GPCR regulation of progenitor cells in pancreatic cancer

描述(申请人提供)：科学问题是寻找胰腺导管腺癌(PDAC)的小分子抑制物。这一方法是在观察到患有2型糖尿病或妊娠期糖尿病的人患PDAC风险增加后采取的。该假说认为，由持续的高胰岛素需求时期激活的胰岛祖细胞是至关重要的。 与遗传易感癌症的糖尿病患者的PDAC的形成有关。然而，胰腺前体细胞对β细胞扩增和PDAC的贡献是有争议的，并受到识别和表征这一独特细胞群的挑战的阻碍。大量的初步数据表明，G蛋白信号调节因子Rgs16的表达是受刺激的胰祖细胞的标志。这种创新的方法利用RGS基因的表达来识别参与PDAC启动和进展的G蛋白偶联受体(GPCRs)的配体。为了实现这些进展，将筛选可能的GPCR配体的焦点文库，以识别在PDAC细胞培养中调节RGS16表达的小合成分子。RNA-Seq分析表明，这些细胞表达导管和内分泌谱系的胚胎祖细胞的标志。HITS将在细胞培养和体内验证对RGS16表达和祖细胞生物学的影响。建议的筛选有四个关键优势：合适的待测试细胞类型、简便的细胞培养分析、合理选择的多样化化合物库以及用于体内验证的小鼠模型。该检测系统使用从表达Rgs16基因绿色荧光蛋白(EGFP)的转基因小鼠获得的PDAC细胞。重要的是，RGS：GFP报告基因受GPCR配体调控。胰腺前体细胞在胚胎发育过程中首次分化时表达GFP，表明它们对GPCR配体有反应。GFP报告在正常成人中检测不到，但在胰腺癌前病变的最早阶段重新激活，并在PDAC肿瘤进展过程中保持。在小鼠基因组的所有GPCRs中，鉴定了在RGS16：GFP+PDAC癌前体细胞、胚胎和正常成人胰腺中表达的GPCRs。其中一种可能的GPCRs激动剂，一种异恶唑衍生物(ISX)，在PDAC培养中诱导RGS16：GFP。ISX作为屏幕的积极控制和原则性证明。目的1确定调节RGS16表达的候选分子，并表征ISX和其他HIT化合物对PDAC细胞生物学的影响，包括诱导或维持祖细胞表型。目的2将确定ISX和其他“HIT”化合物对PDAC进展的体内影响。这种方法是创新的，因为它率先使用RGS基因表达来识别GPCRs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，因为这些目标的成功完成将导致胰腺癌监测的重大进展，并确定治疗干预的可能药物靶点。 公共卫生相关性：在这个项目中，与公共健康相关的是胰腺中表达RGS16的独特细胞群的特征，RGS16是G蛋白信号的调节因子，有助于胰腺发育、糖尿病和胰腺导管腺癌(PDAC)。RGS报告基因的表达将用于确定参与PDAC生成的G蛋白偶联受体的配体。这些GPCRs及其天然配体可用于PDAC的早期检测或新的治疗策略的潜在线索。

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. PUBLIC HEALTH RELEVANCE: The relevance to public health in this project is the characterization of a unique population of cells in the pancreas that express Rgs16, a regulator of G protein signaling, that contributes to pancreas development, diabetes, and pancreatic ductal adenocarcinoma (PDAC). Rgs reporter gene expression will be used to identify ligands of G protein coupled receptors that participate in PDAC generation. These GPCRs and their natural ligands could be used for early detection or potential leads in novel therapeutic strategies for PDAC.