Elucidating the Regulation of Delayed-Early Gene Targets of Sustained MAP Kinase Pathway Activation

阐明持续 MAP 激酶途径激活的延迟早期基因靶标的调节

• 批准号: 9760769
• 负责人: Kali Dale
• 金额: $ 3.29万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760769
• 关键词: ATAC-seq; Adhesions; Aggressive behavior; Apoptosis; BRAF gene; Basement membrane; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Markers; Breast Cancer Cell; C57BL/6 Mouse; CRISPR/Cas technology; Cancer Patient; Cell Adhesion; Cell Cycle; Cell Line; Cell Surface Proteins; Cell physiology; Cell surface; Cells; ChIP-seq; Characteristics; Chimeric Proteins; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Congenic Mice; Cutaneous Melanoma; Cycloheximide; Data; Development; Diagnosis; Disease Progression; Enhancers; Event; Extracellular Matrix; Extracellular Signal Regulated Kinases; Family; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Growth; Growth Factor; Hallmark Cell; Head; Health; Homeostasis; Human; ITGB3 gene; Imagery; Immunocompetent; In Vitro; Induced Mutation; Integral Membrane Protein; Integrin alpha Chains; Integrin beta3; Integrins; Knowledge; Laboratories; Ligand Binding; Link; Logic; Longevity; Luciferases; MAP kinase activator; MEKs; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Mediating; Melanoma Cell; Mentors; Messenger RNA; Metastatic Melanoma; Metastatic to; Methods; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Mutate; Mutation; Neoplasm Metastasis; Normal tissue morphology; Nucleic Acid Regulatory Sequences; Oncogene Activation; Oncogenic; Organ; Pathway interactions; Patients; Pattern; Phenotype; Play; Primary Neoplasm; Protein Synthesis Inhibition; Protein Synthesis Inhibitors; Proteins; Proto-Oncogenes; Publishing; Radial; Regulation; Research; Role; Scientist; Signal Pathway; Signal Transduction; Tissues; Training; Transcriptional Regulation; Transplantation; Universities; cancer cell; cancer survival; cell motility; cell type; improved; in vivo; interest; mRNA Expression; melanocyte; melanoma; migration; mutant; new therapeutic target; receptor; response; supportive environment; therapeutic target; transcription factor; transcriptome sequencing; tumor growth; tumor progression

PROJECT ABSTRACT RAS and its downstream effector, BRAF, are commonly mutated proto-oncogenes in many types of human cancer including melanoma. BRAFV600E is the most common mutation in cutaneous melanoma. This constitutively activating mutation induces sustained signaling of the Mitogen-Activated Protein Kinase (MAPK) pathway through MEKERK to regulate key cancer cell hallmarks such as progress through the cell division cycle, reduced programmed cell death and enhanced cell motility. Amongst the list of RAF-regulated genes are those encoding integrins, alpha-beta heterodimeric transmembrane proteins that regulate cell adhesion to extracellular matrix. Altered integrin expression has been linked to the acquisition of more aggressive behavior by melanoma, lung and breast cancer cells leading to diminished survival of cancer patients. This study aims to elucidate the regulation of integrin beta3 (ITGB3) and determine the biological role it has in the progression of melanoma. We have previously documented the ability of the RAF->MEK->ERK MAP kinase pathway to induce the expression of ITGB3 in several different cell types. RAF-mediated induction of ITGB3 mRNA requires sustained, high-level activation of ERK signaling mediated by oncogene activation and is classified as “delayed- early”, in that it is sensitive to the protein synthesis inhibitor cycloheximide. However, to date, the regulatory mechanisms that allow for induced ITGB3 downstream of sustained, high-level activation of RAF->MEK->ERK signaling remain obscure. To date, we have identified a number of genes using RNA-sequencing, including those expressing additional cell surface proteins, that display similar regulatory characteristics as ITGB3. We aim to relate altered expression of genes to RAF-induced changes in chromatin structure to determine if there is an underlying regulatory logic to the observed effects of activated RAF on delayed-early genes by ATAC-seq. Given the large numbers of human cancers with elevated RAF->MEK->ERK signaling, the mechanisms identified to regulate these genes by this strategy may have potential for use as either biomarkers of metastasis potential or as novel therapeutic targets. Due to the correlative evidence between increased ITGB3 expression and metastatic phenotypes, it is important to find the link between the two. With the use of congenic mouse melanoma cell lines that can be transplanted into C57BL/6 mouse hosts, ITGB3 will be genetically ablated by CRISPR/Cas9 strategies to evaluate the necessity and sufficiency of this gene in the progression of this disease. Results accumulated from the study will lead to further findings that in turn will improve the health of melanoma patients, including expanding the average life span after diagnosis. The applicant, Kali Dale, will trained in a supportive environment for her to become an independent research scientist from formal and informal mentors to be proficient in transcription focused research.

项目摘要 RAS及其下游效应因子BRAF在多种类型的人类中是常见的突变原癌基因 癌症，包括黑色素瘤。BRAFV600E是皮肤黑色素瘤中最常见的突变。这 结构性激活突变诱导丝裂原活化蛋白激酶(MAPK)持续信号传递 途径通过mekERK来调节关键的癌细胞标志，如通过细胞分裂的进展 循环，减少细胞程序性死亡，增强细胞运动性。在受皇家空军控制的基因清单中有 这些编码整合素、α-β异二聚体跨膜蛋白的蛋白调节细胞与 细胞外基质。整合素表达的改变与获得更具攻击性的行为有关 黑色素瘤、肺癌和乳腺癌细胞导致癌症患者的存活率降低。这项研究旨在 阐明整合素Beta3(ITGB3)的调控并确定其在肿瘤发生发展中的生物学作用 黑色素瘤。我们以前已经证明了RAF-&>MEK-&>ERK-ERK-MAPK通路能够诱导 ITGB3在几种不同细胞类型中的表达。RAF介导的ITGB3mRNA的诱导需要 由癌基因激活介导的ERK信号的持续、高水平激活，被归类为“延迟的- 早期“，因为它对蛋白质合成抑制剂放线菌亚胺敏感。然而，到目前为止，监管机构 持续、高水平激活RAF-&MEK-&>ERK下游诱导ITGB3的机制 信号仍然模糊不清。到目前为止，我们已经使用RNA测序确定了一些基因，包括 那些表达额外细胞表面蛋白的基因，表现出与ITGB3相似的调控特征。我们 目的将RAF诱导的染色质结构改变与基因表达改变联系起来，以确定是否存在 ATAC-seq观察到RAF激活对延迟早期基因影响的潜在调控逻辑。 鉴于大量RAF-&GT；MEK-&GT；ERK信号升高的人类癌症，其机制已确定 通过这一策略来调控这些基因可能会被用作转移潜能的生物标志物 或作为新的治疗靶点。由于ITGB3表达增加与 对于转移的表型，找到两者之间的联系是很重要的。利用同源基因小鼠 可移植到C57BL/6小鼠宿主的黑色素瘤细胞株ITGB3将被基因消融 CRISPR/CAS9评估该基因在疾病进展中的必要性和充分性的策略。 这项研究积累的结果将导致进一步的发现，进而改善黑色素瘤的健康 患者，包括延长确诊后的平均寿命。申请者Kali Dale将接受培训 支持她从正式和非正式导师成为独立研究科学家的环境 精通以转录为重点的研究。