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

1798 ORIGINAL RESEARCH Protein phosphatase methylesterase1 (PME1) expression predicts a favorable clinical outcome in colorectal cancer Amanpreet Kaur1,2,3,*, Adam Elzagheid1,4,5,*, Eva-Maria Birkman1, Tuulia Avoranta1,6,7, Ville Kytölä8, Eija Korkeila6, Kari Syrjänen9,10, Jukka Westermarck1,2 & Jari Sundström1 1Department of Pathology, University of Turku, Turku 20520, Finland 2Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku 20520, Finland 3TuBS and TuDMM Doctoral Programmes, Turku 20520, Finland 4Department of Pathology, Faculty of Medicine, Benghazi UniversityPO Box 1308 Benghazi, Libya 5Biotechnology Research Center, Tripoli, Libya 6Department of Oncology and Radiotherapy, University of Turku and Turku University Hospital, Turku 20521, Finland 7Department of Social Services and Healthcare, City of Helsinki, Helsinki 00099, Finland 8BioMediTech, University of Tampere, Tampere 33520, Finland 9Department of Clinical Research, Biohit Oyj, Helsinki 00880, Finland 10Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos 14784-400, Brazil © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Keywords Biomarker, colorectal cancer, PME-1, PP2A, survival, TCGA Correspondence Jari Sundström, Department of Pathology, University of Turku, Turku 20520, Finland. Tel: +358 23131694; Fax: +358 23337459; E-mail: [email protected] Funding Information This study was supported by the Foundation for the Finnish Cancer Institute, Sigrid Jusélius Foundation, and National Agency for Scientific Research, Tripoli, Libya. Received: 22 April 2015; Revised: 19 August 2015; Accepted: 20 August 2015 Cancer Medicine 2015; 4(12): 1798–1808 doi: 10.1002/cam4.541 *Equal contribution. Abstract Colorectal cancer (CRC) accounts for high mortality. So far, there is lack of markers capable of predicting which patients are at risk of aggressive course of the disease. Protein phosphatase2A (PP2A) inhibitor proteins have recently gained interest as markers of more aggressive disease in certain cancers. Here, we report the role of PP2A inhibitor PME1 in CRC. PME1 expression was assessed from a rectal cancer patient cohort by immunohistochemistry, and correlations were performed for various clinicopathological variables and patient survival. Rectal cancer patients with higher cytoplasmic PME1 protein expression (above median) had less recurrences (P = 0.003, n = 195) and better diseasefree survival (DFS) than the patients with low cytoplasmic PME1 protein expression (below median). Analysis of PPME-1 mRNA expression from TCGA dataset of colon and rectal adenocarcinoma (COADREAD) patient cohort confirmed high PPME1 expression as an independent protective factor predicting favorable overall survival (OS) (P = 0.005, n = 396) compared to patients with low PPME1 expression. CRC cell lines were used to study the effect of PME1 knockdown by siRNA on cell survival. Contrary to other cancer types, PME1 inhibition in CRC cell lines did not reduce the viability of cells or the expression of active phosphorylated AKT and ERK proteins. In conclusion, PME1 expression predicts for a favorable outcome of CRC patients. The unexpected role of PME1 in CRC in contrast with the oncogenic role of PP2A inhibitor proteins in other malignancies warrants further studies of cancerspecific function for each of these proteins. Introduction Colorectal cancer (CRC) is the third most common malignant neoplasm in many countries of the western world [1]. Its prognosis has improved gradually as a result of advancements in surgery and adjuvant chemotherapy [2]. However, a significant proportion of the patients still die of the disease [3]. There is a need for biomarkers to predict which patients are at risk of disease recurrence. This would help to direct adjuvant treatments to those patients that gain benefit from them and protect the lowrisk patients from the side effects of therapy. Protein phosphatase2A (PP2A) is a human tumor suppressor, which protects against cellular transformation. It is the major serine–threonine phosphatase, which functions by negatively regulating the activity of numerous signaling Cancer Medicine Open Access 1799 © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. PME1 Predicts Colorectal Cancer OutcomeA. Kaur et al. proteins important for malignant neoplasms [4–6]. Consequently, the neoplasms have developed various mechanisms to oppose the PP2A activity [5, 7]. Among these, three important endogenous PP2A inhibitors have been found: CIP2A, SET, and PME1 [5, 