Membrane Mucin Muc4 promotes blood cell association with tumor cells and mediates efficient metastasis in a mouse model of breast cancer

Mucin-4 (Muc4) is a large cell surface glycoprotein implicated in the protection and lubrication of epithelial structures. Previous studies suggest that aberrantly expressed Muc4 can influence the adhesiveness, proliferation, viability and invasiveness of cultured tumor cells, as well as the growth rate and metastatic efficiency of xenografted tumors. Although it has been suggested that one of the major mechanisms by which Muc4 potentiates tumor progression is via its engagement of the ErbB2/HER2 receptor tyrosine kinase, other mechanisms exist and remain to be delineated. Moreover, the requirement for endogenous Muc4 for tumor growth progression has not been previously explored in the context of gene ablation. To assess the contribution of endogenous Muc4 to mammary tumor growth properties, we first created a genetically engineered mouse line lacking functional Muc4 (Muc4 ko), and then crossed these animals with the NDL (Neu DeLetion mutant) model of ErbB2-induced mammary tumorigenesis. We observed that Muc4 ko animals are fertile and develop normally, and adult mice exhibit no overt tissue abnormalities. In tumor studies, we observed that although some markers of tumor growth such as vascularity and cyclin D1 expression are suppressed, primary mammary tumors from Muc4 ko /NDL female mice exhibit similar latencies and growth rates as Muc4 wt /NDL animals. However, the presence of lung metastases is markedly suppressed in Muc4 ko /NDL mice. Interestingly, histological analysis of lung lesions from Muc4 ko /NDL mice revealed a reduced association of disseminated cells with platelets and white blood cells. Moreover, isolated cells derived from Muc4 ko /NDL tumors interact with fewer blood cells when injected directly into the vasculature or diluted into blood from wild type mice. We further observed that blood cells more efficiently promote the viability of non-adherent Muc4 wt /NDL cells than Muc4 ko /NDL cells. Together, our observations suggest that Muc4 may facilitate metastasis by promoting the association of circulating tumor cells with blood cells to augment tumor cell survival in circulation

Mucin Structure and Function: Insights from Molecular Biology

Mucins are highly O-glycosylated glycoproteins implicated in the protection of cells from extracellular agents. Two general classes of mucins have been described: secreted and membrane. Most, but not all, mucin structures contain a central tandem repeat region which is rich in serine and threonine and is highly variable in size between different mucins; many also contain a cysteine-rich domain. Repeat sequences are often conserved within a single molecule, such as human MUC1 protein, but are poorly conserved between species, e.g. mouse and human MUC1 protein repeats. Many mucins are polymorphic due to variable numbers of repeats, and mucin transcripts are often heterogeneous. Although mucin expression is relatively tissue specific, some mucins, such as the MUC1 and MUC2 proteins, are found in multiple tissues. Moreover, a single tissue may express more than one mucin. Limited studies suggest that regulation of mucin expression is complex. Membrane mucins have been implicated in development and tumor progression, possibly by modulating cell-cell interactions. Some mucin cysteine-rich domains may also play a role in regulatingcell proliferation. Undoubtedly, future studies using recombinant DNA probes will greatly expand our understanding of these complex molecules

Structures of the O-linked oligosaccharides of the major cell surface sialoglycoprotein of MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma

Structures of the principal O-glycosides from the major cell surface sialoglycoprotein (ASGP-1) of the MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma have been determined. Oligosaccharitols were released by alkaline borohydride treatments of ASGP-1 and purified by gel filtration, DEAE-Sephadex ion exchange chromatography, and high performance liquid chromatography. On the basis of carbohydrate composition, methylation analysis, periodate oxidation, and exoglycosidase digestion, the five major oligosaccharides released by mild alkaline borohydride were assigned the following structures: Component II-3: (NeuAcα2→3Galβ1→4GlcNAcβ1→6)Ga1NAcOH(3←1βGa13←2αNeuAc); III-2a: (Ga1β1→4GlcNAcβ1→Ga1NAcOH(3←1βGa13←2αNeuAc); III-2c: (Ga1α1→3Ga1β1→4G1cNAcβ1→6)Ga1NAcOH(3←1βGa13←2αNeuAc); IV-1a: (Ga1β1→4G1cNAcβ1→6)Ga1NAcOH(3←1βGa1); IV-1c: (Ga1α1→3Ga1β1→4G1cNAcβ1→6)Ga1NAcOH(3←1βGa1). Fucosylated derivatives of III-2a, IV-1a, and IV-Ic were found in smaller amounts with the fucose tentatively assigned to the 2-position of the lactosamine galactose. Components II-3, III-2a and the fucosylated derivative of III-2A were found in both MAT-B1 and MAT-C1 sublines. The α-galactosides were found in detectable quantities only in subline MAT-B1. Oligosaccharides from MAT-C1 cells were enriched in sialic acid when compared to those from MAT-B1 cells. These results suggest that the 13762 ascites sublines, which bear different oligosaccharides, will provide models useful for the investigation of mechanisms regulating the expression of structures of the larger O-linked oligosaccharides

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained byducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors

Dual Inhibition Of Sodium-Mediated Proton And Calcium Efflux Triggers Non-Apoptotic Cell Death In Malignant Gliomas

Malignant glioma cells maintain an elevated intracellular pH (pHi) within hypoxic–ischemic tumormicroenvironments through persistent activation of sodium–proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pHi of malignant gliomas by inhibiting isoform 1 of sodium–proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium–calciumexchange. Amiloride\u27s glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na+- dependent calcium efflux by isoform 1.1 of the sodium–calcium exchanger (NCX1.1) , which increases [Ca2+]i and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na+]i) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calciumlevels ([Ca2+]i) also are increased 5-fold. 2′, 4′-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forwardmode of NCX1.1 at ≤1 μM, while dually inhibiting bothNHE1 and NCX1.1 at ≥20 μM. DCB (1 μM) was not cytotoxic to glioma cells,while DCB (20 μM) further increased basal elevated levels of [Ca2+]i in glioma cells thatwas followed by cell demise. Cariporide and SEA0400 are more selective inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase activation