Sam68 Mediates the Activation of Insulin and Leptin Signalling in Breast Cancer Cells

RESEARCH ARTICLE Sam68 Media es he Ac i a ion o Insulin and Lep in Signalling in B eas Cance Cells An onio Pé ez-Pé ez 1 , Flo a Sánchez-Jiménez 1 , Te esa Vila iño-Ga cía 1 , Luis de la C uz 2 , Juan A. Vi izuela 2 , Víc o Sánchez-Ma gale 1 * 1Depa men o Medical Biochemis y and Molecula Biology and Immunology, UGC Clinical Biochemis y, Vi gen Maca ena Uni e si y Hospi al, Uni e si y o Se ille, Se ille, Spain, 2UGC Clinical Oncology, Vi gen Maca ena Uni e si y Hospi al, Se ille, Spain *[email p o ec ed] Abs ac Obesi y is a well-known isk ac o o b eas cance de elopmen in pos menopausal women. High insulin and lep in le els seem o ha e a ole modula ing he g ow h o hese umou s. Sam68 is an RNA-binding p o ein wi h signalling unc ions ha has been ound o be o e exp essed in b eas cance . Mo eo e , Sam68 may be ec ui ed o insulin and lep- in signalling pa hways, media ing i s e ec s on su i al, g ow h and p oli e a ion in di e - en cellula ypes. We aimed o s udy he exp ession o Sam68 and i s phospho yla ion le el upon insulin and lep in s imula ion, and he ole o Sam68 in he p oli e a i e e ec and signalling pa hways ha a e ac i a ed by insulin o lep in in human b eas adenoca ci- noma cells. In he human b eas adenoca cinoma cell lines MCF7, MDA-MB-231 and BT- 474, Sam68 p o ein quan i y and gene exp ession we e inc eased upon lep in o insulin s imula ion, as i was checked by qPCR and immunoblo . Mo eo e , bo h insulin and lep in s imula ion p omo ed an inc ease in Sam68 y osine phospho yla ion and nega i ely egu- la ed i s RNA binding capaci y. siRNA was used o down egula e Sam68 exp ession, which esul ed in lowe p oli e a i e e ec s o bo h insulin and lep in, as well as a lowe ac i- a ion o MAPK and PI3K pa hways p omo ed by bo h ho mones. These e ec s may be pa ly explained by he dec ease in IRS-1 exp ession by down- egula ion o Sam68. These esul s sugges he pa icipa ion o Sam68 in bo h lep in and insulin ecep o signaling in human b eas cance cells, media ing he ophic e ec s o hese ho mones in p oli e a ion and cellula g ow h. In oduc ion Sam68, also known as KHDRBS1 (KH domain-con aining, RNA-binding, signal- ansduc ion- associa ed 1) is a membe o he signal ansduc ion ac i a o o RNA (STAR) amily o RNA- binding p o eins (RBPs). As o he membe s o his amily, Sam68 con ains a GRP33/Sam68/ GLD1 (GSG o STAR) domain o he RNA binding ac i i y [1,2], and can in e ac wi h bo h PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 1/15 a11111 OPEN ACCESS Ci a ion: Pé ez-Pé ez A, Sánchez-Jiménez F, Vila iño-Ga cía T, de la C uz L, Vi izuela JA, Sánchez-Ma gale V (2016) Sam68 Media es he Ac i a ion o Insulin and Lep in Signalling in B eas Cance Cells. PLoS ONE 11(7): e0158218. doi:10.1371/jou nal.pone.0158218 Edi o : Ming Tan, Uni e si y o Sou h Alabama, UNITED STATES Recei ed: Oc obe 30, 2015 Accep ed: June 13, 2016 Published: July 14, 2016 Copy igh : © 2016 Pé ez-Pé ez e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. Funding: The manusc ip has been unded by he Real Maes anza de Caballe ía de Se illa. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: Víc o Sánchez-Ma gale is an Academic Edi o o PLOS ONE. The au ho s ha e decla ed ha no compe ing in e es s exis . RNA a ge s and o he p o eins. Acco ding o he ole o Sam68 as an RNA binding p o ein, i has been desc ibed ha his p o ein modula es se e al s eps o RNA me abolism [3], such as nuclea expo and cy oplasmic u iliza ion o ansla ion o i al and cellula mRNAs [4,5] and egula ion o al e na i e splicing, whe e Sam68 plays a key ole [6]. In addi ion, his p o ein has been desc ibed as a sca old p o ein ec ui ed in a ious signal ansduc ion pa hways [7,8] linking signalling pa hways and RNA me abolism egula ion. Sam68, which was o iginally iden i ied as he i s speci ic a ge o he S c y osine kinase in mi osis [9,10], binds se e al p o eins con aining S c homology 3 (SH3) and S c homology 2 (SH2) domains h ough p oline- ich sequences and y osine-phospho yla ed esidues, espec- i ely. Sam68 splicing ac i i y, RNA binding abili y and localiza ion a e egula ed by phospho - yla ion and o he pos ansla ional modi ica ions [11–15]. Sam68 has been p e iously implica ed in cell p oli e a ion, g ow h and di e en ia ion p o- cesses h ough di e en mechanisms. In his sense, some s udies ha e shown a ole o Sam68 as a necessa y ac o o cellula cycle p og ession [16,17]. Mo eo e , al e na i e splicing o se - e al p oli e a ion- ela ed genes as bcl-x(L), CD44, SGCE, cen ophilin, and cyclin D1 [12,18– 20] ha e been demons a ed o be egula ed by Sam68. Thus, his p o ein has been shown o be ela ed o cance de elopmen and p og ession [21]. Mos speci ically, i has been shown ha Sam68 is up- egula ed in p os a e cance , whe e i s down- egula ion seems o p omo e he inhibi ion o cell p oli e a ion and sensi iza ion o cells o apop osis induced by chemo he a- peu ic agen s [22]. Mo eo e , Sam68 haploinsu iciency delays mamma y umo onse and mul iplici y as i was shown in PyMT ansgenic mice [23]. Silencing o Sam68 in he b eas cance cell lines ha e also shown inhibi ion o cell p oli e a ion and ancho age-independen g ow h, up- egula ion o cyclin-dependen kinase inhibi o s, inc eased FOXO ansc ip ional ac i i y and deac i a ion o he PI3K/Ak pa hway [24]. The y osine phospho yla ion o Sam68 has also been in ol ed in cance de elopmen o p og ession. Thus, Sam68 Ty -phospho yla ion has been epo ed in ce ain cance s whe e y osine kinases a e induced. Indeed, i has been shown ha Sam68 is a downs eam subs a e o he epi helial g ow h ac o EGF h ough BRK phospho yla ion [25] and i s phospho yla ion is also ele a ed in b eas umou s issues and cell lines [14]. Sam68 phospho yla ion has been epo ed in o he sys ems when hey a e s imula ed by mi ogenic and ophic ho mones such as insulin and lep in, whe e i has been linked o cellula g ow h and p oli e a ion h ough i s pa icipa ion in he main pa hways ac i a ed by hese ho mones. Thus, Sam68 has also been ela ed o he insulin-dependen MAPK and PI3K pa hways ac i a ion, whe e i has been shown o be associa ed o G b2, GAP and PI3K egula o y subuni , exe ing a ole in hese pa hways unde insulin and also lep in s imula ion in di e en cellula sys ems [26–28]. Mo e ecen s udies ha e shown ha Sam68 down- egula ion a oided he comple e lep in-dependen ac i a ion o MAPK and PI3K pa hways in cho ioca cinoma JEG3 cells, also p e en ing lep in- dependen cellula g ow h and p oli e a ion [29]. Insulin and lep in a e sugges ed links be ween obesi y and b eas cance isk, as hey may con ibu e o he malignan ans o ma ion o b eas epi helial cells and cance p og ession [30,31]. In his sense, hey p omo e g ow h and p oli e a ion in b eas cance cell lines [32–34]. Mo eo e , bo h hype insulinemia and hype lep inemia a e common indings in obesi y- ela ed b eas cance [35,36]. In his line, o e exp ession o insulin and lep in ecep o ha e been ound in pos menopausal b eas cance [37–39] and may in luence p ognosis and ea men esponse [40,41]. Acco ding o he p e iously desc ibed pa icipa ion o Sam68 in insulin and lep in signal- ling [8], we aim o s udy he ole o his p o ein in he signal ansduc ion pa hways ha a e ac i a ed by lep in and insulin o media e hei p oli e a i e e ec in b eas cance cells. Sam68 Media es Insulin and Lep in Signaling in B eas Cance Cells PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 2/15 Ma e ials and Me hods 2.1. Cell cul u e and ea men s MCF7, MDA-MB-231 and BT-474 cell lines we e o iginally om Ame ican Tissue Type Cul- u e (ATTC) and hey we e ob ained om D . A. Lopez-Ri as (CABIMER, Se ille, Spain) [42]. Since cell lines we e o iginally ob ained be o e 2010, cell lines we e au hen ica ed using STR analysis by he Ins i u o de In es igaciones Biomédicas (IIBM) “Albe o Sols”CSIC-UAM. MCF7 cells om passage-10 o -15 and MDA-MB-231 and BT-474 cells om passage -10 o -12 we e g own in DMEM medium (In i ogen) supplemen ed wi h 10% e al cal se um (FCS), 100 U/mL penicillin, 100 g/mL s ep omycin a 37°C in 5% CO 2 . Cells we e ea ed wi h di e en concen a ions o lep in and insulin (0–10 nM) du ing 10 min o 16h. The ecombi- nan human lep in was p o ided by Sigma (Sigma Chemical); insulin Ac apid was pu chased om No o No disk. 1 nM dose o bo h ho mones was used o he expe imen s, co esponding o he op imal dose- esponse in physiological concen a ions [43,44]. The cell lisa es we e washed wi h cold PBS and solubilized o 30 min a 4°C in lysis bu e con aining 20 mM T is, pH 8, 1% Nonide P-40, 137 mM NaCl, 1mM MgCl 2 , 1mM CaCl 2 , 10% glyce ol, 1mM phenylme hylsul onyl luo ide, and 0.4 mM sodium o ho anada e. To al p o ein le els we e de e mined by he bicinchoninic acid me hod [45] using bo ine se um albumin as s anda d. 2.2. Wes e n blo ing analysis Lysa es o Sam68 siRNA ans ec ed cells we e washed h ee imes wi h lysis bu e . We added 40 uL o SDS-s op bu e con aining 100mM o DTT o he immunop ecipi a es ollowed by boiling o 5 min. The soluble supe na an s we e hen esol ed by 7–15% SDS-PAGE and elec opho e ically ans e ed on o ni ocellulose memb anes. The memb anes we e blocked wi h bu e ed saline–0.05% Tween 20 (PBST) con aining 3% albumin o 1 h a 23°C. The blo s we e hen incuba ed wi h p ima y an ibody o 1 h (an i-β- ubulin 1:5000 and an i-Sam68 (C- 20) 1:2000 om San a C uz Bio echnology; he an i phospho y osine (4G10) 1:1000 om Millipo e; an i-phosphoMEK1/2 (pS217-pS221) and an i-phosphoERK1/2 (pT202-Y204/ pT185-Y187) 1:1500 om Sigma Ald ich; an i-phosphoAKT (pS473) 1.3000 om BD Biosci- ences, Pha migen; an i-P-RPS6KB1 (p70S6K) (pT389) 1:2000 was om Cell Signalling Tech- nology; an i o al insulin ecep o subs a e-1 (an iIRS-1) 1:2000 was om San a C uz. A e he incuba ion wi h p ima y an ibody, he memb anes we e washed in PBST, and u he incu- ba ed wi h he co esponding seconda y an ibodies using ho se adish pe oxidase-linked an i- abbi /an i-mouse 1:10000 immunoglobulin (GE heal hca e). Bound ho se adish pe oxidase was isualized by a highly sensi i e chemiluminescence sys em (Supe Signal om Pie ce). 