Screening strategy for small molecules inhibitors of cancer

描述(由申请人提供)：最初在啮齿动物中发现的进展上调基因-3(PEG-3)，随着癌症变得更具侵袭性而表现出表达上调。控制PEG3表达的最小启动子区域，PEGPROM，已经被分离出来，并被证明在广泛的人类和啮齿动物肿瘤中表达上调，在正常细胞中表达最低。在不同的作用癌基因转化后，或由于未知的遗传因素介导的细胞转化，聚乙二醇亲和蛋白在转录水平上被激活。我们已经确定了这种选择性的机制，它涉及转录因子AP-1和PEA-3，这两种转录因子在几乎所有啮齿动物和人类癌症中都有高水平的表达。此外，在癌症逆转或阻断特定转化癌基因表达的情况下，PEG-Prom的活性降低。在这些背景下，聚乙二醇组蛋白是一种有价值的预测工具和读数，用于识别具有潜在抗肿瘤活性的分子，而不需要事先识别导致转化/致瘤表型的基因变化。这笔赠款的主要目标是识别高通量筛选方法产生的聚乙二醇启动子的化学抑制剂。这些小分子化学探针可能具有潜在的抗癌活性，为新型抗肿瘤药物的开发奠定了基础。首先，我们将使用基于荧光素酶细胞的分析来筛选化学物质文库，HeLa细胞稳定表达萤火虫荧光素酶上游的聚乙二醇启动子。然后，我们将进行基于细胞的二次分析，以验证化学成分。将在包括DU-145、MeWo和H4细胞在内的一系列额外的人类肿瘤细胞中测试化合物抑制PEG-3启动子的能力，以确认除HeLa细胞外，化合物在癌细胞中的活性。确定针对AP-1和/或PEA-3的化学探针的活性。将产生仅表达最小聚乙二醇组蛋白的AP-1或PEA-3位点的HeLa克隆，以鉴定在转化细胞中影响这两种转录因子之一或两者的化合物。为了从机制上确定允许化合物敏感性的特定致癌途径，我们将使用一系列由单个癌基因转化的Fischer大鼠胚胎成纤维细胞(CREF)，包括CREF-ras、CREF-RAF、CREF-Jun、CREF-src、CREF-AdE1A和CREF-HPV细胞，通过Northern Blot分析高水平表达PEG3的mRNA水平。然后，将在一组人类癌细胞和它们的正常对应细胞上测试经过验证的化合物，以确定它们是否能够选择性地抑制肿瘤细胞的生长和诱导肿瘤细胞死亡。如上所述，通过我们的第一次和第二次筛选产生的有希望的候选化合物，我们将在动物模型中通过监测系统递送PEG-Prom Luc质粒48小时后的小鼠生物发光成像(BLI)来评估选择性地抗转移癌细胞的活性。 与公共卫生相关：控制进展上调基因-3(PEG-3)的最小启动子区域，PEG-Prom，已被证明在广泛的人类和啮齿动物肿瘤中表达上调，在正常细胞中表达极低，为识别具有潜在抗肿瘤活性的分子提供了有价值的预测工具和读数，而无需事先确定导致转化/致瘤表型的基因变化。这项建议的主要目标是确定从高通量筛选方法产生的聚乙二醇启动子的化学抑制剂，进行基于细胞的二次检测以验证在一系列额外的人类肿瘤细胞中的化学打击，在一组人类癌细胞株和它们的正常对应物上进行测试以确定癌细胞特异性的生长抑制/杀伤，并利用基于体内的检测方法确定潜在的聚乙二醇-PROM活性和转化抑制剂的效果。这些通过二次筛选验证的小分子化学探针可能具有潜在的抗癌活性，为新型抗肿瘤药物的开发奠定了基础。

DESCRIPTION (provided by applicant): Progression elevated gene-3 (PEG-3), originally identified in rodents, displays elevated expression as cancers become more aggressive. The minimal promoter region controlling PEG-3 expression, PEG-Prom, has been isolated and shown to display elevated expression in a wide range of both human and rodent tumors, with minimal expression in normal cells. The PEG-Prom is transcriptionally activated following transformation by diverse acting oncogenes or as a consequence of unidentified genetic factors mediating cellular transformation. We have determined the mechanism for this selectivity and it involves the transcription factors, AP-1 and PEA- 3, which are expressed at elevated levels in virtually all rodent and human cancers. Moreover, in cases of cancer reversion or blocking expression of specific transforming oncogenes, PEG-Prom activity is decreased. In these contexts, the PEG-Prom represents a valuable predictive tool and readout to identify molecules with potential antitumor activity, without a priori identification of the genetic changes causative of the transformed/tumorigenic phenotype. The primary goal of this grant is to identify chemical inhibitors of the PEG- Promoter generated from high-throughput screening methods. These small molecule chemical probes might have potential anticancer properties and could provide a foundation for the development of novel antitumor drugs. First, we will screen a library of chemicals employing a luciferase cell-based assay with HeLa cells stably expressing the PEG-Promoter upstream of firefly luciferase. Then we will perform cell-based secondary assays to validate chemical hits. Compounds will be tested for their ability to inhibit the PEG-3 promoter in a series of additional human tumor cells, including DU-145, MeWo and H4 cell lines to confirm compound activity in cancer cells in addition to HeLa cells. To define activity of chemical probes toward AP-1 and/or PEA-3. HeLa clones expressing only the AP-1 or the PEA-3 site of the minimal PEG-Prom will be generated to identify compounds affecting either or both of these transcription factors in transformed cells. To mechanistically define specific oncogenic pathways allowing compound sensitivity we will employ a series of cloned Fischer rat embryo fibroblast (CREF) cells transformed by a single oncogene, including CREF-ras, CREF-raf, CREF-Jun, CREF-src, CREF-AdE1A and CREF-HPV cells, which express high levels of PEG-3, by analyzing the mRNA level of PEG-3 by Northern Blot. Validated compounds will then be tested on a panel of human cancer cell lines and their normal counterparts to determine if they are able to suppress cell growth and induce death selectively in tumor cells. With promising candidate compounds generated through our primary and secondary screens as described above, we will evaluate activity selectively against metastatic cancer cells in animal models by monitoring bioluminescence imaging (BLI) in mice 48 h after systemic delivery of PEG-Prom Luc plasmid. PUBLIC HEALTH RELEVANCE: The minimal promoter region controlling progression elevated gene-3 (PEG-3) expression, PEG-Prom, has been shown to display elevated expression in a wide range of both human and rodent tumors, with minimal expression in normal cells, providing a valuable predictive tool and readout to identify molecules with potential antitumor activity, without a priori identification of the genetic changes causative of the transformed/tumorigenic phenotype. Main goals of this proposal are to identify chemical inhibitors of the PEG-Promoter generated from high-throughput screening methods, to perform cell-based secondary assays to validate chemical hits in a series of additional human tumor cells, to test on a panel of human cancer cell lines and their normal counterparts to determine cancer cell specific growth inhibition/killing, and to define the effect of potential inhibitors of PEG-Prom activity and transformation with an in vivo-based assay using PEG-Prom-Luc. These small molecule chemical probes validated from the secondary screens might have potential anticancer properties and could provide a foundation for the development of novel antitumor drugs.