Combined aberrant expression of E-cadherin and S100A4, but not β-catenin is associated with disease-free survival and overall survival in colorectal cancer patients

Paraffin-embedded tissues were obtained from the department of pathology and fresh frozen specimens were provided by the National Biobank of Korea, with the approval of the Ethics Committee of Chungbuk National University Hospital. Three hundred thirty-three CRC patients (male:female, 189:144), who underwent complete resection (R0) and were followed-up for more than 5 years were enrolled in this study. The patients did not receive any chemotherapy or radiation therapy before surgery. Among the enrolled patients, 68 fresh-frozen specimens were obtained for real time RT-PCR. At the time of surgery, tumor tissues and matched normal tissues were immediately sampled from the resected colorectal specimen by pathologists. The tissue was frozen in liquid nitrogen, and kept at −80°C.

Each tumor was re-evaluated by analysis of the medical records and the tissue slide files by one pathologist. Tumor location (right side or left side), the degree of differentiation (well, moderately, and poorly), depth of tumor invasion, lymph node or distant metastasis, and microsatellite status (characterized in 166 cases) were assessed. The stages were defined according to the TNM staging system of AJCC. We also reviewed macroscopic type (polypoid, ulcerofungating, and ulceroinfiltrative), invasion to the lymphatics or vessels, and perineural invasion. Tumor growth was classified as expanding- or infiltrative-type and tumor budding was evaluated by cytokeratin staining. The number of tumor buds was counted in five regions of clusters of tumor cells comprising less than 5 cells at 200x magnification. We divided cases into those with low (median <10) or high budding (median ≥ 10) (Figure 1).

After all slide reviews, 3-mm sized TMAs were constructed. Areas from the center and the invasive margin of the tumor with the lowest degree of differentiation but abundant in cells with high mitotic activity were chosen from the original blocks. These representative areas were marked by an experienced pathologist on hematoxylin and eosin (H&E)-stained slides from selected paraffin blocks. Serial 4-um sections were then cut from the TMA paraffin blocks. The sections were mounted on Capillary Gap plus Slides. Before IHC staining, all sections were deparaffinized and heated in citrate buffer (10 mM/L citric acid, pH 6.0) in a microwave oven. After inactivation by exposure to 3% H₂O₂ for 10 min, the sections were incubated with blocking serum at room temperature for 10 min. IHC staining was carried out using anti-E-cadherin (DakoCytomation, Glostrup, Denmark, 1:400), anti-cytokeratin (LeicaMicrosystems, Wetzlar, Germany, 1:300), anti-β-catenin (DakoCytomation, Glostrup, Denmark, 1:400) and anti-S100A4 (DakoCytomation, Glostrup, Denmark, 1:800) as primary antibodies. After incubation with secondary antibodies, the expression of these markers in cells was detected with diaminobenzidine (DAB; Sigma, St. Louis, MO, USA) by enhancement with a SABC kit (ZSGB-BIO, Beijing, China). Tissue sections stained without primary antibody served as a negative control. The slides were then counterstained with hematoxylin. The results were assessed by two pathologists who were blinded to the patients’ details. E-cadherin immunostaining was evaluated according to a method described previously [17]. E-cadherin staining was classified as grade 0 (preserved) when more than 90% of the CRC cells on a section showed strong membranous staining, or grade 1 (reduced or lost) when less than 90% of the CRC cells showed positive membranous staining. For β-catenin, the percentage of tumor cells with nuclear staining was evaluated and scored as grade 0 (0-30%) or grade 1 (30-100%). To evaluate S100A4 expression, staining intensity was recorded as 0 (no staining), 1 (weak staining), 2 (intermediate staining), or 3 (strong staining). The proportion of stained cells was expressed as a percentage. The staining score was obtained by multiplying the intensity and percentage of stained cells and classified as grade 0 (low expression) and grade 1 (high expression) (Figure 2).

