PLA2G7 associates with hormone receptor negativity in clinical breast cancer samples and regulates epithelial-mesenchymal transition in cultured breast cancer cells

PLA2G7 associa es wi h ho mone ecep o nega i i y in clinical b eas cance samples and egula es epi helial-mesenchymal ansi ion in cul u ed b eas cance cells Lau a Leh inen, 1 * Paula Vainio, 1 Ha ie Wikman, 2 Heini Huh ala, 3 Volkma Muelle , 4 Anne Kallioniemi, 5 Klaus Pan el, 2 Pauliina K onq is , 1 Olli Kallioniemi, 6,8 Olli Ca pe `n 1,9 and K is iina Iljin 7 1 Depa men o Pa hology, Tu ku Uni e si y and Tu ku Uni e si y Hospi al, Tu ku, Finland 2 Ins i u e o Tumou Biology, Cen e o Expe imen al Medicine, Uni e si y Medical Cen e Hambu g-Eppendo , Ge many 3 School o Heal h Sciences, Uni e si y o Tampe e, Tampe e, Finland 4 Depa men o Gynecology, Uni e si y Medical Cen e Hambu g-Eppendo , Hambu g, Ge many 5 BioMediTech, Uni e si y o Tampe e, Tampe e, Finland 6 FIMM, Ins i u e o Molecula Medicine Finland, Uni e si y o Helsinki, Finland 7 VTT Technical Resea ch Cen e o Finland, Espoo, Finland 8 P esen add ess: Depa men o Oncology-Pa hology, Science o Li e Labo a o y, Ka olinska Ins i u e , Solna, Sweden 9 P esen add ess: Depa men o Pa hology, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki, Finland *Co espondence o:Lau a Leh inen, Depa men o Pa hology,Tu ku Uni e si y and Tu ku Uni e si y Hospi al,Tu ku, Finland. E-mail: lauleh@u u. i B eas cance is he leading cause o cance - ela ed dea hs in women due o dis inc cance sub ypes associ- a ed wi h ea ly ecu ence and agg essi e me as a ic p og ession. High lipop o ein-associa ed phospholipase A2 ( PLA2G7 ) exp ession has p e iously been associa ed wi h agg essi e disease and me as asis in p os a e cance . He e, we explo e he exp ession pa e n and unc ional ole o PLA2G7 in b eas cance . Fi s , a bioin- o ma ic analysis o genome-wide gene exp ession da a om 970 b eas samples was ca ied ou o e alua e he exp ession pa e n o PLA2G7 mRNA in b eas cance . Second, he exp ession p o ile o PLA2G7 was s ud- ied in 1042 b eas cance samples including 89 ma ched lymph node me as asis samples using immunohis o- chemis y. Thi d, he e ec o PLA2G7 silencing on genome-wide gene exp ession p o ile was s udied and alida ed in cul u ed b eas cance cells exp essing PLA2G7 a high le el. Las , he exp ession pa e n o PLA2G7 mRNA was in es iga ed in 24 nonmalignan issue samples and 65 p ima y and 7 me as a ic umou samples de i ed om a ious o gans using qRT-PCR. The esul s om clinical b eas cance samples indica ed ha PLA2G7 is o e exp essed in a subse o b eas cance samples compa ed o i s exp ession in benign b eas issue samples and ha high PLA2G7 exp ession associa ed wi h ho mone ecep o nega i i y as well as wi h poo p ognosis in a subse o b eas cance samples. In i o unc ional s udies highligh ed he pu a i e ole o PLA2G7 in he egula ion o epi helial-mesenchymal ansi ion (EMT)- ela ed signalling pa hways, imen in and E-cadhe in p o ein exp ession as well as cell mig a ion in cul u ed b eas cance cells. Fu he mo e, sup- po ing he indings in b eas and p os a e cance , high PLA2G7 mRNA exp ession was associa ed wi h me a- s a ic cance in ou addi ional o gans o o igin. In conclusion, ou esul s indica e ha PLA2G7 is highly exp essed in a subse o me as a ic and agg essi e b eas cance s and in me as a ic samples o a ious issues o o igin and p omo es EMT and mig a ion in cul u ed b eas cance cells. Keywo ds: PLA2G7; epi helial-mesenchymal ansi ion; EMT; imen in; b eas cance ; p ognosis; me as asis; in asion Recei ed 2 Janua y 2017; Accep ed 10 Ma ch 2017 Mic oa ay da a: Da a can be accessed a A ayExp ess (E-MTAB-5397 and E-MTAB-5398) No con lic