In vivo and In vitro Evidence for Transforming Growth Factor-β1-Mediated Epithelial to Mesenchymal Transition in Esophageal Adenocarcinoma

Jonathan R.E Rees,Benjamin A Onwuegbusi,Rebecca C Fitzgerald,Derek Alderson,Vicki E Save

doi:10.1158/0008-5472.can-06-1842

Abstract

There is increasing evidence that epithelial to mesenchymal transition (EMT) is involved in cancer progression. Because local invasion and metastasis occurs early in the pathogenesis of esophageal adenocarcinoma, we hypothesized that EMT may be important in this disease. Using immunohistochemistry in a well-characterized set of adenocarcinoma tissues, we showed down-regulation of epithelial markers (E-cadherin and cytokeratin 18) and up-regulation of mesenchymal markers (vimentin and alpha-smooth muscle actin) with concomitant transforming growth factor-beta1 (TGF-beta1) expression at the invasive margin compared with the central tumor. A panel of esophageal cell lines was examined for the ability of TGF-beta1 to induce EMT in vitro. TE7 cells were selected as a model because TGF-beta1 (0-5 ng/mL) treatment induced morphologic and molecular expression changes suggestive of EMT. In TE7 cells, these TGF-beta1-induced changes were reversed by 100 ng/mL of bone morphogenetic protein 7 (BMP7), another member of the TGF-beta1 superfamily. EMT was mediated via canonical TGF-beta1 signaling with concomitant up-regulation of SMAD-interacting protein 1. Alterations in functional variables (aggregation, wounding, motility, and invasion) following TGF-beta1 treatment were consistent with a more invasive phenotype. These functional changes were reversed by BMP7 and SMAD4 RNA interference in vitro. These data suggest that TGF-beta1-mediated EMT may be relevant in esophageal carcinogenesis.

Highlights

Esophageal adenocarcinoma is increasing in incidence [1]
Treatment with 5 ng/mL TGF-h1 resulted in a significant increase in cell invasion (Matrigel two-chamber invasion assay; P = 1.63 Â 10À6), a statistically significant decrease in aggregation as marked by an increase in the aggregation score, an increase in healing, and promotion of cell motility from 2.6 Am/s in untreated cells to 3.4 Am/s in cells treated with TGF-h1 at 5 ng/mL. These effects were abrogated by the addition of bone morphogenetic protein 7 (BMP7) or SMAD4 RNA interference (RNAi) for each assay: invasion (P = 7.56 Â 10À6), aggregation (P = 1.43 Â 10À7), healing (P = 0.003), and motility (P = 0.0081; Table 1). These in vivo data show immunohistochemical evidence for epithelial to mesenchymal transition (EMT), which is associated with TGF-h1 expression, at the invasion front of esophageal adenocarcinoma
A TGF-h1-induced in vitro model of EMT has shown morphologic, molecular, and functional evidence for this process that was reversible by BMP7 and SMAD4 RNAi

Summary

Introduction

Esophageal adenocarcinoma is increasing in incidence [1]. The majority of patients present with locally advanced or metastatic disease, and as a result, the outcome for these patients is poor with an overall 5-year survival rate of 13% [2].Many molecular and phenotypic events underlie the development of invasion and metastases [3]. Esophageal adenocarcinoma is increasing in incidence [1]. The majority of patients present with locally advanced or metastatic disease, and as a result, the outcome for these patients is poor with an overall 5-year survival rate of 13% [2]. Many molecular and phenotypic events underlie the development of invasion and metastases [3]. These include the loss of proliferative control, enhanced cellular migration and invasion, extracellular matrix degradation, angiogenesis, lymphangiogenesis, and vascular invasion followed by the distant seeding of tumor cells in specific tissues, such as the liver or lung. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

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Conclusion