Targeting mir-21 for the Therapy of Pancreatic Cancer

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1 originl rticle The Americn Society of Gene & Cell Therpy Trgeting mir-21 for the Therpy of Pncretic Cncer Flvie Sicrd 1,2, Mrion Gyrl 1,2, Huert Lulk 1,2, Louis Buscil 1,2 nd Pierre Cordelier 1,2 1 INSERM U137, Cncer Reserch Center of Toulouse, Toulouse, Frnce; 2 Université Pul Stier Toulouse III, Toulouse, Frnce Despite tremendous efforts worldwide from clinicins nd cncer scientists, pncretic ductl denocrcinom (PDA) remins dedly disese for which no cure is ville. Recently, micrornas (mirnas) hve emerged s key ctors in crcinogenesis nd we demonstrted tht microrna-21 (mir-21), oncomir is expressed erly during PDA. In the present study, we sked whether trgeting mir-21 in humn PDA-derived cell lines using lentivirl vectors (LVs) my impede tumor growth. We demonstrted tht LVs-trnsduced humn PDA efficiently downregulted mir-21 expression, oth in vitro nd in vivo. Consequently, cell prolifertion ws strongly inhiited nd PDA-derived cell lines died y poptosis through the mitochondril pthwy. In vivo, mir-21 depletion stopped the progression of very ggressive model of PDA, to induce cell deth y poptosis; furthermore, comining mir-21 trgeting nd chemotherpeutic tretment provoked tumor regression. We demonstrte herein for the first time tht trgeting oncogenic mirna strongly inhiit pncretic cncer tumor growth oth in vitro nd in vivo. Becuse mir- 21 is overexpressed in most humn tumors; therpeutic delivery of mir-21 ntgonists my still e eneficil for lrge numer of cncers for which no cure is ville. Received 23 August 212; ccepted 3 Jnury 213; dvnce online puliction 12 Mrch 213. doi:1.138/mt Introduction The vst mjority of ptients with pncretic ductl denocrcinom (PDA) disply n dvnced disese tht results in low resection rte leding to disml overll medin survivl of 4 6 months. 1 The estimted 5-yer survivl rte is <5%. Although PDA is not mong the most common tumors, it is one of the most frequent cuses of cncer-relted deth with ~28, deths/yer in the United Sttes nd 4,/yer in Europe. 1 The therpeutic rmmentrium ginst PDA consists of conventionl chemotherpeutic gents such s gemcitine nd more recently FOLFIRINOX tht offer mrginl survivl enefit for PDA ptients. 2,3 Moreover, unlike other digestive cncer entities such s colon cncer or gstrointestinl stroml tumors, moleculr-trgeted therpies hve so fr lrgely filed to positively impct ptient survivl in PDA. 2 Consequently, developing new tretments tht my profoundly chnge the therpeutic lndscpe of PDA is urgently needed. For the vst mjority, PDA occurs spordiclly. Milestone genetic studies identified K-rs gene ctivtion in >85% of PDAs, wheres p16 nd TP53 re inctivted in out 95% of cses. 2 Lst, SMAD4 is lost in 55% of PDAs. By contrst, little is known out the susequent moleculr chnges contriuting to this very ggressive cncer. Mny growth signling pthwys re over-ctivted in PDA while ltertion of tumor suppressor gene expression is frequently detected in PDA. Consequently, evsion of poptosis, cell survivl, errnt ngiogenesis, invsion, nd metsttic spred re lndmrks of PDA. In ddition, mny other processes re involved in PDA nd cn contriute to its development. Much progress hs een mde linking the expression of micrornas (mirna) with PDA. 4 Despite their smll size t out 22 nucleotides, these endogenous noncoding RNAs hve n stonishing effect on protein-coding gene expression, nd regulte vrious cellulr events including prolifertion, poptosis, nd differentition. 5 We hypothesized tht errnt mirna expression could impct these processes in pncretic cells, ultimtely contriuting to tumor initition, promotion, nd/or progression. In preliminry work, we identified microrna-21 (mir-21) s overexpressed in erly pncretic cncer lesions, pncretic tumors, nd pncretic cncer-derived cell lines. 6 mir-21 is one of the most cited mirna, nd hs emerged s the mirna most frequently ssocited with poor outcome in cncer, including PDA. 7 In ddition, mir-21 stnds downstrem in mny oncogenic pthwys 8 including ut not restricted to ctivted KRAS 6 nd EGF receptor 6 to trget multiple tumor suppressors. 8 In theory, invlidtion of mir-21 should (i) lunt mny oncogenic pthwys driving tumorogenesis, nd (ii) relieve multiple ntitumorl signls tht could hinder tumor progression. As such, mir-21 is considered s very promising therpeutic trget for cncer, including PDA. However, most studies hve een performed in vitro, nd in vivo studies sed on systemic or loclized trget delivery of nti-mir-21 re still lcking. 8 In the present work, we sked whether depleting mir-21 my impede tumor prolifertion in vitro nd in vivo in very ggressive preclinicl model of PDA. Since vriety of humn cncer cells including PDA hve een shown to overexpress mir-21, development of trgeted therpeutic tht would suppress oncogenic mir-21 would e promising ntitumor therpy ginst cncer. Correspondence: Pierre Cordelier, INSERM U137, Cncer Reserch Center of Toulouse nd Université Pul Stier Toulouse III, Toulouse 31452, Frnce. E-mil: pierre.cordelier@inserm.fr vol. 21 no. 5, my 213

