BMP4 Signaling Is Able to Induce an Epithelial-Mesenchymal Transition-Like Phenotype in Barrett's Esophagus and Esophageal Adenocarcinoma through Induction of SNAIL2.

Christine Kestens,G Johan A Offerhaus,Jantine W P M Van Baal,Peter D Siersema,Michael Klymkowsky

doi:10.1371/journal.pone.0155754

Abstract

BackgroundBone morphogenetic protein 4 (BMP4) signaling is involved in the development of Barrett’s esophagus (BE), a precursor of esophageal adenocarcinoma (EAC). In various cancers, BMP4 has been found to induce epithelial-mesenchymal transition (EMT) but its function in the development of EAC is currently unclear.AimTo investigate the expression of BMP4 and several members of the BMP4 pathway in EAC. Additionally, to determine the effect of BMP4 signaling in a human Barrett’s esophagus (BAR-T) and adenocarcinoma (OE33) cell line.MethodsExpression of BMP4, its downstream target ID2 and members of the BMP4 pathway were determined by Q-RT-PCR, immunohistochemistry and Western blot analysis using biopsy samples from EAC patients. BAR-T and OE33 cells were incubated with BMP4 or the BMP4 antagonist, Noggin, and cell viability and migration assays were performed. In addition, expression of factors associated with EMT (SNAIL2, CDH1, CDH2 and Vimentin) was evaluated by Q-RT-PCR and Western blot analysis.ResultsCompared to squamous epithelium (SQ), BMP4 expression was significantly upregulated in EAC and BE. In addition, the expression of ID2 was significantly upregulated in EAC and BE compared to SQ. Western blot analysis confirmed our results, showing an upregulated expression of BMP4 and ID2 in both BE and EAC. In addition, more phosphorylation of SMAD1/5/8 was observed. BMP4 incubation inhibited cell viability, but induced cell migration in both BAR-T and OE33 cells. Upon BMP4 incubation, SNAIL2 expression was significantly upregulated in BAR-T and OE33 cells while CDH1 expression was significantly downregulated. These results were confirmed by Western blot analysis.ConclusionOur results indicate active BMP4 signaling in BE and EAC and suggest that this results in an invasive phenotype by inducing an EMT-like response through upregulation of SNAIL2 and subsequent downregulation of CDH1.

Highlights

Barrett’s esophagus (BE) is a premalignant condition in which the normal squamous epithelium (SQ) is replaced by intestinal type of epithelium containing goblet cells.[1]
Compared to squamous epithelium (SQ), Bone morphogenetic protein 4 (BMP4) expression was significantly upregulated in esophageal adenocarcinoma (EAC) and BE
Our results indicate active BMP4 signaling in BE and EAC and suggest that this results in an invasive phenotype by inducing an epithelial-mesenchymal transition (EMT)-like response through upregulation of SNAIL2 and subsequent downregulation of CDH1

Summary

Introduction

Barrett’s esophagus (BE) is a premalignant condition in which the normal squamous epithelium (SQ) is replaced by intestinal type of epithelium containing goblet cells.[1] BE is a wellknown risk factor for the development of esophageal adenocarcinoma (EAC). BMPs bind to transmembrane serine-threonine kinase receptors type 1 and 2 and activation of these receptors leads to phosphorylation of SMAD1, 5 and 8, which permits binding to SMAD4. This complex translocates to the nucleus and regulates gene transcription specific for the BMP pathway. Bone morphogenetic protein 4 (BMP4) signaling is involved in the development of Barrett’s esophagus (BE), a precursor of esophageal adenocarcinoma (EAC). BMP4 has been found to induce epithelial-mesenchymal transition (EMT) but its function in the development of EAC is currently unclear

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Results

Conclusion