RAC1 activation mediates Twist1-induced cancer cell migration

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1 A R T I C L E S RAC1 ctivtion meites -inuce cncer cell migrtion Wen-Ho Yng 1, Hsin-Yi Ln 1, Chi-Hung Hung 2,3, Shyh-Kun Ti 4, Cheng-Hwi Tzeng 5, Shou-Yen Ko 6, Kou-Juey Wu 7,8, Mien-Chie Hung 9,1 n Muh-Hw Yng 1,5,8,11,12,13 Epithelil mesenchyml trnsition (EMT), which is chrcterize y the suppression of the hesion protein E-cherin, is crucil process tht promotes metstsis n stem-like properties of cncer cells. However, the issocition of cellulr ggregtes is not sufficient to explin why cncer cells move, n the motile nture of cncer cells unergoing EMT remins elusive. Here, we ientify mechnism in which the EMT inucer elicits cncer cell movement through ctivtion of RAC1. coopertes with BMI1 to suppress let-7i expression, which results in upregultion of n, leing to RAC1 ctivtion n enling mesenchyml-moe movement in three-imensionl environments. Moreover, the suppression of let-7i contriutes to -inuce stem-like properties. Cliniclly, ctivtion of the let-7i xis in he n neck cncer ptients correltes with tumour invsiveness n worse outcome. Our results uncover n essentil mechnism to explin how inuces the motile stem-like cncer cell phenotype eyon simply suppressing E-cherin. EMT, process in which epithelil cells lose their polrity n re converte into mesenchyml phenotype, is regre s criticl event uring emryonic evelopment, orgn firosis n tumour metstsis 1,2. In cncer cells, this event not only isrupts the intercellulr junctions n enhnces migrtion 1,2, ut it lso promotes stem-like properties to fcilitte metsttic coloniztion n tretment resistnce 3,4. However, the mechnisms riving the motility of these isggregte, stem-like cncer cells, which re unergoing EMT, remin uncler. Epithelil cells migrte s multicellulr ggregtes, tht is, collective movement 5,6, or s iniviul cells using either mesenchymlor moeoi-moe movement 7,8. The mesenchyml moe is chrcterize y n elongte shpe with protruing pseuopos, wheres the moeoi moe is chrcterize y roune morphology with memrnous leing 7 9. The movement of iniviul cells cn e clerly visulize in three-imensionl (3D) mtrices 1. Recent stuies hve inicte criticl role for smll GTPses in controlling iniviul cell movement. The ctivtion of Rc promotes mesenchyml-moe movement; in contrst, ctivte Rho coorintes moeoi motility through Rho-ssocite kinse 7,11,12 (ROCK). Although recent reports highlight the importnce of iniviul cell movement in cncer metstsis, the mechnistic links etween iniviul cell movement n other mjor mechnisms uring the metsttic process remin lrgely unknown. The pleiotropic effects of EMT regultors on ggressive cncer phenotypes hve een grully uncovere. We recently emonstrte tht, mster regultor of EMT (refs 13 15), regultes the expression of the polycom group protein BMI1 n cts coopertively with BMI1 to inuce EMT n stem-like properties 16. In this stuy, we ientifie the irect regultion of microrna (mirna) let-7i y, which results in RAC1 ctivtion n corresponing mesenchymlmoe movement in 3D environments. RESULTS Co-repression of let-7i y n BMI1 We initite this stuy y iming to ientify trgets to iscover unknown functions meite y. Owing to the criticl involvement of mirnas in cncer metstsis 19, we focuse on ientifying -regulte mirnas. We resone tht coopertes with BMI1 to suppress mirna trnscription, similr to 1 Institute of Clinicl Meicine, Ntionl Yng-Ming University, Tipei 11221, Tiwn. 2 Tiwn Avnce Biophrm, New Tipei City 2218, Tiwn. 3 Grute School of Biotechnology, Hung-Kung University, Tichung 32, Tiwn. 4 Deprtment of Otolryngology, Tipei Veterns Generl Hospitl, Tipei 112, Tiwn. 5 Division of Hemtology-Oncology, Deprtments of Meicine, Tipei Veterns Generl Hospitl, Tipei 112, Tiwn. 6 Deprtment of Stomtology, Tipei Veterns Generl Hospitl, Tipei 112, Tiwn. 7 Institute of Biochemistry n Moleculr Biology, Ntionl Yng-Ming University, Tipei 11221, Tiwn. 8 He An Neck Cncer Reserch Progrm, Ntionl Yng-Ming University, Tipei 11221, Tiwn. 9 Deprtment of Moleculr n Cellulr Oncology, The University of Texs M.D. Anerson Cncer Center, Houston, Texs 773, USA. 1 The Center for Moleculr Meicine, Grute Institute of Cncer Biology, Chin Meicl University, Tichung 442, Tiwn. 11 Institute of Biotechnology in Meicine, Ntionl Yng-Ming University, Tipei 11221, Tiwn. 12 Present ress: Institute of Clinicl Meicine, Ntionl Yng-Ming University, No. 1, Sec. 2, Li-Nong St., Peitou, Tipei 112, Tiwn. 13 Corresponence shoul e resse to M-H.Y. (e-mil: mhyng2@vghtpe.gov.tw) Receive 13 July 211; ccepte 7 Ferury 212; pulishe online 11 Mrch 212; DOI: 1.138/nc24 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION Mcmilln Pulishers Limite. All rights reserve.

2 A R T I C L E S mirna rry in sh-/ 113 mirnas expresse Fol chnge of luc/β-gl 113 mirnas expresse Upregultion > twofol in oth systems: let-7i, mir-762, mir-1915 qrt PCR vlition let-7i mirna rry in sh-bmi1/ 1,511 1, E3 E2 E (TSS) MIRLET7I luc let7i luca luc let7i lucb luc let7i mlucb pflag CMV pcdna3.1 pflag pcdna BMI1 let7i luca let7i lucb let7i mlucb Reltive let-7i expression Reltive let-7i expression M r (K) sh-bmi1 BMI sh- c M r (K) M r (K) e Control (133 p) Reltive let-7i expression CMV Twist-1 sh--1 sh-bmi1-1 CMV Input IgG CAGTTG - 3,378 p ChIP 331 p (158 p) nti- nti- BMI1 - sh-bmi1 MIRLET7I ChIP Control ChIP Control ChIP Control ChIP Control ChIP Control BMI1 Figure 1 Co-repression of let-7 i y n BMI1. () Schem for ientifiction of the puttive mirna(s) co-represse y n BMI1. () Upper: western lot of n qrt PCR (n = 3) of let-7i in the -knockown clone versus the control (sh- versus ). Lower: western lot of BMI1 n qrt PCR (n = 3) of let-7i in the BMI1-knockown clone versus the control (sh-bmi1 versus ). ws use s loing control for western lotting. Dt represent men ± s.e.m. P <.1 y Stuent s t -test. (c) Western lot of, BMI1 n qrt PCR (n = 3) of let-7i in FDu cells trnsfecte with n empty vector (CMV),, receiving shrna ginst scrmle sequence (-) or BMI1 (-sh-bmi1). ws use s loing control. Dt represent men ± s.e.m. P <.1 y Stuent s t -test. () Upper: schemtic representtion of the promoter region of the the wy tht n BMI1 co-repress E-cherin n p16ink4 (ref. 16). To select equte cell lines for our experiments, we screene the expression levels of n BMI1 n mnifesttion of the EMT phenotype in 4 he n neck squmous cell crcinom (HNSCC) cell lines, FDu, CAL-27, SAS n. ws selecte s the prentl cell line to generte the - n BMI1-knockown clones owing to the high enogenous n BMI1 expression levels n the mesenchyml phenotype in these cells. Conversely, FDu cells were selecte for ectopic expression of ecuse of their low n BMI1 expression levels n epithelil phenotype (Supplementry Fig. S1,). We crrie out mirna microrry nlyses in the - n BMI1-knockown cells versus control cells, n we focuse on the mirnas upregulte y more thn twofol in oth - n BMI1-knockown clones (Fig. 1). MIRLET7I gene n reporter constructs use in trnsient trnsfection ssys. The constructs were wil-type (let7i luca n let7i lucb) or E-ox-mutte (let7i mlucb). E1, E2, n E3 inicte the loction of E-oxes (CANNTG). TSS, trnscription strt site. Lower: promoter ctivity ssy (n = 3). HEK293T cells were co-trnsfecte with promoter construct n expression vector(s) or/n empty vector(s). Luciferse ctivity/ β-glctosise (β-gl) of cells trnsfecte with pflag CMV n pcdna3.1 ws pplie s the seline control for the experiments using the sme promoter. Dt represent men ± s.e.m. P <.1 y Stuent s t -test. (e) ChIP ssy. Schemtic of the esign of ChIP n control primers is shown in the upper pnel. The 158-p frgment contine the -ining sequence, wheres the 133-p frgment i not contin ny -ining sequence. Uncroppe imges of lots re shown in Supplementry Fig. S9. Three mirnas (let-7i, mir-762 n mir-1915) were upregulte in oth - n BMI1-knockown clones (Supplementry Tle S1). However, let-7i, memer of the let-7 mirna fmily tht functions s tumour suppressor n promotes stem cell ifferentition 2,21, ws the only one shown to e co-represse y n BMI1 through vlition (Fig. 1 n Supplementry Fig. S1c). The expression of let-7i ws inversely correlte with n BMI1 mong the HNSCC cell lines (Supplementry Fig. S1), n the suppressive effects of n BMI1 on let-7i were confirme in primry HNSCC cultures (Supplementry Fig. S1). Other mirnas of the let-7 fmily were not consistently influence y or BMI1 (t not shown). In trnsfectnts, the knockown of BMI1 restore let-7i expression (Fig. 1c). n BMI1 synergisticlly represse the let-7i promoter ctivity through n E-ox (Fig. 1). Chromtin 2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 212 Mcmilln Pulishers Limite. All rights reserve.

