The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing

The mul ikinase inhibi o So a enib enhances glycolysis and syne gizes wi h glycolysis blockade o cance cell killing Valen ina Teso i 1 , Anna Chia a Piscaglia 1 , Daniela Samengo 2 , Ma a Ba ba 3 , Camilla Be na dini 3 , Robe o Sca ena 4 , Alessand o Pon oglio 4 , Lau a Cas ellini 5 , Johannes N. Spelb ink 6 , Giuseppe Maulucci 7 , Ma ia Ausilia ice Puglisi 1 , Gio amba is a Pani 2 & An onio Gasba ini 1 1 Ins i u e o In e nal Medicine and Gas oen e ology, Ca holic Uni e si y o he Sac ed Hea School o Medicine, 2 Ins i u e o Gene al Pa hology, Labo a o y o Cell Signaling, Ca holic Uni e si y o he Sac ed Hea School o Medicine, 3 Ins i u e o Human Ana omy and Cell Biology, Ca holic Uni e si y o he Sac ed Hea School o Medicine, 4 Ins i u e o Biochemis y and Clinical Biochemis y, Ca holic Uni e si y o he sac ed Hea School o Medicine, 5 Depa men o Radia ion Oncology, Cen e o Clinical Sciences Resea ch, S an o d Uni e si y School o Medicine, S an o d, CA 94305, USA, 6 Depa men o Pedia ics, Nijmegen Cen e o Mi ochond ial Diso de s, Radboud Uni e si y Medical Cen e, Gee G oo eplein 10, P.O. Box 9101, 6500 HB Nijmegen, The Ne he lands; Ins i u e o Biomedical Technology & Tampe e Uni e si y Hospi al, Pi kanmaa Hospi al Dis ic , Uni e si y o Tampe e, FI-33014, Finland, 7 Ins i u e o Physics, Ca holic Uni e si y o he Sac ed Hea School o Medicine. Al hough he only e ec i e d ug agains p ima y hepa oca cinoma, he mul ikinase inhibi o So a enib (SFB) usually ails o e adica e li e cance . Since SFB a ge s mi ochond ia, cell me abolic ep og amming may unde lie in insic umo esis ance. To cha ac e ize cance cell me abolic esponse o SFB, wemeasu ed oxygen consump ion, gene a ion o eac i e oxygen species (ROS) and ATP con en in a LCSC (Li e Cance S em Cells) -2 cells exposed o he d ug. Genome wide analysis o gene exp ession was pe o med by A yme ix echnology. SFB cy o oxici y was e alua ed by mul iple assays in he p esence o absence o me abolic inhibi o s, o in cells gene ically deple ed o mi ochond ia. We ound ha low concen a ions (2.5–5 mM) o SFB had a ela i ely modes e ec on LCSC-2 o 293 T cell g ow h, bu damaged mi ochond ia and inc eased in acellula ROS. Gene exp ession p o iling o SFB- ea ed cells was consis en wi h a shi owa d ae obic glycolysis and, acco dingly, SFB cy o oxici y was d ama ically inc eased by glucose wi hd awal o he glycoly ic inhibi o 2-DG. Unde me abolic s ess, ac i a ion o he AMP dependen P o ein Kinase (AMPK), bu no ROS blockade, p o ec ed cells om dea h. We conclude ha mi ochond ial damage and ROS d i e cell killing by SFB, while glycoly ic cell ep og amming may ep esen a esis ance s a egy po en ially a ge able by combina ion he apies. Ae obic glycolysis (‘‘Wa bu g e ec ’’) ep esen s one o he dis inc i e ac s (‘‘hallma ks’’) o he malignan pheno ype 1–4 . Al hough ene ge ically less e icien han espi a ion, e men a i e me abolism is ad an - ageous o cell g ow h due o he inc eased a ailabili y o anabolic in e media es and he educed cell dependence on oxygen; mo eo e , by inc easing in acellula educing equi alen s (NADPH and glu a hione) and dec easing mi ochond ia-de i ed ROS, glycolysis p o ec s malignan cells om oxidan -induced senescence and apop osis 5 and con ibu es o he su i al o Cance S em Cell (CSC) 6 . Biochemical di e ences be ween cance ous and no mal cells may help di ec ing a ge ed he apies agains malignan elemen s. Fo ins ance, umo cells a e o en s ongly dependen on glucose (‘‘glucose-addic ed’’) and he e o e exquisi ely sensi i e o he glycoly ic inhibi o 2-deoxyglucose (2DG) 7 . No ably, he link be ween me abolism, oxida i e s ess and cance may be pa icula ly ele an o he li e 8 , ha plays a pi o al ole in he egula ion o glucose homeos asis. Hence li e cance cells, like he hepa ocholangioca cinoma cell line LCSC-2 we’ e ecen ly de i ed om a no el model o ca cinogenesis in a s 9 , appea ideally sui ed o in es iga e biochemical mechanisms and he apeu ic implica- ions o cance cell me abolic ep og amming. The mul ikinase inhibi o So a enib (SFB) (Nexa a , BAY 43-9006) cu en ly ep esen s he p ima y ea men op ion o ad anced hepa ocellula ca cinoma 10 ; SFB p e e en ially inhibi s he cance - associa ed V600E mu an o he se ine- h eonine kinase and Ras-e ec o BRAF, while he wild ype enzyme is pa adoxically ac i a ed by he d ug in he p esence o ac i e Ras signaling 11 ; SFB also a ge s, a concen a ions in he high nanomola ange, a numbe o Recep o Ty osine Kinases (RTK S ) including, Pla ele De i ed G ow h Fac o – b(PDGFR-b), OPEN SUBJECT AREAS: PRECLINICAL RESEARCH TARGETED THERAPIES NUTRIENT SIGNALLING STRESS SIGNALLING Recei ed 11 Augus 2014 Accep ed 20 Feb ua y 2015 Published 17 Ma ch 2015 Co espondence and eques s o ma e ials should be add essed o G.P. (gpani@ m. unica .i ) SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 1 Vascula Endo helial G ow h Fac o -2 (VEGFR-2), and Vascula Endo helial G ow h Fac o -2 (VEGFR-3) 12 . Howe e , addi ional mechanisms likely con ibu e o he ele a ed an icance ac i i y o his compound, and may ha e by ex ension a ole in he equen eme gence o speci ic chemo esis ance 13 . Ini ial e idence poin o mi ochond ial damage and oxida i e s ess as addi ional, kinase-independen mechanisms unde lying cell esponse o So a enib. In no mal ca diomyocy es, o ins ance, SFB was epo ed o inhibi mi ochond ial espi a ion and o dec ease in acellula ATP le els 14 . Along simila lines, SFB has been shown o inc ease he p oduc ion o mi