SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells

RESEARCH Open Access SRC inhibi ion p e en s P-cadhe in media ed signaling and unc ion in basal- like b eas cance cells Ana So ia Ribei o 1,2* , Ana Ri a Nob e 1,2,3 , Nuno Mendes 1,2 ,JoãoAlmeida 1,2,3 , And é Filipe Viei a 1,2 , Bá ba a Sousa 1,2 , Filomena A. Ca alho 4 ,JoanaMon ei o 2 ,An ónioPolónia 1,2,5 , Ma ina Fonseca 6 , João Miguel Sanches 6 , Nuno C. San os 4 ,RaquelSe uca 1,2,5 and Joana Pa edes 1,2,5 Abs ac Backg ound: Basal-like b eas cance (BLBC) is a poo p ognosis subg oup o iple-nega i e ca cinomas ha s ill lack speci ic a ge he apies and accu a e bioma ke s o ea men selec ion. P-cadhe in is equen ly o e exp essed in hese umo s, p omo ing cell in asion, s em cell ac i i y and umo igenesis by he ac i a ion o S c-Family kinase (SRC) signaling. The e o e, ou aim was o e alua e i he ea men o BLBC cells wi h dasa inib, he FDA app o ed SRC inhibi o , would impac on P-cadhe in induced umo agg essi e beha io . Me hods: P-cadhe in and SRC exp ession was e alua ed in a se ies o in asi e B eas Cance and con ingency ables and chi-squa e es s we e pe o med. Cell-cell adhesion measu emen s we e pe o med by A omic Fo ce Mic oscopy, whe e equency his og ams and Gaussian cu es we e applied. 2D and 3D cell mig a ion and in asion, p o eases sec e ion and sel - enew po en ial we e e alua ed in i o. S uden ’s - es s we e used o de e mine s a is ically signi ican di e ences. The cadhe in/ca enin complex in e ac ions we e e alua ed by in si u p oximi y-liga ion assay, and s a is ically signi ican esul s we e de e mined by using Mann-Whi ney es wi h a Bon e oni co ec ion. In i o xenog a mouse models we e used o e alua e he impac o dasa inib on umo g ow h and su i al. ANOVA es was used o e alua e he di e ences in umo size, conside ing a con idence in e al o 95%. Su i al cu es we e es ima ed by he Kaplan-Meie ’s me hod, using he log- ank es o assess signi ican di e ences o mice o e all su i al. Resul s: Ou da a demons a ed ha P-cadhe in o e exp ession is signi ican ly associa ed wi h SRC ac i a ion in b eas cance cells, which was also alida ed in a la ge se ies o p ima y umo samples. SRC ac i i y supp ession wi h dasa inib signi ican ly p e en ed he in i o unc ional e ec s o P-cadhe in o e exp essing cells, as well as hei in i o umo igenic and me as a ic abili y, by inc easing mice o e all su i al. Mechanis ically, SRC inhibi ion a ec s P-cadhe in downs eam signaling, escues he E-cadhe in/p120-ca enin complex o he cell memb ane, eco e ing cell-cell adhesion unc ion. Conclusions: In conclusion ou indings show ha a ge ing P-cadhe in/SRC signaling and unc ional ac i i y may open no el he apeu ic oppo uni ies o highly agg essi e and poo p ognos ic basal-like b eas cance . Keywo ds: P-cadhe in, Dasa inib, Basal-like b eas cance , S c amily kinase * Co espondence: [email p o ec ed] 1 Epi helial In e ac ions in Cance (EPIC), i3S - Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Rua Al edo Allen 208, 4200-135 Po o, Po ugal 2 Ipa imup, Ins i u e o Molecula Pa hology and Immunology o he Uni e si y o Po o, Po o, Po ugal Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2018 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 h ps://doi.o g/10.1186/s12964-018-0286-2 Backg ound Basal-like b eas cance is an in insic molecula sub ype (BLBC) [1,2] associa ed wi h an agg essi e biological beha iou and o a wo se pa ien p ognosis, which ypic- ally does no exp ess ho monal ecep o s and HER2, cons i u ing he as majo i y o iple-nega i e b eas ca cinomas [3,4]. Un il now, a ge ed he apies a e un- a ailable o hese subg oup o b eas cance pa ien s, mainly due o he absence o well-de ined molecula a - ge s, being chemo and adio he apy he only he apeu ic op ions o ea hese pa ien s. Consequen ly, i is u gen o ind speci ic bioma ke s/pa hways ha could impac he e ec i e ea men o hese poo p ognosis b eas ca cinomas o accu a e p edic i e molecula indica o s ha could be used o selec pa ien s o a ge he apies al eady a ailable. In he las yea s, ou esea ch eam has been mainly ocused in iden i ying key molecules ha could be used as pu a i e molecula a ge s in BLBC [5,6]. We and o he s ha e ound ha he majo i y o hese agg essi e b eas ca cinomas exp ess he basal ma ke P-cadhe in (a cell-cell adhesion molecule), which o e exp ession is signi