3 A R T I C L E S FDu SAS CAL D let7i-1 FDuspg-ctrl FDuspg-let7i-1 F-ctin /DAPI F-ctin /DAPI c Rtio of perimeter 2 / 4π to re let7i Rtio of perimeter 2 / 4π to re BMI1 let-7i FDu CAL-27 SAS 3D F-ctin /DAPI F-ctin /DAPI Rtio of perimeter 2 / 4π to re FDuspg-ctrl spg-let7i e 49. nm spg-ctrl 49. nm μm min 1 μm min Elongte Roun Elongte Roun Elongte Roun Elongte Roun let7i-1 μm min 1 μm min let7i-1 FDuspg-ctrl FDuspg-let7i-1 spg-ctrl let7i spg-let7i FDuspg-ctrl FDuspg-let7i-1 Invsion inex (relte to ) nm. nm let7i nm. nm. nm nm nm Invsion inex (relte to spg-ctrl) 49. nm nm. nm spg-let7i-1. nm. nm nm nm Figure 2 Repression of let-7i chnges cellulr morphology, enhnces motility n invsion. () Upper: phse-contrst imges of HNSCC cell lines cultivte on top of thick collgen (2.5D). Scle rs, 5 µm. Lower: quntifiction of cellulr morphology in HNSCC cell lines with ifferent levels of, BMI1 n let-7i. (n = 2 for ech cell line.) P <.1 y Stuent s t -test. () Immunofluorescence microgrphs showing the morphology n ctin orgniztion of cells trnsfecte with the let-7i-expressing vector (let7i-1) or control vector (), n FDu cells expressing the let-7i sponge vector (spg-let7i-1) or control vector (spg-ctrl). The cells were culture in 2.5D or emee in collgen (3D). Green, F-ctin; lue, nuclei. Scle rs, 25 µm (first n thir row) n 1 µm (secon n fourth row). (c) Quntifiction of cellulr morphology (n = 2 for ech stle cell line). P <.1 y Stuent s t -test. () Representtive trjectories n quntifiction of motility spee (n = 1 for ech stle cell line). Left: let7i-1 versus. Right: spg-let7i-1 versus immunoprecipittion (ChIP) confirme the irect ining of n BMI1 to the proximl promoter of let-7i (Fig. 1e). Together, these results support irect co-repression of let-7i y n BMI1. Suppression of let-7i promotes tumour-inititing cpility without ffecting EMT As let-7i is irect trget of BMI1 n the min function of BMI1 is to inuce EMT n stemness, we investigte the role of let-7i in inucing EMT n stem-like properties in HNSCC. To this en, we stly expresse let-7i in low-let7i cells n neutrlize let-7i expression with sponge vector in high-let-7i FDu cells (Supplementry Fig. S1e,f). First, we exmine the involvement spg-ctrl. The upper pnels show the spee of elongte cells versus roune cells, n the lower pnels show the overll spee of ech stle cell line. Elongte cells: the longest imension ws twice the shortest n with t lest one protrusion. The frction of iniviul cells of, let-7i-1, spg-ctrl n spg-let7i-1 ws 9.2%, 92.9%, 92.6% n 83.8%, respectively. P <.1 y Stuent s t -test. (e) 3D invsion ssy. Left: representtive imges of clones tht inve into collgen fter 24 h (upper) n the reltive invsion inex (lower; n = 3). Right: representtive imges of FDu clones tht inve into collgen fter 24 h (upper) n the reltive invsion inex (lower; n = 3). Cells trnsfecte with the control vector ( n spg-ctrl) serve s control. Scle rs, 25 µm. P <.1 y Stuent s t -test. The ox plots in, c n represent smple mximum (upper en of whisker), upper qurtile (top of ox), mein (n in the ox), lower qurtile (ottom of ox) n smple minimum (lower en of whisker). The histogrms in e represent men ± s.e.m. of let-7i in the tumour-inititing cpility of HNSCC. Among the reporte let-7 trgets relte to stem cell properties (H-RAS, LIN28, HMGA2 n c-myc; refs 2 23), H-RAS ws significntly ownregulte y let-7i. However, the influence of let-7i on other reporte trgets ws not s significnt s on H-RAS (Supplementry Fig. S2). In HNSCC, let-7i only slightly influence cellulr prolifertion (Supplementry Fig. S2). However, let-7i significntly suppresse nchorge-inepenent growth (Supplementry Fig. S2c). Furthermore, ectopic let-7i expression in cells ecrese the proportion of cells hrouring the puttive HNSCC stem-like cell mrker CD44 (ref. 24) n ttenute spheroi formtion. Neutrliztion of let-7i in FDu cells enriche the popultion of NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION Mcmilln Pulishers Limite. All rights reserve.

4 A R T I C L E S M r (K) FDuspg-let7i let7i FDuspg-ctrl GTP RAC1 Totl RAC1 GTP Rho Totl Rho p-mlc2 (1) TSS (168) ATG (2,669) TGA (3,676 ~ 3,76) let-7i ining site let7i consensus 3 U UGUCG U GU UUG AU G AUG GAG U5 3 -UTR(wt) 5 ATAG AGCTACCAGGACAT ATCCTGCTC T CTA3 3 UTR(mut) 5 ATAGAGCTACA AGGAC AT ATCCGTATAGATC p p let7i Fol chnge of luc/β-gl pmir--wt pmir--mut MLC2 e (1) (24) (6,114) (7,86 ~ 7,122) TSS ATG TGA let-7i ining site c M r (K) 2 13 GEFs n RAC co-ctivtors 79 mrna ecrese 1 4 in let7i (versus ) let-7i trgets preicte y PicTr TrgetScn, VAV1, ARHGEF15, FARP1 Protein expression nlysis in let7i versus spg-let7i versus spg-ctrl let7i spg- ctrl FDuspglet7i VAV3 ARHGEF15 FARP1 μm min 1 f M r (K).4.2 EV.6 let7i consensus 3 UU GU CGU G U UUGAUGA UG G AG U5 3 -UTR(wt) 5 AATCAGGGAT TT TCA GC A CT AC C T CTG 3 3 UTR(mut) 5 AATCAGGGATTTTCAGC C AG C A AG ATG 3 Fol chnge of luc/β-gl EV p p let7i let7i-1 pcdna3 pcdna3 let7i-1 pmir -wt pmir -mut GTP RAC1 Totl RAC1 μm min 1 g M r (K) let7i-1 Mock Mock pcl-neo HA pcl Neo HA let7i-1 HA GTP RAC1 Totl RAC1 Figure 3 let-7i represses RAC1 ctivity through trgeting n. () Expression of GTP-oun RAC1, totl RAC1, GTP-oun Rho, totl Rho, phosphorylte MLC2 (p-mlc2) n totl MLC2 in n FDu stle cell lines. Totl RAC1 or totl Rho ws pplie s control for ctivte RAC1 or Rho. ws use s loing control. () Schem for ientifying the motility-relte trgets of let-7i. (c) Western lot of,, VAV3, ARHGEF15 n FARP1. () Upper: representtion of the orgniztion of the trnscript, n the wil-type or let-7i-ining-site-mutte 3 -UTR reporter constructs of (pmir--wt n pmir--mut). Lower: luciferse reporter ssy (n = 3 for ech group). Luciferse ctivity/β-glctosise (β-gl) of HEK293T cells trnsfecte with p ws pplie s the seline control for the experiments using the sme reporter. P <.1 y Stuent s t -test. (e) Upper: representtion of the orgniztion of the trnscript, n the wil-type or let-7i-ining-site-mutte 3 -UTR reporter constructs of (pmir--wt n pmir--mut). Lower: luciferse reporter ssy (n = 3 for ech group). P <.1 y Stuent s t -test. (f) Upper: expression of GTP-oun RAC1, totl RAC1 n in let7i-1 cells trnsfecte with pcdna3 or n empty vector (EV). Lower: representtive trjectories n quntifiction of spee in let7i-1 cells trnsfecte with pcdna3 versus EV (n = 1 for ech group). P <.1 y Stuent s t -test. The frction of iniviul cells of let-7i-1 cells trnsfecte with EV or is 9.8% n 89.6%, respectively. (g) Upper: expression of GTP-oun RAC1, totl RAC1 n HA in let7i-1 cells trnsfecte with pcl Neo HA or control. Lower: representtive trjectories n quntifiction of spee in let7i-1 cells trnsfecte with pcl Neo HA or control (n = 1 for ech group). P <.1 y Stuent s t -test. The frction of iniviul cells of let-7i-1 cells trnsfecte with mock or is 9.6% n 96.5%, respectively. The ox plots in f n g represent smple mximum (upper en of whisker), upper qurtile (top of ox), mein (n in the ox), lower qurtile (ottom of ox) n smple minimum (lower en of whisker). The histogrms in n e represent men ± s.e.m. Uncroppe imges of lots re shown in Supplementry Fig. S9. 4 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 212 Mcmilln Pulishers Limite. All rights reserve.