ochond ial ROS (mROS), dec ease educed Glu a hione le els (GSH) and induce cell dea h in HepG2 human hepa oma cells 15 , and se um le els o ad anced oxida ion p o ein p oduc s in So a enib- ea ed HCC pa ien s co ela e wi h clinical e ec i eness o he d ug 16 . Addi ionally, in human panc ea ic cell lines SFB elici s MEK/ERK independen apop osis, h ough he down egula ion o he mi ochond ial an iapop o ic p o ein Mcl-1 17 . P omp ed by hese e idence and by he eme ging in e es owa ds me abolism- a ge ed an icance he apies, we sough o in es iga e he e ec o So a enib on mi ochond ial ac i i y and oxida i e me a- bolism in a hepa ocolangioc cinoma LCSC-2 cells, in sea ch o no el mechanisms o esponse and/o esis ance o li e cance cells o his inc easingly used d ug. Resul s So a enib inc eases in acellula ROS and inhibi s espi a ion in LCSC-2 cells.Sensi i i y o umo cell lines o RTKs inhibi o s is highly a iable, in pa depending on he mu a ional s a us o RAS and RAF amily membe s 18 . Exposu e o a hepa ocolangioca cinoma LCSC-2 cells, ha lack B-RAF ac i a ing mu a ions, o SFB had a modes g ow h inhibi o y e ec as assessed by P opidium Iodide (PI) exclusion o colony o ma ion assay (Fig. 1, a, b and c), especially in he p esence o e al bo ine se um: in ac , unlike epo ed o higly sensi i e cell lines 12 , 50% inhibi ion was a ained in he low mic omola , a he han nanomola ange. O no e, in his ange no ob ious educ ion o phospho yla ed (ac i e) ERK and AkT, was obse ed unde cell s imula ion wi h Hepa ocy e g ow h Fac o (HGF), sugges ing a g ow h inhibi o y mechanism dis inc om RTK o ERK blockade (Fig. 1, d). Consis en wi h p e ious epo s 15,16 , low cy ome y analysis o cells loaded wi h he edox-sensi i e dye H2-DCF-DA e ealed a ma ked and dose dependen inc ease o ROS 12 hou s a e exposu e o 2,5 o 5mM SFB (Fig. 1 e and 1 g); LCSC-2 cells o en appea ed dis ibu ed in wo dis inc subpopula ion peaks based on DCF-DA luo escence in ensi y, bo h o which we e shi ed o he igh (inc eased oxida ion) upon exposu e o he d ug (Fig. 1e). Impo an ly, he p o-oxidan e ec o SFB was cancelled by he cell pe mean hyd ogen pe oxide sca enge and GPX mime ic Ebselen (EBS) (Fig. 1 ). Since mi ochond ia ep esen he main sou ces o ROS in no mal and in umo cells, we easoned ha ROS inc ease may e lec an e ec o SFB on hese o ganelles. Acco dingly, baseline oxygen consump ion ( ou ine espi a ion) measu ed by High Resolu ion Respi ome y was signi ican ly educed a e 12 hou s incuba ion wi h SFB, compa ed o un ea ed con ols (Fig. 2 a), and a simila di e ence was obse ed unde maximum elec on low, as elici ed by he mi ochond ial p o- onopho e Ca bonyl cyanide p-( i- luo ome hoxy) phenyl-hyd azone (FCCP), (Fig. 2 b). In o de o add ess whe he SFB ac s di ec ly on mi ochond ia, we isola ed he o ganelles om a li e and assessed hei ansmem- b ane po en ial in he p esence o he d ug, using he ca ionic luo - escen dye JC-1 ollowed by low cy ome y. As shown in Fig. 2, c, SFB was able o depola ize mi ochond ia in i o, as e ealed by an e iden educ ion o JC-1 luo escence in he FL2 ( ed) channel. Taken oge he , hese indings con i m ha in LCSC-2 cells, as in o he cell models, SFB in e e es wi h mi ochond ial unc ion, and sugges ha his e ec is due, a leas in pa , o a di ec in e ac ion o he d ug wi h he o ganelle. So a enib a ec s ene gy me abolism o LCSC2 cells.To u he cha ac e ize he impac o SFB on cellula ene gy me abolism, we i s measu ed in acellula ATP a e wel e hou s cell exposu e o he d ug. ATP le els we e ma kedly educed (.50%) in SFB- ea ed cells compa ed o un ea ed con ols (Fig. 3 a), indica ing ha LCSC-2 cells ac i ely u ilize mi ochond ia o hei ene gy supply. In keeping wi h he abo e inding, SFB elici ed he phospho yla ion on Th eonine 172 o he AMP ac i a ed p o ein kinase (AMPK), an ene gy senso ha de ec s changes in he in acellula AMP/ATP and igge s me abolic cell adap a ions aimed a es o ing ATP le els a expense o anabolic p ocesses (Fig. 3 b). SFB phospho yla ion o AMPK was po en ia ed by he glycoly ic inhibi o 2DG (Fig. 3 c and Supplemen a y Figu e S5), and was no inhibi ed by Ebselen (Fig. 3 c), al hough AMPK ac i a ion may in some con ex s espond o ROS 19 . To gain insigh in he global me abolic cell esponse o So a enib, we pe o med mic oa ay analysis o gene exp ession. A o al o 322 di - e en ially-exp essed genes wi h $1,5 old change we e iden i ied in LCSC-2 exposed o he d ug. Among hese, 174 genes esul ed up- egula ed and 148 genes we e down- egula ed in LCSC-2 ea ed wi h SFB compa ed o un ea ed con ols (da a accessible a h p://www. ncbi.nlm.nih.go /geo/ unde he ID numbe GSE43053). O no e, mono- sacca ide me abolism (p 50.00462) and cell p oli e a ion (p 5 0.00631) we e among he highes anking biological p ocesses en iched o di e en ly exp essed genes (Supplemen a y Fig. S1 on line). We ocused on genes in ol ed in glucose me abolism. In e es ingly, he exp ession o h ee genes di ec ly in ol ed in glycolysis [ he solu e ca ie amily 2 (slc2a3), Enolase 2 (eno2), and he pla ele phospho- uc okinase (p kp)], was signi ican ly induced by SFB (Fig. 3 d); con- e sely, SFB dec eased he exp ession o acquapo in 9 (aqp9), membe o a amily o wa e -selec i e memb ane channels, and o he mi- ochond ial enzyme py u a e dehyd ogenase (lipoamide) alpha 1 (pdha1), which ca alyzes he i e e sible con e sion o py u a e o ace yl-CoA, hus linking ae obic glycolysis wi h he ica boxylic acid (TCA) cycle in mi ochond ia. Di e ences in he exp ession le els o selec ed genes we e u he alida ed by quan