Protein phosphatase methylesterase-1 (PME-1) expression predicts a favorable clinical outcome in colorectal cancer

1798 ORIGINAL RESEARCH P o ein phospha ase me hyles e ase- 1 (PME- 1) exp ession p edic s a a o able clinical ou come in colo ec al cance Amanp ee Kau 1,2,3,*, Adam Elzagheid1,4,5,*, E a-Ma ia Bi kman1, Tuulia A o an a1,6,7, Ville Ky ölä8, Eija Ko keila6, Ka i Sy jänen9,10, Jukka Wes e ma ck1,2 & Ja i Sunds öm1 1Depa men o Pa hology, Uni e si y o Tu ku, Tu ku 20520, Finland 2Tu ku Cen e o Bio echnology, Uni e si y o Tu ku and Åbo Akademi Uni e si y, Tu ku 20520, Finland 3TuBS and TuDMM Doc o al P og ammes, Tu ku 20520, Finland 4Depa men o Pa hology, Facul y o Medicine, Benghazi Uni e si yPO Box 1308 Benghazi, Libya 5Bio echnology Resea ch Cen e , T ipoli, Libya 6Depa men o Oncology and Radio he apy, Uni e si y o Tu ku and Tu ku Uni e si y Hospi al, Tu ku 20521, Finland 7Depa men o Social Se ices and Heal hca e, Ci y o Helsinki, Helsinki 00099, Finland 8BioMediTech, Uni e si y o Tampe e, Tampe e 33520, Finland 9Depa men o Clinical Resea ch, Biohi Oyj, Helsinki 00880, Finland 10Molecula Oncology Resea ch Cen e , Ba e os Cance Hospi al, Ba e os 14784-400, B azil © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Keywo ds Bioma ke , colo ec al cance , PME-1, PP2A, su i al, TCGA Co espondence Ja i Sunds öm, Depa men o Pa hology, Uni e si y o Tu ku, Tu ku 20520, Finland. Tel: +358 23131694; Fax: +358 23337459; E-mail: [email p o ec ed] Funding In o ma ion This s udy was suppo ed by he Founda ion o he Finnish Cance Ins i u e, Sig id Jusélius Founda ion, and Na ional Agency o Scien i ic Resea ch, T ipoli, Libya. Recei ed: 22 Ap il 2015; Re ised: 19 Augus 2015; Accep ed: 20 Augus 2015 Cance Medicine 2015; 4(12): 1798–1808 doi: 10.1002/cam4.541 *Equal con ibu ion. Abs ac Colo ec al cance (CRC) accoun s o high mo ali y. So a , he e is lack o ma ke s capable o p edic ing which pa ien s a e a isk o agg essi e cou se o he disease. P o ein phospha ase- 2A (PP2A) inhibi o p o eins ha e ecen ly gained in e es as ma ke s o mo e agg essi e disease in ce ain cance s. He e, we epo he ole o PP2A inhibi o PME- 1 in CRC. PME- 1 exp ession was assessed om a ec al cance pa ien coho by immunohis ochemis y, and co ela ions we e pe o med o a ious clinicopa hological a iables and pa ien su i al. Rec al cance pa ien s wi h highe cy oplasmic PME- 1 p o ein exp es- sion (abo e median) had less ecu ences (P = 0.003, n = 195) and be e disease- ee su i al (DFS) han he pa ien s wi h low cy oplasmic PME- 1 p o ein exp ession (below median). Analysis o PPME-1 mRNA exp ession om TCGA da ase o colon and ec al adenoca cinoma (COADREAD) pa ien coho con- i med high PPME1 exp ession as an independen p o ec i e ac o p edic ing a o able o e all su i al (OS) (P = 0.005, n = 396) compa ed o pa ien s wi h low PPME1 exp ession. CRC cell lines we e used o s udy he e ec o PME- 1 knockdown by siRNA on cell su i al. Con a y o o he cance ypes, PME- 1 inhibi ion in CRC cell lines did no educe he iabili y o cells o he exp es- sion o ac i e phospho yla ed AKT and ERK p o eins. In conclusion, PME- 1 exp ession p edic s o a a o able ou come o CRC pa ien s. The unexpec ed ole o PME- 1 in CRC in con as wi h he oncogenic ole o PP2A inhibi o p o eins in o he malignancies wa an s u he s udies o cance - speci ic unc- ion o each o hese p o eins. In oduc ion Colo ec al cance (CRC) is he hi d mos common ma- lignan neoplasm in many coun ies o he wes e n wo ld [1]. I s p ognosis has imp o ed g adually as a esul o ad ancemen s in su ge y and adju an chemo he apy [2]. Howe e , a signi ican p opo ion o he pa ien s s ill die o he disease [3]. The e is a need o bioma ke s o p edic which pa ien s a e a isk o disease ecu ence. This would help o di ec adju an ea men s o hose pa ien s ha gain bene i om hem and p o ec he low- isk pa ien s om he side e ec s o he apy. P o ein phospha ase- 2A (PP2A) is a human umo sup- p esso , which p o ec s agains cellula ans o ma ion. I is he majo se ine– h eonine phospha ase, which unc ions by nega i ely egula ing he ac i i y o nume ous signaling Cance Medicine Open Access 1799 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. PME- 1 P edic s Colo ec al Cance Ou comeA. Kau e al. p o eins impo an o malignan neoplasms [4–6]. Consequen ly, he neoplasms ha e de eloped a ious mechanisms o oppose he PP2A ac i i y [5, 7]. Among hese, h ee impo an endogenous PP2A inhibi o s ha e been ound: CIP2A, SET, and PME- 1 [5, 7–9]. O