Biochemical and Biophysical Research Communications
Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells
Research highlights
āŗ We identified changes in the expression and function of EGFR by anti-MUC1 antibody. āŗ An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. āŗ GP1.4 specifically inhibited ERK signaling triggered EGFāEGFR signaling pathway. āŗ Internalization of EGFR was dependent on the presence of MUC1 on cell surface. āŗ 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.
References (45)
- et al.
Cell-associated episialin is a complex containing two proteins derived from a common precursor
J. Biol. Chem.
(1992) - et al.
Hypoxia enhances MUC1 expression in a lung adenocarcinoma cell line
Biochem. Biophys. Res. Commun.
(2009) - et al.
Transgenic MUC1 interacts with epidermal growth factor receptor and correlates with mitogen-activated protein kinase activation in the mouse mammary gland
J. Biol. Chem.
(2001) - et al.
MUC1 oncoprotein stabilizes and activates estrogen receptor alpha
Mol. Cell
(2006) - et al.
Human MUC1 carcinoma-associated protein confers resistance to genotoxic anticancer agents
Cancer Cell
(2004) - et al.
Human MUC1 oncoprotein regulates p53-responsive gene transcription in the genotoxic stress response
Cancer Cell
(2005) - et al.
Internalization of MUC1 by anti-MUC1 antibody from cell membrane through the macropinocytotic pathway
Biochem. Biophys. Res. Commun.
(2009) - et al.
Activation of cytosolic phospholipase A2alpha by epidermal growth factor (EGF) and phorbol ester in HeLa cells: different effects of inhibitors for EGF receptor, protein kinase C, Src, and C-Raf
J. Pharmacol. Sci.
(2009) - et al.
Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation
Dev. Cell
(2008) - et al.
Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers
Biochim. Biophys. Acta
(2005)
Regulated migration of epidermal growth factor receptor from caveolae
J. Biol. Chem.
MUC1 and the MUCs: a family of human mucins with impact in cancer biology
Crit. Rev. Clin. Lab. Sci.
Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin
Nat. Struct. Mol. Biol.
MUC1 overexpression results in mammary gland tumorigenesis and prolonged alveolar differentiation
Oncogene
Enhancement of metastatic properties of pancreatic cancer cells by MUC1 gene encoding an anti-adhesion molecule
Int. J. Cancer
Contribution of the MUC1 tandem repeat and cytoplasmic tail to invasive and metastatic properties of a pancreatic cancer cell line
Cancer Res.
RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells
Clin. Cancer Res.
Transforming growth factor alpha dependent cancer progression is modulated by Muc1
Cancer Res.
MUC1 is a novel regulator of ErbB1 receptor trafficking
Oncogene
Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies
J. Clin. Oncol.
Epidermal growth factor receptor expression in human pancreatic cancer: significance for liver metastasis
Int. J. Mol. Med.
Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma
Cancer Res.
Cited by (36)
AntiāMUC1 nanobody can synergize the Tamoxifen and Herceptin effects on breast cancer cells by inducing ER, PR and HER2 overexpression
2023, International ImmunopharmacologyTargeting tumor-associated MUC1 overcomes anoikis-resistance in pancreatic cancer
2023, Translational ResearchCitation Excerpt :Fig 8, E is an attempt to summarize the mechanism of action of TAB004 antibody and its antitumor effects. Multiple studies on the mechanism of anti-MUC1 antibodies as monotherapy or in combination with drugs have been published before, showing activation of the apoptotic pathways and blocking of EGFR and PI3K pathways.55,56 However, this is the first time that a tMUC1 antibody (TAB004) has been shown to induce phosphorylation of MUC1-CT by activating c-Src and reverse anoikis-resistance by degrading tMUC1 with an in-depth transcriptomic and proteomic analysis leading to the functional effect.
Anti-cancer effects of pyrazole-platinum(II) complexes combined with anti-MUC1 monoclonal antibody versus monotherapy in DLD-1 and HT-29 colon cancer cells
2022, Translational OncologyCitation Excerpt :Hisatsune et al. [19] stated, that MUC1 on the cancer cells can be internalized by mAb through the macropinocytic pathway, in the process mediated by MUC1-cytoplasmic tail. It was also reported that anti-MUC1 applied in pancreatic cancer cells caused internalization of EGFR, what resulted in suppression of proliferation and migration of cancer cells [20]. In the present study, performed on DLD-1 and HT-29 colon cancer cells, we applied BC2 anti-MUC1 monoclonal antibody, reacting with the MUC1 background protein with low effectiveness for glycosylation.
Surface engineered nanocarriers for the management of breast cancer
2021, Materials Science and Engineering CCitation Excerpt :MUC1 gene amplification has been found in 40% of breast cancers, and it is strongly linked to an increase in MUC1 mRNA and protein levels [82]. MUC1 targeting inhibits EGFR signaling and decreases cancer growth [83]. To target the MUC1, either anti-MUC1 monoclonal antibodies or aptamers can be used as ligands.
Cross-talk between myeloid-derived suppressor cells and Mucin1 in breast cancer vaccination: On the verge of a breakthrough
2020, Life SciencesCitation Excerpt :MUC1 antibody conjugated with yttrium90 on ovarian cancer has confirmed the elimination of tumoral cells through binding to the MUC1 epitope [195]. In concert with these, anti-MUC1 antibody GP1.4 via inhibition of EGFR and ERK pathway arrests the tumoral proliferation [196]. In line with these findings, a recent study on breast cancer has elucidated that the administration of an antibody against the extracellular portion of C-MUC1 curtails the cellular proliferation in the T47D breast cancer line [197].
Fluorescent imaging probes for in vivo ovarian cancer targeted detection and surgery
2024, Medicinal Research Reviews