1. Introduction. Universidade de São Paulo, São Paulo, Brazil. Correspondence should be addressed to Ana Claudia Latronico;

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1 BioMed Reserh Interntionl, Artile ID , 7 pges Reserh Artile Amplifition of the Insulin-Like Growth Ftor 1 Reeptor Gene Is Rre Event in Adrenoortil Adenorinoms: Serhing for Potentil Mehnisms of Overexpression Tmy Cstro Ribeiro, 1 Alexnder Augusto Jorge, 1,2 Mdson Q. Almeid, 3 Betriz Mrinho de Pul Mrini, 1 Mirin Yumi Nishi, 1 Berenie Bilhrinho Mendon, 1 Mri Cndid Brisson Villres Frgoso, 3 nd An Cludi Ltronio 1 1 Unidde de Endorinologi do Desenvolvimento, Lbortório de Hormônios e Genéti Moleulr LIM42, Brzil 2 Unidde de Endorinologi Genéti/LIM25, Brzil 3 Unidde de Suprrrenl, Disiplin de Endorinologi e Metbologi, Hospitl ds Clínis d Fuldde de Mediin d Universidde de São Pulo, São Pulo, Brzil Correspondene should be ddressed to An Cludi Ltronio; nl@usp.br Reeived 27 Mrh 2014; Aepted 16 June 2014; Published 10 July 2014 Ademi Editor: Mriild Plndi de Mello Copyright 2014 Tmy Cstro Ribeiro et l. This is n open ess rtile distributed under the Cretive Commons Attribution Liense, whih permits unrestrited use, distribution, nd reprodution in ny medium, provided the originl work is properly ited. Context. IGF1R overexpression ppers to be prognosti biomrker of metstti peditri drenoortil tumors. However, the moleulr mehnisms tht re implited in its upregultion remin unknown. Aim. To investigte the potentil mehnisms involved inigf1r overexpression. Ptients nd Methods. Westudied 64 drenoortil tumors.igf1r opy number vrition ws determined in ll ptients using MLPA nd onfirmed using rel time PCR. In subgroup of 32 ptients, utomti sequening ws used to identify IGF1R lleli vrints nd the expression of mirornas involved in IGF1R regultion by rel time PCR. Results. IGF1R mplifition ws deteted in n drenoortil rinom tht ws dignosed in 46-yer-old womn with Cushing s syndrome nd viriliztion. IGF1R overexpression ws demonstrted in this se. In ddition, gene mplifition of other loi ws identified in this drenoortil mlignnt tumor, but no IGF1R opy number vrition ws evidened in the remining ses. Automti sequening reveled three known polymorphisms but they did not orrelte with its expression. Expression of mir- 100, mir-145, mir-375, nd mir-126 did not orrelte with IGF1R expression. Conlusion. We demonstrted mplifition nd overexpression of IGF1R gene in only one drenoortil rinom, suggesting tht these ombined events re unommon. In ddition, IGF1R polymorphisms nd bnorml mirorna expression did not orrelte with IGF1R upregultion in drenoortil tumors. 1. Introdution Adrenoortil tumors re rre endorine mlignnies [1]. However, the inidene of these tumors is remrkbly high in Southern Brzil, where it is estimted to be times greter thn the worldwide inidene [2, 3]. These tumors n our in ll ge groups nd hve been hrterized s hving bimodl ge distribution, with the first pek ourring before 5 yers of ge nd the seond between the fourth nd fifth dedes [3]. The insulin-like growth ftor (IGF) signling system plys n importnt role in the growth nd development of mny tissues, inluding the drenl glnd [4]. Insulin-like growth ftors re mitogens tht regulte ell prolifertion, differentition, nd poptosis by interting with the IGF1 reeptor (IGF1R) [5].Thissystem hs lso been implited in vrious pthophysiologil onditions nd is thought to ply prtiulr role in tumorigenesis [6]. The IGF1R pthwy is importnt in promoting onogeni trnsformtion, growth, nd survivl of ner ells [7], nd IGF1R overexpression hs been demonstrted in mny ners [8, 9]. We previously demonstrted IGF1R overexpression in 65% of hildren nd 13% of dults with drenoortil tumors

