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

摘要 胰腺癌(PC)是一种高度转移性和治疗耐药的恶性肿瘤，患者表现为 确诊时的局部和远处转移。像其他上皮性癌症一样，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MUC16是 在正常胰腺中检测不到MUC16的表达，随着MUC16表达的增加，MUC16的表达逐渐增加 在Panin病变、肿瘤和转移的分级上。我们还演示了MUC16和MUC16-CTER 在胰腺癌转移中的关键作用。然而，MUC16在功能和机制上的参与 胰腺癌的转移仍然知之甚少。MUC16是一种多结构域蛋白，可能 在PC的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 随着EMT标志物的改变，细胞生长和迁移减少。因此，我们假设MUC16是 与PC转移有关，这部分是由MUC16-CTER结构域介导的。来检验这一假设并 为了实现上述目标，提出了三个具体目标。First Aim将评估 MUC16在PC细胞转移扩散过程中的细胞间相互作用。在这个目标中，我们将分析 通过分析细胞-细胞和细胞-细胞外基质的相互作用，研究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的研究将描绘出的分子机制(S) MUC16-Cter介导的PC转移，并确定MUC16相互作用的伙伴。由于MUC16-CTER被浓缩 在核染色质结合部分，它对全球基因表达的影响将使用芯片- SEQ分析。此外，各种点突变对N-糖基化、泛素化和 将根据其功能后果来处理磷酸化问题。第三个目标将调查 利用Muc16-/-和Muc16-/-和MUC16与其他已定义的癌基因突变在PC转移中的协同作用 MUC16Cter转基因小鼠。在这方面，重要的是确定MUC16单独和在 与致癌的K-ras和P53结合，在PC发育过程中。以验证我们的体外发现的作用 MUC16在PC中的表达，我们将获得胰腺特异表达的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 产生突变特异性转基因，以了解这些突变在体内的相关性。整体而言，建议的 研究将使我们能够确定MUC16及其Cter促进转移和 为PC的进攻性做出贡献。这些对PC转移潜在机制的新见解 结合新的数据，试剂和模型将为靶向转移的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.