Role of microRNAs in genetic mouse models of pancreatic cancer

描述（由申请人提供）：胰腺导管腺癌（PDAC）是美国第四大癌症死亡原因。由于其对化疗的顽固抵抗，迫切需要开发新的治疗策略来治疗这种致命的癌症。大量研究揭示了K-ras激活突变和p16、p53和Smad4功能突变丧失的重要病因学贡献。由于Cre/LoxP系统，结合这些基因突变的小鼠模型可以在胰腺的背景下进行专门的研究。然而，其他调控途径的潜在作用，无论是关于PDAC的发展和/或其显著的化学耐药的起源，尚未得到彻底的研究。MicroRNAs （miRNAs）是一类新的短链非编码RNA基因[10-12]，是基因表达[13]的转录后负调控因子。mirna介导的肿瘤发生调控正在成为癌症生物学领域的一个新范式。每种miRNA的大量预测靶基因表明，miRNA可以提供一种新的全局机制来调节与癌症相关的全基因组表观遗传和转录变化的基因表达。此外，一些报道表明，特定的mirna表现出与肿瘤抑制因子和癌基因相似的功能。此外，由于Dicer或其他miRNA加工所需酶的缺失，miRNA活性整体下降，导致致瘤潜力增强，反映了miRNA在介导肿瘤抑制功能中的整体作用。我们的初步研究和其他小组的报告已经将一组mirna与PDAC联系起来，基于：1)它们在胰腺细胞系和/或临床标本中的差异表达和/或2)它们与病原学相关的信号通路（如K-Ras， p16和p53）的相互作用。因此，mirna可以作为早期检测、诊断和/或预后的有用生物标志物，促进我们对肿瘤发生分子机制的理解，并作为新的治疗靶点。基于这些观察结果，我们的总体假设是miRNAs在PDAC的起始和进展中发挥着至关重要的作用，并且与当前的治疗药物和这类新型调节rna的合成调节剂联合治疗可能提供一种有效的策略来增加患者对治疗的反应。我们已经建立了几种携带K-ras和/或p16基因条件突变的转基因小鼠菌株，这是PDAC中最常见的突变基因，我们建议对miRNA进行功能分析，旨在评估：1)通过将条件丧失功能的dicer1floxed/floxed动物与携带K-ras和/或p16基因突变等位基因的易发癌症动物杂交，降低全球miRNA活性在疾病进展中的影响。2)通过调控小鼠和人胰腺癌细胞系中miRNA的表达/活性水平，研究个体miRNA在体外胰腺肿瘤发生中的作用。公共卫生相关性：胰腺导管腺癌（PDAC）是一种致命的恶性肿瘤，其死亡率几乎等于发病率，但目前尚无令人满意的治疗方法。越来越多的证据表明，microrna在癌症生物学中起着关键作用。我们假设目前的化学毒性化合物和这种新型调节rna的合成调节剂的联合治疗可能提供一种有效的策略来增加患者对治疗的反应。在本提案中，我们将研究microrna对PDAC的病因学贡献，并对其潜在的治疗应用进行初步评估。

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States. Because of its stubborn resistance to chemotherapy, there is an urgent need to develop novel therapeutic strategies for this deadly cancer. Numerous studies have revealed an important etiological contribution of activated mutations of K-ras and loss of function mutations of p16, p53 and Smad4. Mouse models that combine mutations in these genes have permitted their study specifically in the context of the pancreas thanks to the Cre/LoxP system. However, the potential role of other regulatory pathways, either with respect to the development of PDAC and/or in relation to the genesis of its marked chemoresistance, has not been thoroughly investigated. MicroRNAs (miRNAs) are a new class of short non-coding RNA genes [10-12], which act as post- transcriptional negative regulators of gene expression [13]. MiRNA-mediated regulation of tumorigenesis is emerging as a new paradigm in the field of cancer biology. The large number of predicted target genes for each miRNA suggests that miRNAs could provide a novel and global mechanism to modulate gene expression comparable to genome-wide epigenetic and transcriptional changes associated with cancer. Moreover, several reports indicate that specific miRNAs exhibit functions reminiscent of tumor suppressors and oncogenes. In addition, global decreases in miRNA activity by loss of Dicer or other enzymes required for miRNA processing result in enhanced tumorigenic potential, reflecting on the overall role of miRNAs in mediating tumor suppressive functions. Our preliminary studies and reports from other groups have linked a subset of miRNAs to PDAC based on: 1) their differential expression in pancreatic cell lines and/or in clinical specimens and/or 2) their interaction with signaling pathways etiologically relevant such as K-Ras, p16 and p53. Thus, miRNAs could serve as useful biomarkers for early detection, diagnosis and/or prognosis, advance our understanding of the molecular mechanisms of tumorigenesis, and serve as novel therapeutic targets. Based on these observations, our overall hypothesis is that miRNAs exert a crucial role in the initiation and progression of PDAC and that combinatorial therapy with current therapeutic agents and synthetic modulators of this novel class of regulatory RNAs may provide an effective strategy to increase patient response to treatment. We have established several transgenic murine strains that bear conditional mutations in K-ras and/or p16 genes, the most frequently mutated genes in PDAC, and we propose to conduct a functional analysis of miRNAs aimed at assessing: 1) The effect of decreased global miRNA activity in disease progression by crossing a conditional loss of function dicer1floxed/floxed animal to cancer-prone animals bearing mutated alleles of K-ras and/or p16 genes. 2) The contribution of individual miRNAs to pancreatic tumorigenesis in vitro by manipulating levels of miRNA expression/activity in murine and human pancreatic cancer cell lines. PUBLIC HEALTH RELEVANCE: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy in which mortality virtually equals incidence, yet for which there are no satisfactory therapeutic treatments. Emerging evidence suggests a pivotal role of microRNAs in cancer biology. We hypothesize that combinatorial therapy of current chemotoxic compounds and synthetic modulators of this novel class of regulatory RNAs may provide an effective strategy to increase patient response to treatment. In this proposal, we will investigate the etiological contribution of microRNAs to PDAC and conduct a preliminary assessment of their potential therapeutic application.