7–9]. Overexpression of CIP2A on protein level has been found in many human neoplasms, and it is a marker of poor outcome in several malignant neoplasms including CRC [10, 11]. SET in turn seems to be particularly important in hematological malignancies [12]. PME1 expression has been studied in only a limited number of human neoplasms, such as astrocytic gliomas, and endometrial, lung, and gastric cancers [9, 13, 14]. Elevated amounts of PME1 have been found in endometrial and glial tumors, which are linked to the altered ERK pathway signaling, cell proliferation, and disease progression of gliomas to malignant subtypes [9, 13]. Additionally, a small fraction (3–4%) of the gastric and lung cancer patients shows PPME1 gene amplification, which also corresponds to elevated PME1 protein expression and activation of ERK and AKT survival signaling [14]. These studies have highlighted potential oncogenic role of PME1 in these malignant neoplasms. However, whether oncogenic function of PME1 can be generalized to various human cancer types is as yet unclear. Also, the studies so far have failed to identify any correlation between tumor PME1 expression and the patient survival. In this study, we report the immunohistochemical analysis of PME1 protein expression in the tumor material of a rectal cancer patient cohort, and its correlation to the clinicopathological parameters as well as patient survival. Unexpectedly, in strike contrast with its previously shown oncogenic role in other malignancies, we show that high PME1 expression correlates with superior clinical outcome in CRC. The association of high PME1 expression with better patient survival is confirmed at the mRNA level by using an independent CRC dataset. Finally, consistent with unexpected role for PME1 in CRC, PME1 inhibition in two human colon cancer cell lines fails to show any inhibitory effect on either cell survival or expression of phosphorylated AKT or ERK, shown to be regulated by PME1 in other previously studied cancer types. Materials and Methods Cell culture and siRNA transfections Human colon carcinoma cell lines HCA7 and CW2 (gifted by Prof. Olli Carpén, University of Turku) were cultured in DMEM (SigmaAldrich, Finland Oy, Helsinki, Finland) and RPMI (SigmaAldrich) media, respectively, supplemented with 10% heatinactivated FBS (Gibco, Thermo Fisher Scientific Inc., Rockford, IL, USA), 2 mmol/L lglutamine, and penicillin (50 units/mL)–streptomycin (50 μg/mL) in a humidified atmosphere of 5% CO2 at 37°C. Small interfering RNA (siRNA) transfections were performed with Lipofectamine RNAiMAX reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions with the final siRNA concentration of 50 nmol/L per well. The scrambled (Scr) or control (5′- GUA ACA AUG AGA GCA CGG C3′) and PME1specific (5′- GGA AGU GAG UCU AUA AGC A3′) siRNAs were purchased from Eurofins MWG Synthesis GmbH, Germany. Three days after transfections, cells were harvested for analysis. Western blotting Cells were lysed in 2× SDS sample buffer/Laemmli Buffer, boiled, and resolved by SDSPAGE. Proteins were transferred to PVDF membranes (Millipore, Merck KGaA, Darmstadt, Germany), which were blocked and incubated with required dilution of primary (at +4°C, incubated overnight) and 1:5000 dilution of secondary antibody (at room temperature, for 1 h) in 5% milkTBSTween20, and developed by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA). PME1, clone B12 (sc25278) antibody used at 1:1000 dilution, and phosphorylated AKT1/2/3 (Thr308) (sc16646) antibody used at 1:500 dilution were purchased from Santa Cruz Biotechnology Inc., Dallas, TX, USA. Antibody for phosphorylated ERK1/2 (Thr202/Tyr204) (#4370) used at 1:1000 dilution was purchased from Cell Signalling Technology, Danvers, MA, USA. Loading control antibody for GAPDH (5G46C5) (1:200,000 dilution) was from HyTest Ltd., Turku, Finland. Densitometric analysis of the blots was performed using Image Lab software (Bio-Rad Laboratories Inc., Hercules, CA, USA). Immunofluorescence Cells were seeded on glass coverslips and transfected with siRNA. After 3 days, cells were fixed with 4% paraformaldehyde (SigmaAldrich) and permeabilized with 0.5% Triton X100 (SigmaAldrich) at room temperature for 10 and 5 min, respectively. Immunostainings were carried out with antiPME1 antibody (clone B12, sc25278) at 1:50 dilution in 10% goat serum blocking buffer, overnight at +4°C under constant rocking. Negative control coverslips were incubated with blocking buffer alone (without PME1 antibody). After 2–3 washes with