Sticky sirnas targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases

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1 Kedinger et l. BMC Cncer 213, 13:338 RESEARCH ARTICLE Open Access Sticky sirnas trgeting nd exert n ntitumorl effect on melnom sucutneous xenogrfts nd lung metstses Vlerie Kedinger 1*, Aline Meulle 1, Omr Zouni 1, Mrie-Elise Bonnet 1, Jen-Bptiste Gossrt 1, Elodie Benoit 1, Melnie Messmer 2, Ptthirmn Shnkrnrynn 3, Jen-Pul Behr 1, Ptrick Ercher 1 nd Anne-Lure Bolcto-Bellemin 4* Astrct Bckground: Melnom represents one of the most ggressive nd therpeuticlly chllenging mlignncies s it often gives rise to metstses nd develops resistnce to clssicl chemotherpeutic gents. Although diverse therpies hve een generted, no mjor improvement of the ptient prognosis hs een noticed. One promising lterntive to the conventionl therpeutic pproches currently ville is the inctivtion of proteins essentil for survivl nd/or progression of melnoms y mens of RNA interference. Survivin nd, oth involved in cell survivl nd prolifertion nd frequently deregulted in humn cncers, re good cndidte trget genes for sirna medited therpeutics. Methods: We used our newly developed sticky sirna-sed technology delivered with liner polyethyleneimine (PEI) to inhiit the expression of nd oth in vitro nd in vivo, nd ddressed the effect of this inhiition on B16-F1 murine melnom tumor development. Results: We confirm tht nd downregultion through RNA interference mechnism induces lockge of the cell cycle s well s impired prolifertion of B16-F1 cells in vitro. Most importntly, PEI-medited systemic delivery of sticky sirnas ginst nd efficiently locks growth of estlished sucutneous B16-F1 tumors s well s formtion nd dissemintion of melnom lung metstses. In ddition, we highlight tht inhiition of expression increses the effect of doxoruicin on lung B16-F1 metstsis growth inhiition. Conclusion: PEI-medited delivery of sticky sirnas trgeting genes involved in tumor progression such s nd, either lone or in comintion with chemotherpeutic drugs, represents promising strtegy for melnom tretment. Keywords: Sticky sirna, Delivery, Survivin, Cyclin B1, Tumor inhiition, Melnom Bckground Melnom is considered s one of the most ggressive humn cncers. The mjority of melnom-ssocited deths re cused y metstses, which cn occur in wider vriety of res thn ny other cncer [1]. Among trget orgns, the lung represents common site of metstsis, * Correspondence: vkedinger@polyplus-trnsfection.com; l_olcto@yhoo.com 1 Polyplus-trnsfection SA, Bioprc, BP 918, Boulevrd Séstien Brnt, 6741 Illkirch, Frnce 4 Current ddress: Quintiles, rue Jen Dominique Cssini, BP 5137, 6744 Illkirch Cedex, Frnce Full list of uthor informtion is ville t the end of the rticle mostly ecuse of its ntomic structure nd high vsculriztion. These chrcteristics mke it preferentil pthwy for metsttic seeding nd rich environment for neoplstic growth [2]. Another feture commonly ttriuted to melnom is its chemo-resistnce [3]. During melnom progression, the rekdown of cell deth control leding to resistnce to chemotherpeutic drugs is chieved through the comined ctivtion of nti-poptotic fctors, inctivtion of pro-poptotic effectors nd reinforcement of survivl signls. Trgeting one or more of these different fctors my e key requisite to overcome drug resistnce 213 Kedinger et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Kedinger et l. BMC Cncer 213, 13:338 Pge 2 of 11 nd thus improve clinicl outcome of ptients with melnom. Survivin, memer of the Inhiitor of Apoptosis Protein (IAP) fmily [4], hs emerged few yers go s promising therpeutic trget in cncers ecuse of its overexpression in wide spectrum of tumors, including melnom [5-7]. Additionlly, ws