2 The Americn Society of Gene & Cell Therpy Trgeting mir-21 for Pncretic Cncer Therpy mir-21 expression (2 Ct ) Opticl density t 595 mm c Cell numer (% of control) Control MOI = 1 MOI = 5 MOI = 1 MOI = 5 MOI =.5 MOI = 1 MOI = 5 Tretment d Tretment Tretment Spheroid surfce (mm 2 ) Tretment Figure 1 In vitro trgeting of mir-21 using lentivirl vectors decreses mir-21 cellulr content nd strongly inhiits PDA cell prolifertion nd viility. Mi PC-2 cells were trnsduced y encoding for mir-21 ntisenses t the indicted MOI. Control cells were trnsduced with GFP-encoding lentivirl vectors, or left untrnsduced. () mir-21 expression ws determined y qrt-pcr. Results, expressed s 2 ΔCt, re men ± SEM of five independent experiments performed in duplicte. () Cell viility nd (c) cell prolifertion were determined 72 hours following trnsduction. Results re men ± SEM of five independent experiments performed in triplicte. (d) Cpn-2 cells were stly trnsduced with. Control cells were stly trnsduced with. After 2 weeks in culture, spheroids surfce ws mesured using ImgeJ softwre. Results re men ± SEM of three independent experiments of eight replictes. P <.5; P <.1. GFP, green fluorescent protein; LV, lentivirl vector; MOI, multiplicity of infection; PDA, pncretic ductl denocrcinom; qrt-pcr, quntittive reverse trnscriptse-pcr. Results Trgeting mir-21 inhiits PDA-derived cells prolifertion We previously demonstrted tht the mirna, mir-21 is overexpressed in erly pncretic cncer lesions, pncretic tumors, nd pncretic cncer-derived cell lines. 6 To further study its function in PDA, we generted lentivirl vectors (LV) for the stle nd permnent expression of RNA interference hirpins ntisense to mir-21 (). These vectors trnsduced exponentilly growing Mi PC-2 cells with high efficcy without selection (Supplementry Figure S1), to knockdown mir-21 expression ( 95 ± 4%, P <.1, Figure 1) s compred with controltrnsduced cells. Noteworthy, did not significntly impct the cellulr levels of other mirnas (Supplementry Figure S2). Consequently, PDA cell viility nd cell prolifertion were strongly ntgonized, in dose-dependent mnner in response to tretment (Figure 1,c, respectively). Inhiition of cell prolifertion ws mximl t 72 hours following cell trnsduction (Supplementry Figure S3). In preclinicl investigtions, the multicellulr tumor spheroid system hs provided n pproprite in vitro system to evlute nd predict pncretic tumor cells response to therpeutics. 9 Consequently, we generted spheroids of Cpn-2 cells stly expressing mir-21 ntisense. We found tht trgeting mir-21 significntly inhiited PDA tumor spheroids growth s compred with control-trnsduced cells (Figure 1d). Tken together, we demonstrte tht mir-21 is essentil for the prolifertion of PDA-derived tumor cells oth in monolyer cultures nd in three-dimensionl models. mir-21 protects PDA-derived cells from poptosis We next investigted the moleculr mechnisms involved in the ntiprolifertive effect consecutive to mir-21 depletion in PDA-derived cell lines. Mi PC-2 cells were trnsduced with different doses of nd smpled for cell cycle nlysis. Flow cytometric nlysis of propidium iodide-stined cells showed tht the pre-g/g1 cell popultion significntly increses Moleculr Therpy vol. 21 no. 5 my

3 Trgeting mir-21 for Pncretic Cncer Therpy The Americn Society of Gene & Cell Therpy , MOI =.5 8 1, 334 µm 334 µm , , Insert Cell numer (% of totl) MOI = MOI = 5 34 µm Insert mir-21 expression (.u.) µm Blot for: PARP Cspse-3 Actin + Pre-G/G1 + G/G1 fter exposure to, s compred with control-trnsduced cells (Figure 2). This effect ws dose-dependent nd mximl t multiplicity of infection = 5 (11.3 ± 8.6 fold increse, P <.5; Figure 2, insert). We next performed western lotting for cspse-3 nd PARP in PDA-derived cell lines depleted for S Blot for: Bx Bcl-2 Bim Actin + G2/M Figure 2 mir-21 inhiition induces humn PDA-derived cells deth y poptosis vi the mitochondril pthwy. Mi PC-2 cells were trnsduced y encoding for mir-21 ntisenses t the indicted MOI. Control cells were trnsduced with GFP-encoding lentivirl vectors. () Flow cytometric nlysis of propidium iodide-stined cells. Histogrms re representtive of three independent experiments. Insert: quntifiction of the