2.3. T ans ec ion expe imen s MCF7 cells we e pla ed a a densi y o 2.5×10 5 cells/mL on o six-well dishes con aining 2 mL o DMEM plus 10% FCS. Cells we e incuba ed o 24 h. Medium was eplaced, and ans ec ion o cells was pe o med using siRNA o Sam68 (In eg a ed DNA echnology, Inc). Duplex Sequences: Fo wa d, 5’-CGCAGAACAAAGUUACGAAGGCUAC-3’; e e se, 5’- GUAGCCUUCGUAACUUUGUUCUGCGUA-3’.Typically, 40 pmol o he Sam68 siRNA duplexes o he uni e sal nega i e con ol duplex NC1 we e ans ec ed using 4 uL o Lipo ec- AMINE (Li e Technologies). The medium was eplaced a e 24 h using DMEM wi hou FCS o 24 h mo e. T ans ec ion analyses we e pe o med by duplica e in each o a leas h ee inde- penden expe imen s. Sam68 Media es Insulin and Lep in Signaling in B eas Cance Cells PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 3/15 2.4. Cell p oli e a ion and iabili y assay A comme cial cell p oli e a ion ki based on MTT colo ime ic assay (Roche) was used acco d- ing o he manu ac u e ’s ins uc ions. A e 16 hou s o lep in and insulin s imula ion in he Sam68 siRNA ans ec ed cells, MTT labelling eagen was added o each well ( inal con- cen a ion 0.5 mg/mL). The cells we e incuba ed o 4 hou s and o mazan dye was p oduced only in me abolic ac i e cells. Then, he solubilisa ion solu ion was applied o he samples o e nigh and he abso bance was measu ed a 600 nM and compa ed o he nega i e con ol measu emen . 2.5. RNA ex ac ion and quan i a i e eal- ime-PCR (qRT-PCR) assay Rela i e abundance o Sam68 and IRS-1 mRNA was de e mined by qRT-PCR. To al RNA was ex ac ed om he h ee cell lines cul u es using TRISURE eagen (Chomczynski. 1993). Con- cen a ion and pu i y o he isola ed RNA we e es ima ed spec opho ome ically a 260 and 280 nm. Fo cDNA syn hesis, 5ug o o al RNA was e e se ansc ibed a 55°C du ing 1 h using he T ansc ip o i s S and cDNA syn hesis Ki (Roche). qRT-PCR was pe o med using he ollowing p ime s based on he sequences o he Na ional Cen e o Bio echnology In o ma ion GenBank da abase: cyclophilin: o wa d, 5’-CTTCCCCGATACTTCA-3’; e e se, 5’-TCTTGGTGCTACCTC-3’; Sam68: o wa d, 5’-TTTGTGGGGAAGAT TCTTGG-3’; e e se, 5’-GGGGGTCCAAAGACTTCAAT-3’. IRS-1: o wa d, 5’-ACCATGGGGACAAGCCCGGCG-3’; e e se, 5’-GGGGCTGCTGGTGTTGGAATC- 3’.Quan i a i e RT-PCR Mas e Mix Reagen ki was ob ained om Roche (Fas S a uni- e sal SYBR G een), and PCRs we e pe o med on a Ch omo 4 DNA Engine (Bio-Rad). A ypi- cal eac ion con ained 10 uM o o wa d and e e se p ime , 3ul o cDNA, and he inal eac ion olume was 20 uL. The eac ion was ini ia ed by p ehea ing a 50°C o 2 min, ol- lowed by hea ing a 95°C o 10 min. Subsequen ly, 41 ampli ica ion cycles we e ca ied ou as ollows: dena u a ion 15 s a 95°C and 1 min annealing and ex ension a 58°C. The h eshold cycle (CT) om each well was de e mined by he Op icon Moni o 3 P og am. Rela i e quan i- ica ion was calcula ed using he 2-ΔΔc me hod. Fo he ea ed samples, e alua ion o 2-ΔΔc indica es he old change in gene exp ession, no malized o a housekeeping gene (cyclophilin), and ela i e o he un ea ed con ol. 2.6. Da a analysis Expe imen s we e epea ed sepa a ely a leas h ee imes o assu e ep oducible esul s. Resul s a e exp essed as means ± s anda d de ia ion (SD) in a bi a y uni s (A.U.). A bi a y uni s we e calcula ed as no malized band in ensi y in Wes e n blo analysis. S a is ical analysis was pe o med using he G aph Pad P ism compu e p og am (G aphPad So wa e). S a is ical sig- ni icance was assessed by ANOVA ollowed