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（由适用提供）：上皮组织是粘附细胞的偏光片，它们在不同人体隔室之间产生边界。适当的上皮组织结构由高度极化的极性蛋白网络维持，并在沿顶端轴对齐的相邻细胞之间进行适当的粘合剂。 KRAS是一种癌基因，在所有人类肿瘤的30％和35-40％的结直肠癌中被突变。 KRAS的异常激活调节RAS/BRAF/MAPK信号，这导致Caco-2结肠癌细胞中顶极性的超增殖和破坏。而几项研究表明 在大多数原发性肿瘤中的激活（例如KRASV12和BRAFV600E）的激活导致细胞极性丧失，这是调节该串扰以控制上皮组织极化组装的基本机制。我提出的研究的目标是识别和表征KRASV12和BRAFV600E ONCEGONES破坏细胞极性的分子机制。并探索与KRASV12和BRAFV600E协调的基因以促进原发性肿瘤的侵袭。具体而言，这项研究的主要目的是研究KRASV12和BRAFV600E激活对上皮形态发生的影响。为了阐明候选基因和信号传导途径，致癌性KRAS和BRAF调节根尖极性，并评估RhoGTPases，细胞粘附机制和“推定”基因是否经常在有色肿瘤合作与KRASV12合作中经常突变，以促进对原发性肿瘤的入侵。使用分子和基于细胞的方法的组合，将生成基于他莫昔芬的诱导型细胞系和表达BRAFV600E和KRASV12的诱导细胞系，以评估癌基因激活对囊肿发育不同阶段的极性维持的临时影响。使用钙开关测定，FRAP和光活化（PA）GFP的组合，将进一步探索AJ成分的动态特性的变化，将进一步探索AJ重塑对顶极性蛋白调节剂的形态影响。为了根据我们的DNA微阵列分析确定调节KRAS依赖性极性缺陷的潜在基因，我将使用Lenti-病毒shRNA介导的基因敲低方法来验证〜60个基因，这些基因可能会在激活KRASV12和BRAFV600E的激活后受到调节。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，将进一步表征候选基因和信号通路，以确定它们在细胞极性调节中的作用。最后，将探索细胞粘合剂成分以及Rhogtpase的调节剂在侵袭和KRAS介导的原发性肿瘤的细胞迁移中的作用。