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 脑部良性到恶性的色素病变和皮肤上的黑色素细胞痣在出生时就存在。 发病率估计为1/50,000-1/200,000新生儿。有症状的NCM，据报道占所有NCM的三分之一到一半 病例是致命的，死亡发生在确诊后2-3年内，原因是神经系统和其他异常。由于 人类样本的稀缺性和实验模型的不可获得性，对其起源和 疾病的发展。最近，NRAS(NRAS Q61K/R)的体细胞激活突变被发现 在大多数NCM患者的肿瘤中发现(Kinsler等人，2013年)。在发育早期获得的突变 在胚胎中，神经脊细胞(NCCs)被认为是驱动因素。然而，人们对此一无所知。 NCCs中NRAS激活的分子机制及其致癌过程 肿瘤性转化。我们的初步数据表明，NRAS Q61K在NCC中的表达不足以 在转基因小鼠中启动肿瘤形成。更多证据表明，肝细胞生长因子(HGF) 胚胎发育过程中的信号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。这一建议旨在检验一种假设，即过度活跃的HGF产生的有丝分裂信号在 NRAS Q61K/R突变NCC在发育过程中启动肿瘤转化的协同作用二 不同的转基因小鼠--一个在发育中的NCCs中有条件地表达NRAS Q61K，另一个在NCC中有条件地表达NRAS Q61K 格伦·梅里诺的实验室描述称，在胚胎发育过程中HGF过度表达，将用于发育 NCM转基因小鼠模型。在这两种杂交产生的后代中出现病变 转基因变种将使用组织学分析、荧光IVIS成像和磁共振成像进行表征。至 鉴定NCCS转化的分子决定因素RNA测序方法及其差异 将采用基因表达分析、通路分析和免疫组织化学方法。从这些 本研究旨在阐明NCM的发生和发展所涉及的细胞通路， 通过致癌的NRAS和HGF协同作用。这些实验符合NIH和NCI的使命，因为 它们直接关系到我们对基础发展的机制的理解 罕见的儿科癌症，我们对此知之甚少。此外，他们还开发了一种转基因小鼠模型 研究NRAS中NCCs和Will致癌协同因素的实验平台 为理解NRAS驱动的神经肿瘤的分子基础提供了有用的体内范例 CREST来源的组织在未来的研究中。