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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：
8073535
负责人：
DANIEL BEAUCHAMP
金额：
$ 31.44万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8073535
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 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 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-钙粘蛋白功能丧失是与上皮间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。不当经典 Wnt 通路的激活是从正常上皮细胞转变为上皮细胞的另一个关键事件。结直肠癌的肿瘤表型。尽管人们普遍认为这些扰动途径代表了肿瘤转化的重要标志，其转导机制和人们对正常和病理条件下的调节知之甚少。我们已经高度发展在 SW620 结直肠癌细胞系中重新表达 E-钙粘蛋白的可靠且灵敏的测定通常表达非常低水平的 E-钙粘蛋白。同样，我们还开发了一种强大的生化该测定概括了 Wnt 辅助受体 LRP6 对经典 Wnt 通路的激活。这两个检测已针对 384 孔格式进行了调整。我们现在建议质疑 E-钙粘蛋白的调节使用基于化学遗传学的方法在高通量中表达和典型的 Wnt 信号传导筛选以鉴定诱导 SW620 细胞中 E-钙粘蛋白表达并扰乱的化合物 β-连环蛋白和 Axin 的降解，这是通过经典 Wnt 途径进行信号传导的两个关键调节因子。我们对 6,400 种小分子的初步筛选已经在这两种化合物中鉴定出了几种先导化合物我们建议筛选总共 160,000 种化合物。我们建议验证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，小曲古抑菌素 A 分子是一种组蛋白脱乙酰酶抑制剂，可诱导 SW620 中 E-钙粘蛋白的表达细胞。我们将探讨其在 E-钙粘蛋白表达中的作用及其调节 Wnt 信号传导的潜力。最后，我们将通过测试确定已验证化合物的作用机制和潜在目标它们对候选细胞途径以及生化重建反应的影响。