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.
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.
|Das, Swadesh K; Sarkar, Siddik; Dash, Rupesh et al. (2012) Chapter One---Cancer terminator viruses and approaches for enhancing therapeutic outcomes. Adv Cancer Res 115:1-38|
|Hedvat, Michael; Emdad, Luni; Das, Swadesh K et al. (2012) Selected approaches for rational drug design and high throughput screening to identify anti-cancer molecules. Anticancer Agents Med Chem 12:1143-55|
|Kim, Keetae; Lee, Seok-Geun; Kegelman, Timothy P et al. (2011) Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics. J Cell Physiol 226:2484-93|
|Bhang, Hyo-eun C; Gabrielson, Kathleen L; Laterra, John et al. (2011) Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression. Nat Med 17:123-9|