ican ly associa ed o a wo se disease- ee and o e all pa ien su i al [5,7,8], as well as wi h a p o-in asi e and s em-like cell beha iou [9–14]. Mo e- o e , we demons a ed ha P-cadhe in inhibi ion dec eases in i o cell in asion and sensi izes b eas cance cells o adia ion, as well as dec eases in i o umo igenic abili y, being indica ed as a pu a i e a ge o BLBC ea men [14]. We ha e also shown ha P-cadhe in-induced unc ional e ec s a e due o he sec e ion o ac i e p o eoly ic enzymes, such as MMPs, and o p o-in asi e soluble clea ed- o ms o P-cadhe in [9], as well as o he ac i a ion o α6β4 in eg in [13] and o he inhibi ion o he E-cadhe in supp essi e in asi e unc ion, by he dis up ion o he E-cadhe in/p120c n complex a he cell memb ane [11]. I is well known ha bo h cy oplasmic p120c n and α6β4 in eg in ac i a ion can induce he phospho yla ion o S c Family Kinases (SRC) and ocal adhesion kinase (FAK), as well as he ac- i a ion o Rac1 small GTPase [15–17]. The e a e se e al lines o e idence showing ha y osine phospho yla ion o hese enzymes may play a ole in he dis up ion o cell-cell adhesion, due o loss o cadhe in/ca enin associ- a ion, wi h a huge impac in cance p og ession. Impo - an ly, we ha e desc ibed ha P-cadhe in exp ession induces SRC ac i a ion in b eas cance cells, sugges - ing ha SRC signaling is being ups eam egula ed by his cell-cell adhesion molecule [13,18]. Thus, in o de o e alua e he ole o P-cadhe in/SRC signaling ac i a ion in BLBC cells, we ha e used dasa inib (an FDA app o ed SRC inhibi o ) o block his pa hway and p e en P-cadhe in induced agg essi e beha io o BLBC cells. Me hods Cell cul u e and siRNA ans ec ions Human b eas cance cell (BCC) lines we e ob ained as ollows: MCF-7/AZ was p o ided by P o . Ma c Ma eel (Ghen Uni e si y, Belgium); SUM-149 was kindly p o- ided by D . S ephen E hie (Uni e si y o Michigan, USA); BT20 and MDA-MB-468 we e ob ained om Ame ican Type Cul u e Collec ion (Manassas, VA, USA). The MCF-7/AZ cell line is a a ian o he human BCC line MCF-7 ha exp esses basal le els o P-cadhe in. This cell line was e o i ally ansduced o encode only EGFP (LZRS-IRES-EGFP plasmid, MCF-7/ AZ.Mock cell line) o bo h P-cadhe in and EGFP (LZRS-P-cad-IRES-EGFP plasmid, MCF-7/AZ.P-cad cell line), as p e iously desc ibed [19]. MCF-7/AZ.Mock cell line was used as a con ol. Cells we e ou inely main ained a 37 °C, 5% CO 2 ,in he ollowing media (In i ogen L d., Paisley, UK): 50% DMEM + 50% HamF12 (MCF-7/AZ, SUM149) and DMEM (BT20, MDA-MB-468), supplemen ed wi h 10% hea -inac i a ed e al bo ine se um (FBS), 100 IU/ml penicillin and 100 mg/ml s ep omycin (In i ogen, UK). SUM149 medium was supplemen ed wi h 5 μg/ml o insulin and 1 μg/ml o hyd oco isone (Sigma-Ald ich, S . Louis, MO, USA). T ans ec ions o alida ed small in e e ing RNA (siRNA) speci ic o P-cadhe in (Hs_CDH3_6, GW Vali- da ed siRNA, Qiagen, Camb idge, MA, USA) and SRC (Hs_SRC_1 FlexiTube and Hs_SRC_11 FlexiTube siRNA, Qiagen) was ca ied ou using Lipo ec amine 2000 (In i ogen), in a inal concen a ion o 50 nM (CDH3 and SRC1) and 100 nM (SRC11), acco ding o he manu ac u e ’s ecommended p ocedu es. A e incuba ion o 5 min, he siRNA and Lipo ec amine 2000 solu ions we e mixed, incuba ed o 20 min, and added o cell cul u e medium. A siRNA con ol, wi h no homology o any gene, was also used (Qiagen), o e i y he speci ici y o he esul s ob ained. D ugs and an ibodies Dasa inib was ob ained om Sequoia Resea ch P oduc s (Pangbou ne, UK), dissol ed in DMSO, and cells we e ea ed a a inal concen a ion o 100 nM each 24 h o a o al pe iod o 48 h. PP2 (P0042, Sigma-Ald ich) was dissol ed in DMSO and added o he cells a a inal concen a ion o 1 and 10 μg/ml. Monoclonal an ibodies we e ob ained as ollows: E-cadhe in (WB) (Taka a Bio Inc., Shiga, Japan); P-cadhe in (WB) and p120c n (BD T ansduc ion Labo a o ies, Palo Al o, CA, USA); E-cadhe in (PLA, IF, IHQ), P-cadhe in (IF), o al S c (L4A1) and pSRC(Ty 416) (Cell Signaling Technology); β-ac in (San a C uz Bio echnologies, San a C uz, CA, USA). The de ailed condi ions o each an ibody a e desc ibed on Addi ional ile 1:TableS1. Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 Page 2 o 16 Cell-cell adhesion measu emen s by a omic o ce mic oscopy Cells we e cul u ed in a issue cul u e dish a a low cell densi y concen a ion o ha e only dispe sed cells, wi h- ou eaching cell con luence. On he day o he expe i- men , cells we e washed wice wi h PBS and 1 mL o se um- ee DMEM was added. Fo he cell-cell adhesion expe imen s, ipless a ow TL1 can ile e s (Nanowo ld, Neucha el, Swi ze land) we e used, wi h a nominal sp ing cons an o 0.03 N/m. Can ile e s we e cleaned o 15 min wi h UV ligh and coa ed wi h poly-D-lysine (50 μg/ml) o a leas 30 min. Can ile e s we e le in he poly-D-lysine solu ion un il used. Isola ed cell suspensions we e ob ained a e 1 h o incuba ion in PBS (wi hou ypsin ea men ) o con lu- en cells in a sepa a e dish and subsequen pipe e dispe sion. The cells and he unc ionalized can ile e s we e moun ed on he CellHesion module (JPK Ins u- men s, Be lin, Ge many), wi h a 100-μm z- ange piezo- elec ic scanne , connec ed o he NanoWiza d II a omic o ce misc oscope moun ed on he op o an Axio e 200 in e ed mic oscope. 100 μL o cells suspension we e injec ed in o he cells subs a e dish. Cells we e allowed o se le o 30 s be o e cap u ing by a unc ion- alized can ile e o one cell using he AFM con ac mode. A e 30 s o can ile e p essing on o he cell, he can ile e was aised 100 μm on he z- ange and he a ached cell allowed o es o 1 min be o e ini ia ing he con ac wi h an adhe en cell on he subs a e unde nea h. Cell-cell con ac was es ablished wi h an applied o ce o 300 pN, in cons an heigh and closed-loop mode. The AFM ip esonan equency was main ained a 2 Hz and a cell-cell con ac ime o 5 s was main ained be o e can ile e e ac ion, wi h a z- ange displacemen o 50 μm). Fi e o ce-dis ance cu es we e pe o med on each cell on he subs a e, wi h a 5 s pause be ween hem. A maximum o 8 di e - en adhe en cells on he subs a e we e es ed wi h he same cell a ached o he AFM can ile e . Wes e n blo P o ein lysa es we e p epa ed om cul u ed cells in ca- enin lysis bu e [1% ( / ) T i on X-100 and 1% ( / ) NP-40 (Sigma) in desionized PBS] wi h 1:7 p o eases inhibi o s cock ail (Roche Diagnos ics GmbH). Cells we e washed wice in PBS and we e allowed o lyse in 500 μl o ca enin lysis bu e o 10 min, a 4 °C. Lysa es we e hen submi ed o o ex 3 imes, cen i uged 10 min (14,000 pm) and supe na an s we e collec ed. P o ein concen a ion was de e mined using a B ad o d assay (BioRad p o ein quan i ica ion sys em, BioRad, He cules, CA, USA). P o eins we e dissol ed in Sample Bu e [Laemmli wi h 5% ( / ) 2-β-me cap oe hanol and 5% ( / ) b omophenol blue] and boiled o 5 min a 95 ° C. Samples we e hen sepa a ed by 8% SDS-PAGE. P o- eins we e ans e ed in o ni ocellulose memb anes (Ame sham Hybond ECL, Ame sham biosciences, Buck- inghamshi e, UK) a 130 V o 1 h. Fo immunos aining, memb anes we e blocked wi h 5% (w/ ) non- a d y milk in PBS con aining 0.5% ( / ) Tween-20. Memb anes we e subsequen ly incuba ed wi h p ima y an ibodies o 1 h a RT (E-cadhe in –dilu ion 1:1000; P-cadhe in – dilu ion 1:500; p120c n –dilu ion 1:1000; S c –dilu ion 1:500; pSRC(Ty 416) –dilu ion 1:500 and β-ac in –dilu- ion 1:1000), ollowed by ou 5 min washes in PBS/ Tween-20 and incuba ion wi h ho se adish pe oxidase- conjuga ed seconda y an ibodies (donkey an i-goa , goa an i-mouse, goa an i- abbi –dilu ion 1:2000) o 1 h. Memb anes we e hen washed o 30 min in PBS/ Tween-20. P o eins we e de ec ed using ECL eagen (Ame sham biosciences) as a subs a e. Blo s we e ex- posed o an au o adiog aphic ilm. Band quan i ica ion o wes e n blo s was pe o med using Quan i y One (BioRad). Each immunoblo was pe o med a leas h ee imes, and he ones selec ed o igu es a e ep esen a i e expe imen s. 3D b eas cance cells sphe oids cul u e Agg egoids we e ob ained by pe o ming he slow agg e- ga ion assay, as p e iously desc ibed [19]. B ie ly, 1 × 10 4 cells in 200 μL medium we e seeded on solidi ied aga in a 96-well pla e o 12 h. Medium con aining he o med cellula agg ega es (10–100 cells) we e cen i uged and u he embedded in acid solubilized a ail collagen I gels (1.7 mg/ml collagen I in PBS; 22.5 mM NaHCO 3 ; 8 mM NaOH; 9% o non-supplemen ed cul u e medium). Cellula agg egoids we e pla ed in a 8-well co e slip bo om chambe s (IBIDI, Ge many) ollowed by 30 min incuba ion a 37 °C o allow polyme iza ion. A e ha ime, medium wi h o wi hou dasa inib was added o he cellula 3D agg egoid cul u es. The in i o beha iou was ollowed by ime-lapse mic oscopy (Leica mic oscope - DMI6000B wi h Adap a i e Focus Con ol) du