PBS, coverslips were incubated with Alexa594conjugated goat antimouse secondary antibody (A11005; Invitrogen, Life Technologies 1800 © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. A. Kaur et al.PME1 Predicts Colorectal Cancer Outcome Ltd, Paisley, UK) for 1 h at room temperature. Nuclei were stained with Hoechst 33342 (Invitrogen), 1:2000 dilution in PBS for 10 min. Coverslips were mounted on glass slides over a drop of Mowiol (SigmaAldrich), and images were acquired with AxioVert 200M fluorescence microscope (CarlZeiss Microscopy GmbH, Göttingen, Germany) using 40× objective. Merged images were generated with ImageJ [15] (National Institutes of Health, Bethesda, MD). Cell viability assays Cell viability was determined by two different assays; CellTiterglo (CTG) (Promega Corp., Madison, WI, USA) and cell proliferation reagent WST1 (Roche Diagnostics GmbH, Mannheim, Germany). CTG assay measures the cellular ATP levels as an indicator of metabolically active and viable cells. WST1 assay is dependent on NAD(P) H production by glycolysis and the activity of mitochondrial dehydrogenase enzymes, an indicator of metabolically active viable cells. Both the assays were performed as per the manufacturer’s recommendations. CTG assay was performed in polystyrene 96well plates (Nunc, Thermo Fisher Scientific Inc., Paisley, UK) and luminescence was measured with Synergy H1 hybrid plate-reader (BioTek, Winooski, VT, USA). The WST1 assay was performed in clear bottom 96well plates and the absorbance was measured at 450 nm. Tumor samples Formalinfixed, paraffinembedded tumor samples were collected from patients treated for rectal cancer at Turku University Central Hospital between 2000 and 2009. These included operative samples (n = 210) with tumors of the middle and lower rectum from the archives of the Department of Pathology, Turku University Hospital. Superficial tumors operated by local excision were excluded from the study as well as patients with distant metastases at the time of diagnosis. The permission for using the archival tissue material was granted by the National Supervisory Authority for Welfare and Health, Finland (permission # Dnro 1709/32/300/02, 13 May 2002). For tumor staging, we applied the sixth edition of TNM classification of malignant tumors in use at the time the patients were operated [16]. Treatment was chosen according to preoperative tumor staging, including computerized tomography (CT) or magnetic resonance imaging (MRI) of the rectum, CT of the abdomen, and Xray or CT of the chest. Patients were treated either with shortcourse preoperative radiotherapy (RT) (n = 88), longcourse preoperative (chemo) RT (n = 52), or received no treatment before surgery (n = 70) on the basis of common clinical recommendations [17]. Shortcourse RT was given 5 Gy fractions on 5 days, and the patients were operated on the following week. Longcourse RT was delivered in 1.8 Gy fractions to a total dose of 50.4 Gy in 6 weeks with (n = 43) or without (n = 9) chemotherapy. At that period, patients were operated 5–7 weeks after RT. The cytostatic treatment included either bolus 5fluorouracil (n = 4) or capecitabine (n = 39). The type of surgery was anterior resection among 113 (54%), and abdominoperineal resection among 93 (44%) patients. Four (2%) patients were operated with low Hartmann’s procedure or other type of surgery. The majority of the specimens (n = 154) were screened to detect vascular invasion, which was found in 44 (28.6%) samples. Patients with established highrisk features were treated with adjuvant chemotherapy. The median followup time was 62.5 months. Disease recurrence was observed among 65 (31%) patients, either local or distant one. The clinical information of the patients is shown in Table 1. Immunohistochemistry Four of the 210 patients had no viable cancer cells left after preoperative treatment, classified as pT0. The amount of cancer cells was too scarce for a reliable evaluation of immunohistochemical staining in additional 11 patients. Consequently, samples of 195 patients were included in the final analysis. The most optimal paraffin blocks were selected to get enough tumor material for analyses. Sections of 5 μm were cut. The antigen retrieval was performed with microwave oven twice for 7 min in 10 mmol/L sodium citrate buffer, pH 9. For immunohistochemical staining, monoclonal mouseantihuman PME1 (clone B12) antibody epitope corresponding to amino acids 161–386 of PME1 of human origin (Santa Cruz Biotechnology, sc25278), at a dilution of 1:200 was