2 2 BioMed Reserh Interntionl Tble 1: Clinil hrteristis of 64 ptients with drenoortil tumors. Clinilly benign n =18 Children Clinilly mlignnt n =7 Adenoms n =24 Adults Crinoms n =15 Sex (F : M) 3.5:1 :1 11:1 4:1 Clinil presenttion (n) Cushing Virilizing Mixed Nonfuntioning Feminizing F: femle; M: mle. [10]. Interestingly, this IGF1R upregultion ws preditor of metstses in hildren with drenoortil tumors. Additionlly, seletive IGF1R kinse inhibitor exhibited ntitumor effets in dult nd peditri drenoortil tumor ell lines, suggesting tht IGF1R inhibitors represent promising therpy for humn drenoortil rinom [10]. The IGF1R upregultion ws onfirmed in nother ohort of peditri ptients [11]. The moleulr mehnisms tht led to inresed IGF1R expression in these tumors remin unexplined. In this study, we investigted whether IGF1R gene mplifition nd lleli vritions ould be implited in IGF1R upregultion. In ddition, the expression of four distint mirornas (mirna) tht were involved in IGF1R regultion ws evluted. 2. Ptients The study inluded 64 Brzilin ptients with spordi drenoortil tumors (Tble 1). Written informed onsent, s pproved by the Ethis Committee of the Hospitl of Clinis from the University of São Pulo, São Pulo, SP, Brzil, ws obtined from ll prtiipnts in this study. The onsent ws quired diretly from the subjet if he/she ws n dult or from the prent/gurdin otherwise. Smples of spordi drenoortil tumors were obtined from 25 hildren nd dolesents (18 girls nd 7 boys; 1 to 18 yers of ge) nd 39 dult ptients (34 women nd 5 men; 19 to 73 yers of ge). The dignosis of mlignny in the peditri group ws estblished in 8 of the 25 drenoortil tumors by dvned tumor stge (III or IV) nd/or poor linil outome. Adult drenoortil tumors were lssified ording to the Weiss riteri nd inluded 23 drenoortil denoms (Weiss sore < 3) nd 16 rinoms (Weiss sore 3). IGF1R expression ws previously studied in 45 ptients (24 hildren/dolesents nd 21 dults) using rel time PCR [10], nd the overexpression of this reeptor ws observed in 17 hildren nd 5 dults [10].In32outofthese 45 ptients utomti sequening ws used to identify IGF1R lleli vrints nd the expression of mirornas involved in IGF1R regultion ws determined by rel time PCR. Eight norml drenl glnd orties, whih were obtined from hildren nd dults (rnge of ge: 1 to 72 yers) during renl surgery or utopsies, were used s ontrols. The frgments of drenl orties were properly seleted by n experiened pthologist. 3. Moleulr Anlysis 3.1. DNA Extrtion. Genomi DNA ws extrted from frozen tumor smples using Wizrd Genomi DNA Purifition Kit (Promeg, WI, USA) nd ws stored t 35 C. The onentrtion nd purity of the genomi DNA were mesured, using spetrophotometer t n bsorbne of 260 nd 280 nm Multiplex Ligtion-Dependent Probe Amplifition (MLPA). The IGF1R opy number of the drenoortil tumors smples ws mesured using the SALSA MLPA kit P217IGF1R(MRC-Hollnd,Amsterdm,Netherlnds).This moleulr ssy ws designed to detet deletions/duplitions of one or more exons of the IGF1R (hromosome 15q26) nd IGFBP3 (hromosome 7p13) genes. The P217 IGF1R probe mix ontined 22 speifi probes for IGF1R, 5probesfor IGFBP3, nd 9 ontrol probes. The FGFR4 nd NSD1 genes nlysis ws performed using the SALSA MLPA kit P026-C1 Sotos (MRC-Hollnd, Amsterdm, The Netherlnds), whih ontins two probes for FGFR4 gene nd 24 probes