s o in e es we e decla ed. O iginal A icle V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made. The Jou nal o Pa hology: Clinical Resea ch J Pa h: Clin Res Ap il 2017; 3: 123–138 Published online 14 Ma ch 2017 in Wiley Online Lib a y (wileyonlinelib a y.com). DOI: 10.1002/cjp2.69 In oduc ion B eas cance is he mos equen cance in women wo ldwide accoun ing o 25% o all cance cases among emales [1]. Al hough su i al a es o b eas cance pa ien s ha e imp o ed signi ican ly du ing he las decade b eas cance is s ill he leading cause o cance - ela ed dea hs in women due o dis inc sub ypes o b eas cance ha a e associa ed wi h ea ly ecu ence and an agg essi e me as a ic p o- g ession o he disease [1–4]. Iden i ica ion o bio- ma ke s and de elopmen o a ge ed he apies especially o hese malignan umou s is impo an . Recen ly, he egula o s o me as asis ini ia ing epi helial-mesenchymal ansi ion (EMT) ha e been sugges ed as impo an ac o s and possible diagnos- ic ma ke s o agg essi e and me as a ic b eas can- ce s, including iple nega i e b eas cance s (TNBC) [5–9]. EMT, a i al p ocess du ing emb yogenesis and is- sue epai , is cha ac e ised by speci ic gene exp ession changes, educed in e cellula adhesion, gain o mes- enchymal cha ac e is ics and inc eased cell mobili y o dis an loca ions. Loss o immo ile epi helial pheno- ype and gain o mesenchymal p ope ies ia EMT has ecen ly been shown o be a key me as asis-p omo ing s ep in many cance s [10]. In addi ion o enabling me as asis EMT has been sugges ed o p omo e he gene a ion and unc ion o cance s em cells as well as de elopmen o d ug esis ance [11]. EMT is induced by mul iple oncogenic e en s and signalling pa hways and equi es he exp ession o a a ie y o egula o s, including membe s o he Twis , Snail/Slug and Zeb ansc ip ion ac o amilies. In addi ion, Tiwa i e al. ecen ly epo ed he SOX4-EZH2 pa hway as one o he mas e egula o s o EMT in me as a ic b eas can- ce [12]. Two majo hallma ks o EMT a e he loss o exp ession o he epi helial cell adhesion molecule E- cadhe in (CDH1) and gain o exp ession o he majo mesenchymal cell cy oskele al componen imen in (VIM). The cy oplasmic in e media e ilamen p o ein imen in is a componen o he cy oskele on no mally ound in emb yonic o mesenchymal cells. Howe e , in cance imen in is a ma ke o EMT and is exp essed in neoplas ic epi helial cells wi h me as a ic p ope ies [13–15]. The exp ession o imen in co e- la es wi h in asi eness and poo p ognosis in clinical cance samples [16–18]. PLA2G7, also known as lipop o ein-associa ed phos- pholipase A2 o pla ele -ac i a ing ac o (PAF) ace yl hyd olase, is a phospholipase ha hyd olyses PAF and unca ed phospholipids gene a ed by oxida i e a ack [19]. PLA2G7 was ecen ly epo ed as a no el bioma ke in 50% o p ima y and 70% o me as a ic p os a e cance s and associa ed wi h agg essi e disease in p os a e cance [20]. Fu he mo e, PLA2G7 was iden i ied as a po en ial d ug a ge especially in ERG oncogene posi i e p os a e cance s and he al e a ions induced by PLA2G7 silencing highligh ed he po en ial o PLA2G7 inhibi ion as an an i-p oli e a i e, p o-apo- p o ic and an i-mig a o y he apeu ic app oach [20,21]. In e es ingly, in addi ion o ERG oncogene exp essing p os a e cance s, high PLA2G7 exp ession has also been p oposed o play a causal ole in colon umou i- genesis downs eam o loss-o - unc ion TP53 mu a- ions and ac i a ing RAS mu a ions [22]. Suppo ing his inding, dele ion o PLA2G7 in Apc Min/1 mice has been shown o dec ease bo h in es inal polyposis as well as colon umou igenesis [23]. In ecen yea s, PLA2G7 has also been unde in ensi e esea ch in he a ea o ca dio ascula diseases. High PLA2G7 amoun and enzyme