5 49, nm nm nm 49, nm 49, nm nm 49, nm nm nm nm nm nm 2,11,4 nm 2,11,4 nm 2,11,4 nm 2,11,4 nm A R T I C L E S f pflag CMV pflag p p let7i M r (K) FC FT FTM FTL Invsion inex (reltive to FC) FC FT FTM FTL FC FT FTM FTL GTP RAC1 Totl RAC1 g Rtio of perimeter 2 / 4π to re NSC23766 (μm) Rtio of perimeter 2 / 4π to re M r (K) Rtio of perimeter 2 /4π to re CMV FC FT h FC FT FTM FTL - - sh-bmi1 c sh- M r (K) NSC23766 (μm) NSC23766 (μm) 1 NSC23766 (μm) 1 μm min 1 FC μm min 1 e FC FT FTM FTL μm min 1 FT Figure 4 Repression of let-7i n RAC1 ctivtion is importnt for -inuce movement. () Expression of GTP-oun RAC1, totl RAC1,, n in FDu cells trnsfecte with pflag CMV (FC), pflag (FT), pflag n p (FTM), n pflag n p let7i (FTL). Totl RAC1 ws pplie s control for ctivte RAC1, n ws loing control. () Western lot of n in FDu cells trnsfecte with n empty vector (CMV),, receiving shrna ginst scrmle sequence (-) or BMI1 (-sh-bmi1). (c) Western lot of, n in cells receiving shrna ginst (sh--1, sh--2) or scrmle sequence (). () Quntifiction of morphology (n = 2 for ech stle cell line). P <.1 y Stuent s t -test. (e) Representtive trjectories n quntifiction of spee (n = 1 for ech stle cell line). The frction of iniviul cells of FC, FT, FTM n FTL cells is 86.5%, 82.8%, 9.% n 94.9%, respectively. P <.1 y Stuent s t -test. (f) 3D invsion ssy. CD44 cells, promote the spheroi-forming cpcity n enhnce the tumour-inititing ility in vivo (Supplementry Fig. S2 f). Next, we investigte the impct of let-7i on EMT. To our surprise, let-7i i not chnge the expression of EMT mrkers in 2D cultures (Supplementry Fig. S2g). As 3D cultivtion is superior system for representing the living microenvironments, n certin phenotypes cn e emonstrte only in 3D environment 25,, we investigte the reltionship etween EMT mrkers n let-7i in cells cultivte on thick collgen (2.5D) or emee in collgen (3D). The result rule out the correltion etween let-7i n EMT in either conition (Supplementry Fig. S2h,i). These results inicte tht the repression of let-7i enhnces the tumour-inititing cpility without ffecting EMT. let-7i repression promotes mesenchyml-moe movement As let-7 mitigtes metstsis without cler mechnism 2, we investigte whether the repression of let-7i contriutes to tumour metstsis y influencing iniviul cell movement. First, we exmine the morphology of HNSCC cell lines cultivte in ifferent conitions. Interestingly, when cells were culture on top of thick collgen or Upper: representtive imges of cells tht inve into collgen fter 24 h. Lower: reltive invsion inex (n = 3). Scle rs, 25 µm. P <.1 y Stuent s t -test. (g) Quntifiction of morphology in FC n FT cells trete with NSC23766 or vehicle control (n = 2 for ech group). P <.1 y Stuent s t -test. (h) Representtive trjectories n quntifiction of spee in FC n FT cells trete with NSC23766 or vehicle control (n = 1 for ech group). The frction of iniviul cells of the FC clone trete with or 1 µm of NSC23766 is 88.6% n 91.5%, respectively; n the frction of iniviul cells of the FT clone trete with or 1 µmof NSC23766 is 86.5% n 81.5%, respectively. P <.1 y Stuent s t -test. In, n c, the cells were collecte from 2.5D culture. The ox plots in, e, g n h represent smple mximum (upper en of whisker), upper qurtile (top of ox), mein (n in the ox), lower qurtile (ottom of ox) n smple minimum (lower en of whisker). The histogrm in f represents men ± s.e.m. Uncroppe imges of lots re shown in Supplementry Fig. S9. Mtrigel or emee in collgen, cells with lower let-7i levels (SAS n ) exhiite n elongte shpe with pseuopo protrusions, n cells with higher let-7i levels (FDu n CAL-27) mnifeste roune shpes. However, these morphologic ifferences were not oserve in cells cultivte on either uncote ishes or ishes cote with thin lyer of collgen (Fig. 2 n Supplementry Fig. S3 e). Consistently, in let-7i-mnipulte n FDu clones, let-7i influence the morphology only when culture on top of thick collgen (Supplementry Fig. 3f h). This fining implie tht repression of let-7i my hve n impct on ltering the cellulr morphology to llow mesenchyml movement in 2.5D or 3D environments. Using time-lpse microscopy, we oserve more rpi movement in the elongte low-let-7i SAS n cells in 2.5D environments. Conversely, the high-let-7i FDu n CAL-27 cells mnifeste roune shpes n sustntilly lower spees (Supplementry Movies S1 S4). To confirm the effect of let-7i on 3D motility, experiments were crrie out in the let-7i-mnipulte n FDu cells cultivte in the 2.5D or 3D system. Enforce let-7i expression in cells resulte in roune morphology n corticl ctin rrngement, NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION Mcmilln Pulishers Limite. All rights reserve.

6 A R T I C L E S FCP FLP FC FCP FLP FC FT FTM FTL FT FTM FTL c Numer of lung noules FCPFLP FC FT FTMFTL Reltive let-7i expression (T/N) T2 T3 T4 e Reltive let-7i expression (T/N) Metstsis( ) Metstsis() Figure 5 Repression of let-7i is criticl for -inuce tumour invsion. () Histologicl exmintion of the implnte sites of nue mice receiving injection of FDu cells trnsfecte with p spg-ctrl (FCP), p spg-let7i (FLP), pflag CMV (FC), pflag (FT), pflag p (FTM) n pflag p let-7i (FTL). The lck rrows inicte the orer of implnte tumour, n the re rrows inicte muscle infiltrtion of tumour cells. Scle rs, 4 µm. () Representtive photos of the lungs of severe comine immunoeficient (SCID) mice seven weeks fter til vein injection of ifferent clones of cncer cells. The rrows inicte the lung noules. Scle rs, 5 mm. wheres locking let-7i in FDu cells resulte in elongte morphology with pseuopo protrusions (Fig. 2,c n Supplementry Movie S5). In terms of cellulr movement, ectopic let-7i in cells reuce mesenchyml movement with ecrese spee, n neutrliztion of let-7i in FDu cells inuce mesenchyml movement with increse spee. Although mesenchyml- n moeoi-moe movements re interconvertile in melnom cells 7, in HNSCC cells, let-7i represse mesenchyml movement without ffecting moeoi motility (Fig. 2 n Supplementry Movies S1, S4 n S6 S8). In 3D environments, ectopic let-7i reuce invsiveness in cells, n locking let-7i in FDu cells ugmente invsiveness (Fig. 2e). Together, these results support criticl role for let-7i in cncer cell movement in 3D environments. let-7i reuces RAC1 ctivity without ffecting Rho signlling Next, we investigte how let-7i regultes iniviul cell movement. As Rc ctivtion is responsile for mesenchyml movement, n the Rho ROCK xis inuces moeoi motility 7, we exmine whether let- 7i coul lter the ctivity of Rc or Rho. Ectopic let-7i ttenute RAC1 ctivity in cells, wheres locking let-7i in FDu cells ctivte RAC1. However, the levels of GTP-oun Rho n phosphorylte myosin light chin-2 (MLC2), which re inictors of the ctivity of Rho ROCK signlling 27, were not chnge (Fig. 3). Furthermore, trnsfection of either wil-type or kinse-e ROCK (ref. 28) into let7i cells i not chnge the morphology n motility even when MLC phosphoryltion ws increse (Supplementry Fig. S4). (c) Quntifiction of the numer of pulmonry noules in til vein injection ssys (n = 6 for ech group). P <.1, P <.1 y Stuent s t -test. () Reltive let-7i expression level (tumour/norml rtio) in ifferent T stges of HNSCC (n = 18, 14 n for T2, T3 n T4, respectively). P <.1 y Stuent s t -test. (e) Reltive let-7i expression in ptients with or without lymph noe/istnt metstsis (n = 21, 37 for ptients with or without metstsis, respectively). The ox plots in n e represent smple mximum (upper en of whisker), upper qurtile (top of ox), mein (n in the ox), lower qurtile (ottom of ox) n smple minimum (lower en of whisker). The histogrm in c represents men ± s.e.m. These results inicte tht in HNSCC, let-7i ttenutes RAC1 ctivity without the counter-ctivtion of the Rho signlling pthwy. Rc, not Rho, is the criticl riving force for iniviul cell movement. Time-lpse microscopic oservtion in ifferent HNSCC cell lines showe tht mesenchyml movement is much more evient thn moeoi movement (Supplementry Movies S1 S4), inicting tht mesenchyml-moe, ut not moeoi-moe, motility is the min type of iniviul cell movement in HNSCC. The RAC1 co-ctivtors n gunine nucleotie exchnge fctor re trgets of let-7i Next, we ientifie the puttive trget genes tht meite the reuction in RAC1 ctivtion y let-7i. We focuse on the Rho-fmily gunine nucleotie exchnge fctors (GEFs) n Rc co-ctivtor(s) regulte y let-7i (n = 84). As mirnas suppress trget gene expression through messenger RNA clevge or trnsltionl repression 29, ifferent strtegies were pplie to mine the trgets (Fig. 3). First, complementry DNA microrry ws crrie out in let7i cells versus control cells to efine the trnscripts tht were ownregulte (log 2 rtio < 1) in the let7i cells (Supplementry Tle S2). We then etermine whether the ownregulte genes contine let-7i ining site using mirn softwre. Using this strtegy,, gene encoing memer of the p13cas (Crk-ssocite sustrte) fmily proteins known to ctivte RAC1 (ref. 3), ws highlighte s cnite let-7i trget. Furthermore, we use the mirna-trget-preicting softwre PicTr n TrgetScn 5.1 to screen 6 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 212 Mcmilln Pulishers Limite. All rights reserve.