identified s mrker of poor prognosis in melnom [8]. In ddition to its role in poptosis inhiition, lso plys criticl role in the regultion of cell division y inducing exit from G1 checkpoint rrest nd susequent entry into S phse [9]. Finlly, recent studies hve involved in cell motility, which my underlie role for this protein in promoting melnom metstsis [1-12]. Vrious pproches involving moleculr inhiitors hve een developed to inhiit its expression in tumor cells [6,13-17]. Another potentil therpeutic trget for cncer tretment is represented y, the regultory suunit of cyclin-dependent kinse 1 (cdk1), which plys pivotl role in the trnsition of the cell cycle from G2 phse to mitosis [18]. Altered expression of hs een reported in numerous cncers, where it could contriute to chromosoml instility [19-23]. Furthermore, severl studies hve demonstrted its clinicl significnce s poor prognosis fctor for severl cncer types [24-27], including melnom [28], nd overexpression is responsile for rdiotherpy resistnce in different tumors [29-31]. RNA interference represents powerful pproch for ntitumor therpy y llowing in vivo silencing of essentil genes for tumor progression nd provides promising lterntive to trditionl smll molecule therpies. However, delivery of sirnas still remins the most chllenging step for the development of sirna-sed therpy. The chllenge includes efficient trget gene silencing in the desired tissue while voiding side effects such s immune response, toxicity nd off-trget silencing. In this context, the ctionic liner polyethylenimine (PEI) is well known for its efficiency to trnsfect genes oth in vitro nd in vivo s it is involved in severl clinicl trils for the tretment of ldder cncer ( show/nct59588), pncretic ductl denocrcinom ( nd multiple myelom ( show/nct143572?term=senesco&rnk=1). In this study, we investigted its ility to deliver functionl ntitumorl sirna. To this end, we hve developed sticky sirnas (s) tht mimic gene structure through reversile conctemeriztion rought y sticky 3 -complementry overhngs [32]. These modified sirnas confer higher stility to the complexes formed with liner PEI, thus incresing gene silencing efficiency oth in vitro nd in vivo, compred with stndrd sirnas. We used this new technology to specificlly trget nd cyclin B1 in B16-F1 murine melnom cells. Our results show tht s re efficient to inhiit nd expression in vitro nd tht systemic tretment with s trgeting these two genes is le to reduce oth sucutneous melnom tumors nd their lung metstses. Moreover, inhiition of expression incresed the effect of doxoruicin tretment on melnom lung metstsis. Altogether, our dt re promising towrds development of s ginst nd s new therpeutic strtegy for melnom tretment. Methods Cell line Murine melnom B16-F1 cell line ws otined from ATCC nd cultured in Dulecco s modified Egle s medium (Euroio, Courtoeuf, Frnce), supplemented with 1% fetl ovine serum (Hyclone, Logn, UT, USA), 2 mm Glutmine (Euroio) nd 2 U/ml penicillin / 2 μg/ml streptomycin (Euroio). Sticky sirnas IEX-HPLC-purified nucleic cids were purchsed from Eurogentec (Brussels, Belgium). Anneling ws performed in nneling uffer (Eurogentec), finl concentrtion.1 X for 2 min t 95 C followed y slow cooling. Sequences were s follow: Cyclin B1 sense, 5 -GAGAUGUACCCUCCA GAAAdTdTdTdTdTdTdTdT-3, Cyclin B1 ntisense, 5 -UUUCUGGAGGGUA CAUCUCdAdAdAdAdAdAdAdA-3, Survivin sense, 5 -CCGUCAGUGAAUUCUU GAAdTdTdTdTdTdTdTdT-3, Survivin ntisense, 5 -UUCAAGAAUUCACU GACGGdAdAdAdAdAdAdAdA-3, Negtive control sense, 5 -AUGUCUACUGG CAGUCCUGdTdTdTdTdTdTdTdT-3, Negtive control ntisense, 5 -CAGGACUGC CAGUAGACAUdAdAdAdAdAdAdAdA-3. In vitro nd in vivo trnsfections jetpei nd in vivo-jetpei were from Polyplus-trnsfection (Illkirch, Frnce). For trnsfection with jetpei regent, complexes were prepred s follows: for triplicte experiment, the required mount of nd trnsfection regent were ech seprtely diluted in 15 μl of NCl 15 mm. A volume of 2.4 μl (for 75 nm of, N/P=6) or 3.2 μl (for 5 nm of, N/P=8) of jetpei ws used per μg of sirna. N/P rtio is defined s the numer of nitrogen residues of jetpei per nucleic cid phosphte. The trnsfection regent solution ws dded to the solution nd left for 3 min t room temperture. Avolumeof1μl of complexes ws dded to B16-F1 cells seeded in 24-well pltes t 4, cells/well one dy efore, nd plced in.5 ml of medium without serum just prior to complexes ddition. After 4 h, the medium

3 Kedinger et l. BMC Cncer 213, 13:338 Pge 3 of 11 ws replced y 1 ml of complete medium contining 1% serum. For in vivo delivery with in vivo-jetpei, complexes were prepred s follows: for 1 mouse, the required mount of nucleic cid nd PEI were seprtely diluted in 1 μl of5% glucose solution (finl concentrtion). For injection of.6 mg/kg of,.16 μl of PEI were used per μg of (N/P=8). For the 1 mg/kg injected mount,.25 μl of PEI were used per μg of (N/P=12.5). The trnsfection regent solution ws dded to the solution nd left for t lest 3 min t room temperture. At this stge complexes re stle for more thn 4 h t room temperture. Animl experiments All niml studies were conducted in ccordnce to the French Animl Cre guidelines nd the protocols were pproved y the Direction des Services Vétérinires. Five-weeks old NMRI Nude femle mice were otined from Elevge Jnvier (Le Genset Sint Isle, Frnce). For sucutneous xenogrfts, B16-F1 cells (1 1 6 cells in 1 μl of culture medium without serum) were injected sucutneously on the right flnk of nimls. / PEI complexes were intrvenously injected through the retro-oritl sinus within 2 s. Tretment with complexes strted when tumors reched 5 mm 3 nd were performed every other dy until scrifice of the nimls. Tumors were mesured t ech injection, nd tumor volume ws clculted s v =(π L l 2 )/6. For lung metstsis model, B16-F1 cells (1 1 6 cells in 3 μl of culture medium without serum) were injected intrvenously through the til vein. Brnched DNA ssy QuntiGene ssy (Pnomics, Snt Clr, CA, USA) ws used to quntify the mount of mrna in cells or lungs. Cells were lysed in 6 μl of 1 lysis uffer nd incuted for 3 min t 5 C. Lungs were lysed in 2 ml of tissue nd cell lysis solution (Teu, Le Perry-en-Yvelines, Frnce), supplemented with.15 mg/ml of K Proteinse (Sigm-Aldrich, St Louis, MO, USA) nd incuted three times 5 min t 6 C with 1 s vortexing. A volume of 1 6 μl of cell or lung lyste ws used for rnched DNA (DNA) ssy. Proe set were designed using QuntiGene ProeDesigner softwre. Trget gene expression ws ssyed ccording to mnufcturer recommendtions. Trget expression level ws normlized to corresponding GAPDH expression from the sme cell lyste. Western lot nlysis Cells were lysed in 1 μl of RIPA uffer. Proteins were quntified with the BCA kit (Pierce, Breieres, Frnce). Fifty microgrms of totl protein were sujected to electrophoresis on 1 or 15% crylmide/iscrylmide gel nd trnsferred to poly (vinylidene fluoride) memrne (Millipore, Molsheim, Frnce). A mouse nti- monoclonl ntiody (Cell Signling Technologies, Dnvers,MA,USA)t1/1,,ritnti- polyclonl ntiody (Cell Signling Technologies) t 1/ 1, nd mouse nti-gapdh monoclonl ntiody (Amion, Austin, TX) t 1/1, were used. Antirit or nti-mouse secondry horserdish peroxidseconjugted were purchsed from Millipore nd used t 1/1,. Protein nds were visulized with enhnced chimioluminescence regent (ECL, Amershm, GE Helthcre, Velizy-Villcouly, Frnce). Prolifertion ssy Cell pellets were homogenized in 1 μl of 1:1 PBS/trypn lue (Euroio) nd live cells were