Role of CEACAM1 in Epithelial Cell Polarization

描述（由申请人提供）：CEACAM1是一种同型细胞粘附分子，在上皮细胞极化中起关键作用，包括在3D培养中生长时乳腺和前列腺上皮细胞的管腔形成。长（72AA）和短（12aa）细胞质结构域异构体都是由mRNA剪接产生的，短形式在大多数上皮细胞中占优势，而长形式在t细胞中占优势。此外，CEACAM1基因在大多数癌症中是沉默的，在结肠癌的情况下，早在恶性前增生性息肉和腺瘤中就沉默了。我们已经证明，从短形式衍生的肽直接与肌动蛋白、原肌球蛋白、钙调蛋白和膜联蛋白2相互作用，在与细胞骨架相互作用中发挥作用，当在Thr和Ser残基上磷酸化时，启动线粒体凋亡途径，在管腔形成中发挥作用。为了更全面地剖析这些作用，我们建议确定导致短形式与细胞骨架产生有效相互作用的事件的等级顺序，鉴定导致短形式细胞凋亡的下游激酶和接头蛋白，确定长形式受体信号抑制的机制，并剖析前列腺癌和结肠癌中基因沉默的机制。为了实现前三个目标，我们提出了生物化学和蛋白质组学方法，这些方法可以直接识别相互作用的蛋白质，并使用siRNA、共聚焦和FRET方法在细胞内进行管腔形成过程中测试这些相互作用。体内足迹和蛋白质组学方法将用于鉴定前列腺和结肠细胞系中负责基因沉默的启动子复合物，这些启动子复合物表达或不表达CEACAM1。确定的因素的功能分析将通过使用siRNA方法的因素消耗来执行。这些研究将有助于深入了解CEACAM1在正常分化过程中相关的管腔形成中的功能机制，以及CEACAM1基因沉默在上皮细胞起源性癌症中的作用机制及其后果。

DESCRIPTION (provided by applicant): 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.