Periostin-binding DNA Aptamer Inhibits Breast Cancer Growth and Metastasis

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1 originl rticle The Americn Society of Gene & Cell Therpy Periostin-inding DNA Aptmer Inhiits Brest Cncer Growth nd Metstsis Yu Jin Lee 1,3, Il Shin Kim 1, Soo-Ah Prk 1, Youndong Kim 3, Jeung Eun Lee 1, Dong-Young Noh 2, Kyong-Ti Kim 3, Sung Ho Ryu 3 nd Pnn-Ghill Suh 1 1 School of Nno-Bioscience & Chemicl Engineering, Ulsn Ntionl Institute of Science nd Technology (UNIST), Ulsn, Repulic of Kore; 2 Deprtment of Surgery, Seoul Ntionl University, College of Medicine, Seoul, Repulic of Kore; 3 Division of Moleculr nd Life Science, Pohng University of Science nd Technology (POSTECH), Pohng, Repulic of Kore Periostin is n extrcellulr mtrix (ECM) protein tht is overexpressed in vriety of humn cncers, nd its functions pper to e linked to tumor growth, metstsis, nd ngiogenesis. Recent clinicl evidence suggests tht errnt periostin expression is correlted with poor outcome in ptients with rest cncer. To identify novel tools to regulte the functionl role of periostin, we generted enzyl-d(u)tp-modified DNA ptmers tht were directed ginst humn periostin (PNDAs) nd chrcterized their functionl roles in rest cncer progression. selectively ound to the FAS-1 domin of periostin with nnomolr ffinity nd disrupted the interction etween periostin nd its cell surfce receptors, α v nd α v integrins. mrkedly ntgonized the periostin-induced dhesion, migrtion, nd invsion of rest cncer cells nd locked the ctivtion of vrious components of the α v nd α v integrin signl trnsduction pthwys. In 4T1 orthotopic mouse model, dministrtion significntly reduced primry tumor growth nd distnt metstsis. Thus, our results demonstrted tht periostin-integrin signling regultes rest cncer progression t multiple levels in tumor cells nd the tumor microenvironment. DNA ptmers trgeting periostin my potentilly e used to inhiit rest cncer progression. Received 28 Septemer 212; ccepted 7 Jnury 213; dvnce online puliction 19 Mrch 213. doi:1.138/mt Introduction The progression from solid to mlignnt tumor involves the sequentil cquisition of numer of genetic ltertions to vriety of cellulr functions, including control of cell prolifertion, survivl, motility, cell cell dhesion, nd interctions with the extrcellulr mtrix (ECM). 1 3 In the tumor microenvironment, osteopontin, tenscin C, nd other ECM proteins contriute to metstsis nd modulte the mintennce nd expnsion of norml or cncer stem cells nd metsttic niches. 4 6 Identifying the specific roles of ECM proteins in the tumor microenvironment nd signling cscdes involved in cell mtrix interctions could result in the development of improved strtegies for the prevention nd tretment of metstses. Periostin, n extrcellulr mtrix protein, is secreted protein tht functions s oth cell ttchment protein nd n utocrine or prcrine fctor tht signls through the cell dhesion molecules α v nd α v integrin. 7,8 Periostin is memer of the fsciclin fmily nd hs n NH 2 -terminl signl peptide sequence, cysteinerich domin, four internl homologous repets nd hydrophilic COOH-terminl domin. 9 Periostin is not only involved in norml physiologicl functions, such s hert development, 1 ut lso functions in pthophysiologicl conditions, such s vsculr disese, 11 wound repir, 12 osteogenesis, 13 nd tumorigenesis. 14 Periostin hs ttrcted incresing ttention ecuse it is frequently overexpressed in vriety of epithelil crcinoms, prticulrly rest cncer, nd it is functionlly implicted in multiple steps of mlignnt progression, including metstsis nd ngiogenesis. 15,16 Clinicl studies hve demonstrted tht periostin overexpression or elevted serum periostin levels re ssocited with incresed metsttic tumor urden nd poor ptient outcomes. 17,18 Periostin hs een reported to ctivte the PI-3K/AKT nd FAK-medited signling pthwys y intercting with integrin receptors, leding to the incresed cell survivl, ngiogenesis, invsion, metstsis, nd importntly, epithelil-mesenchyml trnsition of crcinom cells Therefore, periostin is potentilly promising cndidte for the inhiition of tumor growth nd metstsis. Trgeted therpies hve ecome the primry strtegy for cncer tretment over the pst few yers. 22,23 Nucleic cid sed ptmers comprise n emerging clss of trgeted therpeutic molecules. 24,25 Aptmers re single-strnded DNAs or RNAs tht re designed to ind to proteins with similr or etter ffinity nd specificity thn ntiodies or smll molecule-sed regents. By directly inding to trget proteins through their well-defined, complementry three-dimensionl structures, ptmers cn modulte the ctivities nd functions of trget molecules. Furthermore, ptmers hve numer of potentil dvntges over other therpeutic tools, including incresed stility, ese of genertion nd modifiction nd low immunogenicity nd toxicity. 26 Aptmers trgeting cell surfce proteins hve recently een explored s promising delivery vehicles or dignostic tools for trgeting prticulr disese or tissue in cell-type specific mnner. 27,28 Correspondence: Pnn-Ghill Suh, School of Nno-Bioscience & Chemicl Engineering, Ulsn Ntionl Institute of Science nd Technology (UNIST), Ulsn , Repulic of Kore. E-mil: pgsuh@unist.c.kr 14 vol. 21 no. 5, my 213

