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    Awarded Grants
    Analysis of O-glycans Involved in Lung Metastasis of Breast Cancer

    Scientific Abstract:
    Analysis of O-glycans involved in lung metastasis of breast cancer. a) Background. Apical cell surfaces of epithelia are covered by a variety of carbohydrates attached to the membrane proteins and lipids. When epithelial cells are transformed, cell surface carbohydrates alter significantly. Many studies suggest that O-glycans or mucin type carbohydrates play a major role in cancer metastasis by interacting with a carbohydrate-binding protein expressed on the endothelial cells. We have previously identified a series of peptides for selectin ligand mimicry. When one of such peptides, was injected intravenously into a mouse, the peptide inhibited sialyl Lewis X–dependent cancer cells colonization of the lung. However, this peptide bound to the lung vasculatures in the mutant mice lacking both E- and P-selectins, suggesting an existence of a carbohydrate binding receptor distinct from selectins. Our preliminary data identified this endothelial cells receptor or I-peptide receptor (IPR). b) Objective/Hypothesis. We hypothesize that IPR is a carbohydrate binding protein and plays a critical role in the carbohydrate-dependent breast cancer metastasis to the lung. We also hypothesize that O-glycans expressed on the surface of breast cancer cells function as IPR ligand. However, currently it is difficult to determine a specific O-glycan structure for IPR binding activity. In the proposed study, we will develop tools and methods necessary for structural and functional analysis of O-glycans. c) Specific aims. 1. Determine carbohydrate-binding activity and cell surface localization of IPR using recombinant IPR and glycosyltransferase transfected breast cancer cell lines. 2. Determine the ligand carbohydrate structure for IPR by developing O-glycanases and HPLC methods for O-glycans structure analysis. d) Study Design. Binding of recombinant IPR to human breast cancer cell lines will be determined by transfection with defined cDNAs. Breast cancer cell lines each for positive or negative IPR ligand activity will be analyzed for cell surface O-glycans. O-glycanases capable of releasing intact O-glycans will be developed, and fluorescence tagged O-glycans will be analyzed by HPLC . Structure -defined and pure O-glycans will be tested for IPR binding to determine IPR ligand structure. e) Potential Outcomes and Benefits of the Research. The proposed studies will provide us with tools and reagents for analyzing O-glycans, which will form the foundation for IPR-based diagnosis and therapy of breast cancer.

    Lay Abstract:
    Analysis of O-glycans involved in lung metastasis of breast cancer. When cancer cells are released from the primary tumor and enter to the blood stream, cancer cells interact with the wall of blood vessels. This interaction is a critical step for cancer spreading (metastasis) to distant sites. The surfaces of many cancer cells are covered with carbohydrates, and interaction between some cancer cells and blood vessels are carbohydrate-dependent. Several years ago, scientists studying inflammation discovered a group of carbohydrate binding protein, called selectin. Because selectins appear on the surface of blood vessels during inflammation, it was thought that selectins are the molecule to catch circulating cancer cells and help metastasis. This hypothesis was strengthened as poor clinical outcomes of colon cancers correlate well with the selectin-binding carbohydrates on the colon cancers. Breast cancer often metastasizes to the lung through the blood route. However, selectin-based theory is not always consistent with the clinical observations of cancer metastasis, leaving many questions for selectin’s role in cancer metastasis. Previously we identified the peptides that bind to selectin as if they are carbohydrates. However, our peptides bound to the lung blood vessels of the mutant mice lacking selectins. Our studies indicate an existence of a novel carbohydrate binding receptor in the lung blood vessells, and we recently succeeded in identifying this novel receptor. Proposed study is to characterize further this receptor and carbohydrates which are recognized with the receptor. More specifically, we ask support for developing the methods required for analyzing cell surface carbohydrates, called O-glycans, in breast cancer cells. Since it is expected that the receptor binding to breast cancers is unique to metastatic cells, our study is important for future development of efficient and safe therapies to kill dangerous breast cancer cells before they spread to the lung.