The FGF-2-Derived Peptide FREG Inhibits Melanoma Growth In Vitro and In Vivo

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1 originl rticle The Americn Society of Gene & Cell Therpy The FGF-2-Derived Peptide Inhiits Melnom Growth In Vitro nd In Vivo Mri S Aguzzi 1, Deor Frone 1, Dniel D Arcngelo 1, Frncesco De Mrchis 1, Griele Toiett 1, Domenico Ritti 2, Alerto Przzoli 3, Polo Colomo 3, Murizio C Cpogrossi 1 nd Antonio Fcchino 1 1 Lortorio Ptologi Vscolre, Istituto Dermoptico dell Immcolt, IDI-IRCSS, Rom, Itly; 2 Diprtimento Antomi Umn, Bri, Itly; 3 Acceler s.r.l., Miln, Itly Previous dt report tht firolst growth fctor-2 (FGF-2)-derived peptide potently inhiits FGF-2- dependent ngiogenesis in vitro nd in vivo. Here, we show tht inhiits up to 7% in vitro growth nd invsion/migrtion of smooth muscle nd melnom cells. Such inhiition is medited y pltelet-derived growth fctor-receptor- (PDGF-R); in fct, prolifertion nd migrtion were restored upon PDGF-R neutrliztion. Further experiments demonstrted tht intercts with PDGF-R oth in vitro nd in vivo nd stimultes its phosphoryltion. We hve previously shown tht over expressing PDGF-R strongly inhiits melnom growth in vivo; we, therefore, hypothesized tht PDGF-R gonists my represent novel tool to inhiit melnom growth in vivo. To support this hypothesis, ws inoculted intrvenously (i.v.) in mouse melnom model nd mrkedly inhiited pulmonry metstses formtion. Immunohistochemicl nlyses showed less prolifertion, less ngiogenesis, nd more poptosis in metstsized lungs upon tretment, s compred to untreted controls. Finlly, in preliminry cute toxicity studies, showed no toxicity signs in helthy nimls, nd neither microscopic nor mcroscopic toxicity t the liver, kidney, nd lungs level. Altogether, these dt indicte tht systemic tretment strongly inhiits melnom metstses development nd indicte for the first time tht gonists of PDGF-R my control melnom oth in vitro nd in vivo. Received 2 April 21; ccepted 2 Septemer 21; pulished online 5 Octoer 21. doi:1.138/mt INTRODUCTION Melnom incidence is incresing worldwide. 1 Humn mlignnt melnom is highly metsttic nd is resistnt to conventionl chemo- nd rdiotherpies; prevention nd erly dignosis re currently the most effective strtegies ginst this tumor, s melnom risk correltes with complex mix of fctors including skin pigmenttion phenotype, sunlight exposure, nd genetic predisposition. Although primry melnom is effectively treted in most cses y surgicl excision, prognosis ecomes unfvorle in the metsttic phse, lowering survivl to 2 4% t 1 yers, in ptients with cliniclly pprent regionl lymph node metstses. 1,2 Severl fctors control melnom growth, including ngiogenic fctors such s firolst growth fctor (FGF-2) nd pltelet-derived growth fctors (PDGFs). PDGF fmily comprises dimeric growth fctors expressed s five different isoforms, nmely PDGF-AA, BB, CC, DD, nd AB. PDGFs re known to regulte severl functions including ngiogenesis, 3,4 tissue development nd remodeling, 5 stem cells recruitment, 6 mesenchyml cells prolifertion, nd vsculr wll homeostsis. 7 9 PDGFs ind PDGF receptor α α, β β, nd α β with distinct specificity; nmely PDGF-BB inds PDGF-R α α, β β, nd α β; PDGF-DD intercts with PDGF-R β β nd α β; PDGF-CC nd PDGF-AB ind PDGF-Rα α nd α β; more interestingly for this study, PDGF-AA hs pure PDGF-Rα-medited effect nd intercts with the receptor α α only. 5 FGF-2 elongs to lrge fmily of growth fctors; it controls prolifertion/differentition of different cell types nd tissues 1 12 nd is involved in mediting tumor growth, Prkinson s nd Alzheimer s diseses, 17,18 nd vsculr diseses such s therosclerosis nd restenosis. 19,2 Severl evidence currently indicte growth fctors nd their receptors s specific trgets for the novel pproches to the cncer tretment, with prticulr emphsis on PDGF nd FGF fmily. 21,22 We hve previously shown tht PDGF-BB nd FGF-2 reciproclly inhiit their in vitro nd in vivo ctions, most likely vi their high-ffinity direct interction, which my lter the inding to the corresponding receptors In the presence of the respective lignds (PDGF-BB nd FGF-2), PDGF-receptor-α nd FGFreceptor-1 interct on endothelil nd melnom cells, 26 leding to mrked ntiprolifertion effect. Such dt strongly support the hypothesis tht heterodimeriztion of lignds my drive heterodimeriztion of the corresponding receptors, giving rise to n endogenous mechnism controlling cell prolifertion nd ngiogenesis. Severl oservtions presented y us nd others indicte tht PDGF-AA nd its unique receptor PDGF-Rα my exert ntiprolifertion nd ntimigrtion effects on specific cell types such s smooth muscle nd endothelil cells. 23,24,27 29 More recently, we demonstrted tht overexpressing PDGF-Rα mrkedly reduced melnom cell prolifertion oth in vitro nd in vivo, ccompnied y strong ngiogenesis inhiition, 4 suggesting for the fist time Correspondence: Antonio Fcchino, Istituto Dermoptico dell Immcolt, IDI-IRCCS, Lortorio di Ptologi Vscolre, Vi Monti di Cret 14, 167 Rom, Itly. E-mil:.fcchino@idi.it or ntoniofcchino@yhoo.it vol. 19 no. 2, fe. 211