2 The Americn Society of Gene & Cell Therpy DNA Aptmer Inhiition of Periostin Tle 1 Sequences nd inding constnts of isolted PNDAs Clone # Sequence B Mx (%) K d PNDA-1 ACGAGYCYGGYCCYYCCYYYGACGACYAYYGYYYGGYAYCGAYCAACAACAA nmol/l PNDA-2 ACGAGYCACACGYYGAYGACYGGAYGGYAGYYAAAGAGGGYGGGGCAACAA nmol/l ACGAGYYGYCGCAYGYGCGGYYCAGYCYGGYCCYYCAGCACCGYACAACAA nmol/l PNDA-4 ACGAGYCYGGYCCYYCCYCCCYAAYYGCYGYYGAGGYAYCGGCYACAACAA 9 1 nmol/l Dissocition constnts (K d ) nd B mx were clculted following curve fitting, s descried in Mterils nd Methods. Y, Benzly-d(U)TP. Core sequence portions of ptmers in itlics. Frction ound (%) PNDA-1 PNDA-2 PNDA-4 Frction ound (%) c Frction ound (%) Periostin (nmol/l) d cold ptmer (nmol/l) -iotin / PN: (nmol/l) SYPRO ruy SYPRO ruy -iotin -iotin IgG hβig-h3 hpn mpn WB: Anti-periostin A WB: Anti-periostin A Figure 1 Chrcteriztion of periostin-pnda interctions. () Binding ffinities were determined using 32 P-leled PNDAs nd vrying hpn concentrtions. () Competition of PNDA inding to hpn. Proteins were incuted with 32 P-leled nd n unleled control ptmer or for 3 minutes, nd the protein:ptmer complex ws precipitted nd quntified. (c) Binding of to hpn, mpn, hβig-h3, nd higg ws ssyed in triplicte using direct inding ssys. A control tht contined no protein ws lso processed vi the sme method, nd the resultnt vlue ws set s the ckground. Binding ssys were performed in triplicte, nd the mens ± SD re shown. (d) Left, untreted 1% serum uffer (lne 1), iotinylted ptmer tretment (lne 2) or tretment (lne 3) were purified on streptvidin eds nd then stined with SYPRO Ruy nd immunolotted. Lne 4, one-tenth of the hpn ws loded s input (I). Right, dose-dependent inding of to periostin. hpn, humn periostin; mpn, mouse periostin; WB, western lot. In the present study, we generted modified DNA ptmer,, tht is cple of inding to periostin with high ffinity nd inhiiting its function. inding locked the interction etween periostin nd its cell surfce receptors, resulting in significntly decresed ctivtion of the α v nd α v integrindependent signl trnsduction pthwys nd potent inhiition of dhesion, migrtion, nd invsion oth in vitro nd in n in vivo mouse model. These results suggest tht DNA-sed molecule trgeting periostin my e vile cndidte for the inhiition of rest cncer growth nd metstsis. Results Isoltion of periostin-specific DNA ptmer, To chieve efficient, high-ffinity ptmer selection, we used chemiclly modified nucleotides tht mimic mino cid side chins, such s enzylminocronyl-du (Benzyl-dU), t the 5 positions. We screened periostin ptmers from pooled lirry of rndomly modified nucleotides using purified humn periostin (hpn). After eight successive rounds of systemtic evolution of lignds y exponentil enrichment (SELEX), we isolted four rndomized sequences tht ound tightly to periostin. To improve the inding ffinity nd specificity of the PNDAs, we reduced the length of the PNDAs from 8 to 5 nucleotides. The K d vlues of the PNDAs were then determined y direct inding ssy. ws the est cndidte ptmer in this selection, with n pprent K d of 1.7 nmol/l (Tle 1 nd Figure 1). The specific interction etween nd periostin ws further nlyzed in competition ssy. hpn ound to, nd incresing concentrtions of unleled proe effectively competed for periostin inding, wheres the unleled, nonspecific competitor ( ptmer) did not lter the inding of Moleculr Therpy vol. 21 no. 5 my

3 DNA Aptmer Inhiition of Periostin The Americn Society of Gene & Cell Therpy periostin to (Figure 1). Furthermore, the specificity of inding to hpn ws confirmed y compring its inding with tht of the homologous mouse periostin (mpn), hβig-h3 (fsciclin-i protein fmily) nd higg1 proteins. Direct inding ssy nlysis reveled stronger ffinity of for hpn (1.7 nmol/l) thn for mpn (7.4 nmol/l) nd hβig-h3 (3 nmol/l), wheres no inding ws detectle for IgG protein (Figure 1c nd Supplementry Figure S4). We next nlyzed the specific in vivo interction etween nd hpn y ffinity purifiction. Periostin in uffer contining 1% serum ws incuted with iotinylted, nd the periostin: complex ws detected y immunolotting with n nti-periostin ntiody nd SYPRO Ruy stining. As expected, specificlly intercted with periostin in the serum uffer, wheres no inding ws detected with the iotinylted control ptmer (Figure 1d; left). Furthermore, the specific inding etween periostin nd incresed in dose-dependent mnner (Figure 1d; right). These results collectively indicte tht the ptmer specificlly recognizes periostin in high-ffinity mnner in oth its purified form nd under physiologicl conditions. Counts Counts c TIRF Bright field T Intensity 4T Intensity Specific competitor (1 ) Counts PN reltive expression (%) Control sirna Specific competitor (1 ) MCF Intensity PN PN sirna #1 sirna # Periostin β-ctin Nonspecific competitor (1 ) inds to cells expressing periostin Due to the potentil conformtionl differences etween purified nd endogenous proteins, ptmers developed ginst purified proteins my not ind endogenous proteins. 