Abstract B197: Understanding the mechanism of action of small molecules that restore E-cadherin expression.

E-cadherin is a transmembrane protein that maintains intercellular contacts and cellular polarity in epithelial tissues. The down-regulation of E-cadherin is thought to aid in the induction of an epithelial-to-mesenchymal transition (EMT) resulting in an increased potential for invasion into surrounding tissues and entry into the bloodstream. Loss of E-cadherin has been observed in a variety of human tumors resulting from somatic mutations, chromosomal deletions, proteolytic cleavage of E-cadherin, and most commonly silencing of the CDH1 gene promoter. A novel High Throughput Screen was developed to identify small molecules that restored E-cadherin expression in the SW620 cell line followed by medicinal chemistry employing iterative analog library synthesis to better identify the structure-activity relationship (SAR). Preliminary optimization of the screening hit has shown it is possible to synthesize small molecules that have an improved ability to restore E-cadherin expression compared to the initial screening hits. This restoration of protein has been confirmed by visualization of E-cadherin at the membrane via immunofluorescent microscopy. Further biological analysis of profiled analogs has shown a minimal effect on cell proliferation, but a decrease in cellular invasion. Recent endeavors have been taken to elucidate the mechanism of action of these small molecules to restore E-cadherin expression. Quantitative PCR analysis has shown that treatment with selected small molecules increases mRNA expression ∼50 fold after 16 hours suggesting the small molecules are altering transcription of the CDH1 gene. This was supported by experiments conducted using a plasmid construct containing a 1400bp fragment of the E-cadherin promoter and luciferase reporter. After transfection, the cells were treated with selected compounds, lysed, and luciferase activity was measured. It was shown that selected active small molecules had a significant increase in luciferase activity as compared to DMSO or a dead small molecule, which were used as controls. More specifically, this suggests that the small molecules are specifically effecting transcription within the 1400bp promoter region of the CDH1 gene. Future work will include truncating the promoter region within the plasmid to narrow down the portion of the promoter region targeted by these small molecules, in hopes that specific transcription binding sites will be present within the region. Elucidation of the mechanism of action will aid in identifying the novel molecular target. Such information would allow for further development of more efficacious and potent small molecules as well as further research to understand the importance of this interaction in the role of EMT and as a potential therapeutic target. In addition, combinatorial treatments with sub-therapeutic doses of standard of care chemotherapeutics in the clinic and our small molecules will be screened for synergistic effects in proliferation and apoptosis assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B197.