Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

Valgardur Sigurdsson,Bjarni A Agnarsson,Magnus K Magnusson,Ake Borg,Markus Ringnér,Bylgja Hilmarsdottir,Rene Villadsen,Agla J R Fridriksdottir,Hekla Sigmundsdottir,Thorarinn Gudjonsson,Ole William Petersen,Alexander Swarbrick

doi:10.1371/journal.pone.0023833

Abstract

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44high/CD24low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Highlights

Epithelial to mesenchymal transition (EMT) is associated with increased aggressiveness and adverse prognosis in carcinomas [1,2]
In order to test the effects of breast endothelial cells (BRENCs) on growth, and morphogenesis of D492 cells we set up a coculture with BRENCS and D492 cells inside a reconstituted basement membrane (rBM)
That in a 3D coculture model EMT-like cells arise from immortalized breast epithelial cells with stem cell properties upon interaction with breast endothelial cells

Summary

Introduction

Epithelial to mesenchymal transition (EMT) is associated with increased aggressiveness and adverse prognosis in carcinomas [1,2]. Increasing number of factors are known that can induce EMT including transforming growth factor-b (TGF-b), ligands for receptor tyrosine kinases such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and hepatocyte growth factor (HGF) as well as components of the extracellular matrix [3,19]. These signaling events control transcriptional regulatory factors such as Snail, Slug, Twist, ZEB1, ZEB2 and FOXC2 leading to increased and decreased expression of mesenchymal and epithelial markers, respectively. Defining the cellular and microenvironmental cues that trigger EMT during the progression of breast cancers is critical and could provide new therapeutic targets

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Results

Conclusion