e exp ession o CIP2A on p o ein le el has been ound in many human neoplasms, and i is a ma ke o poo ou come in se e al malignan neoplasms including CRC [10, 11]. SET in u n seems o be pa icula ly impo an in hema ological malignancies [12]. PME- 1 exp ession has been s udied in only a limi ed numbe o human neo- plasms, such as as ocy ic gliomas, and endome ial, lung, and gas ic cance s [9, 13, 14]. Ele a ed amoun s o PME- 1 ha e been ound in endome ial and glial umo s, which a e linked o he al e ed ERK pa hway signaling, cell p o- li e a ion, and disease p og ession o gliomas o malignan sub ypes [9, 13]. Addi ionally, a small ac ion (3–4%) o he gas ic and lung cance pa ien s shows PPME1 gene ampli ica ion, which also co esponds o ele a ed PME- 1 p o ein exp ession and ac i a ion o ERK and AKT su i al signaling [14]. These s udies ha e highligh ed po en ial oncogenic ole o PME- 1 in hese malignan neoplasms. Howe e , whe he oncogenic unc ion o PME- 1 can be gene alized o a ious human cance ypes is as ye unclea . Also, he s udies so a ha e ailed o iden i y any co ela ion be ween umo PME- 1 exp ession and he pa ien su i al. In his s udy, we epo he immunohis ochemical analysis o PME- 1 p o ein exp ession in he umo ma e- ial o a ec al cance pa ien coho , and i s co ela ion o he clinicopa hological pa ame e s as well as pa ien su i al. Unexpec edly, in s ike con as wi h i s p e i- ously shown oncogenic ole in o he malignancies, we show ha high PME- 1 exp ession co ela es wi h supe io clinical ou come in CRC. The associa ion o high PME- 1 exp ession wi h be e pa ien su i al is con i med a he mRNA le el by using an independen CRC da ase . Finally, consis en wi h unexpec ed ole o PME- 1 in CRC, PME- 1 inhibi ion in wo human colon cance cell lines ails o show any inhibi o y e ec on ei he cell su i al o exp ession o phospho yla ed AKT o ERK, shown o be egula ed by PME- 1 in o he p e iously s udied cance ypes. Ma e ials and Me hods Cell cul u e and siRNA ans ec ions Human colon ca cinoma cell lines HCA- 7 and CW- 2 (gi ed by P o . Olli Ca pén, Uni e si y o Tu ku) we e cul u ed in DMEM (Sigma- Ald ich, Finland Oy, Helsinki, Finland) and RPMI (Sigma- Ald ich) media, espec i ely, supplemen ed wi h 10% hea - inac i a ed FBS (Gibco, The mo Fishe Scien i ic Inc., Rock o d, IL, USA), 2 mmol/L l- glu amine, and penicillin (50 uni s/mL)–s ep- omycin (50 μg/mL) in a humidi ied a mosphe e o 5% CO2 a 37°C. Small in e e ing RNA (siRNA) ans ec ions we e pe - o med wi h Lipo ec amine RNAiMAX eagen (In i ogen, Li e Technologies, Ca lsbad, CA, USA) acco ding o he manu ac u e ’s ins uc ions wi h he inal siRNA concen- a ion o 50 nmol/L pe well. The sc ambled (Sc ) o con ol (5′- GUA ACA AUG AGA GCA CGG C- 3′) and PME- 1- speci ic (5′- GGA AGU GAG UCU AUA AGC A- 3′) siRNAs we e pu chased om Eu o ins MWG Syn hesis GmbH, Ge many. Th ee days a e ans ec ions, cells we e ha es ed o analysis. Wes e n blo ing Cells we e lysed in 2× SDS sample bu e /Laemmli Bu e , boiled, and esol ed by SDS- PAGE. P o eins we e ans e ed o PVDF memb anes (Millipo e, Me ck KGaA, Da ms ad , Ge many), which we e blocked and incuba ed wi h equi ed dilu ion o p ima y (a +4°C, incuba ed o e nigh ) and 1:5000 dilu ion o seconda y an ibody (a oom empe a u e, o 1 h) in 5% milk- TBS- Tween- 20, and de eloped by enhanced chemilu- minescence (Pie ce Bio echnology, Rock o d, IL, USA). PME- 1, clone B- 12 (sc- 25278) an ibody used a 1:1000 dilu ion, and phospho yla ed AKT- 1/2/3 (Th 308) (sc- 16646) an ibody used a 1:500 dilu ion we e pu chased om San a C uz Bio echnology Inc., Dallas, TX, USA. An ibody o phospho yla ed ERK- 1/2 (Th 202/Ty 204) (#4370) used a 1:1000 dilu ion was pu chased om Cell Signalling Technology, Dan e s, MA, USA. Loading con ol an ibody o GAPDH (5G4- 6C5) (1:200,000 dilu ion) was om HyTes L d., Tu ku, Finland. Densi ome ic analysis o he blo s was pe o med using Image Lab so wa e (Bio-Rad Labo a o ies Inc., He cules, CA, USA). Immuno luo escence Cells we e seeded on glass co e slips and ans ec ed wi h siRNA. A e 3 days, cells we e ixed wi h 4% pa a o m- aldehyde (Sigma- Ald ich) and pe meabilized wi h 0.5% T i on X- 100 (Sigma- Ald ich) a oom empe a u e o 10 and 5 min, espec i ely. Immunos ainings we e ca ied ou wi h an i- PME- 1 an ibody (clone B- 12, sc- 25278) a 1:50 dilu ion in 10% goa se um blocking bu e , o e nigh a +4°C unde cons an ocking. Nega i e con ol co e slips we e incuba ed wi h blocking bu e alone (wi hou PME- 1 an ibody). A