Role of microRNAs in genetic mouse models of pancreatic cancer

描述(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，迫切需要为这种致命的癌症开发新的治疗策略。大量研究表明，K-ras基因的激活突变和p16、P53、Smad4功能突变的缺失在肿瘤的发病机制中具有重要作用。多亏了Cre/loxP系统，结合了这些基因突变的小鼠模型允许他们在胰腺环境中进行专门的研究。然而，其他调控通路在PDAC的发生和/或与其显著的化疗耐药的发生有关的潜在作用还没有得到彻底的研究。MicroRNAs(MiRNAs)是一类新的非编码短小RNA基因[10-12]，是基因表达的转录后负调控因子[13]。MiRNA介导的肿瘤发生调控正成为癌症生物学领域的一个新范式。每个miRNA预测的大量靶基因表明miRNAs可以提供一种新的全球机制来调节基因表达，与与癌症相关的全基因组表观遗传和转录变化相媲美。此外，一些报告表明，特定的miRNAs具有类似于肿瘤抑制因子和癌基因的功能。此外，由于失去Dier或处理miRNA所需的其他酶而导致miRNA活性的全局下降，导致肿瘤发生潜力增强，这反映了miRNAs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研究小组的报告将miRNAs的一个子集与PDAC联系起来，其基础是：1)它们在胰腺细胞系和/或临床标本中的差异表达和/或2)它们与病因相关的信号通路如K-RAS、p16和P53的相互作用。因此，miRNAs可以作为有用的生物标志物用于早期发现、诊断和/或预后，促进我们对肿瘤发生的分子机制的理解，并作为新的治疗靶点。基于这些观察，我们的总体假设是miRNAs在PDAC的启动和进展中发挥关键作用，与当前治疗药物和这类新型调控RNAs的合成调节剂联合治疗可能提供一种有效的策略来提高患者对治疗的反应。我们已经建立了几个携带PDAC中最常见突变基因K-ras和/或p16基因条件性突变的转基因小鼠株，并建议对miRNAs进行功能分析，目的是评估：1)通过使带有K-ras和/或p16基因突变等位基因的动物与携带K-ras和/或p16基因突变等位基因的动物杂交，全球miRNA活性降低在疾病进展中的作用。2)通过调控小鼠和人胰腺癌细胞株中miRNA的表达/活性水平，探讨单个miRNAs在体外胰腺癌发生中的作用。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，但目前还没有令人满意的治疗方法。新出现的证据表明，microRNAs在癌症生物学中起着关键作用。我们推测，目前的化学毒性化合物和这类新型调控RNA的合成调节剂的联合治疗可能会提供一种有效的策略来提高患者对治疗的反应。在这项提案中，我们将调查microRNAs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.