ing 18 h. Fu he , he images aken we e con e ed in o a ime-lapse mo ie, and quan i a i e analysis o he numbe and leng h o p o usi e s uc u es was pe - o med using Leica AF6000 so wa e. Immuno luo escence analysis Cells we e cul u ed on glass co e slips, ixed wi h 4% pa a o maldehyde (20 min), ea ed wi h NH 4 Cl (50 mM) o 10 min, washed wi h PBS, and pe meablilized wi h 0.1% T i on X-100 in PBS o 5 min, a oom empe a u e. Non-speci ic binding was blocked by ea men wi h 5% BSA in PBS, o 30 min a oom empe a u e. Cells we e hen s ained O ange-phalloidin (Sigma, S . Louis, MO, USA) in o de o isualize F-ac in, o 30 min, o wi h he mono- clonal mouse an ibody o p120c n (dilu ion 1:100) and Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 Page 3 o 16 monoclonal abbi an ibody o E-cadhe in (dilu ion 1:100) o 1 h, ollowed by conjuga ed goa an i-mouse and an i- abbi seconda y IgG (Dako Cy oma ion, Ca pin- e ia, CA, USA), o 1 h a oom empe a u e. A e an- o he wash wi h PBS, each sample was moun ed wi h Vec ashield (Vec o Labo a o ies, Inc., Bu lingame, CA, USA) con aining 4,6-diamidine-2-phenylindolendihyd o- ch olide (DAPI) and isualized wi h Leica DM 2000 mic oscope (Leica). Ma igel in asion assays Ma igel in asion assay was pe o med using 8 μm po e size BD BioCoa ™Ma igel In asion Chambe s (BD Bio- sciences), acco ding o manu ac u e ’s ecommenda- ions. B ie ly, 24 h a e siRNA ans ec ion, 5 × 10 4 cells we e added o he uppe compa men o he chambe , wi h o wi hou dasa inib ea men . In he lowe com- pa men , only esh medium supplemen ed wi h 10% FBS was added. A e 24 h (BT20) o 48 h (MCF-7/AZ) o incuba ion a 37 °C, he uppe su ace o he il e was clea ed om non-in asi e cells wi h a co on swab and washed wi h PBS. The in asi e cells we e ixed wi h cold me hanol and s ained wi h DAPI. In asi e cells we e sco ed by coun ing he cells in he il e wi h a luo es- cence mic oscope (Zeiss Image Z1), a 100× o magni i- ca ion. In asion index exp esses he a io be ween in asi e cells om he di e en condi ions ela i e o he con ol cells. Wound-healing assay A e ans ec ion, o 24 h, wi h he espec i e siRNAs, cells we e ipsinized and seeded o ano he 24 h, o allow he cells o o m a comple e monolaye . A e hey became con luen , wounds we e ca e ully in oduced ac oss he cell monolaye , so ha he su ounding cells we e no dis u bed. The medium was eplaced wi h esh comple e medium, wi h and wi hou dasa inib ea men . Pic u es we e aken a 100× o magni ica ion in an in e ed mic oscope (Zeiss), and measu emen s o he dis ance be ween he wound edges we e made a speci ic ime poin s. Zymog aphy The condi ioned medium collec ed om he se e al cell cul u es, which we e g own 6-well pla es coa ed wi h collagen ype I, was analysed o p o einases ac i i y using β-casein zymog aphy. Samples we e mixed wi h sample bu e [0.03% b omophenol blue, 0.25 M T is-HCl pH 6.8, 10% SDS (w/ ) and 4% suc ose (w/ )] and elec opho esed, unde non- educing condi ions, on 10% polyac ilamide gels con aining 0.1% (w/ ) β-casein om bo ine milk (Sigma). A e elec opho esis, gels we e washed wice, o 30 min, in 2% ( / ) T i on X-100 (Sigma) a oom empe a u e, in o de o emo e SDS. β-casein gels we e hen incuba ed in a subs a e eac ion bu e [0.2 M NaCl, 5 mM CaCl 2 , 1% ( / ) T i on X-100 in 50 mM T is-HCl, pH 7.4], du ing 72 h, and inally s ained wi h Coomassie Blue S aining Solu ion [0.1% (w/ ) Coomassie Blue R250 in 10% ( / ) ace ic acid and 40% ( / ) me hanol], o 25 min. The gels des aining was pe o med in a solu ion wi h 20% me hanol and 10% ace ic acid, un il bands s a o become isible. Enzyma ic ac i i y was isualized as a clea band agains he blue backg ound o s ained casein gels, and MMPs we e iden i ied by hei molecula weigh . Quan i ica ion o band densi y was ca ied ou using he Quan i y One so wa e ( e sion 4.0, BioRad, He cules, CA, USA). In si u p oximi y liga ion assay (PLA) Cells we e deposi ed on glass slides and ixed wi h me hanol o 10 min. PLA was pe o med using he Duolink ki (Olink Bioscience, Sweden), acco ding o manu ac u e ’s ecommenda ions. The ollowing com- bina ion o p ima y an i-human an ibodies we e used agains : P-cadhe in (dilu ion 1:50, abbi polyclonal IgG, Cell Signaling), E-cadhe in (dilu ion 1:100, mouse mono- clonal IgG1, clone HECD-1, Taka a Bio Inc., Shiga, Japan; o dilu ion 1:50, abbi monoclonal IgG, Cell