GPCR regulation of progenitor cells in pancreatic cancer

描述（由申请人提供）：科学问题是寻找胰腺导管腺癌（PDAC）的小分子抑制剂。该方法遵循2型或妊娠糖尿病患者患PDAC的风险增加的观察结果。这一假说认为，胰腺祖细胞被持续的高胰岛素需求所激活， 在遗传上易患癌症的糖尿病患者中形成PDAC。然而，胰腺祖细胞对β细胞扩增和PDAC的贡献是有争议的，并且受到鉴定和表征这种独特细胞群体的挑战的阻碍。大量的初步数据表明，表达的Rgs 16，调节G蛋白信号，标志着刺激胰腺祖细胞。该创新方法使用Rgs基因表达来鉴定参与PDAC起始和进展的G蛋白偶联受体（GPCR）的配体。为了实现这些进展，将筛选推定的GPCR配体的集中文库，以鉴定调节PDAC细胞培养物中Rgs 16表达的小合成分子。RNA-Seq分析显示这些细胞表达导管和内分泌谱系的胚胎祖细胞的标记物。将在细胞培养和体内验证命中对Rgs 16表达和祖细胞生物学的影响。拟议的屏幕有四个关键的优势：适当的细胞类型进行测试，简便的细胞培养试验，合理选择的化合物的多样性库，和小鼠模型的体内验证。该测定系统使用从转基因小鼠获得的PDAC细胞，所述转基因小鼠表达来自Rgs 16基因的绿色荧光蛋白（eGFP）。重要的是，Rgs：GFP报告基因受GPCR配体调控。胰腺祖细胞在胚胎发生中首次分化时表达GFP，表明它们对GPCR配体有反应。GFP报告基因在正常成年人中检测不到，但在胰腺中最早的癌前病变中被重新激活，并在整个PDAC肿瘤进展中维持。在小鼠基因组中的所有GPCR中鉴定了在Rgs 16：GFP+ PDAC癌症祖细胞、胚胎和正常成人胰腺中表达的GPCR。这些GPCR之一的推定激动剂异恶唑衍生物（ISX）在PDAC培养物中诱导Rgs 16：GFP。ISX用作筛选的阳性对照和原理证明。目的1将确定候选分子，调节Rgs 16表达和表征ISX和其他'命中'化合物对PDAC细胞生物学的影响，包括祖细胞表型的诱导或维持。目的2将确定ISX和其他“命中”化合物对PDAC进展的体内作用。该方法具有创新性，因为它率先使用Rgs基因表达来鉴定GPCR及其配体，然后在细胞培养和体内测试它们的功能。这项拟议的研究意义重大，因为成功完成这些目标将导致胰腺癌监测的重大进展，并确定治疗干预的假定药物靶点。

DESCRIPTION (provided by applicant): The scientific problem is to find small molecule inhibitors of pancreatic ductal adenocarcinoma (PDAC). The approach follows the observation that people with type 2 or gestational diabetes are at increased risk for PDAC. The hypothesis is that pancreatic progenitor cells activated by sustained periods of high insulin demand are critical to the formation of PDAC in people with diabetes who are genetically predisposed to cancer. However, the contribution of pancreatic progenitor cells to beta cell expansion and PDAC is controversial and has been hampered by challenges in identifying and characterizing this unique cell population. Extensive preliminary data indicates that expression of Rgs16, a regulator of G protein signaling, marks stimulated pancreatic progenitor cells. The innovative approach uses Rgs gene expression to identify ligands of G protein coupled receptors (GPCRs) that participate in the initiation and progression of PDAC. To achieve these advances, focused libraries of putative GPCR ligands will be screened to identify small synthetic molecules that regulate Rgs16 expression in PDAC cell culture. RNA-Seq analysis shows these cells express markers of embryonic progenitors of the ductal and endocrine lineage. Hits will be validated in cell culture and in vivo for effects on Rgs16 expression and progenitor cell biology. The proposed screen has four crucial advantages; appropriate cell types to be tested, facile cell culture assays, the diverse library of rationally chosen compounds, and mouse models for in vivo validation. The assay system uses PDAC cells obtained from transgenic mice that express green fluorescent protein (eGFP) from the Rgs16 gene. Importantly, the Rgs:GFP reporter gene is regulated by GPCR ligands. Pancreatic progenitor cells express GFP as they first differentiate in embryogenesis, indicating they respond to GPCR ligands. The GFP reporter is undetectable in normal adults but is reactivated in the earliest preneoplastic lesions in the pancreas, and maintained throughout PDAC tumor progression. The GPCRs that are expressed in Rgs16:GFP+ PDAC cancer progenitor cells, embryonic and normal adult pancreas were identified among all GPCRs in the mouse genome. A putative agonist for one of these GPCRs, an isoxazole derivative (ISX), induces Rgs16:GFP in PDAC culture. ISX serves as a positive control and proof of principle for the screen. Aim 1 will identify candidate molecules that regulat Rgs16 expression and characterize the effect of ISX and other 'hit' compounds on PDAC cell biology, including the induction or maintenance of a progenitor cell phenotype. Aim 2 will determine the in vivo effects of ISX and other 'hit' compounds on PDAC progression. The approach is innovative because it pioneers using Rgs gene expression to identify GPCRs and their ligands and then tests their function in cell culture and in vivo. The proposed research is significant because successful completion of these aims will lead to major advances in pancreatic cancer surveillance and identify putative druggable targets for therapeutic intervention.