1、Tumour-secreted miR-9 promotes endothelial cellmigration and angiogenesis by activating theJAK-STAT pathwayGuanglei Zhuang,Xiumin Wu,Zhaoshi Jiang,Ian Kasman,Jenny Yao,Yinghui Guan,Jason Oeh,Zora Modrusan,Carlos Bais,Deepak Sampath andNapoleone Ferrara*Genentech Inc.,South San Francisco,CA,USAAngiog
2、enesis plays a crucial role during tumorigenesis andmuch progress has been recently made in elucidating therole of VEGF and other growth factors in the regulation ofangiogenesis.Recently,microRNAs(miRNAs)have beenshown to modulate a variety of physiogical and pathologicalprocesses.We identified a se
3、t of differentially expressedmiRNAs in microvascular endothelial cells co-culturedwith tumour cells.Unexpectedly,most miRNAs were de-rived from tumour cells,packaged into microvesicles(MVs),and then directly delivered to endothelial cells.Among thesemiRNAs,we focused on miR-9 due to the strong morph
4、olo-gical changes induced in cultured endothelial cells.Wefound that exogenous miR-9 effectively reduced SOCS5levels,leadingtoactivatedJAK-STATpathway.Thissignalling cascade promoted endothelial cell migration andtumourangiogenesis.Remarkably,administrationofanti-miR-9 or JAK inhibitors suppressed M
5、V-induced cellmigration in vitro and decreased tumour burden in vivo.Collectively,theseobservationssuggestthattumour-secreted miRNAs participate in intercellular communicationand function as a novel pro-angiogenic mechanism.The EMBO Journal(2012)31,35133523.doi:10.1038/emboj.2012.183;Published onlin
6、e 6 July 2012Subject Categories:RNA;molecular biology of diseaseKeywords:angiogenesis;endothelium;JAK-STAT;microRNA;tumourIntroductionAngiogenesis is a key requirement for tumour growth(Folkman,1995;Chungetal,2010).Muchevidenceindicates that VEGF is a major regulator of angiogenesis(Chung and Ferrar
7、a,2011).Various inhibitors of the VEGFsignalling pathway have been developed and approved fortreatment of cancer or intraocular neovascular disorders(Ferrara et al,2007;Ebos and Kerbel,2011).Recently,microRNAs(miRNAs)have been shown to provide a newlayer of regulation of gene expression in many phys
8、iologicaland pathological processes,including angiogenesis(Wangand Olson,2009;Anand and Cheresh,2011;Small andOlson,2011;Weis and Cheresh,2011).miRNAs are a family of noncoding RNAs B22nt in length,which can suppress gene expression by pairing to the 30untranslated regions(UTRs)of target mRNAs(Barte
9、l,2004,2009).Accumulating evidence indicates that endothelialmiRNAsareinvolvedindevelopmentalandtumourangiogenesis,thusprovidingopportunitiesforfurthercontrol of the tumour vasculature.Ablation of the miRNAprecursor-processing enzyme Dicer in endothelium reducestumour angiogenesis and progression(Su
10、arez et al,2008),suggesting that specific miRNAs in endothelial cells regulateangiogenic processes.In this regard,miR-296 and miR-132,both induced by VEGF,have been proposed to facilitateangiogenic switching(Wurdinger et al,2008;Anand et al,2010).It would be of considerable interest to identify VEGF
11、-independent angiomirs,which might serve as anti-angiogenictargets in combination with current anti-VEGF therapies.We performed a quantitative PCR-based miRNA screen andidentified a set of differentially expressed miRNAs in micro-vascular endothelial cells co-cultured with tumour cells.Unexpectedly,
12、most miRNAs were derived from tumourcells,packaged into microvesicles(MVs),and then directlydelivered to endothelial cells.Tumour secreted miRNAs werefunctional in recipient endothelial cells as assessed by theability to promote migration and neovascularization.Thus,our data support a novel cellular
13、 communication involvingdirectional transfer of miRNAs during tumour angiogenesis.ResultsTumour cells induce upregulation of miRNAs inendothelial cellsTo mimic some of the activation steps in angiogenesis,weestablished tumour-endothelial co-cultures,in which tumourand endothelial cells are separated
