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: 10203862
• 负责人: Surinder K. Batra
• 金额: $ 31.53万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203862
• 关键词: Address; Affect; Affinity Chromatography; Animals; Autopsy; Binding; Blood Platelets; CA-125 Antigen; Carcinoma; Cell Communication; Cell Nucleus; Cells; ChIP-seq; Chromatin; Cleaved cell; 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）是一种高度转移性和耐药性恶性肿瘤，患者出现 诊断时的局部和遥远转移。像其他上皮癌一样，PC的进展为 以异常的粘蛋白过表达为特征。我们先前的研究表明，虽然MUC16是 在正常胰腺中无法检测到的MUC16表达逐渐增加 在Panin病变，肿瘤和转移等级中。我们还证明了MUC16和MUC16-CETER PLAIG 在胰腺癌转移中的关键作用。但是，MUC16在功能和机理参与中 胰腺癌转移仍然知之甚少。 MUC16是一种多域蛋白，可以潜在地 在PC的转移中扮演多方面的角色。在我们的初步研究中，PC细胞中MUC16的沉默导致 细胞生长和迁移以及EMT标记的改变。因此，我们假设MUC16是 与PC转移相关，该转移部分是由MUC16-CETER结构域介导的。检验假设和 达到理性目标，提出了三个具体目标。第一个目标将评估 MUC16在PC细胞转移性传播过程中介导细胞间相互作用中的MUC16。在此目标中，我们将分析 通过分析的细胞细胞和细胞 - 细胞基质相互作用，MUC16的细胞和分子功能。这些 研究将提供有关MUC16在促进肿瘤细胞与内皮相互作用方面的作用的见解 转移过程中的细胞，血小板和白细胞。 AIM 2中的研究将描述分子机制 MUC16-CETER介导的PC转移并识别MUC16相互作用伴侣。由于MUC16-CETER富含 在核中染色质结合的部分中，将使用芯片评估其对全局基因表达的影响 SEQ分析。此外，影响N-糖基化，泛素化和 磷酸化将根据其功能后果来解决。第三目的将调查 MUC16与使用MUC16 - / - 和PC转移中其他定义的致癌突变的合作作用 MUC16CTER转基因小鼠。在这种情况下，重要的是要单独和在 在PC开发过程中，与致癌的K-RAS和p53结合使用。验证我们对角色的体外发现 PC中的MUC16的MUC16，我们将以胰腺比表达式生成MUC16CTER转基因。此外，取决于 在MUC16-CETER N-糖基化，泛素化和/或磷酸化突变体的表型上，我们将 产生突变特异性转基因，以了解这种突变的体内相关性。总体而言 研究将使我们能够定义MUC16及其最喜欢的转移的分子机制 对PC侵略性的贡献。这些对PC转移基本机制的新颖见解 结合新数据，试剂和模型将为靶向转移PC的新途径 未来。