Signaling), p120c n (dilu ion 1:100, mouse monoclonal IgG1, clone 98, BD Biosciences). The nuclei we e coun- e s ained wi h DAPI. Slides we e analyzed wi h luo es- cence mic oscopy (Zeiss Image Z1 mic oscope) o isualiza ion o b igh luo escen ed signals consis en wi h p o ein-p o ein in e ac ion e en s. Ten s acks pe image we e aken and a minimum o i e ields pe expe imen we e e alua ed. The Blob inde V3.2 ee so wa e (Cen e o Image Analysis, Uppsala, Sweden) was used o quan i y he numbe o blobs (o do s) p esen in each condi ion. Fo cell cul u e expe imen s, an a e age o blobs/cells o a leas h ee independen expe imen s was pe o med and was no malized o he con ol condi ion. Fo pa a in embedded umo s, he speci ic numbe o blobs/cell was analysed. The espec - i e nega i e con ols we e used in each expe imen . In e nuclea p o ile analysis A so wa e applica ion was de eloped in o de o gene - a e and analyze he in e nuclea p o ile o p o eins. In each immuno luo escen image, pai s o cells we e selec ed o analysis in a semi-au oma ed manne . The mapping and quan i ica ion o he p o ein exp ession le el was pe o med by compu ing, espec i ely, 1D IN and RD in ensi y p o iles o wo con iguous cells and wi hin one single cell. To cope wi h cell size and shape a iabili y, a geome ic compensa ion algo i hm was de eloped in a Bayesian amewo k. The me hod was designed as an i e a i e algo i hm composed by he ol- lowing s eps: (i) p o ile ex ac ion om selec ed single Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 Page 4 o 16 cells (in case o RD) o pai s o cells (in case o IN); (ii) image map building by s acking luo escence p o iles oge he in columns a e leng h no maliza ion; (iii) denoising o map image (in which mul iplica i e noise desc ibed by a Poisson dis ibu ion is assumed); (i ) geo- me ic compensa ion o each 1D column p o ile minim- izing he o e all a iabili y o he map along he lines (ho izon al di ec ion); and ( ) compu a ion o he a e age and s anda d de ia ion p o iles using he com- pensa ed map. A e he ex ac ion o he da a, he max- imum mean a io (MMR) pa ame e was calcula ed di iding he maximum luo escence alue (nume a o ) by he luo escence mean (denomina o ). Animal s udies N:NIH(S)II-nu/nu mice, s ain p oduced by Ipa imup, we e housed, b eeded and main ained a he CIM-FMUP Animal House, in a pa hogen- ee en i onmen unde con olled condi ions o ligh and humidi y.The expe i- men s consis ed on he o ho opic injec ion in he mam- ma y a pad o emale mice, wi h 6–8 weeks o age, wi h 2 × 10 6 cells om each P-cadhe in-o e exp essing b eas cance models (SUM149PT, n= 17; BT20, n= 15; MDA-MB-468, n= 13) using a 25G needle. When he induced p ima y umou s eached a median olume o 100 mm 3 , mice we e andomized in o wo g oups, and s a ed he o al daily ea men wi h dasa inib, whe eas he o he s we e ea ed jus wi h he d ug ehicle, un il he end o he expe imen . Doses o dasa inib o be ad- minis e ed we e p epa ed weekly by dilu ion in ci a e bu e 80 mM (pH = 3.1). The ci a e bu e was p e- pa ed as ollows: sodium ci a e powde was dilu ed in ci ic acid solu ion 1 M, ob ained by dissol ing ci ic acid in deionized wa e , o a inal concen a ion o 80 nM and wi h pH = 3.1. The ci a e bu e solu ion was made e e y 3 weeks, il e ed in low chambe and kep a 4 °C. E e y week, daily doses o dasa inib we e p epa ed o a inal concen a ion o 10 mg/kg, in o de o ea mice o bo h expe imen al models. Mice (6–8 pe g oup) we e weigh ed, and umo wid h and leng h we e measu ed wi h calipe s, a leas , once a week. Tumo olume was es ima ed by using he equa ion, V =0.5×a×b 2 , whe e V is he olume, a is he leng h o he majo axis o he umo , and b is he leng h o i s mino axis. Tumo s we e su gically emo ed when an a e age olume o 1000 mm 3 was eached as p e iously de- sc ibed. Mice we e main ained o e alua e he impac o dasa inib in mice o e all su i al, o a maximum o 210 days, unless hey showed any signs o disease p o- g ession. Fo ha , mice we e eu hanized acco dingly o he es ablished Endpoin s. Upon ex ac ion, P ima y u- mo s we e ixed in 10% bu e ed o malin and hen em- bedded in pa a in, sec ioned, and s ained wi h haema oxylin and eosin. Immunohis ochemis y o E- and P-cadhe in was pe o med in umo s sec ions. Immunohis ochemis y A se ies o 467 p ima y in asi e b eas ca cinomas we e e ie ed om he Pa hology Depa men , Hos- pi