Nine samples were taken from patients with MPMs, including four cases of epithelioid, four biphasic and one sarcomatoid subtypes, with written informed consent at Toronto General Hospital (Toronto, Canada). For TMA analysis, a total of 53 MPM were obtained from patients who underwent surgery at Hokkaido University Hospital and its affiliated hospitals between February 1990 and April 2012. Patient’s clinical information was extracted from medical records. The protocol was approved by the appropriate institutional review board of Hokkaido University (no. 012–0136). Detailed information about demography and clinical characteristics are summarized in Table I. Postoperative pathological staging evaluation was demonstrated only in curative operative cases (n=21), showing stage I disease in 1 case, stage II disease in 5 cases, and stage III disease in 15 cases. In all, one patient had T1 disease, 7 patients had T2 disease and 13 patients had T3 disease. A total of 9 patients had N0 disease, 5 patients had N1 disease, and 7 patients had N2 disease (Table II). Histological classification of tumors and stage were performed according to the Union for International Cancer Control (UICC) pathological tumor/node/metastasis (pTNM) classification criteria (13).

The human malignant mesothelioma cell lines used were as follows: NCI-H28, -H226, -H2052 and -H2452 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cancer cells were grown in monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). A human adult normal mesothelial cell line (MES-F) was purchased from Zembio Inc. (Research Triangle Park, NC, USA) and was grown in mesothelial cell growth medium (Zembio). All cell lines were maintained at 37°C in atmospheres of humidified air with 5% CO₂.

Tumor samples from MPM patients with surgery were excised and stored at −80°C. QIAzol Lysis reagent (Qiagen, Valencia, CA, USA) and one 5-mm stainless steel bead (Qiagen) were added before homogenizing with a TissueLyser Adapter Set (Qiagen) for 2 min at 20 Hz. Total RNA was then purified using a miRNeasy Mini kit (Qiagen). The amount and purity were measured using a spectrophotometer (NanoDrop; Thermo Fisher Scientific, Wilmington, DE, USA).

cDNA was synthesized from 2 μg total RNA using QuantiTect^® reverse transcription kit (Qiagen). The primers were designed as follows: for PLK1, forward primer, 5′-cccctcacagtcctcaataa-3′ and reverse primer, 5′-tgtccgaatagtccaccc-3′; for SORORIN, forward primer, 5′-cg ccagagacttggaaatgt-3′ and reverse primer, 5′-gtttctgtttctcgggt ggt-3′; for actin, beta (ACTB), forward primer, 5′-gaaatcgtgcgt-gacattaa-3′ and reverse primer, 5′-aaggaaggctggaagagtg-3′. qRT-PCR analysis was performed using LightCycler480^® SYBR-Green I Master Ready-to-use hot start reaction mix and LightCycler480^® system (Roche, South San Francisco, CA, USA). The thermal cycler conditions were as follows: 5 min at 95°C to denature, 45 cycles at 95°C for 10 sec, 56°C for 20 sec, and 72°C for 10 sec for PCR amplification, and 1 min at 65°C for melting. The threshold cycle value was defined as the value obtained in the PCR cycle when the fluorescence signal increased above the background threshold. PCR reactions were carried out in duplicate.

All short interference RNA (siRNA) oligonucleotide sequences for PLK1 and SORORIN siRNAs were purchased from Qiagen for this study. AllStar Negative Control siRNA^® (Qiagen) were used as the negative control (NC-siRNA). The final concentration of 10 nM of siRNAs was incubated with HiPerFect^® Transfection reagent (Qiagen) according to the manufacturer’s instructions. The PLK1 inhibitor BI 6727 was purchased from Selleck Chemicals (Houston, TX, USA). The CellTiter96^® AQueous One Solution Cell proliferation assay (Promega, Madison, WI, USA) was used for the evaluation of the number of viable cells according to the manufacturer’s instructions, using a microplate spectrophotometer (μQuant; BioTek Instruments, Inc., Winooski, VT, USA). Each experiment was performed in triplicate.

Archival slides for all the cases were reviewed to select three representative areas for each sample by an experienced pathologist (K.H.). TMA blocks were then constructed using a manual tissue microarrayer (JF-4; Sakura Finetek Japan Co., Ltd., Tokyo, Japan) with a 1.0-mm diameter needle. The finalized array blocks were sliced into 4-μm-thick sections and mounted on glass slides. To check the histopatho-logical diagnosis and adequacy of tissue sampling, a section from each microarray was stained with regular haematoxylin and eosin (H&E) and calretinin, and examined by a pathologist (K.H.). SORORIN immunostaining were performed using an automated IHC platform (Autostainer plus; Dako, Glostrup, Denmark). Antigen retrieval was performed in the condition of pH 9.0 for 20 min. The detection kit used was the EnVision™+ Dual Link (K4063; Dako), incubation with post primary for 60 min at RT. Anti-SORORIN polyclonal antibody (bs-7717R CDCA5 Sororin antibody; Acris Antibodies GmbH, Herford, Germany, 1/300) was diluted using mixed antibody diluent (Dako:S2022 Antibody diluent). A polymer-based detection system (EnVision™+ Dual Link #K4063) was used with 3′,3-Diaminobenzidine (DAB) as the chromogen. The positive controls included a sample of testis, and normal lung and pleural samples were used as negative controls. Slides were dehydrated and placed on coverslips.

