The Role of p120ctn in Pancreatic Ductal Morphogenesis and Adenocarcinoma

描述（申请人提供）：胰腺导管腺癌（PDAC）的5年生存率低于5%，每年有40，000例患者死于PDAC，自1997年以来，一线治疗没有改善。PDAC中最常见的突变是小G蛋白KRAS的组成性激活变化。当使用胰腺特异性转录因子Pdx 1和KRAS基因上游的LOX-终止-LOX（LSL）盒（Pdx 1-cre~ KRASG 12 D +/fl）将该突变类型靶向胰腺作为氨基酸12处从甘氨酸到天冬氨酸的单个残基变化时，准确地再现了PDAC从前驱病变到浸润性癌症的进展。然而，这种突变本身具有低水平的突变率，这为进一步的病变需要驱动PDAC的想法提供了论据。 p120连环蛋白（p120 ctn）是一种细胞内蛋白质，是粘附连接的组成部分。粘附连接是上皮组织正常组织所需的大分子复合物，其组分之一E-钙粘蛋白是有效的肿瘤抑制剂。p120 ctn是E-钙粘蛋白的已知结合伴侣，并且已知p120 ctn的缺失导致E-钙粘蛋白降解。p120 ctn涉及癌症的几个方面，包括炎症、运动性和增殖。在结肠、食道、皮肤和唾液腺中的条件性敲除模型表明，p120 ctn缺失能够导致从增生到坦率的浸润性癌症的表型。尽管已知p120 ctn在几乎所有的人类癌症（包括PDAC）中丢失或错误定位，但尚未显示p120 ctn扰动对肿瘤进展是偶然的还是中心的。此外，胰腺发育在很大程度上是由小G蛋白Cdc 42介导的，Cdc 42是p120 ctn调节的下游靶点。因此，p120 ctn可能在胰腺的发育和肿瘤发生中起重要作用，并且有证据表明这些作用可能是同种型特异性的。因此，我们建议在这项研究中，研究p120 ctn的胰腺形态发生和肿瘤发生的作用。我们的总体假设是，p120 ctn在胰腺发育中至关重要，其缺失促进PDAC进展。这一假设将通过以下相互关联的具体目标来实现。 在目标1中，我们将使用三维器官型培养和共培养以及衰老，侵袭，迁移和增殖试验的组合，以确定什么样的影响p120 ctn的损失，以及孤立的p120 ctn亚型过表达，对胰腺导管上皮的形态发生。除了这些方法外，我们还将利用一整套分子生物学技术，包括PCR/qPCR、蛋白质印迹、蛋白质活性测定、病毒载体以及共聚焦显微镜和免疫荧光。在目的2中，我们将利用Pdx 1-cre~ LSL-KRASG 12 D +/fl和Pdx 1-cre~ p120 ctnfl/fl小鼠来研究p120 ctn单独和在组成性激活KRASG 12 D突变的情况下的作用。我们还将利用原位移植，流式细胞术，组织学和生化技术来表征p120 ctn在这些小鼠中的作用，无论是在毛，表型水平以及细胞和生化水平。我们的研究将为p120 ctn生物学和p120 ctn在PDAC中的功能作用提供新的机制见解。我们的体外和体内互补方法将为潜在的翻译应用提供一个平台。

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate under 5% with 40,000 patients succumbing to it each year, and no improvement in first-line treatment has occurred since 1997. The most common mutation in PDAC is a constitutively-activating change in the small G protein KRAS. This mutation type, when targeted to the pancreas as a single residue change from glycine to aspartic acid at amino acid twelve using the pancreas-specific transcription factor Pdx1 and a LOX-Stop-LOX (LSL) cassette upstream of the KRAS gene (Pdx1-cre~ KRASG12D+/fl), accurately recapitulates the progression of PDAC from precursor lesions to invasive cancer. This mutation by itself, however, has a low level of penetrance, arguing for the idea that further lesions are needed to drive PDAC. One possible candidate for this role is p120catenin (p120ctn), an intracellular protein that is a component of the adherens junction. The adherens junction is a macromolecular complex required for normal organization of epithelial tissues, and one of its components, E-cadherin, is a potent tumor suppressor. p120ctn is a known binding partner to E-cadherin, and loss of p120ctn is known to lead to E-cadherin degradation. p120ctn has been implicated in several aspects of cancer, including inflammation, motility, and proliferation. Conditional knockout models in colon, esophagus, skin, and salivary gland show that p120ctn loss is able to lead to phenotypes ranging from hyperplasia to frank invasive cancer. Although p120ctn is known to be lost or mislocalized in almost all human carcinomas, including PDAC, it has not been shown whether p120ctn perturbation is incidental or central to tumor progression. Additionally, pancreatic development is in large part mediated by the small G protein Cdc42, a downstream target of p120ctn modulation. p120ctn likely therefore plays important roles in both development and tumorigenesis of the pancreas, and evidence suggests these roles may be isoform-specific. We therefore propose in this study to investigate p120ctn's role in pancreatic morphogenesis and tumorigenesis. Our overarching hypothesis is that p120ctn is critical in pancreatic development and its loss fosters PDAC progression. This hypothesis will be pursued through the following interrelated specific aims. In Aim 1, we will use a combination of 3D organotypic culture and co-culture as well as senescence, invasion, migration, and proliferation assays to determine what effect p120ctn loss, as well as isolated p120ctn isoform overexpression, has on the morphogenesis of pancreatic ductal epithelium. In addition to these methods, we will utilize a full repertoire of molecular biology techniques including PCR/qPCR, Western blots, protein activity assays, viral vectors as well as confocal microscopy and immunofluoresence. In Aim 2, we will utilize the Pdx1-cre~ LSL-KRASG12D+/fl and the Pdx1-cre~ p120ctnfl/fl mice to study the role of p120ctn both alone and in the context of a constitutively activating KRASG12D mutation. We also will utilize orthotopic transplantation, flow cytometry, histology, and biochemical techniques to characterize the role of p120ctn in these mice both on a gross, phenotypic level as well as cellular and biochemical levels. Our studies will provide new mechanistic insights into p120ctn biology and p120ctn's functional role in PDAC. Our complementary in vitro and in vivo approaches will provide a platform for potential translational applications.