Genetic Analysis of Beta-Catenin Function in Human CA

描述（由申请人提供）：β-连环蛋白的致癌性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已经在遗传学上易于处理的模式生物如黑腹果蝇和小家鼠中进行。通过利用基因靶向方面的最新技术进展，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。为了做到这一点，我们已经创建了高效的人类无启动子β-连环蛋白基因靶向载体，并使用它们来创建人类癌细胞的等基因组，其差异仅在于其内源性突变体和/或野生型β-连环蛋白基因的存在或不存在。初步的功能研究揭示了β-连环蛋白在人类癌症中的功能的几个方面。特别是，我们已经表明，激活β-连环蛋白是必要的TCF介导的转录激活在人类癌细胞。我们还证明了激活的β-连环蛋白的缺失导致癌细胞形成圆顶样结构，重现了分化的Caco-2细胞单层中观察到的表型。初步实验表明，致癌β-连环蛋白不是人类结肠癌细胞在免疫缺陷小鼠中形成异种移植肿瘤所必需的。同样令人惊讶的是，我们已经检测到野生型和突变型活化β-连环蛋白的稳态水平和细胞内定位没有可辨别的差异，如通过蛋白质印迹和免疫细胞化学分析所检测到的。最后，我们已经确定和特点BMP-4，TGF-β同系物，作为一个假定的效应器激活β-连环蛋白信号在人类癌症中使用Affytek基因芯片分析。我们建议进一步利用这些同基因人类癌细胞系统来研究活化的β-连环蛋白在人类癌症发病机制中的功能。具体而言，我们计划：1）确定人癌细胞和组织中活化的β-连环蛋白的细胞生物学和病理生物学表型，2）确定β-连环蛋白的致癌活化是否是维持含有多个遗传命中的人癌细胞中的致瘤性所必需的，3）确定人癌症中β-连环蛋白活化的生化机制，和4）鉴定和表征由活化的β-连环蛋白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.