Digital images of IHC-stained TMA slides were obtained using a whole slide scanner (ScanScope CS, Aperio ePathology; Leica Microsystems, Inc., Richmond Hill, ON, Canada). Annotation of tumor regions on whole slides was performed blinded to clinical follow-up data using Aperio’s annotation software (ImageScope Viewing Software: Positive Pixel Count v9.1; Aperio). SORORIN were quantified with IHC scoring, which summated the percentage of area stained at each intensity level multiplied by the weighted intensity reported in other studies (14–16). Initially, the weighted intensity of staining was graded as follows; grade 0 (negative), 1+ (weak positive), 2+ (moderate positive), and 3+ (strong positive) according to the Aperio’s annotation software. According to the total amount of IHC scores, SORORIN expression was then finally divided into two groups each (the threshold leading to the lowest P-value in log-rank test): low-level SORORIN expression (SORORIN-L, with an IHC score <1.2 and high-level SORORIN expression) (SORORIN-H, with an IHC score ≥1.2). We attempted to correlate clinicopathological variables such as age, gender, pathological TNM stage and histological classification with expression levels of SORORIN protein as determined by TMA analysis. Immunoreactivity was assessed for association with clinicopathological variables using the χ² test for variables. Kaplan-Meier method was used to generate survival curves, and survival differences were analyzed with the log-rank test, based on the status of SORORIN expression. Values of P<0.05 were considered statistically significant. All analyses were performed using the StatView version 5.0 software (SAS Institute, Inc., Cary, NC, USA).

Through qRT-PCR screening for molecular targeted genes in MPMs, we identified SORORIN and PLK1 overexpression in a majority of MPM cases (Fig. 1A). We also confirmed a high expression of these genes using four human MPM cell lines and low expression in a normal human mesothelial cell line MES-F (Fig. 1B). qRT-PCR analysis using a cDNA panel containing normal human tissues also showed that these genes are expressed only in the testis and thymus among the variety of normal human organs (data not shown).

To assess whether therapeutic candidate genes are essential for growth and survival of MPM cells, we transfected each specific siRNA against SORORIN (si-SORORIN-#4 and si-SORORIN-#6) and PLK1 (si-PLK1-#2, si-PLK1-#6 and si-PLK1-#7), into human MPM cell lines H28 and H2052 cells, using AllStar siRNA^® as the negative control (NC-siRNA). The mRNA level of transfected cells with independent siRNAs targeting these genes was significantly decreased in comparison to cells transfected with control siRNAs 48 h after transfection (Fig. 2A). Next, to evaluate the relationship between cell proliferation and gene knockdown, we conducted a cell viability assay using the CellTiter 96^® AQueous One Solution Cell Proliferation assay. After siRNA treatment, proliferation of H28 and H2052 cells were significantly suppressed compared with control groups at 4 days after transfection (Fig. 2B), suggesting that upregulation of these candidate genes are related to growth or survival of MPM cells. Typical microscopic images of the cells are shown in Fig. 2C. Compared to control cells or negative control siRNA-treated cells, SORORIN siRNA-treated mesothelioma cells exhibited distended cell bodies with enlarged nuclei, suggesting SORORIN-induced mitotic defects. Next, we sought to determine how inhibition of SORORIN causes the observed proliferation defect. The cell cycle distribution was analyzed by flow cytometry. We found that the G2/M population was remarkably elevated in siRNA against SORORIN-treated cells, suggesting a potential increase in the number of mitotic cells (Fig. 2D). These results demonstrate that inhibition of SORORIN induced mitotic arrest, resulting in a defect in cell cycle progression in mesothelioma cells.

We categorized SORORIN expression on the TMA according to the IHC score described above. Positive staining of tumor cells by SORORIN generally showed a nuclear and cytoplasmic pattern in cancer tissue (Fig. 3A). No staining was observed in benign chronic or fibrous pleuritis. Of the 53 MPM cases examined, SORORIN-H was observed in 27 cases (50.9%) (details are shown in Table III). Of those, 17 epithelioid type (50.0% of 34 cases), 8 biphasic type (61.5% of 13 cases) and 2 sarcomatoid type (40.0% of 5 cases) showed SORORIN-H. We then tried to correlate SORORIN expression with various clinicopathological parameters. When we examined curative surgical cases, survival analysis with Kaplan-Meier method indicated that MPM patients with SORORIN-H had shorter overall 5-year survival tendency than those with SORORIN-L (P=0.1664, by a log-rank test), although this was not statistically significant (Fig. 3B). No significant association was noted between SORORIN expression and other clinicopathological variables in these cases (Table II).

To investigate the effect of PLK inhibitor BI 6727 in mesothelioma cells, we monitored the cell viability of NCI-H28 and H2052 cells treated under various concentrations with BI 6727. Cell growth was significantly reduced in the presence of BI 6727 at concentrations above 10 nM in both cell lines (Fig. 4A); however, cell viability did not decrease at concentrations >50 nM, demonstrating the growth inhibitory effect of BI 6727 in MPM cells. Next, to determine the combinational effect with inhibition of SORORIN and PLK1, we treated MPM cells with both BI 6727 and siRNAs against SORORIN at the same time. We confirmed that suppression of SORORIN showed a significantly growth suppressive effect in cell proliferation when combined with BI 6727 in mesothelioma cells (Fig. 4B).