133 + CD44 + ) in compa ison wi h con ol small in e e ing RNA (Fig. 1A). Thus, glu amine me abolism plays an impo an ole in egula ing he di e en esponses o CSCs o me o min (Kim e al., 2018). Ano he s udy using PDAC cells demons a ed ha a no el noncanonical glu amine pa hway is essen ial o umou g ow h and oxida i e s ess balance in CSCs (Li e al., 2015). He e, glu amine dep i a ion signi ican ly dec eased he exp ession o s emness ma ke s and he sel - enewal po en ial, and inc eased in acellula ROS le els, inducing apop osis. When using glu amine- ee medium supplemen ed wi h oxaloace a e, he p oduc o he noncanonical glu amine me abolism, hese e ec s we e escued; in con as , he p oduc o he canonical glu amine me abolism, αKG, did no escue hese e ec s on CSCs. Addi ionally, CSCs showed inc eased exp ession o GLS and glu ama e-oxaloace a e ansaminases (GOT1 and GOT2), he la e con e ing glu amine-de i ed aspa a e o oxaloace a e (Fig. 1A). Inhibi ion o his pa hway sensi ized CSCs o adia ion, showing ha he combina ion o a glu amine me abolism inhibi o wi h adio he apy migh be a sui able he apy o PDAC (Li e al., 2015). Fa y acid me abolism Besides using glucose as a uel o anabolic p ocesses, cells can de i e hei ene gy om a y acid me abolism. This is essen ially con olled by: (1) a y acid syn hesis (FAS), an anabolic p ocess ha con e s ace yl-CoA o malonyl-CoA and is equi ed o cell g ow h and p oli e a ion; and (2) a y acid oxida ion (FAO), a ca abolic p ocess ha b eaks down a y acids o gene a e ace yl-CoA o anaple osis, as well as NADH o he p oduc ion o ATP (Ca acedo e al., 2013). Haema opoie ic SCs a e main ained h ough an FAO pa hway downs eam o p omyelocy ic leukaemia p o ein (PML) and pe oxisome p oli e a o -ac i a ed ecep o δ(PPARδ). This PML– PPAR–FAO axis is essen ial o haema opoie ic SCs, as i con ols he asymme ic di ision and he a e o hese cells (I o e al., 2008, 2012). Indeed, he SC popula ion can be exhaus ed by pha macological o gene ic inhibi ion o any componen o his pa hway, sugges ing ha FAO is c ucial o s emness (I o e al., 2012). Ano he FAO-associa ed p o ein, li e kinase B1 (LKB1; also known as STK11), was also ound o be essen ial o haema opoie ic SC main enance (Gan e al., 2010). LKB1 ac i a es and phospho yla es AMPK in esponse o a decline in he ATP/adenosine monophospha e (AMP) a io; in u n, AMPK phospho yla es key egula o y p o eins in ol ed in a y acid me abolism o es o e ATP le els (Gan e al., 2010; Gu umu hy e al., 2010). Dele ion o Lkb1 in mice causes apid deple ion o he haema opoie ic SC pool, while also inducing al e a ions in lipid me abolism, deple ion o cellula ATP and mi ochond ial de ec s in Lkb1-de icien bone ma ow cells (Gan e al., 2010; Gu umu hy e al., 2010; Nakada e al., 2010). Adul mu ine neu onal s em and p ogeni o cells also ha e inc eased ac i i y o a y acid syn hase (Fasn), a key enzyme o de no o lipogenesis, in compa ison wi h di e en ia ed p ogenies (Knobloch e al., 2013). Fasn is equi ed o he p oli e a ion o p ogeni o cells, while quiescen , nonp oli e a ing SCs ended o shi om FAS owa ds FAO. Low p oli e a ing SCs selec i ely exp ess he hy oid ho mone- esponsi e p o ein Spo 14 (also known as Th sp), which educes lipid syn hesis and ac s as a molecula b ake on Fasn-dependen lipogenesis. Thus, neu ogenesis is sus ained h ough a igh egula ion o a y