Masanobu Komatsu

AN INTRAMEMBRANE MODULATOR OF THE ERBB2 RECEPTOR TYROSINE KINASE THAT POTENTIATES NEUREGULIN SIGNALING

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.

Reversible Disruption of Cell-Matrix and Cell-Cell Interactions by Overexpression of Sialomucin Complex

Sialomucin complex (SMC) is a large, heterodimeric glycoprotein complex composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits and expressed abundantly on the cell surface of ascites 13762 rat mammary adenocarcinoma cells. We have isolated recombinant cDNAs containing different numbers of ASGP-1 mucin repeats, which can be expressed as protein products with variable lengths. To study the anti-adhesive effect of SMC, these cDNAs were transfected into human cancer cell lines. Using a tetracycline-responsive, inducible expression system, we demonstrated that the overexpression of SMC induces morphology changes, cell detachment, and cell-cell dissociation of transfected A375 human melanoma cells in culture. The transition between the adherent and suspension states of the cells is fully reversible and dependent on the SMC expression level. The anti-adhesion effect of SMC was further analyzed kinetically by measuring the cell adhesion of transfected A375 melanoma and MCF-7 breast cancer cell lines to fibronectin, laminin, and collagen IV, demonstrating that SMC disrupts integrin-mediated cell adhesion to extracellular matrix proteins. The degree of this anti-adhesion effect was dependent on the number of mucin repeats in the SMC molecule as well as the level of cell surface expression.

Muc4/sialomucin complex, an intramembrane modulator of ErbB2/HER2/Neu, potentiates primary tumor growth and suppresses apoptosis in a xenotransplanted tumor

Overexpression of the membrane mucin MUC4/Sialomucin complex (SMC) has been observed during malignant progression of mammary tumors in both humans and rats, suggesting that deregulation of MUC4/SMC expression might facilitate development of these malignancies. As previously reported, overexpression of SMC results in suppression of both cell adhesion and immune killing of tumor cells. SMC also acts as a ligand for ErbB2/Neu, modulating phosphorylation of the receptor tyrosine kinase in the presence and absence of heregulin. The present studies investigated the effect of Muc4/SMC up-regulation on primary tumor growth using a tetracycline-inducible SMC expression system in a xenotransplanted tumor model. SMC up-regulation provoked rapid growth of transfected A375 melanoma in nude mice. Up-regulation of SMC, however, did not significantly increase proliferation of A375 cells in vitro. Instead, a strong suppression of apoptosis was observed in situ in SMC-overexpressing tumors. These data suggest that Muc4/SMC expression promotes tumor growth in vivo at least in part via suppression of tumor cell apoptosis. Importantly, reduction of apoptosis was also observed in vitro, indicating that anti-apoptotic effect of SMC is independent of tumor-host interactions. These findings strongly suggest that SMC up-regulation alters intracellular signaling to favor cell survival, providing for the first time evidence for the regulation of programmed cell death by a gene of the MUC family.

Muc4/sialomucin complex, the intramembrane ErbB2 ligand, induces specific phosphorylation of ErbB2 and enhances expression of p27(kip), but does not activate mitogen-activated kinase or protein kinaseB/Akt pathways.

Muc4/sialomucin complex (SMC) is a multifunctional glycoprotein complex which can repress apoptosis in transfected tumor cells. Its transmembrane subunit acts as an intramembrane ligand for the receptor tyrosine kinase ErbB2 to induce the phosphorylation of ErbB2 and, by acting synergistically with the ErbB3 ligand neuregulin, can potentiate the phosphorylation of ErbB2 and ErbB3. In the present study we show that Muc4/SMC alone robustly induces the phosphorylation of ErbB2 to enhance the tyrosine phosphate epitope (Tyr1248) recognized by anti-phospho-ErbB2. Although this tyrosine phosphorylation has been implicated in cell transformation, it does not activate any of the three mitogen-activated protein kinases (MAPKs) or protein kinase B/Akt of the phosphatidyl inositol 3-kinase pathway. Instead, Muc4/SMC expression induces up-regulation of the cell cycle inhibitor p27kip, consistent with the expression of Muc4/SMC in differentiated, rather than proliferative, epithelial cells. Interestingly, a combination of Muc4/SMC and neuregulin down-regulate p27kip and activate protein kinase B/Akt. These observations suggest that Muc4/SMC acts as a regulator of differentiation by inducing a limited phosphorylation of ErbB2 and a modulator of proliferation when acting synergistically with neuregulin to induce a more extensive phosphorylation on both ErbB2 and ErbB3.

