Tight coordination of protein translation and HSF1 activation supports the anabolic malignant state. PDF
Preview Tight coordination of protein translation and HSF1 activation supports the anabolic malignant state.
Tight Coordination of Protein Translation and HSF1 Activation Supports the Anabolic Malignant State Sandro Santagata et al. Science 341, (2013); DOI: 10.1126/science.1238303 This copy is for your personal, non-commercial use only. 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Copyright 2013 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS. RESEARCH ARTICLE SUMMARY Tight Coordination of Protein READ THE FULL ARTICLE ONLINE http://dx.doi.org/10.1126/science.1238303 Translation and HSF1 Activation Cite this article as S. Santagata et al., Science 341, 1238303 (2013). DOI: 10.1126/science.1238303 Supports the Anabolic Malignant State Sandro Santagata, Marc L. Mendillo, Yun-chi Tang, Aravind Subramanian, Casey C. Perley, Stéphane P. Roche, Bang Wong, Rajiv Narayan, Hyoungtae Kwon, Martina Koeva, FIGURES IN THE FULL ARTICLE Angelika Amon, Todd R. Golub, John A. Porco Jr., Luke Whitesell,* Susan Lindquist* Fig. 1. Inhibiting protein translation inactivates HSF1. Introduction: Ribosome biogenesis is commonly up-regulated to satisfy the increased anabolic Fig. 2. LINCS analysis reveals that targeting demands associated with malignant transformation and tumor growth. Many different oncogenic sig- protein translation inactivates HSF1. naling pathways converge on the ribosome to increase translational fl ux. Despite the detailed under- Fig. 3. Chemical screens reveal that targeting standing of ribosome regulation in cancer, it is not clear whether the net translational activity of the translation control inactivates HSF1. ribosome can itself regulate transcriptional programs that support and promote the malignant state. Fig. 4. Inhibiting translation initiation with Methods: To investigate the transcriptional effects of modulating translational activity in malignant rocaglates ablates HSF1 DNA binding. cells, we used integrated chemical and genetic approaches, including a gene signature–based genetic and chemical screen of more than 600,000 gene expression profi les (LINCS database) and an indepen- Fig. 5. Rocaglates modulate tumor energy dent, reporter-based chemical screen of more than 300,000 compounds. A lead compound was tested metabolism. in several cell lines unifi ed by their increased dependence on HSF1 activation for growth and survival, Fig. 6. Rocaglates selectively target aneuploid and in an in vivo cancer model. cancer cells and nontransformed cells with Results: Inhibiting translation led to large changes in the transcriptome. The single most enriched cancer-associated genetic aberrations. category consisted of genes regulated by the heat-shock transcription factor, HSF1. The most down- Fig. 7. Rocaglates suppress tumor growth, regulated mRNA was HSPA8, which encodes the constitutive HSP70 chaperone that helps to fold HSPA8 mRNA levels, and glucose uptake nascent polypeptides. The expression of many other genes that HSF1 coordinates to support cancer in vivo. was also strongly affected. HSF1 protein levels were unchanged, but HSF1 DNA occupancy was nearly eliminated. Inhibition of the HSF1-regulated gene expression program is thus a dominant transcrip- SUPPLEMENTARY MATERIALS tional effect elicited by inhibiting protein translation. Using a gene signature of HSF1 inactivation to query the LINCS database revealed a strong con- Materials and Methods nection between HSF1 inactivation and perturbations that inhibit protein translation, including a Figs. S1 to S9 broad spectrum of chemical and genetic interventions that target the ribosome, eukaryotic initiation Tables S1 to S5 factors (eIFs), aminoacyl tRNA synthetases, and upstream signaling/regulatory pathways that control References and Notes translation. Our high-throughput small-molecule screen identifi ed rocaglamide A, an inhibitor of translation RELATED ITEMS IN SCIENCE initiation, as the strongest inhibitor of HSF1 activation. An analog of this compound, RHT, increased V. Gandin, I. Topisirovic, Trans-HFS1 express. thioredoxin-interacting protein (TXNIP) mRNA and protein levels and decreased glucose uptake and Science 341, 242-243 (2013). lactate production. Cell-based cancer models characterized by high dependence on HSF1 activation DOI: 10.1126/science.1242359 for growth and survival were highly sensitive to RHT, as were cells derived from diverse hematopoietic malignancies. RHT had a strong antitumor effect—with marked inhibition of HSF1 activity and glucose uptake—against xenografted acute myeloid leukemia cells. Discussion: The ribosome functions as a central information hub in malig- nant cells: Translational fl ux conveys information about the cell’s metabolic status to regulate the transcriptional programs that support it. Multiple unbi- ased chemical and genetic approaches establish HSF1 as a prime transducer of this information, centrally poised to regulate the transcription of genes that support protein folding, biomass expansion, anabolic metabolism, cellular pro- liferation, and survival. Targeting translation initiation may offer a strategy for reversing HSF1 activation, disabling metabolic and