ac i i y ha e been associa ed wi h an inc eased isk o ca dio ascula disease, and PLA2G7 inhibi o da apladib has been s udied in he p e en ion and ea men o co ona y hea disease [24–29]. In his s udy, we ocus on analysing he exp ession pa e n o PLA2G7 in b eas cance and in es iga e he unc ional ole o PLA2G7 in cul u ed b eas can- ce cells using genome-wide gene exp ession analy- sis, immunohis ochemis y and unc ional assays. The esul s indica e a ole o PLA2G7 in he egula ion o me as asis-enabling EMT and cell mig a ion, asso- cia e high PLA2G7 mRNA exp ession wi h ho mone ecep o nega i e agg essi e disease and indica e ha high PLA2G7 p o ein exp ession in lymph node me as ases associa es wi h un a ou able p ognosis in b eas cance . Me hods T ansc ip omics analysis In o de o e alua e he in i o ele ance o PLA2G7 in b eas cance , we analysed PLA2G7 mRNA exp- ession le els in no mal (n 513) and malignan (n 5957) b eas issue samples using GeneSapiens da abase (www.genesapiens.o g) [30]. Su i al in o - ma ion was a ailable o 395 and ecu ence da a o 159 b eas cance samples. Clinical ma e ial B eas umou samples we e ob ained om pa ien s a Hambu g Uni e si y Medical Cen e (Hambu g, Ge many) be ween 1999 and 2007 and Tampe e Uni- e si y Hospi al (Tampe e, Finland) be ween 1990 and V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 124 L Leh inen e al 1999. Pa ien da a and umou cha ac e is ics ha e been desc ibed p e iously [31,32]. In o med consen o he scien i ic use o issue ma e ials was ob ained om he pa ien s and e hical pe mission was ob ained om he local e hical commi ees. All p ocedu es we e in acco dance wi h he Helsinki Decla a ion. The pa ien eco ds con ained in o ma ion on clini- copa hological pa ame e s, p ima y ea men s, si e and ime o umou ecu ences and su i al. The max- imum ollow-up pe iod was 19.8 yea s. Relapse– ee su i al was de ined as ime o any disease ecu ence (local, egional o dis an ). The end poin in b eas can- ce speci ic su i al was dea h om b eas cance and in o e all su i al dea h om any cause. Immunohis ochemis y The cons uc ion o issue mic oa ays (TMA) has been desc ibed p e iously [31,32]. The pa a in embedded b eas cance TMAs con aining in o al 1042 samples om 897 b eas cance pa ien s, in- cluding 89 cases wi h ma ched p ima y and lymph node me as a ic samples, we e s ained wi h PLA2G7 (1:150, abbi polyclonal, Cayman Chemical) an i- body. Immunos aining was pe o med wi h he au o- ma ed immunos aining de ice BenchMa k XT (Roche Diagnos ics/Ven ana Medical Sys ems, Tuc- son, AZ, USA) using ul aView Uni e sal DAB De ec ion Ki (Roche Diagnos ics/Ven ana Medical Sys ems). PLA2G7 p o ein exp ession le el in he epi helial b eas cance cells in each sample was e alua ed independen ly in blinded ashion by wo pa hologis s and g aded in o wo g oups based on s aining in ensi y: low exp ession (no s aining o weak s aining), high exp ession (mode a e o s ong s aining). A e excluding he cases wi hou esidual issue o umou cells, 618 b eas umou s and 65 lymph node me as asis samples emained eligible o e alua ion. Cell cul u e The b eas ca cinoma cell lines MDA-MB-468, MCF-7, MDA-MB-231 and ZR-75-1 cell lines we e om Ame ican Type Cul u e Collec ion (Manassas, VA, USA). BT-549 and KPL-1 cell lines we e om Cell Lines Se ice (Eppelheim, Ge many). All cell lines we e main ained acco ding o he dis ibu o ’s ins uc ions. Gene silencing Fo ansien knockdown o PLA2G7,siRNA (SI00072177; siPLA2G7, Qiagen, Valencia, CA) was ans ec ed in o MDA-MB-468 cells using siLen Fec Lipid Reagen (Bio-Rad Labo a o ies, CA) in Op i- MEM (In i ogen, Ca lsbad, CA) a a inal concen a- ion o 13 nM. Cells we e incuba ed o 48 hou s be o e u he expe