by di e en pos hoc es s, as indica ed in each ig- u e. A p alue <0.05 was conside ed s a is ically signi ican . Resul s Insulin and lep in up- egula e Sam68 mRNA exp ession and p o ein quan i y in MCF7, MDA-MB-231 and BT-474 adenoca cinoma cells Ac i a ion o signal ecep o s posi i ely modula es he exp ession o di e en p o eins ha pa - icipa e in signal ansduc ion. Thus, we ha e p e iously shown an inc ease o Sam68 exp es- sion in esponse o insulin in CHO-IR cells and adipocy es [46] as well as in cho ioca cinoma JEG3 cells in esponse o lep in [47]. Sam68 Media es Insulin and Lep in Signaling in B eas Cance Cells PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 4/15 In o de o check he e ec o lep in and insulin on Sam68 exp ession in he h ee adenoca - cinoma cell lines, hey we e independen ly incuba ed in he absence o se um wi h and wi hou lep in o insulin (1 nM) o 24 h. We e alua ed mRNA exp ession by means o qRT-PCR, using cyclophilin as an in e nal con ol o eac ion e iciency. Simila expe imen s we e pe - o med o assess he e ec o lep in and insulin on Sam68 p o ein amoun by using Wes e n blo analysis and an ibodies ha speci ically ecognize Sam68. The amoun o o al p o ein in e e y sample was con olled using an i-β- ubulin an ibodies. As i is shown in Fig 1a, lep in and insulin s imula ion o 24 h inc eased Sam68 exp ession abou wice as much as he basal le els in MCF-7 cells. As shown in Fig 1b and 1c, bo h insulin and lep in enhanced he exp es- sion o Sam68 in MDA-MB-231 and BT-474 cells, inc easing app oxima ely 25% ha om con ol wi h s a is ical signi icance o bo h insulin and lep in s imulus e sus con ol. Insulin and lep in ea men s s imula e Sam68 y osine phospho yla ion in adenoca cinoma b eas cells We ha e p e iously implica ed Sam68 as a signalling molecule in he LEPR sys em in human monocy es and lymphocy es [48,49] as well as in cho ioca cinoma JEG3 cells [47]. Mo eo e , Sam68 has been conside ed as an insulin ecep o subs a e in CHO cells and in isola ed a adipocy es, whe e i media es he insulin ac ion [26,46,50,51]. Ty -phospho yla ion o Sam68 has been demons a ed in esponse o insulin and lep in [26,48]. We ha e now es ed his possible implica ion o Sam68 in lep in and insulin signalling in adenoca cinoma cells by s udying Ty -phospho yla ion media ed by hese ho mones. Cells we e s imula ed wi h a physiological 1 nM dose o lep in o insulin, o 10 min. To al ex ac s om con ol and ea ed MCF7, MDA-MB-231 and BT-474 cells we e immunop ecipi a ed wi h C- e minal an i-Sam68 an ibodies and analysed by means o immunoblo ing wi h an i- Fig 1. Lep in and insulin ea men o 24 h inc eases Sam68 exp ession measu ed as Sam68 mRNA quan i ica ion and Sam68 p o ein abundance in b eas cance cell lines. Cells we e ea ed wi h 1 nM lep in dose o 1 nM insulin dose du ing 24 h. To al RNA was ex ac ed and Sam68 mRNA was quan i ied wi h qRT-PCR in independen expe imen s. Cyclophilin was used as in e nal s anda d. Resul s shown a e om a ep esen a i e expe imen and a e exp essed as means ±SD o h ee independen expe imen s *p<0.05 e sus con ol. Cells we e lysed and p o eins we e sepa a ed on SDS-PAGE gels. Sam68 p o ein amoun was de e mined by Wes e n blo analysis. To al amoun o p o ein in each sample was con olled by immunoblo ing he same memb anes wi h an i-β- ubulin an ibodies. Fig 1a, b and c) RT-PCR ela i e exp ession esul s and wes e n blo