cytoprotective pathways in malignant cells. HSF1 at the crossroads of protein translation and metabolism. (Left) Cancers activate an HSF1-regulated transcriptional program to adapt to the anabolic demands of relentless biomass expansion. Glucose uptake increases, and expression of TXNIP, an inhibitor of glucose uptake, drops. (Right) Down-regulating translation with rocaglate scaffold initiation inhibitors reverses cancer-associated HSF1 activation. Glucose uptake drops as TXNIP levels rise. The list of author affi liations is available in the full article online. *Corresponding author. E-mail: [email protected] (L.W.); [email protected] (S.L.) 250 19 JULY 2013 VOL 341 SCIENCE www.sciencemag.org Published by AAAS RESEARCH ARTICLE byHSF1weredown-regulatedwhentranslation- alfluxthroughtheribosomewasreduced.These Tight Coordination of Protein genes included drivers of cell proliferation and mitogenicsignaling(suchasCENPA,CKS1B,and PRKCA),transcriptionandmRNAprocessing Translation and HSF1 Activation (such as LSM2 and LSM4), protein synthesis (suchasFXR1andMRPL18),energymetabolism Supports the Anabolic Malignant State (suchasMAT2A,SLC5A3,PGK1,MBOAT7,and SPR),andinvasion/metastasis(suchasEMP2and LTBP1).Inacomplementaryfashion,genesthat Sandro Santagata,1,2,3*Marc L. Mendillo,3,4* Yun-chi Tang,4,5†Aravind Subramanian,6 arenegativelyregulatedbyHSF1wereup-regulated Casey C. Perley,3,4 Stéphane P.Roche,7Bang Wong,6 Rajiv Narayan,6 Hyoungtae Kwon,3,4 whentranslationalfluxthroughtheribosomewas Martina Koeva,3,4 Angelika Amon,4,5 ToddR. Golub,6John A. Porco Jr.,7 reduced.Theseincludedgenesthatpromotedif- Luke Whitesell,3‡Susan Lindquist3,4‡ ferentiation(suchasNOTCH2NL),cellularadhe- sion(suchasEFEMP1andLAMA5),andapoptosis The ribosomeiscentrally situatedtosensemetabolicstates,butwhether itsactivity,inturn, (suchasBCL10,CFLAR,andSPTAN1). coherentlyrewirestranscriptionalresponsesisunknown.Here,throughintegratedchemical-genetic ThiseffectoftranslationinhibitiononHSF1- analyses,we foundthatadominanttranscriptionaleffect ofblockingproteintranslationin regulated transcription led us to examine the cancercellswasinactivationofheatshockfactor1(HSF1),amultifacetedtranscriptionalregulator genome-widepatternofDNAoccupancybyHSF1 ofthe heat-shockresponseandmanyothercellularprocessesessential foranabolic metabolism, inbreastcancercells.Aftera6-hourexposureto cellularproliferation,andtumorigenesis.Theseanalyseslinkedtranslationalfluxtotheregulation cycloheximide,weperformedchromatinimmu- ofHSF1 transcriptionalactivity andtothemodulation ofenergymetabolism.Targetingthislink noprecipitation coupled with massively parallel withtranslation initiationinhibitorssuchasrocaglates deprived cancercellsoftheirenergyand DNAsequencing(ChIP-Seq)usingapreviously chaperone armamentariumand selectivelyimpairedthe proliferationofbothmalignantand validatedantibodyagainstHSF1(8).Despitecy- premalignant cellswithearly-stageoncogenic lesions. cloheximidetreatment,HSF1proteinlevelsthem- selvesremainedunchanged(Fig.1D).Incontrast Regulation of ribosome activity is crit- Results toDNAoccupancybyRNA-polymeraseII(which ical for supporting cellular prolifera- wasnotgloballyreduced),HSF1occupancywas tion.Incancer,ribosomebiogenesisis InhibitingTranslationalFluxInactivatesHSF1 nearlyeliminated(Fig.1,EtoG,fig.S2,andtable commonly increased to satisfy the increased To investigate the transcriptional effects of re- S3). This held true for genes that were either anabolic demands associated with malignant ducingtranslationalfluxthroughtheribosomein positivelyornegativelyregulatedbyHSF1,aswell transformation and tumor growth (1–4). In ad- malignantcells,weanalyzedthemRNAexpres- as for genes shared with the classic heat-shock dition,manydifferentoncogenicsignalingpath- sionprofilesofbreastcancercellsaftertreatment responseandgenesspecifictotheHSF1cancer ways converge on the ribosome to modulate withvariousinhibitorsoftranslationelongation program(tableS3).Together,thesedatapointed itsfunction(5,6).Theseinputsareintegrated, (anisomycin,emetine,cephaeline,andcyclohex- toalinkbetweentheactivityoftheribosomeand and the net ribosomal translational activity is imide).Largechangesinthetranscriptomewere theactivityofHSF1. tunedtoreflectthemetabolicandproliferative highlycorrelatedacrossallfourinhibitors[Pearson state of the cell. Despite the detailed under- correlation coefficient (r) between 0.85 to 0.97 LINCSEstablishes Translationas a Potent standingofribosomeregulationincancer,itis forallpairwisecorrelations].Themoststrongly Regulatorof HSF1 inCancerCells not well understood whether the net trans- enriched category consisted of genes regulated TofurtherinvestigatethelinkbetweentheHSF1 lational activity of the ribosome can itself be by promoters that contain DNA binding motifs activityandtranslationalprogram,weturnedtoan conveyedto regulatetranscriptional programs fortheheat-shocktranscriptionfactorknownas expression-profiling resource created by the Li- that support and promote the malignant state. heatshockfactor1(HSF1)(P=9.87×10–7) braryofIntegratedNetwork-basedCellularSig- Is a coherent and coordinated transcriptional (Fig.1AandtableS1).Ofthe13,258genesmea- natures(LINCS)program(Fig.2andsupplementary response triggered by modulating translation sured, the