Modeling and Circumventing EMT to Suppress Metastasis

描述（由申请人提供）：大多数癌症死亡是由原发性上皮肿瘤的血源性转移引起的，但我们对这一过程的理解有限。上皮-间充质转化（EMT）是细胞命运的基本发育调节变化，其在癌症中的异常激活被认为有助于癌细胞的侵袭性和运动性。然而，鉴于研究人类癌症转移的困难，大多数研究都依赖于细胞系和小鼠模型，EMT在人类癌症中的相关性尚未得到很好的建立。新技术使循环肿瘤细胞（CTC）的分离和分子分析成为可能， 血液，现在提供了一个独特的机会，以确定机制参与人类癌症转移。我们提出了一种CTC的分子分析，以验证EMT在人类乳腺癌中的患病率，使用EMT的仔细滴定的体外模型，以确定可能构成潜在药物靶点的关键效应子，并使用小鼠模型测试其有效性，其中CTC定量提供了人类乳腺癌细胞转移潜力的快速读数。我们的方法有三个目标：在目标1中，我们将确定EMT是否是乳腺癌不同组织学亚型的一致特征，以及这些标记物是否在对治疗的反应或抗性期间动态演变。这将使用我们开发的RNA原位探针来完成，该探针能够对单个细胞内的上皮和间充质标记物进行定量评分。然后将应用RNA测序来鉴定乳腺循环肿瘤细胞内的EMT相关转录物，从而提供对相关生物学途径的深入了解。在目标2中，我们将研究一种诱导型EMT体外模型，它有效地介导了一个受调节的表观遗传开关。我们已经生成了转录和染色质免疫沉淀谱的时间线，这将用于识别EMT的候选效应子，寻找潜在的可药物靶点。在目标3中，我们将建立一个强大的小鼠试验来监测乳腺癌细胞转移的能力，从而测试EMT相关表型的潜在抑制因子。小鼠模型中的CTC计数将用作血管侵袭性的快速和可定量读数。总之，这些实验旨在联合收割机新的分子和生物工程技术，以解决相关的EMT作为一个治疗目标，在抑制人类癌症转移。

DESCRIPTION (provided by applicant): The majority of cancer deaths are caused by blood-borne metastasis from a primary epithelial tumor, but our understanding of this process is limited. Epithelial-to-Mesenchymal Transition (EMT) is a fundamental developmentally regulated change in cell fate, whose aberrant activation in cancer has been proposed as contributing to the invasiveness and motility of cancer cells. However, given the difficulty in studying human cancer metastasis, most studies have relied on cell line and mouse models, and the relevance of EMT in human cancer is not well established. New technologies enabling the isolation and molecular analysis of circulating tumor cells (CTCs), rare cancer cells that are in transit through the bloodstream, now provide a unique opportunity to define mechanisms involved in human cancer metastasis. We propose a molecular analysis of CTCs to validate the prevalence of EMT in human breast cancer, use a carefully titrated in vitro model of EMT to identify key effectors that could constitute potential drug targets, and test their effectiveness using a mouse model in which CTC quantitation provides a rapid readout for the metastatic potential of human breast cancer cells. Our approach has three aims: in Aim 1, we will determine whether EMT is a consistent feature of different histological subtypes of breast cancer and whether these markers evolve dynamically during response or resistance to therapy. This will be accomplished using RNA-in-situ probes that we have developed, capable of scoring quantitative epithelial and mesenchymal markers within individual cells. RNA sequencing will then be applied to identify EMT associated transcripts within breast circulating tumor cells, thus providing insight into the relevant biological pathways. In Aim 2, we will study an inducible in vitro model of EMT, which effectively mediates a regulated epigenetic switch. We have generated timelines of both transcriptionaland chromatin immuno-precipitation profiles, which will be used to identify candidate effectors of EMT, seeking potential drugable targets. In Aim 3, we will establish a robust mouse assay to monitor the ability of breast cancer cells to metastasize and thus test potential suppressors of EMT-related phenotypes. CTC enumeration in the mouse model will be used as a rapid and quantifiable readout for vascular invasiveness. Together, these experiments aim to combine novel molecular and bioengineering technologies to address the relevance of EMT as a therapeutic target in suppressing human cancer metastasis.