Nabet-2017-Exosome RNA Unshielding Couples Str.pdf

ArticleExosome RNA Unshielding Couples StromalActivationtoPatternRecognitionReceptorSignalingin CancerGraphical AbstractHighlightsdCytoplasmic RNA is shielded by RNA binding proteins toprevent RIG-I recognitiondNOTCH-MYC signaling in cancer fibroblasts deploysunshielded RN7SL1 RNA in exosomesdUnshielded RN7SL1 in exosomes acts as a DAMP andactivates breast cancer RIG-IdRNA unshielding couples stromal activation to inflammationand cancer progressionAuthorsBarzin Y.Nabet,Yu Qiu,Jacob E.Shabason,.,Julia Tchou,Joseph Marcotrigiano,Andy J.MinnCorrespondenceandyminnupenn.eduIn BriefStromal cells shed exosomes containingan RNA that,in its protein-free form,drives anti-viral signaling in recipientbreast cancer cells that ultimately resultsin tumor growth as well as therapyresistance.ExosomesStromal FibroblastsStromalActivationNOTCH1InflammatoryResponseInterferon-Stimulated GenesTumor Progression,Metastasis,ResistanceRN7SL1Breast Cancer CellsPOL3MYCRIG-I Signaling5ppp5ppp5ppp5ppp5pppExosomeTransferRN7SL1(shielded)SRP9/14RN7SL1(unshielded)5ppp5pppRIG-I(active)RIG-I(inactive)5ppp5pppNabet et al.,2017,Cell 170,352366July 13,2017 2017 Elsevier Inc.http:/dx.doi.org/10.1016/j.cell.2017.06.031ArticleExosome RNA Unshielding Couples Stromal Activationto Pattern Recognition Receptor Signaling in CancerBarzin Y.Nabet,1,8Yu Qiu,1,8Jacob E.Shabason,1,8Tony J.Wu,1,8Taewon Yoon,1,8Brian C.Kim,1,8Joseph L.Benci,1,8Angela M.DeMichele,2,6,7Julia Tchou,3,7Joseph Marcotrigiano,9and Andy J.Minn1,4,5,6,7,8,10,*1Department of Radiation Oncology2Department of Medicine3Department of Surgery4Institute for Immunology5Parker Institute for Cancer Immunotherapy6Basser Center for BRCA7Abramson Cancer Center8Abramson Family Cancer Research InstitutePerelman School of Medicine,University of Pennsylvania,Philadelphia,PA,USA9Department of Chemistry and Chemical Biology,Rutgers University,Piscataway,NJ,USA10Lead Contact*Correspondence:andyminnupenn.eduhttp:/dx.doi.org/10.1016/j.cell.2017.06.031SUMMARYInteractions between stromal fibroblasts and can-cer cells generate signals for cancer progression,therapy resistance,and inflammatory responses.Although endogenous RNAs acting as damage-associated molecular patterns(DAMPs)for patternrecognition receptors(PRRs)may represent onesuch signal,these RNAs must remain unrecognizedunder non-pathological conditions.We show thattriggering of stromal NOTCH-MYC by breast cancercells results in a POL3-driven increase in RN7SL1,an endogenous RNA normally shielded by RNAbinding proteins SRP9/14.This increase in RN7SL1alters its stoichiometry with SRP9/14 and generatesunshielded RN7SL1 in stromal exosomes.After exo-some transfer to immune cells,unshielded RN7SL1drives an inflammatory response.Upon transferto breast cancer cells,unshielded RN7SL1 activatesthe PRR RIG-I to enhance tumor growth,metastasis,and therapy resistance.Corroborated by evidencefrom patient tumors and blood,these results demon-strate that regulation of RNA unshielding couplesstromal activation with deployment of RNA DAMPsthat promote aggressive features of cancer.INTRODUCTIONThe dynamic interaction between cancer cells and stromal cellsof the tumor microenvironment critically regulates important fea-tures of cancer(Kalluri,2016).Reciprocal signaling betweenthese heterotypic cell types can beparacrine orjuxtracrine in na-ture and includes multiple oncogenic and developmental path-ways.However,howtheinteractionbetweencancerandstromalcells generates and then integrates signals that result in tumorgrowth,metastasis,therapy resistance,and sterile inflammationare not well understood.Across many common human cancers,a large proportion oftumors unexpectedly express high levels of interferon-stimu-lated