The overall goal of this research is to understand the roles of cell surface glycoproteins in breast cancer. This project is focused on a novel cell surface glycoprotein complex (sialomucin complex, SMC), originally isolated from ascites sublines of the highly metastatic 13762 rat mammary adenocarcinoma. SMC is composed of a mucin subunit ASGP-1 (ascites sialoglycoprotein-1) linked to the plasma membrane via an N- glycosylated transmembrane subunit ASGP-2. The complex is encoded by a single gene, expressed as a 9.2 kb transcript and synthesized as a precursor of approximately 300 kDa (pSMC-1). Molecular cloning and sequencing have revealed the complete sequence of the transcript and proteins. The transmembrane subunit has two EGF-like domains can act selectively as a ligand for the receptor tyrosine kinase ErbB2. These results, plus the anti-adhesive activity of the mucin subunit ASGP-1, suggest that the complex is not only heterodimeric, but also bifunctional. In the rat ASGP-2 is found at epithelial apical cell surfaces in the airway, uterus, brain and oral cavity, in secretory granules in Paneth cells in the small intestine and globet cells in the colon, and in lactating, but not in nonlactating, mammary gland. These highly specific localization patterns suggest different functions in different cellular contexts and highly important roles for one or both of the glycoproteins. Immunocytochemical analyses have shown the presence of ASGP-2 in some human breast carcinomas, particularly in cells of highly aggressive tumors obtained from patient body fluids. Such results have led us to suggest that the complex may play a role in tumor progression. Our hypothesis is that the sialomucin complex contributes to breast cancer progression by a combination of its anti- adhesive and receptor ligand functions. This proposal focuses primarily on the role of SMC(ASGP-2) as a receptor ligand. The initial stage is to investigate the mechanism by which ASGP-2 modulates signaling through ErbB2 using in vitro model systems in insect cells, 32D murine myeloid cells and the MCF-7 breast tumor cell line. These studies will examine the effects of ASGP-2 on binding of other ligands, receptor heterodimerization, receptor phosphorylation and downstream signaling. Second, the role of ASGP-2 in tumor cells from breast cancer patient pleural effusions will be investigated to determine whether its behavior in these tumors is similar to that in the rat ascites cells and in vitro model systems. Third, anti-ASGP-2 antibodies will be used for a study by immunohistochemistry of the relationship of SMC and ErbB2 expression to tumor progression in archival breast tumor samples. The initial signaling studies will address our incomplete understanding of the ligands and mechanisms for activation of ErbB2 and its cellular consequences. The results of these combined studies will provide insights in the role(s) of the sialomucin complex in tumor progression and normal function and may ultimately lead to procedures which can exploit the complex for the diagnosis, prognosis and/or therapy of human tumors in the subset of patients whose breast cancers express SMC.
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