counted using n utomtic hemtocyter (TC1, BioRd, Mrnes-l-Coquette, Frnce). For nuclei morphology nlysis, cells were fixed with ice-cold methnol for 1 min, rinsed with 1 PBS nd stined with DAPI (.1 μg/μl) for 15 min. Cells were oserved using Nikon inverted microscope (Nikon Eclipse TE 2-S, Amsterdm, Netherlnd). Cell cycle nd poptosis nlysis Cells were fixed in chilled 5% ethnol for 15 min t 2 C. Pellets were incuted in 1 PBS with.1% Triton X-1 nd 5% BSA for 1 min on ice, nd for 3 min t 37 C in 1 PBS contining 2 μg/ml of RNse A. Propidium iodide (1 μg/ml) ws dded for 1 min t room temperture. Cell pellet ws resuspended in 1 PBS, 5 mm EDTA to otin cell concentrtion lower thn cells/ml. Cells were nlyzed y FACS using Guv pprtus from Millipore (Molsheim, Frnce). 5 -RACE nlysis Totl RNA ws isolted using RNA NOW regent (Biogentex Lortories, Houston, TX, USA) following instructions of the mnufcturer. One μg of RNA ws ligted to GeneRcer RNA Oligo (5 -CGA-CUG-GAG- CAC-GAG-GAC-ACU-GAC-AUG-GAC-UGA-AGG- AGU-AGA-AA-3 ; Life Technologies, Sint Auin, Frnce). Two-hundred nd fifty nnogrms of Oligo were used respectively for or nlysis. Ligted RNA ws reverse trnscried using gene-specific primer (Tle 1). To detect the clevge product, one or two rounds of consecutive PCR (for conditions see Additionl Tle 1 Primers used for Reverse trnscription Nme Sequence Cyclin B1 Rev (CCNBM1548R) 5 -TTC-GAC-AAC-TTC-CGT-TAG-CC-3 Survivin Rev (81R) 5 -AGC-TCT-GGA-CTC-TGG-CCA-CCC-3

4 Kedinger et l. BMC Cncer 213, 13:338 Pge 4 of 11 Tle 2 Primers used for PCR Nme Sequence Cyclin B1 Rev (CCNBM975R) 5 -AGG-GCG-ACC-CAG-GCT-GAA-GT-3 Survivin Rev (81R) 5 -AGC-TCT-GGA-CTC-TGG-CCA-CCC-3 Survivin RevN (743R) 5 -GCC-ACC-TCC-CTG-TGG-ACT-CA-3 file 1: Tle S1) were performed using primers complementry to the RNA Oligo nd to or gene sequence (Tle 2). Histology After mice scrifice, lungs were perfused with 4% prformldehyde, incuted for 16 h in 4% prformldehyde nd processed for prffin emedding. Prffin sections (7 μm) were generted nd Hemtoxylin/Eosin (H&E) stined. Sttisticl nlysis All sttisticl nlyses were performed using GrphPd prism softwre pckge (version 5). P vlues were clculted ccording to Mnn-Withney test nd were considered significnt when lower thn.5. Results nd discussion in vitro s gene silencing efficiency in B16-F1 murine melnom cells We used previously vlidted nd sirna sequences [33,34], designed sticky sirnas with these sequences y dding 3 complementry overhngs nd first tested their silencing efficiency oth t the mrna nd protein levels in B16-F1 cells. A sequence presenting no homology with mrna dtses ws used s negtive control. Trnsfection of B16-F1 cells with jetpei nd different concentrtions of specific s rnging from 25 to 1 nm ws performed nd ll led to significnt reduction of nd mrna control cells negtive control Survivin control cells negtive control c Survivin/GAPDH nm, N/P=8 75 nm, N/P=6 d CyclinB1/GAPDH nm, N/P=8 75 nm, N/P=6 Clevge site 17 KD 24 h 48 h 72 h GeneRcer RNA dptor GR5 mrna 3 clevge product Rev 36 KD GAPDH GRN5 RevN 58 KD 36 KD Cyclin B1 GAPDH Finl 5 -RACE PCR product Figure 1 Inhiition of nd expression in B16-F1 cells. (, ) mrna expression level of nd nlyzed y rnched DNA 16 h fter trnsfection with s nd PEI. Cyclin B1 nd levels re expressed reltive to GAPDH. (c) Protein level of nd nlyzed y Western lot 24, 48 nd 72 h fter trnsfection with negtive control (2) or s ginst or (3) (5 nm, N/P=8). Non trnsfected cells (1) were lso loded. GAPDH ws used s loding control. (d) In vitro 5 -RACE nlysis of RNA extrcted from B16-F1 cells either untrnsfected (1) or 16 h fter trnsfection with negtive control (2), or (3) s nd PEI (5 nm, N/P 8). Top prt, schemtic representtion of the 5 -RACE procedure.