single most down-regulated mRNA materials, materials and methods). The LINCS activity? wasHSPA8,whichencodesaconstitutiveHSP70 databaseisalargecatalogofgene-expression chaperonethatfoldsnascentpolypeptidesasthey profilescollectedfromhumancellstreatedwith emergefromtheribosome(Fig.1BandtableS2) chemical(small-molecule)andgenetic[shorthair- 1DepartmentofPathology,BrighamandWomen’sHospital (BWH), andHarvard Medical School, Boston, MA02215, (7).HSPA1A,acancer-inducedHSP70gene,was pinRNA(shRNA)]perturbations. USA.2DanaFarberCancerInstitute,Boston,MA02215,USA. alsoamongthe10mostdown-regulatedmRNAs. WegeneratedaquerysignatureforHSF1in- 3WhiteheadInstituteforBiomedicalResearch(WIBR),Cam- This transcriptional response suggested that re- activationfromexpressionprofilesofbreastcan- bridge,MA02142,USA.4HowardHughesMedicalInstitute, ducedtranslationalfluxcausesaprofoundshiftin cercellsthathadbeentreatedwithHSF1shRNAs DepartmentofBiology,MassachusettsInstituteofTechnology (MIT),Cambridge,MA02142,USA.5DavidH.KochInstitute theactivityofHSF1. (supplementarymaterials,materialsandmethods) forIntegrativeCancerResearchandHowardHughesMedical Inawiderangeofcancers,HSF1regulatesa (8).Thissignatureincludedbothgenesthatwere Institute,MIT,Cambridge,MA02142,USA.6BroadInstituteof transcriptional network that is distinct from the up-regulatedbyHSF1inactivationanddown- MITandHarvard,Cambridge,MA02142,USA.7Department conventionalnetworkactivatedbythermalstress regulatedbyHSF1inactivation.Wecomparedour ofChemistry,CenterforChemicalMethodologyandLibrary (8). This cancer network includes many classic HSF1 query signature with LINCS expression Development (CMLD-BU), Boston University, Boston, MA 02215,USA. “heat-shock”genes.But,italsoincludesabroad profilesfromninecelllinesthatarecurrentlythe *Theseauthorscontributedequallytothiswork. cadreofothergenesthatplaycriticalrolesinma- most extensively characterized in this database †Presentaddress:InstituteofHealthSciences,ShanghaiInsti- lignancy,someofwhicharepositivelyregulated (Fig.2A).Eightofthesearecancerlinesofdi- tutesforBiologicalSciences,ChineseAcademyofSciences& byHSF1andsomenegativelyregulated.Allfour versehistopathologicorigin.Theselineshavebeen ShanghaiJiaoTongUniversitySchoolofMedicine,20025 inhibitorsoftranslationelongationaffectedgenes treatedindividuallywith3866small-moleculecom- Shanghai,China. ‡Correspondingauthor.E-mail:[email protected](L.W.); intheHSF1cancernetwork(P=0.016)(Fig.1C poundsor16,665shRNAstargeting4219genes. [email protected](S.L.) andfig.S1).Genesthatarepositivelyregulated Thecompoundsusedforthesegeneexpression www.sciencemag.org SCIENCE VOL 341 19JULY2013 1238303-1 RESEARCH ARTICLE profilesencompassedU.S.FoodandDrugAd- signalingpathwaysthatregulateproteintransla- thehigh-throughput384-wellformat(supplemen- ministration(FDA)–approveddrugsandknown tion: phosphatidylinositol 3-kinase (PI3Kinase)/ tarymaterials,materialsandmethods),weused bioactives.TheshRNAsusedweredirectedagainst mammaliantargetofrapamycin(mTOR)inhib- a reporter cell line stably transduced with a the known targets of these compounds, against itors(Fig.2BandtableS4).Ofthenearly200 luminescence-basedreporterandinducedHSF1 genesinrelatedpathways,oragainstothergenes geneontologyclassesanalyzed,theribosomesub- activationwithasimpleproteotoxicstressor(the thathavebeenimplicatedinavarietyofhuman unitfamilywasthesinglemostenriched(Fig.2, proteasomeinhibitorMG132). diseases.Inall,wecomparedourHSF1signa- BandC,andtableS4).Inaddition,eukaryotic Approximately2500hitcompoundsfromthe turewith161,636LINCSsignatures,eachgen- initiation factors (eIFs) and aminoacyl tRNA primaryscreen,whichblockedinductionofthe eratedfromatleastthreereplicates(foratotalof synthetaseswerealsohighlyenriched.Thisun- reporter,werethencounter-screenedwithanin- 614,216profiles.) biasedanalysisusingtheLINCSdatabasedem- dependentdualreportercellline(Fig.3B)soas As expected, the LINCS perturbations that onstratestheconnectionbetweentranslationalflux toeliminatenonselectiveinhibitors.Thissecond negativelycorrelatedwithourHSF1inactivation andthefunctionofHSF1incancer. linehadbeenstablytransducedwithtwoconstructs, signaturewereenrichedforknownactivatorsof oneencodingagreenfluorescentprotein(GFP) HSF1.TheyincludedshRNAsthattargetcom- AnUnbiasedHigh-Throughput Chemical drivenbyHSEsandtheotherencodingaredflu- ponents of the proteasome. They also included Screenfor HSF1Inhibitors orescentprotein(RFP)drivenbyadoxycycline- compoundsthatinhibittheproteasomeandthat TofindalternatewaystoinhibitHSF1,weper- regulatedcontrolpromoter.Compoundsthat inhibitHSP90(Fig.2,BandCandtableS4). formedalargehigh-throughputchemicalscreen. selectivelyinhibitHSF1activityshouldsuppress TheLINCSperturbationsthatpositivelycor- We screened 301,024 compounds through the GFP expression in this cell line but should not relatedwithourHSF1inactivationsignaturewere U.S.NationalInstitutesofHealth(NIH)Molec- suppressdoxycycline-mediatedinductionofRFP. mosthighlyenrichedfortranslationinhibitors ularLibrariesProbeCenterNetwork(MLPCN; Notably, compounds that are thought to selec- (cephaeline,cycloheximide,andemetine)(Fig.2, PubchemAID,2118)(Fig.3A)usinganHSF1- tivelyinhibitHSF1—suchastriptolide,quercetin, BandC,andtableS4).Theseperturbationswere regulatedreporterdrivenbyconsensusheat-shock KNK423, and KNK437 (9)—all suppressed also highly enriched for compounds that target elements(HSEs).Toaccommodateconstraintsof bothreporters(fig.S3).Thus,thesecompounds Fig.1.Inhibitingproteintransla- tioninactivatesHSF1.