DNA content distriution in ech phse of the cell cycle. Results re men ± SEM of three independent experiments. P <.5. () Western lot nlysis of PARP nd cspse-3 clevge, nd of Bx, Bcl-2, nd Bim expression in the trnsduced cells. Results re representtive of three independent experiments. GFP, green fluorescent protein; LV, lentivirl vector; MOI, multiplicity of infection; PDA, pncretic ductl denocrcinom. + Figure 3 Efficient knockdown of mir-21 in PDA tumors using lentivirl vectors. Pncretic tissue ws hrvested 12 dys following gene trnsfer for nlysis of mir-21 expression y in situ hyridiztion. Results re representtive of five different () low ( 4) or () high ( 4) power fields from three different tumors for ech group. Insert: quntifiction of mir-21 expression using ImgeJ softwre. Results re men ± SEM of 1 different fields from three different tumors for ech group. P <.5..u., ritrry unit; GFP, green fluorescent protein; LV, lentivirl vector; PDA, pncretic ductl denocrcinom. mir-21. As shown in the left pnel of the Figure 2, trnsduction with induces cspse-3 nd PARP clevge in PDA-derived cell lines, s compred with control-trnsduced cells. In ddition, we found tht mir-21 knockdown resulted in Bcl-2 inhiition nd induction of Bx nd Bim y western lotting (Figure 2, right pnel) nd immunofluorescence (dt not shown). The ltter findings support tht mir-21 knockdown results in the ctivtion of poptosis through the mitochondril pthwy in PDA-derived cells. Noninvsive trcking of PDA tumor growth We next generted novel model of PDA for the noninvsive trcking of tumor growth sed on the Luci luciferse. Luci elongs to the fmily of secreted luciferse, such s Gussi luciferse, tht cn e smpled in the lood to monitor tumor growth nd response to therpeutics. 1 Mi PC-2 cells expressing Luci were plted in culture dishes nd grown for 7 dys. Luci production ws smpled in the culture medium nd cells were counted. We found tht (i) Luci ccumulted in the cell superntnt, nd tht (ii) Luci levels were correlted to tumor cell numer (R 2 =.91) (Supplementry Figure S4). We next demonstrted tht vol. 21 no. 5 my 213

4 The Americn Society of Gene & Cell Therpy Trgeting mir-21 for Pncretic Cncer Therpy Insert 14 Tumor progression (luci levels, % of control) 1, Gene trnsfer Tumor volume (fold increse) Dys Dys following gene trnsfer 8 15 Fluorescence intensity Fluorescence intensity 1 5 Ki67 Cleved cspse-3 Figure 4 Trgeting mir-21 inhiits PDA tumor growth. () Noninvsive monitoring of PDA tumor growth ws performed for 34 dys efore nd fter intrtumorl gene trnsfer. Results re men ± SEM of Luci levels, expressed s % of dy, in the serum of 12 nimls per group. Insert: tumor volume ws mesured t dy nd dy 12 following intrtumorl gene trnsfer using cliper. Results re men ± SEM of tumor volume progression in 12 nimls per group. P <.1; P <.5. () Pncretic tissue ws hrvested for the nlysis of Ki67 (left) or cleved cspse-3 (right) expression in the tumors y immunofluorescence. Results re men ± SEM of 15 different fields from three different tumors for ech group. P <.1. GFP, green fluorescent protein; LV, lentivirl vector; PDA, pncretic ductl denocrcinom. Mi PC-2 Luci cells were s sensitive s prentl Mi PC-2 cells to mir-21 depletion (Supplementry Figure S4). Lst, Luci monitoring permits the noninvsive quntifiction of mir- 21 knockdown inhiitory effect on PDA cells prolifertion, in time nd dose-dependent mnner (Supplementry Figure S4c). We next engrfted Mi PC-2 Luci cells in the pncres of five severe comined immunodeficiency mice. Luci ws smpled from serum for 63 dys. As expected, Luci levels incresed progressively in the lood of severe comined immunodeficiency mice with intrpncretic tumors (Supplementry Figure S5). However, tumor growth vried considerly etween sujects. In ddition, such slow-growing tumors my not e dequte for the preclinicl testing of therpeutic regimens. Consequently, intrpncretic tumors were removed, mechniclly processed, cultured ex vivo, nd injected in the pncres of recipient mice. As shown in Supplementry Figure S5 nd insert, tumor growth monitored y Luci levels nd tumor volume mesurement ws homogenous, to give rise to tumors 15 dys following tumor cells implnttion (Supplementry Figure S5). Lst, Luci levels were correlted to tumor volume (R 2 =.85) (Supplementry Figure S5c). Tken together, we demonstrte herein for the first time tht Luci lood ssy cn e used for the ex vivo, noninvsive, monitoring of PDA-derived cells tumor growth. In vivo depletion of mir-21 strongly inhiits PDA tumor growth To ssess the therpeutic efficcy of trgeting mir-21 in PDA, LV(/ mir-21) were dministrted with single intrtumorl injection 16 dys following