Molecular dissection of pancreatic ductal adenocarcinoma

描述(申请人提供)：胰腺癌是美国癌症相关死亡的主要原因，估计每年有32,000人死亡。KRAS2癌基因的激活突变，AKT2癌基因的扩增，以及TP53、INK4A和MADH4抑癌基因的丢失，通常发生在本病中。然而，由于这些基因变化而发生的确切的信号扰动仍然知之甚少。例如，虽然KRAS2癌基因的激活突变发生在大约90%的胰腺导管腺癌中，但由Raf、PI3-Kinase和RalGEF介导的各种RAS刺激的信号通路在疾病发病机制中的相对贡献尚不清楚。因此，本应用描述了一种新的胰腺导管腺癌(PDAC)小鼠模型，该模型是通过将编码癌基因的禽类逆转录病毒体细胞导入表达禽类逆转录病毒受体TVA的转基因小鼠的胰腺中产生的，尤其是在胰腺管上皮内，以确定K-RAS刺激的信号通路在肿瘤的启动、维持和进展中的相对贡献。 本提案试图回答以下问题：1)胰管上皮细胞中RAS信号的躯体和零星激活的后果是什么？2)各种RAS激活的信号通路在PDAC中的作用是什么？3)在胰腺肿瘤中，与RAS调节的信号通路激活相关的基因表达特征是什么？ 为了确定Kras基因激活在胰腺管上皮细胞(PDECs)中的直接影响以及单个下游效应通路所起的作用，将编码激活的Kras等位基因或优先导致个别下游通路激活的Kras等位基因的病毒引入胰腺管上皮细胞，并测定其对细胞增殖和凋亡的影响。此外，通过将编码K-RAS或K-RAS效应突变体的病毒导入TVA转基因小鼠的胰腺中，并通过组织学、免疫染色和生化方法分析肿瘤表型，将在体内诱导肿瘤。此外，通过使用小分子抑制剂和RNA干扰方法，将建立持续的肿瘤细胞增殖和生存所需的特定RAS刺激的信号通路。最后，基因表达谱将被用来确定：(I)与K-RAS介导的胰管上皮细胞转化的早期步骤相关的基因表达变化；(Ii)与K-RAS诱导的胰腺导管肿瘤相关的基因表达特征；以及(Iii)与特定RAS刺激的下游通路激活相关的基因表达特征。这样的基因表达特征可能被用作诊断工具，以及作为对靶向治疗干预反应的替代标记。

DESCRIPTION (provided by applicant): Pancreatic cancer is a leading cause of cancer-related mortality in the United States with an estimated 32,000 deaths annually. Activating mutations in the KRAS2 oncogene, amplification of the AKT2 oncogene, and loss of the TP53, INK4A, and MADH4 tumor suppressor genes, commonly occur in this disease. However, the precise signaling perturbations that occur as a consequence of these genetic changes are still poorly understood. For example, although activating mutations in the KRAS2 oncogene occur in approximately 90% of pancreatic ductal adenocarcinomas, the relative contributions of the various Ras-stimulated signaling pathways - mediated by Raf, PI3-kinase, and RalGEFs - to the pathogenesis of the disease are unknown. Therefore, this application describes the use of a novel mouse model for pancreatic ductal adenocarcinoma (PDAC), generated through the somatic introduction of oncogene-encoding avian retroviruses into the pancreata of transgenic mice expressing the avian retroviral receptor, TVA, specifically within the pancreatic duct epithelium, to identify the relative contributions of K-Ras-stimulated signaling pathways to tumor initiation, maintenance, and progression. This proposal seeks to answer the following questions: 1) What are the consequences of somatic and sporadic activation of Ras signaling in the pancreatic duct epithelium? 2) What are the roles of the various Ras-activated signaling pathways in PDAC? And 3) What are the gene expression signatures associated with the activation of Ras-regulated signaling pathways in pancreatic tumors? To identify the immediate consequences of Kras gene activation in pancreatic duct epithelial cells (PDECs) and the roles played by individual downstream effector pathways, viruses encoding an activated Kras allele, or Kras alleles that preferentially lead to the activation of individual downstream pathways, will be introduced into PDECs and the effect on cell proliferation and apoptosis determined. In addition, tumors will be induced in vivo by the introduction of viruses encoding K-Ras or K-Ras effector mutants into the pancreata of TVA transgenic mice, and the tumor phenotypes analyzed by histology, immunostaining and biochemical methods. Furthermore, through the use of small molecule inhibitors and RNA interference methods, the requirement for specific Ras-stimulated signaling pathways for sustained tumor cell proliferation and survival will be established. Finally, gene expression profiling will be performed to identify: (i) Gene expression changes associated with the early steps of K-Ras-mediated transformation of pancreatic duct epithelial cells; (ii) A gene expression signature associated with K-Ras-induced pancreatic ductal tumors; and (iii) Gene expression signatures associated with the activation of specific Ras-stimulated downstream pathways. Such gene expression signatures may potentially be used as diagnostic tools, as well as surrogate markers for response to targeted therapeutic interventions.