for NSD1 gene (5q35.1). MLPA ws performed with totl of 200 ng of genomi DNA for eh smple, s previously desribed [12]. ThePCRprodutswere submittedtopillry eletrophoresison n ABI Prism 310 Geneti Anlyzer (PE Applied Biosystems, The Perkin-Elmer Corportion, CA, USA), nd the MLPA results were nlyzed using the Genesn 3.7 softwre nd further evluted using the Exel spredsheet softwre (Mirosoft Corportion, Redmond, WA, USA). The tumor smple normlized pek height ws then divided by the verge normlized pek height of norml drenls. Dosge quotient res outside the rnge of 0.70 were onsidered bnorml. In ddition, visul omprison of pek profiles ws performed, three ontrol smples were inluded in eh MLPA experiment, nd ll results were onfirmed by two independent tests SYBR Green Rel Time PCR. SYBR Green rel time PCR for the IGF1R gene (GenBnk ession number

3 BioMed Reserh Interntionl 3 NC for genomi nd NM for mrna sequenes) ws used to onfirm the MLPA results for ll of the drenoortil tumors tht were studied. The ndidte gene opy number ws mesured using n ABI Prism 7000 instrument (Applied Biosystems, Forster City, CA, EUA). The Niemnn-Pik disese type C1 gene (NPC1) (GenBnk ession number NC for genomi nd NM for mrna sequenes) ws used s house-keeping gene, nd speifi forwrd nd reverse primers for IGF1R were used. The PCR ws performed in 25 μl ontining 2.5 μl of DNA (50 ng), 10 pmol of eh primer, nd the power SYBR Green PCR mster mix (Applied Biosystems, Forster, CA). The therml yling onditions onsisted of denturtion for 10 min t 95 C, followed by 40 yles of denturtion for 15 se t 95 C nd nneling/extension for 1 min t 60 C. A yle threshold (CT) vlue in the liner rnge of mplifition ws seleted for eh smple in triplite nd normlized to NPC1. The IGF1R dosge ws determined using the 2 ΔΔCT method [13]. The normlized vlue (ΔCT) for eh tumor smple ws then ompred with the men ΔCT of five norml drenls to generte fold hnge rtio (norml = 1) tht ws multiplied by 2 to obtin the opy number (norml = 2) IGF1R Sequene Anlysis. IGF1R sequene nlysis ws performed in 32 (19 hildren-dolesents nd 13 dults; 19 denoms nd 13 rinoms) of 64 ptients with drenoortil tumors. Among these ptients, 18 (52.6%) exhibited IGF1R overexpression. The entire oding region nd exonintron boundries of IGF1R were mplified from genomi DNA using PCR. The oligonuleotide sequenes nd PCR onditions re vilble upon request. The PCR produts were pretreted with ombintion of shrimp lkline phosphtse nd exonulese I (United Sttes Biohemil Corp., Clevelnd, OH) nd diretly sequened using the BigDye TM termintor yle sequening redy retion kit (PE Applied Biosystems, Foster City, CA) in n ABI Prism 310 utomti sequener Expression of MiroRNAs. Some mirornas n regulte IGF-1R expression. Four mirornas tht were relted to IGF1R regultion were seleted: mir-100, mir-145, mir- 375, nd mir-126 [11, 14 16]. MiroRNA nlysis ws performed in 32 smples of drenoortil tumors nd in the sme tumors tht were submitted to utomti sequening. To mesure mirorna expression, RNA ws extrted nd quntittive rel time PCR ws performed. Singlestrnded DNA ws synthesized from 1 mg of totl RNA using Megplex RT Primers, Humn Pool A v2.1 (PN , Applied Biosystems), nd the TqMn MiroRNA Reverse Trnsription Kit (PN , Applied Biosystems). Twenty-five nnogrms of DNA ws used s templte in 20-μL PCR retion, nd the PCR produts were mplified using speifi primers (TqMn MiroRNA Assys: hs-mir100-id: , hs-mir 145-ID: , hs-mir375- ID: , hs-mir126-id: , Applied Biosystems) nd the Tqmn Universl Mster