used. For detection, the EnVisionTM + Dual Link SystemHRP (Dako, Glostrup, Denmark) was utilized. Analysis of PME1 expression Two observes blinded to the clinical data evaluated the cytoplasmic and nuclear IHC staining of PME1 (A.E. all samples and E.M.B. 40 samples). Tissue from human glioblastoma was used as positive control. Both nuclear and cytoplasmic staining was scored on a scale of four intensity levels (+++, ++, +, −). The strong staining intensity (+++) corresponds to the positive control of PME1; weak staining intensity can still be distinguished from the background. Moderate staining intensity is intermediate between the previous ones. After estimating the predominant cytoplasmic and nuclear staining 1801 © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. PME1 Predicts Colorectal Cancer OutcomeA. Kaur et al. intensities, staining indices were analyzed. These include the most intensive cytoplasmic and nuclear indices. To calculate these indices, the area of the most intensive staining in cancer cells was chosen from each sample. After that, the fractions of cancer cells belonging to each staining intensity categories were estimated. The following formula was used to calculate the staining indices: I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where I is the staining index and f0–f3 are the fractions of the cells showing a defined level of staining (from 0 to 3). Theoretically, the index can vary between 0 and 3 [18]. Evaluation of the tumor regression grade Tumor regression grade (TRG) after longcourse RT was estimated by a pathologist (J.S.), using a scale of poor, moderate, or excellent TRG, according to a modified Dworak scale, as described previously [19]. Briefly, poor TRG was defined as minimal or no tumor regression after (chemo) RT. In case of poor response, a considerable amount of tumor cells were still remaining after treatment. In tumors with moderate response, there were only some tumor cells or tumor cell groups left in the Table 1. The clinical characteristics of the rectal cancer patients. Total n = 210 Shortcourse radiotherapy, n (%) Longcourse radiotherapy, n (%) Control, n (%) Sex Male 119 54 (45) 32 (27) 33 (28) Female 91 34 (37) 20 (22) 37 (41) Mean age (years) 65 64 74 Preoperative 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) Postoperative 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) Postoperative 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) Postoperative stage1 Stage I 55 26 (47) 4 (7) 25 (46) Stage II 66 25 (38) 24 (36) 17 (26) Stage III 85 37 (44) 20 (23) 28 (33) No viable tumor left 4 0 (0) 4 (100) 0 (0) Postoperative grade3 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) Circumferential margin 0 18 3 (17) 11 (61) 4 (22) 0≤ crm ≤221 8 (38) 6 (29) 7 (33) >2 120 65 (54) 25 (21) 30 (25) Unknown 51 12 (23) 10 (20) 29 (57) Diseasespecific outcome Alive without recurrence 114 59 (52) 22 (19) 33 (29) Alive with recurrence 9 3 (33) 3 (33) 3 (33) Died of disease 56 16 (28) 20 (36) 20 (36) Died of other causes 31 10 (32) 7 (23) 14 (45) 1T, the extent of tumor invasion; N, nodal status, and stage according to the TNM classification of malignant tumors [16]. 2Includes the T3 tumors with threatened circumferential margin involvement. 3Postoperative tumor differentiation grade. 1802 © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. A. Kaur et al.PME1 Predicts Colorectal Cancer Outcome primary tumor (easy to find). In tumors with excellent response, very few or no tumor cells could be found. Statistical analysis Statistical analyses for rectal cancer dataset were run using IBM SPSS Statistics 22.0.0.1 for Windows (IBM Corporation, Somers, NY) software package. Frequency tables were analyzed using the c2test, with the likelihood ratio (LR) or Fisher’s exact test for categorical variables. Contingency tables (2 × 2) were used to calculate odds ratio (OR) and 95% confidence interval (CI) using the exact method. Fisher’s exact test, Spearman’s correlation, and LR were used to assess the significance of the correlation between individual variables in univariate analysis. Interobserver reproducibility of the assessments was tested with weighted kappa, calculated using the intraclass correlation coefficient (ICC) test, in parallel mode with a twoway random model, using consistency assumption and the averagemeasures option to interpret the ICC (95% CI). The ICC of assessments was very good, with weighted kappa values around 0.8. Univariate survival analysis for diseasefree survival (DFS) and diseasespecific