imen s. To gene a e s able PLA2G7 de i- cien MDA-MB-468 cells (MDA-468/PLA2G7- cells), wo len i i al shRNA cons uc s (NM_005084.2- 1046s1c1; shPLA2G7_1 and NM_005084.2-684s1c1; shPLA2G7_2, Sigma-Ald ich, S . Louis, MO) we e ansduced in o MDA-MB-468 cells as desc ibed [33]. All S a s sc ambled siRNA (siSc , Qiagen) and non- a ge ing cons uc (SHC002V; shSc : Sigma-Ald ich) we e used as nega i e con ol. Quan i a i e RT-PCR The con i ma ion o e ec i e a ge gene silencing was pe o med wi h Taqman e e se ansc ip ase PCR (qRT-PCR). To al RNA om 70 000 cells was ex ac ed using RNeasy ki (Qiagen) acco ding o he manu ac u e ’s p o ocol and p ocessed o cDNA wi h Applied Biosys ems cDNA syn hesis ki . TaqMan gene exp ession p obes and p ime s om he Uni e sal P obe Lib a y (Roche Diagnos ics, Espoo, Finland) we e used o s udy PLA2G7 ( o wa d: ggc c acc agaaccc ga; e e se: gc c gc cg acc ), and b-ac in (ACTB, o wa d: ccaaccgcgagaaga ga; e e se: ccagaggcg acaggga ag) mRNA exp ession. The quan i a i e PCR was done using ABI P ism 7900 (Applied Biosys ems, Fos e Ci y, CA). Quan i- a ion was ca ied ou using he DD CT me hod wi h RQ manage 1.2 so wa e (Applied Biosys ems). A e age exp ession o he con ol samples was con- side ed o he calcula ion o he old changes and b- ac in was used as an endogenous con ol. Th ee o mo e eplica e samples we e s udied o de ec ion o mRNA exp ession. Gene exp ession analysis using bead a ays 500 ng o o al pu i ied RNA was used o ampli ica- ion wi h Illumina RNA To alP ep Ampli ica ion ki (Ambion, Aus in, TX), and bio in labelled cRNA was hyb idised o Sen ix HumanRe -12 s.3 Exp ession Bead Chips (Illumina, San Diego, CA). Analysis was done as desc ibed [20]. Mic oa ay da a a e a ailable in he A ayExp ess da abase (www.ebi.ac.uk/a ay exp ess, siRNA ea men : E-MTAB-5397 and shRNA ea men s: E-MTAB-5398). Two eplica e samples we e s udied o each ea men and gene exp ession p o iles o he PLA2G7 silenced samples we e compa ed wi h he sc ambled con ol- ea ed samples. Ingenui y Pa hway Analysis (IPA) So wa e (Ingenui y Sys ems Inc., Redwood Ci y, CA, USA), DAVID Bioin o ma ics Resou ces [34,35] and PLA2G7 in b eas cance 125 V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 Molecula Signa u es Da abase (MSigDB) Gene Se En ichmen Analysis So wa e [36,37] we e used o assess he en ichmen o unc ional and pa hway anno a ions as well as gene se s o he genes di - e en ially exp essed by PLA2G7 siRNA o bo h PLA2G7 shRNAs. Cell iabili y and apop osis assays The e ec s o PLA2G7 silencing on cance cell iabil- i y and induc ion o apop osis we e assessed wi h he CellTi e-Glo cell iabili y assay and ApoONE apop o- sis assay (P omega Inc, Madison, WI, USA) acco ding o he manu ac u e ’s ins uc ions. The EnVision mul- ilabel pla e eade (Pe kin-Elme , Wal ham, MA) was used o signal quan i ica ion. Sc ambled siRNA o shRNA was used as he nega i e con ol and KIF11 siRNA as he posi i e con ol. Wes e n blo ing Wes e n blo analysis was pe o med using an ibodies agains imen in (1:1000, V6630, Sigma-Ald ich), E- cadhe in (1:1000, Cell Signalling Technology, Dan- e s, MA), EGFR (1:100, Cell Signalling Technolo- gies) and b-ac in (1:5000, Sigma-Ald ich). The signals ob ained we e isualised wi h he enhanced chemiluminescence (ECL) de ec ion sys em (Ame - sham) and no malised o b-ac in signal. Immuno luo escence s aining Fo he immuno luo escence s aining, MDA-468/ PLA2G7- cells we e ixed wi h 4% pa a o maldehyde (PFA) in PBS, pe meabilised wi h 0.2% T i on X- 100, and blocked wi h 3% bo ine se um albumin in PBS. Cells we e s ained wi h imen in an ibody and Alexa-conjuga ed seconda y an ibody was used o seconda y s aining (1:300 dilu ion; Molecula