i a i e eal ime PCR (qPCR) (Fig. 3 e). Mo eo e , consis en wi h me abolic shi owa ds glycolysis in esponse o SFB, sugges ed by he abo e ansc ip ional changes, we obse ed enhanced up ake o he luo escen glucose analog 6NDBG, and inc eased glucose consump ion and L-lac a e elease by LCSC-2 cells exposed o he d ug (Fig. 3 , g and h). So a enib oxici y is inc eased by he glycoly ic inhibi o 2-deoxy- glucose.The abo e indings p op ed us o es whe he he e ec s o SFB could be enhanced by a glycolysis inhibi o , such as he non- me abolizable glucose analogue 2-deoxy-glucose (2DG). In oxici y assays (PI exclusion and MTT), 2DG d ama ically inc eased cell killing by SFB bo h in LCSC-2 cells (Fig. 4 a and b) and in he highly malignan mu ine melanoma cell line B16F10 (Fig. 4 c); a simila e ec was elici ed by glucose wi hd awal om he cul u e medium (Supplemen a y Fig. S2 on line). O no e, he MEK inhibi o PD98059 had ma ginal e ec s on LCSC-2 cells, ha was no enhanced by glucose wi hd awal (Supplemen a y Fig S2 on line). Ins ead, in B16F10 cells, he e ec o So a enib was mimicked by he mi ochond ial Complex I inhibi o Ro enone 20 (Fig. 4 c); mo eo e , deple ion o mi ochond ial DNA in a HEK293 cell line a ian exp essing a doxycycline inducible dominan nega i e mu an o he mi ochond ial DNA polyme ase gamma 1 (POLG1 D890N) 21 nea ly ab oga ed sensi i i y o SFB ( ig. 4d), u he poin ing o a mi ochond ial ac ion o he d ug. The syne gis ic e ec o SFB and 2DG o cell killing was also con i med in a numbe o addi ional human and mu ine cance cell lines, including Ras- ans o med Mouse Emb yonic Fib oblas s (MEF) lacking p53 (Supplemen a y Fig. S3 on line). www.na u e.com/scien i ic epo s SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 2 AMPK links me abolic damage by SFB o he mTOR/au ophagy cascade.To u he cha ac e ize he biochemical e en s elici ed by SFB and i s combina ion wi h 2DG, we in e oga ed nu ien signalling e en s along he AMPK kinase pa hway. In LCSC2 (Fig. 5 a and Supplemen a y Fig. S5) and B16F10 cells (Supplemen a y Fig. S4) SFB and 2DG combina o ially and dose-dependen ly inc eased he phospho yla ion o AMPK on Se 172, consis en wi h p o ound cell de-ene giza ion. E en mo e d ama ically he combina ion inhibi ed he phospho yla ion o he ibosomal S6 p o ein, a downs eam e ec o o he mTOR (mammalian Ta ge o Rapamycin)/S6 Kinase cascade ha is nega i ely egula ed by AMPK unde nu ien dep i a ion 22 . Mo eo e , Simila s udies pe o med on HEK293- POLG1 (D890N) cells showed ha AMPK phospho yla ion by SFB is a ou ed unde glycolysis blockade by ei he 2DG o glucose dep i a ion, and ha i equi es ac i e mi ochond ia, as indica ed by he lack o esponse o he d ug (o he p esence o an opposi e, inhibi o y ac ion) in m DNA-deple ed cells (Fig. 5 d). No iceably, as an addi ional ou pu o AMPK/mTOR signalling modula ion in LCSC2 cells, SFB and SFB 12DG igge ed cell au op- hagy, a sel -ea ing p ocess aimed a cell su i al unde s a a ion 23 ; his was e ealed, biochemically, by inc eased LC3B elec opho e ic mobili y (due o co alen conjuga ion o his phagosome-associa ed p o ein wi h phospha idyl-e hanolamine), and deg ada ion o he au ophagy subs a e p62 (Fig. 5 a and Supplemen a y Fig. S5), and, unc ionally, by an accele a ed au ophagic lux (i.e. au ophagosome- au olysosome ansi ion), moni o ed by con ocal analysis o LCSC-2 cells ansien ly ans ec ed wi h a mRFP-GFP andem luo escen - agged LC3 24 (Fig. 5 b and 5 c). Figu e 1 | G ow h inhibi ion and gene a ion o ROS in LCSC-2 cells exposed o So a enib. (a), (b) and (c) P opidium Iodide exclusion assay (a) and colony o ma ion assay (b and c) showing dose and ime-dependen g ow h-inhibi o y e ec o SFB on LCSC-2 cells. FCS 5 e al cal se um. In b, ba s ep esen pla ing e iciency (nucolonies/nuo pla ed cells); c: ep esen a i e pic u e o colonies s ained wi h Giemsa. In a and b ba s a e mean 6SD o duplica e o iplica e samples. Panels ep esen a i e o se e al independen expe imen s. (d). Wes e n Blo analysis o Ak (Se 473) and p44-42 MAP Kinase (ERK,Th 202/Ty 204) phospho yla ion a e 12 hou s incuba ion wi h he indica ed combina ions o Hepa ocy e G ow h ac o (HGF, 50 ng/ml) and SFB. An i o al ERK immune-s aining con i ms equal p o ein loading h oughou he lanes. Rele an bands a e indica ed by a ows. (e). Rep esen a i e low cy ome y plo e ealing b oad dis ibu ion o H2-DCFDA luo escence in LCSC-2 cells and inc eased signal (oxida ion) in esponse o 2.5 o 5 mM SFB. ( ). E ec o an ioxidan s and GPX mime ic Ebselen on SFB-induced ROS. A shi o cell luo escence p o ile o he le con i ms e ec i e ROS sca enging by he compound. Plo s ep esen a i e o se e al independen analyses. (g). Quan i a ion o ROS inc ease in LCSC-2 cells exposed o 2.5 mM SFB o 12 hou s. Values a e Mean 6SD o mean luo escence a ios (SFB/Dmso) o e n 56 independen expe imen s. p calcula ed on aw luo escence alues by pai ed wo- ailed - es . www.na u e.com/scien i ic epo s SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 3 LCSC-2 cell killing by SFB in ol es ROS and is coun e ac ed by AMPK.In o de o cla i y he mechanis ic ole o ROS and phospho- AMPK, bo h elici ed by SFB, in he cy o oxic ac i i y o he d ug, we i s e alua ed killing e iciency in LCSC2 cells p e- ea ed wi h he ROS sca enge Ebselen. The an ioxidan signi ican ly educed cell dea h in he p esence o 5 and 10 mM SFB (p ,0.01) (Fig. 6 a and Supplemen a y Fig. 6) con i ming SFB-induced oxida i e damage as a necessa y con ibu o o he d ug cy o oxic ac ion. Acco dingly, when LCSC-2 wi h high and low