7 A R T I C L E S for GEFs n RAC1 co-ctivtors regulte y let-7i. Four genes were preicte to e trgets of let-7i: ARHGEF15,, FARP1 n VAV1. We screene the protein expression of these five cnites in the let-7i-mnipulte clones. Only n, GEF known to ctivte RAC1 (ref. 31), were regulte y let-7i (Fig. 3c). Quntittive PCR with reverse trnscription (qrt PCR) confirme the ownregultion of trnscripts y let-7i (Supplementry Fig. S5,). The regultion of n y let-7i ws confirme in cells collecte from 3D cultures (Supplementry Fig. S5c,). A 3 -untrnslte region (UTR) reporter ssy confirme tht n re irect trgets of let-7i (Fig. 3,e). Reconstitution of or expression in let-7i cells restore RAC1 ctivity n mesenchyml movements (Fig. 3f,g n Supplementry Movies S9 n S1). Together, these results inicte tht trgeting n y let-7i contriutes to the reuction of RAC1 ctivity. RAC1 ctivtion is criticl for -inuce mesenchyml-moe movement. We next confirme tht inuces mesenchyml-moe movement in 2.5D or 3D environments through the repression of let-7i n ctivtion of RAC1. First, we investigte the existence of the BMI1 let-7i n RAC1 xis. In FDu cells, ectopic expression increse n expression n RAC1 ctivity, n the reconstitution of let-7i in trnsfectnts rogte n expression n RAC1 ctivity in oth 2.5D n 3D conitions (Fig. 4 n Supplementry Fig. S6). Knockown of BMI1 in trnsfectnts ownregulte n (Fig. 4). In cells, the repression of enogenous mitigte n expression (Fig. 4c). Next, we exmine the phenotypic impct of let-7i reconstitution in - expressing FDu cells. Reconstitution of let-7i i not chnge the morphology in 2D n expression of EMT mrkers in either 2D or 2.5D conitions (Supplementry Fig. S6,c). However, in 2.5D or 3D cultures, re-expression of let-7i rogte the -inuce cellulr protrusions, elongtion, invsion n mesenchyml movement (Fig. 4 f n Supplementry Fig. S6 n Movies S11 S15). Furthermore, the Rc inhiitor NSC23766 converte the elongte cells into roune cells n suppresse their movement. However, the effects of Rc inhiition on the FDu control clones were not s evient s in the trnsfectnts, supporting specific trgeting effect of the Rc inhiitor on -expressing cells (Fig. 4g,h n Supplementry Movie S16). In ition to repressing -inuce motility, let-7i ttenute the tumour-inititing cpility of the trnsfectnts: let-7i reconstitution reuce H-RAS expression, the proportion of CD44 cells n the in vivo tumour-inititing ility of cells (Supplementry Fig. S6e g). We investigte whether let-7i coul e represse y other EMT inucers, resulting in the upregultion of n expression. The results inicte tht lthough the expression of let-7i ws influence y severl EMT regultors, no other EMT regultor coul simultneously suppress let-7i expression n increse oth n expression to the sme extent s (Supplementry Fig. S7). Tken together, these results inicte tht mong the ifferent EMT regultors, let-7i is specificlly regulte y n is criticl for -inuce motility n stemness through n EMT-inepenent mechnism. Repression of let-7i y promotes tumour invsiveness in vivo n in he n neck cncer ptients To etermine the crucil effect of let-7i repression on -inuce invsion in vivo, we exmine the histology of xenotrnsplnte tumours shortly fter tumour formtion. The neutrliztion of let-7i in FDu cells promote invsion of the tumour cells into jcent tissues. In the trnsfectnts, reconstitution of let-7i rogte tumour invsion (Fig. 5). Furthermore, we investigte the effect of let-7i on pulmonry coloniztion of intrvenously injecte tumour cells. In FDu cells, let-7i neutrliztion increse the formtion of lung noules, n the trnsfectnts forme the most pulmonry tumours. Restortion of let-7i in the trnsfectnts prtilly ttenute this effect (Fig. 5,c). To confirm the significnce of let-7i expression in HNSCC ptients, we exmine let-7i expression in 58 cses using qrt PCR. Clerly reuce levels of let-7i expression were oserve in ptients with tumours tht h inve the jcent tissues, tht is, T4 isese 32 (Fig. 5). However, let-7i expression ws not significntly ifferent etween ptients with lymph noe or/n istnt metstsis when compre with those without metstsis (Fig. 5e). These results inicte tht the repression of let-7i in vivo hs higher impct on locl tumour invsion thn on the formtion of tumours t istl sites. Finlly, we investigte the prognostic significnce of the let- 7i n xis in HNSCC ptients. Two inepenent sets of smples were use in this stuy; group A ws exmine y qrt PCR, n group B ws exmine y immunohistochemistry. The chrcteristics of these two groups of ptients re presente in Supplementry Tle S3, n representtive results from the immunohistochemistry nlysis re shown in Fig. 6c. In ptient group A, negtive correltions etween let-7i n let-7i, n positive correltion etween were oserve (Supplementry Tle S4); however, mrna ws not correlte with let-7i, or (t not shown). Prognostic trens of the iniviul mrkers were emonstrte (Supplementry Fig. S8 c), n the comintion of ll three mrkers, tht is, the coexistence of more thn twofol increse in n n ecrese let-7i levels to less thn hlf-fol in tumour, showe n improve preiction of ptient outcome (Fig. 6,). In ptient group B, ws significntly ssocite with oth n (Supplementry Tle S5). The prognostic tren of iniviul mrkers ws lso oserve (Supplementry Fig. S8 f), n the co-expression of, n ws ssocite with the worst survivl (Fig. 6). We propose the following moel to summrize our finings: the overexpression of upregultes BMI1, n they ct coopertively to suppress let-7i expression, leing to increse expression of n n ctivtion of RAC1. In ition, the repression of let-7i y n BMI1 enhnces H-RAS expression. These moleculr events inuce mesenchyml-moe movements n tumour-inititing cpilities of cncer cells (Fig. 6e). DISCUSSION Here, we elucite the regultory mechnism controlling the iniviul movements of cncer cells n provie comprehensive picture of -inuce metstsis. On the sis of the present results n previous mjor finings out, we suggest tht trgets ifferent molecules to ensure the evelopment of cncer metstsis s follows. First, suppresses E-cherin, leing to cellulr isggregtion NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION Mcmilln Pulishers Limite. All rights reserve.

8 A R T I C L E S c let-7i Activtion of let-7i xis (n = 21) Tumour/norml rtio 2 Tumour/norml rtio.5 2 Tumour/norml rtio.5 Non-ctivtion of let-7i xis (n = 37) Proportion of overll survivl OS of group A (PCR group) DFS of group A (PCR group) let-7i xis non-ctivtion let-7i.8.8 xis non-ctivtion let-7i.4 xis ctivtion let-7i.2 xis ctivtion.2 P =.4 P = Months Months Proportion of isese-free Cse 1 Cse 2 e Increse Repression of let-7i Proportion of overll survivl De-repression of n.8.6 OS of group B (IHC group) non-co-expression co-expression Proportion of isese-free DFS of group B (IHC group) non-co-expression.4 co-expression.2.2 P =.1 P < Months Months Increse mesenchyml movements n tumour-inititing cpilities GDP RAC1 mrna mrna GTP RAC1 E-ox H-RAS mrna Increse BMI1 De-repression of H-RAS Figure 6 Activtion of the let-7i signl xis correltes with worse outcome of he n neck cncer ptients, n n illustrtion showing the function of let-7i signlling in cncer cells. () A het mp showing the result of qrt PCR of let-7i, n in HNSCC smples (n = 58). Re, upregultion in tumour tissue (tumour/norml 2); lue, ownregultion in tumour tissue (tumour/norml.5); white: tumour/norml mrna expression rtio etween.5 n 2. () Comprison of the overll survivl perios (OS; left) n isese-free survivl (DFS; right) of HNSCC ptients in group A (exmine y qrt PCR; n = 58) with ctivtion of the let-7i xis (tumour/norml 2, let -7i.5 n 2; n = 21) versus non-ctivtion (n = 37). P vlue ws estimte y log-rnk test n is shown in the left lower qurnt of ech pnel. (c) Representtive pictures of immunohistochemicl stining of, n in two HNSCC cses. Cse 1, triple-negtive cse; n EMT (ref. 14). Secon, regultes BMI1 expression n inuces stemness 16. Thir, rogtes p53- n R-epenent pthwys, resulting in n overrie of oncogene-inuce premture senescence 33. In this stuy, the repression of let-7i y engeners iniviul cell movement n stem-like properties. The pleiotropic functions of in cncer metstsis re therefore emonstrte. Recently, the correltion etween EMT n stemness hs een extensively investigte. However, how the cncer cells unergoing EMT cn possess oth migrtory potentil n stem cell properties, which re usully consiere to inclue lck of motility, remins cse 2, cse with co-expression of, n. The re rrow inictes nucler expression of, n the yellow rrow inictes the cytoplsmic n. Scle rs, 2 µm. () Comprison of the overll survivl perios (OS; left) n isese-free survivl (DFS; right) of HNSCC ptients in group B (exmine y immunohistochemistry (IHC); n = 125) with co-expression of (n = 36) versus non-co-expression (n = 89). P vlue ws estimte y log-rnk test n is shown in the left lower qurnt of ech pnel. (e) An illustrtion showing let-7i signlling in cncer cells. Increse expression of upregultes BMI1, n let-7i is co-represse y n BMI1. Downregultion of let-7i e-represses its trget genes (, n H-RAS), leing to n enhnce motility n tumour-inititing cpility. The re cells represent stem-like cncer cells, n the fusiform cells represent cells moving in mesenchyml moe. elusive. In this stuy, we emonstrte tht the suppression of let-7i y not only promotes cellulr motility ut lso contriutes to the tumour-inititing cpility. This fining unveils possile mechnism for the existence of migrtory cncer stem cells. A recent stuy hs shown tht let-7, nother memer of the let-7 fmily of mirnas, represses the expression of n Snil. In this stuy, we emonstrte tht suppresses let-7i; however, let-7i itself oes not influence EMT. The similrities n isprities in the phenotypic impcts of ifferent memers of the let-7 fmily of mirnas on cncer cells, n whether reciprocl regultion etween 8 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 212 Mcmilln Pulishers Limite. All rights reserve.