(A)Geneset enrichment analysis was performed byusingtheMSigDBwebservicere- leaseversion3.84(www.broadinstitute. org/gsea/index.jsp) on genes nega- tivelyregulatedinbreastcancercells followinga6-hourincubationwithin- hibitors of protein translation elongation. Selected results are displayed; control tubulin after a 6-hour exposure to the indicated concentrations of completeGSEAresultsareprovidedintableS1.(B)Scatterplotoflevelsof cycloheximide(CHX).(E)HeatmapofRNApolymeraseIIChIP-Seqreaddensity mRNAtranscripts(log2)aftera6-hourincubationwiththeindicatedinhibitors inMCF7cellsthatweretreatedwithDMSOor10mMCHXfor6hours.Genomic ofproteintranslationelongationversuscontroldimethylsulfoxide(DMSO).The regionsfrom–2kbto+2kbrelativetothetranscriptionstartsiteforallRefSeq levelsofHSPA1AandHSPA8areindicatedforeachelongationinhibitor.(C) genesareshown.(F)HeatmapofHSF1ChIP-SeqreaddensityinMCF7cellsthat Translation elongation inhibitors alter the basal transcriptional program of weretreatedwithDMSOor10mMCHXfor6hours.Genomicregionsfrom HSF1inbreastcancercells.GenesboundbyHSF1inMCF7wererankedby –1kbto+1kbrelativetothepeakofHSF1bindingforallHSF1-enriched theirdifferentialexpressionbetweencellstreatedwithtranslationelongation regions (union of all HSF1-enriched regions in the four data sets depicted inhibitors(TI)andcontrolDMSO(D).Eachcolumnrepresentsageneandis here)areshown.(G)RepresentativegenesboundbyHSF1inMCF7cells(HSPA8, normalizedacrossthecolumn,withhighexpressioninredandlowexpression HSPA1A,CKS2,andRBM23).Thexaxisdepictsfrom−2kbfromthetranscription inblue.(D)AnimmunoblotshowsthelevelsofHSF1proteinandtheloading startsite(TSS)to5kbfromtheTSSforeachgene. 1238303-2 19JULY2013 VOL341 SCIENCE www.sciencemag.org RESEARCH ARTICLE arefarlessspecificforHSF1thancommonly additional rocaglates (fig. S4). These included itself(Fig.3Dandfig.S6A).However,mRNA assumed. both natural products and totally synthetic ana- levels of Hsp40 (DNAJA1) and Hsp70 genes Moretothepoint,thechemicalscreenresults logs preparedwithphotocycloadditionmethods (HSPA1BandHSPA8)droppedsubstantially.The alsosuggestedalinkbetweenHSF1activationand (12, 13). Five hydroxamate analogs were more mostdramaticallyaffectedwastheconstitutively thetranslationmachinery.Byfarthemostpotent potentthanrocaglamideAatinhibitingtheHSE expressed HSPA8 gene (>90% reduction) (Fig. and selective hit to emerge from the 301,024 reporterwhileretainingsimilarselectivity(table 3D).Thiswasalsothegenethatwehadfound compounds wetestedwastherocaglateknown S5).ThemostpotentinhibitorhadanIC of tobethemoststronglyrepressedbytranslation 50 asrocaglamideA[medianinhibitoryconcentra- ~20nM(tableS5).Wenamedthiscompound elongationinhibitors(Fig.1B). tion(IC )of~50nMfortheheatshockreporter [previouslyreportedas“8e”(13)]Rohinitib(or TheeffectsofRHTwerenotduetoreductions 50 versusIC >1000 nMfor thecontrol reporter] RHT),forRocaglateHeatShock,Initiationof inHSF1proteinlevels,whichremainedconstant 50 (Fig.3C).Thisnaturalproductinhibitsthefunc- TranslationInhibitor. (Fig. 3E and fig. S6B). The sharp decrease in tion of the translation initiation factor eIF4A, a HSP70 mRNAlevels inresponseto RHTheld DEADboxRNAhelicase(10,11).Presumably, Characterizing the Effects ofRHT on trueacrossahistologicallydiversepanelofhu- itpassedcounterscreeninginoursecondaryassay Cancer Cells mancancercelllines(MCF7,breastadenocarci- withthedualreportersystembecausetranslation Tovalidatefindingsfromourengineeredreporter noma;MO91,myeloidleukemia;CHP100,sarcoma; of the doxycycline-regulated RFP control does system,wemeasuredtheeffectsofRHTonthe andHeLa,cervicalcarcinoma)aswellasinar- notrequiretheclassicalcap-dependentinitiation basal expression of several endogenous HSF1- tificiallytransformed293Tkidneycells(Fig.3D complex. regulated transcripts (Fig. 3D and figs. S5 and andfig.S6,AandC).RHThadamuchsmaller To define structure-activity relationships for S6).RHTdidnotreducethetranscriptlevelsof effect on HSP70 mRNA levels in proliferating inhibitionoftheHSEreporterbyrocaglamideA, thecontrolhousekeepinggenesB2MandGAPDH. butnontumorigenicdiploidcells(WI38andIMR90) we used our dual reporter system to test 38 Nor did it reduce the transcript levels of HSF1 (fig.S6C). A 161K unique gene HSF1 query signature of Results ordered by B NES Score expression signatures up/down regulated genes connectivity to HSF1 2.28 Translation inhibitors * 2.00 Ribosome subunits * 1 161k 1 161k 1.71 PI3K/mTOR inhibitors * + + 11..2365 EAImFi nsouabcuynli ttsR *NA synthetases * 0 0 * p < 10-2 - -1.20 Proteasome subunits * - Genes ranked by Analytics calculate Negative Positive change in expression connectivity score connection connection -2.80 Proteasome inhibitors * vs. control -3.05 HSP90 inhibitors * C Total Negative connections Positive connections signatures All 161k perturbagens Null Proteasome 56 inhibitors HSP90 390 inhibitors Translation 142 inhibitors Ribosome 442 subunits -0.70 -0.50 -0.30 -0.10 0.10 0.30 0.50 0.70 Connectivity score Fig. 2. LINCSanalysisrevealsthattargetingproteintranslationin- Normalizedenrichmentscore(NES)ofselectedresultsareplotted(complete activatesHSF1.