2 The Americn Society of Gene & Cell Therpy FGF-2-Derived Peptide Inhiits Melnom Growth Cell numer % versus ctrl FCS ng/ml SCR ng/ml Cell numer % versus ctrl PDGF-BB ng/ml SCR ng/ml c Cell numer PDGF-BB PDGF-BB SCR PDGF-BB Time (hours) Figure 1 inhiits PDGF- induced cell prolifertion. () FCS-induced RASMC prolifertion (48 hours) ws exmined in the presence or in the sence of incresing doses of or the negtive control scrmled peptide (SCR). reduced cell prolifertion in dose-dependent wy reching the plteu t 1 ng/ml, while the SCR ws ineffective t ll tested doses (P <.5 versus FCS). () reduced in dose-dependent wy PDGF-BB (1 ng/ml)-induced cell prolifertion, reching the plteu t 1 ng/ml. The SCR ws ineffective t ll tested doses (P <.5 versus PDGF-BB). (c) (1 ng/ml) reduced PDGF-BB mitogenic ctivity on RASMC in time-dependent wy, while SCR (1 ng/ml) ws ineffective (P <.5 versus PDGF-BB). Dt re expressed s men ± SD of three experiments. ctrl, control; PDGF, pltelet-derived growth fctor. tht low expression levels of PDGF-Rα my medite melnom progression, while high levels of PDGF-Rα induce significnt ntimelnom growth effect. According to such oservtion, inhiiting FGF-2 nd ctivting PDGF-Rα my exert potent controlling effect on endothelil nd melnom growth. An FGF-2-derived peptide nmed hs een previously reported s potent FGF-2 inhiitor, cting s decoy of the whole FGF-2 molecule. 3 Such peptide ws shown to ind FGF-2 nd to interfere with the whole FGF-2 ctivtion process, nmely FGF-2 dimeriztion, heprin inding, receptor inding, receptor phosphoryltion, nd FGF-2 cell internliztion, leding to strong inhiition of ll tested in vitro nd in vivo ctions of FGF-2. peptide hs een shown to mimic most of the FGF-2 ctions; we thus hypothesized tht such peptide my ctivte PDGF-Rαmedited ction similr to the prent whole FGF In this study, is shown to strongly inhiit melnom growth, oth in vitro nd in vivo, with mechnism involving PDGF-Rα. RESULTS inhiits cell prolifertion nd migrtion in PDGF-R-medited wy Previous evidence pulished from our lortory showed tht FGF-2 nd inhiit endothelil cells prolifertion vi PDGF-Rα-medited mechnism. 24,3 Such dt gree with the oservtion tht PDGF-AA, pure PDGF-Rα gonist, inhiits PDGF-BB-induced migrtion in smooth muscle cells. 23,28 In this study, peptide mrkedly inhiited rt ortic smooth muscle cell (RASMC) prolifertion induced y PDGF nd y fetl clf serum (FCS) in dose- nd time-dependent wy (Figure 1 c), indicting tht hs strong inhiitory effects, similr to the inhiitory effects shown under similr experimentl conditions y the prent molecule FGF-2 (ref. 23) nd similr to PDGF-AA. 4 The scrmled peptide (SCR) used s specificity control ws ineffective in ll cses., similr to PDGF-AA, 23 lso reduced PDGF-BB-induced RASMC migrtion, while it did not inhiit fironectin-induced migrtion (Figure 2). Such effect ws completely olished in RASMC overexpressing dominnt-negtive form of PDGF-Rα, indicting tht this inhiition is medited y Cells/field % versus PDGF-BB PDGF-BB SCR Cells/field PDGF-BB Cells/field % versus PDGF-BB Fironectin SCR PDGF-Rα (Figure 2). In order to evlute the iologicl relevnce of these results, different cellulr system ws then investigted; the following experiments were crried out in melnom in vitro nd in vivo setting, in order to investigte the role my ply pcdna 3 DN PDGF-R Figure 2 inhiits PDGF-induced cell migrtion in PDGF-Rdependent wy. () RASMC migrtion induced y PDGF-BB (1 ng/ ml) or fironectin (5 μg/ml) ws exmined in the presence of or SCR (1 ng/ml) fter 5 hours exposure t 37 C. PDGF-BB chemotctic ctivity ws mrkedly inhiited y, while SCR ws ineffective (P <.5 versus PDGF-BB), while neither nor SCR ffected fironectin chemottrctnt ctivity. () effect ws investigted in RASMC trnsfected with the empty vector (pcdna 3 ) or with DN-PDGF-R. showed strong ntichemotctic ction in cells trnsfected with pcdna 3, while in cells trnsfected with DN-PDGF-R this effect ws olished (P <.5 versus ctrl). Dt re expressed s men ± SD of three experiments. PDGF-R, pltelet-derived growth fctor-receptor-. Moleculr Therpy vol. 19 no. 2 fe

3 FGF-2-Derived Peptide Inhiits Melnom Growth The Americn Society of Gene & Cell Therpy Cells numer % versus ctrl FCS ng/ml c 1 Cells/field % versus ctrl Cells numer % versus ctrl 6 in pthologiclly relevnt system. strongly inhiited FCSinduced prolifertion nd invsion of humn melnom cells in dose-dependent wy (Figure 3,c, respectively); inctivting the PDGF-Rα y neutrlizing ntiody (.6 μg/ml) significntly olished the ntiprolifertion ction (Figure 3), further confirming tht inhiitory ction is medited y PDGF-Rα. Altogether, these dt indicte tht inhiits PDGF-BB nd FCS mitogenic/chemotctic effects in PDGF-Rα-medited wy, in different cell types. intercts with PDGF-R To further investigte the s mechnism of ction, we investigted whether directly intercts with PDGF-Rα oth in vitro nd in live melnom cells. Humn recominnt PDGF-Rα ws immoilized onto plstic nd iotin-leled ws incuted t incresing doses. Under such conditions, direct sturle nd concentrtion-dependent interction ws oserved (Figure 4). This finding suggested tht my directly ind PDGF-Rα; to confirm such finding in n experimentl set-up closer to live conditions, dditionl experiments were crried out using melnom cells overexpressing PDGF-Rα nd incuted with iotin-leled. Detection ws chieved with Fluorescein Avidin D y fluorescence microscopy. Figure 4 reports interction with untrnsfected cells (wild type), with pcdna 3 empty