14、 by a transwell membraneinsert(Figure 1A).We paired five human tumour cell lines(H1299,non-small cell lung cancer(NSCLC);MDA435,mel-anoma;Panc1,pancreatic cancer;SF-539,glioblastoma;HM7,colorectal cancer)with microvascular endothelialcells isolated from normal tissues matching each tumourtype examin
15、ed(Figure 1B).After 24h,we profiled endothe-lial miRNAs in the presence or absence of tumour cells usingTaqman micro fluidic cards(Figure 1A).Hierarchical cluster-ing indicated that different endothelial cells have surprisinglydistinct miRNA expression(Figure 1B),while tumour cellstimulation only af
16、fected a subset of miRNAs(SupplementaryTable 1).Volcano plot of rank products analysis showedagroupofmostsignificantlydifferentiallyexpressedmiRNAs among the five endothelial cell types(Figure 1C),the majority of which are highly conserved across speciesand were confirmed by quantitative RTPCR(Figur
17、e 1D;*Corresponding author.Genentech Inc.,1 DNAWay,South SanFrancisco,CA 94080,USA.Tel.:1 650 225 2968;Fax:1 650 225 4265;E-mail:Received:29 February 2012;accepted:14 June 2012;publishedonline:6 July 2012The EMBO Journal(2012)31,35133523|&2012 European Molecular Biology Organization|All Rights Reser
18、ved 0261-4189/12www.embojournal.org EMBO THEEMBOJOURNALTHEEMBOJOURNAL3513&2012 European Molecular Biology OrganizationThe EMBO JournalVOL 31|NO 17|2012SupplementaryFigure1A).Importantly,thesemiRNAsappeared to be selectively activated by tumour cells,sinceSK23 melanoma cells,but not normal skin melan
19、ocytes orfibroblasts,promoted miRNA expression(Figure 1E).To rule out the possibility that our observations may berestricted to a few tumour cell lines,we screened a largepanel of NSCLC lines and found that the vast majority wereable to induce miRNA changes in endothelial cells,althoughto different
20、degrees(Supplementary Figure 1B).Furthermore,human umbilical vein endothelial cells(HUVECs),when co-cultured with human or mouse tumour cells,also exhibitedmiRNA changes comparable to those observed in microvas-cular endothelial cells(Supplementary Figure 1C).Therefore,our observations are consisten
21、t with a general endothelialmiRNA upregulation induced by tumour cells,which mayplay a role in tumour angiogenesis.Tumour-derived miRNAs are delivered into endothelialcells via MVsTo follow-up our observations with tumour-endothelial co-cultures,we tested conditioned media from several tumourcell li
22、nes and found that they also resulted in enhancedexpression of miRNAs in endothelial cells(SupplementaryFigure 2A),raising the possibility that soluble factors areresponsible for this effect.Recombinant VEGF had no effecton the miRNAs identified in this study.Additionally,wild-type(C2P)and VEGF-null
23、(G5)mouse fibrosarcoma cells(Dong et al,2004)had similar abilities to induce thesemiRNAs in HUVECs(Figure 2A).We initially considered the possibility that induction ofmiRNAs in endothelial cells might be mediated by growthfactor(s)other than VEGF.We tested several angiogenicfactors(bFGF,TNFa,HGF,PDG
24、F-A,PDGF-B,PDGF-C andangiopoietin-1)and found that none of these was able to alterthe expression of these miRNAs(Supplementary Figure 2B).We also undertook an initial biochemical characterizationof such activity.We chose SK23 tumour cells as a sourcebecause they elicited a strong induction of endoth
25、elialmiRNAs(Figure 1E)and could also be easily maintained inserum-free medium.The inducing activity was completelyretained by Amicon filters with up to 50kDa MW cutoff(SupplementaryFigure2C),whichindicatesthatthisLungH1299SkinMDA435PancreasPanc1BrainSF539ColonHM7Control+TumourmiR-9miR-9*miR-96miR-18