Mix II, with no UNG (PN , Applied Biosystems). The PCR produts were deteted using the ABI Prism 7000 Sequene Detetion System (Applied Biosystems). RNU 44 (ID: , Applied Biosystems) nd RNU 48 (ID: , Applied Biosystems) were used s house-keeping genes. The reltive expression levels were nlyzed using the 2 ΔΔCT method. A ommeril pool of norml humn drenls RNA ws used s referene smple (Clonteh, Plo Alto, CA) P53 Anlysis. Genomi DNA ws extrted from frozen tumor speimens using QIAmp DNA mini Kit (QIA- GEN, Hilden, Germny). The entire exon 10 ws mplified by PCR using the following introni primers: 5 - GCTGTATAGGTACTTGAAGTGCAG-3 nd 5 -GATGA- GAATGGAATCCTATG-3. The mplifition protool onsisted of denturing t 94 C for 5min, followed by 35 yles onsisting of nneling t 50 Cfor30se,primer extension t 72 Cfor30se,nddenturingt94 Cfor30se. All mplified frgments were exmined on 1% grose gel eletrophoresis. The PCR produts were pretreted with n enzymti ombintion of exonulese I nd shrimp lkline phosphtse (United Stted Biohemil Corp., Clevelnd, OH) nd diretly sequened using the BigDye termintor yle sequening redy retion kit (PE Applied Biosystems, Foster City, CA) in n ABI PRISM 310 utomti sequener (Perkin-Elmer Corp.). 4. Results 4.1. IGF1R Copy Number. IGF1R mplifition ws deteted using MLPA nd onfirmed using rel time PCR in n drenoortil rinom tht ws dignosed in 46-yerold femle ptient with Cushing s syndrome ssoited with linil viriliztion (Figure 1). The tumor s size ws m, nd it ws lssified s ENSAT stge III nd presented Weiss sore of 7 upon histopthologil nlysis. IGF1R mrna levels were 5 times higher in this tumor thn in the norml totl drenl glnd pool. Additionlly, the mplifition of the IGFBP3, FGFR4 (dt not shown), nd NSD1 (dt not shown) genes ws lso deteted in this rinom, but IGF1R mplifition ws not demonstrted in the remining ses with drenoortil tumors IGF1R Sequene Anlysis. Three distint previously desribed exomi polymorphisms were deteted in IGF1R: exon 11 rs (10.5%, genotype frequeny), exon 16 rs (65.8%, genotype frequeny), nd exon 21 rs (7.9%, genotype frequeny). No orreltion between the IGF1R vrints nd their expression ws demonstrted in the drenoortil tumors. In ddition, nother 6 previously known polymorphisms were identified in the introni regions (rs , rs , rs , rs , rs , nd rs ) MiroRNA Expression. Rel time PCR nlysis reveled tht mir-100 (medin 0.3 (from 0.07 to 1.97)) nd 375 (medin 0.05 (from 0.01 to 0.27)) were downregulted in drenoortil tumors. This redution ws observed in benign nd mlignnt tumors nd ws not ssoited with inresed expression of IGF1R. mir-145 (medin 2.2 (from

4 4 BioMed Reserh Interntionl b b b b b b 2 (IA) b b b b b b (IIA) b b b b b b (IB) b b b b b b (IIB) Figure 1: (I) Representtive MLPA pek ptterns of the IGF1R nd IGFBP3 genes. The numbers in the top portion of pnels (IA) nd (IB) represent the frgment size in the pillry eletrophoresis nlysis. (II) Histogrm showing the normlized dt with results of three ontrol individuls nd internl ontrols (referene probes). Normlized reltive vlues, rnging within onfidene intervl of 0.7 to, were estblished by ontrol dt vritions nd orrespond to two gene opies in the genotype. The vlues in y-xis orrespond to the normlized reltive vlue nd the number on the top of eh br represents the normlized vlue for eh probe. (A) Norml drenl glnd. (B) Adrenoortil tumor with IGF1R nd IGFBP3 mplifition. Some referene probes in this tumor re lso mplified. (): twenty-two IGF1R probes.(b): six IGFBP3 probes. (): referene probes.