al Xe al-Cíes, Vigo, Spain, diagnosed be ween 1978 and 1992. The umo s ha e been cha ac e ized o clinical and pa hological ea u es as p e iously desc ibed [11]. IHC was pe o med as p e iously desc ibed [6]. The memb ane s aining ex ension was e alua ed on a scale o 0 o 3 (0–0% o 10%; 1–10% o 25%; 2–25% o 50%; 3–50% o 100%). Fo E-cadhe in and o al S c, sco es o 0 and 1 we e conside ed nega i e and immuno eac i i y o 2 and 3 was sco ed as posi i e. Fo P-cadhe in all umo s p esen ing an unequi ocal memb anous s aining, in a leas 10% o he neoplas ic cells (≥1), we e sco ed as posi i e. Fo pSRC(Ty 416) umo s p esen ing memb ane o cy oplasmic s aining, in a leas 10% o he neoplas ic cells (≥1), we e sco ed as posi i e. Some sam- ples could no be assessed due o co e alling o lack o enough biological ma e ial o analyze. S a is ical analyses All o he s a is ical analyses we e pe o med by G aph Pad P ism e sion 5.0c so wa e (G aph Pad So wa e, San Diego, CA, USA), unless o he wise s a ed. P alues less han 0.05 we e conside ed s a is ically signi ican . Fo he AFM analysis, S uden ’s - es s we e used o de e mine s a is ically signi ican di e ences. F equency his og ams we e pe o med in O igin (O iginLab, No h- amp on, MA, USA) and Gaussian cu es we e applied. Quan i a i e pa ame e s o In e nuclea p o iles (no - malized o a cons an leng h o 100 a bi a y uni s) in P-cadhe in cells we e analyzed using a Mann-Whi ney es wi h a Bon e oni co ec ion. Fo he in i o xenog a assays, ANOVA es was used o e alua e he di e ences in umo size, conside - ing a con idence in e al o 95%. Su i al cu es we e es ima ed by he Kaplan-Meie ’s me hod, using he log- ank es o assess signi ican di e ences o mice o e all su i al. Conce ning he unc ional in i o assays, all we e pe - o med independen ly and in iplica e. Fo s a is ical analysis o he immunohis ochemis y esul s, con ingency ables and chi-squa e es s we e pe o med by SPSS 15.0 so wa e package o Win- dows (SPSS, Inc., USA), o es ima e he ela ionship be ween s aining pa e ns o P-cadhe in and pSRC (Ty 416). All s a is ical es s we e wo-sided. Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 Page 5 o 16 Fig. 1 (See legend on nex page.) Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 Page 6 o 16 Resul s P-cadhe in/CDH3 o e exp ession is signi ican ly associa ed wi h SRC ac i a ion in human b eas cance cells In o de o con i m he associa ion be ween P-cadhe in and SRC ac i a ion, we s a ed by analysing he exp es- sion o CDH3 (P-cadhe in codi ying gene) and SRC asso- cia ed genes (SRC, YES1, FYN, LYN, LCK, CSK)ina public da abase o b eas cance cell lines belonging o di e en molecula sub ypes (Fig. 1a) [CCLE da abase [20]]. In e es ingly, cell lines showing he highes le els o CDH3 also p esen a signi ican ly inc eased exp ession o YES, SRC, LYN and FYN. Acco dingly, mos o hese cell lines we e classi ied as Basal A, which a e he ones ha be e esemble he BLBC sub ype (Fig. 1b-c). We hen alida ed he associa ion be ween P-cadhe in and SRC ac i a ion using wo es ablished in i o b eas cance cell models, whe e P-cadhe in exp ession was manipula ed. P-cadhe in o e exp essing cells showed inc eased le els o pSRC(Ty 416), while P-cadhe in silencing led o a signi ican dec ease o pSRC(Ty 416) le els (Fig. 1d and Addi ional ile 1: Figu e S1A and B). No al e a ions in he o al le els o SRC we e ound by Wes e n blo . E-cadhe in and p120c n we e also e alu- a ed in bo h models and he le el o exp ession and localiza ion o bo h p o eins we e analysed by he luo - escence in e nuclea p o iles [21]. In con as o wha was obse ed o E-cadhe in exp ession, which did no al e upon gene ic manipula ion o P-cadhe in, a de- c ease o p120c n memb ane in ensi y was obse ed in P-cadhe in o e exp essing cells (Fig. 1e- ). Mo eo e , a signi ican inc ease in Rac1 ac i i y, a downs eam a ge o cy oplasmic p120c n, was obse ed in cells wi h P-cadhe in o e exp ession, as well as he silencing o P-cadhe in in BT20 cells was accompanied by a signi i- can dec ease in Rac1 ac i a ion (Fig. 1g). Cell-cell adhe- sion s eng h was also clea ly comp omised in P-cadhe in o e exp essing cells, as demons a ed by (See igu e on p e ious page.) Fig. 1 P-cadhe in o e exp ession associa es wi h SRC ac i a ion co ela ing wi h an agg essi e beha io . aGene exp ession p o ile o SRC amily membe s om BCC