(A)SchematicrepresentationoftheLINCSanalysisused GSEAresultsareprovidedintableS4).(C)Barcodeplotoftheconnectivity toidentifychemicalandgeneticmodulatorsthatarecorrelatedwithHSF1 scoreofalloftheindividualperturbationscomprisingtheindicatedenriched inactivation(supplementarymaterials,materialsandmethods).Pinkrepre- chemical or gene sets. The bagel plot in the center of the barcode plot sentsgeneswhoselevelsincrease,andgreenrepresentsgeneswhoselevels summarizesthepositive,negative,andnull(notconnected)fractionsforthe decrease,aftershRNA-mediatedknockdownofHSF1.(B)GSEAresultsofour indicated enriched class. All perturbations that are positively or negatively HSF1inactivationsignatureLINCSanalysis.Perturbationsignatureswererank- connectedfortheindicatedenrichedclassesareshown.Totalperturbationsin orderedbyconnectivitywiththeHSF1inactivationsignature,andenrichment eachclassareindicatedontherightoftheplot.Bluerepresentsnegatively wasdeterminedforKEGGpathwaygenesetsandATCchemicalclasses(de- connected, and red represents positively connected, classes of enriched tailsareavailableinthesupplementarymaterials,materialsandmethods). perturbations. www.sciencemag.org SCIENCE VOL 341 19JULY2013 1238303-3 RESEARCH ARTICLE To obtain a more direct and global view of (TXNIP)wereup-regulated.TXNIPisapower- increased dependence on HSF1 activation for RHT’seffectsonHSF1activity,weexamined fulnegativeregulatorofglucoseuptakeandisa growth and survival. Although it occurs very genome-widepromoteroccupancybymeansof well-establishedregulatorofcellularenergystatus earlyduringoncogenesis,simplelossofthetu- ChIP-Seqanalysis.RHTvirtuallyabolishedHSF1 (16,17).Itsexpressionisdramaticallyreduced morsuppressorNf1leadstoanincreaseinHSF1 bindingthroughoutthegenome(Fig.4,AandB; inmalignantcells,leadingtoincreasedglucose proteinlevels,nuclearlocalization,andtranscrip- fig. S6D; and table S3). As had occurred with uptake(18).Conversely,increasingTXNIPlev- tionalactivation(19).Wetreatedmouseembryonic cycloheximide,RHTaffectedbothgenesthatare elsleadstoreducedglucoseuptake(16).Thein- fibroblasts (MEFs) in which Nf1 was knocked positivelyregulatedbyHSF1andgenesthatare ductionofTXNIPmRNAbyRHTwasobserved out and wild-type littermate control MEFs in negativelyregulatedbyHSF1(Fig.4A).Further- acrossadiversepaneloftumorcelllines(Fig.5A). whichHSF1wasnotactivated,witheitherRHT more, it affected both classic heat-shock genes TXNIPproteinlevelsalsoincreasedsharplyde- orwithcycloheximide.Thetwocelltypeswere andgenesspecifictotheHSF1cancerprogram spite a marked reduction in the levels of other similarly sensitive to cycloheximide. However, (tableS3).TheeffectsonHSF1DNAoccupancy short-lived proteins, such as p53 (Fig. 5B). Al- Nf1-null MEFs were more sensitive than were occurredatconcentrationsofcycloheximideand though we did not detect HSF1 bound to the wild-typeMEFstoRHT(Fig.6A).Inthismodel RHTthatinhibittheribosomeactivitytoasim- TXNIP locus, HSF1 null cells showed higher foranearlyeventintumorigenesis,targetingtrans- ilarextent(Fig.4C). levelsofTXNIP(fig.S7).Inaddition,HSF1did lationinitiationratherthantranslationelongation directlyregulateagroupofothergenesinvolved seemstoprovideamoreselective,bettertolerated RocaglatesModulateTumorEnergyMetabolism inenergymetabolism(includingMAT2A,SLC5A3, approach for disrupting the link between trans- Whilecharacterizing the effects of RHTon the andPGK1).Atafunctionallevel,theeffectsof lationandHSF1activation. transcriptome,wenotedthattreatedcellsfailedto RHTwereassociatedwithconcentration-dependent Asecondengineeredsystemallowedustoask acidifytheculturemedium(detectedincidentally reductions in both glucose uptake and lactate whether rocaglates would selectively inhibit the by thecolor of thepH indicator phenolred in- production(Fig.5C).Thus,theeffectsofRHTon growthofcellscarryingasimplechromosomal cludedinstandardmedia).Thissuggestedare- proteintranslation,HSF1activation,andenergy aberrationthatmodelsanothercommonearlyevent versalofthe“Warburgeffect,”ametabolicshift metabolism—processes lying at the core of the inthedevelopmentofcancer:aneuploidy.Chro- responsible for increased lactic acid production anabolicstateofcancer—appeartobecoordinated. mosomalimbalancesleadtobothincreasedener- bymanycancers.GeneticcompromiseofHSF1 gyandproteotoxicstress.Thisisreflectedbythe drivesashiftinmetabolisminbothcellculture Rocaglates SelectivelyTargetPre-Malignant elevation of theHSF1-regulated chaperone pro- andanimalmodels(14,15).Hence,thiseffectof Cellswith Early-Stage Oncogenic Lesions teinHSP72,encodedbyHSPA1A(20).Weisolated RHTisconsistentwithinactivationofHSF1. Doesthistightcoordinationcreatevulnerabilities MEFsfrommicecarryingRobertsonianfusions OurmRNAexpressionprofilingofrocaglate- for the malignant phenotype that could be ex- forchromosome13(21).TheseMEFs(TS13)car- treated breast cancer cells also revealed that ploitedasatherapeuticstrategy?Welookedata ryasingleextracopyof120Mbofchromosome mRNAlevelsforthioredoxin-interactingprotein rangeofcell-basedcancermodelsunifiedbytheir 13,introducinganadditionalcopyof843genes. Fig.3.Chemicalscreensrevealthattargeting translationcontrolinactivatesHSF1.