e 2–3 washes wi h PBS, co e slips we e incuba ed wi h Alexa- 594- conjuga ed goa an i- mouse seconda y an ibody (A- 11005; In i ogen, Li e Technologies 1800 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. A. Kau e al.PME- 1 P edic s Colo ec al Cance Ou come L d, Paisley, UK) o 1 h a oom empe a u e. Nuclei we e s ained wi h Hoechs 33342 (In i ogen), 1:2000 dilu ion in PBS o 10 min. Co e slips we e moun ed on glass slides o e a d op o Mowiol (Sigma- Ald ich), and images we e acqui ed wi h AxioVe 200M luo escence mic oscope (Ca l- Zeiss Mic oscopy GmbH, Gö ingen, Ge many) using 40× objec i e. Me ged images we e gen- e a ed wi h ImageJ [15] (Na ional Ins i u es o Heal h, Be hesda, MD). Cell iabili y assays Cell iabili y was de e mined by wo di e en assays; CellTi e - glo (CTG) (P omega Co p., Madison, WI, USA) and cell p oli e a ion eagen WST- 1 (Roche Diagnos ics GmbH, Mannheim, Ge many). CTG assay measu es he cellula ATP le els as an indica o o me abolically ac i e and iable cells. WST- 1 assay is dependen on NAD(P) H p oduc ion by glycolysis and he ac i i y o mi ochon- d ial dehyd ogenase enzymes, an indica o o me abolically ac i e iable cells. Bo h he assays we e pe o med as pe he manu ac u e ’s ecommenda ions. CTG assay was pe - o med in polys y ene 96- well pla es (Nunc, The mo Fishe Scien i ic Inc., Paisley, UK) and luminescence was measu ed wi h Syne gy H1 hyb id pla e- eade (BioTek, Winooski, VT, USA). The WST- 1 assay was pe o med in clea bo - om 96- well pla es and he abso bance was measu ed a 450 nm. Tumo samples Fo malin- ixed, pa a in- embedded umo samples we e collec ed om pa ien s ea ed o ec al cance a Tu ku Uni e si y Cen al Hospi al be ween 2000 and 2009. These included ope a i e samples (n = 210) wi h umo s o he middle and lowe ec um om he a chi es o he Depa men o Pa hology, Tu ku Uni e si y Hospi al. Supe icial umo s ope a ed by local excision we e excluded om he s udy as well as pa ien s wi h dis an me as ases a he ime o diagnosis. The pe mission o using he a chi al issue ma e ial was g an ed by he Na ional Supe iso y Au ho i y o Wel a e and Heal h, Finland (pe mission # Dn o 1709/32/300/02, 13 May 2002). Fo umo s aging, we applied he six h edi ion o TNM classi ica ion o malignan umo s in use a he ime he pa ien s we e ope a ed [16]. T ea men was chosen acco ding o p eope a i e umo s aging, includ- ing compu e ized omog aphy (CT) o magne ic eso- nance imaging (MRI) o he ec um, CT o he abdomen, and X- ay o CT o he ches . Pa ien s we e ea ed ei he wi h sho - cou se p eope a i e adio he apy (RT) (n = 88), long- cou se p eope a i e (chemo) RT (n = 52), o ecei ed no ea men be o e su ge y (n = 70) on he basis o common clinical ecommenda ions [17]. Sho - cou se RT was gi en 5 Gy ac ions on 5 days, and he pa ien s we e ope a ed on he ollowing week. Long- cou se RT was deli e ed in 1.8 Gy ac ions o a o al dose o 50.4 Gy in 6 weeks wi h (n = 43) o wi hou (n = 9) chemo he apy. A ha pe iod, pa ien s we e ope a ed 5–7 weeks a e RT. The cy os a ic ea - men included ei he bolus 5- luo ou acil (n = 4) o capeci abine (n = 39). The ype o su ge y was an e io esec ion among 113 (54%), and abdomino- pe ineal e- sec ion among 93 (44%) pa ien s. Fou (2%) pa ien s we e ope a ed wi h low Ha mann’s p ocedu e o o he ype o su ge y. The majo i y o he specimens (n = 154) we e sc eened o de ec ascula in asion, which was ound in 44 (28.6%) samples. Pa ien s wi h es ablished high- isk ea u es we e ea ed wi h adju an chemo- he apy. The median ollow- up ime was 62.5 mon hs. Disease ecu ence was obse ed among 65 (31%) pa ien s, ei he local o dis an one. The clinical in o ma ion o he pa ien s is shown in Table 1. Immunohis ochemis y Fou o he 210 pa ien s had no iable cance cells le a e p eope a i e ea men , classi ied as pT0. The amoun o cance cells was oo sca ce o a eliable e alua ion o immunohis ochemical s aining in addi ional 11 pa ien s. Consequen ly, samples o 195 pa ien s we e included in he inal analysis. The mos op imal pa a in blocks we e selec ed o ge enough umo ma e ial o analyses. Sec ions o 5 μm we e cu . The an igen e ie al was pe o med wi h mic owa e o en wice o 7 min in 10 mmol/L so- dium ci a e bu e , pH 9. Fo immunohis ochemical s aining, monoclonal mouse- an i- human PME- 1 (clone B12) an ibody epi ope co esponding o amino acids 161–386 o PME- 1 o human o igin (San a C uz Bio echnology, sc- 25278), a a dilu ion o 1:200 was used. Fo de ec ion, he EnVisionTM + Dual Link Sys em- HRP (Dako, Glos up, Denma k) was u ilized. Analysis o PME- 1 exp ession Two obse es blinded o he clinical da a e alua ed he cy oplasmic and nuclea IHC s aining o PME- 1 (A.E. all samples and E.M.B. 40 samples). Tissue om human glioblas oma was used as posi i e con ol. Bo h nuclea and cy oplasmic s aining was sco ed on a scale o ou in ensi y le els (+++, ++, +, −). The s ong s aining in ensi y (+++) co esponds o he posi i e con ol o PME- 1; weak s aining in ensi y can s ill be dis inguished om he backg ound. Mode a e s aining in ensi y is in e media e be ween he p e ious ones. A e es ima ing he p edominan cy oplasmic and nuclea s aining 1801 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. PME- 1 P edic s Colo ec al Cance Ou comeA. Kau e al. in ensi ies, s aining indices we e analyzed. These include he mos in ensi e cy oplasmic and nuclea indices. To calcula e hese indices, he a ea o he mos in ensi e s aining in cance cells was chosen om each sample. A e ha , he ac ions o cance cells belonging o each s aining in ensi y ca ego ies we e es ima ed. The ollowing o mula was used o calcula e he s aining indices: I = 0* 0 + 1* 1 + 2* 2 + 3* 3, whe e I is he s aining index and 0– 3 a e he ac ions o he cells showing a de ined le el o s aining ( om 0 o 3). Theo e ically, he index can a y be ween 0 and 3 [18]. E alua ion o he umo eg ession g ade Tumo eg ession g ade (TRG) a e long- cou se RT was es ima ed by a pa hologis (J.S.), using a scale o poo , mode a e, o excellen TRG, acco ding o a modi ied Dwo ak scale, as desc ibed p e iously [19]. B ie ly, poo TRG was de ined as minimal o no umo eg ession a e (chemo) RT. In case o poo esponse, a conside - able amoun o umo cells we e s ill emaining a e ea men . In umo s wi h mode a e esponse, he e we e only some umo cells o umo cell g oups le in he Table 1. The clinical cha ac e is ics o he ec al cance pa ien s. To al n = 210 Sho - cou se adio he apy, n (%) Long- cou se adio he apy, n (%) Con ol, n (%) Sex Male 119 54 (45) 32 (27) 33 (28) Female 91 34 (37) 20 (22) 37 (41) Mean age (yea s) 65 64 74 P eope a i e T1 T1–2 48 27 (56) 0 (0) 21 (44) T3 68 54 (79) 2 (3) 12 (18) T4 49 1 (2) 45 (92) 3 (6) Tx 45 6 (13) 5 (11) 34 (76) Pos ope a i e T1,2 T1 10 3 (30) 2 (20) 5 (50) T2 65 32 (49) 7 (11) 26 (40) T3 111 49 (44) 26 (24) 36 (32) T4 20 4 (20) 13 (65) 3 (15) T0 4 0 (0) 4 (100) 0 (0) Pos ope a i e N1 N0 124 51 (41) 34 (27) 39 (32) N1 55 25 (46) 14 (25) 16 (29) N2 28 12 (43) 4 (14) 12 (43) Nx 3 0 (0) 0 (0) 3 (100) Pos ope a i e s age1 S age I 55 26 (47) 4 (7) 25 (46) S age II 66 25 (38) 24 (36) 17 (26) S age III 85 37 (44) 20 (23) 28 (33) No iable umo le 4 0 (0) 4 (100) 0 (0) Pos ope a i e g ade3 G1 32 9 (28) 10 (31) 13 (41) G2 134 56 (42) 32 (24) 46 (34) G3 35 21 (60) 3 (9) 11 (31) Gx 9 2 (22) 7 (78) 0 (0) Ci cum e en ial ma gin 0 18 3 (17) 11 (61) 4 (22) 0≤ c m ≤221 8 (38) 6 (29) 7 (33) >2 120 65 (54) 25 (21) 30 (25) Unknown 51 12 (23) 10 (20) 29 (57) Disease- speci ic ou come Ali e wi hou ecu ence 114 59 (52) 22 (19) 33 (29) Ali e wi h ecu ence 9 3 (33) 3 (33) 3 (33) Died o disease 56 16 (28) 20 (36) 20 (36) Died o o he causes 31 10 (32) 7 (23) 14 (45) 1T, he ex en o umo in asion; N, nodal s a us, and s age acco ding o he TNM classi ica ion o malignan umo s [16]. 2Includes he T3 umo s wi h h ea ened ci cum e en ial ma gin in ol emen . 3Pos ope a i e umo di e en ia ion g ade. 1802 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. A. Kau e al.PME- 1 P edic s Colo ec al Cance Ou come p ima y umo (easy o ind). In umo s wi h excellen esponse, e y ew o no umo cells could be ound. S a is ical analysis S a is ical analyses o ec al cance da ase we e un using IBM SPSS S a is ics 22.0.0.1 o Windows (IBM Co po a ion, Some s, NY) so wa e package. F equency ables we e analyzed using he c2- es , wi h he likelihood a io (LR) o Fishe ’s exac es o ca ego ical a iables. Con ingency ables (2 × 2) we e used o calcula e odds a io (OR) and 95% con idence in e al (CI) using he exac me hod. Fishe ’s exac es , Spea man’s co ela ion, and LR we e used o assess he signi icance o he co - ela ion be ween indi idual a iables in uni a ia e analysis. In e obse e ep oducibili y o he assessmen s was es ed wi h weigh ed kappa, calcula ed using he in aclass co - ela ion coe icien (ICC) es , in pa allel mode wi h a wo- way andom model, using consis ency assump ion and he a e age- measu es op ion o in e p e he ICC (95% CI). The ICC o assessmen s was e y good, wi h weigh ed kappa alues a ound 0.8. Uni