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.

 描述（由申请方提供）：上皮组织是粘附细胞的极化层，在不同的身体隔室之间产生边界。适当的上皮组织结构由极性蛋白的高度极化网络和沿顶-基底轴沿着排列的相邻细胞之间的适当粘附来维持。KRAS是一种致癌基因，在30%的人类肿瘤和35-40%的结直肠癌中发生突变。KRAS的异常激活上调RAS/BRAF/MAPK信号传导，这导致Caco-2结肠癌细胞的过度增殖和顶端极性的破坏。虽然一些研究表明， 癌基因如KRASV 12和BRAFV 600 E的激活导致大多数原发性肿瘤中细胞极性的丧失，但调节这种串扰以控制上皮组织的极化组装的潜在机制尚不清楚。我提出的研究目标是确定和表征KRASV 12和BRAFV 600 E癌基因破坏细胞极性的分子机制;并探索与KRASV 12和BRAFV 600 E合作促进原发性肿瘤侵袭的基因。具体而言，本研究的主要目的是研究KRASV 12和BRAFV 600 E激活对上皮形态发生的影响;阐明致癌KRAS和BRAF调节顶端极性的候选基因和信号通路，并评估RhoGTP酶，已知在结直肠肿瘤中频繁突变的细胞粘附机制和“假定的”基因与KRASV 12合作促进原发性结肠癌的侵袭，肿瘤的使用基于分子和细胞的方法的组合，将产生表达BRAFV 600 E和KRASV 12的基于他莫昔芬的诱导型细胞系，以评估在囊肿发育的不同阶段癌基因激活对极性维持的时间效应。使用钙开关测定、FRAP和光活化（PA）GFP的组合，将确定癌基因活化后AJ组分的动态性质的变化。此外，AJ重塑对顶端极性蛋白调节剂的形态学影响将被探索。为了鉴定调节KRAS依赖性极性缺陷的潜在基因，基于我们的DNA微阵列分析，我将使用慢病毒shRNA介导的基因敲减方法来验证约60个在KRASV 12和BRAFV 600 E激活后可能上调的基因。此外，报告基因测定将用于确定信号通路活性的癌基因依赖性变化。使用共聚焦显微镜和生物化学，候选基因和信号通路将进一步表征，以确定它们在细胞极性调节中的作用。最后，将探讨细胞粘附组分和RhoGTP酶调节剂在KRAS介导的原发性肿瘤的侵袭和细胞迁移中的作用。