vector- or PDGF-Rα-trnsfected cells. inding is similr to untrnsfected SK-MEL-11 nd to FCS APDGF-R FCS ng/ml Figure 3 strongly inhiits melnom growth in PDGF-Rdependent wy. () SK-MEL-11 prolifertion induced y FCS 1% (48 hours) ws tested in the presence of incresing concentrtions. inhiited melnom cell growth in concentrtion-dependent mnner (P <.5 versus FCS). () In the presence of the ntiody neutrlizing PDGF-R (.6 μg/ml), the ntimitogenic ction of (1 ng/ml) ws significntly reverted (P <.5 versus FCS; P <.5 versus FCS in the presence of ). (c) FCS-induced melnom cell invsion through collgen IV (1 μg/ml) ws inhiited y in dosedependent wy (P <.5 versus FCS). Dt re expressed s men ± SD of three independent experiments. ctrl, control; FCS, fetl clf serum; PDGF-R, pltelet-derived growth fctor-receptor-. the control pcdna 3 -trnsfected cells (Figure 4 pnels B nd D, respectively), while inding is strongly incresed in PDGF-Rαtrnsfected SK-MEL-11 (Figure 4, pnel F). Such dt indicte tht inds in vitro PDGF-Rα immoilized onto plstic nd strongly increses its inding to cells overexpressing PDGF-Rα, s compred to control cells. induces PDGF-R phosphoryltion The ove reported results strongly support the hypothesis tht peptide my ct s PDGF-Rα gonist. Endogenous PDGF-Rα phosphoryltion ws then monitored in the presence of lone or in the presence of PDGF-AA, used s specific PDGF-Rα lignd. 5 Cells were exposed to PDGF-AA (1 ng/ml) or to (1, 1, nd 1 ng/ml) for 5 minutes. Under these conditions, induced endogenous PDGF-Rα phosphoryltion with rte comprle to PDGF-AA, i.e., the physiologic lignd of PDGF-Rα (Figure 4c). Altogether, the ove reported dt indicte s strong inhiitor of melnom cell prolifertion nd invsion, with mechnism involving (i) PDGF-Rα inding, (ii) PDGF-Rα phosphoryltion, nd (iii) the presence of functionl PDGF-Rα. Preliminry dt collected from four melnom cell lines (nmely mouse B16F1, humn SK-MEL-11, humn SK-MEL-28, nd humn MEWO) suggest tht increses its ntimitogenic effect s function of PDGF-Rα expression (Figure 4d). Such dt gree with previous evidence showing tht PDGF-Rα overexpression strongly inhiits melnom growth in vitro nd in vivo nd, likely s consequence of tht, melnom progression my select cells with reduced expression of PDGF-Rα; in fct PDGF-Rα hs een found to e significntly downregulted in humn melnom iopsies. 4 Such evidence suggest tht PDGF-Rα gonists nd my represent novel pproches to the melnom tretment. We then tested in n in vivo model of murine melnom growth. In vivo lung metstsis ssy Dt presented ove indicte tht inhiits in vitro humn melnom cell prolifertion vi PDGF-Rα. Therefore in vivo ntimelnom ctivity ws tested in murine model; mouse B16F1 melnom cells were shown to express in vitro PDGF-Rα y either western lotting nlysis (Figure 5) or rel time-pcr (RT-PCR; Supplementry Figure S1) s well s in vivo (Figure 6d); therefore B16F1 cells were injected intrvenously (i.v.) in 2 C57BL/6 mice to develop melnom lung metstses. Six dys fter cell inocultion, i.v. inocultion ws initited in 1 nimls ccording to the schedule reported in Mterils nd Methods section, while 1 mice were shm treted. At 15 dys fter cell inocultion (corresponding to 9 dys fter the initition of tretment) mice were killed nd lung metstses were counted. i.v. tretment reduced y >5% the numer of mcroscopic lung metstses t 3 s well s t 6 mg/kg/dy dose (Figure 5,c). Anlysis of vrince, followed y Dunnett s multiple comprison test s post hoc nlysis, indicted tht the effect induced y 6 mg/kg/dy dose is significnt (P <.5) nd the post hoc test for liner trend indicted tht response is significntly relted to the dose (P <.2). Microscopic histologicl nlyses indicted tht tretment significntly reduced the numer vol. 19 no. 2 fe. 211

4 The Americn Society of Gene & Cell Therpy FGF-2-Derived Peptide Inhiits Melnom Growth 1.4 Binding to PDGF-R 7 g/ml 1.2 Binding to plstic , 1,5 2, Biotinylted mol/l Biotinylted OD 45 Wild type c W.B. ptyr W.B. PDGF-R PDGF-AA (ng/ml) pcdna (ng/ml) 1 d effect Spermn correltion index =.9 PDGF-R PDGF-R expression Figure 4 intercts with PDGF-R. () inding to plstic-immoilized PDGF-R (7 μg/ml) ws mesured in solid-phse ssys. Biotinylted (form to 2 mmol/l) ws incuted for 4 hours onto immoilized-pdgf-r. Unound mteril ws wshed wy nd colorimetric ssy ws performed. Biotinylted ound PDGF-R in sturle nd concentrtion-dependent mnner. () SK-MEL-11 untrnsfected or overexpressing PDGF-R were seeded on glss coverslips nd treted with iotinylted (1 ng/ml) for 1 minutes t 4 C nd stined with Fluorescein Avidin D. Br = 4 μm. Biotinylted clerly ound to PDGF-R-trnsfected cells more thn pcdna 3 -trnsfected cells or other controls. (c) Endogenous PDGF-R phosphoryltion ws evluted in SK-MEL-11 treted for 5 minutes with PDGF-AA (1 ng/ml) or with incresing dose of (1, 1, nd 1 ng/ml). PDGF-AA, used s positive control, induced phosphoryltion of its selective receptor; induced PDGF-R phosphoryltion with rte comprle to PDGF-AA. (d) Correltion etween peptide effect nd PDGF-R expression in four melnom cell lines (nmely: mouse B16F1, humn SK-MEL-11, humn SK-MEL-28, humn MEWO). ws used t 1 ng/ml nd its inhiitory effect on cell prolifertion ws correlted to PDGF-R expression mesured y western lot nlysis. Spermn correltion index ws computed