5 Kedinger et l. BMC Cncer 213, 13:338 Pge 5 of 11 levels. The level of inhiition ws optiml t 5 nm, N/P of 8 nd 75 nm, N/P of 6 (Figure 1 nd ). Indeed, oth concentrtions induced 7 to 8% inhiition of the two trget mrna expression compred to control cells. This downregultion of mrna expression induced diminution of the corresponding protein level for t lest 48 h for oth trget proteins, s nlyzed y Western lot (Figure 1c). In order to confirm tht the gene silencing oserved fter trnsfection occurred through RNAi mechnism, we then performed rpid mplifiction of cdna ends (5 -RACE) on RNAs extrcted from B16-F1 cells trnsfected with, or negtive control. A nd corresponding to the predicted clevge product (158 p for nd 33 p for ) ws specificlly detected from RNAs extrcted of oth nd 5 nm, N/P=8 % cell growth/control cells trnsfected cells (Figure 1d). Sequencing nlysis of the PCR products confirmed their specificity (dt not shown). The inhiition of nd expression oserved fter trnsfections ws ccompnied y specific cellulr effect, s cells presented growth inhiition of more thn 8% compred to control cells 48 h post-trnsfection (Figure 2). The prolifertion rte of cells trnsfected with the negtive control ws lso somewht lowered compred to control cells. This diminution cn e ttriuted to the trnsfection itself, which cuses trnsient lockge of the cell cycle progression. To further chrcterize the growth inhiition induced y the down-regultion of oth nd, we nlyzed y flow cytometry B16-F1 DNA content 48 h fter trnsfection of s (Figure 2). cells G1 = 77,4 % G2/M = 17,9 % 75 nm, N/P=6 negtive control G1 = 79,7 % G2/M = 12,6 % G1 = 41,4 % G2/M = 52,9 % 2 G1 = 66,4 % G2/M = 25,4 % negtive control cells d c % Apoptotic cells cells 7. ± 2.44 negtive control 7.1 ± ± ± 4.88 cells negtive control Figure 2 Effect of nd inhiition on B16-F1 cell cycle progression. () Prolifertion ssy 48 h fter trnsfection of s. The prolifertion rte is represented s percentge of cell growth reltive to control untrnsfected cells. () Flow cytometer nlysis of the cell cycle distriution of untrnsfected B16-F1 cells or 48 h fter trnsfection with negtive control, or s with PEI (5 nm, N/P=8). (c) Tle presenting the percentge of cells in poptosis (sug1 popultion) of 3 independent experiments determined fter FACS nlysis in the presence of propidium iodide 48 h fter trnsfection with negtive control nd specific s with PEI (5 nm, N/P=8). (d) DAPI stining, 48 h fter trnsfection with negtive control, or s with PEI (5 nm, N/P=8) showing the presence of meg-nuclei nd chromosoml errtions in cells trnsfected with either or s.

6 Kedinger et l. BMC Cncer 213, 13:338 Pge 6 of 11 Wheres only 17.9 nd 12.6% of nontrnsfected or negtive control -trnsfected cells were locted in G2/M phse, respectively, more thn 5% of cells trnsfected with were rrested in G2/M phse. Comprtively, the effect ws milder fter cyclin B1 inhiition, yet significnt lockge of the cells in G2/M phse ws still present (Figure 2). Moreover, percentge of poptotic cells ws lso incresed following downregultion of nd expression compred to controls (Figure 2c). Finlly, DAPI stining of cells in which or expression ws inhiited showed high proportion of cells presenting meg-nuclei (more thn 5% of the cells), chrcteristic of cell cycle rrest (Figure 2d). Altogether, these results vlidte the pproch to silence nd through mechnism of RNA interference in vitro in B16-F1 murine melnom cells. Systemic tretment with s inhiits growth of estlished sucutneous melnom xenogrfts B16-F1 cells hve the ility to form tumors when injected sucutneously in nude mice [35]. We took dvntge of this model mimicking primry melnom tumor to test the potency nd specificity of PEI-medited delivery of s in vivo. First, in order to confirm RNA interference mechnism of s in vivo, we performed 5 -RACE nlysis fter intr-tumorl injection of (.6 mg/kg, N/P=8) complexed with in vivo-jetpei. As shown in Figure 3, we detected nd specific for the clevge product fter injection of either or s in B16-F1 xenogrfts tht could not e detected in glucose or negtive control injected tumors. We then ssessed the effect of systemic tretment of s delivered with in vivo-jetpei on the growth of B16-F1 xenogrfts. Intrvenous delivery of s t 1 mg/kg (N/P = 12.5) ws performed every other dy. The men tumor size monitored fter ech injection is represented in Figure 3 for ech group. We strted to oserve reduction of tumor growth fter the third injection of ginst (i.e., 5 dys fter tumor cells injection), which persisted until the end of the experiment nd reched significnt inhiition of up to 5% compred to control mice (Figure 3). The effect of ws less pronounced, yet still significnt s it leds to 44% inhiition of tumor growth compred to controls (Figure 3c). The difference Tumor Volume (mm3) Survivin Negtive * ** c Tumor volume (mm3) negtive control ** ** Dys fter the first injection Dys fter the first injection Figure 3 B16-F1 tumor xenogrft growth inhiition upon systemic tretment of mice with s nd PEI. () In vivo 5 -RACE nlysis of RNA extrcted from sucutneous tumors 16 h fter intr-tumorl injection of.6 mg/kg of negtive control (2) nd or (3) nd PEI (N/P=8). As control, 5 -RACE ws lso performed on RNA extrcted from tumor injected with glucose only (1). (, c) Mice ering tumor xenogrfts were treted intrvenously every other dy with glucose (dimond), negtive control (tringle) nd () or (c) (squre) t 1 mg/kg, N/P=12.5. Tumor growth ws monitored fter ech injection nd represented s men tumor volume ± SEM. n = 1 nimls per group. P vlues were clculted etween control (glucose treted) group nd specific groups; * p<.5; **p<.1.