survival (DSS) was based on the Kaplan–Meier method where stratumspecific outcomes were compared using logrank (Mantel–Cox) statistics. To adjust for the covariates, a Cox proportional hazards regression model was used. Covariates (as listed separately in results) were entered in a stepwise backward manner. The TCGA colon and rectum adenocarcinoma (COADREAD) exon expression by RNAseq (Illumina HiSeq) dataset (n = 416) was downloaded from UCSC cancer genomics browser, and analyzed by JMP Pro 11.1.1 (SAS Institute Inc., Cary, NC, USA) software. PPME1 expression distribution was studied and overall survival (OS) estimate curves were generated using Kaplan–Meier method (n = 396). Logrank chisquare test was used to assess the significance of correlation between variables. To study the significance of PPME1 mRNA expression compared to covariate variables, Cox proportional hazards regression model was fitted for the COADREAD dataset. Model fitting was done in R version 3.1.2 [20] using package “Survival v.2.377” [21]. To make the results comparable, a set of covariates corresponding as closely to the clinical variables in the rectal cancer dataset as possible was aggregated from TCGA clinical data. The statistical analyses of western blots was performed with MS excel using twotailed Student’s paired ttest. All statistical tests were twosided and declared significant at a Pvalue of <0.05. Results Validation of the specificity of PME1 antibody in CRC cell lines In order to confirm the specificity of PME1 antibody to be used for immunohistochemistry of patient tumor material, we tested a PME1 antibody in colon cancer cell lines, HCA7, and CW2. PME1specific siRNA was used to knockdown its expression in these cells, which were lysed and subjected to western blotting with PME1 (clone B12) mouse monoclonal antibody (Fig. 1A). This antibody Figure 1. Validation of the specificity of PME1 antibody in colorectal cancer cell lines. (A) Western blot image of protein lysates from HCA7 and CW2 cells transfected with scrambled (S) or PME1 (P) siRNA (for 72 h), and blotted with PME1 (B12) antibody. GAPDH was used as a protein loading control. Black lines denote the location of protein molecular weight marker bands. Immunofluorescence images of HCA7 (B) and CW2 (C) cells transfected with Scr or PME1 siRNA (for 72 h), and incubated with PME1 antibody and visualized with antimouseAlexa594 secondary antibody (red). Hoechst 33342 shows nuclear staining (blue). PME1 and nuclear staining overlay is shown in merge (fuchsia). All images were taken at 40× magnification. siRNA: Scr. PME-1 PME-1 Hoechst 33342 Merge HCA-7 siRNA: Scr. PME-1 PME-1 Hoechst 33342 Merge 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 Authors. Cancer Medicine published by John Wiley & Sons Ltd. PME1 Predicts Colorectal Cancer OutcomeA. Kaur et al. recognized only one band corresponding to the molecular weight of PME1 (44 kDa) in the control (Scr. siRNA transfected) cells. The intensity of this band was greatly reduced in the cells transfected with PME1 siRNA, suggesting that the antibody is specifically recognizing PME1 in these cell lysates. We used the same approach to test the PME1 antibody specificity by immunofluorescence, which corroborated the western blotting results in both HCA7 (Fig. 1B) and CW2 (Fig. 1C) cells. PME1 staining was present evenly in both cytoplasm and the nucleus in CW2 cells; however, the HCA7 cells displayed more intense staining in the nucleus as compared to the cytoplasm (Fig. 1B). PME1 protein expression in rectal cancer samples As observed in the colon cancer cell lines, the PME1 staining was localized both in the cytoplasm and nucleus, also in the sections made from paraffin blocks of clinical tumor samples. The staining intensity varied from one tumor to another, and the median of most intensive PME1 index concerning both cytoplasm and nucleus was 1.2. Normal rectal epithelium was mostly negative, although in some samples there was faint backgroundlike staining in some areas, especially in those near the cancer cells. Examples of negative, weak, moderate, and strong PME1 expression by IHC are illustrated in Figure 2A. PME1 protein expression related to clinicopathological variables Among men, the most intensive cytoplasmic index of PME1 was more often below median than among women (P = 0.027, Pearson χ2 test). The recurrent disease was more common among patients with most intensive cytoplasmic index of PME1 below median than those above median (P = 0.003, Pearson χ2 test). The correlations of these