Epigenetic Silencing DACH1Promotes Esophageal Cancer Growth By Inhibiting TGF-β Signaling

Background&AimsEsophageal cancer is the fifth most malignant disease and has been ranked as thefourth leading cause of cancer related deaths in China. Esophageal squamous cellcarcinoma (ESCC) is the predominant histological type of esophageal cancer, and accountsfor approximately90%of esophageal cancer cases in the northern and central China. Themechanisms of esophageal carcinogenesis remain unclear. Multiple genetic and epigeneticalterations were regarded as important factors for developing esophageal cancer. HumanDachshund homologue1(DACH1) is a major component of the Retinal DeterminationGene Network. Loss of DACH1expression was found in breast, prostate, lung,endometrial, colorectal and hepatocellular cancer. The expression of DACH1wasregulated by promoter region hypermethylation in endometrial, colorectal andhepatocellular cancer. DACH1suppressed human hepatocellular carcinoma by activatingTGF-β signaling. While the epigenetic changes and the function of DACH1in humanESCC remain unclear. In this study, we mainly analyzed the epigenetic changes, functionand mechanism of DACH1on esophageal carcinogenesis.MethodsIn this study,11esophageal cancer cell lines,10cases of normal esophageal mucosa,51cases of different grades of dysplasia and104cases of primary esophageal squamouscancer were employed. The expression and the methylation status of DACH1weredetected by RT-PCR, immunohistochemistry and methylation specific PCR. The functionof DACH1in esophageal carcinogenesis was detected by flow cytometry, colonyformation techniques and xenograft mice model. The effect of DACH1on TGF-β signalingwas analyzed by dual-luciferase reporter assay and western blot. ResultsPromoter region methylation and loss of DACH1expression were found in4(KYSE150, KYSE510, TE1and TE3) of11esophageal cancer cell lines.Re-expression/increasing expression of DACH1was induced by5-aza-2’-deoxycytidinetreatment in these methylated cell lines.18.8%(6of32) of grade1,42.1%(8of19) ofgrade2and grade3dysplasia, and61.5%(64of104) of esophageal cancer weremethylated, but no methylation was found in10cases of normal esophageal mucosa. Themethylation was increased in progression tendency during esophageal carcinogenesis (P <0.01). DACH1methylation was associated with poor differentiation (P <0.05) and latetumor stage (P <0.05). Restoration of DACH1expression inhibited cell growth andactivated TGF-β signaling in KYSE150and KYSE510cells. DACH1suppressed humanesophageal cancer cell tumor growth in xenograft mice.ConclusionDACH1is frequently methylated in human esophageal cancer and methylation ofDACH1is involved in the early stage of esophageal carcinogenesis. DACH1expression isregulated by promoter region hypermethylation. DACH1suppresses esophageal cancergrowth by activating TGF-β signaling.