(A)Flow- chart outlining the steps in the high-throughput MLPCNscreenforinhibitorsofHSF1activation.(B) Schematicofdual-reportercelllineusedtocounter-screenprimaryscreenhits.GFPexpressionisregulatedbyaheatshock–induciblepromoter.RFPexpression isregulatedbyadoxycyclineresponseelement(TetR).(C)EffectofrocaglamideAontheHSE-drivenGFPanddoxycycline-drivenRFPsignalsafterincubation with2.5mMMG132and2mg/mldoxycycline.ChemicalstructureofrocaglamideAisdisplayedintheinset.ErrorbarsindicatemeanTSEMofquadruplicates. (D)EffectofRHTonHSF1-regulatedandcontrolendogenousmRNAtranscriptlevelsinM0-91leukemiacellsmeasuredbymeansofnanostringnCounteraftera 6-hourincubationwithindicatedconcentrationsofRHT.LevelsofendogenoustranscriptareshownaspercentofDMSO-treatedcontrol.Meanofduplicatesare reported.(E)HSF1proteinlevelsarenotaffectedinM0-91leukemiacellstreatedwithRHT.ImmunoblotshowsthelevelsofHSF1proteinandtheloadingcontrol (Tubulin)aftera6-hourexposuretotheindicatedconcentrationsofRHT. 1238303-4 19JULY2013 VOL341 SCIENCE www.sciencemag.org RESEARCH ARTICLE Cycloheximide, as well as conventional cy- transformed colon epithelial cell lines with eu- mice (fig. S9). We assessed aqueous solubility, totoxicchemotherapeutics(taxolandhydroxyurea), ploidchromosomecontentweretheleastsensitive plasmastability,plasmaproteinbinding,hepatic inhibitedthegrowthofbothtrisomicandlitter- ofallthelineswetested(Fig.6F). microsome stability, and cellular permeability matecontrolMEFstoanequalextent(Fig.6B (fig.S9A).Nosevereliabilitieswerefound.We andfig.S8).But,trisomicMEFs(P<0.0001)were Rocaglates Suppressthe Growthof Cancer nextestablishedminimallytoxicparametersfor more sensitive than wild-type MEFs to RHT Cellsand Tumors dosingmicewithRHTandperformedaplasma (Fig.6B).Thus,againinthismodelforanearly Somerocaglateshavepreviouslybeenshownto pharmacokinetic study after administration of neoplasticchangethatactivatesHSF1,targeting exert profound anticancer activity (10, 24–26). 1mg/kgsubcutaneously(fig.S9,BandC).Peak translation initiation seems to provide a better WetestedRHTagainstacollectionofcelllines plasmalevelswerefarinexcessofthoserequired tolerated, more selective approach for targeting including nontransformed diploid lines and forthekeybiologicalactivitieswehaddemon- themalignantstate. cancer cell lines with diverse histopathological stratedincellculture.Moreover,levelsrequired HSF1 activation is even more prominent in originsandoncogeniclesions(Fig.7A).Thenon- foranticanceractivityinvitroweremaintainedin advancedmalignancies(8,22,23).Forexample, transformedcelllineswererelativelyresistantto excessof2hoursinvivo. coloncancersfrequentlyshowimmunohistochem- RHT(IC from100to300nM).Allcancercell Wenextestablishedsubcutaneoustumorxeno- 50 icalevidenceofstrongHSF1activation(Fig.6C) linesweresensitivetoRHT(IC <30nM);the grafts of the human myeloid leukemia cell line 50 andthiscorrelateswithpoorclinicaloutcome(8). hematopoietictumorcelllineswereespecially M091innonobesediabetic(NOD)–severecom- Weminedpubliclyavailableexpressionprofiling sensitive(IC ≤6nM).Weusedoneofthese binedimmunodeficient(SCID)immunocompro- 50 from colon cancer lines with highly aneuploid hematopoietictumorlines,theM0-91cellline misedmice.Whenthemeantumorvolumereached karyotypes[chromosomalinstability(CIN)]and originallyderivedfromapatientwithacutemye- 100mm3,weadministeredRHTat1mg/kgfor4 from colon cancer lines with near-euploid kar- loid leukemia (27), to further characterize the consecutivedayseachweekfor3weeks(Fig.7D). yotypesbutmicrosatelliteinstability(MIN).The effectsofRHT.RHTstronglysuppressedHSPA8 Overthetreatmentperiod,therewasnoevidence CIN lines expressed markedly higher levels of mRNAlevelsinM0-91cellsandinducedTXNIP ofgrosssystemictoxicity(fig.S9D).RHTmediated HSPA1A,whichisconsistentwithgreaterlevels mRNA(Fig.7B).Inaddition,RHTsharplyde- marked,sustainedinhibitionofthegrowthofthis ofproteotoxicstressandgreateractivationofthe creasedglucoseuptakebythesecells(Fig.7C). veryaggressivemyeloidmalignancy(Fig.7D). HSF1-regulated cancer program (Fig. 6, D and AretheeffectsofRHTincellcultureachiev- Wethenpursuedpharmacodynamicsstudies. E).Next,wetestedseveralpatient-derivedcolon ableatdrugexposuresthataresystemicallytol- Micebearingxenograftsweregivenasingledose cancerlineswithCINandseveralpatient-derived erableinanimals?Todirectlyaddressthiscritical of RHT. Tumors were explanted 4 hours later, colon cancer lines with MIN for sensitivity to issueoftherapeuticindex,wefirstusedstandard andHSPA8andTXNIPmRNAlevelsweredeter- inhibition by RHT. The CIN lines were much in vitro assays to test whether RHT had suffi- minedbymeansofreversetranscriptionpolymer- more sensitive than were the MIN lines. Non- ciently drug-like properties to justify testing in asechainreaction(RT-PCR)(Fig.7E).Similarto Fig.4.Inhibitingtranslation initiationwithrocaglates ablatesHSF1DNAbinding. (A)HeatmapofHSF1ChIP- SeqreaddensityinM0-91cells thatweretreatedwithDMSO, 20nMRHT,100nMRHT,or 10mMCHXfor6hours.Ge- nomicregionsfrom–1kbto +1kbrelativetothepeakof HSF1bindingforallHSF1en- richedregions(unionofall HSF1-enrichedregionsinthe sevendatasetsdepictedhere) areshown.(B)Representative HSF1-boundgenesinM0-91 cells(HSPA8,HSPA1B,CKS2, andRBM23).Thexaxisdepictsfrom–2kbfromthetranscriptionstartsite(TSS)to5kbfromtheTSSforeachgene.