7 Kedinger et l. BMC Cncer 213, 13:338 Pge 7 of 11 oserved etween the two trget genes could e explined y the multifunctionlity of compred to, s the first protein is implicted in poptosis, prolifertion nd motility [36], wheres the second one only plys criticl role in the control of the cell cycle progression nd poptosis [18]. The mice treted with negtive control presented minor, sttisticlly nonsignificnt, reduction of tumor size. The difference etween the control nd negtive control groups could reflect the nonspecific effect of trnsfection on the cell cycle previously oserved in vitro (see ove). Melnom lung metstses re reduced following systemic tretment with s Metstses, which represent the leding cuse of mortlity for ptients with melnom, re the most chllenging cells to trget. PEI ws previously shown to preferentilly deliver ctive nucleic cids, plsmids nd sirnas, to the lung fter intrvenous injection [37-4]. We therefore wondered whether systemic tretment of ntitumorl s delivered with PEI could reduce the formtion of melnom lung metstses. When injected into nude mice til vein, B16-F1 cells rpidly give rise to lung metstses. The dvntge of this model is tht it circumvents the initil step of tumor cell migrtion nd llows us to evlute the effect of our tretment on the implnttion of metsttic cells in the host tissue nd their susequent prolifertion. Tretment with s ws performed s descried in Figure 4. At the end of the experiment, lungs were excised nd oserved for metsttic nodules. Tretment with nd specific s led to n importnt reduction of the numer of metstses (lck nodules) compred to controls s illustrted y representtive pictures of lungs (Figure 4). The lung weight c 1.4 ** * i.v. injection of 1x1 6 B16-F1 cells Scrifice of the mice dys Lung weight (% of ody weight) i.v. injections of 1 mg/kg sticky sirna nd PEI N/P=12.5 negtive control d H&E stining negtive control Figure 4 Anti-metsttic therpeutic effect of systemic tretment with s nd PEI. Nude mice were i.v. injected with B16-F1 cells, rndomized nd treted with glucose, negtive control, or s nd PEI (1 mg/kg, N/P=12.5). () Schemtic representtion of the tretment procedure. () Representtive pictures of lungs excised from mice of the different treted groups. (c) Overll men lung weight reltive to the ody weight of the mice. n = 8 nimls per group. P vlues were clculted; * p<.5; **p<.1. (d) Representtive imges of Hemtoxylin-Eosin stined lung sections fter tretment with glucose, or s nd PEI.

8 Kedinger et l. BMC Cncer 213, 13:338 Pge 8 of 11 ws lso used s n indictor of metstsis progression. We determined the men percentge of lung over ody weight in ech group nd oserved significnt decrese in groups treted with nd s compred to control groups (Figure 4c). Histologicl nlysis of lung sections ws performed which further confirmed the drstic diminution of oth nodule size nd numer in oth nd treted lungs (Figure 4d). Even if representtive of the metsttic grde, lung weight evlution is not the est quntittive method, nd proly leds to n underestimtion of the effect produced y the s injection. Indeed, with this method only 2 nd 25% reduction of lung tumor weight were oserved following nd tretment, respectively. We thus developed quntittive ssy sed on MITF dosge. Indeed, s MITF is melnocyte specific trnscription fctor [41], it llows quntifiction of the B16-F1 nodules present in the lungs. When MITF expression is detected in lungs, its level should e directly proportionl to the numer of B16-F1 tumor cells. To verify this hypothesis, we nlyzed MITF mrna level in lungs contining incresing numer of B16-F1 tumors (Figure 5). As expected, no MITF mrna ws oserved in lungs without B16-F1 tumors. In lungs presenting n intermedite numer of B16-F1 tumors, n intermedite level of MITF ws oserved, wheres in lungs with high level of tumor cells, high level of MITF ws oserved (Figure 5). In ddition, we vlidted the linerity of our MITF ssy (see Additionl file 2: Figure S1). We then looked t the MITF mrna level in lungs of -treted mice, nd oserved significnt diminution fter either or systemic tretment compred to the level of MITF present in lungs of glucose or negtive control treted mice (65 nd 56% inhiition compred to negtive control, respectively, Figure 5). These results re in etter greement with our mcroscopic nd microscopic oservtions, nd confirm the nti-metsttic effect of systemic tretment of mice with s trgeting genes implicted in the cell cycle regultion. Alternted tretment with doxoruicin nd induces n dditive diminution of melnom lung metstses The poor prognosis ttriuted to melnoms lrgely results