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

摘要 胰腺癌（PC）是一种高转移性和治疗抗性的恶性肿瘤，患者表现为 诊断时有局部和远处转移。与其他上皮癌一样，PC进展是 以异常的粘蛋白过度表达为特征。我们之前的研究表明，虽然MUC 16是 在正常胰腺中检测不到，MUC 16的表达随着胰腺癌的发生而逐渐增加。 PanIN病变、肿瘤和转移的分级。我们还证明了MUC 16和MUC 16-Cter在细胞内的表达， 在胰腺癌转移中起关键作用。然而，MUC 16在功能上和机制上参与 胰腺癌转移仍然知之甚少。MUC 16是一种多结构域蛋白， 在前列腺癌的转移中起多方面的作用。在我们的初步研究中，PC细胞中MUC 16的沉默导致了 细胞生长和迁移沿着减少，同时EMT标志物发生改变。因此，我们假设MUC 16是 与PC转移相关，其部分由MUC 16-Cter结构域介导。为了验证假设， 为了实现上述目标，提出了三个具体目标。第一个目标将评价 MUC 16在PC细胞转移扩散期间介导细胞间相互作用。为此，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，研究MUC 16的细胞和分子功能。这些 研究将提供MUC 16在促进肿瘤细胞与内皮细胞相互作用中的作用的见解。 细胞、血小板和白细胞。目标2中的研究将描述 MUC 16-Cter介导的PC转移并鉴定MUC 16相互作用伴侣。由于MUC 16-Cter富集 在细胞核中的染色质结合部分中，将使用ChIP评估其对整体基因表达的影响。 Seq分析。此外，还研究了影响N-糖基化、泛素化和 磷酸化将根据其功能后果来处理。第三个目标将调查 使用Muc 16-/-和 MUC 16 Cter转基因小鼠。在这种情况下，重要的是确定MUC 16单独和在肿瘤细胞中的作用。 与致癌K-ras和p53的结合，在PC发展过程中。为了验证我们在体外发现的 将MUC 16在PC中进行表达，我们将获得胰腺特异性表达的MUC 16 Cter转基因小鼠。此外，根据 关于MUC 16-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，拟议 研究将使我们能够确定MUC 16及其Cter促进转移的分子机制， 有助于PC的侵略性。这些新的见解的潜在机制的PC转移 结合新的数据，试剂和模型将为靶向转移性PC提供新的途径， 未来

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.