The present study rigidly adhered to the ethical guidelines of the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. Written informed consent for the use of clinical samples and data was obtained from all patients, as required by the Institutional Review Board of Nagoya University (Nagoya, Japan; approval no. 2014-0044).

ESCC cell lines (TE1, TT and TTn) and a non-tumorigenic epithelial cell line (Het-1A) were obtained from the American Type Culture Collection (Manassas, VA, USA). NUEC2 and WSSC cell lines were established at Nagoya University (17). KYSE510, KYSE590, KYSE890, KYSE1170, KYSE1260 and KYSE1440 cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) (18). Cells were stored at −80°C in a cell preservative (Cell Banker; LSI Medience Corporation, Tokyo, Japan) and cultured in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in an atmosphere containing 5% CO₂ at 37°C. A total of 106 primary ESCC tissues and adjacent normal tissues were acquired from patients who underwent radical esophageal resection at Nagoya University Hospital between October 2001 and January 2016 (19). The tumors were determined to be radically resected when pathologically diagnosed as stage I to III. All tissue samples were histologically diagnosed as ESCC, immediately frozen following resection and stored at −80°C. Specimens were histologically classified using the 7th edition of the UICC staging system for esophageal cancer (20). Patients who received neoadjuvant chemotherapy were excluded. Postoperative follow-up included physical examination, measurement of serum tumor markers every 3 months, and enhanced computed tomography of the chest and abdominal cavity every 6 months. Adjuvant chemotherapy was administered to selected patients according to their condition and at the discretion of the physician.

The expression levels of CPNE5 mRNA were measured using a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. Total RNA isolated using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) from cell lines and 106 pairs of surgically-resected primary ESCCs and adjacent normal tissues served as template for cDNA synthesis. Reverse transcription was performed as follows: 10.5 µl 1 µg/µl RNA, 4 µl of 5X first strand buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 µl 100 mM dithiothreitol (Thermo Fisher Scientific, Inc.), 1 µl 10 mM dNTP mix (Promega Corporation, Madison, WI, USA), 1 µl random primer (Roche Diagnostics, Basel, Switzerland), 1 µl 200 U/µl Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific, Inc.) and 0.5 µl RNase inhibitor (Roche Diagnostics) were mixed and incubated for 60 min at 37°C. GAPDH mRNA expression levels (TaqMan; GAPDH control reagents; Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were quantified, and the data were used to normalize the expression levels. RT-qPCR was performed using the SYBR Green PCR Core Reagents kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) as follows: One cycle at 95°C for 10 min; 40 cycles at 95°C for 5 sec and 60°C for 60 sec without a final extension step. The samples were tested in triplicate, and samples without a template were included in each PCR plate as a negative control (21). Real-time SYBR Green fluorescence was detected using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) (22) and the 2^−ΔΔCq method was used for PCR quantification (23). The expression level of each sample is expressed as the value of the CPNE5 amplicon divided by that of GAPDH. The sequences of the specific primers are listed in Table I.

The protein was extracted from each cell line using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) and protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). For SDS-PAGE, 20 µg protein was added to a NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific, Inc.) and electrophoresed for 35 min a 200 V. A polyvinylidene difluoride membrane was used for blotting and the membrane was blocked with 5% skim milk (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 60 min at room temperature. The CPNE5 protein expression levels in ESCC cell lines were evaluated with a rabbit anti-CPNE5 polyclonal antibody (1:100 dilution and overnight incubation at 4°C; cat. no. HPA031369; Atlas Antibodies AB, Bromma, Sweden) as a primary antibody and anti-rabbit IgG HRP-linked antibody (1:1,000 dilution and 60 min incubation at room temperature; cat. no. 7074S; Cell Signaling Technology, Inc., Danvers, MA, USA) as a secondary antibody. As an internal control, β-actin protein expression was detected with a mouse anti-β-actin polyclonal antibody (1:10,000 dilution and incubated for 60 min at room temperature; cat. no. ab6276; Abcam, Cambridge, UK) as a primary antibody and anti-mouse IgG HRP-linked antibody (1:1,000 dilution and 60 min incubation at room temperature; cat. no. 7076S; Cell Signaling Technology, Inc.) as a secondary antibody. Enhanced Chemiluminescence Western Blot Analysis System (GE Healthcare, Chicago, IL, USA) was used for visualization of the secondary antibody. An ESCC cell line with relatively high CPNE5 mRNA expression (KYSE590) and a low-expression ESCC cell line (TT) were evaluated.

Genomic DNA was isolated from the cell lines using a QIAamp DNA Mini kit (Qiagen GmbH) and treated with bisulfite (2 cycles at 95°C for 5 min and 60°C for 10 min). Bisulfite-modified DNA from ESCC cell lines and a non-cancerous esophageal mucosa cell line (Het-1A) were amplified as follows: One cycle at 94°C for 2 min; and 50 cycles at 94°C for 15 sec, 56°C for 15 sec and 72°C for 30 sec, using specific primers (Table I). Sequencing was performed by Eurofins Genomics Tokyo Co., Ltd. (Tokyo, Japan), using a Big Dye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific, Inc.) and a 3730× l DNA Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.) (24).