a ia e su i al analysis o disease- ee su i al (DFS) and disease- speci ic su i al (DSS) was based on he Kaplan–Meie me hod whe e s a um- speci ic ou comes we e compa ed using log- ank (Man el–Cox) s a is ics. To adjus o he co a ia es, a Cox p opo ional haza ds e- g ession model was used. Co a ia es (as lis ed sepa a ely in esul s) we e en e ed in a s epwise backwa d manne . The TCGA colon and ec um adenoca cinoma (COADREAD) exon exp ession by RNAseq (Illumina HiSeq) da ase (n = 416) was downloaded om UCSC cance genom- ics b owse , and analyzed by JMP P o 11.1.1 (SAS Ins i u e Inc., Ca y, NC, USA) so wa e. PPME1 exp ession dis ibu ion was s udied and o e all su i al (OS) es ima e cu es we e gene a ed using Kaplan–Meie me hod (n = 396). Log- ank chi- squa e es was used o assess he signi icance o co ela- ion be ween a iables. To s udy he signi icance o PPME1 mRNA exp ession compa ed o co a ia e a iables, Cox p opo ional haza ds eg ession model was i ed o he COADREAD da ase . Model i ing was done in R e sion 3.1.2 [20] using package “Su i al .2.37- 7” [21]. To make he esul s compa able, a se o co a ia es co esponding as closely o he clinical a iables in he ec al cance da ase as possible was agg ega ed om TCGA clinical da a. The s a is ical analyses o wes e n blo s was pe o med wi h MS excel using wo- ailed S uden ’s pai ed - es . All s a is ical es s we e wo- sided and decla ed signi ican a a P- alue o <0.05. Resul s Valida ion o he speci ici y o PME- 1 an ibody in CRC cell lines In o de o con i m he speci ici y o PME- 1 an ibody o be used o immunohis ochemis y o pa ien umo ma- e ial, we es ed a PME- 1 an ibody in colon cance cell lines, HCA- 7, and CW- 2. PME- 1- speci ic siRNA was used o knockdown i s exp ession in hese cells, which we e lysed and subjec ed o wes e n blo ing wi h PME- 1 (clone B- 12) mouse monoclonal an ibody (Fig. 1A). This an ibody Figu e 1. Valida ion o he speci ici y o PME- 1 an ibody in colo ec al cance cell lines. (A) Wes e n blo image o p o ein lysa es om HCA- 7 and CW- 2 cells ans ec ed wi h sc ambled (S) o PME- 1 (P) siRNA ( o 72 h), and blo ed wi h PME- 1 (B- 12) an ibody. GAPDH was used as a p o ein loading con ol. Black lines deno e he loca ion o p o ein molecula weigh ma ke bands. Immuno luo escence images o HCA- 7 (B) and CW- 2 (C) cells ans ec ed wi h Sc o PME- 1 siRNA ( o 72 h), and incuba ed wi h PME- 1 an ibody and isualized wi h an i- mouse- Alexa- 594 seconda y an ibody ( ed). Hoechs 33342 shows nuclea s aining (blue). PME- 1 and nuclea s aining o e lay is shown in me ge ( uchsia). All images we e aken a 40× magni ica ion. siRNA: Sc . PME-1 PME-1 Hoechs 33342 Me ge HCA-7 siRNA: Sc . PME-1 PME-1 Hoechs 33342 Me ge CW2 siRNA: PME-1 GAPDH HCA-7CW-2 170 130 100 70 55 40 35 25 S P S P AB C 1803 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. PME- 1 P edic s Colo ec al Cance Ou comeA. Kau e al. ecognized only one band co esponding o he molecula weigh o PME- 1 (44 kDa) in he con ol (Sc . siRNA ans ec ed) cells. The in ensi y o his band was g ea ly educed in he cells ans ec ed wi h PME- 1 siRNA, sug- ges ing ha he an ibody is speci ically ecognizing PME- 1 in hese cell lysa es. We used he same app oach o es he PME- 1 an ibody speci ici y by immuno luo escence, which co obo a ed he wes e n blo ing esul s in bo h HCA- 7 (Fig. 1B) and CW- 2 (Fig. 1C) cells. PME- 1 s ain- ing was p esen e enly in bo h cy oplasm and he nucleus in CW- 2 cells; howe e , he HCA- 7 cells displayed mo e in ense s aining in he nucleus as compa ed o he cy o- plasm (Fig. 1B). PME1 p o ein exp ession in ec al cance samples As obse ed in he colon cance cell lines, he PME- 1 s aining was localized bo h in he cy oplasm and nucleus, also in he sec ions made om pa a in blocks o clinical umo samples. The s aining in ensi y a ied om one umo o ano he , and he median o mos in ensi e PME- 1 index conce ning bo h cy oplasm and nucleus was 1.2. No mal ec al epi helium was mos ly nega i e, al hough in some samples he e was ain backg ound- like s aining in some a eas, especially in hose nea he cance cells. Examples o nega i e, weak, mode a e, and s ong PME- 1 exp ession by IHC a e illus a ed in Figu e 2A. PME- 1 p o ein exp ession ela ed o clinicopa hological a iables Among men, he mos in ensi e cy oplasmic index o PME- 1 was mo e o en below median han among women (P = 0.027, Pea son χ2 es ). The ecu en disease was mo e common among pa ien s wi h mos in ensi e cy- oplasmic index o PME- 1 below