tumor induction, time point t which nimls typiclly hve medium-sized tumors. Control tumors received. We oserved no significnt vritions in ody weight of nimls receiving LVs, underscoring the sfety of trgeting oncogenic mirna using LVs (Supplementry Figure S6). Pncretic tissue ws hrvested 2 weeks lter for nlysis of mirna expression. As expected, humn tumors trnsduced with exhiited high-level expression of mir-21, oth in tumor cells nd their microenvironment (Figure 3). On the other hnd, delivery resulted not only in histologicl evidence of tumor necrosis ut lso inhiition of mir-21 expression in tumor cells (Figure 3 nd insert). These dt demonstrte tht provides n effective mens to trget mir-21 in pncretic tumors. We next monitored orthotopic pncretic tumors progression using lood Luci ssy following or intrtumorl dministrtion. Strikingly, the growth of this very ggressive tumor model ws stopped y dy 2 up to dy 12 following in vivo depletion of mir-21, compred with green fluorescent protein (GFP)-expressing LVs (Figure 4). Animls were killed Moleculr Therpy vol. 21 no. 5 my

5 Trgeting mir-21 for Pncretic Cncer Therpy The Americn Society of Gene & Cell Therpy c 3 RhoB expression (2 Ct ) 2 1 Tretment d 1,2 Vehicle 1,1 Gemcitine 1, Insert 9 + gemcitine CD34 expression (.u.) Tumor progression (luci levels, % of control) # Dys following gene trnsfer Figure 5 Trgeting mir-21 induces tumor ngiogenesis nd potentite gemcitine ntitumorl effect. Pncretic tissue ws hrvested 12 dys following gene trnsfer for nlysis of CD34 expression y immunohistochemistry. Results re representtive of () 5 low nd () 15 high power fields from three different tumors per group. Insert: quntifiction of CD34 expression using Imge J softwre. Results re men ± SEM of 15 different low power fields from three different tumors for ech group. P <.5. (c) Totl RNA ws extrcted from tumors nd nlyzed for RhoB expression y qrt-pcr. Results, expressed s 2 ΔCt, re men ± SEM of three different tumors for ech group. (d) Luci ws quntified in mice serum every 2 3 dys during the course of the experiment. Arrows indicte the intrperitonel injection of gemcitine. Results re men ± SEM of Luci levels, expressed s % of dy, in the serum of 5 1 nimls per group. P <.5; P <.1; P <.5, # P <.5 compring gemcitine versus gemcitine + or versus gemcitine +..u., ritrry unit; GFP, green fluorescent protein; LV, lentivirl vector; qrt-pcr, quntittive reverse trnscriptse-pcr. nd tumor urden ws ssessed 2 weeks following intrtumorl gene trnsfer. Animls treted with control vectors developed fulminnt disese s tumor volume incresed y more thn eightfold during the course of the experiment (Figure 4, insert). In contrst, -treted nimls were drmticlly protected, exhiiting only wek tumor progression (2.9 ±.4 fold increse). In ddition, we oserved (i) mrkedly reduced Ki67 stining nd (ii) induction of poptosis s mesured y cspse-3 clevge in tumors following dministrtion of (Figure 4). We conclude tht trgeting mir-21 uniformly diminished disese progression of experimentl pncretic denocrcinom. Trgeting mir-21 induces tumor ngiogenesis mir-21 is one of the few mirnas involved in ngiogenesis regultion, 11,12 nd cts s n inhiitor of endothelil cell prolifertion nd migrtion in mouse model of choroidl neovsculriztion. 11 We sked whether pncretic tumor vsculriztion ws ffected following tretment. -trnsduced pncretic tumors were wekly vsculrized (Figure 5,). However, mir-21 depletion resulted in the mssive induction of vessels surrounding the tumor (4 ± 1.1 fold increse, P <.5; Figure 5, insert). Interestingly, peripherl lood vessels lso demonstrted mrked reduction in mir-21 levels following gene trnsfer (Supplementry Figure S7). To investigte the mechnisms through which mir-21 down-expression results in pncretic tumor ngiogenesis, we exmined functionl trgets of this mirna. 8,11 While the expression of Sprouty-1 nd Sprouty-2 remined unchnged etween - nd - treted tumors (dt not shown), RhoB expression gretly incresed following mir-21 depletion (Figure 5c), s previously 99 vol. 21 no. 5 my 213