with PRISM 5 softwre for Windows. PDGF-R, pltelet-derived growth fctor-receptor-. of microscopic foci y 33% (P <.2), while size ws reduced y ~5% (Figure 5d,e). Additionlly, immunohistochemicl nlysis reveling Ki-67, ctive cspse-3, nd CD 31 expression levels, chosen s prolifertion, poptosis, nd ngiogenesis mrkers, respectively, showed tht prolifertion is strongly reduced in the lung of treted nimls versus control (25% of positive cells versus 77%). Furthermore, tretment incresed ctive cspse-3 expression versus control (38% positive cells versus 15%), indicting poptosis induction. Finlly, significntly reduced ngiogenesis in treted lungs versus control (8% positive cells versus 3% immunorectivity expression of CD 31 mrker; Figures 6,). Preliminry toxicity studies The in vitro nd in vivo dt reported ove strongly support the hypothesis tht my exert ntimelnom ctivity in vivo. In order to exclude ny specific toxic effect responsile for the oserved effects, preliminry cute toxicity experiments were crried out in vivo; ws dministered in helthy C57BL/6 mice t 6 mg/kg/dy (tretment nd injection schedule were identicl to the highest dose used in the efficcy studies). Totl ody weight of ech mouse ws recorded t dys, 2, 4, 7, 8, nd 9. No significnt differences versus controls were oserved t ny dy (Figure 6c). Upon tretment, nimls showed good generl conditions in ll cses. No tretment-relted chnges such s chronic inflmmtion, extrmedullr hemtopoiesis, incresed mitosis in liver, chronic inflmmtion nd tuulr sophili in kidneys, nd hemorrhge in lungs were oserved microscopiclly in treted nimls s compred to controls, ccording to the nlysis of professionl pthologist evluting slides in lind. Further, dt collected y hemtologicl nd clinicl chemistry nlysis show no sign of toxicity (e.g. no significnt difference ws found in red nd white lood cell content, pltelets, hemogloin, totl protein content, glucose, ure, nd cretinine levels; Supplementry Tles S1 nd S2). According to these dt, we concluded tht the oserved ntimetsttic effect of is unlikely relted to toxic effects. Moleculr Therpy vol. 19 no. 2 fe

5 FGF-2-Derived Peptide Inhiits Melnom Growth The Americn Society of Gene & Cell Therpy PDGF-R Actin Ctrl B16F1 c Metsttic foci (% versus ctrl) (mg/kg/dy) d e 6 Foci numer/slide 4 2 Figure 5 intrvenous (i.v.) tretment inhiited melnom lung metstses in murine melnom model. () In vitro expression of PDGF-R ws investigted in growing B16F1 mouse melnom cell line, y western lotting nlysis. Humn HUVEC cells were used s positive control, ccording to previously reported dt. 26 () B16F1 melnom cells (2 1 5 /2 μl PBS) were injected i.v. into C57BL/6 mice til vein t dy 6. Animls were treted i.v. with 5 μl of (3 or 6 mg/kg/dy) on dys, 2, 4, 7, nd 8. Mcroscopic lung metstses re indicted in representtive lungs. Br = 1 mm. (c) Quntifiction of mcroscopic lung metstses indictes significnt reduction upon tretment. Asterisk () indictes sttisticl significnce (P <.5 versus ctrl) ccording to one-wy nlysis of vrince test followed y Dunnett s multiple comprison test. Liner trend post hoc nlysis indictes significnt dose-relted effect (P <.2). (d) Microscopic metsttic foci upon histologicl nlysis. Representtive imge re reported, indicting representtive foci-size. Br = 25 μm. (e) Quntifiction of numer of microscopic foci per slide indictes significnt reduction upon tretment (P <.5 versus ctrl). ctrl, control; PDGF-R, pltelet-derived growth fctor-receptor-. DISCUSSION In this study, we present dt demonstrting strong ntimelnom effect of, n FGF-2-derived peptide. FGF-2 is one of the most potent mitogenic nd ngiogenic fctors, while its truncted forms exert ntichemotctic nd ntingiogenic ctivity. 31 FGF fmily memers re known to undergo proteolyticl mturtion s physiologicl mechnism to modulte their ctivity 11,32,33 nd FGF-derived frgments re reported to ply specific iologicl ctivities; t lest one FGF-derived frgment hs een recently identified s cncer-relted ntigen 34 nd we hve shown tht FGF-2 loses ctivity nd releses frgments upon thromin-dependent proteolytic digestion. 35 We hve previously reported tht, designed from the region predicted to e t the interfce of the FGF-2/FGF-2 homodimer, strongly ffects in vitro nd in vivo FGF-2 iologicl functions y inhiiting FGF-2 dimeriztion process nd cting s moleculr decoy. 3 More recently, we hve shown tht FGF-2 intercts with PDGF 25 nd tht FGF-R1 heterodimerizes with PDGF-Rα 26 supporting the hypothesis tht FGF-2 nd PDGF, s well s their receptors, my form functionl network structurlly nd functionlly relted. In greement with this scenrio, we hypothesized tht ctivity my e relted to PDGF-Rα. This initil hypothesis ws confirmed y the results depicted in Figures 2 nd 3, showing tht indeed ntimitogenic nd ntichemotctic ctivity is rogted when PDGF-Rα is neutrlized, either y dominnt-negtive molecule or y neutrlizing ntiody, respectively. The mechnism mediting this ction, t lest in prt, is the direct / PDGF-Rα interction nd ws demonstrted y different experimentl pproches, depicted in Figure 4. The ility of to induce PDGF-Rα phosphoryltion, nd therefore to ctivte its downstrem pthwy, definitely proved tht this peptide inhiits smooth muscle cells nd melnom cells prolifertion/migrtion with mechnism medited, t lest in prt, y PDGF-Rα. In vivo experiments crried out in murine metsttic melnom model demonstrted tht systemic inocultion is le to strongly reduce numer nd size of lung metstses s compred to untreted melnom. Doses used were chosen ccording to previous dt indicting tht low moleculr weight peptides with 27 vol. 19 no. 2 fe. 211