acid me abolism, whe e Fasn and Spo 14 play a majo ole in he egula ion o malonyl-CoA le els o he gene a ion o complex a y acids, which in u n egula e he mo e quiescen o commi ed s a e o he neu onal cells (Knobloch e al., 2013). Simila ly o haema opoie ic SCs, quiescen leukaemia-ini ia ing CSCs we e also shown o be egula ed by FAO. Recen epo s e ealed ha a subpopula ion o leukaemia SCs, exp essing he a y acid anspo e CD36, can eside in gonadal adipose issue niches o induce lipolysis and uel FAO, leading o chemo he apy esis ance (Ye e al., 2016). CD36 + cells a e also p esen in o al squamous cell ca cinomas and comp ise a popula ion o slow- cycling cells exp essing he s em cell ma ke CD44, while also exp essing high le els o lipid me abolism genes and being associa ed wi h me as asis (Pascual e al., 2017). CD36 + cells we e addi ionally ound in o he umou s, namely in melanoma and b eas cance , whe e hey also associa e wi h me as a ic po en ial (Pascual e al., 2017). These s udies sugges ha a subse o highly agg essi e CSCs ob ain hei ene gy h ough FAO, hus e ealing a 7 REVIEW Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms speci ic me abolic p o ile equi ed du ing me as asis ha migh be a po en ial a ge o he e adica ion o CSCs. Fu he mo e, in hepa ocellula ca cinoma, Nanog induces a me abolic ep og amming o CSCs: a dec ease in mi ochond ial espi a ion and an enhanced eliance on glycolysis and, addi ionally, an up egula ion o FAO genes o suppo he sel - enewal o hese cells (Chen e al., 2016) (Fig. 1A). In con as , de no o lipid syn hesis is inc eased in glioma CSCs compa ed wi h ma u e glioma cells, owing o high Fasn exp ession. Fasn inhibi ion educes s em cell ma ke exp ession while inc easing di e en ia ion ma ke s and dec easing he p oli e a ion and mig a ion o CSCs (Yasumo o e al., 2016) (Fig. 1A). O he lipid me abolism enzymes, such as a achidonic acid 5-lipoxygenase (ALOX5), which is in ol ed in he syn hesis o leuko ienes (Box 1) om a achidonic acid, and acyl-CoA syn he ase e y-long-chain 3 (ACSVL3; also known as SLC27A3), a key enzyme in a y acid ac i a ion o he o ma ion o a y acyl- CoA, we e also shown o suppo glioblas oma CSCs sel - enewal and o induce umou xenog a o ma ion (Wang e al., 2011; Sun e al., 2014). Finally, colo ec al CSCs ha e high le els o lipid d ople s, which co ela e wi h he exp ession o s em cell ma ke s. CSCs wi h mo e lipid d ople s showed a highe umou igenic capaci y upon xeno ansplan a ion, sugges ing ha lipid me abolism can media e s emness in colo ec al cance (Ti ina o e al., 2015). O he me abolic ea u es Mu a ions in genes ha encode me abolic enzymes ha e e ealed ye ano he mechanism o cance s emness egula ion by me abolic ep og amming. In leukaemia, mu a ions in isoci a e dehyd ogenase 1and2(IDH1 and IDH2, espec i ely) we e speci ically associa ed wi h CSC egula ion. These mu a ions al e he no mal IDH1/2- media ed con e sion o isoci a e o αKG in o an abe an con e sion o αKG o he analogue, 2-hyd oxyglu a a e (2-HG). This me aboli e accumula es in acellula ly and inhibi s e me hylcy osine dioxygenase 2 (TET2) unc ion by compe ing wi h i s co ac o αKG. In he p esence o mu an IDH1/2o TET2 deple ion, he sel - enewal po en ial o haema opoie ic SCs inc eases and di e en ia ion is impai ed, which sugges s a p o-leukaemic pheno ype (Figue oa e al., 2010; Cimmino