exp ession analysis o h ee di e en b eas cance cells lines (MCF7, MDA-MB-231 and BT-474 espec i ely). Bands densi ome ies a e shown in a ep esen a i e expe imen o h ee independen ones o each cell line. Densi og ams wi h SD a e shown. *p<0.05 e sus con ol. doi:10.1371/jou nal.pone.0158218.g001 Sam68 Media es Insulin and Lep in Signaling in B eas Cance Cells PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 5/15 phospho y osine an ibodies. As shown in Fig 2a, 2b and 2c, Sam68 was y osine phospho y- la ed when he cells we e incuba ed wi h bo h ho mones, showing simila phospho yla ing e ec in MDA-MB-231 and BT-474 cells, and e en highe han wo old inc ease in MCF7 cells. The amoun o p o ein immunop ecipi a ed in e e y lane was con olled by immunoblo - ing wi h he same immunop ecipi a ing an ibody. Inhibi ion o Sam68 exp ession by Sam68 siRNA p e en s lep in and insulin s imula ion o cellula g ow h in adenoca cinoma MCF7 cells Biological e ec s o lep in and insulin in p omo ing g ow h and p oli e a ion ha e been ex en- si ely epo ed in many di e en cell sys ems, including b eas cance cells [33,34]. Mo eo e , he unc ional ole o Sam68 pa icipa ing in lep in-dependen cellula g ow h and p oli e a- ion has p e iously been sugges ed in ophoblas ic cho ioca cinoma cells [29]. To es he cellula e ec s o Sam68 down- egula ion on he lep in and insulin ophic e ec s, we used MCF7 as he mos esponsi e o ho mone ea men cells. Besides, MCF7 cells ha e been success ully used as a pa hophysiological app oach o insulin and lep in- ela ed b eas cance in p e ious wo ks [33,34]. Cells we e incuba ed in he absence o p esence o bio- logical dose o bo h ho mones (1 nM) o 16 h a e 48 h ea men s wi h Sam68 siRNA o NC- duplexes siRNA as con ol. As shown in Fig 3, when Sam68 exp ession was dec eased wi h siRNA, he e ec s o lep in and insulin ac i a ing he cellula me abolism we e also impai ed. The e ec o Sam68 siRNA alone wi hou ho monal s imulus also esul ed in a dec eased cell su i al and p oli e a ion as compa ed o con ol. We ha e also con i med hese esul s in he o he wo b eas cance cell lines, whe e we ha e ound e y simila esul s. We a e including he e ec o Sam68 down egula ion on bo h lep in- and insulin-s imula ed cell p oli e a ion in MDA-MB-231 and BT-474 cells in S1 Fig. Lowe ing he exp ession o Sam68 p e en s he p oli e a i e e ec o bo h insulin and lep in. Fig 2. Lep in and insulin ea men s o h ee di e en adenoca cinoma cellula lines inc ease Sam68 phospho yla ion. Cells we e incuba ed in he p esence o lep in 1 nM o insulin 1 nM o 10 min, lysed and he soluble cla i ied cell lysa es we e subjec ed o immunop ecipi a ion wi h an i-Sam68 an ibodies. Immunop ecipi a es we e esol ed by SDS-PAGE and Wes e n blo wi h an i-phospho y osine an ibodies. The lysa es we e analyzed by immunoblo using he an i-Sam68 an ibodies o con ol he amoun o p o ein in e e y lane. Fig 2a, 2b and 2c) Immunop ecipi a ion esul s o , espec i ely, MCF7, MDA-MB-231 and BT-474 b eas cance cells. doi:10.1371/jou nal.pone.0158218.g002 Sam68 Media es Insulin and Lep in Signaling in B eas Cance Cells PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 6/15 Sam68 down- egula ion p e en s lep in and insulin ac i a ion o signaling pa hways ha media es hei g ow h e ec s in adenoca cinoma MCF7 cells Lep in and insulin a e well known p omo ing ac o s o cellula g ow h and p oli e a ion h