baseline le els o ROS (as e ealed by he luo escen dye DCF-DA) we e sepa a ed by luo escence- ac i a ed cell so ing, he wo popula ion displayed di e en sensi i- i y (sensi i e he o me , esis an he la e ) o he d ug (Fig. 6 b and c). In e es ingly, howe e , Ebselen had no e ec on massi e cell killing by he SFB 12DG combina ion (Fig. 6A and Supplemen a y Fig. 6), implying a ROS independen oxic mechanisms (ene gy deple ion?) o he la e syne gis ic e ec . On he o he hand, AMPK deple ion by siRNA echnology (Fig. 6 d and e) po en ia ed cell killing by 5 mMSFB bo h alone and in combina ion wi h 2DG (p ,0.05 and p ,0.01, espec i ely), in pa allel wi h impai ed inhibi ion o mTOR/S6K signaling (Fig. 6 e). A cell p o ec i e ole o AMPK in esponse o SFB was u he co obo a ed by pha macological modula ion o he kinase wi h he cell pe mean ac i a o 5-Aminoimidazole-4- ca boxamide 1-b-D- ibo u anoside (AICAR, 5 mM, p ,0.05), and he speci ic inhibi o Compound C (20 mM, p ,0.01). Thus, AMPK ac i a ion likely con ibu es o esis ance o So a enib a leas in LCSC-2 cells. Discussion Cance cell me abolism elies on a delica e balance be ween he glycoly ic pa hway, conside ably ampli ied o p o ide o he needs o a apid and in asi e g ow h, and oxida i e phospho yla ion, which emains ac i e, o con ibu e o he umo ene gy needs 1,25 . In e e ence wi h his unique me abolic se ing may open new pos- sibili ies o a ge ed an icance he apy. We ha e he e in es iga ed he me abolic e ec s o So a enib, a mul ikinase inhibi o , epo ed o p e e en ially a ge mu an (V600E) BRAF and a numbe o cance - ele an y osine kinase ecep o s 12 . These e ec s, ha include inhibi ion o mi ochond ial espi a ion, educed ATP p oduc ion, and ele a ion o in acellula ROS, a e in many cell lines, including he a hepa ocolangioca ci- noma cell line LCSC-2, la gely insu icien o d i e cell dea h; we he e sugges ha such ine ec i eness is due, o an e icien cell ep og am- ming owa ds ae obic glycolysis. The d ama ic inc ease o SFB cy o- oxici y by glucose wi hd awal o he glycoly ic inhibi o 2DG clea ly suppo s his iew, opening o he possibili y o employing SFB in no el me abolism-based combina ion he apies. Impo an ly, SFB po en ia ion by glycolysis blockade was no limi ed o li e -de i ed cells, bu also ex ended o melanoma cells and o he cell ypes, and is Figu e 2 | So a enib inhibi s cellula espi a ion and depola izes isola ed mi ochond ia. (a) and (b). Respi ome ic Analysis o LCSC-2 cells exposed o 2, 6 and 12 hou s o 2.5 mM SFB o Dmso as ehicle con ol. The wo his og ams illus a e he inhibi o y e ec o he d ug on Rou ine Respi a ion (A), and uncoupled espi a ion (B) (see me hods o expe imen al de ails). As e isks deno e s a is ical signi icance ( wo- ailed S uden - es , compa ed o un ea ed con ol). (c). Flow cy ome y analysis o mi ochond ial s aining wi h he po en iome ic dye JC-1. Dec eased ed (FL-2) luo escence o mi ochond ia exposed o SFB indica es loss o ans-memb ane po en ial (y). Plo ep esen a i e o wo independen expe imen s. www.na u e.com/scien i ic epo s SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 4 likely o be independen om he umo supp esso p53 (Supple- men a y Fig. S3). No ewo hy, signaling s udies (Fig. 1 d) indica e ha me abolic ac ions o So a enib a e independen om inhibi ion o RTK signal- ing, sugges ing ins ead a di ec in e ac ion o he compound wi h mi ochond ia. Since mi ochond ia ep esen a no el p omising sub- s a e o a ge ed d ugs 26 , molecula in e ac ions o SFB wi h his o ganelle ce ainly dese e o be u he in es iga ed. In keeping wi h he idea ha SFB me abolically ep og ams a ge cells, mic oa ay da a a e consis en wi h a cell shi owa ds ae obic glycolysis, wi h a signi ican up- egula ion o genes in ol ed in he glycoly ic pa hway, such as scl2a3 (also known as GLUT-3, a glucose anspo e 27 ), p kp (Phospho uc okinase, pla ele iso o m 28 ) and eno2 (Enolase 2, a pa o he phosphopy u a e hyd a ase complex 29 ) (Supplemen a y Fig. S1). Impo an ly, all hese h ee genes ha e been p e iously linked o human malignancy 30–32 . Con e sely, among SFB-down egula ed genes ela ed o me abolism, he mi ochond ial enzyme py u a e dehyd ogenase (lipoamide) alpha 1 (pdha1) ca a- lyzes he i e e sible con e sion o py u a e o ace yl-CoA, linking he glycolysis wi h he ica boxylic acid (TCA) cycle and espi a- ion 33 . Thus, o e all, his gene signa u e poin s o a glycoly ic adap - i e esponse o mi ochond ial ailu e, as i is also consis en wi h e idence o enhanced glucose consump ion and lac a e p oduc ion in SFB- ea ed LCSC-2 cells (Fig. 3D). Glycolysis p o ides, beside ATP and anabolic in e media es, also educing equi alen s ( h ough he Pen ose Phospha e Pa hway, PPP) ha main ain he GSH/GSSG edox bu e , and inc eased glycolysis may coun e ac SFB ac ion, in pa , by imp o ing he cell an ioxidan capaci y 34 . In suppo o his iew, LCSC-2 cells wi h cons i u i ely low le els o ROS a e in insically esis an , unlike cells wi h high ROS, o he d ug ac ion (Fig. 6 b and c). In e es ingly, a simila Low- ROS p o ile iden i ies pu a i e s em cells om no mal and umo b eas cance cell popula ions 6 , sugges ing he possibili y ha esis - ance o SFB and a low oxidan con en cha ac e izes a subse LCSC-2 o cells wi h he p ope ies o Cance S em Cells. By ex ension, es- is ance o CSC o SFB may explain he limi ed cu a i e capaci y o Figu e 3 | So a enib modula es ene gy me abolism. (a). ATP measu emen in LCSC-2 cells ea ed wi h SFB. Values a e in ligh uni s no malized o p o ein con en . Ba s a e Mean 6SD o duplica e