e al., 2011; Ka s e al., 2014). Pu ine syn hesis is ano he me abolic mechanism ha con ols s emness in b ain CSCs. Wang e al. obse ed ha CSCs show up egula ion o enzymes in ol ed in pu ine syn hesis o he p oduc ion o pu ine nucleo ides ha se e as building blocks o DNA and RNA (Wang e al., 2017b). These include he enzymes phospho ibosyl py ophospha e syn he ase 1 (PRPS1) and phospho ibosyl py ophospha e amido ans e ase (PPAT) o syn hesize inosine monophospha e (IMP), adenylosuccina e lyase (ADSL) and adenylosuccina e syn hase (ADSS) o syn hesize AMP, and guanine monophospha e syn hase (GMPS) and IMP dehyd ogenase 1 (IMPDH1) o syn hesize guanosine monophospha e (GMP). Gene ic pe u ba ions o hese enzymes caused a dec ease in CSC g ow h and main enance and ab oga ed umou o ma ion in immunode icien and immunocompe en mouse models, by deple ing he in acellula pools o pu ine nucleo ides. In con as , a ge ing pu ine biosyn hesis did no a ec di e en ia ed glioblas oma cells, which collec i ely suppo s he selec i e dependence o b ain CSCs on he pu ine syn hesis pa hway (Wang e al., 2017b). Lysine ca abolism was also ound o be essen ial o p omo e he sel - enewal o colo ec al CSCs and induce li e me as asis. Colo ec al CSCs exp essing he h ombopoie in-binding ecep o CD110 (also known as MPL) signal h ough h ombopoie in by ac i a ing lysine deg ada ion. This gene a es ace yl-CoA, which is used o he ace yla ion o he LDL ecep o - ela ed p o ein 6 (LRP6). This ace yla ion in u n ec ui s casein kinases ha phospho yla e LRP6 o he ac i a ion o WNT p o ein signalling and egula ion o CD110 + CSC sel - enewal. Fu he mo e, lysine ca abolism in hese cells p omo ed he gene a ion o glu ama e, which enhances cys eine up ake and GSH syn hesis. Thus, CD110 + CSCs a e able o modula e hei edox s a us by lysine ca abolism, p omo ing sel - enewal, d ug- esis ance and li e me as asis (Wu e al., 2015). No mal SCs seem o ha e a mo e consis en me abolic pheno ype and con ol o hei molecula pa hways (I o and Suda, 2014). As discussed in his sec ion, CSCs, al hough lacking a common me abolic pa e n ac oss cance ypes, clea ly ha e a dis inc i e me abolic pheno ype compa ed wi h hei ma u e cance cell coun e pa s. The numbe o publica ions add essing he me abolism o CSCs is s ill small, and disc epancies, such as di e en en i onmen al s imuli in he expe imen al se ing, migh explain he con adic o y esul s. As an example, many o he s udies discussed in his Re iew a ou glycolysis by g owing cells in high glucose and high oxygen cul u e condi ions ha ail o ecapi ula e issue homeos asis and TME condi ions in i o. Indeed, he TME is an impo an ac o o cellula me abolism, as i c ea es a symbio ic sys em whe e, o example, highly glycoly ic s omal cells gene a e me abolic p oduc s ha can be used by cance cells; hese, in u n, shi hei me abolism owa ds OXPHOS and can po en ially igge a ep og amming o mo e s em-like s a es (Pa lides e al., 2009; Migneco e al., 2010; Ma inez-Ou schoo n e al., 2011; Nakajima and Van Hou en, 2013; Da idson e al., 2016). Such o ganiza ion canno be ep oduced when using es ablished cance cell lines in in i o sys ems ha lack a sui able TME, esul ing in inconsis en indings and majo di e ences conce ning he me abolic pheno ypes in in i o and in i o se ings. Impo an ly, he e y de ini ion o CSCs and he expe imen al designs