Sialomucin Complex, a Heterodimeric Glycoprotein Complex EXPRESSION AS A SOLUBLE, SECRETABLE FORM IN LACTATING MAMMARY GLAND AND COLON

Ascites 13762 rat mammary adenocarcinoma cells express abundantly on their cell surfaces a heterodimeric glycoprotein complex composed of a sialomucin ascites sialoglycoprotein (ASGP)-1 and a transmembrane subunit ASGP-2. The latter, which contains two epidermal growth factor-like domains, binds the receptor tyrosine kinase p185neu, suggesting that the complex is bifunctional as well as heterodimeric. Immunoblot analyses using monoclonal antibodies prepared against the complex demonstrate high levels of expression in rat lactating mammary gland and colon. Immunolocalization studies with anti-ASGP-2 indicate that ASGP-2 is present in these two tissues in the apical regions of secretory epithelial cells. Both mammary gland and colon contain a soluble, secretable form of ASGP-2, which is not found in the ascites cells; milk and mammary gland also have the membrane form. Immunoblot analyses using a COOH-terminal-specific polyclonal antibody indicate that the soluble form of ASGP-2 is missing its COOH-terminal domains. Both the soluble and membrane forms of ASGP-2 are similar to the membrane-associated form from the 13762 adenocarcinoma with respect to Mr, antigenicity, and association with ASGP-1. The presence of ASGP-1 in milk suggests that it is a candidate for the uncharacterized high Mr milk mucin, MUCX. ASGP-2 expression is up-regulated in mammary gland during pregnancy, because it is undetectable in virgin and early pregnant rats but abundant in the gland from late pregnant and lactating animals. However, compared with the lactating mammary gland, the 13762 ascites cells overexpress ASGP-2 by more than 100-fold, which may contribute to their malignancy. These combined results indicate that sialomucin complex is a unique secreted product in the mammary gland and colon, whose behavior is different from that in the mammary ascites tumors, and which may play important roles in mammary and intestinal physiology.

Muc4/sialomucin complex, the intramembrane Er6B2 ligand, in cancer and epithelia: To protect and to survive

The membrane mucin Muc4, also called sialomucin complex (SMC), is a heterodimeric complex of two subunits, ASGP-1 and ASGP-2, derived from a single gene. It is produced by multiple epithelia in both membrane and soluble forms and serves as a protective agent for the epithelia. The membrane form of Muc4 acts as a steric barrier to the apical cell surface of epithelial or tumor cells. An important example is the uterus of the rat, in which Muc4 expression is downregulated for blastocyst implantation. The soluble form facilitates the protection and lubrication of epithelia by mucous gels composed of gel-forming mucins, as in the airway, where Muc4 is proposed to participate in mucociliary transport as a constituent of the periciliary fluid. The soluble form is also found in body fluids, such as milk, tears, and saliva. The transmembrane subunit ASGP-2 acts as an intramembrane ligand and activator for the receptor tyrosine kinase ErbB2. Formation of this ligand-receptor complex is proposed to repress apopotosis in epithelial and cancer cells in which the ligand-receptor complex is formed, providing a second type of cell protective mechanism. Muc4 expression is regulated in epithelial tissues in a cell- and tissue-specific manner during epithelial differentiation. In stratified epithelia, it is predominantly in the most superficial, differentiated layers, often coincident with ErbB2. Dysregulation of Muc4 expression may contribute to cell and tissue dysfunction, such as the proposed contribution of Muc4 to mammary tumor progression. These observations clearly show that Muc4 has multiple roles in epithelia, which may provide insights into aberrant behaviors of these tissues and their derivative carcinomas.

Potentiation of metastasis by cell surface sialomucin complex (rat MUC4), a multifunctional anti-adhesive glycoprotein

Sialomucin complex (SMC), a rat homologue of the human mucin MUC4, is a large membrane‐bound mucin complex, originally isolated from highly metastatic ascites 13762 mammary adenocarcinoma cells. When overexpressed, SMC exerts potent anti‐adhesive effects, which sterically disrupt molecular interactions for cell‐cell and cell‐ECM adhesions. SMC similarly suppresses anti‐tumor immunity by inhibition of interactions between cytotoxic lymphocytes and target tumor cells. Previously, recombinant cDNAs for SMC were transfected and inducibly expressed in A375 human melanoma cells using a tetracycline‐responsive expression system. In the current studies, we investigated the role of MUC4/SMC in tumor metastasis by regulating SMC expression of tumor transplants in vivo. Intravenous injection of SMC‐overexpressing cells resulted in substantially greater lung metastasis than injection of SMC‐repressed cells. Injection of SMC‐overexpressing cells followed by in vivo downregulation of SMC did not lower the frequency of lung metastasis. Growth of the micrometastatic lesions was the same for all 3 cases in short‐term (3‐week) assays. Further, subcutaneous injection of A375 cells followed by in vivo induction of SMC overexpression within the solid tumor resulted in spontaneous distant metastasis. These studies suggest that SMC potentiates metastasis by contributing to the establishment of metastatic foci. These studies directly demonstrate for the first time that tumor metastasis can be modulated by the regulation of MUC4/SMC expression. Int. J. Cancer 87:480–486, 2000.