genes(ISGs)that are typically associated with anti-viralsignaling(Weichselbaum et al.,2008).We reported that theseISGs can be induced in a subset of breast cancer cells uponcell-cell contact with stromal fibroblasts(Boelens et al.,2014).This subset of breast cancer cells is denoted as ISG responders(ISG-R)and are predominantly basal/triple-negative breast can-cers(TNBC).In contrast,ISG non-responders(ISG-NR)fail toupregulate ISGs and primarily belong to the luminal/ER-positivesubtypes.ISG induction results from the transfer of stromal-derived exosomes,which are small extracellular vesicles impli-cated in a myriad of processes related to cancer progression(Becker et al.,2016).These exosomes contain RNA(exoRNA)that is enriched in non-coding transcripts.Upon transfer toISG-R breast cancer cells,the exoRNA stimulates the viralRNA pattern recognition receptor(PRR)RIG-I,resulting inSTAT1 activation and ISG induction.STAT1 amplifies theNOTCH3 transcriptional response,resulting in expansion of tu-mor-initiating cells and therapy resistance(Figure 1A).Consis-tent with these experimental findings,patients with tumors ex-pressing high levels of ISGs are more likely to relapse afterchemotherapy or radiation therapy.Similar examples of PRRsrecognizing exoRNA in the tumor microenvironment have beenreported to influence cancer progression(Liu et al.,2016).How-ever,given that cancer-associated anti-viral signaling is occur-ring in a sterile microenvironment,this raises questions on thenature of the endogenous RNA that is activating RIG-I and theextent to which it influences the multitude of effects that stromalcells exert on cancer progression and therapy response.There are many properties that RIG-I utilizes to distinguish selffromnon-selfRNA.Typically,RIG-Irecognizescytoplasmicdou-ble-stranded RNA that is 50-triphosphorylated,short(300 bp)and has a blunt 50end(Schlee and Hartmann,2016).For viral352Cell 170,352366,July 13,2017 2017 Elsevier Inc.RNAs,polyuridine motifs can favor recognition(Saito et al.,2008),while RNA modifications such as 20-O-methylation cancritically prevent RIG-I binding to 50capped cellular RNAs(De-varkar et al.,2016;Schuberth-Wagner et al.,2015).However,much of the RNA features and requirements for optimal RIG-Iactivation are based on synthetic and/or artificial RNAs in vitro.Emerging evidenceindicates thatendogenousRNAcan functionas a damage-associated molecular pattern(DAMP)to activatePRRs under a variety of stress and pathological conditions,such as after chemotherapy(Chiappinelli et al.,2015;Rouloiset al.,2015;Sistigu et al.,2014)or radiation(Bernard et al.,2012;Ranoa et al.,2016),or in autoimmunity(Eckard et al.,ABCEFGDFigure 1.Stromal RNA Is Transferred to Breast Cancer Cells by Exosomes(A)Summary of ISG-R and ISG-NR breast cancer cells and differential exosome transfer and RIG-I activation upon interaction with stromal fibroblasts.(B)MRC5fibroblastsexpressingaCD81-RFPexosome reporter wereco-culturedwithCFSE-labeledISG-R1833orISG-NRMCF7breastcancercells.Exosometransfer is quantitated(right)and representative transfer is shown(arrows).(C)Schema for measuring RNA transfer from stromal to breast cancer cells utilizing the uridine analog EU for fluorescence microscopy(green)or 4sU forstreptavidin pull-down(orange).(D)MRC5 fibroblasts were labeled with EU and co-cultured with DiD lipid-labeled 1833 breast cancer cells.EU-positive 1833 cells(yellow arrows)and quan-titation are shown.(E)Relative transfer of 4sU RNA to mono-cultured 1833 breast cancer cells after addition of conditioned media(CM)isolated from 4sU-labeled MRC5 fibroblastsgrown in mono-culture(Stroma,orange)or from 1833 ISG-R co-culture(Co-cx,blue).Co-culture CM depleted of exosomes(Co-cx Exo(?)CM)is shown as acontrol for exosome-dependency(n=5).