29 Therefore, ptmers were ssyed for their ility to ind to 4T1 (periostin-expressing cells) nd MCF7 rest cncer cells (periostin-deficient cells) y flow cytometry. Western lot nlysis confirmed the production of periostin in 4T1 nd MCF7 cell lystes nd medi (Supplementry Figure S1). Cy3- leled ut not the control ptmer tht ound 4T1 cells in dose-dependent mnner (Figure 2; left). However, neither the control ptmer nor the exhiited inding greter thn the ckground inding oserved with the MCF7 negtive control cell line (Figure 2; right). Accordingly, the specific inding of to 4T1 or MDA-MB-231 cells ws rogted y depletion of endogenous periostin with specific smll interfering RNA (Figure 2 nd Supplementry Figure S5). Thus, the ptmer ppers to e cple of recognizing periostin on the cell surfce. To confirm the cell surfce-specific inding of to periostin on living cells, we exmined Cy3-leled inding on the cell surfce using totl internl reflection fluorescence (TIRF) microscope. TIRF microscopy is suitle for oserving cell surfce molecule inding or dynmics (usully <2 µm cn e oserved). 3 After the ddition of Cy3 to the culture medium of 4T1 cells, red fluorescent spots ppered on the cell surfce (Figure 2c; ). Bckground cellulr utofluorescence (oserved efore the ddition of Cy3 ) nd the ckground fluorescence cused y Cy3 in the medium were not detected due to the low excittion depth of TIRF microscopy (dt not shown). The inding of Cy3 to the cell surfce ws completely inhiited y the simultneous ddition of excess unleled (Figure 2c; Specific competitor). However, the unleled control ptmer did not lter periostin inding to on the cell surfce (Figure 2c; Nonspecific competitor). These dt demonstrte the specific inding of Cy3 to the surfce of periostin-expressing cells. locks the inding of periostin to integrins nd inhiits the periostin-induced signling pthwys Periostin is lignd for integrins α V nd α V nd stimultes the downstrem signling molecules tht regulte vrious cellulr Figure 2 specificlly intercts with periostin. () A Cy3-leled control (pink, 1 nmol/l; green, 5 nmol/l) or (lue, 1 nmol/l; ornge, 5 nmol/l) ws incuted with periostin-overexpressing (4T1, left) nd periostin-negtive cells (MCF7, right) nd nlyzed y flow cytometry. A control tht contined no DNA ws lso included (lck). Counts represent the numer of cells counted. () Left, inding of 5 nmol/l Cy3-leled to 4T1 cells following 72-hours trnsfection with specific periostin sirna (PN sirna #1, green) or nonrelted sirna (control sirna, pink). Right, lystes from 4T1 cells following 72-hour trnsfection with PN sirna or control sirna were quntified y quntittive reltime PCR nd western lotting with n nti-periostin ntiody. The lots shown re representtive of t lest three independent experiments, nd nti-β-ctin ntiodies were used s n internl control. P <.1; P <.5 compred with control sirna. (c) The cell surfce inding of to 4T1 cells ws nlyzed y TIRF microscopy. A specific competitor (unleled ) or nonspecific competitor (unleled control ptmer) ws incuted with 1 nmol/l Cy3-leled- for 15 minutes. Imges were cquired fter wshing. All imges were cptured using the sme exposure. Representtive imges from five rndom fields of t lest three independent experiments re shown. Scle r: 2 μm. PN, periostin; TIRF, totl internl reflection fluorescence vol. 21 no. 5 my 213

4 The Americn Society of Gene & Cell Therpy DNA Aptmer Inhiition of Periostin Biotin-/SA eds SP EMI FAS I FAS II FAS III FAS IV FAS I FAS II FAS III FAS IV COOH FL COOH N1 (kd) NT FL N1 N2 N3 N4 N5 WB: Anti-GFP FAS II FAS III FAS IV FAS III FAS IV FAS IV COOH N2 COOH N3 COOH N4 COOH N Totl cell lystes WB: Anti-GFP 48 WB: β-ctin c ptmer (5 nmol/l) (1 nmol/l) (5 nmol/l) Integrin α v Integrin α v d ptmer (5 nmol/l) (1 nmol/l) (5 nmol/l) Integrin α v Integrin α v Periostin: Periostin: p-fak p-fak FAK FAK p-src p-src Src Src β-ctin β-ctin 4T1 MDA-MB-231 Figure 3 inhiits periostin-dependent signling. () Schemtic representtion of the full-length periostin nd deletion constructs used in this study. () Biotinylted ws mixed with cell lystes from 293T cells overexpressing mutnt periostins, nd pull-down ssy ws performed with streptvidin eds, followed y western lotting with the indicted ntiodies. (c) 4T1 nd (d) MDA-MB-231 cells were serum strved, untreted, or treted for 3 hours with, control ptmer or nti-integrin As nd then stimulted for 9 minutes with periostin (1 ng/ml) in the presence or sence of ech ptmer or A. Cell lystes were prepred, nd the ctivted forms of phospho-fak (Tyr397) nd phospho-src were nlyzed y western lotting. The totl frction of FAK nd Src ws used s the experimentl control, nd β-ctin ws used s loding control. The lots shown re representtive of three experiments. WB, western lot. functions. 