A total of 107 paraffin-embedded tumor tissue samples from patients who underwent esophagectomy at Qilu Hospital of Shandong University (Jinan, China) were collected between January 2008 and October 2008. All cases were confirmed as ESCC by three people as stated in the immunohistochemical (IHC) analysis section. Samples were excluded from the study as follows: i) If the patient received radiation therapy or chemotherapy before surgery; ii) if the patient died or was untraceable during follow-up and iii) if the patient was diagnosed with more than one primary tumor. The clinicopathological data, including age, sex, history of smoking and alcohol consumption, differentiation degree, invasion depth (T stage), lymph node metastasis (N stage), pathological TNM (pTNM) (20), and adjuvant treatment after surgery were obtained from the clinical or pathological records. The present study was approved by The Ethics Committee of Qilu Hospital. All patients in the study were anonymous and provided written informed consent.

In the first 2 years after surgery, patients were contacted by telephone every 3 months to enquire about their recovery and assess the level of recurrence, if any. More detailed instructions were provided according to tumor progression if recurrence occurred. After 2 years, information regarding the patients was collected every 6 months until November 2013, unless they were either untraceable or had died.

Tumor tissues of patients with ESCC were fixed in 10% formalin for 12 h at 4°C and embedded in paraffin. The paraffin-embedded tissues were cut into 5 µm thick sections, and IHC staining was performed using the Streptavidin-BiotinComplex kit according to the manufacturer's protocol (cat. no. SA1022; Wuhan Boster Biological Technology Co., Ltd.). Tissue sections were mounted on to the microslides and incubated for 1 h at 60°C before de-waxing in xylene and hydrated in graded concentrations of alcohol (95, 90, 85, 80 and 75%). Then the sections were placed in a solution of sodium citrate (pH 6.0) and were heated to 93°C, and maintained at 90°C for 15 min to retrieve the antigen. The solution was cooled to room temperature and 3% H₂O₂ was subsequently added at room temperature for 10 min, to block the non-specific protein binding sites and inactivate the endogenous peroxidase. After blocking with 5% BSA (Wuhan Boster Biological Technology Co., Ltd.) for 20 min at room temperature, the sections were incubated overnight at 4°C with mouse polyclonal primary antibody against YAP (dilution 1:300; cat. no. 4912; Cell Signaling Technology, Inc.). Then, the sections were incubated with biotinylated goat anti-rabbit antibody from the kit for 20 min at 37°C. Finally, the slides were counterstained with hematoxylin at 25°C for 2 min dehydrated in graded alcohol solution (75, 80, 85, 90, 95 and 100%) and xylene, and covered with coverslips using neutral balsam.

The IHC images were scored for positive staining intensity in five high-power fields using a light microscope (magnification, ×400) independently by three people, including a pathologist and two authors of the current study who did not participate in the IHC staining (LZ and YJ). The intensity score was graded as follows: i) None, 0; ii) mild, 1; iii) moderate, 2; and iv) intense, 3. The percentage of positive tumor cells was assessed according to the following patterns: i) No staining, 0; ii) ≤10%, 1; iii) 10–50%, 2; iv) and >50% 3. The staining intensity was assessed as follows: i) Negative, 0; ii) weak, 1; iii) moderate, 2; and iv) strong, 3. The final score was the combination of the intensity score and the positive percentage score for each section. A score of 1–5 was designated as low expression and an overall score of 6–9 was designated as high expression of YAP in ESCC tissues (21).

Human esophageal squamous carcinoma cell lines Eca109, TE-10 and TE-11 were obtained from Procell Life Science & Technology Co., Ltd. The Kyse150 cell line was purchased from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Kyse140 was kindly provided by Professor Xinyuan Guan (Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China). The cells were grown in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), and supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin antibiotic solution. Human esophageal squamous carcinoma Eca109 cells were cultured in RPMI-1640 supplemented with only 10% FBS. The cells were incubated at 37°C in a humidified incubator containing 5% CO₂.

Eca109 and Kyse150 cell lines were transfected with YAP-specific siRNA oligonucleotides synthesized by GenePharma using EndoFectin™ MAX (GeneCopoeia Inc.). YAP-specific siRNA oligonucleotides were synthesized according to the following target sequences: YAP#1, 5′-CAGGTGATACTATCAACCAAA-3′ and YAP#2, 5′-GACCAATAGCTCAGATCCTTT-3′. Non-targeting siRNA (silencer negative control siRNA, siNC, forward: 5′-CCCAUUCAUUGUUGUCACUTT-3′, reverse: 5′-AGUGACAACAAUGAAUGGGTT-3′) was also transfected into Eca109 and Kyse150 cell lines as the negative control. The final concentration of the siRNA used was 50 nM. After transfection for 48 h, the cells were used in the subsequent experiment.

After transfection for 48 h, cells were lysed, and proteins were extracted using RIPA (cat. no. P0013C; Beyotime Institute of Biotechnology) and determined by a BCA protein assay. The proteins (25 µg/lane) were separated by SDS-PAGE (5% gel for concentration and 10% gel for separation). Then, the separated proteins were transferred onto polyvinylidene difluoride membranes. The membrane was blocked with 5% skimmed milk in TBST (pH 7.4) at room temperature for 1 h, and incubated with primary antibodies against YAP (dilution 1:1,000), vimentin (dilution 1:1,000; cat. no. 5741; Cell Signaling Technology, Inc.), E-cadherin (dilution 1:1,000; cat. no. ab15148; Abcam), N-cadherin (dilution 1:1,000; cat. no. 22018-1-AP; ProteinTech Group, Inc.) and GAPDH (dilution 1:1,000; cat. no. sc-47724; Santa Cruz Biotechnology, Inc.) overnight at 4°C, then incubated with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. s0001) and goat anti-mouse (cat. no. s0002) immunoglobulin G secondary antibodies (diluted 1:5,000; Affinity Biosciences) for 1 h at 25°C. Immunoreactivity was detected using an enhanced chemiluminescence reaction kit (Pierce, Thermo Fisher Scientific, Inc.) and the bands were quantified by densitometry using ImageJ software (version 1.8.0; National Institutes of Health). GAPDH was used as the loading control.