6 The Americn Society of Gene & Cell Therpy FGF-2-Derived Peptide Inhiits Melnom Growth Ki-67 Active cspse-3 CD 31 Immunorectivity (% on totol cell numer) c d Body weight (g) Dys Ki-67 Active cspse-3 CD 31 PDGF-R Actin Tumor numer Ctrl ct 2 1 B16F Tumor numer Figure 6 Immunohistochemicl nlyses were crried out on mice injected intrvenously with B16F1 melnom cells nd treted with (6 mg/kg/dy) t dys, 2, 4, 7, nd 8. () The routine hemtoxylin eosin stining nd Ki-67, ctive cspse-3, nd CD 31 leling were performed on prffin-emedded lungs. Br = 25 μm. () Quntifiction of percentge of immunorectivity expression indictes significnt reduction of cell prolifertion (Ki-67 stining), mrked induction of poptosis (cspse-3 ctivtion), nd significnt decrese of ngiogenesis (CD 31 stining) upon tretment (P <.5 versus control). (c) Totl ody weight ws recorded in helthy C57BL/6 mice t dys, 2, 4, 7, 8, nd 9 in mice treted with 6 mg/kg/dy intrvenously. No significnt differences versus control were oserved t ny dy indicting no gross cute toxicity upon tretment. (d) PDGF-R expression ws detected y western lotting (top) nd y quntittive rel-time PCR nlysis (ottom) in 5 mouse primry melnoms (otined y s.c. injection of B16F1 melnom cells in C57BL6 mle mice). The experiments were performed in triplicte; dt re reported s verge nd SD. PDGF-R, pltelet-derived growth fctor-receptor-. short circultion time re quickly metolized in vivo, requiring high doses nd repeted inocultions, to otin cceptle ntimetsttic effects in vivo. 2,36,37 Upon tretment immunohistochemicl nlyses showed strong inhiition of prolifertion, mesured y Ki-67 stining of lung sections, nd mrked ntingiogenic effect, y CD 31 stining in lung sections, s compred to untreted melnom, suggesting tht ntimetsttic ction in vivo my derive from the comined ntingiogenic effect trgeting endothelil cells 24 nd the ntimitogenic effect trgeting melnom cells. Tissue strvtion deriving from reduced ngiogenesis my lso explin, t lest in prt, the oserved strong increse of ctive cspse-3 stining, mrker of incresed poptosis. Finlly, it should e highlighted tht no toxicity signs were evidenced in helthy nimls treted with t the sme dose nd inocultion schedule used in the melnom model, suggesting tht hs no specific toxic ction. Although indicting for the first time tht synthetic PDGF-Rα gonist my exert potent ntimelnom effects, dt presented in this study gree with previous dt pulished y our l nd y others indicting FGF-2 nd PDGF-Rα s negtive modultors of cell prolifertion nd ngiogenesis, under prticulr experimentl conditions. 4,23,24,26 Interestingly PDGF-Rα hs een shown to medite inhiitory effects on vsculr smooth muscle cell migrtion, 23,27 while it cn hve opposing effects on the stroml tissues. 38,39 In conclusion, this study demonstrtes tht PDGF-Rα my represent relevnt trget for novel ntimelnom pproches nd indictes s potent PDGF-Rα gonist with significnt in vitro nd in vivo ntimelnom growth ctivity. MATERIALS AND METHODS Peptides synthesis. peptide hs the sequence DPHIKLQLQAE, corresponding to region of humn FGF-2 (ccession numer P938). (Previously, ws referred to s (ref. 3) ccording to ccession numer XP_55784, currently removed from NCBI protein dtse t scrmled (KHIAQLDEPLQ, here referred to s SCR) ws used s sequence specificity control. Both peptides were custom synthesized y Polypeptide Lortories (Strsourg, Frnce). Peptides purity, ssessed y reversephse high-performnce liquid chromtogrphy nd mss spectrometry, Moleculr Therpy vol. 19 no. 2 fe

7 FGF-2-Derived Peptide Inhiits Melnom Growth The Americn Society of Gene & Cell Therpy ws higher thn 95% in ll cses. Different tches of nd SCR were tested nd gve similr results in ll iologicl ssys. Cell isoltion nd cell cultures. Humn metsttic melnom cells (SK-MEL-11) 4 were cultured t 37 C in 5% CO 2 tmosphere in Dulecco s modified Egle s medium (DMEM; Hyclone, Logn, UT) supplemented with 2 mmol/l l-glutmine (Gico, Invitrogen, Crlsd, CA), 1 IU/ml penicillin streptomycin (Gico, Invitrogen, Crlsd, CA), nd 1% het-inctivted FCS (Hyclone). Primry RASMCs were otined s previously reported 23 nd grown in the sme medium. Three different preprtions were crried out nd the purity, evluted y α-ctin immuno stining, ws consistently higher thn 95%. Proliferting RASMCs ( pssges 3 6) t 7% confluence were used in ll ssys. Migrtion ssys. Migrtion ssys were crried out in modified Boyden chmers (Corning Life Sciences, Lowel, MA) s descried. 