Exploitation of RAS signaling to develop therapy and early detection strategies for PDA

描述（由申请人提供）：我们解决的治疗问题是如何抑制IPMN（导管内乳头状粘液瘤），PanIN（胰腺上皮内瘤变）和PDA（胰腺导管腺癌）的发展和进展。生物学问题是确定启动和传播IPMN、PanIN和PDA的关键信号通路。该领域的一个核心争论是PDA是起源于胰腺祖细胞还是起源于实质腺泡细胞向祖细胞样导管细胞的去分化，这一过程被称为腺泡到导管化生（ADM）。转录因子Master Regulators （MRs）在腺泡细胞识别过程中的功能至关重要，但其在翻译后通过受体酪氨酸激酶（RTK）和G蛋白偶联受体（GPCR）信号传导进行的活性调控尚不清楚。G蛋白信号转导（RGS）蛋白的调节因子整合来自rtk和gpcr的多种胞内信号，反馈调节Gi和Gq蛋白信号转导。我们开发了一种GFP报告基因（Rgs16:GFP），使我们能够从IPMN， PanIN和PDA的体内小鼠模型的早期阶段监测肿瘤的发生和肿瘤的形成。将Rgs16:GFP报告基因与IPMN、PanIN和PDA的小鼠模型结合，为这些肿瘤的发展提供了新的见解，并为该过程的抑制剂提供了一种新的、快速的体内筛选方法。我们发现RGS基因抑制IPMN向浸润性癌的进展。基于使用这些模型的初步分析，我们发现IPMN和PanIN在胰腺腺泡或内分泌室的应激反应中在离散的导管祖细胞中启动。这一解释挑战了目前最受欢迎的范式，即PDA起源于经历ADM的细胞，而我们提出慢性ADM刺激导管中已有祖细胞的增殖。我们发现RTK Axl在胰管中表达，并在原代培养的PDA祖细胞中与Rgs16:GFP高表达。我们的假设是，在基质中表达的Axl配体Gas6唤醒了上皮祖细胞中的Axl信号传导，并有助于KrasG12D的激活、MR水平和/或活性的翻译后控制、PDA的启动和进展。在我们抑制PDA进展的新疗法的初步体内筛选中，我们确定华法林（阻断Gas6诱导的Axl活化）与标准护理吉西他滨和Abraxane （GA）联合，可显着减少PDA进展。本提案的具体目的是：1)确定最佳药物组合，包括Axl信号抑制剂，以抑制PDA。2)确定血浆中IPMN、PanIN和PDA的早期标志物。3)确定PDA中Gas6-Axl的功能。该项目如果成功，将通过以下途径对癌症产生重大影响：1)识别PDA的早期血液标志物，可能还有其他上皮性癌症；2)引入一种新的、通用的、快速的体内筛选技术，以发现抑制PDA进展的新药物组合；3)提供新的遗传工具，以确定PDA是否在胰管祖细胞中启动；4)识别以前未知的MR转录因子的调节机制。

DESCRIPTION (provided by applicant): The therapeutic problem we address is how to inhibit the development and progression of IPMN (intraductal papillary mucinous neoplasm), PanIN (Pancreatic Intraepithelial Neoplasia) and PDA (pancreatic ductal adenocarcinoma). The biological problem is to identify key signaling pathways that initiate and propagate IPMN, PanIN and PDA. A central debate in the field is whether PDA initiates in pancreatic progenitor cells or with dedifferentiation of parenchymal acinar cells to progenitor-like ductal cells, in a process termed acinar to ductal metaplasia (ADM). The function of the transcription factor Master Regulators (MRs) of acinar cell identity is critical to this process but post-translational regulaton of their activity through receptor tyrosine kinase (RTK) and G protein coupled receptor (GPCR) signaling is completely unexplored. Regulators of G protein signaling (RGS) proteins integrate multiple intracellular signals from RTKs and GPCRs to feedback regulate Gi and Gq protein signaling. We have developed a GFP reporter (Rgs16:GFP) that allows us to monitor neoplasia and tumor formation from early stages in in vivo mouse models of IPMN, PanIN and PDA. Combining the Rgs16:GFP reporter with mouse models for IPMN, PanIN and PDA is providing insights into development of these tumors and a novel and rapid in vivo screen for inhibitors of this process. We discovered RGS genes that suppress progression of IPMN to invasive carcinoma. Based on preliminary analyses using these models, we find that IPMN and PanIN initiate in discrete ductal progenitor cells in response to stress in the acinar or endocrine compartments of the pancreas. This interpretation challenges the currently favored paradigm that PDA initiates with cells that underwent ADM whereas we propose that chronic ADM stimulates proliferation of pre-existing progenitor cells in ducts. We find that the RTK Axl is expressed in pancreatic ducts and is highly expressed with Rgs16:GFP in PDA progenitor cells in primary culture. Our hypothesis is that the Axl-ligand Gas6 expressed in stroma evokes Axl signaling in epithelial progenitor cells and contributes to activation of KrasG12D, post-translational control of MR levels and/or activity, PDA initiation and progression. In our preliminary in vivo screens of new therapeutics to inhibit PDA progression, we determined that warfarin, which blocks Gas6 induced activation of Axl, combined with the standard of care Gemcitabine and Abraxane (GA), significantly reduced PDA progression. The Specific Aims of this proposal are 1) Identify optimal drug combinations, including inhibitors of Axl signaling, to inhibit PDA. 2) Identify early markers of IPMN, PanIN and PDA in plasma. 3) Determine Gas6-Axl function in PDA. The proposed project, if successful, will have a major impact on cancer by i) identifying early blood markers of PDA, and perhaps other epithelial cancers, ii) introducing a novel, general and rapid in vivo screening technique to find novel drug combinations that inhibit PDA progression, iii) providing novel genetic tools to determine if PDA initiates in progenitor cells in the pancreatic duct, Iv) identify previously unknown regulatory mechanisms of the MR transcription factors.