ESCC cells were resuspended in serum-free RPMI-1640 medium and added to the upper chamber of Transwell inserts (8-µm pore size, 6.5-mm diameter; Costar; Corning, Inc.) at a density of 3.0×10⁵ cells/ml. The lower chamber contained RPMI-1640 medium with 15% FBS. After incubation for 24 h, a cotton-tipped swab was used to swab the cells on the upper chamber. The migrated cells, which were attached to the lower surface of the membrane, were fixed with pure methanol for 30 min at 25°C and stained with 0.1% crystal violet (Sigma-Alrich; Merck KGaA) for 20 min at 25°C. The numbers of migrated cells were counted (5 fields per filter) using an inverted light microscope at a magnification of ×100 and the mean number was subsequently calculated. The experiments were performed in triplicate and repeated three times. The procedure for the Transwell invasion assay was similar to the migration assay except that the membrane was precoated with Matrigel (Corning, Inc.) and the time of incubation was increased to 36 h.

χ² test was performed to evaluate the association between YAP expression and clinicopathological factors in ESCC. Kaplan-Meier analysis and log-rank test was used to calculate the survival rate and assess the difference between the two subgroups (the YAP overexpression and YAP low expression groups), respectively. Cox regression model was used in univariate and multivariate analyses, to identify significant independent prognostic factors associated with ESCC. For cell experiments, the data are presented as the mean ± standard deviation and three individual experiments were performed in triplicate. One-way analysis of variance followed by Tukey's post hoc test was used to compare the data from different groups. All statistical analyses were performed using SPSS v17.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Clinicopathological characteristics of the 107 patients with ESCC are shown in Table I. Out of the 107 patients, a total of 20 (18.7%) were females and 87 (81.3%) were males, with a median age of 61 years, ranging from 32 to 84 years. A total of 54 (50.5%) patients had a former or current cigarette smoking history and 49 (45.8%) had a history of alcohol consumption. The median follow-up time was 49.50 months (range, 4.40–0.40 months).

YAP expression level in the tumor tissues was investigated using IHC staining and shown in brown. As shown in Fig. 1A and B, YAP was mainly positively expressed in the cytoplasm, but it was also observed in the nucleus. IHC staining showed that YAP was overexpressed in 39.3% (42/107) of the samples and its expression was low in 60.7% (65/107) of the samples.

To better understand the role of YAP expression level in the progression of ESCC, the data was analyzed using χ² test. Overexpression of YAP was significantly associated with increased N stage of ESCC (P=0.029). However, there were no significant associations with the other clinicopathological features (P>0.05; Table II).

The mean survival time of all 107 patients was 43.20±22.83 months (range 4.40–70.40 months). Kaplan-Meier analysis was performed along with log-rank test to investigate the relationship between YAP expression and prognosis. Compared to low expression of YAP, its overexpression was significantly associated with decreased DFS (P=0.004) and OS (P<0.001; Fig. 1C and D). Furthermore, the prognostic significance of YAP expression in patient subgroups stratified by pTNM stage (I and II vs. III) was also investigated, and the results revealed significant associations between YAP overexpression and poor survival in patients with late stage ESCC, but not in early stage ESCC (I and II; Fig. 2).

To further identify the role of YAP expression, univariate and multivariate analyses were performed. Univariate analysis showed that age, N stage, pTNM and YAP expression were statistically significantly associated with OS and DFS (P<0.05). Since previous studies reported that sex, smoking status, alcohol consumption, T stage, differentiation degree, and adjuvant treatment might play critical roles in prognosis of ESCC patients (22,23), all factors were included in the multivariate analysis, and the result indicated YAP overexpression is an independent prognostic factor for OS (HR, 2.727; 95% CI 1.556–4.780; P<0.001) and DFS (HR, 2.161; 95% CI, 1.223–3.818; P=0.008; Table III).

Five common ESCC cell lines (TE-10, TE-11, Kyse150, Kyse140 and Eca109) were used to screen for cell lines with high YAP expression. Since Kyse150 and Eca109 expressed higher levels of YAP than other three cell lines, they were selected for further experiments. The results of the screening by western blot are shown in Fig. S1.

To investigate the function of YAP in ESCC, siRNA was used to specifically knockdown YAP expression in ESCC cells. YAP siRNA and siNC were successfully transfected into the cells, and western blot analysis was performed. The analysis showed that YAP expression was successfully downregulated by siYAP but not by siNC, at the protein level (Fig. 3). Western blot analysis was also performed to detect levels of EMT-related proteins. Compared with the control group, levels of vimentin and N-cadherin were significantly reduced when YAP was downregulated, while that of E-cadherin was significantly increased, in both cell lines (Eca109 and Kyse150). Western blotting results showed that YAP could regulate levels of EMT-related proteins in ESCC cells.

To identify whether YAP affects the ability of cell migration and invasion in Eca109 and Kyse150 cells, transwell migration and invasion assays were performed. Compared with the control group, cell migration and invasion were both significantly decreased by the downregulation of YAP after siYAP#1 and siYAP#2 transfection (P<0.001), in both cell lines. However, no significant difference was found between the control and siNC groups (Fig. 4).