(C)AutoradiographofS35-labeledprotein lysatesfromMCF7cellstreatedfor6hourswiththeindicatedconcentrationsofRHTorCHX.Graphsshowthecountsperminutefromacetoneprecipitationof proteinsineachsample,quantitatedbyusingascintillationcounter. www.sciencemag.org SCIENCE VOL 341 19JULY2013 1238303-5 RESEARCH ARTICLE theeffectsweobservedincellculture,RHTcaused Althoughcancercellsoftenco-optpowerful, were used to ensure that additional karyotypic astrongdecreaseinHSPA8transcriptlevelsanda adaptive nononcogene systems for their benefit changeshadnotyetoccurred.Twoprimaryhu- strong increase in TXNIP transcript levels. In a (40),itnowappearsthatbyco-optingthelinkbe- mancelllines(CCD112CoNandCCD841CoN), separateexperiment,wemonitoredtheuptakeof tweentheribosomeandHSF1,cancersbecome five MIN lines (HCT-116, HCT-15, DLD-1, fluorescentlylabeled2-deoxyglucose48hours especiallyvulnerabletoagentsthattargettransla- SW48, and LoVo), and five CIN lines (Caco2, afterRHTdosing.RHTstronglysuppresseduptake tionanditsupstreamregulatorypathways.Inthis HT-29,SW403,SW480,andSW620)wereob- of this glucose analog by these tumors (Fig. regard, our animal experiments suggest that tar- tainedfromATCC.Chromosomenumberand 7F).Clearly,theeffectsofRHTachievedincell getingtranslationinitiationmayofferastrategy karyotypeinformationwereobtainedfromtheNa- culturecouldalsobeachievedinwholeanimals. forreversingHSF1activationanddisablingthe tionalCancerInstitutedatabaseandtheCOSMIC metabolicandcytoprotectiveaddictionsofma- datasetattheSangerInstitute.M0-91cellswere Discussion lignantcells. previously described (27). The M0-91 cell line HSF1providesessentialsupportforthemalig- usedinthisstudywereestablishedfromexplanted nantstatebyblockingapoptoticresponsesand Materials andMethods M0-91tumorsthathadbeenxenograftedoncein promoting protein synthesis, anabolic energy mice.Allcellculturesweremaintainedunder5% metabolism,mitogenicsignalingpathways,and CellLines CO inmediaaccordingtotheirspecifications. 2 pathways that facilitate invasion and metastasis WI38,CHP100,HeLa,293T,PC3,MCF7,and (8,14,15,19,23,28–30).Here,weshowthatthe NIH3T3 cells were purchased from American mRNAExpressionProfilingandAnalysis abilityofHSF1tomaintainthiscancerprogram Type Culture Collection (ATCC). Immortalized ExpressionprofilesforMCF7cellstreatedfor isexquisitelysensitivetotranslationalactivity. Nf1knockoutMEFsandlittermatewild-typecon- 6hourswithanisomycin(15mM),emetine(7mM), Ourworkestablishesthattheribosomecould trol MEFs were kind gifts from K. Cichowski. cephaeline (6 mM), and cycloheximide (14 mM) function as a central information hub: Transla- Littermate-derivedeuploidandtrisomicprimary were previously deposited in the Connectivity tionalfluxconveysinformationaboutthecell’s MEFsweredescribedpreviously(20).RHTtreat- Map(41).MCF7cellsweretreatedwith200nM metabolicstatustoregulatethetranscriptional mentsexperimentswereperformedbyusingchro- rocaglamideAor50nMRHTfor6hours,and programsthatsupportit.Thespecificmolecular mosome 13 trisomic cell lines and littermate RNAwas then purified after extraction with mechanismsarelikelytobemultifaceted,butHSF1 control euploid cell lines that carried a single TRIzolreagent[Invitrogen(Carlsbad,CA),cata- isclearlyalinchpininthisprocess.Oneplausible Robertsoniantranslocation.Early-passageMEFs logno.15596-026].Geneexpressionanalysiswas mechanismfortheeffectsoftranslationinhibitors onHSF1activitycouldinvolvethetranslationof mRNA for a modifier of HSF1 transcriptional activitythatissensitivetosubtlechangesintrans- lationactivity,involvingeIF4Aand/orotherinitia- tionfactors.BecauseHSF1regulatestheexpression of genes encoding for ribosomal subunits and otherregulatorsoftranslation(8,15,31),theex- istenceofafeedforwardregulatorycircuitinvolv- ingtheproteintranslationmachineryandHSF1 isalsopossible. HSF1 is centrally poised to support protein foldingandbiomassexpansionaswellasmany other functions to which malignant cells are addicted(8,14,15,32).Wepostulatethatthe translation/HSF1 link we have uncovered in cancermayderivefromancientsystemsgeared toalignandsynchronizeessentialcellularfunc- tionsforgrowthandsurvival.Incomparison,inthe nematodeHSF1isalongevityfactor,andinyeast itisanessentialgenethatparticipatesincotransla- tionalqualitycontrol(33–35). Inman,thetranslation/HSF1linkisparticu- larlyimportantinsupportingthemalignantphe- notypebecauseitcanrespondtovariedmetabolic Fig.5.Rocaglatesmodulate inputsthatarecommonlydysregulatedincancer (5,6,36–38).Thistranslation/HSF1linkallows tumorenergymetabolism. (A)TXNIPmRNAtranscriptlev- thesemetabolicinputstobolsterthecytoprotec- elsinapanelofcancercell tivemilieu,helpingtumorcellstoaccommodate linesmeasuredbynanostring thedrasticinternalimbalancesarisingduringon- nCounteraftera6-hourincu- cogenesisaswellasthesevereexternalstresses bationwith50nMRHT.Mean arisingfromtherapeuticinterventions (39). The ofduplicatesarereported.(B) tightcoordinationofproteintranslationandHSF1 Immunoblot showing TXNIP activation,togetherwiththemanywaysthatcells levelsintheindicatedcancer integrate the derangements of malignancy with celllinesaftera6-hourincu- translationalactivity,suggeststhatunifyingprin- bationwiththeindicatedcon- ciplesdriveHSF1activationacrosstheextraor- centrationofRHT.b-actinistheloadingcontrol.Theeffectonp53,ashorthalf-lifeprotein,isshown.