Chemical Genetics Approach to Interrogate & Regulate E-Cadherin Expression

E-钙粘附素功能丧失是与上皮向间充质转化相关的关键事件， 人类结直肠癌和其他癌症的侵袭性和转移表型。不适当 规范的Wnt途径的激活是从正常上皮向 结直肠癌的肿瘤表型。尽管人们普遍认为，这些因素的扰动 通路是肿瘤转化的重要标志，它们的转导机制和 人们对正常和病理条件下的调节知之甚少。我们已经高度发展了 可靠和敏感的E-钙粘蛋白在SW620结直肠癌细胞系中重新表达的检测方法 正常情况下，E-钙粘附素的表达水平很低。同样，我们也开发了一种强大的生化 重述由Wnt辅助受体LRP6激活规范的Wnt途径的实验。这两个都是 化验方法已被调整为384井格式。我们现在建议询问E-钙粘附素的调控 用基于化学遗传学的方法在高通量中表达和规范Wnt信号 筛选诱导SW620细胞E-钙粘附素表达及其干扰的化合物 β-连环蛋白和轴蛋白的降解，这两个关键的信号调节因子通过典型的Wnt途径。 我们对6400个小分子的初步筛查已经在这两种化合物中发现了几种先导化合物 我们建议筛选出总共160,000个化合物。我们建议验证先导化合物来自 这一初步筛选是在各种体外和体内试验中进行的。我们的初步研究表明，小型 组蛋白脱乙酰酶抑制剂曲古抑素A诱导SW620细胞E-钙粘附素表达 细胞。我们将探索它在E-钙粘蛋白表达中的作用以及它调节Wnt信号的可能性。 最后，我们将通过测试来确定有效化合物的作用机制和潜在的靶点 它们对候选细胞途径以及生化重组反应的影响。

Loss of E-cadherin function is a critical event that is associated with epithelial-to-mesenchymal transition, invasiveness and the metastatic phenotype in human colorectal cancer and other carcinomas. Inappropriate activation of the canonical Wnt pathway is another critical event in the transition from normal epithelium to the neoplastic phenotype in colorectal cancer. Although it is well accepted that perturbation of these pathways represent important hallmarks of neoplastic tranformation, their mechanisms of transduction and regulation under normal and pathological conditions are poorly understood. We have developed highly reliable and sensitive assays for re-expression of E-cadherin in the SW620 colorectal cancer cell line that normally expresses very low levels of E-cadherin. Similarly, we have also developed a robust biochemical assay that recapitulates activation of the canonical Wnt pathway by the Wnt coreceptor, LRP6. Both of these assays have been adapted for a 384-well format. We now propose to interrogate regulation of E-cadherin expression and canonical Wnt signaling using a chemical genetics-based approach in a high-throughput screen to identify compounds that induce expression of E-cadherin in SW620 cells and that perturb degradation of beta-catenin and Axin, two key regulators of signaling through the canonical Wnt pathway. Our initial screen of 6,400 small molecules has already identified several lead compounds in both of these assays and we propose screen a total of 160,000 compounds. We propose to validate lead compounds from this initial screen in a variety of in vitro and in vivo assays. Our preliminary studies indicate that the small molecule trichostatin A, a histone deacetylase inhibitor, acts to induce E-cadherin expression in SW620 cells. We will explore its role in E-cadherin expression as well as its potential for regulating Wnt signaling. Finally, we will identify the mechanisms of action and potential targets of validated compounds by testing their effects on candidate cellular pathways as well as in biochemically reconstituted reactions.