Altered Egfr Localization and Degradation in Human Breast Cancer Cells with an Amphiregulin/Egfr Autocrine Loop

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  • Topic: Signal transduction, Cell biology, Breast cancer
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  • Published : May 15, 2013
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Cell Signal. Author manuscript; available in PMC 2010 February 1. Published in final edited form as: Cell Signal. 2009 February ; 21(2): 212–219. doi:10.1016/j.cellsig.2008.10.003.

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Altered EGFR Localization and Degradation in Human Breast Cancer Cells with an Amphiregulin/EGFR Autocrine Loop Nicole E. Willmartha,b, Andrea Baillob, Michele L. Dziubinskib, Kristy Wilsonc, David J. Riese IIc, and Stephen P. Ethierb,1 a Department of Cancer Biology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA, USA 19107 b Breast Cancer Program, Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, Detroit, MI, USA 48201 and c Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA 47907

Abstract
The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein than MCF10A cells cultured in EGF. Pulse chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell-cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.

Keywords Amphiregulin; EGFR; degradation; localization; breast cancer

1To whom correspondence should be addressed: Karmanos Cancer Institute, 4100 John R, Detroit, MI, 48201, Tel. 313 576-8335; Fax. 313 576-8626; E-mail: ethier@karmanos.org. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Willmarth et al.

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Introduction
The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase [1]. The seven different ligands that can bind the EGFR are epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), amphiregulin (AR), heparin binding EGF (HBEGF), betacellulin (BTC), epiregulin (EPR), and epigen [2–8]. Once ligand binds, the receptor dimerizes and becomes activated, leading to propagation of downstream...
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