6 The Americn Society of Gene & Cell Therpy Trgeting mir-21 for Pncretic Cncer Therpy descried in other models. 11 Altogether, these dt document tht mir-21 represses the expression of RhoB, providing one mechnism through which trgeting this mirna enhnces PDA tumor ngiogenesis. Comining mir-21 trgeting nd chemotherpeutic tretment provokes pncretic tumor regression Despite its contested efficcy in preclinicl models nd clinicl trils for PDA ptients, gemcitine ecme the stndrd tretment for dvnced disese 15 yers go fter showing superiority over fluorourcil. Since then, mny phse III trils of newer cytotoxic or iologic gents comined with gemcitine hve not shown ny survivl improvement compred with gemcitine lone. 2 To ssess whether trgeting mir-21 my trnslte into clinicl prctice to mnge PDA, we compred the efficcy of our pproch with stndrd gemcitine tretment. Consequently, nd vectors were injected into exponentilly growing pncretic tumors. In prllel, high dosge of gemcitine (125 mg/kg) ws dministrted twice weekly y intrperitonel injection. Blood smples were collected every 2 3 dys during the course of the experiment. We found tht gemcitine slightly inhiited PDA tumor progression, s compred with vehicle ( 21%, P <.5, Figure 5d). On the other hnd, mir-21 depletion strongly inhiited PDA tumor growth, s compred with -trnsduced tumors ( 75%, P <.1, Figure 5d). Becuse the dt presented herein support tht depletion of mir-21 stimultes ngiogenesis in this experimentl model of PDA, we hypothesized tht trgeting mir-21 my ugment tumor drug delivery to enhnce the efficcy of systemic therpies. The results presented Figure 5d demonstrte tht comining gemcitine tretment nd trnsduction with stopped tumor progression (Figure 5d) nd induced tumor shrinkge ( 4 ± 5%, P <.1 dt not shown), s compred with control tumors. Thus, we demonstrte for the first time tht (i) trgeting mir-21 is more effective thn stndrd chemotherpeutic tretment to inhiit PDA tumor growth, nd tht (ii) comining oth therpeutic pproches provokes tumor regression in this very ggressive experimentl model of pncretic cncer. Discussion PDA is highly heterogeneous disese. 13 Lrge-scle genetic nlysis recently shed light on the numerous exomic ltertions detected in this cncer in diverse signling pthwys. 14 In ddition, primry tumors re intrinsiclly heterogeneous, nd specific cellulr suclones cn lso e identified in metstsis. 15 Interestingly, the type nd numer of genomic rerrngements in DNA vry considerly etween ptients, while they seem to occur erly during tumor development. 16 This heterogeneity is elieved to e mjor clinicl ostcle to the successful tretment of PDA. PDA hs developed sophisticted networks of iologicl ctivities to mintin self-sufficiency in growth signl, resistnce to endogenous ntiprolifertive signls, evsion from poptosis, limitless replictive potentil, nd tissue invsion nd metstsis. Noteworthy, mirnas re demonstrted to prticipte in ech one of these processes. This feture renders mirna highly ppeling s therpeutic trget, considering tht modultion of single mirna my ffect mny pthwys simultneously to chieve clinicl enefit. Such pproch is likely to reduce the emergence of resistnt clones since mny concomitnt muttions would e required to suvert the effects of mirna expression modultion. Altertions in mirna expression re not exceptionl ut rther common in humn cncer. 5 We were mong the first to demonstrte tht mir-21, the mirna which is most frequently ssocited with poor outcome in humn cncer, 7 is expressed erly during pncretic crcinogenesis. 6 Compelling evidences indicte tht mir-21 prticiptes in mny cncerous pthwys, such s cncer cell prolifertion, migrtion, invsion, metstsis, nd resistnce to cytotoxic chemotherpeutic gents. 8 In PDA, inhiition of mir-21 decreses prolifertion, mtrigel invsion, nd chemoresistnce to gemcitine, in vitro. 17 Tken together, these findings stem for the use of mir-21 s therpeutic trget in PDA. To dte, most trnsltionl in vivo studies trgeting mirnas fced severl concerns tht need to e ddressed efore dvncing to medicl prctice. 5 In the present study, we found tht locked nucleic cid ntgomirs successfully inhiited mir-21 function in vitro, ut filed to trget this mirna in vivo (dt not shown). Our findings strongly suggest tht the lck of stility nd the incpcity of these molecules to overcome the tumor microenvironment re mjor hurdles for direct mirna-sed therpy of pncretic tumors. While the use of synthetic oligonucleotide inhiitors is promising nd will remin fruitful re of investigtion, rpid progress must e mde to chieve effective delivery of mirna inhiitors in trget tissues. Consequently, we selected virlsed vectors originlly developed for gene therpy to express nti-mir-21 moleculr sponges. We recently demonstrted tht pncretic cncer gene therpy strongly relies on the delivery vector From our expertise, HIV-1 sed LVs outshine other vectors (PEI, denovirus, SV4 ) in delivering therpeutic genes into pncretic cncer cells, in vitro nd in vivo. 18 In ddition, LVs hve proven to e effective to chieve stle mirna knockdown, ex vivo. 21 The results descried herein demonstrte for the first time tht mirna ntgonists re highly efficient in trgeting mir-21, when delivered y LVs oth in vitro nd in vivo, without impcting on endogenous mirna iogenesis. However, it is mndtory to underline the possile genotoxicity ssocited with the use of LVs. 22 In our study, the delivered mteril is integrted in the host DNA with n unpredictle risk of insertionl mutgenesis, ctivtion of proto-oncogenes in helthy cells or even genertion of errnt trnscripts. 