21 Efficient inhiition of the interction etween periostin nd integrins my inhiit cell invsiveness nd tumor metstsis. We next sought to identify the periostin domins involved in inding. Periostin deletion mutnts (Figure 3) were overexpressed in 293T cells, nd the inding of iotinylted to the mutnt periostin proteins ws detected y ffinity purifiction. Although full-length periostin ws detected in the iotin-:protein complex, inding ws significntly diminished in complexes contining ΔN4 or ΔN5 (Figure 3). Thus, the essentil nd miniml domin tht is required for inding is the third FAS-1 domin, which is criticl for integrin inding. To elucidte the moleculr mechnism underlying the inhiitory effect of on cell surfce inding, we performed western lot nlysis of the constituents of the α V nd α V integrin pthwys in rest cncer cells. We first exmined the phosphoryltion sttus of FAK (Tyr 397) nd Src s mrkers of integrin signling nd cell spreding. 31 The western lot nlysis reveled incresed levels of FAK nd Src phosphoryltion in cells exposed to periostin. or nti-integrin As (α V nd α V ) tretment drsticlly reduced FAK nd Src phosphoryltion levels in 4T1 nd MDA-MB-231 cells, wheres no effects were oserved in the presence of the control ptmer (Figure 3c,d). Furthermore, the dose-dependent inhiition of FAK nd Src phosphoryltion ws correlted with the concentrtion in 4T1 nd MDA-MB-231 cells. Interestingly, nti-α V A-treted cells exhiited decresed FAK nd Src ctivtion, similr to tht oserved in treted cells, nd the nti-α V A prtilly locked (Figures 3c,d). These results suggest tht ctivtion of the FAK/ Src pthwy y periostin is primrily medited y α V integrin Moleculr Therpy vol. 21 no. 5 my

5 DNA Aptmer Inhiition of Periostin The Americn Society of Gene & Cell Therpy Numer of migrted cells c Cell dhesion (% BSA) Numer of invded cells MCF7 4T1 MDA-MB-231 MCF7 4T1 MDA-MB-231 MCF7 4T1 Adhesion Migrtion Invsion MDA-MB-231 BSA PN PN + PN + PN + α V PN + α V BSA PN PN + PN + PN + α V PN + α V BSA PN PN + PN + PN + α V PN + α V Figure 4 Inhiitory effects of on rest cncer cell dhesion, migrtion, nd invsion. () In the dhesion ssy, rest cncer cells were preincuted with, the control ptmer (1 nmol/l) or nti-integrin As (5 μg/ml) nd then dded to periostin-coted 96-well pltes. Adherent cells were quntified in microplte reder t 57 nm. BSA-treted cells were used s negtive control (set s 1%). () The effect of on the migrtion of rest cncer cells ws nlyzed using trnswell migrtion ssy in the presence of PNDA, control ptmer or nti-integrin As for 3 hours towrd periostin, which ws used s migrtion inducer. (c) Cell invsion through Mtrigel towrd periostin ws performed in the presence of, control ptmer or nti-integrin As for 24 hours. (,c) The cells tht migrted or invded were stined with Hoechst nd counted with n inverted microscope (1 ojective). The vlues represent the mens ± SD of five independent experiments. P <.1; P <.5 compred with the BSA control. BSA, ovine serum lumin; PN, periostin. in rest cncer cells nd tht the inding of to periostin results in the inhiition of downstrem integrin signl trnsduction. Periostin inding to integrins lso ctivted the PI3-K/AKT survivl pthwy. 32 Therefore, we exmined whether could interfere with AKT ctivtion vi periostin inhiition. AKT phosphoryltion ws not stimulted y exogenous periostin, nd tretment hd no pprecile effect on the levels of phospho-akt (Supplementry Figure S3). These dt estlish tht my lock cell dhesion/migrtion/invsion-relted signling pthwys vi the FAK/Src pthwy. inhiits periostin-medited rest cncer cell dhesion, migrtion, nd invsion in vitro We investigted the effects of periostin nd on the metsttic potentil of severl rest cncer cell lines using in vitro dhesion, migrtion, nd Mtrigel invsion ssys (See Supplementry Mterils nd Methods). As cell dhesion molecule, periostin efficiently induced the dhesion of the rest cncer cells compred with the negtive control (ovine serum lumin). In the presence of, dhesion ws reduced y 6% (MDA-MB-231 nd 4T1) nd 45% (MCF7), wheres the nonspecific control ptmer hd no effect (Figure 4). As shown in Figure 4, treting cells with reduces MCF7, 4T1, nd MDA-MB-231 cell migrtion stimulted y periostin (etween 4 nd 67% s compred with the control ptmer, P <.1). The specific invsion towrd periostin ws 1.8-fold (MCF7), 2.3-fold (4T1), nd 3.3-fold (MDA-MB-231) greter thn tht towrd ovine serum lumin, tht is significntly suppressed y tretment with (etween 4 nd 65% s compred with the control ptmer, P <.5) (Figure 4c). In greement with the effects of on the periostin-induced signling pthwy, the nti-integrin α V neutrlizing A prtilly locked the metsttic potentil of rest cncer cells, wheres tretment with the nti-integrin α V A decresed the metsttic potentil more significntly (Figure 4 c). Finlly, we exmined the cytotoxicity ssocited with tretment in rest cncer cells. As ssessed y MTT ssy, did not cuse ny visile