Functional analysis of genetic alterations occuring in uveal melanoma

本提案中描述的实验旨在分析葡萄膜黑色素瘤中发现的遗传改变。目的是在体外和体内检查最近发现的GNAQ和GNA 11基因突变的功能，并建立相应的小鼠模型。 黑素细胞中GNAQ和GNA 11 Q209 L癌基因的特征目前对GNAQ和GNA 11癌基因在葡萄膜黑色素瘤发生中的功能了解甚少。为了实现这一点，该基因的生物学功能将以以下方式进行研究：1。确定肿瘤形成所必需的蛋白质结构域和信号效应子，2. GNAQ/11癌基因诱导蛋白磷酸化和RNA表达的详细分析。分析GNAQ/11诱导的衰老或凋亡，4.研究GNAQ/11癌基因转化细胞的转移特性，5.其他遗传因子如BAP 1、MYC、PTEN和p53参与GNAQ/11癌基因诱导的肿瘤和转移。第一个项目的目标是确定可能用于开发有效治疗策略的功能机制。 建立GNAQ/11癌基因小鼠模型与皮肤黑色素瘤的各种癌基因（BRAF、HRAS、KIT）的现有小鼠模型的广泛列表相反，目前没有GNAQ/11癌基因的现有小鼠模型。利用这些生理性癌基因建立葡萄膜黑色素瘤模型，从基础科学和转化肿瘤学的角度来看都是有价值的。申请人在美国博士后期间设计和克隆了各种转基因和敲入构建体，为该项目奠定了基础。这些小鼠将允许GNAQ/11癌基因在不同组织中的不同时间点表达。计划：1。测试这种表达对黑素细胞和其他组织中的肿瘤发生以及胚胎发育的影响。2.通过不同的交叉，在体内分析影响BAP 1、CDKN 2A、p53、MYC和PTEN与GNAQ/11癌基因表达组合的其他基因改变的作用。3.应用小鼠模型来测试有效抑制肿瘤生长和转移的各种不同治疗方法。

The experiments described in this proposal aim to analyze the genetic alterations found in uveal melanoma. The goal is to examine the function of the recently discovered gene mutations in GNAQ and GNA11 both in vitro and in vivo as well as establish corresponding mouse models.1) Characterization of GNAQ and GNA11 Q209L oncogenes in melanocytesThe function of the oncogenes GNAQ and GNA11 in the development of uveal melanomas is currently poorly understood. To achieve this, the biological function of this gene is to be studied in the following fashion: 1. Determine the protein domains and signal effectors that are essential for tumor formation, 2. A detailed analysis of GNAQ/11 oncogene induce protein-phosphorylations and RNA expression, 3. Analysis of GNAQ/11 induced senescence or apoptosis, 4. Studying the metastasis profile of GNAQ/11 oncogene transformed cells, 5. Participation of other genetic factors i.e. BAP1, MYC, PTEN and p53 in GNAQ/11 oncogene induced tumors and metastasis. The goal of this first project aim is to identify functional mechanisms that could be of potential use for developing effective therapeutic strategies.2) Establishing GNAQ/11 oncogene mouse modelsIn contrast to an extensive list of existing mouse models with various oncogenes (BRAF, HRAS, KIT) for cutaneous melanomas, there are currently no existing mouse models of GNAQ/11 oncogenes. Establishing a model of uveal melanoma using these physiological oncogenes would be valuable both from a basic science as well as a translational oncology perspective. With the design and cloning of various transgenic and knock-in constructs during his postdoc in the US, the applicant has laid the groundwork for this project. These mice will allow the expression of GNAQ/11 oncogenes at various timepoints in different tissues. It is planned to: 1. Test the effect of this expression, both on tumor genesis as well as embryonic development in both melanocytic and other tissue. 2. Through different crossings, analyze in vivo the role of additional gene alterations affecting i.e. BAP1, CDKN2A, p53, MYC and PTEN in combination with GNAQ/11 oncogene expression. 3. Apply the mouse model to test a variety of different therapeutic approaches for effectively inhibiting tumor growth and metastasis.