sample. S a is ics a e by wo- ailed - es . His og am ep esen a i e o wo independen expe imen s. (b). Immunode ec ion o phospho-(Th 172) AMPK phospho yla ion unde he indica ed s imuli; an i AMPK ( o al) immunoblo ing was used as loading con ol. (c). An i p-AMPK immunoblo showing no inhibi o y e ec o Ebselen on AMPK phospho yla ion by SFB o SFB 12DG. To al AMPK was used as loading con ol. Rele an bands a e indica ed by a ows. Pic u e ep esen a i e o a leas wo independen expe imen s. Black colums a e Mean 6SD o densi ome ic alues om 2–3 independen expe imen s, no malized o a e age band indesi y. (d). Glycolysis- ela ed genes modula ed by So a enib in LCSC-2 cells. Fold change e e s o he un ea ed (Dmso) sample. Up egula ed genes a e highligh ed in ed, down egula ed genes in g een. (e). Real Time PCR alida ion o h ee selec ed genes om panel C, a. Fold change (SFB/Dmso) o each gene was calcula ed by he DC me hod (see Supplemen a y Me hods); ba s a e mean 6SD o h ee independen eac ions. S a is ics a e by wo- ailed - es . ( ), (g) and (h). Ba g aphs displaying enhanced 6NBDG up ake and inc eased glucose me abolism in LCSC-2 cells a e 48 hou s exposu e o SFB. Ba s a e Mean SD o 2–3 independen samples. *p,0.05 (ANOVA); *** p,0.0001 ( - es ). www.na u e.com/scien i ic epo s SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 5 his d ug 10 .Wi h his espec i is no ewo hy ha glycolysis blockade o e comes SFB esis ance in ashion ha is ROS-independen (Fig. 6 a and Supplemen a y Fig. 6) and he e o e ci cum en s, unlike SFB alone, po en ial an ioxidan s-based su i al s a egies implemen ed by cance (s em) cells. LCSC-2 esis ance o So a enib also in ol es AMP kinase. Dose dependen induc ion o AMPK phospho yla ion by SFB is mos likely a consequence o cell de-ene giza ion, and appea s o be inde- penden o ROS ( ig. 3 c) bu equi es ac i e mi ochond ia, as shown by lack o esponse in HEK-293 cells g own in high glucose o de oid o mi ochond ial DNA by a POLG1 dominan nega i e mu an ( ig. 5 e and Supplemen a y Fig. S5). Mo eo e , a p o ec i e ole o he kinase agains SFB-induced cell dea h is s ongly sugges ed by expe i- men s o gene ic and pha macologic inhibi ion o he enzyme ( ig. 6 d– ) and by co ela i e e idence o cons i u i e AMPK phospho yla- ion and esis ance o SFB in HEK-293T POLG1 D890N cells ( igs. 4 d, 5e and Supplemn a y Fig. S5). Mul iple p o ec i e mechanisms, including down egula ion o mTOR signaling wi h educed ATP consump ion 35 , induc ion o au ophagy ( ig. 5 b and c), and gene ic ep og amming (Supplemen a y Fig. S1) may accoun o his e ec . While his aspec needs o be u he cla i ied, he abo e obse a ions a e ansla ionally ele an o he possibili y o a ge ing AMPK in combina ion wi h SFB o inc ease cell esponse o he d ug. Mo eo e , simila o o he mi ochond ia- a ge ing d ugs like Biguanides, umo sensi i i y o SFB may be p edic ed by loss o unc ion o he umo supp esso and AMPK ac i a ing kinase LKB1 36 , and boos ed by lim- i ed glucose a ailabili y in he umo mass 37 . O no e, while his manu- sc ip was in p epa a ion, i was epo ed ha SFB an i umo ac ion is media ed by AMPK/mTOR in b eas cance cells 38 , indica ing ha oles o AMPK downs eam o his d ug may be umo - and con ex - dependen . In conclusion, we ha e he e p o ided no el e idence o impo - an me abolic e ec s o he mul ikinase inhibi o So a enib on li e cance cells. Ou obse a ions, al hough wi h he in insic limi a ions Figu e 4 | So a enib oxici y is inc eased by he glycoly ic inhibi o 2-deoxy-glucose. (a). Cy o oxic e ec o SFB (5 mM) and 2DG (20 mM), applied as single agen s o in combina ion, in LCSC-2 cells. Pe cen age o dead (P opidium Iodide-pe mean ) cells was de e mined by low cy ome y a e 24 hou s exposu e o he d ugs. The dashed line indica es he expec ed alue o simple addi i i y, clea ed o backg ound cell dea h (Dmso), calcula ed by he Bliss Independence Model (see Supplemen a y Me hods). A whi e ci cle in he SFB 12DG ba indica es he amoun o cell dea h en i ely a ibu able o he d ug combina ion, clea ed o he backg ound. ** indica es p ,0.01 compa ed o ehicle; in e ac ion deno es a signi ican ‘‘cell ( ow 3column) e ec ’’ in he wo-way ANOVA es . His og am ep esen a i e o se e al independen expe imen s (b). MTT Assay unde he same condi ions as in a. Ba s a e mean 6SD o h ee independen expe imen s, each in duplica e. Line and s a is ics a e as in panel a. (c). E alua ion o SFB oxici y and SFB 12DG in e ac ion in B16 mouse melanoma cells. Cells we e analysed o PI exclusion as in a, a e 24 hou s exposu e o he d ugs. Ro enone (Ro , 5 mM), mimics he e ec o SFB (2.5 mM), and syne gizes wi h 2DG. Ba s a e mean 6SD o duplica e samples. *5p,0.05 and ** 5p,0.01 compa ed o Dmso. S a is ics as in panel a. Panel ep esen a i e o wo independen expe imen s. (d) MTT assay showing lack o esponse o SFB (5 mM) o HEK293 –POLG1 (D890N) cells g own in doxycycline 50 ng/ml o 10 days o deple e m DNA (Dox1); Dox- a e non-induced cells ha e ain m DNA. Pic u e ep esen a i e o wo independen expe imen s. S a is ics as in panel a. www.na u e.com/scien i ic epo s SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 6 o s udies in i o, indica e in mi ochond ia an impo an a ge o he d ug’s ac ion, in ol e ROS in SFB-dependen cy o oxici y, and iden i y mul iple (AMPK, me abolic ep og amming, an ioxidan capaci y) po en ial mechanisms o d ug esis ance, o be ci cum- en ed by no el and mo e e ec i e combina ion he apies. Me hods Reagen s and an ibodies.Mos o he Chemicals we e ob ained om Sigma-Ald ich (Milan, I