Auto-Feedback Regulation of Beta-catenin in Human Cancer

描述（由申请人提供）：β-连环蛋白是Wnt信号传导靶基因的关键激活剂。β-连环蛋白通过其负调节因子的突变失活而异常激活，这与几种常见人类癌症的发病机制有关。本实验室最近的研究表明，在大肠腺瘤性息肉病（APC）肿瘤抑制蛋白介导的调节机制因突变而失效后，β-连环蛋白受到自反馈调节。通过消减法富集β-连环蛋白诱导基因构建的cDNA文库的功能筛选导致了β-连环蛋白的靶基因--支架蛋白Axin 2的鉴定。作为传导蛋白或小鼠Axil的同源物，Axin 2先前被其他几个实验室鉴定为Wnt/β-连环蛋白信号传导的负调节因子。我们的瞬时表达实验揭示了Axin 2在破坏β-连环蛋白中的一种新功能，这种功能与APC介导的调节机制截然不同。我们假设Axin 2对β-catenin的自反馈调节是经典Wnt/β-catenin信号传导的重要机制。基于这一假设，我们认为：1）β-连环蛋白的下调需要APC和Axin 2的协同作用; 2）Axin 2介导的自反馈调节的失败将导致β-连环蛋白的异常激活，从而导致肿瘤转化。我们建议使用遗传和生物化学方法的组合来测试这一假设，如以下具体目标所述。目标一：使用一组基因工程结肠癌细胞系作为体外模型系统，评估APC和Axin 2之间的协同合作是否是β-连环蛋白下调所必需的;以及AIM II：使用携带杂合Axin 2等位基因的基因工程突变小鼠，测试Axin 2的自反馈功能的丧失是否会导致β-连环蛋白的异常激活和体内肿瘤发生。AIM III：确定单个蛋白质结构域对Axin 2肿瘤抑制功能的贡献，并使用这些信息描述Axin 2介导的β-连环蛋白破坏的独特特征。这些拟议研究的长期目标是获得关于β-连环蛋白在人类癌症中的发病机制的新的机制信息，目的是确定对合理的癌症预防和治疗有用的关键细胞靶点。

DESCRIPTION (provided by applicant): Beta-catenin is a key activator of Wnt signaling target genes. Aberrant activation of beta-catenin through mutational inactivation of its negative regulators has been implicated in the pathogenesis of several common human cancers. Recent studies from our laboratory have demonstrated that beta-catenin is subject to an autofeedback regulation after the adenomatous polyposis coli (APC) tumor suppressor protein mediated regulatory mechanism failed due to mutation. Functional screening of a cDNA library constructed by enriching beta-catenin induced genes through subtraction has lead to the identification the scaffolding protein Axin2, a target gene of beta-catenin. A homolog of Conductin or mouse Axil, Axin2 was previously identified as a negative regulator of Wnt/beta-catenin signaling by several other laboratories. Our transient expression experiments revealed a novel function of Axin2 in the destruction of beta-catenin that is uniquely different from that of the APC-mediated regulatory mechanism. We hypothesize that auto-feedback regulation of beta-catenin by Axin2 is an important mechanism of canonical Wnt/beta-catenin signaling. Based on this hypothesis, we suggest: 1) downregulation of beta-catenin requires a synergetic effect contributed by both APC and Axin 2; and 2) failure of Axin2-mediated auto-feedback regulation will lead to aberrant activation of beta-catenin and consequently neoplastic transformation. We propose to test this hypothesis using a combination of genetic and biochemical approaches as outlined in the following specific aims. AIM I: To assess whether synergistic cooperation between APC and Axin2 is required for the downregulation of beta-catenin using a set of genetically engineered colon cancer cell lines as the in vitro model system; and AIM II: To test whether loss of the auto-feedback function of Axin2 will cause aberrant activation of beta-catenin and tumorigenesis in vivo using genetically engineered mutant mice harboring heterozygous Axin2 allele. AIM III: To define the contribution of individual protein domains to the Axin2 tumor suppressor function, and use these information to delineate the unique features in Axin2-mediated destruction of beta-catenin. The long term goal of these proposed studies is to gain new mechanistic information concerning the pathogenesis of beta-catenin in human cancer, with the objective of identifying key cellular targets useful for rational cancer prevention and therapy.