Up-regulation Of Twist Suppresses E-Cadherin And Predicts Poor Clinical Outcome Of Patients With Oesophageal Squamous Cell Carcinoma

Objectives: To identify the expression of the bHLH factor Twist in oesophageal squamous cell carcinoma (ESCC) and analyze its correlation with patient survival, and to explore its possible role in promoting tumor metastasis by suppressing E-Cadherin.Methods: In experiment 1, immunohistochemical staining using polyclonal Twist antibody was performed on paraffin embedded specimens from 112 patients with ESCC, and 30 specimens of non-neoplastic oesophageal epithelium were used as control. The staining intensity was stratified as absent (-), weak (+), moderate (++), and strong (+++). Its association with clinicopathologic data and prognosis of ESCC was analyzed. In experiment 2, another cohort of 32 fresh-frozen specimens from 20 patients was investigated by western blot to detect the expression of Twist and E-Cadherin, including 8 metastatic ESCC (M1 group), 12 non-metastatic ESCC (M0 group) and their paired 12 non-neoplastic oesophageal epithelium at the resection margin (control group). And the correlation between Twist and E-Cadherin expression was evaluated.Results:In experiment 1, Twist staining gave a diffuse cytoplasmic pattern in both ESCC and some of non-neoplastic oesophageal epithelium, nevertheless, the protein level was significantly increased in the ESCC specimens compared with the non-neoplastic oesophageal epithelium (P<0.001). The Twist expression was positively related with pTNM stage (P=0.035) and distant metastasis after operation (P=0.001), but no significant distinction of staining was found among different groups of age, sex, tumor location, T/N stage and histological grade (P>0.05). Kaplan-Meier analysis showed that up-regulated expression of Twist was significantly associated with lower survival rates of patients after operation (log-rank, P<0.001). In addition, mono-variate analysis revealed that T/N classification, pTNM stage, distant metastasis and Twist expression were significantly correlated with mortality risk of patients after operation (P<0.05). By Multi-variate analysis, Twist expression (RR=2.394, P<0.001) and N classification were identified as the independent prognostic factors of patient survival.In experiment 2, the expression of Twist was gradually increased in the control, M0 and M1 groups (P<0.001), whereas the expression of E-Cadherin was progressively reduced. Spearman's rank test was applied to correlate between Twist and E-Cadherin expression level. The result showed a significant negative correlation between Twist and E-Cadherin expression (r=0.820, P<0.001).Conclusions:1. Twist protein was located in cytoplasm of ESCC cells and may play a vital role in carcinogenesis of ESCC.2. Twist may promote distant metastasis of post-operation patients with ESCC.3. Up-regulation of Twist was identified as an independent prognostic factor of ESCC,which associated with lower survival rates and higher mortality risk of post-operation patients with ESCC.4. Twist may promote metastasis of ESCC by suppressing E-Cadherin expression.