Diversificiation of MUC1 Function by Alternate Splicing in Pancreatic Cancer

描述（由申请人提供）：本申请提出了将探索MUC 1（一种细胞表面相关粘蛋白）的可变剪接变体有助于胰腺癌中MUC 1功能多样化的分子机制的实验。先前的工作已经确定MUC 1在各种腺癌中过表达和差异糖基化，并且这种过表达与胰腺癌的侵袭性形式相关。大多数研究集中在全长MUC 1亚型，包含VNTR和所有7个外显子，但MUC 1的选择性剪接产生至少26种亚型，其中几种形式与卵巢癌，宫颈癌和乳腺癌的恶性状态有关。具体地说，剪接变体A、D、X、Y和Z在恶性卵巢肿瘤中比良性肿瘤更频繁地表达。一项关注MUC 1在乳腺癌中表达的有趣研究表明，MUC 1/SEC和MUC 1/Y同种型共表达，MUC 1的分泌形式MUC 1/SEC与MUC 1/Y相互作用，诱导胞质尾区磷酸化状态的变化并改变细胞形态。MUC 1胞质尾区（MUC1.CT）的磷酸化状态最近已被证明在细胞信号传导中是重要的，并且响应于细胞外环境。MUC1.CT是GSK 3、c-Src、EGFR、PDGFR、c-Met和HGF的已知底物，并在Wnt信号传导中发挥作用。MUC1.CT磷酸化的调节和由此产生的信号传导尚未得到很好的理解，MUC 1同种型的改变，包括MUC 1 VNTR的存在或缺失和/或包含细胞表面上存在的特异性MUC 1剪接变体所特有的序列，可能在定义MUC 1如何通过其胞质尾区响应其细胞外环境发出信号方面具有重要作用。我们最初的工作集中在MUC 1选择性剪接已经开发了一种剪接敏感的寡核苷酸阵列来分析亚型表达，并显示在中度分化的胰腺细胞系中3种不同的剪接变体的表达增加，这是高度转移的。本申请中提出的实验将提供对胰腺癌中MUC 1剪接变体表达的了解，并将寻求将特异性同种型表达与疾病侵袭性相关联。我们还将通过分析MUC 1胞质尾区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改变MUC 1与其微环境的相互作用。这些实验有可能提供新的见解MUC 1选择性剪接产物在胰腺肿瘤的发生和转移的功能不同的角色，以及作为未来的研究模型到多样化的蛋白质功能的基因与多种选择性剪接产物。公共卫生相关性：本申请提出的实验将探索MUC 1（一种细胞表面相关粘蛋白）的选择性剪接变体有助于胰腺癌中MUC 1功能多样化的分子机制。本申请将研究哪些MUC 1亚型在胰腺癌中表达，以及这些可变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.