9 A R T I C L E S let-7 mirnas n exists, s for let-7 n LIN28 (ref. 35), require further investigtion. Different types of iniviul cell movement ply istinct roles in cncer metstsis. Mesenchyml-moe movement epens on the egrtion of the extrcellulr mtrix n is responsile for locl invsion; in contrst, moeoi-moe movement is proteseinepenent n is responsile for istnt metstsis 36. In this stuy, we showe tht the mesenchyml moe, ut not the moeoi moe, is the min route of motility in HNSCC, n the principl role of let-7i is to suppress locl invsion rther thn istl issemintion. Our finings provie resonle explntion for the clinicl oservtion tht HNSCC results in severe locl tissue estruction with reltive low frequency of istl orgn metstsis 37. However, our preliminry t inicte tht the let-7i xis lso exists in other types of cncer (t not shown). Further in-epth investigtion of the role of let-7i in ifferent types of cncer is require to uncouple the mechnisms responsile for locl invsion versus istnt metstsis. Our stuy provies crucil mechnism for -inuce cellulr movement. These results will e vlule for unerstning the nture of EMT-erive migrtory cncer stem cells n provie n iel trget for locking -inuce metstsis through the inhiition of RAC1 ctivity. METHODS Methos n ny ssocite references re ville in the online version of the pper t Note: Supplementry Informtion is ville on the Nture Cell Biology wesite ACKNOWLEDGEMENTS We thnk M. F. Olson (The Beston Institute for Cncer Reserch, UK) for proviing the ROCK ER-WT n ROCK ER-K121G plsmis, n H. Kimur (Ntionl Institute of Neuroscience, Tokyo, Jpn) for the pcl neo HA plsmi. We thnk T. Z. Hung, Y. B. Khotsky, C. W. Lee n S. S. Chng (M. D. Anerson Cncer Center, Texs, USA) for criticl comments on the mnuscript. We thnk C. H. Lin n H. W. Liu (Ntionl Yng-Ming University, Tiwn) for supporting the time-lpse microscopy techniques. This work ws supporte y the Ntionl Helth Reserch Institutes (NHRI-EX1-137BI to M-H.Y.; NHRI-EX1-9931BI to K-J.W.); the Ntionl Science Council (NSC B-1-7-MY3 n B-1-15 to M-H.Y.; NSC B-39-2 to M-C.H.); Tipei Veterns Generl Hospitl (VGH 1-C-88, 11-C-5 to M-H.Y.); Veterns Generl Hospitls University System of Tiwn Joint Reserch Progrm (VGHUST11-G7-4-1 to M-H.Y.); grnt from the Ministry of Euction, Aim for the Top University Pln (to M-H.Y.); grnt from the Deprtment of Helth, Center of Excellence for Cncer Reserch (DOH1-TD-C to M-H.Y; DOH11-TD-C to M-C.H.); n the Sister Institution Fun of Chin Meicl University n Hospitl n MD Anerson Cncer Center. AUTHOR CONTRIBUTIONS M-H.Y. n W-H.Y. conceive n esigne the experiments. W-H.Y. n H-Y.L. crrie out the experiments with the ssistnce of C-H.H for in vivo work. M-H.Y., W-H.Y. n H-Y.L. nlyse the t. M-H.Y. wrote the pper with ssistnce from M-C.H. n K-J.W. The tretment n smple collection of HNSCC ptients were crrie out y S-K.T., C-H.T., S-Y.K. n M-H.Y. COMPETING FINANCIAL INTERESTS The uthors eclre no competing finncil interests. Pulishe online t Reprints n permissions informtion is ville online t 1. Klluri, R. & Weinerg, R. A. The sics of epithelil-mesenchyml trnsition. J. Clin. Invest. 119, (29). 2. Thiery, J. P., Acloque, H., Hung, R. Y. & Nieto, M. A. Epithelil-mesenchyml trnsitions in evelopment n isese. Cell 139, (29). 3. Mni, S. A. et l. The epithelil-mesenchyml trnsition genertes cells with properties of stem cells. Cell 133, (28). 4. Gupt, P. B. et l. Ientifiction of selective inhiitors of cncer stem cells y high-throughput screening. Cell 138, (29). 5. Wolf, K. et l. Multi-step pericellulr proteolysis controls the trnsition from iniviul to collective cncer cell invsion. Nt. Cell Biol. 9, (27). 6. Friel, P. & Wolf, K. Plsticity of cell migrtion: multiscle tuning moe. J. Cell Biol. 188, (21). 7. Snz-Moreno, V. et l. Rc ctivtion n inctivtion control plsticity of tumor cell movement. Cell 135, (28). 8. Snz-Moreno, V. & Mrshll, C. J. The plsticity of cytoskeletl ynmics unerlying neoplstic cell migrtion. Curr. Opin. Cell Biol. 22, (21). 9. Wyckoff, J. B., Pinner, S. E., Gschmeissner, S., Coneelis, J. S. & Shi, E. ROCKn myosin-epenent mtrix eformtion enles protese-inepenent tumor-cell invsion in vivo. Curr. Biol. 16, (26). 1. Wolf, K. et l. Compenstion mechnism in tumor cell migrtion: mesenchymlmoeoi trnsition fter locking of pericellulr proteolysis. J. Cell Biol. 16, (23). 11. Ymzki, D., Kurisu, S. & Tkenw, T. Involvement of Rc n Rho signling in cncer cell motility in 3D sustrtes. Oncogene 28, (29). 12. Belgiovine, C. et l. Reuce expression of the ROCK inhiitor Rn3 is ssocite with increse invsiveness n metsttic potentil in mesenchyml tumor cells. PLoS One 3, e14154 (21). 13. Furlong, E. E., Anersen, E. C., Null, B., White, K. P. & Scott, M. P. Ptterns of gene expression uring Drosophil mesoerm evelopment. Science 293, (21). 14. Yng, J. et l. Twist, mster regultor of morphogenesis, plys n essentil role in tumor metstsis. Cell 1, (24). 15. Yng, M. H. et l. Direct regultion of TWIST y HIF-1α promotes metstsis. Nt. Cell Biol. 1, 2 35 (28). 16. Yng, M. H. et l. Bmi1 is essentil in -inuce epithelil-mesenchyml trnsition. Nt. Cell Biol. 12, (21).. M, L., Teruy-Felstein, J. & Weinerg, R. A. Tumour invsion n metstsis initite y microrna-1 in rest cncer. Nture 449, (27). 18. Gregory, P. A. et l. The mir-2 fmily n mir-25 regulte epithelil to mesenchyml trnsition y trgeting ZEB1 n SIP1. Nt. Cell Biol. 1, (28). 19. Dykxhoorn, D. M. MicroRNAs n metstsis: little RNAs go long wy. Cncer Res. 7, (21). 2. Yu, F. et l. let-7 regultes self renewl n tumorigenicity of rest cncer cells. Cell 131, (27). 21. Ryk, A. et l. A feeck loop comprising lin-28 n let-7 controls pre-let-7 mturtion uring neurl stem-cell commitment. Nt. Cell Biol. 1, (28). 22. Smpson, V. B. et l. MicroRNA let-7 own-regultes MYC n reverts MYC-inuce growth in Burkitt lymphom cells. Cncer Res. 67, (27). 23. Lee, Y. S. & Dutt, A. The tumor suppressor microrna let-7 represses the HMGA2 oncogene. Genes Dev. 21, (27). 24. Prince, M. E. et l. Ientifiction of supopultion of cells with cncer stem cell properties in he n neck squmous cell crcinom. Proc. Ntl Ac. Sci. USA 14, (27). 25. Evn-Rm, S. & Ym, K. M. Cell migrtion in 3D mtrix. Curr. Opin. Cell Biol., (25).. Slorch, E. M., Chou, J. & Wer, Z. Zeppo1 is metstsis promoter tht represses E-cherin expression n regultes p12-ctenin isoform expression n locliztion. Genes Dev. 25, (211). 27. Amno, M. et l. Phosphoryltion n ctivtion of myosin y Rho-ssocite kinse (Rho-kinse). J. Biol. Chem. 271, (1996). 28. Croft, D. R. & Olson, M. F. Conitionl regultion of ROCK-estrogen receptor fusion protein. Methos Enzymol. 46, (26). 29. Brtel, D. P. MicroRNAs: genomics, iogenesis, mechnism, n function. Cell 116, (24). 3. Coi, S., el Pilr Cmcho-Lel, M., Di Stefno, P. & Defilippi, P. Integrin signlling ptors: not only figurnts in the cncer story. Nt. Rev. Cncer 1, (21). 31. Nmekt, K., Enokio, Y., Iwsw, K. & Kimur, H. MOCA inuces memrne spreing y ctivting Rc1. J. Biol. Chem. 279, (24). 32. Greene, F. L. et l. Americn Joint Committee on Cncer (AJCC) Cncer Stging Mnul 6th en 68 (Springer, 22). 33. Ansieu, S. et l. Inuction of EMT y twist proteins s collterl effect of tumorpromoting inctivtion of premture senescence. Cncer Cell 14, (28).. Chng, C. J. et l. Let-7 functions s regultor of epithelil-mesenchyml trnsition n chemoresistnt property in orl cncer. Oncol. Rep., (211). 35. Peter, M. E. Let-7 n mir-2 micrornas: gurins ginst pluripotency n cncer progression. Cell Cycle 8, (29). 36. Pnková, K., Rösel, D., Novotný, M. & Bráek, J. The moleculr mechnisms of trnsition etween mesenchyml n moeoi invsiveness in tumor cells. Cell Mol. Life Sci. 67, (21). 37. Grvello, W., Ciro, A., Sprefico, R. & Gini, R. M. Risk fctors for istnt metstses in he n neck squmous cell crcinom. Arch. Otolryngol. He Neck Surg. 132, (26). NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION Mcmilln Pulishers Limite. All rights reserve.