ough phospha idylinosi ol-3 kinase (PI3K) and he mi ogen-ac i a ed p o ein kinase (MAPK) signalling pa hways in di e en biological sys ems. To check he e ec o Sam68 down- egula ion on insulin and lep in-dependen phospho yla ion o he main p o eins o hese signalling pa hways, we used adenoca cinoma MCF7 cells as i was shown o ha e be e esponse o insulin and lep in. Cells we e i s ly ans ec ed using Sam68 siRNA o NC1 nega- i e con ol siRNA duplexes and incuba ed in he absence o p esence o insulin/lep in du ing 10 minu es as p e iously indica ed in Ma e ial and Me hods. Bo h an i-Sam68 and an i-β- ubulin an ibodies we e used as con ol o Sam68 down- egula ion and loading con ol espec- i ely. We measu ed he ac i a ion o MAPK pa hway by employing an ibodies ha speci ically ecognize he phospho yla ed o ms o he kinases MEK1-2 and ERK1-2. As shown in Fig 4, he insulin and lep in-media ed MEK1-2 Se phospho yla ion was signi ican ly educed in cells whe e Sam68 was down- egula ed. A simila e ec was obse ed in he lep in and insulin ac i- a ion o ERK1–2 p o eins by Th /Ty phospho yla ion, which was almos comple ely abol- ished by dec easing he exp ession o Sam68. Fig 3. Lep in and insulin e ec s on cellula p oli e a ion is impai ed in Sam68 down- egula ed adenoca cinoma MCF7 cells. MCF7 cells we e ans ec ed wi h Sam68 o NC1-sc ambled nega i e con ol siRNA duplexes du ing 48 h. Cells we e cul u ed o ano he 16 h in he p esence o absence o 1 nM lep in o insulin. A e ha , he MTT eagen was added as indica ed in Ma e ials and Me hods sec ion. Da a a e exp essed as means ±SD om ou independen expe imen s, *P<0.05 e sus “Con ol”,#P<0.05 e sus he co esponding non s imula ed pai . “Con ol”: nega i e con ol siRNA ans ec ed cells; “Sam68 siRNA”: Sam68 siRNA ans ec ed cells wi hou s imulus, “I”: nega i e duplex siRNA ans ec ed and insulin s imula ed cells; “Sam68 siRNA + I”: Sam68 siRNA ans ec ed and insulin-s imula ed cells; “L”: nega i e con ol siRNA ans ec ed, lep in s imula ed cells; “siRNA + L”: Sam68 siRNA ans ec ed and lep in- s imula ed cells. doi:10.1371/jou nal.pone.0158218.g003 Sam68 Media es Insulin and Lep in Signaling in B eas Cance Cells PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 7/15 We also measu ed he ac i a ion o he cen al kinase o PI3K pa hway, i.e. AKT, s udying he e ec s o Sam68 down- egula ion on he insulin and lep in media ed AKT phospho yla- ion. In addi ion, lep in and also insulin,s imula e P70S6K phospho yla ion downs eam o AKT ac i a ion, o p omo e he s imula ion o p o ein syn hesis. As shown in Fig 4, bo h P70S6K and AKT phospho yla ion by insulin and lep in we e impai ed in cells ha we e ea ed wi h Sam68 siRNA o down- egula e Sam68 exp ession. Simila esul s we e obse ed in he o he b eas cance cell lines employed in he p esen s udy. The insulin and lep in ac i a ion o bo h AKT and ERK signalling pa hways a e p e- en ed by down egula ion o Sam68 exp ession in MDA-MB-231 and BT-474 (S2 Fig). Fig 4. Sam68 down- egula ion by Sam68 siRNA p e en s he lep in and insulin-dependen ac i a ion o PI3K and MAPK pa hways in MCF7 cells. MCF7 cells we e ans ec ed wi h Sam68 o NC1-sc ambled nega i e con ol siRNA duplexes, du ing 24 h p io o s imula ion wi h 1nM insulin o lep in o 10 min. Cells we e lysed and soluble cla i ied cell lysa es we e sepa a ed by SDS–PAGE. A wes e n blo analysis was pe o med by using an i-P-MEK1-2 and an