Su1181 A Diagnostic DNA FISH Biomarker Assay Identifies HGD or EAC in Barrett Esophagus

Abstract

examine the effect of acid on Wnt/β-catenin signaling activation and on regulating the expression of Dickkopf1 (DKK1), an inhibitor of the Wnt. Methods: Normal esophageal squamous cells lines (EPC1-hTERT, EPC2-hTERT), a non-dysplastic Barrett's esophageal cell line (CP-A) and an esophageal adenocarcinoma cell line (OE33) were exposed to acidic media (pH 4.0) in a pulsive manner. Human esophageal mucosal biopsies in triplicate from healthy (n=1), NERD (n=1), GERD (n=1) and Barrett's (n=1) patients were obtained and cultured in acidic (pH 4.0) or non acidic media. Localization and levels of β-catenin were determined by Immunofluorescence staining and Western blot. Wnt-activity was assessed by Luciferase assay following transfection with β-catenin-LEF/TCF-sensitive (TOP) or βcatenin-LEF/TCF insensitive (FOP) reporter vector. Immunofluorescence was used for βcatenin and E-Cadherin co-localization. DKK1 and β-catenin gene expression was resolved by qRT-PCR. DKK1 secretion in cells culture media and organ culture media was quantified by ELISA assay.Results: Acid destabilized E-cadherin/β-catenin complex in cell-cell junctions and resulted in β-catenin translocation to nucleus. Wnt-activity correlated with nuclear translocation of β-catenin. Cytosolic shuttling of β-catenin occurred in a rapid and transient manner after acid withdrawal. Chronic pulsed acid exposure increased DKK1 expression in normal squamous cells but not in metaplastic columnar cells. DKK1 overexpression correlated with a significant degradation of β-catenin. Mucosal biopsies from patients with NERD/ GERD secreted significantly higher levels of Dkk1 than healthy and metaplastic mucosa biopsies. Conclusions: These findings suggest that acid reflux induces the activation of Wnt-signaling pathway and that the overexpression of DKK1 is the long term response in the normal squamous esophageal tissue but not in the Barrett's esophagus. These findings also suggest a homeostatic role for DKK1 in GERD and its dysfunction to be a potential mechanism for progression to Barrett's metaplasia.