o hei isola ion and cha ac e iza ion a y ac oss s udies, which also con ibu es o he inconsis ency o esul s. Al hough hese indings s ill lack obus ness and u he alida ions, he speci ic me abolic ea u es o CSCs ha e been es ed as po en ial he apeu ic a ge s and should be aken in o accoun o u u e cance he apies. Me abolism as a he apeu ic a ge o CSCs Cance cell plas ici y and he acquisi ion o a quiescen s a e a e hough o be impo an d i e s o d ug esis ance. Ac ually, se e al indings suppo he ac ha esidual do man clones, which esis he an ip oli e a i e chemo he apeu ic ea men , can become dominan and cause umou elapse (Chen e al., 2012; K eso e al., 2013; Ku o a e al., 2015; Liau e al., 2017). Along wi h he eme gen epo s aiming a cha ac e izing he molecula mechanisms ha go e n s emness in cance , se e al he apeu ic app oaches ha e been de eloped and es ed o he elimina ion o CSCs. Howe e , no an i-CSC he apy has shown su icien e ec i eness in o de o be app o ed o clinical use. Thus, he apies a ge ing he me abolic ne wo ks ha media e cance cell s emness could be an inno a i e and e icien s a egy o a ge his cell popula ion. Se e al s udies using mouse models o cance ha e shown ha a ge ing oxida i e me abolism, he main sou ce o ene gy o CSCs in hese models, sensi izes his popula ion o chemo he apies, hus leading o hei deple ion. One example is he popula ion o slow- cycling JARID1B + cells in melanoma ha has an up egula ion o OXPHOS enzymes. T ea men o melanoma cells wi h se e al d ugs, including cispla in and emu a enib, an inhibi o o mu an 8 REVIEW Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms BRAF, causes an en ichmen o he JARID1B + popula ion and subsequen he apy esis ance. When inhibi ing OXPHOS using ei he ATP-syn hase inhibi o s (oligomycin and Bz-423) o complex I inhibi o s ( o enone and phen o min), JARID1B + cells we e sensi ized o he an icance agen s ha ini ially ailed o elimina e hem (Fig. 2). E en among unc ionally and gene ically he e ogeneous melanomas, his combined app oach o ea men was e ec i e, because cy o oxic agen s mos ly elimina e he apidly di iding cells, while he me abolic inhibi o s could a ge and sensi ize slow-cycling cells (Roesch e al., 2013). Ano he example is PDAC, whe e umou igenesis is essen ially d i en by mu an KRAS. Ta ge ing his oncogene only leads o umou sh inkage, while spa ing a ac ion o cells wi h CSC ea u es ha ha e p ominen exp ession o genes go e ning mi ochond ial unc ion and s ong eliance on OXPHOS o cellula ene ge ics. These CSCs showed high sensi i i y o OXPHOS inhibi o s (p e e en ially o oligomycin) ha , when combined wi h a ge ed he apy o he KRAS pa hway, could elimina e he umou and p e en ecu ence (Viale e al., 2014). In o he s udies, howe e , he mi ochond ial inhibi o me o min was no enough o elimina e some CSC clones, possibly due o he he e ogenei y and plas ici y o PDAC cells and hei in e media e glycoly ic/ espi a o y pheno ype. These CSC clones showed an up egula ion o MYC, which egula es PGC-1αle els and, subsequen ly, con ols he me abolic pheno ype o esis an CSC clones. Gene ic o pha macological a ge ing o MYC e e sed his pheno ype by inc easing he CSC dependency on OXPHOS and sensi izing hem o me o min (Sancho e al., 2015) (Fig. 2). Fu he mo e, he commonly used inhibi o o hymidine syn hesis, 5- luo ou acil (5-FU), was shown