Immunohistochemical staining was performed to determine the difference in CPNE5 protein expression between cancerous tissue and non-cancerous tissues in 45 representative clinical cases. Sections were incubated for 16 h at 4°C with a rabbit polyclonal antibody raised against CPNE5 (cat. no. HPA031369; Atlas Antibodies AB) diluted 1:100 in Antibody Diluent (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Sections were incubated with secondary antibody (SignalStain^® Boost IHC Detection Reagent labelled with HRP; Cell Signaling Technology, Inc.) for 30 min at room temperature. Antigen antibody complexes were visualized by exposure with liquid 3,3′-diaminobenzidine (Nichirei, Tokyo, Japan) for 2 min. A total of two independent observers evaluated the specimens using an optical microscope with ×400 magnification as follows: Cancerous tissue >non-cancerous tissue; equivalent; or cancerous tissue <non-cancerous tissue.

Quantitative data are presented as the mean ± standard deviation. Patients were divided into low and high CPNE5 groups according to the median levels of CPNE5 mRNA expression in the cancerous tissues. The differences between CPNE5 mRNA expression values in the two groups (differentiated cell lines vs. undifferentiated cell lines, or cancerous tissues vs. non-cancerous tissues) were compared using the Mann-Whitney test. The χ² test was used to analyze the association between CPNE5 mRNA expression levels and clinicopathological characteristics. Overall survival (OS) and disease-free survival (DFS) rates were calculated using the Kaplan-Meier method. The Cox proportional hazards model was used to compare survival rates, and multivariable regression analysis was used to identify prognostic factors. Statistical analysis was performed using JMP 10 software (SAS Institute Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

CPNE5 mRNA expression levels differed among the 11 ESCC cell lines (Fig. 1A), and were lower compared with those of Het-1A cells, except for KYSE590 and KYSE1440. There were no significant differences in CPNE5 mRNA expression levels between differentiated (0.00197±0.00172) and undifferentiated (0.00161±0.00133) cell lines (P=0.897). ESCC cell lines established from metastatic sites, including TT and TTn, and those established from lymph node metastases, including KYSE1170 and KYSE1260, expressed low levels of CPNE5 mRNA. Western blot analysis using an anti-CPNE5 antibody illustrated high CPNE5 protein expression in an ESCC cell line with high CPNE5 mRNA expression (KYSE590) and low CPNE5 protein expression in a low-expression ESCC cell line (TT) (Fig. 1B). Bisulfite sequencing analysis of CPNE5 revealed that the CpG sites in the CPNE5 DNA promotor region in all ESCC cell lines and Het-1A were completely methylated. The bisulfite sequencing results for Het-1A, KYSE590 and TT cells are presented as representative cell lines expressing high and low levels of CPNE5 mRNA, respectively (Fig. 1C).

The median age of the 106 patients was 65 years (range, 44–84 years), and the female: Male ratio was 20:86. According to the UICC staging system (7th edition), 24, 29 and 53 patients were diagnosed with disease at pathological stages I, II and III, respectively. Adjuvant chemotherapy was administered to 36 patients (34%). The median duration of follow-up was 34.1 months, during which 44 patients (42%) experienced recurrence and 39 patients (37%) succumbed to the disease.

The mean normalized CPNE5 mRNA expression level was significantly lower in ESCC tissues (0.0107±0.0138) compared with the corresponding non-cancerous adjacent mucosal tissues (0.00829±0.0123; P=0.003; Fig. 2A).

There was no significant association between the low and high CPNE5 expression groups with their clinicopathological characteristics (sex, tumor size and depth, lymphatic involvement, vascular invasion and pathological stage). By contrast, the percentage of patients who received adjuvant chemotherapy was significantly higher in the low CPNE5 group (Table II).

Patients in the low CPNE5 expression group experienced a significantly shorter OS time compared with those in the high CPNE5 expression group (the 5-year OS rates were 55.6 and 77.3% for the low and high expression groups, respectively; P=0.016; Fig. 2B). Though the difference was not statistically significant, there was a similar trend observed in DFS (P=0.052; Fig. 2C). Univariate analysis revealed that tumor depth, lymph node metastasis, undifferentiated tumor phenotype, lymphatic involvement, postoperative chemotherapy and low CPNE5 expression were significantly associated with lower survival rates. Multivariable analysis identified low CPNE5 expression and lymphatic involvement as independent prognostic factors for OS (hazard ratio, 2.55; 95% confidence interval, 1.21–5.68; P=0.014; and hazard ratio, 6.28; 95% confidence interval, 1.70–40.6; P=0.004, respectively; Table III).

Differences between recurrence patterns were predicted, since the Kaplan-Meier analysis revealed fold-differences between OS and DFS. Among patients with recurrence, a significant number were members of the low CPNE5 group (P=0.018). Among patients with local recurrence, significantly more were members of the low CPNE5 group (P=0.027; Fig. 2D). A similar tendency was observed in patients with lymph node recurrence (P=0.250; Fig. 2D). By contrast, the rates of distant metastasis to tissues including the lung/pleura and liver were not significantly different between groups (Fig. 2D).

The expression patterns of CPNE5 protein were evaluated using immunohistochemical staining. Among the 45 clinical samples, CPNE5 protein expression was suppressed in the cancerous tissue in 14 (31%) samples, equally expressed in cancerous and non-cancerous tissue in 19 (42%) samples and overexpressed in cancerous tissue in 12 (27%) samples. Representative examples of suppression in cancerous tissue, and an example of equivalent expression, are presented (Fig. 3).