26、2miR-183miR-9miR-9*miR-96miR-182miR-183miR-9miR-9*miR-96miR-182miR-18300122344566810Relative miRNA expression(log2)log10P-valueRelative miRNA expression(log2)LungEC-H1299SkinEC-MDA435*Control00123456224646SK23 melanomaSkin melanocyteSkin fibroblastCo-cultureExperiment overviewVolcano plotlogFCmiR-12
27、25-3pmiR-374b*miR-372miR-9miR-183miR-451miR-182miR-96miR-642miR-9*miR-135bmiR-154miR-544miR-523Tumour cellsEndothelial cellsRNA purificationTaqMan arrayACDEBFigure 1 Tumour cells induce upregulation of miRNAs in endothelial cells.(A)Experimental overview.(B)Hierarchical clustering of miRNAsin contro
28、l or tumour-stimulated endothelial cells.(C)Volcano plot of miRNA changes across five pairs of tumour-endothelial cells.(D)Verification of tumour-induced miRNAs by quantitative RTPCR.Lung endothelial cells co-cultured with H1299 and skin endothelialcells co-cultured with MDA435 for 24h are shown.The
29、 experiment was repeated three times.*Po0.05,Students t-test.(E)Quantitative PCRofmiRNAs in skin microvesicular endothelial cells stimulated with SK23 melanoma cells,normal skin melanocytes or fibroblasts.The experimentwas repeated twice.*Po0.05,Students t-test.Secreted miR-9 regulates tumour angiog
30、enesisG Zhuang et al3514The EMBO JournalVOL 31|NO 17|2012&2012 European Molecular Biology Organizationfactor(s)has a relatively large molecular mass.We thensubjected the tumour cell conditioned media to anion ex-change chromatography on a Hi-Trap Q column,but themiRNA inducing activity did not bind
31、to the column andwasalmostcompletelyrecoveredintheflow-through(Figure 2B).In contrast,a heparin-sepharose(HS)columnboundthe activity,which was eluted by 0.5M NaCl(Figure 2C).These results prompted us to hypothesize thatendothelial miRNAs could be stimulated by tumour-derivedsoluble factors with hepa
32、rin-binding properties.However,in conflict with this hypothesis,we did not detectany significant changes in endothelial pri-miRNA or pre-miRNA in response to conditioned medium or HS fractions.Therefore,to determine the source of upregulated miRNAs,we knocked down Drosha,which is required for miRNAb
33、iogenesis.Surprisingly,Drosha knockdown in tumour cellsimpaired miRNA induction(Supplementary Figure 2D).Accordingly,when we individually inhibited miR-9 or miR-183 in SK23 melanoma cells,tumour cells were less capableof inducing endothelial miR-9 or miR-183,respectively(Supplementary Figure 2D).The
34、 level of inhibition inducedby siDrosha or antagomirs on endothelial miRNAs wasB50%.This incomplete inhibition may be due to the residualmiRNAs in knockdown conditions.These results suggest thatVEGFControlmiR-9miR-96miR-182miR-183SK23 CMSK23 MV4T1 MVC2PG5miR-9miR-9*miR-96miR-182miR-183SF539H1299MDA4
35、35Panc1HM7SK23C2PMC38G5EMT-6TC1miR-451miR-135bmiR-9Control1:41:101:251:41:21:101:25ControlInputFlow-throughmiR-9*miR-96miR-182miR-183miR-135bmiR-154miR-132miR-296miR-126000264810110100100010 000122344566788910101214Relative miRNA expression(log2)CM miRNA concentration(pM)Relative miRNA expression(lo
36、g2)Relative miR-9 expression(log2)0264810Relative miR-9 expression(log2)*PKH67-labelled SK23 MV30K filtrateControlInputFlow-throughFractionsFractions0.1 M1 MNaCl0.1 M1 M0.5 MNaClDiI-labelledSK23 co-culture Mouse cancer linesHuman cancer linesACEFDB30K filtrateMV of DiI-labelled SK23 Figure 2 Tumour-
37、derived miRNAs are delivered to endothelial cells via microvesicles.(A)Quantitative PCR of miRNAs in HUVECs stimulatedwith VEGF(50ng/ml),or co-cultured with parental(C2P)or VEGF null(G5)fibrosarcoma cells.*Po0.05,Students t-test.(B)Quantitative PCRofmiR-9 in HUVECs co-cultured with different fractio
38、ns of SK23 conditioned medium after anion exchange chromatography.The experiment wasrepeated twice.(C)Quantitative PCRof miR-9 in HUVECs co-cultured with different fractions of SK23 conditioned medium after heparin-sepharosechromatography.The experiment was repeated twice.(D)Conditioned medium was c