clinicopathological variables and PME1 staining are shown in Table 2. There was no significant correlation between PME1 most intensive cytoplasmic staining index in relation to age, the nodal status, postoperative T, Figure 2. PME1 expression correlates with better survival of colorectal cancer patients. (A) Representative images of PME1 immunohistochemical staining from rectal carcinoma patient samples used for correlation analysis (scored 0–3). (B) Kaplan–Meier survival curve for diseasefree survival (DFS) (in months) analysis by PME1 most intensive cytoplasmic staining index in rectal cancer patient cohort (n = 195). (C) Kaplan–Meier survival curve for overall survival (OS) (in days) by PPME1 gene expression (RNAseq exon array) in TCGA colon and rectal adenocarcinoma (COADREAD) patients (n = 396). AB C 0.0 0.2 0.4 0.6 0.8 1.0 050 100 150 200 Cummulative survival Disease-free survival (months) Kaplan-Meier Survival estimates, by PME-1 most intensive cytoplasmic staining index n = 57 n = 138 PME-1 index (below median) PME-1 index (above median) Log-Rank test, P = 0.007 0.0 0.2 0.4 0.6 0.8 1.0 1000 2000 3000 4000 Overall survival (OS) Analysis time (days) Kaplan-Meier Survival estimates, by PPME1 RNAseq exon expression (COADREAD database) n = 249 n = 147 0 PPME1expression (high) PPME1expression (low) 0 1 2 3 Log-Rank test, P = 0.005 1804 © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. A. Kaur et al.PME1 Predicts Colorectal Cancer Outcome postoperative stage, postoperative grade, circumferential margin, vascular invasion, or postradiotherapy (RT) TRG (Table S1). PME1 expression is related to DFS of rectal cancer patients In the univariate survival analyses for the whole cohort, tumors with the most intensive cytoplasmic index of PME1 under median were linked to shorter DFS than those over median (110.7 vs. 116.7 months, P = 0.007, logrank test; Fig. 2B). The multivariate (Cox) proportional hazards regression for DFS was performed for the patients with following covariates: treatment group, sex, age (70 years as cutoff), postoperative N (positive/negative), vascular invasion (positive/ negative), circumferential margin (2 mm as cutoff), and the most intensive cytoplasmic index of PME1 (median as cutoff). The following remained as independent factors predicting a poor DFS of rectal cancer patients: sex if male (hazard ratio [HR] 4.12; 95% CI 0.25–0.98; P = 0.042), postoperative N (HR 10.33; 95% CI 1.68–8.40; P = 0.001), circumferential margin (HR 6.13; 95% CI 1.19–4.53; P = 0.013), and the most intensive cytoplasmic index of PME1 (HR 9.28; 95% CI 1.78–14.31; P = 0.002). The multivariate (Cox) Table 2. Association of clinicopathological variables of rectal cancer patients with PME1 protein expression (most intensive cytoplasmic index). Variable Total n = 1951 PME1 most intensive cytoplasmic index Pvalue2 Below median, n (%) Above median, n (%) Sex Female 85 53 (62) 32 (38) 0.027 Male 110 85 (77) 25 (23) Recurrence Yes 57 49 (86) 8 (14) 0.003 No 138 89 (64) 49 (36) 1PME1 most intensive cytoplasmic staining index could be analyzed from 195 patients only. 2Pearson chisquare test. Table 3. Multivariate survival analysis of rectal cancer patient samples and TCGA colon and rectal adenocarcinoma (COADREAD) patient samples using Cox proportional hazards regression models. Variables Rectal cancer (n = 142)1 Diseasefree survival Rectal cancer (n = 114)1 Diseasespecific survival TCGA COADREAD (n = 347)1 Overall survival HR 95% CI Pvalue HR 95% CI Pvalue HR 95% CI Pvalue 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 years 1 1 1 >70 years 1.38 0.32–1.33 0.240 4.46 0.24–0.95 0.035 1.91 1.16–3.16 0.011 Circumferential margin ≤2 mm 1 1 >2 mm 6.13 1.19–4.53 0.013 1.63 0.78–3.20 0.201 Postoperative N2 Negative (N0) 1 1 1 Positive (N12) 10.33 1.68–8.40 0.001 0.79 0.54–4.99 0.374 3.15 1.79–5.53 <0.001 Vascular invasion3 Negative 1 1 1 Positive 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 recurrence No 1 Yes 21.62 15.50–840.54 <0.001 PME1 expression4,5 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, hazard ratio; 95% CI, 95% confidence interval. Statistically significant P-values are indicated in bold. 1Only 142 and 114 Rectal cancer patients for diseasefree survival and diseasespecific survival, respectively, and 347 TCGA COADREAD patients for overall survival could be analyzed for which complete data were available for all the covariates. 2Postoperative nodal status according to the TNM classification of malignant tumors [16]. 