P obes, The mo Fishe Scien i ic, Wal ham, MA, USA). Nuclei we e s ained wi h Vec ashield moun ing medium (Vec o Labo a o ies, Bu lingame, CA) con- aining DAPI. Images we e aken wi h Zeiss Axio e 200M luo escence mic oscope (Ca l Zeiss AG, Obe kochen, Ge many). Wound healing in asion assay MDA-468/PLA2G7- cells we e u ilized in he wound healing in asion assay using he IncuCy e eal- ime imaging sys em (Essen BioScience, Ann A bo , Michigan, USA). The cells we e pla ed on 96-well pla es (ImageLock pla e, Essen BioScience, MI) coa ed wi h 50 ml 10% G ow h Fac o Reduced Ma igel (BD Biosciences) a e which he cells we e allowed o a ach o e nigh a 1378C. A wound was sc a ched ac oss each well (Wound Make , Essen BioScience) and he g ow h medium was emo ed. The cells we e hen ca e ully co e ed wi h 50 ml 25% Ma igel in no mal g ow h medium andincuba edin378C o 2–3 hou s o allow gel- ling, a e which 100 mlo g ow hmediumwas ca e ully added o each well. The a e o in asion (wound closu e h ough he ma ix) was moni o ed hou ly wi h Incucy e imaging so wa e (Essen Bio- Science) o 72 hou s. In asion e iciency was de e - mined as pe cen age o he ela i e wound con luence compa ed o espec i e nega i e con ol ( ega ded as 100%). cDNA umou panel TissueScan TM Cance Su ey cDNA A ay 96 – I (O igene, Rock ille, MD, USA) consis ing o 96 samples om 65 p ima y and 7 me as a ic umou samples as well as 24 non-malignan issue samples de i ed om 8 di e en p ima y o gans and p o ided wi h clinical in o ma ion was u ilised acco ding o he manu ac u e ’s ins uc ions o analyse he exp es- sion pa e n o PLA2G7 mRNA in mul iple malignan- cies. The exp ession o PLA2G7 was quan i ied using qRT-PCR. S a is ical analysis S a is ical analysis o immunohis ochemical samples was ca ied ou using he SPSS p og am (SPSS Inc., Chicago, IL). Compa isons be ween ca ego ical a ia- bles we e pe o med using he 2 es . Kaplan-Meie cu es we e used o es ima e su i al p obabili y and pa ien su i al di e ences we e analysed by a log- ank es . O he s a is ical analyses we e pe o - med using S uden ’s - es (*p <0.05; **p <0.01; ***p <0.001) and he Pea son co ela ion coe icien . The esul s om cul u ed cells a e p esen ed as he mean 6SD o a leas h ee eplica es unless o he - wise s a ed. Resul s Co ela ion o PLA2G7 mRNA exp ession wi h ho mone ecep o exp ession and p ognosis in b eas cance To e alua e he exp ession pa e n and possible asso- cia ion o PLA2G7 wi h poo su i al in b eas can- ce , he GeneSapiens da abase was u ilised o analyse PLA2G7 mRNA exp ession in 13 no mal b eas 126 L Leh inen e al V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 samples and 957 b eas cance samples [30]. Com- pa ison o PLA2G7 exp ession le els in no mal b eas issues and b eas cance samples indica ed high PLA2G7 mRNA exp ession in a subse o b eas cance s (Figu e 1A). Co-exp ession analysis indica ed PLA2G7 mRNA o be o e exp essed especially in b eas cance samples wi h low oes ogen and p oges- e one ecep o (ESR1 and PGR) exp ession (p 50 and p 53.4E-10, espec i ely; Figu e 1B) whe eas he associa ion be ween high PLA2G7 mRNA and low HER2/ERBB2 exp ession was no s a is ically signi ican (supplemen a y ma e ial, Figu e S1). To in es iga e whe he PLA2G7 may ha e p ognos ic signi icance, Kaplan–Meie analysis was pe o med. The esul s e ealed a signi ican associa ion be ween high PLA2G7 mRNA exp ession and poo b eas can- ce su i al (p 50.004) as well as inc eased isk o disease elapse (p 50; Figu e 1C). To alida e he associa ion be ween high PLA2G7 exp ession and ho mone ecep o nega i i y as well as poo p ognosis in b eas cance , wo addi ional independen se s o clinical b eas cance issue mic oa ays we e s udied