morphologicl chnges or reductions in cell viility in the three different rest cncer cell lines used in this study (Supplementry Figure S2). Collectively, these results suggest tht cn effectively nd significntly inhiit the in vitro correltes of rest cncer cell dhesion, migrtion, nd invsion vi α V integrin-fak/src ctivtion. inhiits tumor growth nd metstsis The efficcy of ws exmined in in vivo growth nd metstsis models. We used xenogrft model of 4T1 cell implnttion into the mmmry ft pds of femle BALB/c mice. The control ptmer,, or vehicle ws intrtumorlly injected into primry tumors every 2 dys. The primry tumor volume ws significntly decresed in the tretment group y 58% t dy 16 reltive to the control groups (P <.1) (Figure 5). There ws no weight loss in the ll mice groups, suggesting tht the ptmer ws not toxic during tretment (Figure 5). On dy 2 following injection, necropsy tissues reveled similr tumor distriution in ll mice implnted with rest tumor cells, including n extensive tumor mss nd distnt metstses in the lymph nodes, liver, spleen, nd lung. Strikingly, severl nodules nd lrger metsttic foci ( 2 mm) were oserved in the lung tissue of the vehicle- nd control ptmer-treted groups, wheres tretment significntly reduced the nodules nd metsttic foci (P <.5; Figure 5c,d; Tle 2). Blood vessel mrker (CD31) nd Ki-67 stining were performed to determine whether 18 vol. 21 no. 5 my 213

6 The Americn Society of Gene & Cell Therpy Averge weight (g: men ± SEM) Tumor volume (mm3) DNA Aptmer Inhiition of Periostin Dy fter injection c Numer of nodules d Dy fter injection Lung (2 ) Lung (4 ) Figure 5 inhiits rest cncer growth nd metstsis in vivo. () inhiited tumor growth in syngeneic rest cncer model employing periostin-positive 4T1 cells (58% t dy 16 compred with or the control group); P <.1. Dy mrks the first dy of injection. The dt re expressed s the mens ± SD (n = 1 tumors). () Men ody weights of the nimls. (c) Necropsy ws performed on ll of the mice, nd representtive imges of treted nd untreted mice re shown. White rrows indicte lung nodules (left). The r grphs demonstrte the numer of lung nodules. (n = 1; P <.5) (right). (d) Representtive lung tissue sections stined with hemtoxylin nd eosin demonstrte metsttic foci in the vehicle-, control ptmer-, nd treted groups. The lck rrows nd circles denote metsttic foci in the lung. Scle rs: 2 μm (top) nd 1 μm (ottom). inhiits tumor growth y suppressing the occurrence of tumor-ssocited cells.21,33 Interestingly, tumors from the treted group presented decresed numer of proliferting Ki-67 positive cells compred with the vehicle- or control ptmer-treted groups (Figure 6,). Moreover, tretment of tumors with resulted in strongly reduced microvsculr density (Figure 6,c). Accordingly, periostin levels were lower in the primry tumor regions of xenogrfts treted with thn those treted with the control ptmer or vehicle (Figure 6). Tken together, these dt suggest tht suppresses tumor progression nd metstsis in n orthotopic Moleculr Therpy vol. 21 no. 5 my 213 loction in n immunocompetent host nd my t lest prtilly reduce the growth of cncer or tumor-ssocited cells, such s endothelil cells, y inhiiting ngiogenesis. We thus confirmed whether the inding specificity of for periostin-expressing cells is preserved in vivo following intrvenous dministrtion of the ptmer (See Supplementry Mterils nd Methods). We treted the mice s previously descried with single intrvenous injection of 5 µg/kg Cy3-leled or control ptmer. After killing the nimls t the indicted times, the tumors were excised, frozen, nd sectioned for confocl microscopic nlysis nd fluorescence quntifiction. In tissues otined 19

7 DNA Aptmer Inhiition of Periostin The Americn Society of Gene & Cell Therpy Tle 2 Summry of nlysis for metstses nd primry tumor size in rest cncer mouse model using BALB/C mice Mice with metstses Numer 1/1 9/1 3/1 Lung 1/1 9/1 2/1 Liver 2/1 /1 /1 Spleen 5/1 3/1 1/1 Lymph nodes 8/1 7/1 2/1 Primry tumor size, mm 3 Men (95% CI) Compred with vehicle, tretment with not only significntly reduced tumor urden (P <.2), ut lso decresed tumor lung metstses. CI, confidence intervl. Ki-67 positive cell (%) PN CD31 Ki-67 H&E c Figure 6 In vivo effects of on tumor growth nd ngiogenesis. () Representtive imges of primry tumor sections from the vehicle-, control ptmer-, nd treted groups, which were stined s indicted. (,c) Quntifiction of the Ki-67 () nd CD31 (c) stining of proliferting nd endothelil cells, respectively. Percent vlues represent the mens ± SD of 1 rndomly selected fields. P <.5. The originl imges were digitlly cptured. Scle r: 1 μm. H&E, hemtoxylin nd eosin. 