39、ollected from each cancer cell line and miRNAsconcentration was measured by quantitative RTPCR against spike-in synthetic oligonucleotides.(E)Quantitative PCR of miRNAs in HUVECstreated with SK23 conditioned medium,SK23 microvesicles or 4T1 microvesicles.Three biological replicates were used for eac
40、h condition.*Po0.05,Students t-test.(F)SK23 microvesicles were labelled with PKH67(green),concentrated with Amicon filters(30KDa)and incubatedwith HUVECs for 24 hours(left panel).SK23 cells were labelled with DiI(red)and co-cultured with HUVECs(middle panel).Purified microvesiclesfrom DiI-labelled S
41、K23 were concentrated with Amicon filters(30KDa)and added to HUVECs(right panel).The experiment was repeated twice.Secreted miR-9 regulates tumour angiogenesisG Zhuang et al3515&2012 European Molecular Biology OrganizationThe EMBO JournalVOL 31|NO 17|2012theobservedincreasesinendothelialmiRNAslargel
42、yoriginate within tumour cells in the co-culture system.Indeed,we were able to detect miRNAs in tumour condi-tioned media by RTPCR(Figure 2D).Considering that ourdata suggest these miRNAs are in relatively large molecularweight complexes,we hypothesized that tumour-secretedMVs might be the carriers(
43、Balaj et al,2011;Lee et al,2011;Chen et al,2012),transporting miRNAs into endothelial cells.In agreement with this hypothesis,isolated MVs from eitherSK23 or 4T1 cells induced miRNAs in endothelial cells(Figure 2E).To further verify that the miRNAs were indeedassembled within the MVs,we treated puri
44、fied MVs withRNase and found that miRNAs were substantially protectedfrom RNase digestion.Pre-incubation with 0.5%SDS,but notwith proteinase K,significantly reduced miRNA levels afterRNase treatment(Supplementary Figure 2E).To determinewhether MVs are truly taken up by recipient endothelial cells,we
45、 labelled isolated MVs with PKH67 green fluorescence andvisualized them while internalized into endosome-like struc-tures by HUVECs(Figure 2F,left panel).Similar results wereobserved when we incubated SK23 cells with DiI and co-cultured the labelled cells with HUVECs(Figure 2F,middlepanel).Furthermo
46、re,we verified that purified MVs from DiI-labelled tumour cells are also transferred into HUVECs(Figure 2F,right panel).Following miR-9 knockdown inSK23 cells,we observed a concurrent decrease in miR-9 levelsinpurifiedMVsorHUVECsco-culturedwithMVs(Supplementary Figure 2F).Taken together,these data d
47、e-monstrate that miRNAs are secreted by tumour cells and aredelivered into endothelial cells via a specific population ofMVs that bind heparin.Tumour secreted miR-9 promotes endothelial cellmigrationTo determine whether MVs carrying miRNA contribute toangiogenesis,we assessed endothelial cell migrat
48、ion in realtime using the xCELLigence system.Consistent with previousreports(Skog et al,2008;Grange et al,2011),both SK-23tumour conditioned media and isolated MVs led to enhancedmigration of HUVECs(Figure 3A).Following knockdown ofDrosha in tumour cells,tumour-derived MVs showed reducedability to p
49、romote endothelial cell migration,indicating thatmiRNAs in MVs play an important role in this process(Figure 3B).To identify functionally important miRNAs,we overex-pressed each miRNA found to be upregulated in endothelialcells and determined its ability to affect endothelial cellmotility.We found t
50、hat the vast majority of miRNAs had noobvious effect(data not shown).However,miR-9 inducedstriking morphological changes in HUVECs,including elon-gated cellular shape,fewer stress fibres and increased lamel-lipodia at the cell edge(Figure 3C).These observationssuggested a potential role for miR-9 in
