To study the molecular oncogenesis of congenital Neurocutaneous melanocytosis in a preclinical transgenic mouse model

研究临床前转基因小鼠模型中先天性神经皮肤黑素细胞增多症的分子肿瘤发生

• 批准号: 10651336
• 负责人: Dipanjan Basu
• 金额: $ 7.51万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651336
• 关键词: Address; Alleles; Automobile Driving; Benign; Biochemical; Birth; Brain; CRISPR/Cas technology; Cell Proliferation; Cells; Cessation of life; Congenital Disorders; Crossbreeding; Data; Detection; Development; Developmental Biology; Diagnosis; Diffuse; Disease; Embryo; Embryonic Development; Etiology; Experimental Models; Exposure to; Fluorescence; Future; Gene Expression; Gene Expression Profiling; Genetic; Global Change; Growth Factor; Growth Factor Overexpression; HGF gene; Histologic; Human; Hydrocephalus; Hyperplasia; Imaging Techniques; Immunohistochemistry; In Vitro; Inbred C57BL Mice; Incidence; Infiltration; Interleukin-2; Intracranial Hypertension; KRAS oncogenesis; Knock-in; Knowledge; Laboratories; Leptomeninges; Lesion; MET gene; Magnetic Resonance Imaging; Malignant - descriptor; Malignant Childhood Neoplasm; Melanocytic nevus; Methods; Mission; Mitogen-Activated Protein Kinases; Mitogens; Molecular; Morphogenesis; Mus; Mutation; Neoplastic Cell Transformation; Neural Crest; Neural Crest Cell; Neuroepithelial, Perineurial, and Schwann Cell Neoplasm; Neurologic; Neurologic Symptoms; Oncogene Activation; Oncogenes; Oncogenic; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Pattern; Physiological; Pigments; Play; Pre-Clinical Model; Process; Prognosis; Property; Receptor Protein-Tyrosine Kinases; Reporting; Role; Sampling; Seizures; Signal Pathway; Signal Transduction; Skin; Sorting; Structure; Testing; Tissue-Specific Gene Expression; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; Vertebrates; base; blastomere structure; cell transformation; cell type; embryo cell; experimental study; fluorescence imaging; genetic variant; in vivo; in vivo imaging system; melanocyte; mouse model; mutant; neoplastic; neoplastic cell; novel; offspring; overexpression; permissiveness; pre-clinical; rare cancer; response; spatiotemporal; stem-like cell; transcriptome sequencing; transcriptomics; tumor; tumorigenesis; tumorigenic

PROJECT SUMMARY: Neurocutaneous Melanocytosis (NCM) is a rare congenital disorder characterized by benign to aggressively malignant pigmented lesions in the brain and melanocytic nevi on skin present at birth. Incidence is estimated at 1/50,000 – 1/200,000 births. Symptomatic NCM, reported to be a third to half of all cases is fatal, death occurring within 2-3 years of diagnosis due to neurological and other abnormalities. Due to the rarity of human samples and unavailability of experimental models, very little is known about the origin and development of the disease. Recently, somatic activating mutations in NRAS (NRAS Q61K/R) have been identified in majority of NCM patients’ tumors (Kinsler et.al., 2013). Mutation acquired early during development in the embryonic neural crest cells (NCCs) is thought to be the driving factor. However, nothing is known about the molecular mechanisms underlying NRAS activation in NCCs and oncogenic processes involved in their neoplastic transformation. Our preliminary data indicate that NRAS Q61K expression in NCCs is not sufficient to initiate tumorigenesis in a transgenic mouse. Additional evidence suggests that Hepatocyte Growth Factor (HGF) signaling during embryogenesis may be an important component in driving leptomeningeal and skin melanocytosis. This proposal aims to test the hypothesis that mitogenic signaling from overactive HGF plays a cooperative role in initiating neoplastic transformation of NRAS Q61K/R mutant NCCs during development. Two different transgenic mice – one conditionally expressing NRAS Q61K in developing NCCs and the other described by Glenn Merlino’s laboratory as overexpressing HGF during embryogenesis will be used to develop a transgenic mouse model of NCM. Lesions developed in the offspring resultant from cross breeding these two transgenic variants will be characterized using histological analyses, fluorescence IVIS imaging and MRI. To identify the molecular determinants of NCCs transformation RNA-Sequencing approaches along with differential gene expression analysis, pathway analysis and immunohistochemical methods will be employed. From these data, this study aims to elucidate the cellular pathways involved in the origin and progression of NCM, induced cooperatively by oncogenic NRAS and HGF. These experiments fit the mission of the NIH and NCI because they have direct relevance to furthering our understanding of the mechanisms underlying the development of a rare pediatric cancer about which we know very little. Furthermore, they develop a transgenic mouse model as an experimental platform to investigate cooperative factors in NRAS initiated oncogenesis from NCCs and will serve as useful in vivo paradigm for understanding molecular underpinnings of NRAS driven tumors of neural crest derived tissues in future studies.

项目摘要：神经皮肤黑素细胞增多症 (NCM) 是一种罕见的先天性疾病，其特征是 出生时大脑中存在良性至恶性的色素病变和皮肤上的黑素细胞痣。 有症状的 NCM 发病率估计为 1/50,000 – 1/200,000，据报道占所有新生儿的三分之一到一半。 病例是致命的，死亡发生在诊断后 2-3 年内，由于神经系统和其他异常所致。 由于人类样本的稀有性和实验模型的不可用，人们对起源和研究知之甚少。 最近，NRAS (NRAS Q61K/R) 的体细胞激活突变已被证实。 在大多数 NCM 患者的肿瘤中发现了这种突变（Kinsler et.al., 2013）。 胚胎神经嵴细胞（NCC）中的这种现象被认为是驱动因素，但目前尚不清楚。 NCC 中 NRAS 激活的分子机制及其涉及的致癌过程 我们的初步数据表明，NCC 中的 NRAS Q61K 表达不足以导致肿瘤转化。 其他证据表明肝细胞生长因子 (HGF) 会引发转基因小鼠的肿瘤发生。 胚胎发生过程中的信号传导可能是驱动软脑膜和皮肤的重要组成部分 该提案旨在检验过度活跃的 HGF 的促有丝分裂信号发挥作用的假设。 在 NRAS Q61K/R 突变型 NCC 发育过程中启动肿瘤转化中的合作作用 两个。 不同的转基因小鼠——一只在发育 NCC 时条件性表达 NRAS Q61K，另一只 Glenn Merlino 实验室将其描述为在胚胎发生过程中过度表达 HGF，将用于开发 NCM 转基因小鼠模型是由这两种动物杂交产生的后代产生的病变。 将使用组织学分析、荧光 IVIS 成像和 MRI 来表征转基因变异。 确定 NCC 转化的分子决定因素 RNA 测序方法以及差异 将采用基因表达分析、通路分析和免疫组织化学方法。 数据，本研究旨在阐明参与 NCM 起源和进展的细胞途径，诱导 这些实验符合 NIH 和 NCI 的使命，因为 NRAS 和 HGF 合作进行。 它们与加深我们对发展的机制的理解有直接关系。 我们对此知之甚少的罕见儿童癌症此外，他们开发了一种转基因小鼠模型。 一个实验平台，用于研究 NRAS 中启动 NCC 肿瘤发生的合作因素，并将 作为有用的体内范例，用于分子理解 NRAS 驱动的神经肿瘤的基础 未来研究中的嵴衍生组织。