CEACAM1 is a homotypic cell adhesion molecule that plays a critical role in epithelial cell polarization, including lumen formation for breast and prostate epithelial cells when grown in 3D culture. Both long (72AA) and short (12 AA) cytoplasmic domain isoforms are produced by alternative mRNA splicing, with the short form predominant in most epithelial cells, and the long form predominant in T-cells. In addition, the CEACAM1 gene is silenced in most cancers, and in the case of colon cancer, it is silenced as early as pre-malignant hyperplastic polyps and adenomas. We have shown that peptides derived from the short form interact directly with actin, tropomyosin, calmodulin, and annexin 2, playing a role in interactions with the cytoskeleton, and that when phosphorylated on Thr and Ser residues, initiate a mitochondrial pathway of apoptosis, playing a role in lumen formation. In order to dissect these roles more fully, we propose to determine the hierarchal sequence of events that lead to productive interactions of the short form with the cytoskeleton, to identify the downstream kinases and adaptor proteins that lead to apoptosis by the short form, to determine the mechanism of receptor signaling inhibition by the long form, and to dissect the mechanism of gene silencing in prostate and colon cancers. To achieve the first three aims we propose biochemical and proteomic approaches that allow the direct identification of interacting proteins and the testing of these interactions in vivo in cells undergoing lumen formation using siRNA, confocal, and FRET approaches. In vivo foot printing and proteomic approaches will be used to identify the promoter complexes responsible for gene silencing in prostate and colon cell lines that do or do not express CEACAM1. Functional analyses of identified factors will be performed by factor depletion using siRNA approaches. These studies should provide a mechanistic insight into the function of CEACAM1 in a relevant biological process, namely lumen formation in normal differentiation, and the mechanism of CEACAM1 gene silencing in cancers of epithelial cell origin and the consequences thereof. ? ? ?
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