3For TCGA data, vascular invasion indicates combined lymphatic, and/or venous invasion status. 4PME1 most intensive cytoplasmic index (protein expression) measured by IHC (cutoff median). 5PPME1 mRNA expression measured by RNA sequencing exon array Illumina HiSeq (cutoff −0.075). 1805 © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. PME1 Predicts Colorectal Cancer OutcomeA. Kaur et al. proportional hazards regression for DSS was performed for patients with the similar covariates as that for DFS added with disease recurrence. The following remained as independent factors predicting a poor DSS: age if over 70 years (HR 4.46; 95% CI 0.24–0.95; P = 0.035) and disease recurrence (HR 21.62; 95% CI 15.50–840.54; P < 0.001). The multivariate (Cox) proportional hazards regression results for DFS and DSS have been presented in Table 3. PME1 gene expression correlates with OS of CRC patients In an independent colon and rectum adenocarcinoma (COADREAD, n = 396) RNA sequencing dataset (exon array IlluminaHiSeq) available from TCGA [22], we analyzed the correlation between OS and PPME1 gene expression using UCSC Cancer Genomics Browser [23, 24]. Although the PPME1 expression was normally distributed among this dataset, two distinct groups can be clearly isolated using cutoff value of −0.075 (Figure S1A). Based on this cutoff value, the data were categorized into two groups, low PPME1 (expression below −0.075) and high PPME1 (expression above −0.075) (Fig. 2C). This analysis revealed a similar trend at the mRNA expression level as was seen for PME1 protein expression in our rectal cancer dataset. The patient group with high PPME1 gene expression (n = 249) showed better OS (P = 0.005 logrank χ2 test) than the patients with low PPME1 expression (n = 147). Similar results were seen using median PPME1 expression (0.07014) as a cutoff (Fig. 1B and C). The multivariate analysis of TCGA COADREAD panel was performed by Cox proportional hazards regression models to assess the effect of other possible variables contributing to the prognosis of CRC patients in the Kaplan–Meier survival analysis. This analysis was carried out in 347 patients, for which full data was available for the following covariates: sex, age (cutoff 70 years), pathologic N (positive/negative), vascular invasion (positive/ negative), and PPME1 gene expression (cutoff −0.075). This analysis revealed low PPME1 expression as an independent highrisk factor (HR 2.22; 95% CI 1.32–3.72; P=0.002) predicting poor OS of CRC patients. Additionally, male gender (HR 1.68; 95% CI 1.01–2.79; P = 0.046), high age (>70 years) (HR 1.91; 95% CI 1.16–3.16; Figure 3. Modulation of survival signaling by PME1 in colorectal cancer cells. The viability of HCA7 and CW2 cells transfected with Scr. or PME1 siRNA (for 72 h) was analyzed by CellTiterglo (CTG) (A) and WST1 (B) assays. Bars represent foldchange values calculated over respective values for Scr. siRNAtransfected cells (n = 3). Western blot analysis of protein lysates from siRNAtransfected HCA7 (C) and CW2 (D) cells, using antibodies to phosphorylated forms of AKT and ERK. The phosphoprotein expression normalized with GAPDH (loading control) is shown as foldchange values over Scr. siRNAtransfected cells (n = 3). *P = 0.003 by Student’s paired ttest. 0.00 0.20 0.40 0.60 0.80 1.00 1.20 Scr PME-1 Scr PME-1 Cell vialibity (Fold change) HCA-7CW-2 siRNA: WST-1 assay 0.00 0.20 0.40 0.60 0.80 1.00 1.20 Scr PME-1 Scr PME-1 Cell vialibity (Fold change) HCA-7CW-2 siRNA: CTG assay AB PME-1 siRNA: PME-1Scr GAPDH CW-2 pAKT pERK PME-1 siRNA: PME-1Scr GAPDH HCA-7 pAKT pERK CD 0 0.5 1 1.5 2 pAKT pERK Normalized expression fold change over Scr. Scr siRNA PME-1 siRNA 0 0.5 1 1.5 2 2.5 pAKT pERK N ormalized expression fold change over Scr. Scr siRNA PME-1 siRNA * 1806 © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. A. Kaur et al.PME1 Predicts Colorectal Cancer Outcome P = 0.011), and pathologic N positivity (HR 3.15; 95% CI 1.79–5.53; P < 0.001) were significant risk factors predicting poor OS of CRC patients. These results have been presented in Table 3. The multivariate analysis for TCGA COADREAD panel carried out using above mentioned covariates and median PME1 expression as cutoff also showed similar results (Table S2). Modulation of survival signaling by PME1 in CRC cells PME1 silencing has been shown to inhibit viability and reduce the phosphorylated AKT and ERK levels in the glioblastoma cells [9] as well as in PME1 amplified gastric and lung cancer cells [14]. To study the alterations in survival signaling by PME1 in colon cancer, PME1 