aly). So a enib (Nexa a , BAY 43-9006) was kindly p o ided by Baye Pha maceu icals. An ibodies and siRNA a e lis ed in SI. Plasmids.p LC3 (mRFP-GFP andem luo escen - agged LC3) was a gi om Tamo su Yoshimo i (Addgene plasmid # 21074). Cell lines and ans ec ions.The a hepa ocholangioca cinoma cell line LCSC-2 was kindly p o ided by D . Thomas D. Shupe and D . B yon E. Pe e sen (Dep . o Pa hology, Uni e si y o Flo ida, Gaines ille, FL). Cells we e main ained in Dulbecco’s modi ied Eagle medium (DMEM 4.5 g/L d-glucose)/Ham’s F12 medium 50550, supplemen ed wi h 8% FBS and 1% an ibio ics (LCSC-medium). Flp-In TM T- REx TM HEK-293 cells s ably exp essing he D890N dominan nega i e mu an o he mi ochond ial Polyme ase gamma (POLG1) unde a Te acycline-inducible p omo e ha e been p e iously desc ibed 21 . Cell we e ou inely main ained in s anda d DMEM (4.5 g/L d-glucose). Induc ion o he ecombinan POLG1-myc p o ein was eadily de ec able a e 24 hou s ea men wi h 50 ng/ml Doxycycline (Sigma), and was main ained o 10 days in o de o se e ely deple e mi ochond ial DNA. Addi ional cell lines a e desc ibed in SI. T ans ec ion o cDNA and siRNA in o LCSC-2 cells was pe o med wi h Lipo ec amineH2000 (Li e Technologies) and HiPe Fec (QIAGEN), espec i ely, acco ding o he manu ac u e ’s ecommenda ions. Cy o oxici y assays.Fo MTT assay, cells we e seeded in 24-well pla es (2 310 5 /well) o 96 well pla es (5 310 4 /well) and challenged wi h SFB o he s imuli in se um- ee medium. A e 24 hou s o incuba ion MTT solu ion (1510 dilu ion o he 5 mg/ml s ock) was added o cells and incuba ed a 37uC o h ee addi ional hou s. The MTT- Figu e 5 | AMPK links me abolic damage by SFB o he mTOR/au ophagy cascade. (a). Wes e n blo analysis o p o ein lysa es om LCSC-2 s imula ed o 4–6 hou s wi h he indica ed combina ions o SFB and 2DG. Phospho yla ion o AMP (Se 172) and o he mTOR e ec o S6 (Se 235/236) we e moni o ed by phospho-speci ic an ibodies. To al AMPK is also displayed. In he lowes wo panels, appea ance o a as mig a ing LC3B band (LC3B II) and dec eased an i-p62 signal in SFB and SFB 12DG ea ed samples deno e enhanced au ophagy. Pic u e ep esen a i e o h ee independen expe imen s wi h compa able esul s. (b). Con ocal analysis o LCSC-2 cells ansien ly ans ec ed wi h a andem ed-g een lu escen agged LC3 and exposed o SFB o 6 hou s. Red punc a (au olysosomes) and yellow punc a (au ophagosomes) we e coun ed and he a io calcula ed o each cell. a. Ba s a e mean 6SD o n 551 (Dmso) and n 532 (SFB); s a is ics by - es . (c). ep esen a i e luo escen mic opho og aphs displaying enhanced au ophagic lux (p e alence o ed o e yellow do s) in he sample exposed o SFB. (d). Exp ession o myc- agged POLG1 (D890N) in HEK293 cells en days a e cul i a ion in he p esence o 50 ng/ml doxycyclin. (e). Induced (Dox1) and non induced (Dox2) HEK-293 POLG1 (D890N) cells we e s imula ed o 4 hou s as indica ed (SFB 155mM) and phospho yla ion o AMPK and S6 e alua ed by immunoblo ing as in a. To al AMPK is also depic ed. In he la e panel, appea ance o a ain slow mig a ing band abo e he main one con i ms AMPK hype phospho yla ion. SFB has li le e ec on AMPK in his highly glycoly ic cell line, unless glycolysis is inhibi ed (2DG and No Glucose). Mi ochond ia deple ion cancels SFB s imula o y e ec on AMPK. www.na u e.com/scien i ic epo s SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 7 con aining medium was hen emo ed, and he con e ed dye solubilized wi h 500 ml (24 well pla e) o 100 ml (96 well pla e) o acidic isop opanol (0.04 M HCl in absolu e isop opanol o Isop opanol/Dmso 151 / . Abso bance was ead a 570 nm. Fo P opidium Iodide exclusion assay, medium con aining loa ing cells was emo ed and a ached cells we e ypsinized and pooled wi h he loa ing ones in low cy ome y es ubes. Few seconds a e addi ion o P opidium Iodide (1 mg/ml inal concen a ion) cells we e analysed o ed luo escence by low cy ome y (Exc. 488 nm, ed luo escence channel FL-3). Pe cen age o PI posi i e (dead) cells was de e mined, a e exclusion o cell deb is. Colony o ma ion assay Fo he de e mina ion o he colony o ma ion capabili y, cells we e seeded in 6-well pla es (500/well) in comple e cul u e medium. A e wo weeks cells we e ixed wi h 3,7% Fo malin o i e hou s and s ained wi h 0.02% Giemsa. In acellula ATP measu emen .Fo in acellula ATP de ec ion, cells we e seeded in 12 well pla es (4 310 5 /well/ml) in p esence o in absence o ele an s imuli. A e 12 h cells we e p ocessed and ATP was de ec ed by using P omega ki , ENLITENHATP Assay Sys em Bioluminescence De ec ion, acco ding o he p o ide ’s ecommenda ions 39 . High esolu ion espi ome y on in ac cells.A suspension o LCSC-2 in DMEM/ F12, p ocessed espec i ely wi h DMSO (c l) and So a enib 2.5 mM, was added o each Oxyg aph Chambe (Chambe A and Chambe B), a a densi y o 0.5 3 10 6 cells/ml. The expe imen s began wi h he measu emen o he Rou ine espi a ion (i.e. he espi a ion o d ug- ea ed cells esuspended in he medium wi hou he addic ion o subs a es), ollowed o abou 10 minu es (STATE R). Then, 2ml o 4 mg/ml Oligomycin, an ATP syn hase blocke , we e added in each chambe (STATE L) and espi a ion was eco ded o 6 minu es. Subsequen ly, 5 mg/ml o FCCP (a p o onopho e - H 1 ionopho e - and uncouple o oxida i e phospho yla ion) we e added (STATE E) and es- pi a ion was eco ded o 3 minu es; highe FCCP concen a ions o wide / b oade ime in e als exe an inhibi o y e ec on cellula espi a ion depending on mi ochond ia. All inhibi o s and uncouple s used in he p o ocol a e able o c oss