10 M E T H O D S DOI: 1.138/nc24 METHODS Cell lines n plsmis. Four humn HNSCC cell lines (FDu, SAS, n CAL-27) n humn emryonic kiney cell line (HEK293T) were use in this stuy. The expression vectors pflag, pcdna3 BMI1, pcdna3 Snil, pcdna3 Slug, pcl neo HA (without 3 -UTR), wil-type ROCK ER-WT n kinse-e ROCK ER-K121G were escrie previously 16,28,38 4. The p let7i, pcdh Ze1 n pcdh Ze2 expression vectors were generte y inserting the full-length cdna (let-7i: NR_29661; Ze1: NM_ ; Ze2: NM_147) into p or pcdh CMV MCS EF1 puro. The pcdna3 ws generte y inserting cdna (NM_643; without 3 -UTR) into pcdna3.1. The let-7i sponge n control vectors (p spg-let7i n p spg-ctrl) were constructe ccoring to previous report 41. The insert of p spg-let7i contins 7 repets of the 5 -AACAGCACAAGUCGCUACCUCA-3 sequence, which is complementry to the see region of let-7i with ulge. The insert of p spg-ctrl contins 7 repets of the sequence 5 -AAGTTTTCAGAAAGCTAACA-3, which is not complementry to ny known mirna. The plsmis for short-hirpin RNA (shrna) experiments were generte y inserting n oligonucleotie contining specific trget sequence or scrmle sequence into psuper.puro, n the sequences re shown in Supplementry Tle S7. The promoter region of MIRLET7I, which ws efine ccoring to previous report 42, ws clone into pgl4.2 to generte let7i luca n let7i lucb, n site-irecte mutgenesis ws pplie to generte the E-ox-mutte let7i mlucb. The full-length 3 -UTRs of (NM_643) n (NM_4947) were clone into pmir-reporter to generte pmir--wt n pmir--wt, n site-irecte mutgenesis ws use to generte the let-7i-ining-site-mutte pmir--mut n pmir--mut. Stle trnsfection. We use stle trnsfection in ll in vitro n in vivo experiments in HNSCC cells. The stle cells were selecte ccoring to the selection mrkers of ifferent plsmis. G418 (3 µg ml 1 ) or puromycin (.2 µg ml 1 ) ws use to select FDu cells, n G418 (6 µg ml 1 ) or puromycin (.4 µg ml 1 ) ws pplie in cells. The urtion for stle cell selection ws t lest 4 weeks. All of the cells use in this stuy were stle clones with the exception of using stle popultion pools in the n reconstitution experiments. mirna n cdna microrry nlyses, n ccession numers of microrry t. An Agilent humn mirna Microrry R14 V2 ws use for mirna microrry nlysis, n n Affymetrix HG-U133 plus 2. whole genome rry ws use for cdna microrry nlysis. The microrry t were eposite in the NCBI GEO tse (GSE239 n GSE286). Reverse trnscription for mirna. Reverse trnscription for mirnas ws crrie out with specific stem-loop oligonucleoties. Clculting the reltive expression of the trget trnscripts in ptient smples. We use GAPDH or U6 s the internl control to normlize the expression level of trget molecules in ech smple (2 CT ), n then clculte the tumour/norml rtio to represent the reltive expression of ech trnscript (2 CT ). 5-romo-2 -eoxyuriine incorportion ssy. The 5-romo-2 -eoxyuriine (BrU) incorportion ssy ws crrie out using Cell Prolifertion ELISA (Roche Applie Science) to mesure the rte of DNA synthesis. Culturing cells on top of thin or thick lyer of collgen, thick lyer of Mtrigel or emee in collgen, n collection of cells from collgen mtrices. PureCor ovine collgen solution (Avnce Biomtrix) n Mtrigel were use for these experiments. For preprtion of cells for culturing on top of collgen, Mtrigel or emeing in collgen, we first culture the cells on plstic ishes to 5% confluency. Then we collecte the cells y trypsinizing them with.1% trypsin in EDTA t 37 C for 5 min, suspene the cells to ml 1 n confirme tht the suspene cells were single cells y microscopic exmintion. A 1.7 mg ml 1 collgen solution (3 ml) ws prepre y mixing 1.7 ml of 3 mg ml 1 PureCor ovine collgen solution,.6 ml of 5 meium n 2 µl of 1 M NOH, n ing wter to totl volume of 3 ml. The collgen ws llowe to polymerize in the tissue culture incutor t 37 C for 1 h. For culturing cells on top of thick collgen (2.5D), we prepre the thick collgen-cote ishes first, n then plte the cells on top of the collgen n e n pproprite mount of serum-contining culture meium. For emeing cells in collgen (3D), we replce the superntnts of cell suspensions with 1.7 mg ml 1 chille collgen solution, n mixe the chille collgen solution with the cells thoroughly. Then we confirme the cells suspene in collgen were single cells y microscopic exmintion, n covere the cell collgen mixture with n pproprite mount of serum-contining meium. For culturing cells on top of thick Mtrigel, 5 µl of non-ilute Mtrigel ws plce into ech well of 24-well plte, n cells were seee on top of the Mtrigel. For culturing cells on thin lyer of collgen, we ilute the collgen to 5 µg ml 1 in.1 M HCl n cote the ishes. After incution for 2 h, the remining solution ws spirte. The cells were seee on the cote ishes n llowe to here. For collection of cells from 2.5D or 3D culture for experiments, we use collgense D (1 mg ml 1 ; Roche Applie Science) mixe with cell-contining collgen solutions t 1:1 rtio, n incute t 37 C for 2 3 h, n then collecte the cells y centrifugtion. The experiments inicting cellulr signlling in 2.5D or 3D conitions (Figs 3 n 4 n Supplementry Figs S2 n S4 S7) were crrie out using cells collecte from 2.5D or 3D cultures. Immunofluorescence microscopy n reconstruction of imges from confocl microscopy. For immunofluorescent stining of F-ctin in 2.5D culture, we cote ech well of L-Tek II chmer slie (no ) with.5 ml of the 1.7 mg ml 1 collgen solution, then ispense cells on the collgen, covere with.5 ml of meium, n llowe to here for 16 h. After incution, the cells were fixe, permeilize n stine with Alex-488 couple to phlloiin (Invitrogen). Imges were cpture y lser confocl microscopy (Olympus FV1). For imging of cells in 3D culture, we mixe cells with.4 ml of the 1.7 mg ml 1 collgen solution, seee them onto L-Tek chmere coverglss system (no. 1383), n then covere them with.4 ml meium. Sequentil Z sections were otine y confocl microscopy, n imges were reconstructe y Olympus FV1-ASW 1.7 softwre. DAPI ws use for nucler stining. Anlysis of cell morphology. We nlyse the cell morphology ccoring to previous report 44. The re n the perimeter of the cells were efine y rwing roun the cell shpe in phse-contrst imges n etermine y MetMorph softwre (Moleculr Devices). The morphology inex ws clculte s the perimeter 2 /4π re. For roun cell, the rtio is 1, n n elongte cell hs n elevte inex. For ech clone, the men vlue of the inex ws etermine from 2 cells. Time-lpse microscopy n quntifiction of the spee of motile cells. Time-lpse microscopic oservtions of cell motility were crrie out s escrie previously 7,45. For 2.5D culture, we covere 3.5-cm-imeter ish with 1 ml of the 1.7 mg ml 1 collgen solution, seee cells on top of the collgen n then covere them with 1 ml of meium. Cells were llowe to here for 16 h, n were then oserve in humiifie, CO 2 -equilirte chmer with motorize-stge-equippe Leic DM IRBE microscope (Leic Microsystems) for 24 h. For 3D culture, we mixe cells with.4 ml of the 1.7 mg ml 1 collgen solution, seee them onto L-Tek chmere coverglss system (no. 1383) n then covere them with.4 ml meium. Cells were llowe to here for 16 h, n were then oserve in humiifie, CO 2 -equilirte chmer with Zeiss LSM7 confocl microscope (Crl Zeiss) for 8 h. The imges were processe n reconstructe with ZEN 29 Light Eition softwre. To quntify the spee of cells in the 2.5D system, we trcke the movements of iniviul cells y MetMorph softwre (Moleculr Devices) in rnomly selecte high-power fiel. The cell motility spee ws clculte n is presente s micrometres per minute. Only iniviul cells were trcke for quntifiction of spee in 2.5D. All of the frctions of iniviul cells for spee quntifiction were more thn 8%. The frctions of iniviul cells for ech clone in spee quntifiction experiments re provie in the legens of figures. 3D invsion ssy. The 3D invsion ssy ws crrie out s escrie previously 7, with minor moifictions. Cells were plte for ttchment, the superntnt ws remove, n the cells were covere with the 1.7 mg ml 1 collgen solution. Meium contining 15% FBS ws e s chemottrctnt, n the pltes were incute t 37 C in 1% CO 2 for 24 h. After incution, the cells were fixe n stine with Alex-488 couple to phlloiin. Confocl Z slices were collecte from ech well t 5 µm n from the ottom of the well with lser confocl microscope, n imges of sequentil Z sections were otine n reconstructe y Olympus FV1-ASW 1.7 softwre. The invsion inex ws clculte s the numer of cells t 3 µm ivie y the totl numer of cells n normlize y the prolifertion inex of ech clone. The t re presente s the percentge of the invsion inex of the control clone inicte in ech pnel. Smll-GTPse-pullown ssys. Active RAC1 n Rho pullown kits (Pierce Biotechnology) were use to ssy the ctive forms of RAC1 n Rho. The pulle 212 Mcmilln Pulishers Limite. All rights reserve. NATURE CELL BIOLOGY

11 DOI: 1.138/nc24 M E T H O D S own ctive RAC1 n Rho were elute n sujecte to western lot nlysis, n the expression levels of totl RAC1 n Rho were use s the controls. Smple collection from HNSCC cses. This stuy ws pprove y the Institutionl Review Bor of Tipei Veterns Generl Hospitl. We use two inepenent sets of smples for the experiments. Smple group A contine pire microissecte tumour n contrlterl norml orl epithelium from 58 HNSCC ptients who receive tretments t the Deprtment of Otolryngology, Tipei Veterns Generl Hospitl etween Jnury 25 n Decemer 28. Totl RNA ws extrcte for RT PCR nlysis. Smple group B comprise 125 prffin-emee tumour tissues from HNSCC ptients receiving tretments t the Deprtment of Stomtology, Tipei Veterns Generl Hospitl etween Jnury 26 n Decemer 28. These smples were constructe into tissue microrry for immunohistochemicl nlysis. In ition, tumour smples from 2 HNSCC ptients were collecte for primry cultivtion to crry out the ectopic expression n shrna experiments. Immunohistochemistry scoring. The slies were inepenently score y two iniviuls ccoring to the Immunorective Score 46 (IRS) metho. For, only nucler expression ws consiere positive, wheres oth nucler n cytoplsmic expression were consiere positive for n. A moerte to strong level (IRS > 4) ws consiere to e positive, n wek or sent expression (IRS 4) ws consiere to e negtive. Sttisticl nlysis. The two-tile inepenent Stuent t-test ws use to compre the continuous vriles etween two groups. The chi-squre test ws pplie to compre the ichotomous vriles. The Kpln Meier estimtion n the log-rnk test ws use to compre the survivl ifference etween ptient groups. All t were erive from inepenent experiments, n the cells were not poole cross experiments. The level of sttisticl significnce ws set t.5 for ll tests. qpcr, western lot, luciferse reporter ssy, ChIP, flow cytometry, spheroi formtion ssy, soft-gr colony formtion ssy, Boyen chmer migrtion ssy, immunohistochemistry, xenotrnsplnttion n til vein injection ssys. These experiments were crrie out s previously escrie 15,16. The primers n ntioies use in the experiments re liste in Supplementry Tles S6 n S7, respectively. 38. Hsu, D. S. et l. Regultion of excision repir cross-complementtion group 1 (ERCC1) y Snil contriutes to cispltin resistnce in he n neck cncer. Clin. Cncer Res. 16, (21). 39. Hung, C. C. et l. Regultion of memrne-type 4 mtrix metlloproteinse (MT4-MMP) y SLUG contriutes to hypoxi-meite metstsis. Neoplsi 11, (29). 4. N Nmekt, K., Enokio, Y., Iwsk, K. & Kimur, H. MOCA inuces memrne spreing y ctivting Rc1. J. Biol. Chem. 279, (24). 41. E Eert, M. S., Neilson, J. R. & Shrp, P. A. MicroRNA sponges: competitive inhiitors of smll RNAs in mmmlin cells. Nt. Methos 4, 1 7 (27). 42. O Hr, S. P. et l. NFkppB p5-ccaat/enhncer-ining protein et (C/EBPβ)- meite trnscriptionl repression of microrna let-7i following microil infection. J. Biol. Chem. 285, (21).. Chen, C. et l. Rel-time quntifiction of micrornas y stem loop RT PCR. Nucleic Acis Res. 33, e9 (25). 44. Pinner, S. & Shi, E. PDK1 regultes cncer cell motility y ntgonising inhiition of ROCK1 y RhoE. Nt. Cell Biol. 1, (28). 45. Wilkinson, S., Pterson, H. F. & Mrshll, C. J. Cc42-MRCK n Rho-ROCK signlling cooperte in myosin phosphoryltion n cell invsion. Nt. Cell Biol. 7, 2 1 (25). 46. Remmele, W. & Schicketnz, K. H. Immunohistochemicl etermintion of estrogen n progesterone receptor content in humn rest cncer. Computer-ssiste imge nlysis (QIC score) vs. sujective gring (IRS). Pthol. Res. Prct. 189, (1993). NATURE CELL BIOLOGY 212 Mcmilln Pulishers Limite. All rights reserve.

