Colorectal cancer is a major cause of cancer-related mortality, and mucinous tumors are typically associated with a poor prognosis. The overall aim of this project is to define the expression and molecular processing of mucin-associated antigens. Mucins are large glycoproteins consisting of oligosaccharide side chains on apomucin peptide backbones. Oligosaccharides of colon cancer mucin carry different (""""""""cancer-associated"""""""") epitopes than those of normal colonic mucin, often as a consequence of incomplete glycosylation revealing truncated epitopes. This aberrant glycosylation not only exposes internal carbohydrate antigens, but also exposes underlying apomucin sequences which then become immunogenic, eliciting both humoral and cell-mediated immune responses. The regulatory mechanisms accounting for the aberrant glycosylation, however, are not well understood. The applicant's previous studies disclosed that the sialosyl-Tn (STn) antigen, a mucin-associated disaccharide, was not expressed in normal colonic mucosa but became exposed in the vast majority of colon cancers where it was associated with a poor prognosis independent of tumor stage. Because of this finding, and because STn antigen can offer a """"""""handle"""""""" on mucin glycosylation, he established clonal cell lines of human colon cancer that differ in STn expression. The STn(+) cell line (LS-C) expresses only very truncated oligosaccharides (Tn, STn) whereas the STn(-) line (LS-B) expresses longer side chains bearing complex carbohydrates. Studies during the last project period revealed the mechanism for why LS-C cells express only short, truncated structures: these cells lack a critical glycosyltransferase (core 1 beta-3-galactosyltransferase) activity which would otherwise permit synthesis of longer structures. The core 1 transferase is a pivotal enzyme in O-linked glycosylation, and its absence in LS-C cells makes these cells a unique system to clone this enzyme by expression cloning, one of the aims of this application. The applicant has also inserted mini-mucin gene constructs into LS-C and LS-B cells and has proven that the same apomucin is glycosylated differently in the two cell lines depending upon the cell's intrinsic glycosylation machinery. Since the glycosyltransferase repertoire of these cells is now known, this system becomes a powerful tool for exploring glycosylation of specific mucin peptides, another aim of this application. Preliminary data in the last project period disclosed that STn antigen in normal colon mucosa occurs as a monomeric epitope, while in colon cancer tissues there is a change to include cluster STn epitopes. The concept of cancer-associated expression of cluster oligosaccharide epitopes and their mechanism of formation have heretofore been underappreciated and therefore represent the third area which the applicant plans to pursue.
Showing the most recent 10 out of 26 publications