i-P-MAPK1-2 an ibodies o s udy lep in and insulin ac i a ion o he MAPK signaling pa hway. An i-P-AKT and an i-P-P70S6K an ibodies we e also used o s udy he lep in o insulin-dependen ac i a ion o he PI3K pa hway. A wes e n blo analysis was pe o med by using an i- Sam68 an ibodies o con ol Sam68 down- egula ion. Sample p o ein loading was con olled by using an i-β- ubulin an ibodies. We show he co esponding densi ome ic analysis o h ee independen expe imen s as means ±SD, *p<0.05 e sus con ol “0”,#p<0.05 e sus lep in o insulin s imula ed; “0”, nega i e duplex siRNA ans ec ed, non-s imula ed cells; “siRNA”, Sam68 siRNA ans ec ed non-s imula ed cells; “I”, nega i e duplex siRNA ans ec ion and insulin-s imula ed cells; “siRNA+I”, Sam68 siRNA ans ec ed insulin-s imula ed cells; “L”, nega i e duplex siRNA ans ec ed lep in-s imula ed cells; “siRNA+L”, Sam68 siRNA ans ec ed lep in-s imula ed cells. doi:10.1371/jou nal.pone.0158218.g004 Sam68 Media es Insulin and Lep in Signaling in B eas Cance Cells PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 8/15 Since bo h insulin and lep in ac i a e he same signalling pa hways, we wan ed o s udy whe he bo h ho mones migh ha e a syne gis ic e ec on he same signalling pa hway. As shown in Fig 5, subop imal concen a ions (0.1 nM) o bo h lep in and insulin p oduced li le phospho yla ion signal in AKT and ERK signalling pa hways. Howe e , when bo h ho mones we e p esen a 1 nM concen a ion a he same ime a g ea e e ec was obse ed, sugges ing ha insulin and lep in ac syne gis ically in MCF7 cells. Sam68 down- egula ion esul s in a dec ease o mRNA and p o ein abundance o he Insulin Recep o Subs a e-1 in b eas cance cells To connec Sam68 exp ession wi h he mechanis ic e ec ha exe s o e he main pa hways ha media es g ow h and p oli e a ion unde lep in and insulin s imula ion, we nex ocus on he e ec o siRNA Sam68 down- egula ion on IRS-1 basal exp ession in he h ee b eas can- ce cell lines: MCF7, MDA-MB-231 and BT-474 cells. As i was demons a ed using qRT-PCR as well as immunoblo ing analysis wi h an i-IRS-1 an ibodies (Fig 6), down- egula ion o Sam68 in he h ee cell lines, signi ican ly dec eased he exp ession o IRS-1, as compa ed o nega i e con ol siRNA ans ec ed cells. Discussion A unc ion o Sam68 in cance has p e iously been sugges ed [21]. Mo e p ecisely, Sam68 has been in ol ed in b eas cance umo igenesis [52] and cell p oli e a ion, whe e Sam68 up egu- la ion in a la ge coho o high p oli e a ing b eas umou s has also been ound [24]. Since we Fig 5. Lep in and insulin ac syne gis ically ac i a ing PI3K and MAPK pa hways in MCF7 cells. MCF7 cells we e incuba e in he p esence o absence o subop imal concen a ions o insulin o lep in (0.1 nM) alone o simul aneously o 10 min. Cells we e lysed and soluble cla i ied cell lysa es we e sepa a ed by SDS–PAGE. A wes e n blo analysis was pe o med by using an i-P-IRS-1, an i-P-AKT and an i-P-ERK1-2 an ibodies o s udy lep in and insulin ac i a ion o signaling pa hways. A wes e n blo analysis was pe o med by using an i-β- ubulin an ibodies o con ol sample p o ein loading. We show he co esponding densi ome ic analysis o h ee independen expe imen s as means ±SD, *p<0.05 e sus lep in o insulin alone. doi:10.1371/jou nal.pone.0158218.g005 Sam68 Media es Insulin and Lep in Signaling in B eas Cance Cells PLOS ONE | DOI:10.1371/jou nal.pone.0158218 July 14, 2016 9/15