o selec i ely a ge CSCs in colon cance . These cells unde go a me abolic ep og amming a ou ing OXPHOS and dec easing he PPP, a mechanism ound o be esponsible o 5-FU esis ance. Thus, combined ea men wi h 5-FU and me o min abolished d ug esis ance and e ec i ely diminished he popula ion o CSCs (Denise e al., 2015). In ag eemen wi h hese indings, ea men o CSCs om epi helial o a ian cance wi h di e en inhibi o s o he elec on anspo chain, namely oligomycin, an imycin and o enone, could lead o apop osis o CSCs (Pas ò e al., 2014) (Fig. 2). Ta ge ing glycoly ic enzymes also p esen ed p omising esul s in he mesenchymal sub ype o glioblas oma CSCs ha o e exp ess ALDH1A3. Radia ion ea men o p oneu al and mesenchymal sub ypes o hese cells has shown ha mesenchymal CSCs a e esis an and highly agg essi e compa ed wi h p oneu al CSCs. Fu he mo e, i adia ion o p oneu al CSCs esul s in an up egula ion o mesenchymal-associa ed ma ke s, and his e ec could be a enua ed only when inhibi ing ALDH1A3. The e o e, he subse o glioblas oma pa ien s wi h a mesenchymal signa u e migh bene i om ALDH inhibi ion o he e adica ion o highly agg essi e CSCs (Mao e al., 2013). In ano he s udy, he combina ion o con en ional an icance agen s, such as doxo ubicin, wi h a de i a i e o 3-b omopy u a e ha inhibi s glycolysis, e ec i ely killed glioblas oma CSCs in i o and inhibi ed umou o ma ion in i o (Zhou e al., 2011) (Fig. 2). Fu he mo e, he me abolic shi om glycolysis o mi ochond ial espi a ion, caused by dichlo oace a e ea men , inc eased ROS and induced apop osis in glioblas oma CSCs, bo h in i o and in i o (Michelakis e al., 2010) (Fig. 2). Lipid me abolism has been also es ed as a p omising a ge o he e adica ion o d ug- esis an CSCs. E omoxi , an inhibi o o he ca ni ine-dependen anspo e CPT1 (also known as CPT1A) and FAO, was able o e adica e ∼50% o quiescen leukaemia SCs in p ima y human myeloid leukaemia samples, and sensi ize Glu Lac a e Py u a e Glucose 6-phospha e Glucose HK2 PDH Fa y acids FAO TCA cycle Ace yl-CoA III III IV V CPT1 Phen o min Ro enone An imycin Oligomycin E omoxi Me o min DCA 3BP Glycolysis Fig. 2. Me abolic a ge s o cance s em cells. In gene al, me abolic inhibi o s can sensi ize cance s em cells o s anda d an icance he apies (highligh ed in yellow ec angles), leading o hei e adica ion. Speci ically, in models in which cance s em cells a e mo e elian on glycolysis, 3-b omopy u a e (3BP) o dichlo oace a e (DCA) can ep og am he me abolism o hese cells and sensi ize hem o chemo he apeu ic agen s. In cance s em cells ha exhibi inc eased oxida i e phospho yla ion (OXPHOS), inhibi ion o mi ochond ial espi a ion by me o min, phen o min, o enone, oligomycin o an imycin can igge apop osis. Inhibi ion o a y acid oxida ion (FAO) by e omoxi , which inhibi s he ca ni ine-dependen anspo e 1 (CPT1), leads o sensi iza ion o cance s em cells o apop osis- inducing agen s. Glu , glucose anspo e ; HK2, hexokinase 2; PDH, py u a e dehyd ogenase; I-V, mi ochond ial espi a o y chain complex I (NADH dehyd ogenase subuni ), complex II (succina e dehyd ogenase subuni ), complex III (ubiquinol- cy och ome c educ ase complex subuni ), complex IV (cy och ome c oxidase subuni ) and complex V (ATP syn hase subuni ). 9 REVIEW Disease Models & Mechanisms (2018) 11, dmm033464. doi:10.1242/dmm.033464 Disease Models & Mechanisms