Identifying and manipulating molecular mechanisms controlling cancer stem cell metastatic potential in a human oral cancer model.

肿瘤转移是导致大多数癌症死亡的原因，它导致了远处器官中的继发性肿瘤。癌症干细胞驱动肿瘤转移。为了做到这一点，他们经历了上皮-间充质转化(EMT)，采用了一种迁移性间充质表型，这种表型可以从原发肿瘤扩散并迁移到次要部位。一旦到达继发部位，它们就会经历间充质-上皮转化(MET)，重新获得增殖性上皮表型，从而形成继发性肿瘤。这一过程被称为转移级联。要做到这一点，原发肿瘤中的上皮癌干细胞必须具备将EMT转化为间充质表型的能力，而间质表型又保留了在次级部位接受MET的能力-这种转换表型的能力被称为‘可塑性’。从治疗上控制上皮癌干细胞的可塑性以防止转移级联反应的能力将从根本上阻止肿瘤的转移。我们假设，在原发肿瘤的上皮性癌症干细胞群体中存在变异--只有这些癌症干细胞中的一些具有启动转移级联所需的可塑性。我们进一步假设，有可能在治疗上操纵上皮癌干细胞的可塑性，从而防止转移过渡。我们建议在使用具有良好特性的EMT诱导剂TGFbeta治疗的人类口腔癌模型中测试这些假设。新的单细胞方法是解剖细胞群体内变异的强大工具，并提供了一种手段来探索对TGFbeta具有不同反应的离散上皮癌干细胞亚群的存在。我们将使用单细胞RNAseq来鉴定不同的上皮性癌症干细胞亚群，并推断这些亚群经历EMT的能力转变为间充质表型，而间质表型又保留了接受MET的能力。我们将使用这些信息来识别控制这些癌症干细胞亚群的候选分子通路，并使用CRISPR筛选方法结合单细胞RNAseq来灭活这些通路，以确定其失活可以防止转移级联启动的分子靶点。在选择靶点时，我们将重点放在关键路径内的可用药节点。然后，我们将使用人类病理标本来测试这些靶点在转移中的重要性，以及在我们实验室开发的新的工程化转移模型中，灭活这些靶点是否可以防止转移。口腔癌是全球十大癌症之一，每年有超过30万例，而且全球和英国的发病率都在上升(在英国，发病率在过去十年中增加了23%)。口腔癌是一种经常转移的致命疾病，是预后不良的唯一最重要的预测因素。这项研究项目将产生控制口腔癌转移的分子途径的重要知识，鉴于EMT在转移中的核心作用，这可能适用于其他类型的肿瘤。从这项研究中出现的靶点将被用于开发新的靶向治疗以防止转移。

Tumour metastasis, which seeds secondary tumours in distant organs, causes the majority of cancer deaths. Cancer stem cells drive tumour metastasis. To do this, they undergo epithelial-mesenchymal transition (EMT) to adopt a migratory mesenchymal phenotype that can disseminate from the primary tumour and migrate to secondary sites. Upon reaching secondary sites, they then undergo mesenchymal-epithelial transition (MET) to re-gain a proliferative epithelial phenotype that can form a secondary tumour. This process is known as the metastatic cascade. For this to occur, epithelial cancer stem cells within the primary tumour must possess the ability to undergo EMT into a mesenchymal phenotype that in turn retains the ability to undergo MET at a secondary site - this ability to switch phenotype is termed 'plasticity'. The ability to therapeutically control the plasticity of epithelial cancer stem cells to prevent initiation of the metastatic cascade would stop tumour metastasis at its root. We hypothesise that there is variation within the epithelial cancer stem cell population in the primary tumour - only some of these cancer stem cells possess the plasticity required to initiate the metastatic cascade. We further hypothesise that it is possible to therapeutically manipulate the plasticity of epithelial cancer stem cells, and thereby prevent metastatic transitions. We propose to test these hypotheses in a human oral cancer model treated with the well-characterised EMT-inducer TGFbeta. New single cell approaches are a powerful tool for dissecting variation within cell populations, and provide a means to probe the existence of discrete epithelial cancer stem cell sub-populations with differing responses to TGFbeta. We will use single cell RNAseq to identify distinct epithelial cancer stem cell sub-populations and infer the ability of these sub-populations to undergo EMT into a mesenchymal phenotype that in turn retains the ability to undergo MET. We will use this information to identify candidate molecular pathways controlling these cancer stem cell sub-populations, and inactivate these pathways using a CRISPR screening methodology combined with single cell RNAseq in order to determine molecular targets whose inactivation can prevent initiation of the metastatic cascade. In selecting targets, we will focus on druggable nodes within key pathways. We will then test the importance of these targets in metastasis using human pathological specimens, and whether inactivating these targets can prevent metastasis in new engineered metastasis models that we have developed in our lab. Oral cancer is one of the top ten cancers worldwide, with over 300,000 cases annually, and incidence is increasing both worldwide and in the UK (in the UK, incidence has increased by 23% over the past decade). Oral cancer is a deadly disease with frequent metastatic spread, which is the single most important predictor of poor outcome. This research project will generate important knowledge of the molecular pathways controlling metastasis in oral cancer and, given the central role of EMT in metastasis, this may be generalizable to other tumour types. Targets emerging from this study will be taken forward for the development of new targeted therapies to prevent metastasis.