he cell mem- b ane and do no equi e a p io cells pe meabiliza ion 40 . Con ocal analysis o au ophagic lux in LCSC-2 cells.Quan i a i e assessmen o au ophagosome o-au olysosome ma u a ion dynamic he was pe o med acco ding o 24 using a luo escen agged LC3 p obe. Figu e 6 | LCSC-2 cell killing by SFB in ol es ROS and is coun e ac ed by AMPK. (a) PI exclusion assay depic ing he e ec o Ebselen (2 mM, g ay ba s) on LCSC-2 cell g ow h inhibi ion by SFB and SFB 12DG. Ba s a e mean 6SD o duplica e o iplica e samples. S a is ics a e by wo-way ANOVA. ** p,0.01 compa ed o ehicle. Signi icance o he in e ac ion SFBxEBS is also indica ed. Rep esen a i e o wo independen expe imen s. (b). Iden i ica ion and so ing o LCSC-2 cell popula ions wi h low and high con en o ROS, based on H2-DCFDA luo escence dis ibu ion on g een luo escence FL-1 his og am. (c). g ow h inhibi ion e alua ed by MTT assay. Values a e pe cen ages o Dmso/PBS. Ba s a e mean 6SD o duplica e samples. S a is ics o ele an compa isons by wo-way ANOVA a e indica ed. Signi icance o he in e ac ion e ec (ROS x SFB) is indica ed. Pic u e ep esen a i e o wo independen expe imen s. (d). Knock-down o AMPKaby siRNA sensi izes LCSC-2 cells o SFB. (e). PI exclusion assay on mock (siC l) and AMPK-silenced (siAMPKa) LCSC-2 cells exposed o SFB alone o in combina ion wi h 2DG o 24 hou s. Signi ican di e ences (AMPK e sus C l) a e indica ed by as e isks. *p,0.05; **p,0.01 by wo-way ANOVA. Ba s a e mean 6SD o duplica e samples. Pic u e ep esen a i e o wo independen expe imen s. (e). wes e n blo analysis con i ming AMPK down egula ion and impai ed pS6 inhibi ion/LC3B lipida ion in siAMPK- ea ed cells exposed o SFB. Rep esen a i e o wo independen expe imen s. ( ). PI exclusion assay illus a ing LCSC-2 sensi i i y o 5 mM SFB upon pha macological modula ion o AMPKa; inhibi o s we e added o cells 2 hou s p io o SFB, and iabili y was assessed 22 hou la e . *p,0.05 ** p,0.01 by one-way ANOVA. Values a e mean 6SD o duplica e samples. Pic u e ep esen a i e o wo independen expe imen s. www.na u e.com/scien i ic epo s SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 8 Isola ion o mi ochond ia om a li e .Mi ochond ia we e isola ed om he li e o an adul emale Wis a a , using he ki MITOISO1 om Sigma. De e mina ion o o ganelle ansmemb ane po en ial wi h JC-1 was pe o med acco ding o he ki ins uc ions, as desc ibed elsewhe e 41 . 1. Bonuccelli, G. e al. Ke ones and lac a e ‘‘ uel’’ umo g ow h and me as asis: E idence ha epi helial cance cells use oxida i e mi ochond ial me abolism. Cell Cycle 9, 3506–3514 (2010). 2. DeBe a dinis, R. J., Lum, J. J., Ha zi assiliou, G. & Thompson, C. B. The biology o cance : me abolic ep og amming uels cell g ow h and p oli e a ion. Cell Me ab 7, 11–20 (2008). 3. Hanahan, D. & Weinbe g, R. A. Hallma ks o cance : he nex gene a ion. Cell 144, 646–674 (2011). 4. WARBURG, O. On espi a o y impai men in cance cells. Science 124, 269–270 (1956). 5. Pani, G., Giannoni, E., Galeo i, T. & Chia ugi, P. Redox-based escape mechanism om dea h: he cance lesson. An ioxid. Redox. Signal. 11, 2791–2806 (2009). 6. Diehn, M. e al. Associa ion o eac i e oxygen species le els and adio esis ance in cance s em cells. Na u e 458, 780–783 (2009). 7. Liu, H., Sa a aj, N., P iebe, W. & Lampidis, T. J. Hypoxia inc eases umo cell sensi i i y o glycoly ic inhibi o s: a s a egy o solid umo he apy (Model C). Biochem. Pha macol. 64, 1745–1751 (2002). 8. Lin, Z. Y. & Chuang, W. L. Genes esponsible o he cha ac e is ics o p ima y cul u ed in asi e pheno ype hepa ocellula ca cinoma cells. Biomed. Pha maco he . 66, 454–458 (2012). 9. Piscaglia, A. C. e al. Es ablishmen o cance cell lines om a hepa ocholangioca cinoma and assessmen o he ole o g anulocy e-colony s imula ing ac o and hepa ocy e g ow h ac o in hei g ow h, mo ili y and su i al. J. Hepa ol. 51, 77–92 (2009). 10. Robe s, L. R. So a enib in li e cance --jus he beginning. N. Engl. J. Med. 359, 420–422 (2008). 11. Ha zi assiliou, G. e al. RAF inhibi o s p ime wild- ype RAF o ac i a e he MAPK pa hway and enhance g ow h. Na u e 464, 431–435 (2010). 12. Wilhelm, S. M. e al. BAY 43-9006 exhibi s b oad spec um o al an i umo ac i i y and a ge s he RAF/MEK/ERK pa hway and ecep o y osine kinases in ol ed in umo p og ession and angiogenesis. Cance Res. 64, 7099–7109 (2004). 13. Naza ian, R. e al. Melanomas acqui e esis ance o B-RAF(V600E) inhibi ion by RTK o N-RAS up egula ion. Na u e 468, 973–977 (2010). 14. Will, Y. e al. E ec o he mul i a ge ed y osine kinase inhibi o s ima inib, dasa inib, suni inib, and so a enib on mi ochond ial unc ion in isola ed a hea mi ochond ia and H9c2 cells. Toxicol. Sci. 106, 153–161 (2008). 15. Chiou, J. F. e al. So a enib induces p e e en ial apop o ic killing o a d ug- and adio- esis an Hep G2 cells h ough a mi ochond ia-dependen oxida i e s ess mechanism. Cance Biol. The . 8, 1904–1913 (2009). 16. Co ia , R. e al. So a enib-induced hepa ocellula ca cinoma cell dea h depends on eac i e oxygen species p oduc ion in i o and in i o. Mol. Cance The . 11, 2284–2293 (2012). 17. Uli i, P. e al. Role o RAF/MEK/ERK pa hway, p-STAT-3 and Mcl-1 in so a enib ac i i y in human panc ea ic cance cell lines. J. Cell Physiol 220, 214–221 (2009). 18. Soli , D. B. e al. BRAF mu a ion p edic s sensi i i y o MEK inhibi ion. Na u e 439, 358–362 (2006). 19. Zmijewski, J. W. e al. Exposu e o hyd ogen pe oxide induces oxida ion and ac i a ion o AMP-ac i a ed p o ein kinase. J. Biol. Chem. 285, 33154–33164 (2010). 