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很常见。与许多癌症相关基因一样，β-连环蛋白生物学中一些最有价值的研究是在遗传易驯化的模式生物中进行的，例如黑腹果蝇和小鼠。通过利用基因打靶方面的最新技术进步，现在可以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，我们创造了高效的人类无启动子β-连环素基因靶向载体，并利用它们来创建人类癌细胞的等基因组，这些等基因组仅在存在或不存在内源性突变和/或野生型β-连环素基因的情况下有所不同。初步的功能研究揭示了β-连环蛋白在人类癌症中的几个方面的功能。特别是，我们已经证明了激活的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-连环蛋白的缺失导致癌细胞形成穹顶状结构，概括了在分化Caco-2细胞单层中看到的表型。初步实验表明，致癌的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瘤所必需的。同样令人惊讶的是，通过Western Blot和免疫细胞化学分析，我们没有发现野生型和突变型激活的β-连环蛋白在稳态水平和细胞内定位方面的明显差异。最后，通过Affymetrix基因芯片分析，我们已经确定并表征了BMP-4，一种转化生长因子-β同源物，可能是人类癌症中激活的β-连环蛋白信号的效应因子。我们建议进一步利用这些同基因的人类癌细胞系统来研究激活的β-连环蛋白在人类癌症发病机制中的作用。特别是，我们计划：1)确定激活的β-连环蛋白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是否需要β-连环蛋白的致癌激活来维持其致瘤性；3)确定人类癌症中β-连环蛋白激活的生化机制(S)，以及4)识别和表征激活的β-连环蛋白转录因子调控的基因。

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.