(F)Same as in(E)except CM was isolated from MRC5 or BJ 4sU-labeled fibroblasts grown in mono-culture or co-cultured with the indicated ISG-R or ISG-NRbreast cancer cells.Shown is relative 4sU RNA transfer after CM addition toeachbreast cancer cell mono-culture(n=3).Transfer is relative to mock 4sU labelingusing DMSO.(G)Allelic frequency of exoRNA SNPs from exosomes isolated from 1833 breast cancer(BrCa),MRC5 fibroblasts(Stroma),or from co-culture of both cell types(Co-cx).Analysis is based on SNPs present in exoRNA from breast cancer cells and not present in fibroblasts.Error bars are SEM of biological replicates.See also Figure S1.Cell 170,352366,July 13,20173532014;Hung etal.,2015).Howendogenous RNAs can functionasDAMPs to activate PRRs while avoiding recognition under non-pathological conditions is not well understood.RESULTSStromal RNA Is Transferred to Breast Cancer Cells byExosomesIncreased exosome production and transfer to breast cancercells only occurs when stromal cells are co-cultured with ISG-Rbreast cancer cells(ISG-R co-culture)but not when co-culturedwith ISG-NR breast cancer cells(ISG-NR co-culture)(Figure 1A).To examine whether the transfer of stromal exosomes also re-sults intransferofstromal RNA,wefirstlabeled MRC5stromal fi-broblasts with a stably expressed CD81-RFP exosome reporter(Figure 1B).This confirmed a high level of exosome transferfrom stromal cells to 1833 ISG-R breast cancer cells,which is ametastatic derivative of MDA-MB-231(Kang et al.,2003).Incontrast,ISG-NR co-cultures show minimal stromal exosometransfer.To examine if RNA from stromal cells accompaniesexosome transfer,we metabolically labeled stromal RNA with5-ethynyl uridine(EU)prior to co-culture with ISG-R 1833 breastcancer cells that were fluorescently marked with lipid dye(Fig-ure 1C).After 24 hours,over 40%of breast cancer cells acquiredstromal cell RNA as measured by EU-modification by azide-linked fluorescein(Figure 1D).Moreover,when stromal cellRNA was similarly labeled with 4-thiouridine(4sU)prior toISG-R co-culture(Figures 1C and S1),application of the exo-some-containing conditioned media(CM)to mono-culturedbreast cancer cells also resulted in stromal RNA transfer,asdeterminedbystreptavidinpull-downofbiotinylated4sU-labeledstromal RNA(Figure 1E).In contrast,stromal RNA was not trans-ferred when exosomes were depleted from the CM,consistentwith our previous findings that the ability of CM to induce ISGsis strictly exosome-dependent(Boelens et al.,2014).Exosome-mediated transfer of stromal RNA was also observed usinganother ISG-R breast cancer cell line,MDA-MB-436,and usingBJfibroblastcellsinco-culture(Figure1F).MarkedlylessstromalRNA was transferred by exosomes using CM from co-cultureswith the ISG-NR breast cancer cell line MCF7.To corroborate the transfer of stromal RNA by exosomes,wealso performed exoRNA SNP analysis using exosomes frommono-cultures of either ISG-R 1833 breast cancer cells orMRC5 stromal cells and compared SNP allelic frequencies totheexoRNAfromco-culture(Figure1G).MultipleSNPs,primarilyfrom mitochondrial RNA,were discovered to have an allelic fre-quency of near one in the exoRNA from breast cancer cells butnear zero in stromal exoRNA.Examination of exoRNA from co-culturerevealedthatmostoftheseSNPsmaintainedafrequencycloser to zero,consistent with the exoRNA primarily originatingfrom stromal cells.In total,these results suggest that cellularRNAs are transferred from stromal to breast cancer cells in anexosome-dependent manner.Stromal RNA Polymerase III Generates 50ppp ExoRNAthat Activates RIG-I in Breast Cancer CellsClassificationofnon-ribosomalexoRNAtranscriptsfromISG-R co-cultures reveals an enrichment in non-coding RNAscompared to cellular