12 SUPPLEMENTARY INFORMATION DOI: 1.138/nc24 Reltive let-7i expression Reltive let-7i expression FDu CAL-27 SAS sh- sh-bmi1 Reltive let-7i expression pflag-cmv pflag- Cse 1 Cse 2 BMI1 E-cherin vimentin Reltive migrtory ility e CMV FDu SAS p p-let7i p-spg-ctrl p-spg-let7i let-7i reporter Fol chnge of Luc/β-gl FDu CAL CAL-27 SAS microrna ining sites poly(a) UGAU 3 -UUGUCGUGUU GAUGGAGU-5 let-7i Sponge 5 -AACAGCACAA CUACCUCA-3 GUCG c Reltive expression Reltive expression f mir Reltive let-7i expression Fol chnge of Luc/β-gl CMV mir-1915 CMV FDuspg-ctrl mir-762 sh- sh- BMI1 let7i mir-1915 sh- spg-let7i sh- BMI1 Figure S1 Suppression of let-7i y -BMI1 in HNSCC cells, n genertion of the let-7i-mniputte stle clones in HNSCC cell lines. () Upper: western lot of, BMI1, E-cherin n vimentin in FDu, CAL-27, SAS n. -ctin ws use s loing control. Lower: reltive expression level of let-7i in the four cell lines (compre with FDu; n=3 for ech cell line). P <.1 y Stuent s t-test. () Upper: phse contrst imges of HNSCC cell lines in 2D culture. Scle r=5µm. Lower: Boyen chmer migrtion ssy (compre with FDu; n=3 for ech cell line). P <.1 y Stuent s t-test. (c) Quntittive RT-PCR for mir-762 n mir-1915 in FDu cells trnsfecte with -expressing vector () or control vector (CMV), or in cells trnsfecte with shrna ginst (sh-), BMI1 (sh-bmi1) or scrmle sequence (). n=3 for ech group. P <.1 y Stuent s t-test. () Quntittive RT-PCR for let-7i expression in two primry HNSCC cultures. Upper: primry HNSCC culture trnsfecte with shrna ginst (sh-), BMI1 (sh-bmi1), or scrmle control (); lower: primry HNSCC culture trnsfecte with or control vector. n=3 for ech group. P <.1 y Stuent s t-test. (e) Upper: schemtic representtion of the let-7i sponge vector; lower: vlition of the let-7i sponge vector y reporter ssy in HEK-293T cells. Luciferse ctivity/glctosise of cells trnsfecte with p n let-7i reporter ws pplie s the seline control for the experiments. n=3 for ech group. P <.1 y Stuent s t-test. (f) Upper: quntittive RT-PCR nlysis of let-7i expression in let-7i-overexpresse clones (let7i)(n=3). Trnsfection of n empty vector ws use to generte the control clone (). Lower: vlition of the neutrlizing effect of let-7i in FDu cells stly expressing the let-7i sponge vector (spg-let7i) y reporter ssy (n=3). Trnsfection of control vector (spg-ctrl) ws use s control. P <.1 y Stuent s t-test. The r chrts in pnel ()-(f) represent men ± S.E.M Mcmilln Pulishers Limite. All rights reserve.

13 SUPPLEMENTARY INFORMATION let7i c e No. of colony let7i spg-let7i spg-ctrl No. of colony let7i Spg-ctrl 3.E63 2.5E6 2.5 Cell numer(x1 6 ) 2.E62 1.5E6 1.5 H-RAS E61 LIN28 5.E C-MYC.E HMGA2 spg-ctrl Fol chnge of BrDu positive cells spg-let7i Om1 let7i-1 OL5 let7i-2 OL12 4.E54 Cell numer(x1 5 ) 3.E53 2.E52 E51 CS3 spg-ctrl 7iS7 spg-let7i-1 7is22 spg-let7i-2.e Dy Dy2 Dy3 Dy4 Dy5 Dy Dy2 Dy3 Dy4 Dy5 SSC let7i-1 spg-let7i-1 f let7i IgG 5% 2% CD44 nti-cd44 56% spg-ctrl % Fol chnge of BrDu positive cells FDuspg-ctrl SSC FDuspg-let7i-1 FDuspg-ctrl IgG 3% 3% spg-let7i CD44 nti-cd44 16% 45% clone Dose spgctrl spglet7i-1 No. of spherois/ 1 cells g let7i 13 No. of spherois/ 1 cells FDuspg-ctrl spg-let7i 13 spg-let7i-1 1, /6 2/6 1, /6 2/6 1, 2/6 4/6 let7i let7i E-cherin h Non-cote ish culture (2D) On top of thick collgen culture (2.5D) Emee in collgen culture (3D) E-cherin i 13 let7i E-cherin 13 vimentin spg-let7i spg-let7i spg-let7i spg-ctrl spg-ctrl spg-ctrl E-cherin 13 vimentin E-cherin 13 vimentin E-cherin vimentin vimentin vimentin Figure S2 Suppression of let-7i promotes tumor-inititing cpility without ffecting the expression of EMT mrkers. () Western lot nlysis of H-RAS, LIN28, c-myc n HMGA2 in OECM1 cells stly trnsfecte with let-7i expression vector (let7i) versus control vector (), or in FDu cells trnsfecte with let-7i sponge vector (spg-let7i) or control vector (spg-ctrl). -ctin ws use s loing control. () Growth curve nlysis (upper pnels; n=3) n 5-romo-2 -eoxyuriine incorportion ssy (lower pnels; n=3) in n FDu stle cell lines. (c) Soft gr colony formtion ssy (n=3). P <.1 y Stuent s t-test. () Flow cytometry nlysis of CD44. The percentges of CD44 cells were shown in the right upper qurnt of ech pnel. Isotype IgG ws use s control. (e) Spheroi formtion ssy. Upper: representtive pictures of the spheroi in ifferent cells. Scle r = 1 µm. Lower: quntifiction of spherois (n=3). P <.1 y Stuent s t-test. (f) Nue mice xenotrnsplnttion ssy. Left: representtive picture of nue mice receiving sucutneous injections. Blck rrow inictes the formtion of xenotrnsplnte tumor. Cell ose: 1 x 1 4 / mouse. Right: tle showing the result of xenotrnsplnttion stuy. (g-i) Western lot nlysis of EMT mrkers (E-cherin n vimentin) in let7i vs. n spg-let7i vs. spg-ctrl cultivte on non-cote ishes (2D culture)(g), on top of thick collgen (2.5D culture) (h), n emee in collgen (3D culture)(i). -ctin ws use s loing control. The r chrts in pnel (), (c), (e) represent men ± S.E.M Mcmilln Pulishers Limite. All rights reserve.

14 SUPPLEMENTARY INFORMATION Non-cote ish culture (2D) On top of thin collgen culture c FDu CAL-27 FDu CAL-27 On top of thick collgen culture (2.5D) FDu CAL-27 SAS SAS SAS FDu On top of thick Mtrigel culture CAL-27 e Emee in collgen culture (3D) FDu CAL-27 SAS SAS f Non-cote ish culture (2D) g On top of thin collgen culture h spg-ctrl spg-ctrl On top of thick collgen culture (2.5D) spg-ctrl let7i-1 spg-let7i-1 let7i-1 spg-let7i-1 let7i-1 spg-let7i-1 Figure S3 Phse contrst imges of HNSCC cells culture in ifferent conitions. (-e) Imges of four HNSCC lines (FDu, CAL-27, SAS, n ) culture in non-cote ishes (2D) (), thin collgen-cote ishes (), on top of thick collgen (2.5D)(c), on top of thick Mtrigel (), or emee in collgen (3D)(e). (f-h) Imges of let7i vs. n spg-let7i vs. spg-ctrl culture in non-cote ishes (2D) (f), thin collgen-cote ishes (g), n on top of thick collgen (2.5D) (h). Scle r=5µm Mcmilln Pulishers Limite. All rights reserve.

15 SUPPLEMENTARY INFORMATION let7i-1 let7i- ROCK-ER-KD let7i- ROCK-ER-WT 4-OHT p-mlc2 let7i- ROCK-ER-KD phse F-ctin/DAPI F-ctin/DAPI F-ctin/DAPI (single section) (3D reconstruction) MLC2 let7i- ROCK-ER-WT c Rtio of perimeter 2 / 4π to re let7i- ROCK-ER-KD let7i- ROCK-ER-WT µm/min let7i- ROCK-ER-KD let7i- ROCK-ER-WT Figure S4 let-7i reuces motility of elongte cells without enhncing roune cell movement, n ROCK ctivtion chnges neither cellulr shpe nor motility of let-7i-expressing cells. () Western lot nlysis of phosphorylte MLC2 (p-mlc2) n MLC2 in let7i-1 cells, let7i-1 cells trnsfecte with the 4-hyroxytmoxifen (4-OHT)-inucile vector which expresses kinse-e ROCK-estrogen receptor fusion protein (ROCK-ER-KD) or wil-type ROCK-estrogen receptor fusion protein (ROCK- ER-WT), with or without 4-OHT inuction. -ctin ws use s loing control. The cells were hrvest from on-top thick collgen culture (2.5D) for experiments. () Imges of cells in 2.5D culture. Representtive section of phse contrst microscopy (scle r=25µm), confocl microscopy with low power fiel (scle r=25µm), high power fiel (scle r=1µm), n the reconstructive imges (scle r=1µm) in let7i trnsfecte with the vector expressing kinse e (upper pnels) or wil-type ROCK-ER fusion protein (lower pnels) fter 4-OHT inuction for 24 hr. The green color inictes the F-ctins stine y n nti-phlloiin ntioy, n lue color inictes the nuclei stine y DAPI. (c) Quntifiction of cellulr morphology in let7i expressing ROCK-ER-KD or ROCK- ER-WT fter 4-OHT inuction. The morphologic inex ws clculte s perimeter 2 /4πre (n=2 for ech group). () Representtive trjectories n quntifiction of motility spee in let7i expressing ROCK- ER-KD or ROCK-ER-WT fter 4-OHT inuction in 2.5D culture (n=1 for ech group). The spee ws clculte s µm/min. The frction of iniviul cells of let-7i cells trnsfecte with ROCK-ER-KD n ROCK-ER-WT is 92.8% n 91.6%, respectively. The ox plots in pnel (c), () represent smple mximum (upper en of whisker), upper qurtile (top of ox), mein (n in the ox), lower qurtile (ottom of ox), n smple minimum (lower en of whisker) Mcmilln Pulishers Limite. All rights reserve.

