Screening strategy for small molecules inhibitors of cancer

描述（由申请人提供）：最初在啮齿动物中鉴定的进展升高基因-3（PEG-3）随着癌症变得更具侵袭性而显示出升高的表达。控制PEG-3表达的最小启动子区域PEG-Prom已被分离，并显示在广泛的人类和啮齿动物肿瘤中表现出升高的表达，在正常细胞中表达最小。PEG-Prom在不同作用癌基因转化后或作为介导细胞转化的未鉴定遗传因子的结果被转录激活。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，它们在几乎所有啮齿动物和人类癌症中的表达水平升高。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活性降低。在这些情况下，PEG-Prom代表了一种有价值的预测工具和读数，用于鉴定具有潜在抗肿瘤活性的分子，而无需先验鉴定导致转化/致瘤表型的遗传变化。这项资助的主要目标是鉴定由高通量筛选方法产生的PEG-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肿瘤活性，为开发新型抗肿瘤药物奠定了基础。首先，我们将采用基于荧光素酶细胞的测定，用稳定表达萤火虫荧光素酶上游的PEG-启动子的HeLa细胞筛选化学品库。然后，我们将进行基于细胞的二次检测，以验证化学命中。将测试化合物在一系列另外的人肿瘤细胞（包括DU-145、MeWo和H4细胞系）中抑制PEG-3启动子的能力，以证实化合物在除HeLa细胞之外的癌细胞中的活性。确定化学探针对AP-1和/或PEA-3的活性。将产生仅表达最小PEG-Prom的AP-1或PEA-3位点的HeLa克隆，以鉴定影响转化细胞中这些转录因子之一或两者的化合物。为了机械地定义允许化合物敏感性的特定致癌途径，我们将采用一系列由单个癌基因转化的克隆Fischer大鼠胚胎成纤维细胞（CREF），包括CREF-ras、CREF-raf、CREF-Jun、CREF-src、CREF-AdE 1A和CREF-HPV细胞，其通过北方印迹分析PEG-3的mRNA水平来表达高水平的PEG-3。然后将在一组人类癌细胞系及其正常对应物上测试验证的化合物，以确定它们是否能够抑制细胞生长并选择性地诱导肿瘤细胞死亡。利用通过如上所述的我们的初级和二级筛选产生的有希望的候选化合物，我们将通过在全身递送PEG-Prom Luc质粒后48小时监测小鼠中的生物发光成像（BLI）来评估选择性地针对动物模型中的转移性癌细胞的活性。 公共卫生相关性：控制进展升高的基因-3（PEG-3）表达的最小启动子区域PEG-Prom已显示在广泛的人类和啮齿动物肿瘤中显示升高的表达，在正常细胞中具有最小表达，提供了有价值的预测工具和读数以鉴定具有潜在抗肿瘤活性的分子，而无需先验鉴定引起转化/致瘤表型的遗传变化。该提案的主要目标是鉴定由高通量筛选方法产生的PEG-启动子的化学抑制剂，进行基于细胞的二次测定以验证一系列另外的人肿瘤细胞中的化学命中，对一组人癌细胞系及其正常对应物进行测试以确定癌细胞特异性生长抑制/杀伤，并通过使用PEG-Prom-Luc的基于体内的测定来确定PEG-Prom活性和转化的潜在抑制剂的作用。这些小分子化学探针可能具有潜在的抗癌活性，为开发新型抗肿瘤药物提供了基础。

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.