3 minutes fter injection of, intense tumor stining ws consistent with ccumultion in the perivsculr spce (dt not shown), wheres no stining ws oserved in the control groups. Within 6 hours, the ptmer ws no longer confined to the site of CD31 positive re (%) its initil inding nd hd diffused throughout the tumor strom (Supplementry Figure S7). To quntittively study ptmer distriution, we mesured the fluorescence intensity of the hrvested tissues. After 3 minutes, the liver nd kidney were predominntly oserved fluorescence intensity, reflecting the two mjor clernce pthwys. At 24 hours, the fluorescent signl hd lmost entirely clered the ody, nd the tumor ws the rightest structure visulized until 72 hours (Supplementry Figure S7). Tken together, these results indicte tht recognize periostin expressed in tumor microenvironment nd hmper periostin-dependent tumor progression. Discussion In the present study, we identified nd chrcterized, novel DNA ptmer tht is cple of inding to nd inhiiting periostin. We demonstrted tht inds periostin in its secreted form on the tumor cell surfce with high ffinity (K d, of 1.7 nmol/l). locked periostin-induced signling y inhiiting the interction etween periostin nd integrins nd suppressed rest cncer growth nd metstsis. High levels of periostin expression hve een oserved in mny types of cncer, including rest cncer, nd hve een suggested to enhnce metstsis, ngiogenesis, nd dvnced mlignncies (one metstsis). 34 Recently, periostin hs ttrcted ttention s ridge etween cncer stem cells nd their metsttic niches. 35 In ddition, periostin promotes the incorportion of tenscin C into the ECM nd orgnizes the meshwork rchitecture of the ECM. 36 Therefore, these results provide strong support for the development of periostin inhiitors for use s nticncer therpies. Severl As directed ginst periostin hve een proposed for use s nticncer drugs. Notly, As trgeting periostin ffected in vitro cncer cell migrtion, invsion, nd dhesion nd hd ntiprolifertive nd ntimetsttic effects in vivo. 37,38 However, the low ffinity (K d in the μmol/l rnge) nd unfvorle phrmcokinetics (e.g., poor tumor penetrtion nd rpid clernce) of these As limit their potentil ppliction in trgeted delivery, thus highlighting the need for high-ffinity molecule with improved phrmcokinetics nd serum stility. 39,4 By contrst, DNA ptmers hve mjor dvntge over other therpeutics ecuse they exhiit high inding specificities nd ffinities while mintining low moleculr weight of ~1 kd, re esy to modify for in vivo use nd hve etter tumor tissue penetrtion Becuse ntive oligonucleotides re susceptile to hydrolysis y nucleses, the stiliztion of the phosphte ckone hs een mjor priority for iomedicl pplictions. Although modifiction with neutrl groups (i.e., methyl phosphonte or phosphormidte), incresed the resistnce to nucleses, these nlogs demonstrted lower inding ffinities thn unmodified oligonucleotides. To improve inding ffinities nd reduce the off rtes, we nlyzed modified nucleotides with diverse functionl groups using SELEX. 44,45 Our dt demonstrted tht enzyl-modified exhiited high-ffinity inding to periostin (1.7 nmol/l) nd minimum cross rectivity with other proteins. Despite species differences etween hpn nd mpn, ound to oth proteins, possily due to the sequence nd structurl homology of hpn nd mpn (91%). Therefore, the speciesindependent inding nd specificity of to periostin 11 vol. 21 no. 5 my 213

8 The Americn Society of Gene & Cell Therpy DNA Aptmer Inhiition of Periostin enles thorough chrcteriztion of this lignd in vriety of experimentl niml models. Secreted periostin functions s cell ttchment protein nd cytokine tht signls through the cell dhesion molecules integrins α V, α V, nd α 6 β 4. Periostin inds to integrins, ctivting the recruitment of the epiderml growth fctor receptor nd the AKT/PKB nd FAK-medited signling pthwys, leding to incresed cell survivl, ngiogenesis, nd metstsis. 31,32 Structurlly, βig-h3 nd periostin re secretory proteins with 4 FAS-1 domins, nd stilin-1 nd stilin-2 re memrne proteins with 7 FAS-1 domins. Although the iologicl functions of these proteins re not completely understood, they ll my function to regulte cell mtrix nd cell cell interctions ecuse ll of the FAS-1 domins contin potentil integrin-intercting YH motifs. In this study, we demonstrted tht inds to the third or fourth FAS-1 domin in periostin nd locks the interction etween periostin nd integrins. In ddition, our study lso ddressed the effects of inding to cell surfce periostin, which primrily occurs through α V -medited signl trnsduction in rest cncer cells. In greement with these results, in 4T1 nd MDA-MB-231 cells, which express periostin, strongly reduced periostin-induced FAK/Src phosphoryltion. However, tretment did not ffect AKT phosphoryltion in rest cncer cells. Thus, the ility of to inhiit FAK/ Src ctivtion my e explined y n ptmer-specific inhiition of the periostin-integrin complex. Periostin-integrin inding directly medites tumor cell dhesion, migrtion nd invsion nd susequently enhnces metstsis nd ngiogenesis. In ccordnce with the involvement of periostin in cncer invsiveness, we demonstrted tht strongly inhiits rest cncer cell dhesion, migrtion nd invsion y 5 6%. In xenogrft mouse model, specificlly trgeted periostin-expressing tumor region