median han hose abo e median (P = 0.003, Pea son χ2 es ). The co ela ions o hese clinicopa hological a iables and PME- 1 s aining a e shown in Table 2. The e was no signi ican co ela ion be ween PME- 1 mos in ensi e cy oplasmic s aining index in ela ion o age, he nodal s a us, pos ope a i e T, Figu e 2. PME- 1 exp ession co ela es wi h be e su i al o colo ec al cance pa ien s. (A) Rep esen a i e images o PME- 1 immunohis ochemical s aining om ec al ca cinoma pa ien samples used o co ela ion analysis (sco ed 0–3). (B) Kaplan–Meie su i al cu e o disease- ee su i al (DFS) (in mon hs) analysis by PME- 1 mos in ensi e cy oplasmic s aining index in ec al cance pa ien coho (n = 195). (C) Kaplan–Meie su i al cu e o o e all su i al (OS) (in days) by PPME1 gene exp ession (RNAseq exon a ay) in TCGA colon and ec al adenoca cinoma (COADREAD) pa ien s (n = 396). AB C 0.0 0.2 0.4 0.6 0.8 1.0 050 100 150 200 Cummula i e su i al Disease- ee su i al (mon hs) Kaplan-Meie Su i al es ima es, by PME-1 mos in ensi e cy oplasmic s aining index n = 57 n = 138 PME-1 index (below median) PME-1 index (abo e median) Log-Rank es , P = 0.007 0.0 0.2 0.4 0.6 0.8 1.0 1000 2000 3000 4000 O e all su i al (OS) Analysis ime (days) Kaplan-Meie Su i al es ima es, by PPME1 RNAseq exon exp ession (COADREAD da abase) n = 249 n = 147 0 PPME1exp ession (high) PPME1exp ession (low) 0 1 2 3 Log-Rank es , P = 0.005 1804 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. A. Kau e al.PME- 1 P edic s Colo ec al Cance Ou come pos ope a i e s age, pos ope a i e g ade, ci cum e en ial ma gin, ascula in asion, o pos adio he apy (RT) TRG (Table S1). PME- 1 exp ession is ela ed o DFS o ec al cance pa ien s In he uni a ia e su i al analyses o he whole coho , umo s wi h he mos in ensi e cy oplasmic index o PME- 1 unde median we e linked o sho e DFS han hose o e median (110.7 s. 116.7 mon hs, P = 0.007, log- ank es ; Fig. 2B). The mul i a ia e (Cox) p opo ional haza ds eg ession o DFS was pe o med o he pa ien s wi h ollowing co- a ia es: ea men g oup, sex, age (70 yea s as cu o ), pos - ope a i e N (posi i e/nega i e), ascula in asion (posi i e/ nega i e), ci cum e en ial ma gin (2 mm as cu o ), and he mos in ensi e cy oplasmic index o PME- 1 (median as cu - o ). The ollowing emained as independen ac o s p edic ing a poo DFS o ec al cance pa ien s: sex i male (haza d a io [HR] 4.12; 95% CI 0.25–0.98; P = 0.042), pos ope a i e N (HR 10.33; 95% CI 1.68–8.40; P = 0.001), ci cum e en ial ma gin (HR 6.13; 95% CI 1.19–4.53; P = 0.013), and he mos in ensi e cy oplasmic index o PME- 1 (HR 9.28; 95% CI 1.78–14.31; P = 0.002). The mul i a ia e (Cox) Table 2. Associa ion o clinicopa hological a iables o ec al cance pa ien s wi h PME- 1 p o ein exp ession (mos in ensi e cy oplasmic index). Va iable To al n = 1951 PME- 1 mos in ensi e cy oplasmic index P- alue2 Below median, n (%) Abo e median, n (%) Sex Female 85 53 (62) 32 (38) 0.027 Male 110 85 (77) 25 (23) Recu ence Yes 57 49 (86) 8 (14) 0.003 No 138 89 (64) 49 (36) 1PME- 1 mos in ensi e cy oplasmic s aining index could be analyzed om 195 pa ien s only. 2Pea son chi- squa e es . Table 3. Mul i a ia e su i al analysis o ec al cance pa ien samples and TCGA colon and ec al adenoca cinoma (COADREAD) pa ien samples using Cox p opo ional haza ds eg ession models. Va iables Rec al cance (n = 142)1 Disease- ee su i al Rec al cance (n = 114)1 Disease- speci ic su i al TCGA COADREAD (n = 347)1 O e all su i al HR 95% CI P- alue HR 95% CI P- alue HR 95% CI P- alue Sex Female 1 1 1 Male 4.12 0.25–0.98 0.042 0.34 0.39–1.66 0.560 1.68 1.01–2.79 0.046 Age ≤70 yea s 1 1 1 >70 yea s 1.38 0.32–1.33 0.240 4.46 0.24–0.95 0.035 1.91 1.16–3.16 0.011 Ci cum e en ial ma gin ≤2 mm 1 1 >2 mm 6.13 1.19–4.53 0.013 1.63 0.78–3.20 0.201 Pos ope a i e N2 Nega i e (N0) 1 1 1 Posi i e (N1- 2) 10.33 1.68–8.40 0.001 0.79 0.54–4.99 0.374 3.15 1.79–5.53 <0.001 Vascula in asion3 Nega i e 1 1 1 Posi i e 1.93 0.82–3.16 0.165 0.42 0.36–1.66 0.519 1.47 0.86–2.48 0.162 Disease ecu ence No 1 Yes 21.62 15.50–840.54 <0.001 PME- 1 exp ession4,5 High 1 1 1 Low 9.28 1.78–14.31 0.00240.1 2.75–2.53 0.75142.22 1.32–3.72 0.0025 HR, haza d a io; 95% CI, 95% con idence in e al. S a is ically signi ican P- alues a e indica ed in bold. 1Only 142 and 114 Rec al cance pa ien s o disease- ee su i al and disease- speci ic su i al, espec i ely, and 347 TCGA COADREAD pa ien s o o e all su i al could be analyzed o which comple e da a we e a ailable o all he co a ia es. 2Pos ope a i e nodal s a us acco ding o he TNM classi ica ion o malignan umo s [16]. 