5 BioMed Reserh Interntionl 5 Expression levels (fold hnge to norml drenl ortex) 10,000 1, mir-145 mir-100 mir-375 mir-126 IGF1R norml expression (n =14) P=ns mir-145 mir-100 mir-375 IGF1R overexpression (n =18) Figure 2: Expression of mirornas 145, 100, 375, nd 126 in drenoortil tumor smples with norml nd inresed IGF1R expression. No differene of mirorna expression ws observed for ll mirornas. (mir-145 P = 0.19, mir-100 P = 0.61, mir-375 P = 0.49, nd mir-126 P = 0.81). 0.01to12.6))hdvribleexpressioninthedrenoortil tumors tht were evluted; however, these vlues were not orrelted with the expression vlues of IGF1R in drenoortil denoms nd rinoms. mir-126 (medin (from 30.8 to )) ws overexpressed in benign nd mlignnt drenoortil tumors nd ws not relted to IGF1R expression (Figure2) Sttistil Anlysis. Sttistil nlysis ws performed using SPSS Softwre (PASW version 19.0; SPSS In., Chigo, IL). Continuous dt re expressed s medin vlues (from minimum to mximum). Differenes in expression levels between two groups were nlyzed by mens of the two-tiled Mnn-Whitney U test. 5. Disussion IGF1R hs potent mitogeni, ntipoptoti, nd trnsforming tivities [17]. Norml growth nd differentition re result of fine blne between the proess of ell prolifertion nd deth, nd the disruption of one or more ftors involved in these tions n result in pthologi phenotypes, inluding mlignny [17]. Indeed, IGF1R is overexpressed in severl primry tumors nd ner-derived ells [18, 19]. It hs been demonstrted tht IGF1R expression is fundmentl prerequisite for ellulr trnsformtion beuse enhned IGF1R levels nd IGF1 signling re onsidered key ftors for the ell to dopt the prolifertive nd onogeni pthwys [20]. Inresed expression of the humn IGF1 reeptor promotes lignd-dependent neoplsti trnsformtion in different ells mir-126 [6, 21]. On the other hnd, the bsene or derese of levels of the IGF1 reeptor prevents mlignnt growth nd trnsformtion [22]. We identified IGF1R overexpression in metstti peditri drenoortil tumors nd found tht it ws prognosti biomrker of dvned tumors in these hildren [10]. In present study, we hypothesized tht IGF1R overexpression in drenoortil tumors ould be used by gene mplifition, lleli vrints, nd dysregulted mirorna expression. In ft, IGF1R mplifition hs been reported in mlignnt melnoms [23], brest ners [24], pnreti denorinoms [25], gstri ell lines [26], rhbdomyosroms [27], Wilms tumors [28],ndgstrointestinl stroml tumors [29]. Moreover, IGF1R mplifition ws ssoited with ellulr trnsformtion nd tumor progression [30]. The IGF1R opy number in 64 drenl tumor smples ws nlyzed using MLPA in this study. Amplifition of IGF1R ws deteted in only one drenoortil rinom (Weisssore7)thtwsdignosedinwomnwhohdn endorine hyperfuntion tht ws hrterized by hyperortisolism nd hyperndrogenism. This ptient ws treted with mitotnendshehdfvorbleevolutionovertheprevious 10 yers. As expeted, the drenoortil tumor with IGF1R mplifition dignosed in this ptient hd higher IGF1R mrna levels ompred to norml drenl smples. MLPA nlysis lso demonstrted multiplegene mplifition, suggesting potentilneuploidy. IGF1R gene mplifition ws not demonstrted in the remining drenoortil tumors, whih suggested tht other mehnisms ould be implited in IGF1R upregultion. Additionlly only the previously known exomi nd introni polymorphisms were observed in tumors with or without IGF1R