Epigenetic Silencing Of EYA4 Induces Epithelial-Mesenchymal Transition In Esophageal Squamous Cell Carcinoma

Esophageal cancer has become the sixth leading cause of death and the eighth most frequently diagnosed cancer worldwide. Esophageal squamous cell carcinoma is predominant in most parts of the world, especially in high risk regions such as China where it accounts for over 90% of the total cases of esophageal cancer. Despite significant advances in the diagnosis, staging and treatment of esophageal squamous cell carcinoma in recent decades, few significant improvements in overall survival have been achieved: the 5-year overall survival rate remains below 14%. Local recurrences and distant organ metastases are the leading cause of treatment failure and poor prognosis. Therefore, it is imperative to explore the underlying mechanism of metastasis and identify additional biomarkers and therapeutic targets for esophageal squamous cell carcinoma.EYA4 is an atypical, dual-functioning protein phosphatase that functions in mediating DNA repair, cell apoptosis and angiogenesis. EYA family members have been reported to exhibit abnormal expression in various malignant tumors, but their functions in carcinomas are controversial. In this study, we examined whether epigenetic changes in EYA4 occurred in ESCC and explored the role of EYA4 in tumor invasion and metastasis in human ESCC.The methylation status of EYA4 gene was detected by MSP and BSP in 50 ESCC surgical tissue samples and 8 ESCC cell lines. We found EYA4 is methylated in 80%(40/50) of ESCC tissues and 58%(29/50) of adjacent non-tumor tissues. Of 8 ESCC cell lines, EYA4 is unmethylated in only KYSE30 and totally methylated or partially in the other 7 cell lines. We found that EYA4 expression was regulated by promoter region hypermethylation and was enhanced with 5-aza-dC treatment in ESCC cell lines.We showed that knockdown of EYA4 with shRNA promoted the migration and invasion abilities of KYSE30 in vivo and in vitro. On the contrary, overexpression of EYA4 inhibited the migration and invasion ability of KYSE180 and KYSE450. Furthermore, EYA4 inhibited migratory and invasive potential of ESCC cell lines as well as the expression of vimentin and slug through suppressing AKT/GSK3β/Slug signaling pathways.TGF-β1 is major inducer of EMT in ESCC. We found TGF-β1 induced epithelial-mesenchymal transition in ESCC cell lines, including changes in cell morphology and induction of mesenchymal marker expression. Accompanied with the EMT, EYA4 expression was decreased by TGF-β1 treatment in a concentration and time dependent manner. TGF-β1 may induce the methylation of EYA4 gene by upregulating the expression of DNA methyltransferase DNMTl and DNMT3A, which reduced the expression of EYA4. Furthermore, EYA4 overexpression in ESCC cell lines significantly abolished TGF-β1-induced EMT, together with the increase of epithelial markers expression and the decrease of mesenchymal markers expression.In addition, EYA4 expression in ESCC cell lines suppressed cisplatin-induced cell apoptosis, promoted DNA repair and maintained genomic stability.In conclusion, these studies suggest that EYA4 is frequently hypermethylated in ESCC tissues and cell lines, plays an important role in the development of ESCC and might be a potential anti-metastasis target in future clinical practice.