lines e ie ed om he CCLE da abase [20]. Unsupe ised hie a chical clus e ing based on classical cadhe ins exp ession (CDH1,CDH2,CDH3), subdi ide he BCC lines in o hei molecula sub ype (Luminal, Basal A and Basal B). bBox-plo ep esen a ion o CDH1, CDH2 and CDH3 gene exp ession alues in he di e en b eas cance molecula sub ypes. cBox-plo o SRC associa ed genes exp ession in he Luminal and Basal A BCC sub ypes. P- alues indica e signi ican ly di e ences obse ed in gene exp ession p o ile om Luminal and Basal A BCC lines. dWes e n blo analysis o P-cadhe in, E-cadhe in, p120c n, pSRC(Ty 416), o al SRC and β-ac in p o eins a e P-cadhe in o e exp ession and P-cadhe in silencing, espec i ely. eDual immuno luo escence con ocal images using an an i-p120c n an ibody (g een), F-ac in ( ed) and DAPI (blue). In e nuclea p o ile o p120c n and E-cadhe in, ob ained h ough he compu a ional analysis o immuno luo escence images om MCF-7/AZ BCC model. P- alue indica e signi ican di e ences obse ed in he memb ane mean luo escen in ensi y o p120c n be ween MCF-7/AZ.Mock e sus MCF-7/AZ.Pcad cells. gQuan i ica ion o he ac i e Rac1-GTP, measu ed by a G-Lisa assay a e P-cadhe in o e exp ession and P-cadhe in silencing, espec i ely. hA e age alues o Wo k (J), ep esen ing he cell-cell adhesion s eng h, using AFM Fo ce Spec oscopy analysis. S uden ’s - es was used o de e mine s a is ically signi ican di e ences, and P- alues a e indica ed in he igu e. iRep esen a i e Images o ime-lapse mic oscopy o BCC sphe oids embedded in collagen ype I. Scale ba = 50 μm. jBox-plo quan i ica ion o he ex ension (μm) o in asi e p o usions. kBox-plo quan i ica ion o he numbe o in asi e p o usions. lRep esen a ion o he pe cen age o cases posi i e o bo h pSRC(Ty 416) and P-cadhe in exp ession. P- alue was calcula ed by Chi-squa e es in o de o es ima e he ela ionship be ween s aining pa e ns o P-cadhe in and pSRC(Ty 416) Table 1 Cha ac e iza ion o he umo se ies, conse ning umo pa hological ea u es and molecula ma ke s N% His ological g ade 1 75 18.0 2 117 28.1 3 204 49.0 Missing 20 4.8 ERαNega i e 140 33.7 Posi i e 274 65.9 Missing 2 0.5 HER2 Nega i e 352 84.6 Posi i e 60 14.4 Missing 4 1.0 EGFR Nega i e 395 95.0 Pos i e 21 5.0 Missing 0 0.0 CKS Nega i e 357 85.8 Posi i e 59 14.2 Missing 0 0.0 CK14 Nega i e 394 94.7 Pos i e 22 5.3 Missing 0 0.0 Vimen in Nega i e 345 82.9 Pos i e 69 16.6 Missing 2 0.5 CD44 Nega i e 205 49.3 Posi i e 210 50.5 Missing 1 0.2 Sub ype Luminal 303 72.8 He 2 29 7.0 T iple-Nega i e 84 20.2 Missing 0 0.0 Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 Page 7 o 16 Fig. 2 (See legend on nex page.) Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 Page 8 o 16 a omic o ce mic oscopy (AFM) measu emen s (Fig. 1h), as well as a signi ican inc ease in he p o usi e and in asi e beha io (Fig. 1i-kand Addi ional ile 1: Figu e S1C) o b eas cance cells was p omo ed, by he e alu- a ion o 3D sphe oids embedded in collagen ype I ma ix by ime-lapse mic oscopy. These indings, oge he wi h he ac ha P-cadhe in a ec s he E-cadhe in/p120c n complex memb ane s abiliza ion, and ha i s jux amemb ane domain is c u- cial o i s p o-in asi e unc ion [11], led us o p esume ha P-cadhe in could be modi ying cell-cell adhesion and in asion capaci y h ough p120c n delocaliza ion occu ing downs eam o SRC ac i a ion signaling. Finally, o alida e in i o he associa ion be ween pSRC(Ty 416) and P-cadhe in, a se ies o 416 p ima y b eas umo s was s udied by immunohis ochemis y (Table 1). A signi ican associa ion was ound be ween he p esence o bo h p o eins, whe e 44% (18 in 41 cases) o he umo s wi h pSRC(Ty 416) we e also posi- i e o P-cadhe in (Fig. 1l). SRC ac i i y supp ession wi h dasa inib inhibi s he in i o unc ional ac i i y induced by P-cadhe in o e exp ession Taking in o accoun he esul s showing a posi i e asso- cia ion be ween P-cadhe in exp ession and SRC ac i a- ion, we specula ed i he downs eam SRC signalling could be an e ec i e a ge o poo p ognos ic P-cadhe in-o e exp essing b eas cance . This s a egy would o e come he lack o FDA-app o ed d ugs di ec ly a ge ing P-cadhe in. Thus, we examined he e ec o SRC inhibi ion in he p e iously de ined in i o unc ional ac i i y induced by P-cadhe in exp ession, by ea ing cells wi h 100 nM o dasa inib (concen a ion ha does no a ec he me abolic s a e and iabili y o b eas