A High-Throughput Model for Human Melanoma

描述(申请人提供)：黑色素瘤是美国增长最快的恶性肿瘤。虽然它只占所有皮肤癌的4%，但它导致了近80%的皮肤癌死亡。如果及早发现，这种疾病很容易得到治疗；然而，一旦疾病扩散到遥远的地方，它通常对辅助治疗不起作用，五年存活率不到20%。随着治疗进入靶向治疗时代，人们越来越需要了解黑色素瘤的遗传复杂性和细胞信号的异质性。了解导致肿瘤形成和维持的遗传和信号特征，应该允许识别适当的分子靶点，并选择最有可能对特定抑制剂有反应的患者群体。为了更好地了解黑色素瘤的形成、进展和转移，我们建立了黑色素瘤的体细胞基因传递小鼠模型。该系统允许 除了快速验证人类黑色素瘤样本中发现的突变基因外，还可以鉴定这种疾病中改变的基因。该系统的主要优势是能够通过将多个癌基因导入体内的同一细胞来模拟多步骤的致癌过程，而不需要花费与交配多个品系的小鼠相关的费用。在这一竞争性修订中，我们建议扩大当前拨款的特定目标的范围，以定义可能的基因改变(S)，该基因改变介导在长时间潜伏期后发展的肿瘤的体内生长，并定义导致体内传代的肿瘤细胞与亲代细胞系相比潜伏期缩短的差异。为此，我们收集了这些肿瘤的组织样本并建立了细胞系。由于这些肿瘤中的CDKN2a基因缺失，我们怀疑它们在基因组上是不稳定的。我们已经通过流式细胞仪的DNA含量分析对两个已建立的细胞系进行了非整倍体鉴定，发现超过50%的细胞是非整倍体。这些结果将通过中期分裂过程中的染色体分析得到验证。将使用阵列比较基因组杂交(ACGH)以高分辨率检测拷贝数变化。如果检测到得失，将使用Western印迹分析和荧光原位杂交(FISH)对特定基因进行研究。突变的存在将通过以下方式进行评估 直接测序。所有检测到的改变都将通过RNA干扰(RNAi)和过度表达来验证它们在肿瘤进展中的作用。我们还将对肿瘤样本进行表达谱分析，并验证任何重大变化。由于我们已拥有进行这项分析所需的样本，有关研究可在建议的时间内完成。我们的长期目标是将从这些研究中获得的知识转化为治疗晚期黑色素瘤的分子靶向治疗的改进。 公共卫生相关性：黑色素瘤发病率的增加，特别是在年轻人和中年人中，是一个重大的公共卫生问题。这项研究的长期目标是使用一种新的黑色素瘤小鼠模型来识别黑色素瘤形成和生长所需的关键蛋白质，这些蛋白质可能成为开发治疗这种疾病晚期的更有效药物的潜在靶点。

DESCRIPTION (provided by applicant): Melanoma is the most rapidly increasing malignancy in the United States. While it accounts for only 4% of all skin cancers, it is responsible for nearly 80% of all skin cancer deaths. If detected early, the disease is easily treated; however, once the disease has spread to distant sites it does not generally respond to adjuvant therapies and the five-year survival is less than 20%. There is a growing need to understand the genetic complexity and cellular signaling heterogeneity of melanoma as treatments move into the era of targeted therapy. Understanding the genetic and signaling profiles that result in tumor formation and maintenance should allow for the identification of appropriate molecular targets and selection of patient populations most likely to respond to specific inhibitors. To better understand melanoma formation, progression, and metastasis we have developed a somatic cell gene delivery mouse model of melanoma. This system allows for the identification of genes altered in this disease in addition to the rapid validation of mutant genes identified in human melanoma samples. The major advantage of this system is the ability to model the multi-step process of carcinogenesis through the introduction of multiple oncogenes into the same cells in vivo without the expense associated with mating multiple strains of mice. In this competitive revision, we propose to expand the scope of the specific aims of the current grant to define the putative genetic alteration(s) that mediate the in vivo growth of the tumors that develop after a long latency and to define the differences that lead to a decreased latency of the in vivo passaged tumor cells compared to the parent cell lines. To this end, we have collected tissue samples and established cell lines for these tumors. Due to the loss of the Cdkn2a locus in these tumors, we suspected that they are genomically unstable. We have already characterized two of the established cell lines for aneuploidy by analysis of DNA content by flow cytometry and found that greater than 50% of the cells were aneuploid. These results will be validated by analysis of chromosomes through metaphase spreads. Copy number changes will be detected at high resolution using array comparative genomic hybridization (aCGH). Specific genes will be investigated using Western blot analysis and fluorescent in situ hybridization (FISH) if gains and/or losses are detected. The presence of mutations will be assessed by direct sequencing. All detected alterations will be validated for their role in tumor progression by both RNA interference (RNAi) and overexpression. We will also perform expression profiling on the tumor samples and validate any significant changes. Because we already possess the samples necessary for this analysis, the studies can be completed within the time frame proposed. Our long-term goals are to translate the knowledge gained from these studies into improvements in molecular targeted therapies for the treatment of advanced melanoma. Public Health Relevance: The increasing incidence of melanoma, in particular among young to middle-aged adults, is a significant public health problem. The long-term goal of this research is to use a novel mouse model of melanoma to identify key proteins required for melanoma formation and growth that could serve as potential targets for the development of more effective drugs for the treatment of advanced stages of this disease.