wi h immunohis ochemical s aining. PLA2G7 exp ession was analysed in a o al o 683 b eas cance samples, including 65 samples de i ed om lymph node me as ases (Figu e 2A). High PLA2G7 exp ession was de ec ed in 21.7% (n 5148) o all b eas cance samples. Associa ion be ween high PLA2G7 exp ession and s anda d clini- copa hological pa ame e s is p esen ed in Table 1. In acco dance wi h he esul s om bioin o ma ic analy- sis o PLA2G7 mRNA exp ession, high PLA2G7 exp ession showed signi ican associa ion wi h ho - mone ecep o nega i i y in p ima y he b eas cance samples analysed. Fu he mo e, e en hough no sig- ni ican associa ion wi h su i al o elapse was seen in he samples ob ained om p ima y umou s, high PLA2G7 p o ein exp ession was associa ed wi h poo Figu e 1. Figu e 1. High PLA2G7 mRNA exp ession is associa ed wi h ho - mone ecep o nega i i y and poo p ognosis in clinical b eas cance samples. (A) Box plo analysis o no malized PLA2G7 mRNA exp ession alues in no mal and malignan issues. The box e e s o qua ile dis ibu ion (25–75%) ange, wi h he median shown as a e ical line. Da a obse a ions which lie mo e han 1.5*in e -qua ile ange highe han hi d qua ile a e conside ed as ou lie s and indica ed sepa a ely. (B) The mRNA co-exp ession pa e n o PLA2G7 and oes ogen ecep o (ESR1) o p oges e one ecep o (PGR) in b eas cance samples (n 5957). (C) Kaplan-Meie plo o b eas cance speci ic su - i al and isk o elapse based on PLA2G7 mRNA exp ession in b eas cance . A log- ank es was pe o med be ween samples wi h high (50%) and low (50%) exp ession o e alua e he p ognos ic signi icance o PLA2G7 mRNA exp ession le el. PLA2G7 in b eas cance 127 V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 Figu e 2. PLA2G7 is exp essed in a subse o b eas cance s and associa es wi h poo p ognosis in b eas cance lymph node me as- asis samples. (A) S aining in ensi y o PLA2G7 in p ima y b eas cance and b eas cance lymph node me as asis was sco ed as ol- lows: Low exp ession (0 o 1), high exp ession (11 o 111). Rep esen a i e sec ion o s aining in ensi ies is p esen ed. The a eas p esen ed a highe magni ica ion ha e been indica ed in he co e images. (B) Kaplan-Meie cu e p esen a ion o o e all su i al and isk o elapse in he pa ien g oups wi h no o weak PLA2G7 s aining (n 557) o posi i e PLA2G7 s aining (n 58) in he lymph node me as asis samples. 128 L Leh inen e al V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 su i al (p 50.002) as well as inc eased isk o dis- ease elapse (p 50.005) in he samples ob ained om lymph node me as ases (Figu e 2B). No signi ican di e ence was de ec ed in PLA2G7 exp ession be ween he pai ed p ima y umou and me as asis samples. A s aining esul was ob ained o 39 pai ed p ima y umou s and lymph node me as ases and 72% (28) o he pai s showed simila s aining in ensi y in p ima y and me as a ic samples. PLA2G7 mRNA exp ession in cul u ed b eas cance cells In o de o ind a sui able b eas cance cell model o unc ional expe imen s he mRNA exp ession le el o PLA2G7 was s udied in six b eas cance cell lines wi h qRT-PCR. Th ee o he cell lines s udied we e ER-posi i e (MCF-7, KPL-1 and ZR-75-1) and h ee we e ER-nega i e (MDA-MB-231, BT-549 and MDA-MB-468). The esul s om qRT-PCR analysis a e in acco dance wi h he clinical da a and show high PLA2G7 exp ession in wo ho mone ecep o nega i e cell lines, MDA-MB-468 and BT-549 (Fig- u e 3A). The e ec o PLA2G7 silencing on genome-wide gene exp ession in MDA-MB-468 b eas cance cells To in es iga e he molecula p ocesses egula ed by PLA2G7 in b eas cance cells, he e ec o PLA2G7 silencing on genome-wide gene exp ession p o ile was s udied in MDA-MB-468 cells exp essing PLA2G7 a high le el. The