3Fo TCGA da a, ascula in asion indica es combined lympha ic, and/o enous in asion s a us. 4PME- 1 mos in ensi e cy oplasmic index (p o ein exp ession) measu ed by IHC (cu - o median). 5PPME1 mRNA exp ession measu ed by RNA sequencing exon a ay Illumina HiSeq (cu o −0.075). 1805 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. PME- 1 P edic s Colo ec al Cance Ou comeA. Kau e al. p opo ional haza ds eg ession o DSS was pe o med o pa ien s wi h he simila co a ia es as ha o DFS added wi h disease ecu ence. The ollowing emained as inde- penden ac o s p edic ing a poo DSS: age i o e 70 yea s (HR 4.46; 95% CI 0.24–0.95; P = 0.035) and disease ecu - ence (HR 21.62; 95% CI 15.50–840.54; P < 0.001). The mul i a ia e (Cox) p opo ional haza ds eg ession esul s o DFS and DSS ha e been p esen ed in Table 3. PME- 1 gene exp ession co ela es wi h OS o CRC pa ien s In an independen colon and ec um adenoca cinoma (COADREAD, n = 396) RNA sequencing da ase (exon a ay IlluminaHiSeq) a ailable om TCGA [22], we ana- lyzed he co ela ion be ween OS and PPME1 gene ex- p ession using UCSC Cance Genomics B owse [23, 24]. Al hough he PPME1 exp ession was no mally dis ibu ed among his da ase , wo dis inc g oups can be clea ly isola ed using cu - o alue o −0.075 (Figu e S1A). Based on his cu - o alue, he da a we e ca ego ized in o wo g oups, low PPME1 (exp ession below −0.075) and high PPME1 (exp ession abo e −0.075) (Fig. 2C). This analysis e ealed a simila end a he mRNA exp ession le el as was seen o PME- 1 p o ein exp ession in ou ec al cance da ase . The pa ien g oup wi h high PPME1 gene exp es- sion (n = 249) showed be e OS (P = 0.005 log- ank χ2 es ) han he pa ien s wi h low PPME1 exp ession (n = 147). Simila esul s we e seen using median PPME1 exp ession (0.07014) as a cu o (Fig. 1B and C). The mul i a ia e analysis o TCGA COADREAD panel was pe o med by Cox p opo ional haza ds eg ession models o assess he e ec o o he possible a iables con ibu ing o he p ognosis o CRC pa ien s in he Kaplan–Meie su i al analysis. This analysis was ca ied ou in 347 pa ien s, o which ull da a was a ailable o he ollowing co a ia es: sex, age (cu o 70 yea s), pa ho- logic N (posi i e/nega i e), ascula in asion (posi i e/ nega i e), and PPME1 gene exp ession (cu o −0.075). This analysis e ealed low PPME1 exp ession as an in- dependen high- isk ac o (HR 2.22; 95% CI 1.32–3.72; P=0.002) p edic ing poo OS o CRC pa ien s. Addi ionally, male gende (HR 1.68; 95% CI 1.01–2.79; P = 0.046), high age (>70 yea s) (HR 1.91; 95% CI 1.16–3.16; Figu e 3. Modula ion o su i al signaling by PME- 1 in colo ec al cance cells. The iabili y o HCA- 7 and CW- 2 cells ans ec ed wi h Sc . o PME- 1 siRNA ( o 72 h) was analyzed by CellTi e - glo (CTG) (A) and WST- 1 (B) assays. Ba s ep esen old- change alues calcula ed o e espec i e alues o Sc . siRNA- ans ec ed cells (n = 3). Wes e n blo analysis o p o ein lysa es om siRNA- ans ec ed HCA- 7 (C) and CW- 2 (D) cells, using an ibodies o phospho yla ed o ms o AKT and ERK. The phosphop o ein exp ession no malized wi h GAPDH (loading con ol) is shown as old- change alues o e Sc . siRNA- ans ec ed cells (n = 3). *P = 0.003 by S uden ’s pai ed - es . 0.00 0.20 0.40 0.60 0.80 1.00 1.20 Sc PME-1 Sc PME-1 Cell ialibi y (Fold change) HCA-7CW-2 siRNA: WST-1 assay 0.00 0.20 0.40 0.60 0.80 1.00 1.20 Sc PME-1 Sc PME-1 Cell ialibi y (Fold change) HCA-7CW-2 siRNA: CTG assay AB PME-1 siRNA: PME-1Sc GAPDH CW-2 pAKT pERK PME-1 siRNA: PME-1Sc GAPDH HCA-7 pAKT pERK CD 0 0.5 1 1.5 2 pAKT pERK No malized exp ession old change o e Sc . Sc siRNA PME-1 siRNA 0 0.5 1 1.5 2 2.5 pAKT pERK N o malized exp ession old change o e Sc . Sc siRNA PME-1 siRNA * 1806 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d. A. Kau e al.PME- 1 P edic s Colo ec al Cance Ou come P = 0.011), and pa hologic N posi i i y (HR 3.15; 95% CI 1.79–5.53; P < 0.001) we e signi ican isk ac o s p edic ing poo OS o CRC pa ien s. These esul s ha e been p esen ed in Table 3. The mul i a ia e analysis o TCGA COADREAD panel ca ied ou using abo e men- ioned co a ia es and median PME- 1 exp ession as cu o also showed simila esul s (Table S2). Modula ion o su i al signaling by PME- 1 in CRC cells PME- 1 silencing has been shown o inhibi iabili y and educe he phospho yla ed AKT and ERK le els in he glioblas oma cells [9] as well as in PME- 1 ampli ied gas ic and lung cance cells [14]. To