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: 10240590
• 负责人: Kali Dale
• 金额: $ 2.74万
• 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 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240590
• 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; Immunocompetent; In Vitro; Induced Mutation; Integral Membrane Protein; Integrin alpha Chains; 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; Visualization; cancer cell; cancer survival; cell motility; cell type; improved; in vivo; interest; mRNA Expression; melanocyte; melanoma; migration; mutant; new therapeutic target; progression marker; 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及其下游效应子BRAF是多种人类肿瘤中常见的突变原癌基因， 癌症包括黑色素瘤。BRAFV 600 E是皮肤黑色素瘤中最常见的突变。这 组成型激活突变诱导有丝分裂原激活蛋白激酶（MAPK）的持续信号传导 通过MEK途径，ERK调节关键的癌细胞标志，如通过细胞分裂的进展 周期，减少程序性细胞死亡和增强细胞运动性。RAF调节基因的列表中有 那些编码整联蛋白、α-β异二聚体跨膜蛋白的蛋白质，其调节细胞粘附至 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生存率下降。本研究旨在 阐明整合素β 3（ITGB 3）的调节，并确定其在肿瘤进展中的生物学作用。 黑素瘤我们先前已经证明了RAF->MEK->ERK MAP激酶途径诱导细胞凋亡的能力。 ITGB 3在几种不同细胞类型中的表达。RAF介导的ITGB 3 mRNA诱导需要 持续的、高水平的ERK信号传导激活，由癌基因激活介导，并被归类为“延迟- 早期”，因为它对蛋白质合成抑制剂放线菌酮敏感。然而，到目前为止，监管 在RAF->MEK->ERK的持续、高水平激活下游允许诱导ITGB 3的机制 信号仍然模糊。到目前为止，我们已经确定了一些基因使用RNA测序，包括 那些表达额外的细胞表面蛋白，显示出与ITGB 3相似的调节特性。我们 目的是将基因表达的改变与RAF诱导的染色质结构的变化联系起来，以确定是否存在 通过ATAC-seq观察到的激活RAF对延迟早期基因的影响的潜在调控逻辑。 鉴于大量的人类癌症具有升高的RAF->MEK->ERK信号传导， 通过这种策略调节这些基因可能有潜力用作转移潜力的生物标志物， 或作为新的治疗靶点。由于ITGB 3表达增加与肿瘤细胞增殖之间的相关性证据， 转移表型，重要的是要找到两者之间的联系。使用同类小鼠 可以移植到C57 BL/6小鼠宿主中的黑色素瘤细胞系中，ITGB 3将被遗传消融， CRISPR/Cas9策略，以评估该基因在该疾病进展中的必要性和充分性。 从研究中积累的结果将导致进一步的发现，反过来将改善黑色素瘤的健康 患者，包括延长诊断后的平均寿命。申请人Kali Dale将在一个 支持她成为一个独立的研究科学家从正式和非正式的导师环境 精通转录研究。