Diversificiation of MUC1 Function by Alternate Splicing in Pancreatic Cancer

描述（由申请人提供）：本申请提出的实验将探索MUC1（一种细胞表面相关的粘蛋白）的可选剪接变体在胰腺腺癌中促进MUC1功能多样化的分子机制。先前的研究已经证实，MUC1在各种腺癌中过表达和差异糖基化，并且这种过表达与侵袭性胰腺癌有关。大多数研究都集中在包含VNTR和所有7个外显子的全长MUC1异构体上，但MUC1的选择性剪接产生至少26种异构体，其中几种形式与卵巢癌，宫颈癌和乳腺癌的恶性状态有关。具体来说，剪接变体A、D、X、Y和Z在卵巢恶性肿瘤中的表达频率高于良性肿瘤。一项关注MUC1在乳腺癌中表达的有趣研究表明，MUC1/SEC和MUC1/Y亚型共表达，MUC1的分泌形式MUC1/SEC与MUC1/Y相互作用，诱导细胞质尾部磷酸化状态的变化并改变细胞形态。MUC1细胞质尾部的磷酸化状态（MUC1. ct）最近被证明在细胞信号传导中很重要，并且对细胞外环境有反应。MUC1。CT是已知的GSK3¿、c-Src、EGFR、PDGFR¿、c-Met和HGF的底物，并在Wnt信号传导中发挥作用。MUC1的调控。CT磷酸化和由此产生的信号传导尚不清楚，MUC1异构体的改变，包括MUC1 VNTR的存在或缺失和/或细胞表面存在的特定MUC1剪接变体所特有的序列的包含，可能在定义MUC1如何通过其细胞质尾部发出信号以响应其细胞外环境方面发挥重要作用。我们最初的工作重点是MUC1选择性剪接，开发了一种剪接敏感寡核苷酸阵列来分析异构体表达，并显示在中度分化的高度转移的胰腺细胞系中，3种不同剪接变体的表达增加。本应用程序中提出的实验将深入了解胰腺癌中MUC1剪接变异体的表达，并将寻求特定异构体表达与疾病侵袭性的关联。我们还将通过分析MUC1细胞质尾部的信号级联和磷酸化状态来确定剪接变体表达的变化是否会改变MUC1与其微环境的相互作用。这些实验有可能为MUC1替代剪接产物在胰腺肿瘤发生和转移中的功能不同作用提供新的见解，并为未来研究具有多个替代剪接产物的基因的蛋白质功能多样化提供模型。公共卫生相关性：本申请提出的实验将探索MUC1（一种细胞表面相关的粘蛋白）的可选剪接变体在胰腺腺癌中促进MUC1功能多样化的分子机制。该应用程序将研究哪些MUC1亚型在胰腺癌中表达，以及这些选择性剪接蛋白参与信号转导、肿瘤进展和转移的机制。

DESCRIPTION (provided by applicant): This application proposes experiments that will explore the molecular mechanisms by which alternative splice variants of MUC1, a cell surface associated mucin, contribute to the diversification of MUC1 function in pancreatic adenocarcinoma. Previous work has established that MUC1 is overexpressed and differentially gycosylated in various adenocarcinomas and that this overexpression is associated with aggressive forms of pancreatic cancer. Most study has focused on the full-length MUC1 isoform that contains the VNTR and all seven exons, but alternative splicing of MUC1 yields a minimum of 26 isoforms and several of these forms have been linked to malignancy state in ovarian, cervical, and breast cancer. Specifically, splice variants A, D, X, Y and Z were more frequently expressed in malignant ovarian tumors than benign tumors. One intriguing study focusing on MUC1 expression in breast cancer demonstrated that MUC1/SEC and MUC1/Y isoforms were coexpressed and that the secreted form of MUC1, MUC1/SEC, interacts with MUC1/Y, inducing a change in the phosphorylation status of the cytoplasmic tail and altering cell morphology. The phosphorylation status of the MUC1 cytoplasmic tail (MUC1.CT) has more recently been shown to be important in cell signaling and is responsive to the extracellular environment. The MUC1.CT is a known substrate for GSK3¿, c-Src, EGFR, PDGFR¿, c-Met, and HGF and has a role in Wnt signaling. The regulation of MUC1.CT phosphorylation and resultant signaling is not well understood and alterations in the MUC1 isoform including the presence or absence of the MUC1 VNTR and/or the inclusion of sequences unique to specific MUC1 splice variants present on the cell surface could have an important role in defining how MUC1 signals through its cytoplasmic tail in response to its extracellular environment. Our initial work focusing on MUC1 alternative splicing has developed a splice-sensitive oligonucleotide array to analyze isoform expression and has shown increases in expression of 3 distinct splice variants in a moderately differentiated pancreatic cell line that is highly metastatic. The experiments proposed in this application will provide insight into MUC1 splice variant expression in pancreatic cancer and will seek to correlate specific isoform expression with disease aggressiveness. We will also determine if changes in splice variant expression alters MUC1 interaction with its microenvironment through the analysis of signaling cascades and phosphorylation status of the MUC1 cytoplasmic tail. These experiments have the potential to provide novel insights into the functionally distinct roles MUC1 alternative splice products have in pancreatic tumor oncogenesis and metastasis, as well as to serve as model for future studies into the diversification of protein function for genes with multiple alternate splice products. PUBLIC HEALTH RELEVANCE: This application proposes experiments that will explore the molecular mechanisms by which alternative splice variants of MUC1, a cell surface associated mucin, contribute to the diversification of MUC1 function in pancreatic adenocarcinoma. This application will investigate which MUC1 isoforms are expressed in pancreatic cancer and the mechanisms through which these alternatively spliced proteins are involved in signal transduction, tumor progression and metastasis.