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.

神经皮肤黑素细胞增多症（NCM）是一种罕见的先天性疾病， 出生时大脑中的良性至恶性色素病变和皮肤上的黑色素细胞痣。 发病率估计为1/50，000 - 1/200，000。据报道，NCM症状占所有NCM症状的三分之一至一半， 病例是致命的，由于神经系统和其他异常，在诊断后2-3年内死亡。由于 由于人类样本的稀有性和实验模型的不可用性，人们对这种疾病的起源和 疾病的发展。最近，NRAS中的体细胞激活突变（NRAS Q61 K/R）已被发现。 在大多数NCM患者的肿瘤中鉴定（Kinsler等，2013年）。在发育早期获得的突变 在胚胎神经嵴细胞（NCCs）被认为是驱动因素。然而，我们对 NRAS在NCC中激活的分子机制以及与其相关的致癌过程 肿瘤性转化我们的初步数据表明NRAS Q61 K在NCC中的表达不足以 启动转基因小鼠的肿瘤发生。其他证据表明，肝细胞生长因子（HGF） 在胚胎发育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， 黑素细胞增多症。该建议旨在验证过度活化的HGF的促有丝分裂信号在细胞增殖中起作用的假说。 在NRAS Q61 K/R突变型NCC发育过程中启动肿瘤转化的协同作用。两 不同的转基因小鼠-一种在发育中的NCC中条件性表达NRAS Q61 K，另一种在发育中的NCC中条件性表达NRAS Q61 K。 由Glenn梅尔利诺的实验室描述为在胚胎发生期间过表达HGF的细胞将用于开发 NCM的转基因小鼠模型。这两种动物杂交后， 将使用组织学分析、荧光IVIS成像和MRI来表征转基因变体。到 确定NCC转化的分子决定因素RNA测序方法沿着差异 采用基因表达分析、通路分析和免疫组织化学方法。从这些 数据，本研究旨在阐明参与NCM起源和进展的细胞途径，诱导 通过致癌NRAS和HGF协同作用。这些实验符合NIH和NCI的使命， 它们直接关系到我们进一步理解发展的机制， 一种我们知之甚少的罕见儿科癌症此外，他们开发了一种转基因小鼠模型， 一个实验平台，以研究NRAS中的协同因子启动NCC的肿瘤发生，并将 作为有用的体内范例，用于理解NRAS驱动的神经系统肿瘤的分子基础。 在未来的研究中，脊衍生组织。