The generation and maintenance of hybrid epithelial-mesenchymal (E-M) cell phenotypes.

胰腺癌是一种致命的疾病，因为癌细胞在全身迅速扩散，导致转移，也因为对治疗的抗药性，即使在表面上成功治疗后，也会导致复发。扩散到全身的癌细胞被称为循环肿瘤细胞(CTCs)。它们的作用类似于癌症干细胞(CSCs)，因为它们可以产生在其他器官或复发病例中在胰腺形成肿瘤所需的所有癌细胞。CTCs和CSCs在胚胎中有许多共同的细胞属性，这表明它们受相同的蛋白质调控。此外，就像某些细胞在胚胎中成簇迁移一样，它们具有混合的细胞形状，具有两种主要细胞类型-上皮细胞和间充质细胞的混合特征，因此它们被称为具有混合上皮-间充质(E-M)表型的细胞。这就是为什么分析这些CTC和CSCs可以更好地了解患者的生存情况，它们具有开发新疗法的潜力。但目前我们正处于利用从CTCs和CSCs获得的任何信息转化到临床的最早阶段。最近，来自胰腺癌患者的CTCs被发现表达一种名为Wilms‘s Tumour Protein(WT1)的蛋白质，这是相当有趣的。首先，我们早期的工作表明，它调节胚胎中具有混合E-M表型的细胞。尽管已知在成人中只调节少数几种细胞类型，但它们都表现出混合的E-M表型。最后，我们用小鼠胰腺癌模型进行的分析支持了WT1可以影响肿瘤中上皮细胞和间充质细胞数量的观点，可能是通过控制产生多少具有E-M混合表型的细胞。因此，在这个项目中，我们将测试WT1是否对混合E-M表型以及随后胰腺癌中CSCs和CTCs的产生起重要作用。我们将使用被证明能够真实地概括正常胰腺癌进展的人胰腺细胞模型来模拟胰腺癌。它也是有用的，因为我们可以使用它来首先研究在细胞中强制表达或丢失WT1的影响，以及这如何通过细胞培养、三维“培养皿中的微型胰腺”或通过小鼠移植来影响杂交E-M表型的产生。其次，我们将找出WT1是如何制造和保持混合E-M表型的细胞的。为了实现这一点，我们将使用我们之前在实验室中使用的协议，然后是下一代测序(NGS)，确定WT1结合和调节的DNA和RNA。我们还将确定染色质修饰物的靶点，我们证明了它对胚胎中WT1的功能是重要的。修饰物和WT1不仅通过结合DNA或RNA，而且还通过影响染色体和细胞核的表观遗传环境来影响基因的表达方式。改变基因的表观遗传背景对于细胞改变命运和成为干细胞样细胞很重要，WT1也可能影响这些因素。最终，我们将有一个全面的想法，哪些基因和表观遗传标记受WT1调控，特别是围绕那些影响杂交E-M表型并产生CSCs或CTCs的基因。与我们的合作者合作，我们将优先考虑导致和维持WT1下游混合E-M表型的最有可能的基因和相关的表观遗传机制。在项目结束时，我们将测试在我们的人类胰腺癌模型中是否存在这种情况。长期目标是充分理解这些发现和产生的数据的翻译潜力。在这个项目中，我们的目标是提供我们从混合E-M表型的WT1调控中学到的东西，作为一个范例来理解其他胚胎调控因子如何在癌症组织中错误表达，重新唤醒休眠的胚胎过程，促进转移、耐药和复发，并导致致命后果。

Pancreatic cancer is a fatal disease because of the rapid spread of cancer cells throughout the body leading to metastasis and also because of resistance to therapies which leads to relapse even after apparent successful treatment. The cancer cells spreading around the body are called circulating tumour cells (CTCs). And they act like cancer stem cells (CSCs) because they can produce all cancer cells necessary to form tumours in other organs or in the pancreas in relapse cases. CTCs and CSCs share many attributes of cells in the embryo which indicate that they are regulated by the same proteins. Also, like certain cells that migrate in the embryo in clusters, they have hybrid cell shapes with mixed traits from two major cell types, epithelial and mesenchymal cells, hence they are called cells with hybrid epithelial-mesenchymal (E-M) phenotypes.That is why analysing these CTCs and CSCs can give us a better picture on the survival of patients, and they hold the potential for developing novel therapies. But at present we are at the earliest stages of exploiting any information we gain from CTCs and CSCs to translate in the clinic.Recently, CTCs from pancreatic cancer patients were found to express a protein called the Wilms' tumour protein (WT1) which is quite interesting. For a start, our earlier work suggest that it regulates cells with hybrid E-M phenotypes in the embryo. And although it is known to regulate only few cell types in the adult, they all exhibit hybrid E-M phenotypes. And finally our analysis using mouse models for pancreatic cancer supported the notion that WT1 can affect the amount of epithelial and mesenchymal cells in the tumours, probably by controlling how many cells with hybrid E-M phenotypes are made. So in this project, we will test whether WT1 is important for hybrid E-M phenotypes and consequently the generation of CSCs and CTCs in pancreatic cancer.We will model pancreatic cancer using a human pancreatic cell model that was shown to be faithful in recapitulating normal pancreatic cancer progression. It is also useful because we can use it to study first the impact of forcibly expressing or losing WT1 in cells and how this affect the production of hybrid E-M phenotypes using: cell culture, three dimensional "mini pancreas in a dish" or by transplantation in mice. Second, we will find out how WT1 makes and keeps the cells in the hybrid E-M phenotypes. To achieve this, we will identify the DNAs and RNAs that WT1 binds and regulates using protocols that we previously used in our lab followed by next generation sequencing (NGS). We will also identify targets of a chromatin modifier that we showed to be important for WT1 function in the embryo. The modifier and WT1 affect how genes are expressed not only by binding DNA or RNA but also by affecting the epigenetic environment in the chromosome and in the nucleus. Changing the epigenetic context of genes is important for cells to change fate and become stem-like-cells and WT1 may affect these too.At the end we will have a comprehensive idea of which genes and epigenetic marks are regulated by WT1, and especially around those genes that affect the hybrid E-M phenotypes and give rise to CSCs or CTCs. Working with our collaborators, we will prioritise the most likely genes and associated epigenetic mechanisms that lead and maintain the hybrid E-M phenotypes downstream of WT1. At the end of the project, we will test whether this is the case in our human pancreatic cancer model. The longer-term objective being to fully appreciate the translational potential of the findings and the data generated.In this project we aim to provide what we learn from WT1 regulation of the hybrid E-M phenotype as a paradigm to understanding how other embryonic regulators mis-expressed in cancer tissues reawaken dormant embryonic processes to promote metastasis, drug resistance and relapse with fatal consequences.