s udy he al e a ions in su i al signaling by PME- 1 in colon cance , PME- 1 speci ic siRNA was employed o knockdown i s exp es- sion and he iabili y was analyzed in CW- 2 and HCA- 7 cell lines. Nei he o he cell lines displayed signi ican di e ence in iabili y upon PME- 1 silencing, con i med by using wo di e en cell iabili y assays (Fig. 3A and B). Fu he immunoblo ing analysis e ealed ha in con as o o he cance ypes, PME- 1 silencing did no inhibi exp ession o ac i e phospho yla ed o ms o se - ine– h eonine- speci ic p o ein kinases AKT- 1/2/3 (p- AKT) and ERK- 1/2 (p- ERK) in ei he o he colon cance cell lines (Fig. 3C and D). In ac , he exp ession o p- AKT in HCA- 7 cells was signi ican ly highe ( wo old, P = 0.003) in PME- 1 silenced cells as compa ed o he con ol (Sc . siRNA) cells (Fig. 3C); and e en hough he o he e ec s did no each he s a is ical signi icance, he e was a clea end owa d highe p- AKT also in CW- 2 cells and highe p- ERK in bo h he cells lines upon PME- 1 inhibi ion. These indings demons a e ha opposi e o o he cane ypes PME- 1 may e en dec ease su i al signaling in CRC cells and his may be linked o be e pa ien ou come o ec al cance pa ien s wi h highe PME- 1 exp ession. Discussion PME- 1 exp ession on p o ein le el was s udied in 195 ec al cance pa ien s. Among hese pa ien s, 52 we e ea ed wi h long- cou se (chemo) RT, 88 wi h sho - cou se RT, and 70 wi h su ge y only. Ou aim was o es PME- 1 as a po en ial ma ke o p edic he ou come o ec al cance pa ien s. The e is li le in o ma ion a ailable conce ning he ole o PME- 1 o pa ien ou come in human malignancies. Based on i s molecula unc ion as a PP2A inhibi o p o- ein such as CIP2A [8], i would be expec ed ha PME- 1 would also ac as a human oncop o ein. I has been shown ha PME- 1 is linked wi h he malignan p og ession o as ocy ic gliomas [9] and endome ial cance s [13]. Fo his eason i is su p ising ha PME- 1 p o ein exp ession seems o ac in an opposi e way in ec al cance . High p o ein exp ession o PME- 1 is a ma ke o a o able ou come in uni a ia e analysis o DFS o he whole co- ho , and i emained an independen p ognos ica o o DFS oge he wi h sex, pos ope a i e N s a us, and ci - cum e en ial ma gin. Impo an ly, umo s wi h high PME- 1 p o ein exp ession we e associa ed wi h ewe ecu ences and a be e disease ou come han hose wi h low PME- 1 p o ein exp ession. The impo ance o hese indings was u he highligh ed by he obse a ion ha in addi ion o he ec al cance , colon cance pa ien s om an in- dependen coho (COADREAD) could be ca ego ized in o wo g oups based on PPME1 mRNA exp ession, which co espond o di e en ial OS ou come in bo h uni a ia e and mul i a ia e su i al analyses. This in o ma ion can be use ul o es ablishing PCR es s based on PPME1 exp ession as a bioma ke o p edic he su i al o CRC pa ien s [25, 26]. PME- 1 exp ession is known o be associa ed wi h inc eased cell p oli e a ion and su i al signaling in human malignan gliomas and endome ial cance s [9, 13]. We s udied hese cellula unc ions in CRC cells. Since PP2A inhibi ion by PME- 1 has been p e iously shown o p omo e phospho yla ion o he AKT and ERK p o eins, i is su p ising ha in CRC cells hese p osu i al signaling e en s a e la gely una ec ed o a ec ed in an opposi e manne as compa ed o he o he ypes o cance . Mechanis ically, PME- 1 p omo es ERK pa hway signaling mainly a a le el ups eam o Ra [9]. Howe e , a signi ican p opo ion o he CRCs con ain ac i a ing mu a ions in K- Ras (50%) and B- Ra (10%), which p omo e ERK signaling independen o he ups eam s imuli [27]. In addi ion, he TGFα- media ed au oc ine eedback loop can u he inc ease he Ras- Ra - ERK pa hway signaling [27, 28]. These al e a ions may ende he CRC cells insensi i e o PME- 1- media ed egula ion. Fu he mo e, inac i a ing mu a ions and/o al e ed exp ession o a ious PP2A componen s [29, 30], speci ically he B- subuni s egu- la ed by PME- 1, migh a ec he ac i i y (phospho yla- ion) o speci ic a ge p o eins. To his end, among he PP2A B- subuni s o PPP2R2 (B55) amily, which a e exclusi ely sensi i e o PP2A C- subuni Leu309 me hyla ion e e sibly emo ed by PME- 1 [31], he PPP2R2B (B55β) subuni is epigene ically silenced by DNA hype me hyla ion in >90% o CRCs [32]. The loss o his B- subuni p omo es d ug esis ance in CRC by ac i a ing he su i al signaling in a PI3K- AKT independen manne [32]. I may be specula ed ha ou su p ising esul o a a o able ou come wi h high PME- 1 exp ession is ela ed o hese al e ed pa hways