Molecular Studies on MUC4 Mucin Gene

描述（由申请人提供）：本研究计划的总体目标是确定MUC4粘蛋白在胰腺癌（PC）发病机制中的多方面作用。在过去的资助周期中，我们已经取得了相当大的进展，从从人胰腺肿瘤cDNA文库中克隆MUC4 cDNA开始，建立其完整的基因组组织，以了解其在PC细胞中的功能和调控。在我们最近的研究中，我们已经证明MUC4与致瘤性和转移性胰腺癌表型直接相关，并为MUC4在PC细胞生长和转移特性中的功能作用提供了实验证据。我们还证明了MUC4在NIH3T3小鼠成纤维细胞中的致癌潜力。有趣的是，我们的研究还揭示了MUC4通过转录后机制调节HER2的表达。我们已经证明MUC4和HER2在PC细胞中相互作用。在本次更新应用中，我们旨在建立MUC4在PC中病理作用的机制基础，并精确定义其在胰腺癌发病过程中与其他遗传畸变的协同作用。我们假设MUC4的多结构域特征使其能够通过改变正常的细胞-细胞和细胞- ecm（细胞外基质）相互作用，同时形成新的癌症特异性相互作用，在功能上参与PC细胞的早期发育和恶性过程。为了验证我们的假设，我们提出了三个具体目标。在Aim 1中，我们将研究MUC4中负责与HER2相互作用的特定结构域或基序，并确定相互作用的特异性和性质（直接或间接）。此外，我们将明确MUC4调控HER2表达的机制。目的2将探讨MUC4在肿瘤生长和转移中的多种作用的分子机制。在Aim 3中，我们将通过产生具有条件胰腺特异性表达的MUC4转基因小鼠，研究MUC4与其他确定的致癌突变在胰腺癌早期发展过程中的协同作用。此外，我们将通过杂交研究MUC4和K-rasG12D（致癌）在小鼠模型中的协同作用。我们还将利用培养的小鼠胰腺导管细胞（PDCs）、携带K-ras突变的PDCs和/或p53失活的PDCs来研究MUC4在同基因系统中的功能。综上所述，这些研究将为MUC4在胰腺癌早期和晚期进展中的作用建立机制基础。公共卫生相关性：第二个竞争性研究项目已进入第10个年头，旨在了解MUC4在致死性胰腺癌（PC）发病机制中的作用机制基础。在之前的周期中，在PC细胞中进行了与MUC4结构、功能和调控相关的研究。MUC4在大多数胰腺癌病例中异常表达，而在正常胰腺中未检测到。拟建的研究将利用人类胰腺癌细胞系以及转基因和同基因动物模型来研究MUC4及其结构域的确切作用。

DESCRIPTION (provided by applicant): The overall objective of this research proposal is to define the multifaceted role of the MUC4 mucin in the pathogenesis of pancreatic cancer (PC). During past funding cycles, we have made considerable progress, beginning with the cloning of the MUC4 cDNA from human pancreatic tumor cDNA libraries, establishing its complete genomic organization to understanding its function and regulation in PC cells. In our recent studies, we have shown a direct association of MUC4 with the tumorigenic and metastatic pancreatic cancer phenotype and provided experimental evidence for a functional role of MUC4 in growth and metastatic properties of PC cells. We have also demonstrated the oncogenic potential of MUC4 in NIH3T3 mouse fibroblast cells. Interestingly, our studies have also revealed that MUC4 regulates the expression of HER2 by post-transcriptional mechanism(s). We have shown that both MUC4 and HER2 physically interact with each other in PC cells. In this renewal application, we aim to establish the mechanistic basis for the pathological role of MUC4 in PC and to precisely define its cooperative action with other genetic aberrations during the course of pancreatic cancer pathogenesis. We hypothesize that the multi-domain structural features of MUC4 enable it to functionally participate in the early development and malignant process of PC cells via altering the normal cell-cell and cell-ECM (extra-cellular matrix) interactions, while simultaneously forming novel, cancer-specific interactions. To test our hypothesis, we propose three specific aims. In Aim 1, we will investigate the specific domain(s) or motif(s) in MUC4 responsible for its interaction with HER2, and determine the specificity and nature (direct or indirect) of interaction. In addition, we will define the mechanisms by which MUC4 regulates HER2 expression. Aim 2 will investigate the molecular mechanisms implicated in defining the multiple roles of MUC4 in tumor growth and metastasis. In Aim 3, we will investigate the cooperative action of MUC4 in combination with other defined oncogenic mutations during the early development of pancreatic cancer by generating MUC4-transgenic mice with conditional pancreas-specific expression. Additionally, we will investigate the cooperative action of MUC4 and K-rasG12D (oncogenic) in mouse models by cross-breeding. We will also utilize cultured mouse pancreatic ductal cells (PDCs), PDCs harboring K-ras mutations, and/or p53 inactivation to study MUC4 function(s) in syngeneic system. Taken together, these studies will establish the mechanistic basis for the role of MUC4 in early and late stages of pancreatic cancer progression. PUBLIC HEALTH RELEVANCE: The proposed second competitive research program in its 10th year aims to understand the mechanistic basis of the role of MUC4 in the pathogenesis of lethal pancreatic cancer (PC). During previous cycles, studies pertinent to MUC4 structure, function and regulation were performed in PC cells. MUC4 is aberrantly expressed in majority of pancreatic cancer cases compared to no detection in the normal pancreas. The proposed studies will investigate the precise role of MUC4 and its domains using human pancreatic cancer cell lines as well as in transgenic and syngeneic animal models.