23 Consequently, we re now designing integrse-deficient LVs for the sfe nd efficient gene delivery of mirna inhiitors in PDA-derived cells. We found tht tumor cell prolifertion nd tumor progression re strongly inhiited following mir-21 depletion in very ggressive model of pncretic cncer. Interestingly, mir-21 inhiition lso decresed PDA-derived tumor cells prolifertion in three-dimensionl models, strongly suggesting tht this mirna my fvor the tumor-inititing cpcity of pncretic cncer stem cells. Although the moleculr sis of this inhiitory effect requires further investigtion, we demonstrte herein tht mir-21 depletion provokes PDA cell deth y poptosis s determined y PARP nd cspse-3 clevge ssys. We further demonstrte tht nti-mir-21 tretment is ssocited with downregultion of Bcl-2 nd upregultion of Bx expression, 24,25 respectively, nd provide for the first time evidences of Bim induction in response Moleculr Therpy vol. 21 no. 5 my

7 Trgeting mir-21 for Pncretic Cncer Therpy The Americn Society of Gene & Cell Therpy to mir-21 depletion in cncer cells. Our results re in greement with previous reports suggesting tht mir-21 is n oncogene tht plys key role in resisting progrmmed cell deth in cncer cells, prticulrly through the mitochondril pthwy. 8 In the present study, the expression levels of PTEN nd PDCD4, two cnonicl trgets or mir-21, were not ltered following mir-21 depletion in oth Mi PC-2 nd Cpn-2 cells (dt not shown). While protein levels of PTEN re incresed in PDA-derived HS766T cells y ntisense to mir-21, 26 direct trgeting of this tumor suppressor gene y mir-21 in PDA remins to e fully chrcterized. 8 Studies re undergoing to identify direct trgets of mir-21 in PDA-derived cells. We next estlished the principle tht trgeting mir-21 severely impirs pncretic tumor growth. We used for the first time secreted luciferse s mrker for the noninvsive monitoring of PDA growth in mice. We demonstrted tht lood levels of Luci luciferse produced y PDA cells perfectly correltes with cliper mesurement of orthotopic tumors volume. Unlike commonly used mesuring devices, Luci levels my e vlule indictor of tumor viility. Using this unique model, we demonstrte tht therpeutic delivery of mirna inhiitors in highly proliferting nd very ggressive pncretic tumors cn result in tumor growth inhiition. The drmtic sensitivity of the tumor cells to the specific reduction in mir-21 levels underscores the contriution of gin-of-function of this mirna to pncretic tumorigenesis. Tumor cells stopped to proliferte nd underwent poptosis following mir-21 depletion. We found tht our pproch surpsses the therpeutic efficcy of stndrd tretments for this disese. Wht remins to e investigted is whether mir-21 is rtionle trget tht my hve eneficil effects on metsttic disese. Surprisingly, we found tht trgeting mir-21 increses the numer of lood vessels in the surrounding tissue. In order to elorte puttive mechnism for mir-21 trgeting to induce ngiogenesis, we focused on the identifiction of the trgets of this mirna oth in the tumor nd its microenvironment. Indeed, we found tht peripherl lood vessels lso demonstrted mrked reduction in mir-21 following gene trnsfer. Our results support role for the Rho GTPse RhoB, key regultor of ngiogenesis through the modultion of vsculr permeility, extrcellulr mtrix remodeling, migrtion nd prolifertion of endothelil cells, in the prongiogenic process following mir-21 depletion. Interestingly, RhoB hs lso een extensively descried in tumor cells where it seems to ct s tumor suppressor gene. Whether RhoB my lso prticipte in the inhiition of tumor cell prolifertion following mir-21 trgeting remins to e investigted. Bsed on the finding tht mir-21 silencing enhnces lood vessel growth, we hypothesized tht depleting mir-21 my provoke etter drug delivery in pncretic tumors. We demonstrte tht gemcitine nd mir-21 trgeting re synergistic, s this cotretment led to very impressive ntitumorl effect tht is rrely chieved in this experimentl model. Tken together, we demonstrte tht mir-21 is suitle therpeutic trget in PDA nd tht comining epigenetic silencing nd chemotherpeutic tretment induce unexpected pncretic tumor regression. The present study design involved the tretment of existing tumors with mir-21 inhiitors, prdigm closely relted to the clinicl scenrios in which this pproch my e employed. Indeed, we