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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：
7813816
负责人：
DANIEL BEAUCHAMP
金额：
$ 31.28万
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/7813816
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 Malignant Neoplasms 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 -钙粘蛋白功能的丧失是与上皮细胞向间质细胞转化相关的关键事件。