Genetic Analysis of Breast Cancer Progression in Mice Using Inducible Transposition

项目摘要 尽管通过对人类乳腺癌基因组测序积累了令人印象深刻的突变目录， 到目前为止，大多数被破译的基因组来自早期疾病，这可能会留下许多遗传事件 推动疾病发展的未知因素。正在努力将更复杂的基因组从更多 晚期和耐药乳腺癌前景看好，但在这些癌症中，较高的突变负担 癌症使区分肇因司机事件和无害乘客的困难任务复杂化 事件。提供乳腺癌期间获得的突变的全基因组视图的动物模型 进展可以提供跨物种验证，这对验证候选司机基因至关重要。 借鉴乳腺癌基因组学图景和我们自己的小鼠建模工作，我们制定了 假设乳腺癌的进展取决于在疾病阶段获得的驱动基因突变- 具体的方式，被不断积累的乘客突变所掩盖。基于这一假设，我们 制定了以下长期目标：通过执行以下操作来发现乳腺癌进展的新驱动因素 利用定时动员转座子(所谓的“跳跃”)对小鼠乳腺癌模型进行基因筛选 基因“)处于离散的疾病阶段。 在未发表的初步研究中，我们设计了新的小鼠模型来发现和验证 候选乳腺癌癌基因。为了能够发现癌基因，我们生成了一个可诱导版本的 睡美人(SB)转座系统，使小鼠乳房中的转座子能够定时动员。 我们证实，该系统提供了一个有效的癌症基因发现平台 与Wnt通路激活协同驱动体内乳腺肿瘤发生的新癌基因。至 配合这个高通量的癌症基因发现平台，我们设计了一种新的策略，高效地 乳腺生长监测验证候选癌基因并探讨其作用机制 3D培养中生长的组织碎片(有机物质)。这一策略使用活细胞成像来定量 对最终导致乳腺细胞过度生长的癌基因驱动事件进行评分。 我们将通过完成三个具体目标来解决我们的假设。在目标1中，基于转座子的基因 这一发现将被用于表达已知乳腺癌的小鼠模型-- 相关癌基因。我们的目标是找出驱动乳腺转变的新候选基因 从乳腺增生到局灶性乳腺癌。在目标2中，延迟的转座子动员将在 已建立的乳腺癌发生在经典的MMTV-Neu乳腺癌模型中。我们的目标是找出 候选基因驱动对拉帕替尼的耐药性，拉帕替尼是一种临床上重要的药物，可以阻断Her2/Neu信号转导。在……里面 目标3，我们将优化我们的活细胞成像平台，并将其应用于测试是否以及如何候选癌症 基因赋予了在3D培养中生长的乳腺细胞恶性的能力。

Project Summary Despite the impressive catalog of mutations amassed from sequencing human breast cancer genomes, most genomes decoded so far derive from early-stage disease, which likely leaves many genetic events driving disease progression undiscovered. Ongoing efforts to decode the more complex genomes from more advanced and drug-resistant breast cancers hold great promise, but the higher mutation burden in these cancers complicates the difficult task of distinguishing causative driver events from innocuous passengers events. Animal models that offer a genome-wide view of the mutations acquired during breast cancer progression can provide cross-species validation crucial for validating candidate driver genes. Drawing on the breast cancer genomics landscape and our own mouse modeling work, we formulated the hypothesis that breast cancer progression depends on driver mutations acquired in a disease stage- specific manner, obscured by continuously accruing passenger mutations. Based on this hypothesis, we developed the following long-term goal: discover novel drivers of breast cancer progression by performing genetic screens in mouse breast cancer models using timed mobilization of transposons (so-called “jumping genes”) at discrete disease stages. In unpublished preliminary studies, we engineered new mouse models for discovering and validating candidate mammary oncogenes. To enable oncogene discovery, we generated an inducible version of the Sleeping Beauty (SB) transposition system, which enables timed transposon mobilization in the mouse breast. We confirmed that this system provides an efficient cancer gene discovery platform by identifying known and novel oncogenes that cooperate with Wnt pathway activation to drive mammary tumorigenesis in vivo. To complement this high-throughput cancer gene discovery platform, we designed a novel strategy for efficiently validating candidate oncogenes and probing their mechanisms of action by monitoring the growth of mammary tissue fragments (organoids) grown in 3D culture. This strategy employs live-cell imaging for quantitative scoring of the oncogene-driven events that culminate in mammary cell overgrowth. We will address our hypothesis by completing three Specific Aims. In Aim 1, transposon-based gene discovery will be employed in the context of mouse models engineered to express known breast cancer- relevant oncogenes. Our goal is to identify novel candidate genes that drive the transition from mammary hyperplasia to focal mammary cancer. In Aim 2, delayed transposon mobilization will be initiated within established mammary cancers arising in the classic MMTV-Neu breast cancer model. Our goal is to identify candidate genes that drive resistance to Lapatinib, a clinically important drug that blocks Her2/Neu signaling. In Aim 3, we will optimize our live-cell imaging platform and apply it to test whether and how candidate cancer genes confer malignant capabilities to mammary cells grown in 3D culture.