cance cells –Addi ional ile 1: Figu e S1D). B eas cance cells ea ed wi h dasa inib showed a dec ease o pSRC(Ty 416) exp ession (Fig. 2a and Addi ional ile 1: Figu e S1E); mo eo e , his ea - men led o a dec ease in cell mig a ion (Fig. 2b) and cell in asion (Fig. 2c), associa ed wi h a dec ease o MMP2 ac i i y and sP-cad exp ession in he condi ioned media (Fig. 2d), as well as wi h a dec eased mammosphe e o m- ing e iciency (MFE) (Fig. 2e). Addi ionally, cell-cell adhesion was highly p omo ed in P-cadhe in o e exp essing cells ea ed wi h dasa inib, as obse ed by he b igh ield images and quan i ied by AFM in bo h cell lines a e SRC inhibi ion (Fig. 2 ). Addi ionally, when we e alua ed he in i o 3D in asion abili y o P-cadhe in o e exp essing cells a e dasa inib ea men , we obse ed a signi ican dec ease in he numbe and leng h o p o usi e and in asi e s uc u es in o he collagen ype I ma ix (Fig. 2g-i). As a con- ol, we ha e pe o med dasa inib ea men in P-cadhe in low b eas cance cells (MCF-7/AZ.mock and BT20 siC ) and, as expec ed, he impac o he dasa inib ea men is e y modes , since he pa hway is no as ac i a ed as in P-cadhe in o e exp essing cells (Addi ional ile 1:Figu eS2A-C)[22]. Al oge he , he esul s suppo ha SRC ac i i y sup- p ession by dasa inib is able o p e en he unc ional e - ec s induced by P-cadhe in o e exp ession in b eas cance cells. Dasa inib ea men p omo es he eco e y o cell-cell adhesion unc ion by s abilizing E-cadhe in/p120c n complex a he cell memb ane We ha e p e iously shown ha P-cadhe in o e exp es- sion in an E-cadhe in wild- ype con ex weakens cell-cell adhesion by dis up ing he E-cadhe in/ca enins complex [11]. Taking his in o accoun , we e alua ed he e ec o dasa inib a his molecula le el. As obse ed in Fig. 3a, dasa inib p omo ed an inc ease in memb ane exp ession o p120c n, as obse ed by i s in e nuclea p o ile (Fig. 3b), which was associa ed wi h a dec ease in he downs eam ac i a ion o Rac 1 (Fig. 3c). Once again, E-cadhe in exp ession and localiza ion was no al e ed (Fig. 3b), al hough dasa inib ea men p omo ed a signi ican eco e o he E-cadhe in/p120c n complex, mainly a he cell memb ane (Fig. 3d and Addi ional ile 1: Figu e S3A and B). Bo h esul s show ha ac i- a ion o SRC signaling in e e es wi h E-cadhe in/ (See igu e on p e ious page.) Fig. 2 Dasa inib ea men inhibi s he in i o unc ional ac i i y induced by P-cadhe in exp ession. aWes e n blo ing o pSRC(Ty 416), o al S c, P-cadhe in, E-cadhe in and p120c n in P-cadhe in o e exp essing cells a e dasa inib ea men (100 nM) o 48 h. P o ein le els o β-ac in we e analyzed and used as he loading con ol. bRep esen a i e expe imen om a wound healing mig a ion assay, in bo h P-cadhe in o e exp essing BCC (MCF-7/AZ.Pcad and BT20), ea ed wi h 100 nM o dasa inib o DMSO o 24 h. cFold change in he numbe o in asi e cells, e alua ed by he ma igel in asion assay o bo h MCF-7/AZ.Pcad and BT20 ea ed wi h DMSO o 100 nM o dasa inib. dZymog aphy o MMP2 ac i i y and Wes e n blo ing o sP-cad, using he condi ioned medium om cells ea ed wi h DMSO o 100 nM o dasa inib o 48 h, in bo h MCF-7/AZ.Pcad and BT20 BCC models. eMammosphe e o ming assay was pe o med o bo h cell models. The P- alues indica e he s a is ically signi ican di e ence be ween DMSO and dasa inib ea ed cells, in bo h P-cadhe in BCC models. A e age alues o Wo k (J), ep esen ing he cell-cell adhesion s eng h, o bo h BCC cance cell models ea ed wi h DMSO and dasa inib, using AFM Fo ce Spec oscopy analysis. gBox-plo quan i ica ion o he leng h (μm) and hnumbe o in asi e p o usions o MCF-7/AZ.Pcad sphe oids, ea ed wi h DMSO o 100 nM dasa inib, o 24 h. P- alues < 0.05 we e conside ed s a is ically signi ican . iRep esen a i e Images o ime-lapse mic oscopy o BCC sphe oids embedded in collagen ype I, o MCF-7/AZ.Pcad ea ed wi h DMSO and dasa inib. Scale ba = 50 μm Ribei o e al. Cell Communica ion and Signaling (2018) 16:75 Page 9 o 16 21. Sanches JM, Figuei edo J, Fonseca M, Du aes C, Melo S, Esmenio S, Se uca R. Quan i ica ion o mu an E-cadhe in using bioimaging analysis o in si u luo escence mic oscopy. A new app oach o CDH1 missense a ian s. Eu J Hum Gene . 2015;23:1072–9. 22. Ribei o AS, Ca alho FA, Figuei edo J, Ca alho R, Mes e T, Mon ei o J, Guedes AF, Fonseca M, Sanches J, Se uca R, e al. 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