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，即使在明显成功的治疗后也会导致复发。在体内扩散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的胰腺中形成肿瘤所需的所有癌细胞。ctc和CSCs具有胚胎细胞的许多特性，这表明它们受相同的蛋白质调节。此外，与胚胎中成群迁移的某些细胞一样，它们具有混合两种主要细胞类型（上皮细胞和间充质细胞）的混合特征的杂交细胞形状，因此它们被称为上皮-间充质（E-M）杂交表型细胞。这就是为什么分析这些ctc和CSCs可以让我们更好地了解患者的生存情况，并且它们具有开发新疗法的潜力。但目前，我们正处于利用从ctc和CSCs获得的任何信息并将其应用于临床的早期阶段。最近，来自胰腺癌患者的CTCs被发现表达一种叫做Wilms肿瘤蛋白（WT1）的蛋白质，这很有趣。首先，我们早期的工作表明，它调节胚胎中具有杂交E-M表型的细胞。虽然已知它只调节成人体内的几种细胞类型，但它们都表现出杂交的E-M表型。最后，我们对胰腺癌小鼠模型的分析支持了这样一种观点，即WT1可以影响肿瘤中上皮细胞和间充质细胞的数量，可能是通过控制混合E-M表型细胞的数量来实现的。因此，在本项目中，我们将测试WT1是否对混合型E-M表型以及胰腺癌中CSCs和ctc的产生很重要。我们将使用人类胰腺细胞模型来模拟胰腺癌，该模型被证明是忠实于正常胰腺癌进展的再现。它也很有用，因为我们可以用它来研究细胞中强行表达或失去WT1的影响，以及这如何影响杂交E-M表型的产生，使用细胞培养、三维“迷你胰腺”或通过小鼠移植。其次，我们将发现WT1是如何使细胞保持E-M杂交表型的。为了实现这一目标，我们将使用我们之前在实验室中使用的协议识别WT1结合和调节的dna和rna，然后进行下一代测序（NGS）。我们还将确定一个染色质修饰子的靶标，我们已经证明该修饰子对胚胎中WT1的功能很重要。修饰子和WT1不仅通过结合DNA或RNA来影响基因的表达，还通过影响染色体和细胞核内的表观遗传环境来影响基因的表达。改变基因的表观遗传环境对于细胞改变命运并成为干细胞很重要，而WT1也可能影响这些。最后，我们将对哪些基因和表观遗传标记受WT1调控有一个全面的了解，特别是那些影响杂交E-M表型并产生CSCs或ctc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下游杂交E-M表型的最可能的基因和相关的表观遗传机制。在项目结束时，我们将在我们的人类胰腺癌模型中测试情况是否如此。长期目标是充分认识研究结果和所产生的数据的转化潜力。在这个项目中，我们的目标是提供我们从杂交E-M表型的WT1调控中获得的知识，作为一个范例，以了解癌症组织中错误表达的其他胚胎调控因子如何重新唤醒休眠的胚胎过程，从而促进转移、耐药和复发，并带来致命的后果。

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.