Contribution of Membrane Mucins to Tumor Progression Through Modulation of Cellular Growth Signaling Pathways

Kermit L. Carraway, III; Melanie Funes; Heather C. Workman; Colleen Sweeney    UC Davis Cancer Center, Sacramento, California 95817

I Mucin Structure, Function, and Involvement in Tumor Progression

Mucins and mucin-like proteins comprise a family of large transmembrane or secreted glycoproteins that are commonly associated with epithelial tissues (Strous and Dekker, 1992), but are also present at the surfaces of other selected cell types. Mucins are the major glycoprotein components of the mucous layer coating the cells lining the respiratory, digestive, and urogenital tracts, and are also prominently expressed by the ocular epithelium. Mucins possess specific domains that promote their oligomerization into viscous solutions or gels. In addition, their extensive O-glycosylation contributes to a high negative charge and a rigid and extended protein conformation, resulting in steric and charge repulsion of cells or other molecules (Jentoft, 1990). These properties allow mucins to function in protecting epithelial tissues from infection, dehydration, and physical or chemical assault, and to serve as a lubricant for tissues exposed to mechanical stresses (Perez-Vilar and Hill, 1999). The observed expression of mucins by vascular endothelial cells (Zhang et al., 2005) suggests that they may contribute to the formation of poorly adhesive surfaces in the lumen of blood vessels, augmenting blood flow by suppressing the counterproductive binding of blood components. On the other hand, some mucin glycosyl groups can serve as ligands for selectins and other cell adhesion molecules, promoting cell–cell interactions. In this context, mucin expression on the leading edge of activated T cells (Correa et al., 2003) suggests that mucins could also play a role in extravasation during inflammation.

A Mucin Structure

The unifying structural feature of all mucins and mucin-like proteins is that they express O-linked glycans in serine- and threonine (Ser/Thr)-rich clusters in their extracellular regions. A major subset of the mucins, a subfamily of proteins encoded by at least 20 distinct genes (designated MUC1–MUC20), contain Ser/Thr-rich tandem repeats of 8 to 59 amino acid residues in length, depending on the identity of the mucin (Baldus et al., 2004a). Repeats are heavily O-glycosylated, such that typically greater than 50% of mucin mass is O-linked oligosaccharide. N-linked glycans are also often present in mucins but to a much lesser degree. The number of Ser/Thr-rich repeats encoded by a particular mucin gene can vary markedly among individuals due to genetic polymorphism, and variable number tandem repetition (VNTR) polymorphisms in mucins are associated with susceptibility to various diseases (Fowler et al., 2001). Moreover, mucin glycosylation patterns are tissue and cell type dependent, and can be altered with cellular differentiation state or with neoplastic transformation. Aberrant mucin glycosylation occurs in essentially all types of human cancers and is associated with tumorigenecity and metastasis (Moniaux et al., 2004). Many glycosyl epitopes are tumor-associated antigens and have been used in diagnosis and immunotherapy.

Mucins are divided into three subgroups: the gel-forming, soluble, and transmembrane mucins. The gel-forming mucins, composed of MUC subfamily proteins MUC2, MUC5AC, MUC5B, MUC6, and MUC19, are characterized by the presence of several cysteine-rich domains in their extracellular regions (Chen et al., 2004). A single C-terminal cysteine-knot (CTCK) domain may be involved in the dimerization of these mucins. Trypsin inhibitor-like (TIL) domains and von Willebrand factor C (VWC) or D (VWD) domains are commonly found in a variety of extracellular proteins and may be involved in specific protein–protein interactions that contribute to the gel-forming properties of these mucins. Gel-forming mucins are typically expressed by specialized glands or goblet cells and are major contributors to the viscoelastic properties of mucus secretion. For example, MUC5AC and MUC5B mucins are the predominant gel-forming glycoproteins in airway mucus (Thornton and Sheehan, 2004). The soluble mucin MUC7 is significantly smaller than the gel-forming mucins and has no recognizable domains in its extracellular region. It is most abundantly expressed in the less viscous secretions of salivary (Bobek et al., 1993) and lachrymal glands (Gipson, 2004).

Membrane mucins, including MUC1, MUC3A, MUC3B, MUC4, MUC11, MUC12, MUC13, MUC17, and MUC20 are defined by the presence of a single hydrophobic transmembrane domain that secures them to the cell surface. With the exception of MUC4, all membrane mucins contain a SEA (Sea urchin sperm protein, Enterokinase, and Agrin) domain in their extracellular regions that may be involved in binding carbohydrate moieties. Several of the membrane mucins also contain EGF-like domains that may be involved in protein–protein interactions. Membrane mucins are usually found at the apical surface of epithelial cells, providing protection to the epithelia. Membrane mucins can also be found as soluble molecules as a result of either splice removal of exons encoding the transmembrane and intracellular domains exons (Moniaux et al., 2000; Williams et al., 1999), or by proteolytic removal of the mucin from its transmembrane tether (Boshell et al., 1992; Wang et al., 2002). The soluble forms of membrane mucins can be found in fluids such as milk, tears, and saliva, or may remain loosely bound to the cell surfaces as part of the protective mucin barrier. While their functional roles remain to be fully elucidated, it is possible that soluble forms of membrane mucins could aid in the protection of epithelia from microbes. For example, it has been suggested that the presence of soluble membrane mucins in mothers' milk could provide the newborn with protection from enteric pathogens (Newburg et al., 2005; Ruvoen-Clouet et al., 2006).

B Membrane Mucins and Tumor Progression

Intense interest has developed in understanding the role of aberrantly expressed mucins in the genesis and progression of tumors. MUC1 is highly expressed in the vast majority (>90%) of human breast tumors, and mislocalization of MUC1 (nonapical localization) predicts poor patient outcome (Rakha et al., 2005). MUC1 is overexpressed and differentially glycosylated in a number of other human tumors as well, including pancreatic, gastrointestinal, and lung, where it is associated with metastatic and invasive potential and has been used as a diagnostic and prognostic factor (Taylor-Papadimitriou et al., 1999). Several assays to detect tumor-associated antigens in serum samples are based on antibodies directed against circulating MUC1 antigens (Baldus et al., 2004a), and are used in the postoperative monitoring of breast and ovarian carcinoma patients. MUC4 overexpression may also eventually serve as a diagnostic and prognostic marker for numerous cancers, including pancreatic tumors (Swartz et al., 2002), where its expression is associated with metastatic phenotype (Singh et al., 2004), lung adenocarcinomas (Llinares et al., 2004), and mass-forming type intrahepatic cholangiocarcinoma, where its coexpression with ErbB2 correlates with short survival time (Shibahara et al., 2004). MUC4 expression was shown to be increased during the pathologic process of squamous dysplastic transformation of exocervical epithelium, and could possibly be used as a marker in this process (Lopez-Ferrer et al., 2001).

Of the membrane mucins, human MUC1 and rat MUC4 have been studied in the greatest detail functionally and biochemically. Both are present at the cell surface as heterodimers formed from high molecular weight precursors, proteolytically cleaved early in their transit to the cell surface (Ligtenberg et al., 1992; Sheng et al., 1990). Both provide protection to the cell surface by virtue of their rigid and...