from resistnce to conventionl chemotherpy [3,42]. Doxoruicin, topoisomerse II inhiitor, hs een used for yers to tret diverse types of cncers, nd it is one of the most effective nticncer drugs currently known. However, its clinicl use is limited y dosedependent toxicity, low specificity ginst cncer cells nd emergence of resistnce [43]. Indeed, melnom tumors MITF level (x1 5 ) AU MITF level (x1 5 ) AU control *** re known to e prtilly refrctory to doxoruicin [3], nd high doses with susequent toxic effects re necessry to induce tumor regression. Since ws shown to e key fctor in chemo-resistnce, we hypothesized tht tretment with could enhnce the effect of doxoruicin on the inhiition of melnom lung metstsis growth. In order to nswer this question, we first estlished the optiml dose of doxoruicin, which would induce tumor growth inhiition with the lest toxicity. To this end, three doxoruicin tretments (t dy 4, 8 nd 1) t three different doses (1, 2.5 nd 1 mg/kg) were performed following B16-F1 tumor cells injection. Tumor growth inhiition ws evluted y lung weight nd toxicity * negtive control Figure 5 Quntifiction of the B16-F1 lung metstses using dosge of MITF melnocyte specific trnscription fctor expression. () Brnched DNA nlysis of RNA extrcted from wild-type or B16-F1 tumor-ering lungs for determintion of MITF trnscription fctor mrna expression level. () MITF RNA level ws nlyzed y rnched DNA in B16-F1 tumor-ering lungs untreted (control) or fter systemic tretment with nd or negtive control delivered with in vivo-jetpei (1 mg/kg, N/P=12.5). n = 6 to 9 nimls per group. P vlues were clculted; * p<.5; ***p<.1.

9 Kedinger et l. BMC Cncer 213, 13:338 Pge 9 of 11 ws determined y ody weight loss during the course of the experiment. The tretment with doxoruicin t 1 mg/ kg ws not toxic t ll ut hd no effect on tumor growth either. On the other end, the 1 mg/kg dose ws very efficient on lung tumors ut induced drstic ody weight loss, lmost 15% t the end of the experiment, ccompnied y generl posture of the mice which ws chrcteristic of high toxicity (Figure 6 nd ). We thus retined the intermedite dose of 2.5 mg/kg which hd only mild effect on tumor growth inhiition ut lso shows miniml toxicity for the nimls. We then evluted the effect of n lternting tretment of doxoruicin nd compred to tretment with either doxoruicin or lone. The tretment with doxoruicin nd ws performed every lternte dy s illustrted on Figure 6c. Doxoruicin (2.5 mg/kg) nd (1 mg/kg) hd n dditive effect on lung metstsis growth inhiition (Figure 6d). Wheres doxoruicin lone t 2.5 mg/kg ws poorly efficient, its comintion with induced significnt metstsis inhiition s determined y lung weight decrese compred to controls, nd this without producing ny toxicity. A strong diminution of the numer nd size of tumor nodules ws oserved visully (dt not shown). Moreover, dosge of MITF mrna level showed 87% inhiition of tumor growth (MITF level, / )followingthe lternting tretment of doxoruicin nd compred to controls (MITF level, / ) which ws significntly higher thn the conditions without doxoruicin (54% inhiition compred to control, MITF level, / ). These results re very encourging for the development of s new strtegy to circumvent the inherent chemo-resistnce of melnoms or to enhnce the chemosensitivity of melnoms. This comintion therpy using s nd chemotherpy offers novel strtegy for cncer tretment nd is confirmed in other cncers [44,45]. Conclusion RNA interference is evolving s promising strtegy for cncer tretment. However, delivery of sirnas in vivo 1.5 Doxoruicin 2.5 mg/kg Doxoruicin 1 mg/kg Doxoruicin 1 mg/kg Lung weight (% of ody weight) 1..5 Body weight (g) c i.v. injection of 1x1 6 B16-F1 cells Scrifice of the mice i.v. injections of 2.5 mg/kg doxoruicin dys d Lung weight (% of ody weight) Dys fter cell injection ns * **.6 i.v. injections of 1 mg/kg sticky sirna nd PEI N/P=12.5 Figure 6 Additive effect of nd doxoruicin on the tretment of lung metstses. () Dose dependent effect of doxoruicin (1, 2.5 or 1 mg/kg) on lung metstsis growth ssessed y the overll men lung weight reltive to the ody weight of the mice. n = 9 nimls per group. () Body weight mesurement from the eginning to the end of the tretment ws evluted to determine the toxicity of doxoruicin tretments. (c) Schemtic representtion of the tretment procedure with doxoruicin lone, lone, or oth. (d) Overll men lung weight reltive to the ody weight of the mice fter n lternted tretment of doxoruicin nd with PEI, compred to tretment with doxoruicin or lone. n = 9 nimls per group. P vlues were clculted; * p<.5; **p<.1.