(C) dinarilywiderangeofhumancancersinwhich EffectsoftheindicatedamountofRHTon[H3]-2-deoxyglucoseuptake(left)andlactateproduction(right) thatactivationoccurs(8,22). inapanelofcancercelllinesafter8hoursofincubation.ErrorbarsindicatemeanTSEMoftriplicates. 1238303-6 19JULY2013 VOL341 SCIENCE www.sciencemag.org RESEARCH ARTICLE performedbyusingAffymetrix(SantaClara,CA) positedinapublicdatabase[NationalCenterfor aftertreatmentofMCF7cellswithtranslation GeneChipHTHumanGenomeU133A96-Array Biotechnology Information Gene Expression elongation inhibitors was performed by using Plates,anddatawasanalyzedaspreviouslyde- Omnibus(GEO)pending].Genesetenrichment thegenesetenrichmentanalysis(GSEA)Web scribed (8). All microarray raw data were de- analysis of the differentially expressed genes site release version 3.84 (42). Enrichment for Fig. 6. Rocaglatesselectivelytargetaneuploidcancercellsandnontransformed cellswithcancer-associatedgeneticaberrations.(A)PhotomicrographsofNf1wildtype andNf1nullMEFsthatweretreatedfor14dayswith25nMRHT.Therelativeviablecell numberofRHT-treated(middle)andCHX-treated(right)areshown.Errorbarsindicate meanTSEMofn=6replicates.(B)Effectof72hourstreatmentwitheitherRHT(left)or cycloheximide(right)ontheproliferationofMEFscarryingasingleextracopyof120Mbofchromosome13(TS13)comparedwithMEFsderivedfromlittermate controls(WT),[meanTSD,n=3replicates,***P<0.001,two-wayanalysisofvariance(ANOVA)].(C)Photomicrographsofnormalcolonepithelialcellsand invasive colon adenocarcinoma (hematoxylin and eosin stains and HSF1 immunohistochemistry) from the same section of a human tumor resection (im- munostainedsimultaneously).HSF1-expressingcellsstainbrown,andHSF1-negativecellsstainbluefromthetoluidinebluecounterstain.Scalebar,50mm.(D) HSPA1AmRNAtranscriptlevelsareelevatedincolorectaladenocarcinomaswithhigh-gradeaneuploidkaryotypes.DatafromthreeMINandnineCINcolon cancercelllinesfromtheGSKCancerCellLineGenomicProfilingDataasdescribedinthemethods.Boxplot,barismedian,andwhiskersareminandmax(three celllinesinMINcategoryintriplicate9celllinesinCINcategoryintriplicate).(E)RT-PCRanalysisofHSPA1AmRNAlevelsintheindicatedeuploidnontransformed celllines,andMINandCINcancercelllines.ErrorbarsindicatemeanTSDoftriplicates.(F)EffectofRHTontheproliferationofapanelofcelllineswithhigh-grade aneuploidkaryotypes(CINlines:Caco2,HT29,SW403,SW480,andSW620);near-euploidkaryotypeswithmicrosatelliteinstability(MINlines:HCT-116,HCT-15, DLD-1,SW48,andLoVo);ornontransformedcolonepithelialcelllineswithaeuploidchromosomalnumber(CCD112CoNandCCD841CoN),(meanTSD,n=3 replicates,***P<0.001,two-wayANOVA)treatedfor72hours. www.sciencemag.org SCIENCE VOL 341 19JULY2013 1238303-7 RESEARCH ARTICLE HSF1-boundgenesamongthegenesdifferen- EvaluationofHSPA1AmRNAlevelswasper- ChiP-Seq andChIP-PCR tiallyexpressedaftertreatmentofMCF7cellswith formedbyusingdatafromtheGlaxoSmithKline Describedinsupplementarymaterials,materials translationelongationinhibitorswasconducted (GSK)CancerCellLineGenomicProfilingData andmethods. byusingGSEAv2.08software(42).HSF1-bound https://cabig.nci.nih.gov/community/caArray_GSK- genesinMCF7cellsweredefinedasthosegenes data.MINlinesusedwereHCT15,LS174T,SW48. Immunoblot boundinatleasttwoofthefourdatasets[two CINlinesusedwereNCIH508,NCIH747,SW1116, Describedinsupplementarymaterials,materials datasetsfromthisstudyandtwofrom(8)]. SW1417,SW403,SW480,SW620,T84,andSW948. andmethods. Fig. 7. Rocaglates suppress tumor growth, HSPA8 mRNA levels and RHT(1mg/kg),ondaysmarkedbydownwardpointingarrows].Eightmicewerein glucoseuptakeinvivo.(A)ScatterplotofIC valuesofthegrowthofadiverse eachtreatmentgroup(errorbarsindicatemeanTSEM;P<0.0001).(E)RT-PCR 50 panelofcelllinestreatedwithRHT.Cellsweretreatedfor5days.Redindicates analysisofHSPA8andTXNIPmRNAlevelsfromtumorxenograftsafterasingle hematopoieticcancerlines,darkblueindicatessolidtumorcelllines,andlight treatmentofeithervehicleorRHT(1mg/kg,subcutaneous;fivemiceineachgroup). blueindicateseuploidnontransformedcells.(B)mRNAlevelsofHSPA8,TXNIP, Tumorswereharvested4hoursaftertreatment(errorbarsindicatemeanTSEM; andcontrolhousekeepinggenesinM0-91cellstreatedwithRHT.Meanofdu- HSPA8,P<0.005;TXNIP,P<0.0005,two-tailedttest,n=5replicates).(F) plicatesarereported.(C)GlucoseuptakeofofIRDye800CW2-deoyglucose(2-DG) RepresentativeimageofepifluorescenceofIRDye800CW2-deoxyglucose(2-DG) inM0-91celllinestreatedwithRHT.ImagingwasperformedbyusingLICOR. uptakeinM0-91xenografts.Micebearingtumorsweretreatedwithvehicleor (Right)Quantitationofmeasuredintensity(errorbarsindicatemeanTSEM;P< RHT (1 mg/kg) as described in the supplementary materials, materials and 0.005,two-tailedttest,n=4replicates).(D)PlotofthetumorvolumeofM0-91 methods;fourmiceineachgroup.Imageswereacquired48hoursafterthelast acutemyeloidleukemiaxenograftstreatedwithvehicleorRHT.Themeantumor treatment. (Right) Quantitation of measured radiant efficiency from epifluo- volume(incubicmillimeters)isplottedovertime.Miceweretreatedwithsub- rescenceofIRDye800CW2-DGfromimagesofM0-91xenografts(errorbars cutaneousinjectionsstartingonday18afterimplantation[eithervehiclealoneor indicatemeanTSEM;P=0.031,two-tailedttest,n=4replicates). 1238303-8 19JULY2013 VOL341 SCIENCE www.sciencemag.org
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