20. Bull, V. H., Rajalingam, K. & Thiede, B. So a enib-induced mi ochond ial complex I inac i a ion and cell dea h in human neu oblas oma cells. J. P o eome. Res. 11, 1609–1620 (2012). 21. Wan ooij, S., Go a , S., Pohjoismaki, J. L., Yasukawa, T. & Spelb ink, J. N. Exp ession o ca aly ic mu an s o he m DNA helicase Twinkle and polyme ase POLG causes dis inc eplica ion s alling pheno ypes. Nucleic Acids Res. 35, 3238–3251 (2007). 22. Inoki, K., Zhu, T. & Guan, K. L. TSC2 media es cellula ene gy esponse o con ol cell g ow h and su i al. Cell 115, 577–590 (2003). 23. Mizushima, N. & Koma su, M. Au ophagy: eno a ion o cells and issues. Cell 147, 728–741 (2011). 24. Kimu a, S., Noda, T. & Yoshimo i, T. Dissec ion o he au ophagosome ma u a ion p ocess by a no el epo e p o ein, andem luo escen - agged LC3. Au ophagy. 3, 452–460 (2007). 25. Dang, C. V. Links be ween me abolism and cance . Genes De . 26, 877–890 (2012). 26. Yagoda, N. e al. RAS-RAF-MEK-dependen oxida i e cell dea h in ol ing ol age-dependen anion channels. Na u e 447, 864–868 (2007). 27. Mueckle , M. e al. Sequence and s uc u e o a human glucose anspo e . Science 229, 941–945 (1985). 28. E o, K. e al. Cloning o a comple e p o ein-coding sequence o human pla ele - ype phospho uc okinase isozyme om panc ea ic isle . Biochem. Biophys. Res. Commun. 198, 990–998 (1994). 29. Oli a, D., Cali, L., Feo, S. & Giallongo, A. Comple e s uc u e o he human gene encoding neu on-speci ic enolase. Genomics 10, 157–165 (1991). 30. Ku a a, T., Ogu i, T., Isobe, T., Ishioka, S. & Yamakido, M. Di e en ial exp ession o acili a i e glucose anspo e (GLUT) genes in p ima y lung cance s and hei li e me as ases. Jpn. J. Cance Res. 90, 1238–1243 (1999). 31. Moon, J. S. e al. K uppel-like ac o 4 (KLF4) ac i a es he ansc ip ion o he gene o he pla ele iso o m o phospho uc okinase (PFKP) in b eas cance . J. Biol. Chem. 286, 23808–23816 (2011). 32. Selga, E., Mo ales, C., Noe, V., Peinado, M. A. & Ciudad, C. J. Role o ca eolin 1, E- cadhe in, Enolase 2 and PKCalpha on esis ance o me ho exa e in human HT29 colon cance cells. BMC. Med. Genomics 1, 35 (2008). 33. Kaplon, J. e al. A key ole o mi ochond ial ga ekeepe py u a e dehyd ogenase in oncogene-induced senescence. Na u e 498, 109–112 (2013). 34. Scha e , Z. T. e al. An ioxidan and oncogene escue o me abolic de ec s caused by loss o ma ix a achmen . Na u e 461, 109–113 (2009). 35. Choo, A. Y. e al. Glucose addic ion o TSC null cells is caused by ailed mTORC1- dependen balancing o me abolic demand wi h supply. Mol. Cell 38, 487–499 (2010). 36. Shackel o d, D. B. e al. LKB1 inac i a ion dic a es he apeu ic esponse o non- small cell lung cance o he me abolism d ug phen o min. Cance Cell 23, 143–158 (2013). 37. Bi soy, K. e al. Me abolic de e minan s o cance cell sensi i i y o glucose limi a ion and biguanides. Na u e 508, 108–112 (2014). 38. Fuma ola, C. e al. E ec s o so a enib on ene gy me abolism in b eas cance cells: ole o AMPK-mTORC1 signaling. B eas Cance Res. T ea . 141, 67–78 (2013). 39. McEl oy, W. D. & DeLuca, M. A. Fi e ly and bac e ial luminescence: basic science and applica ions. J. Appl. Biochem. 5, 197–209 (1983). 40. Pes a, D. & Gnaige , E. High- esolu ion espi ome y: OXPHOS p o ocols o human cells and pe meabilized ibe s om small biopsies o human muscle. Me hods Mol. Biol. 810, 25–58 (2012). 41. Ree s, M., Smi h, T. W. & Chen, L. B. J-agg ega e o ma ion o a ca bocyanine as a quan i a i e luo escen indica o o memb ane po en ial. Biochemis y 30, 4480–4486 (1991). Acknowledgmen s We wish o hank D . Thomas D. Shupe and P o . B yon Pe e sen (Depa men o Pa hology, Immunology and Labo a o y Medicine, College o Medicine, Uni e si y o Flo ida, Gaines ille, Flo ida, USA), o ha ing kindly p o ided he LCSC-2 cell line. This wo k was pa ially suppo ed by Baye Pha maceu ical ( o A.G.), by he I alian associa ion o Cance Resea ch (AIRC, g an IG8634/2009 o G.P.) and by he I alian Minis y o Uni e si y and Resea ch (MIUR, PRIN 2008, g an nu2008T7WRTM_003 o G.P.). JNS is suppo ed by he Academy o Finland [CoE unding] and he Ne he lands O ganiza ion o Scien i ic Resea ch [NWO: VICI g an 865.10.004]. Au ho con ibu ions G.P. and A.G. concei ed and supe ised he s udy; G.P. and V.T. ca ied ou he expe imen s and analyzed da a; D.S. pa icipa ed in expe imen al p ocedu es; G.M. pe o med con ocal analysis o au ophagic lux; A.C.P. and G.M. analyzed da a; M.B., L.C. and C.B. ca ied ou he Mic oa ay Analysis; R.S. and A.P. pe o med he oxyme y expe imen s, J.N.S. con ibu ed undamen al eagen s and M.A.P. con ibu ed o da a analysis. G.P., V.T. and A.C.P. we e in ol ed in w i ing he pape . All he au ho s had inal app o al o he submi ed e sion. Addi ional in o ma ion Supplemen a y in o ma ion accompanies his pape a h p://www.na u e.com/ scien i ic epo s Compe ing inancial in e es s: V.T.’s ellowship was pa ially unded by Baye Pha maceu ical. The au ho s decla e no o he po en ial compe ing inancial in e es s. How o ci e his a icle: Teso i, V. e al. The mul ikinase inhibi o So a enib enhances glycolysis and syne gizes wi h glycolysis blockade o cance cell killing. Sci. Rep. 5, 9149; DOI:10.1038/s ep09149 (2015). This wo k is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in he c edi line; i he ma e ial is no included unde he C ea i e Commons license, use s will need o ob ainpe mission om he license holde in o de o ep oduce he ma e ial.To iew a copy o his license, isi h p://c ea i ecommons.o g/licenses/by/4.0/ www.na u e.com/scien i ic epo s SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 9