The MUC4 membrane-bound mucin regulates esophageal cancer cell proliferation and migration properties: Implication for S100A4 protein

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Abstract

MUC4 is a membrane-bound mucin known to participate in tumor progression. It has been shown that MUC4 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplastic lesions to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is the gastro-esophageal reflux, and MUC4 was previously shown to be upregulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC4 plays a role in biological properties of human esophageal cancer cells. For that stable MUC4-deficient cancer cell lines (shMUC4 cells) were established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of shMUC4 cells were analyzed. Our results show that shMUC4 cells were less proliferative, had decreased migration properties and did not express S100A4 protein when compared with MUC4 expressing cells. Absence of MUC4 did not impair shMUC4 invasiveness. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC4. Altogether, these data indicate that MUC4 plays a key role in proliferative and migrating properties of esophageal cancer cells as well as is a tumor growth promoter. MUC4 mucin appears thus as a good therapeutic target to slow-down esophageal tumor progression.

Highlights

► Loss of MUC4 reduces proliferation of esophageal cancer cells. ► MUC4 inhibition impairs migration of esophageal cancer cells but not their invasion. ► Loss of MUC4 significantly reduces in vivo tumor growth. ► Decrease of S100A4 induced by MUC4 inhibition impairs proliferation and migration.

Introduction

Mucins are large O-glycoproteins that can be secreted in the lumen where they participate in mucus formation and in epithelia protection. Mucins may also be membrane-bound and participate in cell signaling and cell–cell or cell–extracellular matrix interactions [1], [2]. Membrane-bound mucins, to which MUC4 belongs, are normally expressed at the apical membrane of epithelial cells. Because of this localization and their extremely long extracellular domain (2 μm) these mucins are considered as receptors and sensors of the external environment. Because of their capacity to interact with oncogenic receptors they also play roles in signal transduction and modulate cellular biological properties [3]. It has also been shown that membrane-bound mucins are often overexpressed in cancer and that they promote tumor progression [4], [5].

MUC4 is a type-I transmembrane glycoprotein of 930 kDa composed of two subunits: one extracellular highly glycosylated, MUC4α, and one transmembrane subunit, MUC4β, that contains three EGF-like domains [2]. An aberrant pattern of expression of MUC4 has been reported in numerous cancers [6] and its overexpression is often correlated with a poor prognosis [7]. In the normal human esophagus the membrane-bound mucin MUC4 is expressed at a very low level whereas in esophageal cancer its expression is progressively increasing from the early metaplastic steps till adenocarcinoma (ADK) [8], [9].

Esophageal ADK incidence has been increasing for the last 40 years [10] and up to now, curative treatment is mainly based on extended esophageal resection, in combination with radiation and/or chemotherapy for locally advanced tumors [11]. It is also known that esophageal ADK is induced by a chronic exposure of the distal esophagus to the duodeno-gastro-esophageal reflux [12], [13], [14]. This exposure induces the initiation of early carcinogenetic lesions called Barrett esophagus (BE), characterized by an intestinal and/or gastric metaplasia [15]. Under the effects of reflux, these lesions may evolve to low-grade then high-grade dysplasia and finally to ADK [16]. Incidently, MUC4 has been shown in our laboratory to be upregulated by the main bile acids present in the reflux (cholic, taurocholic and glycocholic acids) in human esophageal adenocarcinomatous cells [17], [18].

In order to improve patient prognosis and therapy, it is now mandatory to better identify actors and molecular mechanisms involved in this carcinogenesis. MUC4 altered pattern of expression and the fact that membrane-bound mucins are known to play a role in tumor progression and metastasis formation in other type of cancers [4], [5], [7], [19] led us to hypothesize that this mucin could also play a key role in esophageal ADK initiation and/or progression.

Having shown a link between reflux, bile acids and MUC4 overexpression in esophageal ADK progression, we thus undertook to study MUC4 effects on the biological properties of human esophageal adenocarcinomatous cells.

Section snippets

Cell culture

The OE33 esophageal adenocarcinomatous cell line was cultured as described previously [17]. OE33 cells knocked-down for MUC4 (shMUC4) were established by stable retro-viral infection using a pRetroSuper plasmid (SABiosciences™) containing a sequence targeting MUC4 (5′-AAGTGGAACGAATCGATTCTGTCTCTTGAACAGAATCGATTCGTTCCACTT-3′). The control cells [20] were infected with the corresponding empty vector. Results presented in this study are the means of two representative clones, shMUC4-1 and shMUC4-2,

Characterization of shMUC4 OE33 cells

To study the effects of MUC4 mucin on esophageal cancer cells properties, a stable OE33 shMUC4 cell line was established using shRNA approach (Fig. 1). MUC4 decreased expression in shMUC4 cells compared to Mock clones was confirmed at the protein level (Fig. 1A).

Proliferation

MUC4 role in proliferation was studied by measuring shMUC4 cells capacity to proliferate. MUC4 deficient cells showed decreased proliferation compared to Mock cells as of day 3 (Fig. 1B). This decrease was significant as of day 4 (p = 

Discussion

In this report we show that decreased expression of MUC4 in OE33 cells strongly impaired their in vitro proliferation and migration properties whereas invasion was unchanged. In vivo SC tumors deficient for MUC4 were also smaller than those expressing MUC4. These results confirm the fact that MUC4 is an oncoprotein involved in many epithelial cancers as it has been shown that MUC4 could regulate biological properties of other cell type as pancreatic cancer cells [6], [22], [23], [24]. Aberrant

Acknowledgments

We thank Dr. Jean-Luc Desseyn (Inserm U995, Lille, France) and Dr. David Bernard (Inserm UMR1052/CNRS5286, Centre Léon Bérard, Lyon, France) for their help in establishing the stable MUC4 deficient cell lines. This work was supported by La Fondation pour l’Avenir and La Fondation pour la Recherche Médicale (Equipe FRM 2010, Christophe Mariette). Emilie Bruyère is the recipient of a Ph.D. Fellowship from Le Centre Hospitalier Régional Universitaire de Lille and Région Nord-Pas de Calais. Nicolas

References (32)

  • T. Tabata et al.

    RNA interference targeting against S100A4 suppresses cell growth and motility and induces apoptosis in human pancreatic cancer cells

    Biochem. Biophys. Res. Commun.

    (2009)
  • K.L. Carraway et al.

    Cell signaling through membrane mucins

    BioEssays

    (2002)
  • N. Jonckheere et al.

    The ever growing family of membrane-bound mucins

  • D.W. Kufe

    Mucins in cancer: function, prognosis and therapy

    Nat. Rev. Cancer

    (2009)
  • M.A. Hollingsworth et al.

    Mucins in cancer: protection and control of the cell surface

    Nat. Rev. Cancer

    (2004)
  • P. Chaturvedi et al.

    Structure, evolution, and biology of the MUC4 mucin

    FASEB J.

    (2008)
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