Roles of MiR-17-92 Cluster MicroRNAs in K-Ras-Induced Pancreatic Tumorigenesis

描述(申请人提供)：胰腺导管腺癌(PDAC)是美国癌症相关死亡的第四大原因，五年存活率不到5%，确诊后中位存活率为6个月。这些统计数字反映了缺乏有效的早期发现和缺乏对系统性疾病的有效治疗。在开发更有针对性和有效的治疗方法之前，需要更多地了解PDAC启动和进展的潜在生物学知识。这项拟议的工作将探索PDAC(突变激活的KrasG12D)的驱动突变与miR-17~92 microRNAs簇之间的潜在关系，特别是讨论Kras在胰腺内的增殖、促生存和致瘤表型对miR-17~92的要求以及这种关系背后的分子机制。为了确定miR-17~92 miRNAs是否是KrasG12D诱导的细胞表型所必需的，我们将分离原代导管上皮细胞(PDECs)，并检测miR-17~92缺失对KrasG12D诱导的细胞增殖和存活的影响。MiRNA簇的组成部分将被外源表达，以评估特定miRNAs对这些表型的潜在挽救作用。缺失miR-17~92簇对体内PDAC前体病变形成的影响将在内源性表达KrasG12D并特异性地在胰腺中缺失miR-17~92簇的动物身上进行评估。动物将在4、8和12个月龄时被处死，并检查胰腺组织切片，以确定前驱病变的存在、数量和级别。利用另外合并了TP53肿瘤抑制基因杂合性缺失的小鼠模型，将确定miR-17~92缺失对PDAC发生和进展的影响。这些研究是特定目标1的重点。本研究还将确定miR-17~92簇与激活的Kras相互作用的机制。通过使用多靶点预测算法和来自表达KrasG12D的PDECs信使RNA微阵列的数据，将识别在PDECs中表达的miR-17~92簇的可能靶点。Western印迹将评估miR-17~92野生型和空PDECs的表达水平，并通过将靶3‘UTRs与荧光素酶融合并测量有和没有miRNA簇的细胞中的发光来证实miRNA介导的抑制。将在簇缺失的PDEC中测量Kras下游通路的活性，以确定17~92是否通过调节特定的下游效应通路来调节KrasG12D诱导的表型。这些研究是特定目的2的重点。总之，本申请中提出的研究将阐明miR-17~92 miRNA簇在胰腺癌发生发展中的作用，可能为开发新的治疗方法奠定基础。

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with a five-year survival rate of less than 5%, and a median survival after diagnosis of 6 months. These statistics reflect the lack of effective early detection and the absence of effective treatments for systemic disease. Greater knowledge of the underlying biology of PDAC initiation and progression is required before more targeted and effective therapies can be developed. The proposed work will explore the potential relationship between the driving mutation of PDAC (mutationally activated KrasG12D) and the miR-17~92 cluster of microRNAs, specifically addressing the requirement for miR-17~92 in Kras' proliferative, pro-survival and tumorigenic phenotypes within the pancreas and the molecular mechanism behind this relationship. To determine whether miR-17~92 miRNAs are required for KrasG12D-induced cellular phenotypes, primary ductal epithelial cells (PDECs) will be isolated and the effect of miR-17~92 deletion on KrasG12D-induced proliferation and survival in the presence of apoptotic stimuli will be determined. Components of the miRNA cluster will be expressed exogenously to assess potential rescue of these phenotypes by specific miRNAs. The effect of deletion of the miR-17~92 cluster on PDAC precursor lesion formation in vivo will be assessed in animals that endogenously express KrasG12D and have deleted the miR-17~92 cluster specifically in the pancreas. Animals will be sacrificed at 4, 8, and 12 months of age and histologic sections of the pancreas examined for the presence, quantity and grade of precursor lesions. Using a mouse model that additionally incorporates heterozygous deletion of the TP53 tumor suppressor gene, the impact of miR-17~92 deletion on PDAC development and progression will be ascertained. These studies are the focus of Specific Aim 1. This study will also identify the mechanism of interaction between the miR-17~92 cluster and activated Kras. Through the use of multiple target prediction algorithms and data from a messenger RNA microarray profile of PDECs expressing KrasG12D, probable targets of the miR-17~92 cluster that are expressed in PDECs will be identified. Expression levels will be evaluated in miR-17~92 wild type and null PDECs by Western blot, and miRNA-mediated suppression will be confirmed by fusing target 3'UTRs to luciferase and measuring luminescence in cells with and without the miRNA cluster. Downstream pathway activity of Kras will be measured in cluster-null PDECs to address whether 17~92 modulates KrasG12D-induced phenotypes through the regulation of specific downstream effector pathways. These studies are the focus of Specific Aim 2. Together, the studies proposed in this application will shed light on the role of the miR-17~92 miRNA cluster in pancreatic cancer initiation and progression, potentially laying the foundation for the development of novel therapeutic approaches.