re currently conducting first-in-mn phse I gene therpy clinicl tril in 24 ptients dignosed with dvnced pncretic cncer (Thergp clinicl tril, CliniclTrils.gov identifier NCT ). We demonstrted during this tril, the fesiility nd the sfety of trnsfecting PDA tumors with nonvirl vectors using endoscopic ultrsound (dt not shown). While groundreking, this unique clinicl tril will strongly enefit from the chrcteriztion of new delivery vehicles nd new moleculr trgets tht my help llevite the disml prognosis of this disese. Becuse mir-21 is overexpressed in most humn tumors, 7 therpeutic delivery of mir-21 ntgonists my still e eneficil for lrge numer of cncers for which no cure is ville. While there clerly remins significnt work to e done, our findings highlight the therpeutic promise of this pproch. Mterils nd Methods Cell culture nd genertion of multicellulr tumor spheroids. Humn PDA-derived Cpn-2 cells were grown in RPMI medium supplemented with 1% fetl clf serum, l-glutmine, ntiiotic nd ntimycotic cocktil (Life Technologies SAS, St Auin, Frnce) nd Plsmocin (Cyl- INVIVOGEN EUROPE, Toulouse, Frnce). Mi PC-2 cells were grown in Dulecco s modified Egle s medium contining 4.5 g/l glucose (Invitrogen), 1% fetl clf serum, l-glutmine, ntiiotics, Fungizone, nd Plsmocin (InvivoGen). Spheroids were prepred following the hnging-drop method with minor modifictions. 9 Briefly, 2 µl drops contining 5 Cpn-2 cells were suspended from the lids of 24-well culture dishes nd trnsferred to 24-well culture dishes, se-coted with gr for 2 weeks. Cell lines nd spheroids were grown in humidified incutor t 37 C in 5% CO 2. Cell cycle nlysis y flow cytometry. Mi PC-2 cells were collected, rinsed once in phosphte-uffered sline (PBS) nd fixed in ice-cold 7% ethnol overnight t 4 C. Cells were collected y centrifugtion t 1,g nd rinsed with PBS. Cells were incuted with propidium iodide following mnufcturer s recommendtion (Invitrogen). Cell cycle distriution ws determined using BD FACS Cliur pprtus nd Cell quest pro softwre (Becton Dickinson, Le Pont de Clix, Frnce). Gene expression nlysis. Totl RNA ws isolted from cell lines with TRIzol Regent (Invitrogen) ccording to supplier s instructions nd RNA concentrtion ws mesured with the ND-1 NnoDrop spectrophotometer (Nnodrop, Wilmington, DE). mirnas were quntified from 1 µg totl RNA using the miscript PCR System (Qigen, COURTABOEUF, Frnce). U6 nd 5S RNAs were used s internl controls. cdna smples were diluted 1 into 1 for mirna detection or U6 nd 1 into 1, for 5S RNA detection. RhoB ws quntified s reported previously. 11 DNA smples were diluted 1 into 1 for RhoB detection or U6 nd 1 into 1, for GAPDH nd ctin RNA detection. Duplicte quntittive reverse trnscriptse-pcr ssys were crried out in StepOnePlus Rel-Time PCR System (Life Technologies SAS) with SYBR Green PCR Mster Mix. Reltive mounts of were clculted y the comprtive threshold cycle (CT) method s 2 ΔCT, where ΔCT = CT (gene of interest) CT (geometric men of control genes). Cell viility nd cell prolifertion nlysis. Mi PC-2 cells were seeded t cells per well in 96-well dish nd grown in complete medium. Twenty-four hours lter, cells were trnsduced with LV(/ mir-21) indicted multiplicity of infection. Control cells were trnsduced with. Numer of vile cells ws determined y colorimetric method using CellTiter 96 AQueous Non-Rdioctive Cell Prolifertion Assy (Promeg, Chronnieres, Frnce) ccording to mnufcturer s instructions 72 hours following trnsduction. Cell prolifertion ssys vol. 21 no. 5 my 213

8 The Americn Society of Gene & Cell Therpy Trgeting mir-21 for Pncretic Cncer Therpy were performed in 35-mm dishes Mi PC-2 cells were cultured in complete medium for 24 hours (2 ml per dish). The next dy, cells were trnsduced with t the indicted multiplicity of infection (Supplementry Mterils nd Methods). Control cells were trnsduced with. Cell growth ws mesured t dys 1, 2, nd 3 following trnsduction y cell counting using Coulter counter model ZM (Beckmn Coulter, Roissy, Frnce). All experiments were conducted with different tches of LVs. Trnsduced cells were not selected in this study. Western lotting. Proteins were extrcted from trnsduced cells or tumors, resolved on SDS-polycrylmide gels, nd trnsferred to nitrocellulose memrne. After room temperture locking for 1 hour, lots were incuted overnight t 4 C with ntiodies purchsed from Cell signling Technology (St Quentin Yvelines, Frnce) diluted ccording to the mnufcturer s recommendtions. Secondry horserdish peroxidse-conjugted ntiodies (dilution 1:1,; Perio Science, Breières, Frnce) were dded, nd lots were incuted for 1 hour t room temperture. Immunorective proteins were visulized