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年存活率低于5%，每年有40,000名患者死于它，自1997年以来一线治疗没有任何改善。PDAC中最常见的突变是小G蛋白KRAS的结构性激活变化。当使用胰腺特异的转录因子Pdx1和KRAS基因上游的LOX-STOP-LOX(LSL)盒(Pdx1-cre~KrasG12D+/fl)针对胰腺时，这种突变类型作为氨基酸12位从甘氨酸到天冬氨酸的单一残基变化，准确地概括了PDAC从前体病变到侵袭性癌症的进展。然而，这种突变本身具有低水平的外显性，这证明了需要进一步的损伤来驱动PDAC的想法。一个可能的候选角色是p120catenin(P120ctn)，这是一种细胞内蛋白，是粘连连接的一个组成部分。黏附连接是上皮组织正常组织所必需的大分子复合体，其成分之一E-钙粘附素是一种有效的肿瘤抑制因子。P120ctn是已知的E-钙粘蛋白的结合伙伴，p120ctn的缺失会导致E-钙粘蛋白的降解。P120ctn与癌症的多个方面有关，包括炎症、运动和增殖。结肠、食道、皮肤和唾液腺的条件性基因敲除模型表明，p120ctn缺失能够导致从增生到直接浸润性癌症的各种表型。尽管已知p120ctn在几乎所有人类肿瘤中丢失或定位错误，包括PDAC，但尚未显示p120ctn的干扰是肿瘤进展的附带或中心。此外，胰腺的发育在很大程度上是由小G蛋白CDC42介导的，CDC42是p120ctn调控的下游靶标。因此，p120ctn可能在胰腺的发育和肿瘤发生中发挥重要作用，有证据表明这些作用可能是异构体特异性的。因此，在这项研究中，我们建议研究p120ctn在胰腺形态发生和肿瘤发生中的作用。我们的主要假设是p120ctn在胰腺发育中起关键作用，它的缺失促进了PDAC的进展。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，我们将结合3D器官培养和共培养以及衰老、侵袭、迁移和增殖试验来确定p120ctn缺失以及单独的p120ctn亚型过表达对胰腺导管上皮形态发生的影响。除了这些方法外，我们还将利用一整套分子生物学技术，包括聚合酶链式反应/定量聚合酶链式反应、蛋白质印迹、蛋白质活性分析、病毒载体以及共聚焦显微镜和免疫荧光。在目标2中，我们将利用pdx1-cre~ls1-KrasG12D+/f1和pdx1-cre~p120ctnf1/f1小鼠来研究p120ctn在单独和在组成性激活KrasG12D突变的背景下的作用。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p120ctn在大体、表型以及细胞和生化水平上在这些小鼠中的作用。我们的研究将为p120ctn生物学和p120ctn在PDAC中的功能作用提供新的机制见解。我们在体外和体内的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.