overexpression. Stimultory nd inhibitory trnsription ftors determine the level of IGF1R expression, nd, onsequently, the prolifertive ell sttus. The IGF1R gene is ontrolled by number of tumor suppressors, inluding p53, whih is the most frequently mutted moleule in humn ner. P53 ws ble to suppress IGF1R promoter tivity by pproximtely 90%, wheres its mutnt forms were ssoited with IGF1R upregultion [31]. A speifi germintive muttion (p.r337h) tht ffeted the tetrmeriztion domin of p53 hs been identified in high frequeny in Brzilin hildren with drenoortil tumors [32, 33]. This unique p53 defet ws deteted in 22 of the 64 (34.3%) ptients of the Brzilin ohort tht ws studied here (dt not shown). Nevertheless, IGF1R overexpression nd gene mplifition were not ssoited with the presene of this p53 muttion. In ddition, the drenoortil rinom with IGF1R mplifition did not hrbor the p.r337h muttion in blood or tumor tissue DNA (dt not shown). MiroRNA bnormlities re potentil mehnisms tht mybessoitedwith IGF1R overexpression. Clin et l. [34] demonstrted tht more thn 50% of mirorna genes were loted in ner-ssoited genomi regions. In the present work, we demonstrted tht the expression of mir-100 nd 375 ws deresed in benign nd mlignnt drenoortil tumors; however, no orreltion ws found between the seleted mirornas nd IGF1R expression. mir-145 lso did not orrelte with IGF1R expression vlues. Our dt

6 6 BioMed Reserh Interntionl orroborte with those from previous studies tht showed tht mir-100 nd 375 expression ws deresed in drenoortil tumors nd ould be tumor suppressor mirornas [11, 35]. In ddition, Doghmn et l. [11] showedthtmir- 100 regulted the expression of IGF1R nd mtor signling in drenoortil ells by modulting the expression of key proteins involved in these pthwys [11]. mir-375 is lsodownregultedingstrinersndheptoellulr rinoms [15]. Previous studies in esophgus rinom ells showed tht mir-375 hs tumor suppressor effet by inhibiting IGF1R [15]. Shi et l.[14] identified tht IGF1R nd its doking protein, the insulin reeptor substrte-i, re trgets of mir-145. mir-126 ws overexpressed in benign nd mlignnt drenoortil tumors. This mirorna suppresses endothelil migrtion by trgeting set of genes in ner ells tht drive endothelil migrtion, by tivting IGF1R, whih is promoter of endothelil migrtion, nd inhibiting the endothelil MERTK reeptor, whih is suppressor of endothelil migrtion [16]. In onlusion, we deteted onurrent IGF1R gene opy number vrition nd IGF1R overexpression in single funtioning mlignnt drenoortil tumor. This dt suggests tht the IGF1R overexpression, whih is ommon in peditri drenoortil tumors, is not driven by inresed gene opy number. In ddition, IGF1R polymorphisms nd the bnorml expression of the mirornas mir-100, mir- 145, mir-375, nd mir-126 did not orrelte with IGF1R overexpression in these tumors. Conflit of Interests The uthors delre no dulity of finnil interest or diret or indiret onflit of interests on the prt of ny uthor of this pper. Aknowledgments This work ws supported by Fundção de Ampro àpesquis do Estdo de São Pulo (FAPESP) Grnt nos. 2008/ nd 2010/ (to Tmy Cstro Ribeiro) nd Brzilin Grnts from Conselho Nionl de Desenvolvimento Científio e Tenológio (CNPq no /2012 for Alexnder Augusto Jorge nd no / to An Cludi Ltronio). Referenes [1] F. M. Brlskr nd G. D. 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