nd sufficiently locked tumor cell dissemintion to other orgns. We lso demonstrted tht despite its poor effects on in vitro cell growth, efficiently inhiited tumor growth in vivo. This pprent difference my e ttriuted to the function of periostin in tumor microenvironment. Indeed, periostin hs een reported to ply role in the regultion of cncer nd tumor-surrounding cells, such s endothelil cells. Periostin-induced ngiogenesis is medited in prt y upregultion of the vsculr endothelil growth fctor receptor 2 in endothelil cells vi the integrins-fak signling pthwy. To ddress this possiility, we determined the in vitro nd in vivo ntingiogenic effects of. In vitro tue formtion ssy nlysis nd tumor sections from treted mice exhiited reduced ngiogenesis compred with the vehicleor control ptmer treted conditions (Supplementry Figure S6). Thus, my influence not only cncer cell function ut lso endothelil nd other tumor-ssocited cells in the tumor microenvironment. In conclusion, our dt demonstrte for the first time tht the inhiition of periostin with DNA ptmer my significntly inhiit the metstsis nd growth of periostin-positive rest cncers. Furthermore, comined with nnotechnology, 46 represents s new clss of nticncer drugs tht my ecome verstile iomedicl tools trgeting cncers tht overexpress periostin. Mterils nd Methods In vitro selection of periostin DNA ptmers. To select periostin-specific ptmers, modified DNA SELEX procedure ws used s conducted previously. 47,48 Briefly, n ssdna lirry templte consisting of 4-nucleotide rndom region (N4) flnked y two constnt regions ws prepred nd immoilized on streptvidin-coted eds (Pierce, Rocklnd, MA) vi its 5 -OH-end iotin. A primer extension ws then performed using the datp, dctp, dgtp, nd enzyl-dutp nucleotides. The modified DNA lirry ws detched from the templte using high ph conditions nd then incuted with His-tgged periostin (R&D Systems, Minnepolis, MN), prtitioned y Dyneds TALON (Invitrogen, Crlsd, CA) nd mplified y conventionl PCR using 5 -OH terminl iotinylted reverse primer. A primer extension ws then performed, nd n enriched pool ws prepred for the next round. After eight rounds of SELEX, the enriched DNA pool ws cloned nd sequenced ccording to stndrd procedures. After ech round of SELEX, inding ssys were performed to mesure the dissocition constnt (K d ) vlue of the ptmer pool to ensure tht the K d vlue exhiited decresing trend. Binding specificities nd dissocition constnts. The PNDA-1, 2, 3, nd 4 ptmers were ssyed for their ility to ind recominnt periostin (R&D Systems). Some of the 1 nmol/l [α- 32 P]-ATP leled PNDAs (3, Ci/ mmol, 1 mci/ml; Perkin Elmer, Wlthm, MA) were incuted with different recominnt periostin concentrtions (1 nmol/l 1 pmol/l) for 3 minutes t 37 C in selection uffer (4 mmol/l HEPES, ph 7.2, 12 mmol/l NCl, 5 mmol/l mol/lgcl 2, 5 mmol/l KCl). PNDA:protein complexes were incuted with Dyneds TALON (Invitrogen) nd loded onto on vcuum mnifold (Millipore, Milford, MA). Smple wells were wshed three times with 2 μl of selection uffer, nd the nitrocellulose nd nylon filters were dried nd visulized using n FLA5 (GE Helthcre, Pisctwy, NJ). The rw inding dt were corrected for the nonspecific (ckground) inding of the rdioleled DNA to the nitrocellulose filter. Dissocition constnts were clculted y plotting ound PNDA versus the protein concentrtion using the following eqution: Y = B mx X/(K d + X), where B mx is the extrpolted mximl mount of the PNDA:protein complex ound. To determine the inding specificity, 1 nmol/l [α- 32 P]-ATP leled PNDAs were incuted with 1 nmol/l mpn, hpn, hβig-h3, nd higg (Sigm-Aldrich, St Louis, MO) for 3 minutes t 37 C in selection uffer. The inding ssys were performed s descried ove. All proteins were from R&D Systems. Cell culture. The MCF7 nd MDA-MB-231 humn rest cncer cell lines nd 293T humn emryonic kidney cells were otined from the Americn Type Culture Collection (Mnsss, VA). 4T1 cells were kind gift from Dr. Jung Hn Yoon prk t Hllym University in the Repulic of Kore. MCF7 cells were cultured in RPMI 164 (Lonz, Bsel, Switzerlnd) nd supplemented with 1% fetl ovine serum (Gico BRL, Grnd Islnd, NY), penicillin (1 units/ml; Gico), nd streptomycin (1 units/ml; Gico) t 37 C nd 5% CO 2. The MDA-MB-231, 4T1, nd 293T cells were cultured in Dulecco s modified Egle s medium (Lonz) supplemented with 1% fetl ovine serum (Gico) nd ntiiotics (Gico). Cell surfce inding of PNDAs. Approximtely MCF7 nd 4T1 cells were incuted with 1 nmol/l Cy3-leled in 2 μl of selection uffer for 1 hour t 4 C. The cells were then wshed three times with 2 μl of selection uffer nd resuspended in 5 μl of 4% prformldehyde. The smples were nlyzed in the FL3-H chnnel of FACSCliur flow cytometer (BD Biosciences, Sn Jose, CA). The extent of inding to the cell surfce ws ssessed y TIR fluorescence microscopy t 1 mgnifiction (Cell R; Olympus, Tokyo, Jpn) t the UNIST-Olympus Biomed Imging Center (UOBC). For inding competition, cells were incuted with unleled or ptmers for 3 minutes efore incution with Cy3- leled. All imges were cquired under the sme exposure conditions to compre the inding. Moleculr Therpy vol. 21 no. 5 my

