(PQB6) Accurate Comparative Genome-wide Analysis of Primary Tumors and Metastases

描述(申请人提供)：大多数癌症死亡不是由原发肿瘤引起的，而是由远处转移引起的。惰性原发肿瘤进展为转移的过程涉及肿瘤细胞基因表达和染色质修饰模式的戏剧性重新编程。有效地分析整个基因组中的这些变化，并确定强大的促转移基因表达和染色质修饰特征，将是了解肿瘤进展和治疗人类癌症的关键。近年来，我们在全基因组范围内描绘表观遗传和基因表达模式的能力有了巨大的进步。然而，由于难以分离出不受基质污染的纯肿瘤细胞群体，因此适用于小鼠癌症模型变得复杂起来。我们将开发专门从小鼠实体瘤中分离出无间质和其他污染细胞类型的肿瘤细胞的技术。这将使得对原发肿瘤和转移瘤的核的纯群体进行准确的比较。具体地说，我们将：1)开发生物素标记人类细胞系中的核膜蛋白的方法，以及从裸鼠移植瘤中分离生物素标记的核的方法。作为原则的证明，我们将测试我们是否可以对这些模型中的原发肿瘤和转移进行全基因组比较。2)建立和测试Cre重组酶可诱导核膜生物素化盒表达的基因工程小鼠模型。当与人类癌症的小鼠模型相结合时，其中肿瘤是由CRE介导的癌基因的激活或肿瘤抑制基因的缺失驱动的，这将允许分离和分析纯肿瘤细胞核群体。这项工作将产生广泛适用于研究人类癌症小鼠模型中肿瘤进展和转移的工具和方法。

DESCRIPTION (provided by applicant): The majority of deaths due to cancer are caused not by the primary tumor but by distant metastases. The process by which an indolent primary tumor progresses to become metastatic involves a dramatic reprogramming of the tumor cell's gene expression and chromatin modification patterns. Effectively analyzing these changes across the entire genome, and identifying robust pro-metastatic gene expression and chromatin modification signatures, will be essential to understanding tumor progression and treating human cancer. Our ability to profile epigenetic and gene expression patterns on a genome-wide scale has advanced enormously in recent years. However, the applicability to mouse models of cancer is complicated by the difficulties in isolating pure populations of tumor cells that are free from contaminating stroma. We will develop technology for specifically profiling tumor cells from solid tumors in mice, free from stroma and other contaminating cell types. This will allow the accurate comparison of pure populations of nuclei from primary tumors and metastases. Specifically, we will: 1) Develop methods for biotin tagging a nuclear envelope protein in human cells lines and for isolating the biotin tagged nuclei from a xenograft tumor in nude mice. As proof of principle, we will test whether we can perform a genome-wide comparison of primary tumors and metastases from these models. 2) Generate and test a genetically engineered mouse model in which expression of a nuclear envelope biotinylation cassette can be induced by the Cre recombinase. When combined with mouse models of human cancer in which the tumor is driven by Cre-mediated activation of an oncogene or deletion of a tumor suppressor, this will allow for the isolation and analysis of pure populations of tumor cell nuclei. This work will generate tools and methods that will be widely applicable to the study of tumor progression and metastasis in mouse models of human cancer.