Genetic Analysis of Beta-Catenin Function in Human CA

描述（由申请人提供）：β -连环蛋白的致癌激活在多种人类癌症的发病机制中是常见的。与许多癌症相关基因的情况一样，β -连环蛋白生物学中一些最有价值的研究是在遗传上易于处理的模式生物中进行的，如黑腹果蝇和小家鼠。通过利用基因靶向方面的最新技术进步，现在有可能对人类癌细胞本身的基因功能进行类似的研究。为此，我们创建了高效的人类无启动子β -连环蛋白基因靶向载体，并利用它们创建了人类癌细胞的等基因组，这些癌细胞仅在存在或不存在内源性突变和/或野生型β -连环蛋白基因方面存在差异。最初的功能研究揭示了-连环蛋白在人类癌症中的几个方面的功能。特别是，我们已经证明激活的β -连环蛋白是tcf介导的人类癌细胞转录激活所必需的。我们还证明了激活的β -连环蛋白的缺失会导致癌细胞形成圆顶状结构，再现了在Caco-2细胞单层分化中所见的表型。初步实验表明，人类结肠癌细胞在免疫缺陷小鼠体内形成异种移植物肿瘤并不需要致癌的β -连环蛋白。同样令人惊讶的是，我们通过Western Blot和免疫细胞化学分析发现，野生型和突变型活化β -连环蛋白在稳态水平和细胞内定位方面没有明显差异。最后，我们使用Affymetrix基因芯片分析鉴定并表征了tgf - β同源物BMP-4，作为人类癌症中活化的β -连环蛋白信号的假定效应物。我们建议进一步利用这些等基因人类癌细胞系统来研究活化β -连环蛋白在人类癌症发病机制中的功能。特别是，我们计划：1)确定活化的β -catenin在人类癌细胞和组织中的细胞生物学和病理生物学表型，2)确定在含有多个基因命中的人类癌细胞中，β -catenin的致癌激活是否需要维持致瘤性，3)确定β -catenin在人类癌症中活化的生化机制，4)鉴定和表征活化的β -catenin转录因子调节的基因。

DESCRIPTION (provided by applicant): Oncogenic activation of beta-catenin is common to the pathogenesis of a wide range of human cancers. As is the case for many cancer-related genes, some of the most valuable studies in beta-catenin biology have been undertaken in genetically tractable model organisms such as Drosophila melanogaster and Mus musculus. By exploiting recent technological advances in gene targeting it is now possible to undertake similar studies of gene function in human cancer cells themselves. To do this, we have created high-efficiency human promoterless beta-catenin gene-targeting vectors and employed them to create isogenic sets of human cancer cells that differ only in the presence or absence of their endogenous mutant and/or wild-type beta-catenin genes. Initial functional studies have shed light on several aspects of the function of beta-catenin in human cancer. In particular, we have shown that activated beta-catenin is necessary for TCF-mediated transcriptional activation in human cancer cells. We have also demonstrated that deletion of activated beta-catenin causes cancer cells to form dome-like structures, recapitulating a phenotype seen in differentiating Caco-2 cell monolayers. Pilot experiments have suggested that oncogenic beta-catenin is not required for the human colon cancer cells to form xenograft tumors in immunodeficient mice. Also surprisingly, we have detected no discernible differences in the steady state levels and intracellular localization of wild-type and mutant, activated beta-catenin, as detected by Western Blot and immunocytochemistry analysis. Finally, we have identified and characterized BMP-4, a TGF-beta homolog, as a putative effector of activated beta-catenin signaling in human cancer using Affymetrix Genechip analysis. We propose to further utilize these isogenic human cancer cell systems to study the functions of activated beta-catenin in the pathogenesis of human cancer. In particular, we plan to: 1) Determine the cell biological and pathobiological phenotypes of activated beta-catenin in human cancer cells and tissues, 2) Determine whether oncogenic activation of beta-catenin is required for maintenance of tumorigenicity in human cancer cells containing multiple genetic hits, 3) Determine the biochemical mechanism(s) of beta-catenin activation in human cancer, and 4) Identify and characterize genes regulated by the activated beta-catenin transcription factor.