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. This complex 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 approx. 300 kDa (pSMC-1), which is cleaved to its subunits early in its transit to the cell surface. Molecular cloning and sequencing have revealed the complete sequence of the transcript and proteins. Interestingly, the transmembrane subunit has two EGF-like domains which contain all of the consensus residues of active growth factors. Moreover, experiments with membranes containing the EGF receptor and p185neu demonstrate that ASGP-2 can modulate the phosphorylation of these receptor kinases. These results, plus earlier studies linking the mucin ASGP-1 to metastasis and resistance to immune killing, suggest that the complex is not only heterodimeric, but also bifunctional. RT-PCR, immunocytochemical and immunoblot analyses have shown the presence of ASGP-2 in some human breast cancer cell lines and breast carcinomas, particularly in cells of highly invasive tumors obtained from patient body fluids, and in human ovarian cancer ascites fluid. Such results have led to suggest that the complex may play a role in tumor progression. In the rat ASGP-2 is found at epithelial apical cell surfaces in the lung, brain and oral cavity, in secretory granules in Paneth cells in the small intestine and goblet cells in the colon, and in lactating, but not 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. Moreover, immunocytochemical analyses of embryos indicate that expression of ASGP-2 is developmentally regulated, RT-PCR analyses show differential splicing of ASGP-2 transcripts in different tissues. Furthermore, ASGP-1 and ASGP-2 in the intestine and mammary gland appear to be different from the glycoproteins int he 13762 ascites tumor cells. Our hypothesis is that the sialomucin complex contributes to tumor progression in the rat mammary carcinoma (and human breast carcinomas) because it is aberrantly expressed and/or processed in the tumors. This proposal therefore focuses on the mammary tumors and mammary gland. To test the hypothesis that overexpression of ASGP-1/ASGP-2 can promote tumor formation and progression, we will transfect intact ASGP-1/ASGP-2 precursor pSMC-1 or its truncated analogs (e.g. pSMC-1 minus ASGP-2 extracellular domain) into cells in culture. The transfected cells will be analyzed for expression of the glycoproteins, for changes in properties involved in growth and invasiveness and for tumorigenicity and metastasis in nude mice. We will investigate the structures, localization and function of ASGP-1 and ASGP-2 in the lactating mammary gland using biochemical, immunological and molecular biological analyses, including antisense studies. We will analyze the factors involved in the regulation of the expression of the sialomucin complex gene in rat mammary tumors and mammary gland, using both biochemical and molecular biological protocols to identify cis and trans elements involved in its control. The results of these combined studies will provide insights into 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.
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