The RAS and P13K Pathways in Pancreatic Adenocarcinoma

本项目的重点是表征K-Ras下游的信号转导途径，介导 胰腺导管腺癌（PDAC）的发病机制。我们的工作假设是K-Ras依赖 磷酸肌醇3-激酶（PI 3 K）的活化在这种癌症中起关键作用。我们推测 PI 3 K下游通路在PDAC进展的沿着每一步都发挥着不同的生物学作用 流程？特异性地影响快速细胞增殖、抗凋亡、侵袭性运动原性 行为和血管生成。此外，我们建议，详细确定上游和 该信令网络的下游组件将提供关于关键目标的信息，以改进 治疗学Ras/PI 3 K/Akt信号通路组分的活化状态将在 在项目1中开发的用于多阶段PDAC的各种小鼠模型。这些模型包括 突变型K-Ras在胰腺中的表达与多种肿瘤抑制因子的生殖细胞丢失有关 基因（INK 4a/Arf，p53;和/或SMAD 4）。磷酸特异性抗体将用于蛋白质印迹、组织 染色和细胞免疫荧光以确定Ras下游蛋白激酶的活化状态， 包括ERK、AKT、tuberin、FOXO家族成员、p70 S6 K、S6和4 EBP 1作为肿瘤的功能 在项目1和4中生成和表型表征的小鼠模型中的进展。在 此外，这些相同的组分将在人组织和细胞系中进行评价， 生物库核心。这些途径的激活将与在基因组学中确定的基因谱进行比较。 项目1是为了将基因组扰动与生化反应相关联。重点将是 这些激活事件如何与PanIN和肿瘤生物学分级的增加相关， 晚期PDAC肿瘤的特性，包括与项目3合作的血管生成。同样的 分析将用于表征项目4中定义的潜在PDAC癌症干细胞的性质。 此外，人PDAC细胞系中致瘤性生长对PI 3 K信号传导组分的需求 将使用诱导型shRNA载体进行评估。最后，一个严格的遗传方法来评估 PI 3 K信号传导在PDAC发病机制中的功能作用将通过杂交小鼠来进行， 将条件性缺乏PI 3 K亚基的小鼠模型与上述小鼠模型进行比较。关键的识别 K-Ras依赖性肿瘤发生的介质在明确的肿瘤背景下将照亮未来 药物靶点，并查明支撑这种致命的生物学进展的关键分子， 疾病

The focus of this project is characterize signal transduction pathways downstream of K-Ras that mediate the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Our working hypothesis is that K-Rasdependent activation of phosphoinositide 3-kinase (PI3K) plays a critical role in this cancer. We speculate that pathways downstream of PI3K play diverse biological roles at every step along the PDAC progression process?specifically effecting rapid cell proliferation, resistance to apoptosis, aggressive motogenic behavior and angiogenesis. Further we propose that the detailed identification of both the upstream and downstream components of this signaling network would provide information on critical targets for improved therapeutics. The activation state of components of the Ras/PI3K/Akt signaling pathway will be assessed in the various mouse models for multistage PDAC being developed in Project 1. These models include expression of mutant K-Ras in the pancreas in the context of germline loss of various tumor suppressor genes (INK4a/Arf, p53; and/or SMAD4). Phospho-specific antibodies will be utilized in western blots, tissue staining and cytoimmunoflourescence to define the activation state of protein kinases downstream of Ras, including ERK, AKT, tuberin, FOXO family members, p70S6K, S6 and 4EBP1 as a function of tumor progression in the mouse models that are generated and phenotypically characterized in Projects 1 and 4. In addition these same components will be evaluated in human tissues and cell lines available from the Biobank Core. The activation of these pathways will be compared to the genetic profiles determined in Project 1 in order correlate genomic perturbations with biochemical responses. A strong emphasis will be placed on how these activation events relate to increasing grades of PanIN and the tumor biological properties of advanced PDAC tumors including angiogenesis in collaboration with Project 3. This same analysis will be used to characterize the properties of potential PDAC cancer stem cells defined in Project 4. In addition, the requirement for PI3K signaling components for tumorigenic growth in human PDAC cell lines will be evaluated using inducible shRNA vectors. Finally, a rigorous genetic approach to assess the functional role of PI3K signaling in PDAC pathogenesis will be undertaken by crossing mice that are conditionally deficient in PI3K subunits to the aforementioned mouse models. The identification of the crucial mediators of K-Ras-dependent tumorigenesis across the well-defined tumor contexts will illuminate future drug targets and pinpoint the critical molecules underpinning the biological progression of this deadly disease.