Muc4/Sialomucin Complex in the Mammary Gland and Breast Cancer

MUC4 is a one of the membrane mucins of the mucin gene (MUC) family, characterized by mucin tandem repeat domains and a transmembrane domain which associates it with the cell plasma membrane. Although MUC4 is encoded by a single gene, it is produced by epithelial cells as a heterodimer through a proteolytic cleavage mechanism. This heterodimer is found in both membrane and soluble forms associated with epithelia. Functionally, MUC4 is proposed to provide a protective mechanism for vulnerable epithelia, such as those of the airway, eye, female reproductive tract and mammary gland. The protective mechanism(s) may be highjacked by some carcinomas, such as those of the breast, to increase tumor progression. Two mechanisms are proposed to contribute to the MUC4 functions. First, MUC4 acts as an anti-adhesive or anti-recognition barrier at epithelial or tumor cell surfaces. Second, MUC4 can bind the receptor tyrosine kinase ErbB2 and alter its cellular signaling. Expression of MUC4 in mammary gland is repressed by posttranscriptional mechanisms involving basement membrane and TGF-β, which are relieved during pregnancy to permit secretion of MUC4 into milk. These mechanisms are also abrogated in some breast cancers, providing a scenario for promotion of tumor progression. These observations imply important functions for MUC4 in both normal mammary function and in breast cancer.

SIALOMUCIN COMPLEX IN TUMORS AND TISSUES

Sialomucin complex (SMC) is a high Mr glycoprotein heterodimer, originally discovered on the cell surfaces of ascites sublines of the highly metastatic 13762 rat mammary adenocarcinoma, and composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. SMC is encoded by a single gene and synthesized as a large precursor protein which is cleaved into its subunits early in its transit to the cell surface. SMC exhibits behavior typical of both membrane and secreted mucins. In the ascites cells, it is found only in the membrane form, creating a protective barrier at the cell surface to reduce cell adhesiveness and protect the tumor cell from immune killing. Normal tissues express both the membrane formand a non-membrane form, which may be secreted by either constitutive or regulated, secretory granule mechanisms. This soluble form is proposed to contribute to multilayer mucus gels which protect epithelia, though it may also play other roles. ASGP-2 contains two EGF-like domains, one of which binds the receptor tyrosine kinase ErbB-2. Thus, SMC may be a bifunctional protein, the mucin serving a protective function and the transmembrane domain possibly playing a role in the proliferation of metastatic tumor cells or repair processes necessary for the maintenance of damaged epithelia.

Synthesis and secretion of Muc4/sialomucin complex: implication of intracellular proteolysis

Muc4/sialomucin complex (SMC) is a heterodimeric glycoprotein complex implicated in epithelial protection and overexpressed in some tumours. It is encoded by a single gene, and the two subunits are produced by proteolytic cleavage at a time before substantial O-glycosylation, near the time of transit from the endoplasmic reticulum to the Golgi. Although Muc4/SMC is translated as a membrane protein, it is produced as a soluble form in many epithelia. Transfection experiments using Cos-7, HBL-100 human epithelial, MCF-7 human breast tumour and HC11 mouse mammary cell lines showed that soluble rat Muc4/SMC is produced by a proteolytic cleavage mechanism and released by secretion from multiple cell lines, including both human and mouse normal epithelial cells and tumour cells. Similar transfection experiments demonstrated the same mechanism for the human analogue MUC4. Gel electrophoresis analyses of deglycosylated membrane and soluble transmembrane subunits and of the membrane-associated cleavage fragment indicated a released cleavage product of 25 kDa, resulting from cleavage between two epidermal growth factor-like domains. Further evidence for this site was obtained from deletion mutants removing this region of the protein, which blocked secretion. Finally, pulse-chase analyses of Muc4/SMC biosynthesis indicated no kinetic difference between the timing of the cleavage to release the soluble form and that to produce the two subunits, indicating that the soluble form is created early in transit to the cell surface. These studies provide the first clear evidence that membrane mucins can be released from cells by an intracellular proteolytic mechanism that leads to secretion of the soluble form of the mucin.