4 RASMC or SK-MEL-11 (2 1 5 ) were seeded in the upper portion of the Boyden pprtus, onto 8-μm pores polycronte filters (Corning Life Sciences) previously coted with porcine skin geltin (5 mg/l; Sigm-Aldrich, St Louis, MO) or with murine collgen type IV (1 μg/ml; Becton Dickinson, Bedford, MA). Humn recominnt fctors used s chemottrctnts were PDGF-BB (1 ng/ml; R&D Systems, Minnepolis, MN) or fironectin (5 μg/ml; Gico, Invitrogen, Crlsd, CA) for RASMC, nd FCS 1% for SK-MEL-11. or SCR were dissolved in DMEM.1% ovine serum lumin (BSA; Sigm-Aldrich) nd were dispensed in the upper portion of the Boyden chmer. Assys were then crried out t 37 C in 5% CO 2 for 4 hours; filters were then removed, fixed with solute ethnol, nd stined with Giems (Merck, Drmstdt, Germny). Cells migrted cross the filters were counted t 4 mgnifiction; 1 fields/filter were evluted nd the verge numer of cells/field ws reported. All experiments were performed t lest three times in duplicte. Cell trnsfection with wild-type nd dominnt-negtive PDGF receptor. Plsmid coding for wild-type PDGF-Rα, dominnt-negtive form of PDGF-Rα (DN-PDGF-Rα; generous gifts of C.H. Heldin, Ludwig Institute for Cncer Reserch, Uppsl, Sweden) or pcdna 3 (Gico, Invitrogen) empty vectors were trnsfected in RASMC or in SK-MEL-11 s previously reported. 23 Cells were cotrnsfected with pegfp-n1 (Clontech, Mountin View, CA) reporter vector (molr rtio 3:1). Trnsfection ws crried out with Lipofectmine plus regent (Gico, Invitrogen, Crlsd, CA) diluted in DMEM, for 3 hours t 37 C in 5% CO 2 tmosphere. Medium ws then replced with DMEM 1% FCS nd then cells were used for ssys. Prolifertion ssys. SK-MEL-11 prolifertion ws induced for 48 hours with FCS 1% nd cells were treted with in the presence or in the sence of neutrlizing PDGF-Rα ntiody (.6 μg/ml; R&D Systems). PDGF-BB- or FCS-induced RASMC prolifertion ws ssyed in time course or t 48 hours in dose-dependent experiments s descried 23 in the presence or in the sence of or SCR. Cells were then hrvested nd counted with hemcytometer. All experiments were crried out t lest three times in duplicte. PDGF-R phosphoryltion nd expression. SK-MEL-11 grown to 7% confluence were strved for 24 hours in serum-free DMEM. Five-minute exposure to PDGF-AA (1 ng/ml) or (1, 1, nd 1 ng/ ml) ws then crried out. Cells were then rinsed with ice-cold phosphte-uffered sline (PBS), scrped, nd lysed for 5 minutes with 1% Triton X-1, 1 mmol/l NCl, 1% glycerol, 2 mmol/l HEPES, ph 7.4, 5 mmol/l EDTA, 1 μmol/l N 3 VO 4, 1 mmol/l sodium fluoride, 2 mmol/l phenyl methylsulfonyl fluoride nd proteses inhiitors cocktil (Sigm-Aldrich). Lystes were centrifuged t 14, r.p.m. for 1 minutes nd 5 μg totl proteins were nlyzed y sodium dodecyl sulfte polycrylmide gel electrophoresis. Proteins were electrotrnsferred to nitrocellulose memrne nd locked with.1% Tris PBS nd 5% nonft milk. Ponceu S stining (Sigm-Aldrich) ws performed to verify the equl loding. Western lot nlysis ws crried out y proing the nitrocellulose memrne with the ntiphosphotyrosine ntiody (1 μg/ml, sc-72; Snt Cruz Biotechnology, Alex, CA) overnight nd with nti-pdgf-rα (1:2, sc-431; Snt Cruz Biotechnology), followed y wshing nd incution with horserdish peroxidse conjugted secondry ntiody (Pierce Endogen, Rockford, IL). Bnds were then reveled y ECL detection system (Amershm, Buckinghmshire, UK). All experiments were performed t lest three times. For PDGF-Rα expression studies, western lot nlysis of PDGF-Rα ws crried out onto four melnom cell lines (nmely mouse B16F1, humn SK-MEL-11, humn SK-MEL-28, nd humn MEWO) nd onto five mouse primry melnoms (excised 2 weeks fter sucutneous injection of B16F1 melnom cells in C57BL6 mle mice). PDGF-R interction nlyzed y solid-phse ssy. Biotinylted interction to recominnt PDGF-Rα ws evluted y solid-phse ssy ccording to procedure previously descried 26 with modifictions. Briefly, microtiter pltes (Corning Life Sciences) were coted with recominnt PDGF-Rα (.7 μg; R&D Systems) diluted in AC7.5 uffer (5 mmol/l Tris HCl, ph 7.5, 1 mmol/l KCl, 3 mmol/l MgCl 2, 1 mmol/l CCl 2 ) for 4 hours t room temperture (RT). After locking in 3 mg/ ml BSA (3 μl/well) AC7.5 uffer overnight t 4 C nd wshing three times with AC7.5T/BSA uffer (AC7.5 uffer contining.1% Tween-2 nd.15 mg/ml BSA), wells were incuted for 4 hours t RT with incresing doses of iotinylted. They were then wshed four times with AC7.5T/BSA uffer, incuted for 1 hour t RT with 1 μl/well Vectstin ABC Regent (Vector Lortories, Burlingme, CA) nd stined with the ELISA Amplifiction System (Invitrogen, Crlsd, CA) ccording to mnufcturer s instructions. Asorption t 495 nm (A 495 ) ws determined. Fluorescence microscopy. Wild-type SK-MEL-11, or pcdna 3 empty vector trnsfected cells, or PDGF-Rα-trnsfected cells were mplified on glss coverslips nd treted with iotinylted (1 ng/ml), for 1 minutes t 4 C. Cells were then wshed three times with PBS nd fixed with fresh 2% prformldehyde for 1 minutes t RT. They were then wshed three times with PBS, incuted with PBS BSA 2% for 3 minutes t RT. Binding of iotinylted to the cells ws nlyzed y Fluorescein Avidin D stining (Vector Lortories) for 1 hour t RT. The nlysis ws then crried out on Zeiss Axiopln 2 microscope (r = 4 μm). Quntittive rel-time PCR of PDGF-R mrna expression. Totl RNA (2 mg/smple) extrcted from HUVEC cells, murine B16F1, nd primry mouse firolsts ws prepred using TRIzol (Invitrogen) ccording to the mnufcturer s instructions. mrnas levels were nlyzed using the SYBR- GREEN qpcr method Qigen. Quntittive RT-PCR ws performed on complementry DNAs otined following retrotrnscription of mrnas. Complementry DNAs were used s templtes for Tqmn qrt-pcr with ABI Assys-on Demnd on n ABI Prism 7 sequence detection system (Applied Biosystems, Foster City, CA). Primers for murine nd humn PDGF-Rα were designed with the Primer Express softwre, version 2. (Applied Biosystems, Foster City, CA). Glycerldehyde-3- phosphte dehydrogense ws mplified on the sme plte for ech smple for normliztion purposes. In vivo melnom growth: lung metstsis ssy. According to n ccepted niml study protocol, murine B16F1 melnom cells (2 1 5 /2 μl PBS) were injected i.v. t dy 6 into 12-week-old mle C57BL/6 mice (1 mice/group) for experimentl metstsis studies, ccording to procedure previously descried. 