Understanding the role of TWIST1 in colorectal cancer progression and metastasis

项目摘要： 尽管几乎所有的结直肠癌（CRC）死亡率都是由于转移，但目前没有药物可以预防 或阻止转移。更好地了解结直肠癌进展和转移的基础生物学， 导致下一代的治疗方法，旨在阻断转移级联反应中的步骤。以前的研究 提供了一个框架，以了解肿瘤转移，通过癌细胞侵入的顺序阶段， 基底膜、迁移到脉管系统、循环、外渗和远处器官的定殖。 每一步都严重依赖于不同的细胞间通信和微环境，而微环境却没有 在以前的体外和体内模型系统中得到了很好的概括，以研究CRC转移。但 CRISPR工程化的3D结肠直肠癌类器官（CRCO）和结肠镜引导的最新发展 原位CRC移植模型为研究转移提供了更生理学的模型系统。 CRCO是用在大多数人CRC中发现的致癌突变精确工程化的，例如 Apc、Kras和P53基因（AKP）突变。此外，这些工程CRCO被移植到 小鼠结肠，它们在其天然环境中生长并转移到肝脏：最常见的 人的CRC转移部位。先前使用这些模型系统的研究将Twist 1确定为 与原发性肿瘤相比，在CRC肝转移中显著上调。Twist 1是一个众所周知的 参与促进上皮-间质转化（EMT）的转录因子。EMT是指上皮细胞 失去它们的细胞粘附并获得更能动的间充质表型。目前还不清楚Twist 1扮演什么角色 CRC进展和转移。目的1将验证与对照组相比，Twist 1在肝转移瘤中上调。 原位移植小鼠中AKP CRCO的原发性肿瘤， RNAscope和RT-qPCR。目的1还将评估Twist 1是否是肝转移形成所必需的 通过CRISPR-Cas9介导的AKP类器官损失和白喉毒素受体细胞消除模型。 此外，为了评估Twist 1的过表达是否增加转移潜能，将使用AKP CRCO。 使用互补DNA进行工程化以过表达Twist 1。因此，Aim 1将确定Twist 1是否 需要CRC转移。TWIST 1在人类结直肠癌进展和转移中的作用将是研究的重点 目标2目的2将筛选人CRC组织微阵列中TWIST 1和其他EMT的表达 标记。每个组织中的TWIST 1将被定量并与患者肿瘤部位、阶段、基因型和肿瘤大小相关。 结果。此外，为了验证TWIST 1在人CRC转移中更高表达， 将其移植到免疫缺陷小鼠中并通过免疫组织化学测定TWIST 1的表达， RNAscope和RT-qPCR。TWIST 1在CRC转移中的作用的这种知识可以阐明肿瘤转移的靶点。 第一个转移特异性治疗，并提高该领域对EMT和转移的理解。

Project Summary: Although almost all colorectal cancer (CRC) mortality is due to metastasis, there are currently no drugs to prevent or halt metastasis. A better understanding of the underlying biology of CRC progression and metastasis could lead to the next generation of therapeutics aimed at blocking steps in the metastatic cascade. Previous research provided a framework to understand tumor metastasis through sequential stages of cancer cell invasion into the basement membrane, migration into vasculature, circulation, extravasation, and colonization of distant organs. Each step is heavily dependent on diverse cell-cell communications and the microenvironment, which has not been well recapitulated in previous in vitro and in vivo model systems to study CRC metastasis. However, the recent development of CRISPR-engineered 3D colorectal cancer organoids (CRCOs) and colonoscopy-guided orthotopic CRC transplantation models have provided a more physiological model system to study metastasis. The CRCOs are precisely engineered with oncogenic mutations found in the majority of human CRC, such as mutations in the Apc, Kras, and P53 genes (AKP). Moreover, these engineered CRCOs are transplanted into the murine colon, where they grow in their native environment and metastasize to the liver: the most common site of CRC metastasis in humans. Previous research using these model systems identified Twist1 as significantly upregulated in CRC liver metastases compared to the primary tumor. Twist1 is a well-known transcription factor involved in promoting epithelial-mesenchymal transition (EMT). EMT is when epithelial cells lose their cell adhesions and acquire a more motile mesenchymal phenotype. It is unclear what role Twist1 plays in CRC progression and metastasis. Aim 1 will verify that Twist1 is upregulated in liver metastases compared to the primary tumor from AKP CRCOs in orthotopically transplanted mice using immunohistochemistry, RNAscope, and RT-qPCR. Aim 1 will also assess whether Twist1 is necessary for liver metastasis formation through CRISPR-Cas9-mediated loss in AKP organoids and a diphtheria toxin receptor cell-elimination model. Moreover, to evaluate if overexpression of Twist1 increases metastatic potential, the AKP CRCO will be engineered to overexpress Twist1 using complementary DNA. Thus, Aim 1 will determine whether Twist1 is required for CRC metastasis. The role of TWIST1 in human CRC progression and metastasis will be the focus of Aim 2. Aim 2 will screen human CRC tissue microarrays for expression of TWIST1 and other EMT markers. TWIST1 in each tissue will be quantified and correlated with patient tumor site, stage, genotype, and outcome. Moreover, to validate TWIST1 is more highly expressed in human CRC metastases, a human CRCO will be transplanted into immunodeficient mice and assayed for expression of TWIST1 by immunohistochemistry, RNAscope, and RT-qPCR. Such knowledge of TWIST1’s role in CRC metastasis can illuminate targets for the first metastasis-specific therapies and improve the field’s understanding of EMT and metastasis.