Conserved coamplification event in HER2+ breast cancer increases metastasis

项目总结 乳腺癌是女性中最常见的癌症，估计是最常见的癌症。 世界各地女性的死因。大约五分之一的乳腺癌患者肿瘤过度表达 人表皮生长因子受体2(HER2)蛋白；这是长链上扩增事件的结果 人类17号染色体的臂，导致HER2/Neu基因(ERBB2)的高拷贝数增加。转移性 HER2+乳腺癌比其他亚型乳腺癌更具侵袭性，对一般乳腺癌的反应较差 化疗，而且总是致命的。缺乏对支撑这一现象的分子机制的了解 转移性级联导致治疗选择有限，临床结果不佳。研究其作用机制。 在HER2+乳腺癌中的转移，我们利用了一个过度表达Neu的转基因小鼠模型，即大鼠 HER2的异构体，在小鼠乳腺肿瘤病毒(MMTV)启动子的控制下，此后被称为 MMTV-Neu。有趣的是，我们最近的论文描述了MMTV-Neu初级患者的全基因组测序 肿瘤显示出以小鼠染色体11qD为中心的共扩增事件，位于ERBB2附近，在大多数 样本。将这些结果与通过癌症基因组图谱(TCGA)获得的人类基因组数据进行比较，揭示了 在高度同线的人类染色体17q21.33上的类似扩增事件，接近，但与 ERBB2扩增子。在25%的HER2+患者中发现这种次级扩增子，在所有HER2+患者中发现9%的人存在这种次级扩增子 乳腺癌，并包含一些候选基因，这些基因可能介导转移。初步数据 阐述了这些候选基因在介导转移方面的潜力，但 这是如何发生的仍是个未知数。这一提议的中心假设是三个基因的上调 在该扩增子中，胶原1α1链(Col1A1)、软骨粘附素(乍得)和禁止素(PHB)， 通过改变肿瘤微环境和致癌信号通路增加转移。这 假设将使用三个独立的目标进行评估，这三个目标将共同确定关键事件和 HER2+转移级联中的依赖性。目标1确定受影响的转移阶段 17q21.33扩增片段的敲除/敲除及三个候选基因在HER2+/Neu中的过表达 将过表达的细胞系注射到同基因小鼠体内。目的2利用肿瘤之间的转录差异， 免疫组织化学(IHC)和免疫共沉淀法(Co-IP)确定信号网络差异 在肿瘤+/-候选基因和肿瘤内基因的空间表达之间。目标3决定 扩增子中哪些基因是必需的或充分的，可以通过过度表达候选基因来增加转移 单独或串联的基因，并测量对迁移和转移的影响。长期目标和 这一假说驱动的提案的广泛目标是提供对为什么有些乳房 癌症会发生转移，最终会改善患者的临床结果。

PROJECT SUMMARY Breast cancer is the most common cancer diagnosed among women and is estimated to be the leading cause of death for women worldwide. Approximately one fifth of breast cancer patients’ tumors overexpress the human epidermal growth factor receptor 2 (HER2) protein; this is the result of an amplification event on the long arm of human chromosome 17, leading to high copy number gains of the HER2/Neu gene (ERBB2). Metastatic HER2+ breast cancer is more aggressive than other subtypes of breast cancer, responds poorly to general chemotherapy, and is invariably fatal. Lack of understanding of the molecular mechanisms that underpin the metastatic cascade leads to limited therapy options and poor clinical outcomes. To study the mechanisms of metastasis in HER2+ breast cancer, we utilize a transgenic mouse model which overexpresses Neu, the rat isoform of HER2, under control of the mouse mammary tumor virus (MMTV) promoter, henceforth known as MMTV-Neu. Interestingly, our recent paper describing whole genome sequencing (WGS) of MMTV-Neu primary tumors revealed a coamplification event centered on mouse chromosome 11qD, near Erbb2, in a majority of samples. Comparing these results to human genomic data through The Cancer Genome Atlas (TCGA) reveals an analogous amplification event on the highly syntenic human chromosome 17q21.33 near, but separate from, the ERBB2 amplicon. This secondary amplicon was found to be present in 25% of HER2+ patients, 9% of all breast cancers, and contains a number of candidate genes that putatively mediate metastasis. Preliminary data addresses the potential for these candidate genes in mediating metastasis, but the molecular mechanisms for how this occurs remains unknown. The central hypothesis of this proposal is that upregulation of three genes in this amplicon, collagen type 1 alpha 1 chain (Col1A1), chondroadherin (CHAD), and prohibitin (PHB), increases metastasis through alterations to the tumor microenvironment and oncogenic signaling pathways. This hypothesis will be evaluated using three independent aims that will collectively identify key events and dependencies in the HER2+ metastatic cascade. Aim 1 identifies the stage of metastasis affected by the 17q21.33 amplicon by knockout/knockdown and overexpression of the three candidate genes in HER2+/Neu overexpressing cell lines injected into syngeneic mice. Aim 2 utilizes transcriptomic differences between tumors, immunohistochemistry (IHC), and co-immunoprecipitation (Co-IP) to determine signaling network differences between tumors +/- for candidate genes and spatial expression of genes within the tumor. Aim 3 determines what genes in the amplicon are necessary or sufficient to increase metastasis by overexpressing candidate genes alone or in tandem and measuring effects on migration and metastasis. The long-term objectives and broad goals of this hypothesis driven proposal are to provide an increased understanding of why some breast cancers become metastatic, ultimately to improve patient clinical outcomes.