specific siRNA was employed to knockdown its expression and the viability was analyzed in CW2 and HCA7 cell lines. Neither of the cell lines displayed significant difference in viability upon PME1 silencing, confirmed by using two different cell viability assays (Fig. 3A and B). Further immunoblotting analysis revealed that in contrast to other cancer types, PME1 silencing did not inhibit expression of active phosphorylated forms of serine–threoninespecific protein kinases AKT1/2/3 (pAKT) and ERK1/2 (pERK) in either of the colon cancer cell lines (Fig. 3C and D). In fact, the expression of pAKT in HCA7 cells was significantly higher (twofold, P = 0.003) in PME1 silenced cells as compared to the control (Scr. siRNA) cells (Fig. 3C); and even though the other effects did not reach the statistical significance, there was a clear trend toward higher pAKT also in CW2 cells and higher pERK in both the cells lines upon PME1 inhibition. These findings demonstrate that opposite to other caner types PME1 may even decrease survival signaling in CRC cells and this may be linked to better patient outcome of rectal cancer patients with higher PME1 expression. Discussion PME1 expression on protein level was studied in 195 rectal cancer patients. Among these patients, 52 were treated with longcourse (chemo) RT, 88 with shortcourse RT, and 70 with surgery only. Our aim was to test PME1 as a potential marker to predict the outcome of rectal cancer patients. There is little information available concerning the role of PME1 for patient outcome in human malignancies. Based on its molecular function as a PP2A inhibitor protein such as CIP2A [8], it would be expected that PME1 would also act as a human oncoprotein. It has been shown that PME1 is linked with the malignant progression of astrocytic gliomas [9] and endometrial cancers [13]. For this reason it is surprising that PME1 protein expression seems to act in an opposite way in rectal cancer. High protein expression of PME1 is a marker for favorable outcome in univariate analysis of DFS for the whole cohort, and it remained an independent prognosticator of DFS together with sex, postoperative N status, and circumferential margin. Importantly, tumors with high PME1 protein expression were associated with fewer recurrences and a better disease outcome than those with low PME1 protein expression. The importance of these findings was further highlighted by the observation that in addition to the rectal cancer, colon cancer patients from an independent cohort (COADREAD) could be categorized into two groups based on PPME1 mRNA expression, which correspond to differential OS outcome in both univariate and multivariate survival analyses. This information can be useful for establishing PCR tests based on PPME1 expression as a biomarker to predict the survival of CRC patients [25, 26]. PME1 expression is known to be associated with increased cell proliferation and survival signaling in human malignant gliomas and endometrial cancers [9, 13]. We studied these cellular functions in CRC cells. Since PP2A inhibition by PME1 has been previously shown to promote phosphorylation of the AKT and ERK proteins, it is surprising that in CRC cells these prosurvival signaling events are largely unaffected or affected in an opposite manner as compared to the other types of cancer. Mechanistically, PME1 promotes ERK pathway signaling mainly at a level upstream of Raf [9]. However, a significant proportion of the CRCs contain activating mutations in KRas (50%) and BRaf (10%), which promote ERK signaling independent of the upstream stimuli [27]. In addition, the TGFαmediated autocrine feedback loop can further increase the RasRafERK pathway signaling [27, 28]. These alterations may render the CRC cells insensitive to PME1mediated regulation. Furthermore, inactivating mutations and/or altered expression of various PP2A components [29, 30], specifically the Bsubunits regulated by PME1, might affect the activity (phosphorylation) of specific target proteins. To this end, among the PP2A Bsubunits of PPP2R2 (B55) family, which are exclusively sensitive to PP2A Csubunit Leu309 methylation reversibly removed by PME1 [31], the PPP2R2B (B55β) subunit is epigenetically silenced by DNA hypermethylation in >90% of CRCs [32]. The loss of this Bsubunit promotes drug resistance in CRC by activating the survival signaling in a PI3KAKT independent manner [32]. It may be speculated that our surprising result of a favorable outcome with high PME1 expression is related to these altered pathways