9 DNA Aptmer Inhiition of Periostin The Americn Society of Gene & Cell Therpy Anlysis of the inding etween nd periostin domin- deletion vrints. pcdna 3.1 periostin-his constructs were kind gifts from Xio Fn Wng 2 (Duke University, Durhm, NC) nd Dennis J Slmon (University of Cliforni t Los Angeles, Los Angeles, CA). 49 Periostin deletion mutnts were cloned into the pegfp-n1 mmmlin expression vector (Clonetech, Plo Alto, CA) using conventionl PCR methods. The sequences of ll constructs were verified y direct sequencing. To test the inding of with periostin deletion vrints, 293T cells were trnsfected using Lipofectmine 2 (Invitrogen). After 48 hours, the trnsfected cells were wshed with phosphte-uffered sline (Lonz) nd lysed in lysis uffer. Biotinylted (1 nmol/l) ws incuted with lystes from cells overexpressing domin frgment constructs for 2 hours t 4 C. The complexes were collected using streptvidin eds (Pierce) nd wshed four times with cold lysis uffer. Precipittes were detected y immunolotting using nti-periostin (Acm, Cmridge, MA) nd nti-green fluorescent protein ntiodies (Snt Cruz Biotechnology, Snt Cruz, CA). Animl experiments. Six-week-old, femle BALB/c mice were otined from Chrles River Breeding Lortories (Boston, MA) nd housed under specific pthogen-free conditions t the niml fcility of the Pohng University of Science nd Technology. All experimentl niml procedures were pproved y the institutionl niml cre nd use committee of the Pohng University of Science nd Technology. 4T1 cells (1 1 4 ) were suspended in Hnk s lnced slt solution (Gico) nd implnted t the L4 position in mouse mmmry ft pds (1 per group). Thirty nonnecrotic tumors pproximtely.5 cm 3 in dimeter were rndomly divided into three groups of 1 mice s follows: group 1: vehicle; group 2: ptmer-treted; nd group 3: treted. The control nd ptmers (5 μg/kg) were intrtumorlly injected in totl volume of 5 μl three times week for 16 dys. During ech experiment, mice were monitored dily for signs of suffering. Tumors were mesured every 2 dys with clipers, nd the tumor volume ws clculted s follows: V = (1/2) long dimeter short dimeter 2. Growth curves were plotted s the men tumor volume ± SD (n = 1). Histology nd immunohistochemistry. Tumors were emedded in prffin nd sectioned t 5 μm. To inhiit endogenous peroxidses, the sections were treted with.5% H 2 O 2 in 1% methnol for 15 minutes t room temperture. For histologicl exmintion, seril prffin sections were stined with hemtoxylin nd eosin to confirm the dignosis of the tumors. To evlute the prolifertive ctivity of neoplstic cells, the sections stined with n nti-ki-67 ntiody (Dko, Crpinteri, CA). Blood vessels nd periostin expression in primry tumors were detected using the nti-cd31 (BD Phrmingen, Sn Diego, CA) nd nti-periostin (Acm) ntiodies, respectively. Stining ws performed ccording to stndrd protocol. The slides were counterstined with hemtoxylin, dehydrted, nd mounted using synthetic mounting medium (Dko). The slides were oserved nd nlyzed using digitl virtul microscope (dotslide; Olympus) nd MetMorph imge softwre (Moleculr Devices, Sunnyvle, CA). Sttisticl nlysis. All dt re presented s the men ± SD of t lest triplicte smples. Anlyses of differences etween groups were performed using Student s t-test. A P vlue of <.5 or.1 ws considered significnt. SUPPLEMENTARY MATERIAL Figure S1. Periostin expression in rest cncer cell lines. Figure S2. Effect of inding on cell viility. Figure S3. Effect of on cell prolifertion. Figure S4. Binding constnts for. Figure S5. specificlly intercts with periostin. Figure S6. inhiits in vitro tue formtion in HUVECs. Figure S7. In vivo distriution of in mice ering xenogrft tumors derived from rest cncer 4T1 cells. Supplementry Mterils nd Methods. ACKNOWLEDGMENTS We thnk Kyun Heo (Ntionl Cncer Center, Repulic of Kore) nd Mi Jin Kwon (UNIST, Repulic of Kore) for his useful suggestions nd criticl review of the mnuscript. We lso thnk to Jong Hun Im nd Sun-Hk Lee (POSTECH, Repulic of Kore) for technicl ssistnce. This work ws supported y the grnt of the Kore Helth technology R&D Project, Ministry of Helth & Welfre, Repulic of Kore (A8422), nd y the Ntionl Reserch Foundtion of Kore Grnt funded y the Koren Government (No ). The uthors declred no conflict of interest. REFERENCES 1. Friedl, P nd Wolf, K (23). Tumour-cell invsion nd migrtion: diversity nd escpe mechnisms. Nt Rev Cncer 3: Yokot, J (2). Tumor progression nd metstsis. Crcinogenesis 21: Jcks, T nd Weinerg, RA (22). Tking the study of cncer cell survivl to new dimension. Cell 111: Chmers, AF, Groom, AC nd McDonld, IC (22). Dissemintion nd growth of cncer cells in metsttic sites. Nt Rev Cncer 2: Mlnchi, I, Sntmri-Mrtínez, A, Susnto, E, Peng, H, Lehr, HA, Delloye, JF et l. 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