41 Mice were treted with i.v. inocultions of 5 μl of (two doses, 3 or 6 mg/kg/dy) on dys, 2, 4, 7, nd 8. Mice were killed 9 dys fter strting tretment nd mcroscopic lung metstses were counted t surgicl microscope ( 2). The routine hemtoxylin eosin stining s well s Ki-67 (Acm, Cmridge, MA), ctive cspse-3 (Acm), nd CD 31 (Serotec, Oxford, UK) leling were performed on prffin-emedded lungs vol. 19 no. 2 fe. 211

8 The Americn Society of Gene & Cell Therpy FGF-2-Derived Peptide Inhiits Melnom Growth In vivo toxicity studies. Preliminry cute toxicity studies were crried out on totl of 12 helthy nimls, y injecting i.v. t 6 mg/kg/dy (tretment nd injection schedule were identicl to the highest dose used in the efficcy studies) in helthy C57BL/6 mle mice, in volume of 1 ml/ kg. The control group received vehicle lone (.5% wt/vol dextrose solution). All nimls in the toxicity study were killed on dy 9 s in the efficcy study. Mortlity, ehvior, nd generl conditions were monitored dily. Body weight ws recorded on dys, 2, 4, 7, 8, nd 9. Hemtologicl nd clinicl chemistry exmintions were performed on dy 9 from dominl ort lood. Postmortem mcroscopic nd microscopic exmintion were performed on dy 9. For the microscopic exmintion, liver, kidneys, nd lungs collected t necropsy nd fixed in 1% uffered formlin were trimmed nd emedded in prffin. Five-μm histologicl sections were stined with hemtoxylin nd erythrosine nd exmined y ord-certified pthologist with Zeiss Axioskop 2 plus microscope (Crl Zeiss, Chester, VA). Sttisticl nlysis. All in vitro nd in vivo efficcy experiments were performed t lest three times in duplictes. Student s t-test nd one-wy nlysis of vrince test (followed y Dunnett s multiple comprison test s post hoc nlysis nd y the test for liner trend to investigte the role of the dose response) were crried out with PRISM softwre (Grph Pd Softwre, L Joll, CA); P <.5 ws considered to e significnt. SUPPLEMENTARY MATERIAL Figure S1. Quntittive rel-time polymerse chin rection of PDGF-R mrna expression in B16F1 mouse melnom cell line. Tle S1. Within the cute toxicity studies hemtologicl nlysis ws crried out in helthy mice upon tretment. Non significnt toxicity signs were oserved. Tle S2. Within the cute toxicity studies clinicl-chemistry nlyses wre performed in helthy mice upon tretment. Non significnt toxicity signs were oserved. ACKNOWLEDGMENTS We thnk Bioinformtics/Proteomics Fcility t CNR (Avellino) nd Fcility for Complex Protein Mixture Anlysis t the Diprtimento di Emtologi, Oncologi e Medicin Molecolre, ISS (Rome), Itly, for the support in collecting nd nlyzing dt. The uthors declred no conflict of interest. This study ws supported in prt y grnt from Itlin Ministry of Helth Contrct (RF7 Onc-25/3) nd y Progetto Oncoproteomic Itli USA 527B/2A/5 to A.F. REFERENCES 1. Gre, C, Peris, K, Huschild, A, Sig, P, Middleton, M, Sptz, A et l. (21). Dignosis nd tretment of melnom: Europen consensus-sed interdisciplinry guideline. Eur J Cncer 46: Gndini, S, Ser, F, Cttruzz, MS, Psquini, P, Aeni, D, Boyle, P et l. (25). Met-nlysis of risk fctors for cutneous melnom: I. Common nd typicl nevi. Eur J Cncer 41: Zhng, D, Ouyng, J, Wng, N, Zhng, Y, Bie, J nd Zhng, Y (29). Promotion of PDGF-induced endothelil cell migrtion y phosphorylted VASP depends on PKA nchoring vi AKAP. Mol Cell Biochem 335: Frone, D, Aguzzi, MS, Toiett, G, Fcchino, AM, Fcchino, F, Mgent, A et l. (29). Pltelet-derived growth fctor-receptor strongly inhiits melnom growth in vitro nd in vivo. Neoplsi 11: Hoch, RV nd Sorino, P (23). Roles of PDGF in niml development. Development 13: Long, JS, Yokoym, K, Tigyi, G, Pyne, NJ nd Pyne, S (26). Lipid phosphte phosphtse-1 regultes lysophosphtidic cid- nd pltelet-derived-growth-fctorinduced cell migrtion. Biochem J 394(Pt 2): Heldin, CH nd Westermrk, B (1999). Mechnism of ction nd in vivo role of pltelet-derived growth fctor. Physiol Rev 79: Heldin, CH, Ostmn, A nd Rönnstrnd, L (1998). Signl trnsduction vi plteletderived growth fctor receptors. Biochim Biophys Act 1378: F Andre, J, Gllini, R nd Betsholtz, C (28). Role of pltelet-derived growth fctors in physiology nd medicine. Genes Dev 22: Ornitz, DM nd Itoh, N (21). Firolst growth fctors. Genome Biol 2: REVIEWS Sperinde, GV nd Nugent, MA (2). Mechnisms of firolst growth fctor 2 intrcellulr processing: kinetic nlysis of the role of heprn sulfte proteoglycns. Biochemistry 39: Beenken, A nd Mohmmdi, M (29). The FGF fmily: iology, pthophysiology nd therpy. Nt Rev Drug Discov 8: Brillri, G, Sgdri, C, Fiorelli, V, Smniego, F, Colomini, S, Mnzri, V et l. (1999). The Tt protein of humn immunodeficiency virus type-1 promotes vsculr cell growth nd locomotion y engging the 51 nd v3 integrins nd y moilizing sequestered sic firolst growth