Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

https://doi.org/10.1016/j.bbrc.2011.01.029Get rights and content

Abstract

MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalized by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.

Research highlights

ā–ŗ We identified changes in the expression and function of EGFR by anti-MUC1 antibody. ā–ŗ An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. ā–ŗ GP1.4 specifically inhibited ERK signaling triggered EGFā€“EGFR signaling pathway. ā–ŗ Internalization of EGFR was dependent on the presence of MUC1 on cell surface. ā–ŗ GP1.4 significantly inhibited EGF-dependent cancer cell proliferation and migration.

Introduction

MUC1 is present on the surface of various mucosal epithelial cells and hematopoietic cells and is overexpressed in various adenocarcinomas [1]. MUC1 is synthesized as a single peptide and then undergoes autocleavage into two subunits, subsequently forming a stable non-covalent heterodimer consisting of an extracellular domain and a cytoplasmic tail [2], [3]. The extracellular domain of MUC1 is composed of variable number tandem repeats (VNTR) that consist of 20-amino acids enriched in serine, threonine, and proline residues modified by extensive O-glycans, which is thought of as a physical barrier against the extracellular milieu. Enhanced expression of MUC1, by tumor microenvironment such as hypoxia [4], has been associated with tumor progression [5]. In vitro studies demonstrated that the expression of MUC1 in cancer cells is involved in the invasion [6], [7], [8], potentiation of cellular signaling [9], [10], and resistance to genotoxic anticancer reagents [11], [12], suggesting that the expression of MUC1 is closely associated with malignancy of tumor, leading to poor prognosis.

MUC1 has been shown to interact with ErbBs on the cell surface [9], [13], leading to the sustenance of cell signaling by ligand binding [14]. The EGF receptor (EGFR) is a member of the ErbB family and a transmembrane tyrosine kinase protein that are overexpressed in as many as 80% of pancreatic ductal adenocarcinomas [15], [16]. Stimulation of EGFR in cancer cells results in the activation of multiple intracellular signaling cascades, including ERK and Akt, which markedly increase cellular proliferation [17], [18]. In fact, inhibition of ErbB pathways with targeted agents, such as monoclonal antibodies or tyrosine kinase inhibitors, suppresses the activation of downstream signaling pathways, and restores normal cell proliferation control and induces apoptosis in vitro and xenograft models [19]. Since the reduction of MUC1 expression resulted in the rapid downregulation of surface EGFR in EGF treated cells, compared to MUC1 positive cells [14], it is thought that the expression of MUC1 on the cell is crucial for the expression of EGFR and EGFR signaling which might lead to cancer progression.

We previously showed that MUC1 was internalized by the anti-MUC1 antibody via the macropinocytotic pathway [20]. Since cell signaling through EGFR can be modulated by its expression level on the cell surface, we hypothesize that the anti-MUC1 antibody might reduce EGFR signaling by the alteration of EGFR expression on the cell surface. In this study, we demonstrate that the anti-MUC1 antibody triggered the translocation of EGFR from the cell surface to the intracellular region, leading to the desensitization to its ligand and the reduction of cellular signaling through EGFR.

Section snippets

Materials

All the chemicals and reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated. The following antibodies were obtained commercially: anti-MUC1 mouse monoclonal antibody GP1.4 (Lab Vision, Fremont, CA) and C595 (AbD Serotec, Oxford, UK), anti-EGF receptor mouse monoclonal antibody, non-immune normal mouse IgG (Santa Cruz, Santa Cruz, CA), anti-Ī²-actin antibody (Sigma), anti-phosphorylated ERK, ERK rabbit polyclonal antibody (Cell Signaling, Danvers, MA). Cells used in this

Results

We examined the cellular trafficking of EGF receptor (EGFR) following an interaction with MUC1 and anti-MUC1 antibody in human pancreatic Panc-1 cells. EGFR were localized on the cell surface of Panc-1 cells when cells were treated with vehicle (Fig. 1A) or non-immune control IgG for 1Ā h at 37Ā Ā°C (Supplementary Fig. S1A). EGFR and MUC1 were notably localized in the cytoplasmic region and on the cell surface, when cells were treated with 4Ā Ī¼g/ml of GP1.4 for 1Ā h at 37Ā Ā°C (Fig. 1B), and most signals

Discussion

In this study, immunocytochemical analysis demonstrated that when pancreatic cancer Panc-1 cells were treated with anti-MUC1 antibody GP1.4, EGFR were mostly localized in the cytoplasmic region, but not by another anti-MUC1 antibody C595 (Fig. 1), which was confirmed by the FACS analysis (Supplementary Fig. S2). In addition, GP1.4, but not C595, inhibited ERK phosphorylation by EGF, due to the lack of EGFR expression on cell surface (Fig. 2). It suggested that GP1.4 induced internalization of

Competing financial interests

The authors declare no competing financial interests.

Acknowledgment

We are very grateful to Dr. Mary Ann Suico (Kumamoto University, Graduate School of Pharmaceutical sciences) for excellent editing throughout entire manuscript.

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