Understanding the role of TWIST1 in colorectal cancer progression and metastasis

了解 TWIST1 在结直肠癌进展和转移中的作用

• 批准号: 10642694
• 负责人: Joseph Charles Sedlak
• 金额: $ 5.52万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10642694
• 关键词: 3-Dimensional; Ablation; Basement membrane; Biological Assay; Biological Models; Biology; CDH1 gene; CRISPR/Cas technology; Cancer Biology; Cancer Etiology; Cancer Model; Cell Adhesion; Cell Communication; Cells; Cessation of life; Circulation; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Colon; Colonoscopy; Colorectal Cancer; Complementary DNA; Complex; Development; Diphtheria; Distant; Engineering; Environment; Epithelial Cells; Epithelium; Evaluation; Extravasation; Future; Genetic Engineering; Genotype; Harvest; Human; Human Engineering; Immunocompromised Host; Immunodeficient Mouse; Immunohistochemistry; In Situ Hybridization; In Vitro; Individual; Invaded; Knock-out; Knowledge; Link; Literature; Liver; Mediating; Mesenchymal; Metastatic Neoplasm to the Liver; Modeling; Mus; Mutation; Neoplasm Metastasis; Oncogenic; Organ; Organoids; Outcome; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Play; Primary Neoplasm; Process; Quality of life; Receptor Cell; Research; Role; Sampling; Site; Stains; TP53 gene; TWIST1 gene; Testing; Therapeutic; Therapeutic Intervention; Tissue Microarray; Tissues; Transplantation; United States; Work; cancer cell; cell motility; colon cancer patients; colorectal cancer metastasis; colorectal cancer progression; differential expression; diphtheria toxin receptor; epithelial to mesenchymal transition; improved; in vivo Model; insight; loss of function; metastatic colorectal; metastatic process; migration; mortality; mouse model; novel therapeutics; organoid transplantation; overexpression; physiologic model; post-transplant; prevent; receptor; transcription factor; transplant model; tumor

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.

项目总结： 虽然几乎所有的结直肠癌(CRC)的死亡都是由转移引起的，但目前还没有预防的药物 或者阻止肿瘤转移。更好地了解大肠癌进展和转移的潜在生物学机制可能 导致下一代旨在阻断转移级联步骤的治疗方法。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袭到 基底膜、向血管系统的迁移、循环、外渗和远处器官的定植。 每一步都严重依赖于不同的细胞间通讯和微环境，而微环境并没有 已在以前的体外和体内模型系统中很好地概括了用于研究结直肠癌转移的研究。然而， CRISPR基因工程三维结直肠癌有机化合物(CRCOs)及结肠镜引导的研究进展 结直肠癌原位移植模型为研究转移提供了更具生理学意义的模型体系。 CRCO精确地利用在大多数人类结直肠癌中发现的致癌突变进行工程设计，例如 APC、Kras和P53基因突变(AKP)。此外，这些经过改造的CRCO被移植到 小鼠的结肠，它们在自然环境中生长并转移到肝脏：最常见的 人类结直肠癌转移的部位。之前使用这些模型系统的研究将Twist1识别为 与原发灶相比，结直肠癌肝转移灶的表达显著上调。Twist1是一家知名的 转录因子参与促进上皮-间充质转化(EMT)。EMT是指上皮细胞 去除细胞黏附，获得更活跃的间充质表型。目前还不清楚Twist1扮演什么角色 在结直肠癌的进展和转移中。目标1将验证Twist1在肝转移瘤中表达上调 用免疫组织化学方法检测AKP CRCOs在小鼠原位移植的原发肿瘤， RNAScope和RT-qPCR。Aim 1还将评估Twist1是否是形成肝转移所必需的 通过CRISPR-Cas9介导的AKP类有机物丢失和白喉毒素受体细胞消除模型。 此外，为了评估Twist1的过度表达是否会增加转移潜能，AKP CRCO将 设计成使用互补DNA过表达Twist1。因此，目标1将确定Twist1是否 是结直肠癌转移所必需的。Twist1在结直肠癌进展和转移中的作用将成为关注的焦点 Aim 2将筛查人类结直肠癌组织微阵列中Twist1和其他EMT的表达 记号笔。每个组织中的Twist1将被量化，并与患者的肿瘤部位、分期、基因和 结果。此外，为了验证Twist1在人结直肠癌转移瘤中的高表达，人CRCO 将移植到免疫缺陷小鼠体内，用免疫组织化学方法检测Twist1的表达， RNAScope和RT-qPCR。了解Twist1‘S在结直肠癌转移中的作用可以为研究肿瘤转移的靶点提供帮助。 首先是转移的特异性治疗，并提高该领域对EMT和转移的理解。