Modeling and Circumventing EMT to Suppress Metastasis

描述(申请人提供)：大多数癌症死亡是由原发上皮性肿瘤的血液转移引起的，但我们对这一过程的了解有限。上皮向间充质转化(EMT)是一种发育调节的细胞命运变化，其在癌症中的异常激活被认为与癌细胞的侵袭性和运动性有关。然而，考虑到研究人类癌症转移的难度，大多数研究都依赖于细胞系和小鼠模型，EMT与人类癌症的相关性还没有得到很好的确立。能够分离和分子分析循环肿瘤细胞(CTC)的新技术，CTC是一种罕见的癌细胞，通过 血流现在提供了一个独特的机会来确定与人类癌症转移有关的机制。我们建议使用CTC的分子分析来验证EMT在人类乳腺癌中的流行程度，使用仔细滴定的EMT体外模型来识别可能构成潜在药物靶点的关键效应因子，并使用CTC定量提供快速读出人类乳腺癌细胞转移潜力的小鼠模型来测试其有效性。我们的方法有三个目标：在目标1中，我们将确定EMT是否是不同组织学亚型乳腺癌的一致特征，以及这些标记物在治疗反应或抵抗期间是否动态演变。这将使用我们开发的RNA原位探针来完成，该探针能够在单个细胞内标记定量的上皮和间质标记。然后将应用RNA测序来识别乳腺循环肿瘤细胞中与EMT相关的转录本，从而提供对相关生物途径的洞察。在目标2中，我们将研究一种可诱导的EMT体外模型，该模型有效地调节表观遗传开关。我们已经生成了转录和染色质免疫沉淀图谱的时间表，将用于识别EMT的候选效应，寻找潜在的可用药靶点。在目标3中，我们将建立一种可靠的小鼠实验来监测乳腺癌细胞的转移能力，从而测试EMT相关表型的潜在抑制因子。小鼠模型中的CTC计数将被用作血管侵袭性的快速和可量化的读数。总之，这些实验旨在结合新的分子和生物工程技术，以解决EMT作为抑制人类癌症转移的治疗靶点的相关性。

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.