To investigate the molecular and cellular mechanisms through which KRAS and BRAF oncogenes regulate cell polarity during epithelial morphogenesis

研究 KRAS 和 BRAF 癌基因在上皮形态发生过程中调节细胞极性的分子和细胞机制

• 批准号: 9326319
• 负责人: Ridhdhi R Desai
• 金额: $ 6.1万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9326319
• 关键词: Actins; Adherens Junction; Adhesions; Affect; Apical; Architecture; BRAF gene; Biochemistry; Biological Assay; Caco-2 Cells; Calcium; Cancer Patient; Candidate Disease Gene; Carcinoma; Cell Adhesion; Cell Adhesion Molecules; Cell Culture System; Cell Line; Cell Polarity; Cell division; Cells; Characteristics; Colon; Colon Adenocarcinoma; Colon Carcinoma; Colorectal Cancer; Colorectal Neoplasms; Confocal Microscopy; Cyst; DNA Microarray Chip; Data; Defect; Development; Dimensions; E-Cadherin; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Event; Extracellular Matrix; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Goals; Human; Immunofluorescence Immunologic; Intercellular Junctions; Investigation; KRAS2 gene; Lead; Link; Luciferases; MAP Kinase Gene; Maintenance; Malignant Neoplasms; Mediating; Membrane; Metabolism; Microarray Analysis; Molecular; Morphogenesis; Morphology; Mutate; Mutation; Oncogene Activation; Oncogenes; Oncogenic; Pathway interactions; Primary Neoplasm; Property; Proteins; Proto-Oncogene Proteins c-myc; Regulation; Regulator Genes; Reporter Genes; Research; Role; Severities; Signal Pathway; Signal Transduction; Stem cells; Structure; System; Tamoxifen; Tissues; Transgenes; Up-Regulation; base; c-myc Genes; cancer cell; cell motility; experimental study; genome sequencing; knock-down; lentiviral-mediated; migration; neoplastic cell; protein complex; public health relevance; rho GTP-Binding Proteins; small hairpin RNA; three dimensional cell culture; three dimensional structure; tumor; whole genome

 DESCRIPTION (provided by applicant): Epithelial tissues are polarized sheets of adherent cells that generate boundaries between different body compartments. Proper epithelial tissue architecture is maintained by a highly polarized network of polarity proteins and proper adhesion between neighboring cells that are aligned along the apical-basal axis. KRAS is an oncogene that is mutated in 30% of all human tumors and in 35-40% of colorectal cancers. Abnormal activation of KRAS up regulates RAS/BRAF/MAPK signaling, which results in hyper proliferation and disruption of apical polarity in Caco-2 colon cancer cells. While several studies indicate that activation of oncogenes such as KRASV12 and BRAFV600E result in loss of cell polarity in most primary tumors, the underlying mechanisms that regulate this crosstalk to govern polarized assembly of epithelial tissue is not known. The goals of my proposed research are to identify and characterize the molecular mechanisms through which KRASV12 and BRAFV600E oncogenes disrupt cell polarity; and to explore genes that cooperate with KRASV12 and BRAFV600E to promote invasion of primary tumors. Specifically, the main aims of this research are to investigate the effects of KRASV12 and BRAFV600E activation on epithelial morphogenesis; to elucidate gene candidates and signaling pathways through which oncogenic KRAS and BRAF regulate apical polarity, and to assess whether RhoGTPases, the cell adhesion machinery and 'putative' genes known to be frequently mutated in colorectal tumors cooperate with KRASV12 to promote invasion of primary tumors. Using a combination of molecular and cell-based approaches, tamoxifen-based inducible cell lines expressing BRAFV600E and KRASV12 will be generated to assess the temporal effects of oncogene activation on polarity maintenance at different stages of cyst development. Using a combination of calcium switch assay, FRAP and photoactivatable (PA) GFP, changes in the dynamic properties of AJ components upon oncogene activation will be determined. Further, the morphological effects of AJ remodeling on regulators of apical polarity proteins will be explored. To identify potential genes that regulate KRAS-dependent polarity defects, based on our DNA microarray analysis, I will use a lenti-viral shRNA-mediated gene knockdown approach to validate ~60 genes that are potentially up regulated upon activation of KRASV12 and BRAFV600E. Further, reporter gene assays will be used to determine oncogene-dependent changes in the activity of signaling pathways. Using confocal microscopy and biochemistry, candidate genes and signaling pathways will be further characterized to determine their role in the regulation of cell polarity. Finally, the role of cell adhesion components, and modulators of RhoGTPases in invasion and cell migration of KRAS-mediated primary tumors will be explored.