RNA(Figure 2A).These non-coding RNAsinclude repeat and transposable elements,small nuclear RNA(snRNA),signal recognition particle RNA(srpRNA),and others,but no viral RNAs were detected.Previously,we demonstratedthat upon transfection this exoRNA activates RIG-I to induceISGs in recipient breast cancer cells,and this activity requiresa50-triphosphate(50ppp)RNAend.Toconfirmthisnotion,weuti-lized CRISPR/Cas9 to knock out RIG-I in breast cancer cells andre-expressed either wild-type(WT)RIG-I or RIG-I with alaninesubstitution mutations in key lysine residues(K858 and K861)that make contacts with the 50ppp motif(RIG-IK858/861A)(Wanget al.,2010)(Figures S2A and S2B).Co-culture-derived exo-somes were purified(Figures S2C and S2D)and transfection ofthe exoRNA failed to induce ISGs in RIG-I KO breast cancer cells(Figure 2B).Re-expression of WT RIG-I rescued this defectwhereas RIG-IK858/861Awas markedly less effective at restoringactivity.Incontrast,cellularRNAfailedtoinduceISGsregardlessof RIG-I status.Thus,these results provide evidence that 50pppexoRNA from stromal cells activates RIG-I.In the absence of viral infection,the main source of endoge-nous 50ppp RNA is from RNA polymerase III(POL3)transcription(White,2011).Therefore,we sought to examine if stromal POL3generates the exoRNA that is transferred to breast cancercells to activate anti-viral signaling.Indeed,the POL3 subunitPOLR3G was upregulated in stromal cells after ISG-R co-culture(Figures 2C and S2E).Knockdown of POL3 using a small inter-fering RNA(siRNA)to the POLR3F subunit(Figure S2F)revealedthatinhibitingPOL3instromalcells,butnotinbreastcancercellsalone,significantly blunted breast cancer ISG induction(Fig-ure 2D).Interrogation of functional consequences revealed thatthe ability of stromal cells to protect breast cancer cells afterradiation was impaired with stromal POL3 knockdown,butunchanged after breast cancer POL3 knockdown(Figures 2Eand S2G).Consistent with these findings,treatment with aPOL3 small-molecule inhibitor(Wu et al.,2003)also bluntedstroma-mediated resistance and ISG induction in breast cancercells after co-culture(Figures S2H and S2I).To confirm that exo-somes are responsible for the effects resulting from inhibitingstromal POL3,we isolated exosome-containing CM fromISG-R co-culture treated with or without the POL3 inhibitor.CM isolated from co-culture both induced ISGs when added tomono-cultured breast cancer cells(Figure 2F)and re-estab-lished stroma-mediated radiation resistance that was abrogatedby POL3 inhibition(Figures 2G and S2J).In contrast,CM fromISG-R co-culture treated with POL3 inhibitor failed to induceISGs,but expression of unrelated genes such as IFI16 was notaffected(Figure 2F).To test if exoRNA can directly activateRIG-I in a POL3-dependent manner,we examined the abilityof exoRNA to stimulate ATP hydrolysis of recombinant RIG-I.Indeed,addition of exoRNA from ISG-R co-culture,but notexoRNA from POL3 inhibitor-treated co-culture or equimolaramounts of cellular RNA,stimulates RIG-I ATP helicase activity(Figure 2H).Thus,these results suggest that stromal POL3 gen-erates exoRNA that directly activates breast cancer RIG-I andpromotes stroma-mediated protection against DNA damage.To characterize the exoRNA generated by stromal POL3,wedeveloped an approach to identify 50ppp RNA by sequencing.For this,we utilized a set of enzymatic reactions to sequentially354Cell 170,352366,July 13,2017modify the 50end of RNA prior to library construction to depleteRNA lacking a 50ppp modification(50ppp-seq).Many coding andnon-coding RNAs were depleted by?10-fold or greater,consis-tent with the absence of a 50ppp(Figure 2I,left).Examination ofRNA classes that maintained or increased abundance revealedmany exoRNA transcripts known to