Wnt/ß-Catenin Signaling Activation beyond Robust Nuclear ß-Catenin Accumulation in Nondysplastic Barrett's Esophagus: Regulation via Dickkopf-1.

ABSTRACT

INTRODUCTION: Wnt/ß-catenin signaling activation has been reported only during the late steps of Barrett's esophagus (BE) neoplastic progression, but not in BE metaplasia, based on the absence of nuclear ß-catenin. However, ß-catenin transcriptional activity has been recorded in absence of robust nuclear accumulation. Thus, we aimed to investigate the Wnt/ß-catenin signaling in nondysplastic BE. METHODS: Esophageal tissues from healthy and BE patients without dysplasia were analyzed for Wnt target gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Esophageal squamous (EPC1-& EPC2-hTERT), BE metaplastic (CP-A), and adenocarcinoma (OE33) cell lines were characterized for Wnt activation by qRT-PCR, Western blot, and luciferase assay. Wnt activity regulation was examined by using recombinant Wnt3a and Dickkopf-1 (Dkk1) as well as Dkk1 short interfering RNA. RESULTS: Wnt target genes (AXIN2, c-MYC, Cyclin D1, Dkk1) and Wnt3a were significantly upregulated in nondysplastic BE compared with squamous mucosa. Elevated levels of dephosphorylated ß-catenin were detected in nondysplastic BE. Nuclear active ß-catenin and TOPflash activity were increased in CP-A and OE33 cells compared with squamous cells. Wnt3a-mediated ß-catenin signaling activation was abolished by Dkk1 in CP-A cells. TOPFlash activity was elevated following Dkk1 silencing in CP-A but not in OE33 cells. Dysplastic and esophageal adenocarcinoma tissues demonstrated further Dkk1 and AXIN2 overexpression. CONCLUSIONS: Despite the absence of robust nuclear accumulation, ß-catenin is transcriptionally active in nondysplastic BE. Dkk1 overexpression regulates ß-catenin signaling in BE metaplastic but not in adenocarcinoma cells, suggesting that early perturbation of Dkk1-mediated signaling suppression may contribute to BE malignant transformation.