gene exp ession analysis was conduc ed using one siRNA and wo di e en shRNA molecules silencing PLA2G7 e icien ly com- pa ed o he espec i e sc ambled con ol (Figu e 3B and supplemen a y ma e ial, Figu e S2A). Fo MDA- 468/PLA2G7- cells he genes uni o mly di e en ially egula ed by bo h o he shRNAs we e selec ed o u he analysis. DAVID Bioin o ma ics Resou ces and Ingenui y Pa hway Analysis (IPA) so wa e was used o assess en ichmen o unc ional and pa hway anno a ions o Table 1. PLA2G7 exp ession and s anda d clinicopa hological pa ame e s in 618 b eas cance samples. TMA 1 TMA 2 (p ima y umou s) Va iable All (n) All (%) High PLA2G7 (n) High PLA2G7 (%) pAll (n) All (%) High PLA2G7 (n) High PLA2G7 (%) p All samples 400 100 84 21.0 218 100 56 25.7 Tumou ype 309 218 ILC 135 43.7 21 15.6 0.028 27 12.4 5 18.5 0.048 IDC 174 56.3 45 25.9 165 75.7 43 26.1 Ho mone ecep o s a us 328 218 nega i e 53 16.2 16 30.2 0.041 44 20.2 18 40.9 0.010 posi i e 275 83.8 51 18.0 174 79.8 38 21.8 HER2 exp ession 330 187 nega i e 285 86.4 54 18.9 0.061 160 85.6 41 25.6 0.706 posi i e 45 13.6 14 31.1 27 14.4 6 22.2 WHO g ade 260 213 I 81 31.2 12 14.8 0.244 10 4.7 4 40.0 0.018 II 126 48.5 27 21.4 116 54.5 21 18.1 III 53 20.4 14 26.4 87 40.8 30 34.5 pT s age 342 218 pT1 218 63.7 51 23.4 0.227 109 50.0 29 26.6 0.973 pT2 98 28.7 14 14.3 91 41.7 23 25.3 pT3 12 3.5 1 8.3 10 4.6 2 20.0 pT4 14 4.1 3 21.4 8 3.7 2 25.0 Lymph node me as asis 322 217 pN0 205 63.7 38 18.5 0.538 129 59.4 31 24.0 0.590 pN1 117 36.3 25 21.4 88 40.6 24 27.3 Me as asis 341 214 M0 330 96.8 68 20.6 0.350 205 95.8 50 24.4 0.543 M1 11 3.2 1 9.1 9 4.2 3 33.3 Recu ence 345 208 no 316 91.6 67 21.2 0.065 161 77.4 40 24.8 0.211 yes 29 8.4 2 6.9 47 22.6 16 34.0 ILC, in asi e lobula ca cinoma; IDC, in asi e duc al ca cinoma. Numbe s in bold indica e s a is ically signi ican P alues. PLA2G7 in b eas cance 129 V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 he genes di e en ially exp essed by PLA2G7 im- pai men . In acco dance wi h ea lie unc ional s ud- ies in p os a e cance cells [20], in ansien ly silenced PLA2G7 siRNA ans ec ed b eas cance cells, he gene exp ession p o iling highligh ed he e ec o PLA2G7 on p oli e a ion (cell cycle) and apop osis in addi ion o cell mo ion and adhesion (supplemen a y ma e ial, Table S1). Howe e , unc- ional cell iabili y and apop osis assays wi h siRNA ea ed MDA-468 b eas cance cells showed only a mino dec ease in cell iabili y a he 48 hou s ime poin (supplemen a y ma e ial, Figu e S2B). In e es - ingly, he esul s om genome-wide gene exp ession p o iling using MDA-468/PLA2G7- cells indica ed ha PLA2G7 silencing dec eases he exp ession o genes associa ed wi h egula ion o cell mig a ion, epi helial cell di e en ia ion and EMT (Table 2). Suppo ing he ole o PLA2G7 in he egula ion o EMT as well as mig a ion and in asion, PLA2G7 impai men induced gene exp ession changes associa ed wi h IL-17A (in e leukin 17A), No ch and oncos a in M signalling. These p ocesses ha e all been p e iously associa ed wi h umou me as asis o EMT [38–42]. Valida ion o he e ec o PLA2G7 impai men on imen in, E-cadhe in and EGFR exp ession Two majo hallma ks o EMT a e he loss o exp es- sion o he epi helial cell adhesion molecule E- cadhe in and gain o exp ession o he majo mesen- chymal cell cy oskele al componen imen in. To alida e he ole o PLA2G7 in EMT, MDA-468/ PLA2G7- cells we e analysed o imen in and E- cadhe in p o ein exp ession. The esul s indica ed clea ly educed imen in and inc eased E-cadhe in p o ein le els (Figu e 4A). In addi ion, immuno- luo escence s aining o MDA-468/PLA2G7- cells showed a clea dec ease in imen in s aining in en- si y in compa ison o he co esponding con ol (Fig- u e 4B). Figu e 3. PLA2G7 mRNA is highly exp essed in iple nega i e b eas cance cell lines. (A) PLA2G7 mRNA exp ession in oes o- gen ecep o posi i e and oes ogen ecep o nega i e b eas cance cell lines. (B) Valida ion o s able PLA2G7 gene silencing in MDA-MB-468 b eas cance cells a he mRNA le el. Table 2. The e ec o s able PLA2G7 silencing on gene exp es- sion p o ile in MDA-MB-468 b eas cance cells Down (n 519) Biological p ocesses P alue Epi helial cell di e en ia ion 6.3E-3 Regula ion o cell mig a ion 9.4E-3 Epide mis de elopmen 1.1E-2 Regula ion o cell mo ion 1.2E-2 Ec ode m de elopmen 1.3E-2 Canonical Pa hways Bile Acid Biosyn hesis, Neu al Pa hway 1.28E-2 Regula ion o he Epi helial-Mesenchymal T ansi ion Pa hway 1.36E-2 Me hylglyoxal Deg ada ion III 1.48E-2 IL-17A Signalling in Gas ic Cells 2.45E-2 No ch Signalling 3.7E-2 Up (n 539) Biological p ocesses Immune esponse 2.5E-3 Canonical Pa hways P alue In e e on Signalling 1.35E-13 Role o Pa e n Recogni ion Recep o s in Recogni ion o Bac e ia and Vi uses 2.34E-5 Ac i a ion o IRF by Cy osolic Pa e n Recogni ion Recep o s 1.83E-4 Oncos a in M Signalling 1.63E-3 Role o IL-17A in Pso iasis 2.26E-2 The unc ional gene on ology and pa hway anno a ions we e analysed o he se s o uni o mly di e en ially exp essed genes (FC >1.5 o FC <0.666) in esponse o wo shRNAs. The analysis was conduc ed using DAVID Bioin- o ma ics Resou ces and Ingenui y Pa hway Analysis so wa e 130 L Leh inen e al V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 Figu e 4. PLA2G7 impai men dec eases he p o ein exp ession o imen in and inc eases he p o ein exp ession o E-cadhe in. (A) The e ec o s able PLA2G7 silencing on imen in and E-cadhe in p o ein exp ession in MDA-MB-468 cells. b-ac in exp ession was used as an endogenous con ol. (B) Immuno luo escence s aining o MDA-MB-468 cells wi hou (shSc ambled) o wi h s able PLA2G7 silencing (shPLA2G7) using imen in an ibody ( ed). DAPI s aining (blue) was used o isualize nuclei. 633objec i e magni ica ion. PLA2G7 in b eas cance 131 V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138 62. Reina z S, Finke nagel F, Adhika y T, e al. A ansc ip ome- based global map o signaling pa hways in he o a ian cance mic oen i onmen associa ed wi h clinical ou come. Genome Biol 2016; 17: 108. 63. Low HB, Png CW, Li C, e al. Monocy e-de i ed ac o s includ- ing PLA2G7 induced by mac ophage-nasopha yngeal ca cinoma cell in e ac ion p omo e umou cell in asi eness. Onco a ge 2016; 7: 55473–55490. SUPPLEMENTARY MATERIAL ONLINE Supplemen a y igu e legends Table S1. The e ec o ansien PLA2G7 silencing on b eas cance cell gene exp ession p o ile Figu e S1. The mRNA co-exp ession pa e n o PLA2G7 and HER2/neu (ERBB2) in b eas cance samples (n 5957) Figu e S2. (A) Valida ion o ansien PLA2G7 gene silencing in MDA-MB-468 b eas cance cells a he mRNA le el. (B) The e ec o 48 h PLA2G7 and KIF11 silencing on MDA-MB-468 cell iabili y. (C) The e ec o 48 h PLA2G7 and KIF11 silencing on induc ion o apop osis in MDA-MB-468 cells Figu e S3. The e ec o s able PLA2G7 silencing on EGFR p o ein exp ession in MDA-MB-468 cells. ac in exp ession was used as an endog- enous con ol Figu e S4. The e ec o s able PLA2G7 silencing and shSc ambled exp ession on MDA-MB-468 cell iabili y a 48 h a e pla ing equal num- be s o cells Figu e S5. Rela i e PLA2G7 mRNA exp ession in non-malignan and malignan o a ian, p os a e and hy oid issue samples based on TissueS- can TM Cance Su ey cDNA panel 138 L Leh inen e al V C2017 The Au ho s The Jou nal o Pa hology: Clinical Resea ch published by The Pa hological Socie y o G ea B i ain and I eland and John Wiley & Sons L d J Pa h: Clin Res Ap il 2017; 3: 123–138