Reversible interconversion and maintenance of mammary epithelial cell characteristics by the ligand-regulated EGFR system

Shinji Fukuda,Hiroyuki Kubota,Hironao Nakayama,Koh-Ichi Nakashiro,Daisuke Nanba,Hisayo Nishida-Fukuda,Shigeki Higashiyama

doi:10.1038/srep20209

Shinji Fukuda, Hiroyuki Kubota + Show 5 more

Open Access

https://doi.org/10.1038/srep20209

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Abstract

Epithelial cell plasticity is controlled by extracellular cues, but the underlying mechanisms remain to be fully understood. Epidermal growth factor (EGF) and amphiregulin (AREG) are high- and low-affinity ligands for EGF receptor (EGFR), respectively. EGFR signaling is known to promote epithelial-mesenchymal transition (EMT) by the activation of ERK and the induction of an EMT transcription factor, ZEB1. Here, we demonstrate that ligand-switching between EGF and AREG at equivalent molarity reversibly interconverts epithelial and mesenchymal-like states of EGFR signal-dependent mammary epithelial cells. The EGF- and AREG-cultured cells also differ in their epithelial characteristics, including the expression of cell surface markers, the mode of migration and the ability for acinus-formation. The ligand-switching between EGF and AREG temporally alters strength of the shared EGFR-ERK signaling. This alteration inverts relative expression levels of ZEB1 and its antagonizing microRNAs, miR-205 and miR-200c, those are critical determinants of the epithelial phenotype. Further, AREG-induced EGFR accumulation on the plasma membrane compensates for the weak association between AREG and EGFR. The EGFR dynamics enables AREG to support proliferation as efficiently as EGF at equivalent molarity and to maintain epithelial characteristics. Our findings reveal a role of EGFR ligands-generated signal strength in the regulation of mammary epithelial cell plasticity.

Highlights

Epithelial cell plasticity is controlled by extracellular cues, but the underlying mechanisms remain to be fully understood
The results indicated that AREG was able to support cell proliferation as efficiently as Epidermal growth factor (EGF) after several subcultures
Two groups have reported that EGF and AREG induced uni-directional differentiation of human mammary epithelial cells using a 3D organoid culture of breast tissues[48] or 2D culture of telomerase reverse transcriptase-immortalized stem/progenitor cells[49]

Summary

Introduction

Epithelial cell plasticity is controlled by extracellular cues, but the underlying mechanisms remain to be fully understood. The ligand-switching between EGF and AREG temporally alters strength of the shared EGFR-ERK signaling This alteration inverts relative expression levels of ZEB1 and its antagonizing microRNAs, miR-205 and miR-200c, those are critical determinants of the epithelial phenotype. EMT and the subsequent MET have been shown to play critical roles in the metastatic process[3,4] These findings illustrate the fundamental importance of epithelial cell plasticity regulated by the repeated EMT/MET cycles. The removal of TGFβ 1 triggers the inversion of the relative expression levels of ZEB1 and its antagonizing miRNAs, by which mesenchymal state can be reversed to epithelial state[8] These results suggest that the negative feedback loop involving EMT transcription factors and www.nature.com/scientificreports/. MiRNAs establishes the EMT/MET regulatory signaling network, and it could act as the molecular switch to determine the epithelial or mesenchymal phenotypes[9]

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