Role of CEACAM1 in Epithelial Cell Polarization

描述(申请人提供)：CEACAM1是一种同型细胞黏附分子，在上皮细胞极化中发挥关键作用，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。长的(72aa)和短的(12aa)胞质结构域的异构体都是通过交替的mRNA剪接产生的，在大多数上皮细胞中以短的形式为主，在T细胞中以长的形式为主。此外，CEACAM1基因在大多数癌症中都是沉默的，对于结肠癌来说，它早在癌前增生性息肉和腺瘤中就被沉默了。我们已经证明，来自短形式的多肽直接与肌动蛋白、原肌球蛋白、钙调蛋白和膜联蛋白2相互作用，在与细胞骨架的相互作用中发挥作用，并且当苏氨酸和丝氨酸残基被磷酸化时，启动线粒体的凋亡途径，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相互作用的事件的层级序列，通过短形式识别导致细胞凋亡的下游激酶和接头蛋白，确定长形式抑制受体信号的机制，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，我们提出了生化和蛋白质组学方法，允许直接鉴定相互作用的蛋白质，并使用siRNA、共聚焦和FRET方法在进行管腔形成的细胞中在体内测试这些相互作用。在活体足印和蛋白质组学方法将被用来确定在前列腺和结肠细胞系中负责基因沉默的启动子复合体，这些细胞系表达或不表达CEACAM1。对已确定的因子进行功能分析将通过使用siRNA方法进行因子耗竭。这些研究将为CEACAM1在相关的生物学过程中的作用提供一个机械性的见解，即正常分化中的管腔形成，以及CEACAM1基因沉默在上皮细胞起源的癌症中的机制及其后果。

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.