Tumor and matched normal tissues were homogenized and total RNA was isolated using an RNeasy Mini kit (Qiagen, Tokyo, Japan) following the manufacturer’s instructions. All samples were treated with RNase-free DNase (Qiagen). RNA quality control and quantification were carried out on an Agilent 2100 Bioanalyzer using a RNA 6000 Pico LabChip (Agilent Technologies, Tokyo, Japan). First-strand complementary DNA was made from total RNA using the Prime Script RT-reagent kit (Amersham Biosciences Europe GmbH, Freiburg, Germany) according to the manufacturer’s instructions. To quantify the expression levels of E-cadherin, β-catenin, N-cadherin, and S100A4, real-time PCR amplification was performed with a Rotor Gene 6000 instrument (Corbett Research, Mortlake, Australia). Real-time PCR assays using SYBR Premix EX Taq (TAKARA BIO INC., Otsu, Japan) were carried out in micro-reaction tubes (Corbett Research, Mortlake, Australia). The PCR reaction was performed in a final volume of 10 μl, consisting of 5 μl of 2x SYBR premix EX Taq buffer, 0.5 μl of each 5’- and 3’- primer (10 pmol/μl), and 1 μl of sample cDNA. The product was 10-fold serially diluted from 100 pg/μl to 0.1 pg/μl. Dilution series of PCR products were used to establish standard curves for real-time PCR. Spectral data were captured by using Rotor-Gene Real-Time Analysis Software 6.0 Build 14 (Corbett Research, Mortlake, Australia). All samples were run in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous RNA reference gene. Gene expression was normalized to the expression of GAPDH. Primers and product sizes for E-cadherin, N-cadherin, β-catenin, S100A4, and GAPDH are summarized in Table 1.

Differences were compared using Fisher’s exact test or Pearson’s test for qualitative variables and Student’s t-test or analysis of variance for continuous variables. Prognosis was determined by disease-free survival and overall survival. Prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards model). All statistical tests were two sided, and statistical significance was accepted at the P < 0.05 level. All analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA).

Clinicopathologic characteristics of the patients showed in Table 2. The relation between clinicopathological parameters and aberrant expression of E-cadherin, β-catenin and S100A4 in the invasive margin was observed. Loss of E-cadherin was related with cell grade (p = 0.050), macroscopic type (p = 0.014), perineural invasion (p = 0.037) and high tumor budding (p = 0.010). Nuclear β-catenin expression was higher in microsatellite stable (MSS) than microsatellite instable (MSI) tumors (p = 0.004) and left CRCs than in right CRCs (p = 0.037). S100A4 expression was increased in infiltrative growth (p = 0.017), macroscopic type (p = 0.024), T-stage (p = 0.010), nodal stage (p = 0.002), AJCC stage (p = 0.001), ratio of metastatic lymph nodes (p = 0.019), lymphovascular invasion (p = 0.034), and perineural invasion (p = 0.050). It was also related with TLI (p = 0.001) (Table 3).

The aberrant expression of EMT-related proteins was higher in the invasive margin than in the tumor center; loss of E-cadherin, 17.9% versus 31.8% (p < 0.0001), nuclear expression of β-catenin, 10.6% versus 12.5% (p < 0.0001), and gain of S100A4, 10.2% versus 15.5% (p < 0.0001), respectively. Aberrant expression of each protein was related to the others: S100A4 versus E-cadherin (r = 0.312, p = 0.050), S100A4 versus β-catenin (r = 0.166, p = 0.004) and E-cadherin versus β-catenin (r = 0.152, p = 0.009).

Disease-free survival was associated with AJCC stage (p < 0.0001, Figure 3A), tumor budding (p = 0.007, Figure 3B), tumor growth type (p = 0.003, Figure 3C), and perineural invasion (p = 0.004, Figure 3D). Aberrant expression of the E-cadherin (p = 0.007, Figure 3E), S100A4 (p = 0.004, Figure 3F), and combination of both proteins in the invasive margin were related to disease free survival (p = 0.005, Figure 3G), whereas β-catenin was inversely related (p = 0.032, Figure 3H). Multivariate Cox analysis showed that combination of E-cadherin and S100A4 expressions in the invasive margin and the AJCC stage are independent risk factor for disease-free survival (Table 4). Overall survival was associated with AJCC stage (p < 0.0001, Figure 4A), tumor budding (p = 0.01, Figure 4B), tumor growth type (p = 0.01, Figure 4C), perineural invasion (p = 0.02, Figure 4D), and TLI (p = 0.005, Figure 4E). Aberrant expression of E-cadherin (p = 0.002, Figure 4F), S100A4 (p = 0.003, Figure 4G), and combination of both proteins in the invasive margin were associated with overall survival time (p = 0.0002, Figure 4H), whereas β-catenin was not. Cox regression analysis showed that the combination of E-cadherin and S100A4 in the invasive margin, the AJCC stage and perineural invasion were independent risk factors of overall survival time (Table 5).

Transcript levels of β-catenin and S100A4 were correlated with IHC findings at the tumor invasive margin; β-catenin (r = 0.369, p = 0.003) and S100A4 (r = 0.504, p < 0.0001). However, there was no relation between the RT-PCR data and the IHC findings for E-cadherin. E-cadherin and N-cadherin showed a weak inverse relation without statistical significance (r = -0.169, p = 0.084). mRNA levels of N-cadherin were higher in recurrent or mortality cases (p = 0.040 and p = 0.042, respectively).