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Chemical Genetics Approach to Interrogate & Regulate E-Cadherin Expression

化学遗传学询问方法

基本信息

批准号：
7245692
负责人：
DANIEL BEAUCHAMP
金额：
$ 28.55万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7245692
关键词：
Action Potentials Adherens Junction Binding Biochemical Biological Biological Assay Biological Models Biology Cancer cell line Carcinoma Cell membrane Cells Chemicals Chemistry Colon Carcinoma Colorectal Cancer Complex Condition Core Facility Development E-Cadherin Epigenetic Process Epithelial Cells Epithelium Event Future Genes Growth and Development function Histone Deacetylase Inhibitor Histones Human In Vitro Institutes Lead Libraries Loss of E-cadherin Expression Maintenance Manuscripts Methodology Molecular Target Mutation Neoplasm Metastasis Neoplastic Processes Pathway interactions Personal Satisfaction Phenotype Play Preparation Reaction Regulation Repression Reproduction spores Role Scaffolding Protein Signal Pathway Signal Transduction Staging Testing Therapeutic Intervention Tight Junctions Transcription Coactivator Trichostatin A Universities Validation base beta catenin cancer cell chemical genetics chemical synthesis chemotherapeutic agent epithelial to mesenchymal transition high throughput screening in vivo insight malignant phenotype metaplastic cell transformation neoplastic neoplastic cell novel protein E receptor reconstitution small molecule success tumorigenesis

项目摘要

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.

E-钙粘蛋白功能的丧失是与上皮向间质转化相关的关键事件，在人结肠直肠癌和其它癌中的侵袭性和转移表型。不适当经典Wnt通路的激活是从正常上皮细胞向正常上皮细胞转变的另一个关键事件。结直肠癌的肿瘤表型。虽然人们普遍认为，这些扰动通路代表了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。我们高度发展在SW620结肠直肠癌细胞系中E-钙粘蛋白再表达的可靠和灵敏的测定，通常表达非常低水平的E-钙粘蛋白。同样，我们也开发了一种强大的生化通过Wnt辅助受体LRP 6再现经典Wnt途径的活化的测定。这两测定法已经适用于384孔形式。我们现在建议询问调节E-钙粘蛋白表达和规范的Wnt信号转导，使用基于化学遗传学的方法，在高通量筛选以鉴定在SW620细胞中诱导E-钙粘蛋白表达并干扰E-cadherin表达的化合物。 β-连环蛋白和Axin的降解，这是通过经典Wnt途径的信号传导的两个关键调节因子。我们对6,400个小分子进行了初步筛选，已经在这两种物质中发现了几种先导化合物。分析，我们建议筛选总共160，000种化合物。我们建议验证先导化合物在多种体外和体内测定中进行该初始筛选。我们的初步研究表明，组蛋白去乙酰化酶抑制剂- 细胞我们将探讨其在E-cadherin表达中的作用以及其调节Wnt信号传导的潜力。最后，我们将通过测试确定验证化合物的作用机制和潜在靶点它们对候选细胞途径以及生化重建反应的影响。