Defining the Rho-kinase PKN2 as a 1p22 encoded tumour suppressor in colorectal cancer.

肠癌是英国第四大常见癌症，也是导致癌症死亡的第二大原因。预防和治疗这些疾病是一个巨大的未得到满足的临床需求。肠道是我们身体中最具挑战性的器官之一，通过暴露在物理压力、摄入的毒素、肠道细菌和消化过程中，所有这些都会导致组织损伤和炎症。为了保持健康并替换受损的细胞和组织，肠道经历了持续的细胞生长，这为癌症的发展提供了一个理想的环境。为了限制癌症的发展，肠道中的细胞生长受到非常严格的控制。只有一小部分被称为干细胞的细胞具有替代受损组织的能力，而这些细胞最有可能转变为癌细胞。干细胞部分由被称为肿瘤抑制基因的基因控制。正常情况下，这些基因会阻止干细胞生长，除非给它们提供了允许肠道修复的非常具体的指令。在癌症中，这些肿瘤抑制基因发生突变或丢失，导致生长失控，导致肠癌。利用人类癌症和结肠癌小鼠模型的生物信息学数据，我们已经确定了一种全新的肿瘤抑制基因，称为蛋白激酶N2(PKN2)。在人类癌症中，PKN2通常通过多种机制被抑制。例如，在30%的侵袭性结肠癌中，PKN2基因所在的1号染色体称为1p22的区域丢失。此外，在一些结肠癌中存在PKN2的基因突变，这可能表明在癌症的发展中发挥了作用。此外，我们还表明，在从结肠扩散到身体其他器官的癌症样本中，PKN2的表达减少。使用遗传小鼠模型，我们已经发现，特定地移除PKN2基因会导致小鼠变得对结肠癌高度敏感。这提供了有力和直接的证据，证明PKN2是一种肿瘤抑制因子，可以防止结肠癌的发生。在这项授权中，我们希望探索PKN2如何阻止肠癌的发生。初步实验表明，PKN2可能会防止组织损伤和炎症，这是众所周知的在结肠癌发展中发挥重要作用的因素。我们的数据表明，PKN2对于加强肠道衬里细胞之间的连接非常重要，这可以解释为什么PKN2的丢失与肠道损伤和癌症发生的易感性有关。我们将使用我们的PKN 2小鼠模型来展示PKN 2的基因缺失如何增强肠道中的组织损伤和炎症。我们还将使用实验室癌细胞株和被称为有机类化合物的‘迷你肠道’模型来研究正常肠道细胞中由PKN2调控的分子机制。接下来，我们希望在小鼠遗传模型中检查结肠癌。人类结肠癌几乎总是由一种重要的肿瘤抑制基因突变引起的，这种基因被称为结肠腺瘤性息肉病(APC)。我们将用一只APC基因突变的小鼠来培育我们的PKN2基因小鼠模型。这些小鼠会发生自发的肠道肿瘤，我们预计删除PKN2将使肿瘤以更快和更具侵袭性的方式发展。这将帮助我们了解PKN2如何在人类结肠癌中发现的最常见突变驱动的癌症进展中起作用。最后，我们将研究PKN2在人类结肠癌患者发展的迷你肠道培养中的作用。我们将使用CRISPR-Cas9技术删除PKN2，看看这如何改变一组癌症患者衍生的迷你肠道的生长和侵袭性。这将提供关键信息，帮助将我们的发现转化为临床益处。总而言之，这项研究将有助于定义一种令人兴奋的新的肿瘤抑制途径，它可以帮助防止结肠癌，以及潜在的其他癌症的发展。

Bowel cancer is the 4th most common cancer in the UK and the second largest cause of cancer deaths. Preventing and treating these diseases represents a huge unmet clinical need. The bowel is one of the most challenged organs in our body, through exposure to physical stresses, ingested toxins, gut bacteria and digestive processes, all of which can induce tissue damage and inflammation. In order to remain healthy and replace damaged cells and tissue, the bowel undergoes constant cell growth, and this presents an ideal environment for cancers to develop. To limit cancer development, cell growth in the bowel is under very tight control. Only a small group of cells, called stem cells, have the ability to replace damaged tissues, and these cells are the most likely to change into cancer cells. Stem cells are partly controlled through genes called tumour suppressors. Normally, these genes stop stem cells growing unless they are provided with very specific instructions to allow the bowel to repair. In cancers these tumour suppressor genes are mutated or lost, leading to uncontrolled growth, resulting in bowel cancer. Using bioinformatic data from human cancers and a mouse model of colon cancer, we have identified an entirely new tumour suppressor gene, called protein kinase N2 (PKN2). In human cancers, PKN2 is commonly suppressed through a variety of mechanisms. As an example, a region of chromosome 1 called 1p22, where the gene for PKN2 sits, is lost in 30% of aggressive colon cancers. In addition, there are genetic mutations in PKN2 in some colon cancers, which can indicate a role in cancer development. Further, we show that PKN2 is reduced in samples from cancer that has spread from the colon to other organs in the body. Using a genetic mouse model, we have found that specifically removing the PKN2 gene results in mice becoming highly susceptible to colon cancer. This provides strong and direct evidence that PKN2 is a tumour suppressor, which can prevent colon cancer from developing.In this grant we wish to explore how PKN2 stops bowel cancers from developing. Preliminary experiments suggest that PKN2 may prevent tissue damage and inflammation, which are known to play important roles in cancer development in the colon. Our data suggest that PKN2 is important for strengthening the connections between the cells lining the intestine, which could explain why loss of PKN2 is associated with a greater susceptibility to intestinal injury and cancer development. We will use our PKN2 mouse model to show how genetic deletion of PKN2 enhances tissue damage and inflammation in the bowel. We will also use laboratory cancer cell lines and 'mini-gut' models called organoids to examine the molecular machinery regulated by PKN2 in normal intestinal cells. Next, we wish to examine colon cancer in a genetic mouse model. Human colon cancers are almost always initiated by mutation of an important tumour suppressor gene called adenomatous polyposis coli (APC). We will breed our PKN2 genetic mouse model with a mouse in which the APC gene is mutated. These mice develop spontaneous bowel tumours and we expect that deleting PKN2 will make tumours develop more rapidly and in a more aggressive way. This will help us understand how PKN2 contributes to cancer progression driven by the most common mutation found in human colon cancer. Finally, we will examine the role of PKN2 in mini-gut cultures developed from human colon cancer patients. We will delete PKN2 using CRISPR-Cas9 technology and see how this changes the growth, and invasiveness of a panel of cancer-patient derived mini-guts. This will provide crucial information to help translate our findings to clinical benefit. Together, this research will help define an exciting new tumour suppressor pathway, which can help prevent colon cancer, and potentially other cancers, from developing.