Project 1: The Role and Mechanism(s) of MUC16 and MUC16-Cter in Potentiating PC Metastasis

项目1：MUC16和MUC16-Cter在增强PC转移中的作用和机制

• 批准号: 10413938
• 负责人: Surinder K. Batra
• 金额: $ 30.89万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-08 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10413938
• 关键词: Address; Affect; Affinity Chromatography; Animals; Autopsy; Binding; Blood Platelets; CA-125 Antigen; Carcinoma; Cell Communication; Cell Nucleus; Cells; ChIP-seq; Chromatin; Cytoplasmic Tail; DNA Binding Domain; Data; Development; Diagnosis; Disease; Distant Metastasis; Down-Regulation; Ectopic Expression; Endothelial Cells; Exhibits; Extracellular Matrix; Future; Galactose Binding Lectin; Glycoproteins; Golgi Apparatus; Human; In Vitro; KRAS oncogenesis; KRAS2 gene; KRASG12D; Lesion; Leukocytes; Light; Malignant Neoplasms; Malignant neoplasm of pancreas; Mass Spectrum Analysis; Mediating; Membrane; Mitochondria; Modeling; Molecular; Molecular Weight; Mucins; Mutation; Neoplasm Metastasis; Nuclear; Oncogenic; Organoids; PTK2 gene; Pancreas; Pancreatic Ductal Adenocarcinoma; Pancreatic Intraepithelial Neoplasia; Pathway interactions; Patients; Phenotype; Phosphorylation; Play; Point Mutation; Post-Translational Protein Processing; Prognosis; Property; Proteins; Reagent; Role; Sampling; TP53 gene; Tertiary Protein Structure; Testing; The Cancer Genome Atlas; Time; Transgenic Mice; Transgenic Organisms; Tumor Tissue; Up-Regulation; base; cell growth; cell motility; epithelial to mesenchymal transition; functional outcomes; glycosylation; in silico; in vivo; insight; mesothelin; mouse model; mutant; neoplastic cell; novel; overexpression; pancreatic cancer cells; pancreatic ductal adenocarcinoma model; premalignant; programs; therapy resistant; tool; transcriptome; tumor; tumor progression; tumorigenic

Abstract Pancreatic cancer (PC) is a highly metastatic and therapy-resistant malignancy with patients presenting with local and distant metastases at the time of diagnosis. Like other epithelial cancers, PC progression is characterized with aberrant mucin overexpression. Our previous study indicated that while MUC16 was undetectable in the normal pancreas, there was a progressive increase in MUC16 expression with the increase in grade of PanIN lesions, tumor and metastasis. We also demonstrated that MUC16 and MUC16-Cter play critical roles in metastasis of pancreatic cancer. However, functional and mechanistic involvement of MUC16 in pancreatic cancer metastasis remains poorly understood. MUC16 is a multi-domain protein that can potentially play multifaceted role in metastasis of PC. In our preliminary studies, silencing of MUC16 in PC cells resulted in reduced cell growth and migration along with alterations in EMT markers. We thus hypothesize that MUC16 is associated with PC metastasis, which, in part, is mediated by MUC16-Cter domain. To test the hypothesis and achieve the aforementioned objectives, three specific aims are proposed. First aim will evaluate the role of MUC16 in mediating cell-to-cell interactions during metastatic spread of PC cells. In this aim, we will analyze the cellular and molecular functions of MUC16 by analyzing cell-cell and cell-extracellular matrix interactions. These studies will provide insights into the role of MUC16 in facilitating the interaction of tumor cells with the endothelial cells, platelets and leukocytes during metastasis. Studies in Aim 2 will delineate the molecular mechanism(s) of MUC16-Cter-mediated PC metastasis and identify MUC16 interacting partners. Since MUC16-Cter is enriched in the chromatin bound fraction in the nucleus, its effect on global gene expression will be evaluated using ChIP- Seq analyses. Further, the effect of various point mutations affecting N-glycosylation, ubiquitylation and phosphorylation will be addressed in the light of its functional consequences. Third aim will investigate the cooperative action of MUC16 with other defined oncogenic mutations in the metastasis of PC using Muc16–/–and MUC16Cter transgenic mice. In this context, it is important to determine the role of MUC16, alone and in combination with oncogenic K-ras and p53, during PC development. To validate our in vitro findings of the role of MUC16 in PC, we will generate MUC16Cter transgenic with pancreas specific expression. Further, depending on the phenotypes of MUC16-Cter N-glycosylation, ubiquitylation and/or phosphorylation mutants, we will generate mutant specific transgenic to understand the in vivo relevance of such mutations. Overall the proposed studies will allow us to define the molecular mechanisms by which MUC16 and its Cter facilitate metastasis and contribute to the aggressiveness of PC. These novel insights into the underlying mechanisms of PC metastasis combined with the new data, reagents and models will provide novel avenues for targeting metastatic PC in future.

抽象的 胰腺癌（PC）是一种高度转移且难治性的恶性肿瘤，患者表现为 诊断时的局部和远处转移。与其他上皮癌一样，PC 的进展是 以异常粘蛋白过度表达为特征。我们之前的研究表明，虽然 MUC16 在正常胰腺中无法检测到，MUC16 表达随着胰岛细胞增多而逐渐增加。 PanIN 病变、肿瘤和转移的分级。我们还证明了 MUC16 和 MUC16-Cter 可以发挥作用 在胰腺癌转移中发挥重要作用。然而，MUC16 的功能和机制参与 胰腺癌的转移仍知之甚少。 MUC16 是一种多结构域蛋白，有可能 在PC转移中发挥多方面的作用。在我们的初步研究中，PC 细胞中 MUC16 的沉默导致 细胞生长和迁移减少以及 EMT 标记的改变。因此我们假设 MUC16 是 与 PC 转移相关，部分由 MUC16-Cter 结构域介导。为了检验假设并 为实现上述目标，提出了三个具体目标。第一个目标将评估的作用 MUC16 在 PC 细胞转移扩散过程中介导细胞间相互作用。为了这个目标，我们将分析 通过分析细胞-细胞和细胞-细胞外基质相互作用来了解 MUC16 的细胞和分子功能。这些 研究将深入了解 MUC16 在促进肿瘤细胞与内皮细胞相互作用中的作用 转移过程中的细胞、血小板和白细胞。目标 2 的研究将描述其分子机制 MUC16-Cter 介导的 PC 转移并鉴定 MUC16 相互作用伙伴。由于MUC16-Cter被富集 在细胞核中染色质结合部分中，其对整体基因表达的影响将使用 ChIP-进行评估 序列分析。此外，各种点突变影响N-糖基化、泛素化和 将根据其功能后果来讨论磷酸化。第三个目标将调查 使用 Muc16–/– 和 MUC16 与其他确定的致癌突变在 PC 转移中的协同作用 MUC16Cter转基因小鼠。在这种情况下，确定 MUC16 单独和在其中的作用非常重要。 在 PC 开发过程中与致癌 K-ras 和 p53 结合。验证我们的体外研究结果 在PC中导入MUC16，我们将产生具有胰腺特异性表达的MUC16Cter转基因。此外，取决于 关于MUC16-Cter N-糖基化、泛素化和/或磷酸化突变体的表型，我们将 产生突变体特异性转基因以了解此类突变的体内相关性。总体而言，建议 研究将使我们能够定义 MUC16 及其 Cter 促进转移和 有助于PC的攻击性。这些对 PC 转移的潜在机制的新见解 结合新的数据、试剂和模型将为靶向转移性 PC 提供新途径 未来。