fctor. Blood 94: Wng, H, Ruin, M, Fenig, E, DeBlsio, A, Mendelsohn, J, Yhlom, J et l. (1997). Bsic firolst growth fctor cuses growth rrest in MCF-7 humn rest cncer cells while inducing oth mitogenic nd inhiitory G1 events. Cncer Res 57: Chndler, LA, Sosnowski, BA, Greenlees, L, Aukermn, SL, Bird, A nd Pierce, GF (1999). Prevlent expression of firolst growth fctor (FGF) receptors nd FGF2 in humn tumor cell lines. Int J Cncer 81: Crmeliet, P (2). Firolst growth fctor-1 stimultes rnching nd survivl of myocrdil rteries: gol for therpeutic ngiogenesis? Circ Res 87: Jensen, P, Pedersen, EG, Zimmer, J, Widmer, HR nd Meyer, M (28). Functionl effect of FGF2- nd FGF8-expnded ventrl mesencephlic precursor cells in rt model of Prkinson s disese. Brin Res 1218: Bellucci, C, Lilli, C, Broni, T, Prnetti, L, Sori, S, Emilini, C et l. (27). Differences in extrcellulr mtrix production nd sic firolst growth fctor response in skin firolsts from spordic nd fmilil Alzheimer s disese. Mol Med 13: Ngo, S, Muro, K, Imchi, H, Co, WM, Yu, X, Li, J et l. (26). Pltelet derived growth fctor regultes ABCA1 expression in vsculr smooth muscle cells. FEBS Lett 58: Chen, PY, Simons, M nd Friesel, R (29). FRS2 vi firolst growth fctor receptor 1 is required for pltelet-derived growth fctor receptor -medited regultion of vsculr smooth muscle mrker gene expression. J Biol Chem 284: Wng, Z, Kong, D, Li, Y nd Srkr, FH (29). PDGF-D signling: novel trget in cncer therpy. Curr Drug Trgets 1: Schwertfeger, KL (29). Firolst growth fctors in development nd cncer: insights from the mmmry nd prostte glnds. Curr Drug Trgets 1: Fcchino, A, De Mrchis, F, Turchetti, E, Fcchino, F, Guglielmi, M, Denro, A et l. (2). The chemotctic nd mitogenic effects of pltelet-derived growth fctor-bb on rt ort smooth muscle cells re inhiited y sic firolst growth fctor. J Cell Sci 113 (Pt 16): De Mrchis, F, Ritti, D, Gimpietri, C, Lentini, A, Frone, D, Scoccinti, M et l. (22). Pltelet-derived growth fctor inhiits sic firolst growth fctor ngiogenic properties in vitro nd in vivo through its receptor. Blood 99: Russo, K, Rgone, R, Fcchino, AM, Cpogrossi, MC nd Fcchino, A (22). Pltelet-derived growth fctor-bb nd sic firolst growth fctor directly interct in vitro with high ffinity. J Biol Chem 277: Frone, D, Aguzzi, MS, Rgone, G, Russo, K, Cpogrossi, MC nd Fcchino, A (26). Heterodimeriztion of FGF-receptor 1 nd PDGF-receptor-: novel mechnism underlying the inhiitory effect of PDGF-BB on FGF-2 in humn cells. Blood 17: Plumo, R, Getno, C, Antonini, A, Pompilio, G, Brcco, E, Rönnstrnd, L et l. (22). Different effects of high nd low sher stress on pltelet-derived growth fctor isoform relese y endothelil cells: consequences for smooth muscle cell migrtion. Arterioscler Throm Vsc Biol 22: Koym, N, Hrt, CE nd Clowes, AW (1994). Different functions of the plteletderived growth fctor- nd - receptors for the migrtion nd prolifertion of cultured oon smooth muscle cells. Circ Res 75: Heldin, CH (1997). Simultneous induction of stimultory nd inhiitory signls y PDGF. FEBS Lett 41: Fcchino, A, Russo, K, Fcchino, AM, De Mrchis, F, Fcchino, F, Ritti, D et l. (23). Identifiction of novel domin of firolst growth fctor 2 controlling its ngiogenic properties. J Biol Chem 278: Levin, EG, Sikor, L, Ding, L, Ro, SP nd Srirmro, P (24). Suppression of tumor growth nd ngiogenesis in vivo y truncted form of 24-kd firolst growth fctor (FGF)-2. Am J Pthol 164: Meddhi, A, Lemdjr, H, Cruelle, JP, Brritult, D nd Horneeck, W (1995). Inhiition y dextrn derivtives of FGF-2 plsmin-medited degrdtion. Biochimie 77: Grie, TA nd Burgess, WH (2). The mitogenic ctivity of firolst growth fctor-1 correltes with its internliztion nd limited proteolytic processing. J Cell Physiol 184: Hnd, K, Yewdell, JW nd Yng, JC (24). Immune recognition of humn renl cncer ntigen through post-trnsltionl protein splicing. Nture 427: Tott, P, De Cristofro, R, Gimpietri, C, Aguzzi, MS, Frone, D, Cpogrossi, MC et l. (29). Thromin-medited impirment of firolst growth fctor-2 ctivity. FEBS J 276: Strieth, S, Eichhorn, ME, Sutter, A, Jonczyk, A, Berghus, A nd Dellin, M (26). Antingiogenic comintion tumor therpy locking (v)-integrins nd VEGFreceptor-2 increses therpeutic effects in vivo. Int J Cncer 119: Go, YJ, Cheng, JK, Zeng, Q, Xu, ZZ, Decosterd, I, Xu, X et l. (29). Selective inhiition of JNK with peptide inhiitor ttenutes pin hypersensitivity nd tumor growth in mouse skin cncer pin model. Exp Neurol 219: Andererg, C, Li, H, Fredriksson, L, Andre, J, Betsholtz, C, Li, X et l. (29). Prcrine signling y pltelet-derived growth fctor-cc promotes tumor growth y recruitment of cncer-ssocited firolsts. Cncer Res 69: Ogw, Y, Kwmur, T, Furuhshi, M, Tsukmoto, K nd Shimd, S (28). Improving chemotherpeutic drug penetrtion in melnom y imtini mesylte. J Dermtol Sci 51: Aguzzi, MS, Gimpietri, C, De Mrchis, F, Pdul, F, Get, R, Rgone, G et l. (24). RGDS peptide induces cspse 8 nd cspse 9 ctivtion in humn endothelil cells. Blood 13: Ws, H, Cichon, T, Smolrczyk, R, Rudnick, D, Stop, M, Chevlier, C et l. (26). Overexpression of heme oxygense-1 in murine melnom: incresed prolifertion nd viility of tumor cells, decresed survivl of mice. Am J Pthol 169: Moleculr Therpy vol. 19 no. 2 fe

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