Tight coordination of protein translation and HSF1 activation supports the anabolic malignant state.

12 Pages·2014·2.82 MB·English

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
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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).
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