16 SUPPLEMENTARY INFORMATION Fol chnge of mrna expression let7i-1 let7i-2 FARP1 ARH GEF15 VAV3 Fol chnge of mrna expression spg-ctrl spg-let7i-1 spg-let7i-2 FARP1 ARH GEF15 VAV3 c Collgen emee 3D culture Collgen emee 3D culture let7i spg-let7i spg-ctrl 2 GTP-RAC1 Totl RAC1 GTP-RAC1 Totl RAC1 Figure S5 let-7i ownregultes the expression level of mrna n protein. (, ) Quntittive RT-PCR nlysis of FARP1, ARHGEF, VAV3, n expression in cells trnsfecte with the let-7i expression vector (let7i) or control vector () (), FDu trnsfecte with let-7i sponge vector (spg-let7i) or control sponge vector (spg-ctrl)(). n=3 for ech group. P <.1 y Stuent s t-test. (c, ) Expression of GTP-oun RAC1, totl RAC1, n in let7i vs. (c) n spg-let7i vs. spg-ctrl (). The cells were hrveste from collgen-emee culture (3D culture). Totl RAC1 ws pplie s control of ctivte RAC1, n -ctin ws use s loing control. The r chrts in pnel (), () represent men ± S.E.M Mcmilln Pulishers Limite. All rights reserve.

17 SUPPLEMENTARY INFORMATION 2 Collgen emee 3D culture FC FT FTM FTL Non-cote ish culture (2D) FC FT FTM FTL GTP-RAC1 Totl RAC1 13 FC FT FTM FTL E-cherin vimentin c FC FT FTM FTL On top of thick collgen culture (2.5D) FC FT FTM FTL 2.5D 13 E-cherin vimentin F-ctin /DAPI F-ctin /DAPI e FC FT FTM FTL H-RAS 3D F-ctin /DAPI F-ctin /DAPI f FC IgG 1% nti-cd44 14% FTM IgG 2% nti-cd44 31% g clone ose FC FT FTM FTL SSC FT 3% 33% SSC FTL 1% 18% 1, 5/6 6/6 6/6 4/6 1, 3/6 5/6 4/6 3/6 CD44 CD44 1, /6 3/6 3/6 /6 Figure S6 Phenotypic impct of -let-7i-, n H-RAS xis on HNSCC cells uner ifferent environments. () Expression of GTP-oun RAC1, totl RAC1,, n in FDu cells trnsfecte with pflag-cmv (FC), pflag- (FT), pflag- n (FTM), n pflag- n p-let7i (FTL). The cells were hrveste from collgen-emee culture (3D culture). Totl RAC1 ws pplie s control of ctivte RAC1, n -ctin ws use s loing control. () Upper: phse contrst imges of FC, FT, FTM n FTL cells in 2D culture. Scle r=5µm. Lower: western lot of E-cherin n vimentin in FC, FT, FTM n FTL cells hrveste from 2D culture. -ctin ws use s loing control. (c) Western lot of E-cherin n vimentin in FC, FT, FTM n FTL cells hrveste from on-top thick collgen culture (2.5D). -ctin ws use s loing control. () Immunofluorescence to show the cellulr morphology n ctin orgniztion of FC, FT, FTM, n FTL cells in 2.5D or 3D culture. The green color inictes the F-ctin stine y n nti-phlloiin ntioy, n lue color inictes the nuclei stine y DAPI. Scle r = 25µm (first n thir row), 1µm (secon n fourth row). (e) Western lot nlysis of H-RAS in FC, FT, FTM, n FTL cells. -ctin ws use s loing control. (f) Flow cytometry nlysis of CD44. The percentges of CD44 cells were shown in the right upper qurnt of ech pnel. Isotype IgG ws use s control. (g) A tle showing the results of nue mice xenotrnsplnttion ssy Mcmilln Pulishers Limite. All rights reserve.

18 SUPPLEMENTARY INFORMATION FDu FDu FDu FDu EV Snil Snil EV Slug Slug 13 EV Ze1 Ze1 13 EV Ze2 Ze2 Reltive expression of let-7i sh-snil Snil Reltive expression of let-7i sh-slug Slug Reltive expression of let-7i sh-ze1 Ze1 Reltive expression of let-7i sh-ze2 Ze Reltive expression of let-7i Reltive expression of let-7i Reltive expression of let-7i Reltive expression of let-7i Figure S7 Expression of EMT regultors Snil, Slug, Ze1 or Ze2 oes not significntly ffect the expression of let-7i, n in HNSCC cells. () Upper: western lot nlysis of the expression of the EMT regultor (Snil, Slug, Ze1 or Ze2), n in FDu cells trnsfecte with the EMT regultor expression vector or n empty vector (EV). The cells were hrveste from on-top thick collgen culture (2.5D culture); lower: quntittive RT-PCR nlysis of let-7i expression in FDu cells trnsfecte with the EMT regultor expression vector or n empty vector. n=3 for ech group. P <.1 y Stuent s t-test. () Upper: western lot nlysis of the expression of the EMT regultor (Snil, Slug, Ze1 or Ze2), n in cells receiving shrna ginst ifferent EMT regultors or scrmle sequence. The cells were hrveste from on-top thick collgen culture (2.5D culture); lower: quntittive RT-PCR nlysis of let-7i expression in cells receiving shrna ginst ifferent EMT regultors or scrmle sequence. n=3 for ech group. P <.1 y Stuent s t-test. The r chrts in pnel (), () represent men ± S.E.M Mcmilln Pulishers Limite. All rights reserve.

19 SUPPLEMENTARY INFORMATION Proportion of overll survivl P= OS fol chnge < 2x (n=3) fol chnge 2x (n=28) Proportion of isese-free DFS fol chnge < 2x (n=3) fol chnge 2x (n=28).2 P= Proportion of overll survivl P= OS fol chnge < 2x (n=24) fol chnge 2x (n=) Proportion of isese-free DFS fol chnge < 2x (n=24) fol chnge 2x (n=).2 P= c Proportion of overll survivl P= OS let-7i fol chnge >.5x (n=32) let-7i fol chnge.5x (n=) Proportion of isese-free DFS let-7i fol chnge >.5x (n=32) let-7i fol chnge.5x (n=).2 P= Proportion of overll survivl P= OS -negtive (n=64) -positive (n=61) Proportion of isese-free P=.6 1 DFS -negtive (n=64) -positive (n=61) e Proportion of overll survivl P=.8 OS -negtive (n=7) -positive (n=) Proportion of isese-free P= DFS -negtive (n=7) -positive (n=) f Proportion of overll survivl P=.7 OS -negtive (n=45) -positive (n=8) Proportion of isese-free P= DFS -negtive (n=45) -positive (n=8) Figure S8 Survivl nlysis of two inepenent sets of HNSCC ptients. (-c) Anlysis of HNSCC ptients group A exmine y quntittive RT-PCR (n=58). () Comprison of the overll survivl (OS; left) n isese-free survivl (DFS; right) of ptients with or without n increse mrna expression (tumor/norml 2 fols). () Comprison of the OS (left) n DFS (right) of ptients with or without n increse mrna expression (tumor/norml 2 fols). (c) Comprison of the OS (left) n DFS (right) of ptients with or without ecrese let-7i expression (tumor/norml.5 fol). (-f) Anlysis of HNSCC ptients group B exmine y immunohistochemistry (n=125). () Comprison of the overll survivl (OS; left) n isese-free survivl (DFS; right) of ptients with or without positive expression. (e) Comprison of the OS (left) n DFS (right) of ptients with or without positive expression. (f) Comprison of the OS (left) n DFS (right) of ptients with or without positive expression. The ptients numer (n) of ech group is shown in ech pnel. P vlue ws estimte y log-rnk test n is shown in the left lower qurnt of ech pnel Mcmilln Pulishers Limite. All rights reserve.

20 SUPPLEMENTARY INFORMATION sh- IB: Figure 1 sh-bmi1 IB: BMI1 CMV Figure 1c - IB: IB: BMI1 - sh-bmi1 IB: IB: IB: Figure 3 let7i let7i Spg-ctrl spg-let7i Spg-ctrl spg-let7i IB: GTP-RAC1 IB: p-mlc2 IB: GTP-RAC1 IB: p-mlc2 IB: totl RAC1 IB: GTP-Rho IB: MLC2 IB: IB: totl RAC1 IB: GTP-Rho IB: MLC2 IB: IB: totl Rho IB: totl Rho let7i IB: IB: IB: VAV3 IB: ARHGEF15 let7i 13 IB: FARP1 IB: Figure 3c Spg-ctrl spg-let7i IB: IB: IB: VAV3 IB: ARHGEF15 spg-let7i Spg-ctrl 13 IB: FARP1 IB: Figure S9 Uncroppe films showing the key experiments isplye in the min figures Mcmilln Pulishers Limite. All rights reserve.

21 SUPPLEMENTARY INFORMATION Figure 3f Figure 4 13 let7i-1 pcdna3- EV IB: IB: 2 13 let7i-1 HAmock IB: HA IB: 1 1 FC FT FTM FTL IB: GTP RAC1 IB: Totl RAC FC FT FTM FTL IB: IB: 1 IB: GTP-RAC1 1 IB: GTP-RAC1 IB: IB: 1 IB: Totl RAC1 IB: Totl RAC1 Figure 4c sh- Figure 4 CMV - - sh-bmi1 IB: IB: IB: 2 13 IB: 2 IB: IB: IB: Figure S9 continue Mcmilln Pulishers Limite. All rights reserve.