10 Kedinger et l. BMC Cncer 213, 13:338 Pge 1 of 11 still remins the mjor issue for the development of successful sirna-sed therpy. Our present study highlights n emerging RNAi technology, sed on the use of sticky sirnas delivered with PEI. These modified sirnas, y mimicking gene structure, enhnce sirna delivery into cells nd consequently led to etter inhiition of genes involved in mlignncies. In order to vlidte this new technology, we chose to design sticky sirnas ginst two well-known plyers of the tumor progression process, nd [19,46,47]. The results presented in this work demonstrte tht PEI-medited systemic delivery of sticky sirnas ginst nd led to n efficient inhiition of tumor growth. Moreover, we showed in previous study tht no mjor inflmmtion is induced y liner PEI-medited nucleic cid delivery in vivo, s neither pro-inflmmtory cytokines nor heptic enzymes were produced [48]. Altogether, these dt re very encourging for the clinicl development of such therpies which could represent promising pproch for melnom tretment. Additionl files Additionl file 1: Tle S1. PCR conditions for 5 RACE nlysis. Additionl file 2: Figure S1. Linerity of MITF ssy. Brnched DNA nlysis of RNA extrcted from wild-type or B16-F1 tumor-ering lungs to determine MITF mrna expression level. Different volumes of lung extrct (.1;.5; 1 nd 5 μl) were nlyzed, showing good linerity of the ssy. Arevitions : Sticky sirna; PEI: Polyethyleneimine; N/P: PEI mine over nucleic cid phosphte chrge rtio. Competing interests All the uthors except Ptthirmn Shnkrnrynn re employees of the Polyplus-trnsfection compny, they declre no competing interests. Authors contriutions VK crried out in vivo nd in vitro experiments, prticipted in the design of the study nd drfted the mnuscript, AM crried out in vivo experiments nd prticipted in the design of the study, OZ crried out in vivo nd in vitro experiments, MEB crried out in vivo nd in vitro experiments, JBG crried out in vivo nd in vitro experiments, EB crried out in vivo experiments, MM crried out in vitro experiments, PS gve some mteril nd criticlly revised the mnuscript, JPB prticipted in the design of the study nd criticlly revised the mnuscript, PE conceived the study, coordinted nd helped to drft the mnuscript, ALBB conceived the study, prticipted in its design nd coordinted nd helped to drft the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgments We thnk Dr Clire Wrtel-Weil nd Gerldine Guerin-Peyrou for criticl reding of the mnuscript. This work ws supported y OSEO Innovtion nd Conseil Régionl d Alsce. Author detils 1 Polyplus-trnsfection SA, Bioprc, BP 918, Boulevrd Séstien Brnt, 6741 Illkirch, Frnce. 2 Current ddress: IBMC, 15 rue René Descrtes, 6784 Strsourg, Frnce. 3 IGBMC, 1 rue Lurent Fries, 674 Illkirch, Frnce. 4 Current ddress: Quintiles, rue Jen Dominique Cssini, BP 5137, 6744 Illkirch Cedex, Frnce. Received: 26 Ferury 213 Accepted: 1 July 213 Pulished: 9 July 213 References 1. Nguyen DX, Bos PD, Mssgue J: Metstsis: from dissemintion to orgn-specific coloniztion. Nt Rev Cncer 29, 9(4): Zheng Y, Fernndo HC: Surgicl nd nonresectionl therpies for pulmonry metstsis. The Surg Clin North Am 21, 9(5): Soengs MS, Lowe SW: Apoptosis nd melnom chemoresistnce. Oncogene 23, 22(2): Amrosini G, Adid C, Altieri DC: A novel nti-poptosis gene,, expressed in cncer nd lymphom. Nt Med 1997, 3(8): Grossmn D, McNiff JM, Li F, Altieri DC: Expression nd trgeting of the poptosis inhiitor,, in humn melnom. 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