using ECL immunodetection (Immoilon; Millipore, Billeric, MA). Immunostining for Ki67, CD34, nd cleved cspse-3. Mi PC-2 tumors were hrvested nd fixed in formlin. Four micrometer thick sections were prepred from prffin-emedded sections nd rehydrted. Following ntigen retrievl, sections were incuted for 1 minutes in Protein Block, Serum-free regent to reduce ckground stining (DkoCytomtion). Slides were next incuted overnight t 4 C with nti-ki67 or CD34 ntiodies (DkoCytomtion, Les Ulis, Frnce; dilution: 1:1), or cleved cspse-3 ntiody (Cell signling Technology; dilution: 1:25) developed in rits. Antiody incutions were done in Antiody diluents (DkoCytomtion). For CD34 stining, slides were wshed nd incuted in 3% H 2 O 2 for 3 minutes t room temperture for endogenous peroxidse inhiition. Slides were quickly rinsed in distilled wter, wshed twice in PBS, nd incuted for 3 minutes t room temperture with Envision+ system-hrp (DkoCytomtion). After wshing in distilled wter, slides were incuted in AEC+ regent nd counterstined with Myer s hemtoxylin. For Ki67 nd cleved cspse-3 stining, slides were wshed nd incuted for 1 hour, protected from light with Got nti-rit CY3 conjugted secondry ntiodies (1:5; Jckson ImmunoReserch, Suffolk, UK). After wshing in PBS, slides were mounted with Vectshield contining DAPI (Vector Lortories, Burlingme, CA). Immunostining ws recorded with n opticl microscope, nd quntified using VisioL2 imge nlyzer (Biocom, Les Ulis, Frnce). In situ hyridiztion for mir-21. In situ hyridiztion for mir-21 ws conducted s descried elsewhere. 6 Briefly, in situ hyridiztion ws performed on PDA tumors using LNA proes for mir-21 (Exiqon, Vedek, Denmrk). Prffin-emedded tumors were deprffinized, treted y proteinse K, nd fixed in prformldehyde. Digoxigenin-leled LNA proe ws hyridized overnight. Slides were rinsed nd incuted with nti-digoxigenin F frgment (Roche dignostics, Meyln, Frnce) overnight. The detection rection ws performed using NBT/BNI Redy-to-use tlets (Roche dignostics) nd slides were mounted with cover slip using Glycergel mounting medium (DkoCytomtion). Slides were oserved with Nikon E4 opticl microscope, coupled to n imge nlyzer (VisioL2; Biocom). For ech smple, 15 fields were nlyzed. Experimentl protocol. Sixteen dys following implnttion of Mi PC-2 Luci F1 cells (Supplementry Mterils nd Methods), tumors mesured ~4 mm 3 in volume nd mice were rndomized into the following tretment groups (n = 12) regrdless of the level of ioluminescence mesured in the serum: (i) trnsduction with 15 ng of p24 of ; (ii) trnsduction with 15 ng of p24 of in level 2 niml sfety fcility s previously descried. 18 For the gemcitine study, we used five nimls per group. Gemcitine (125 mg/kg) ws dministrted twice weekly y intrperitonel injection. Blood smples were collected every 2 3 dys during the course of the experiment. The nimls were killed 12 dys fter gene trnsfer nd the tumor tissue ws (i) formlin-fixed nd prffin-emedded for immunohistochemistry nd routine hemtoxylin nd eosin stining ws performed, nd (ii) snp frozen in liquid nitrogen nd stored t 8 C. Hemtoxylin nd eosin stining confirmed the presence of tumor(s) in ech pncres. Luci production ws mesured in 5 µl of serum using coelenterzine (5 µmol/l) s sustrte. Sttisticl nlysis. Results re expressed s men ± SE. Dt were compred y unpired t-tests (P <.5, P <.1; P <.5) or one-wy nlysis of vrince with Bonferroni s multiple comprison test ( # P <.5) using Grphpd Prism softwre (Grphpd Softwre, L Joll, CA). P <.5 ws considered significnt. SUPPLEMENTARY MATERIAL Figure S1. Lentivirl vectors trnsduce humn PDA-derived cell lines with high efficcy. Figure S2. Trgeting mir-21 does not impct on other cellulr mirna expression. Figure S3. Kinetics of inhiition of cell prolifertion following trgeting of mir-21 in PDA-derived cells. Figure S4. Luci expression correltes with tumor cell prolifertion nd response to tretment. Figure S5. Noninvsive trcking of pncretic tumor growth using Luci luciferse. Figure S6. Tumor growth nd intrpncretic injection of lentivirl vectors re well tolerted in SCID mice. Figure S7. mir-21 is downregulted in peripherl lood vessels following gene trnsfer. Mterils nd Methods. ACKNOWLEDGMENTS This work ws supported y grnts from INSERM, Region Midi-Pyrenees (15131), nd l Fondtion de l Avenir (ET2-656). F.S. ws supported y postdoctorl fellowship from the Inniosnté Foundtion. REFERENCES 1. Jeml, A, Bry, F, Center, MM, Ferly, J, Wrd, E nd Formn, D (211). Glol cncer sttistics. CA Cncer J Clin 61: Torrisni, J, Bournet, B, Cordelier, P nd Buscil, L (28). [New moleculr trgets in pncretic cncer]. 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