Abstract 1589: Identification of EMT-selective cytotoxic compounds

Abstract

Abstract Background: Epithelial to mesenchymal transition (EMT) is a cellular phenotype which may contribute to metastasis and therapeutic resistance in cancer. Therapeutic targeting of EMT-like phenotypes in cancer cells, thus may afford an approach to treating metastatic disease and/or thwarting therapeutic resistance. Methods: We have developed a cell-based high-throughput screening protocol using the high content Operetta imaging platform to identify EMT cytotoxic compounds. This screen was performed on PC3E and TEM 4-18 cell lines which were originally isolated form a parental PC3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer. A library of 2,640 compounds (Microsource) was screened on a co-culture of epithelial cells (PC3E GFP cells) and EMT-like cells (TEM 4-18 mCherry cells). After 72h exposure to compounds, relative numbers of GFP- and mCherry-positive cells were quantitated using the Operetta system. Dose-response curves were established for each compound exhibiting a greater toxicity against EMT-like cells. The results were validated on PC3E and TEM 4-18 cell lines cultured separately with the CellTiter Blue viability assay. Results: Among the 2,640 compounds tested, monensin and salinomycin, both monovalent cation ionophores, exhibited a relatively greater toxicity against EMT-like cells. Nigericin, compound closely related to monensin but absent from the Microsource library, was also evaluated. The highest potency and selectivity for the EMT-like cells was obtained with nigericin (IC50 357nM for PC3E vs 7nM for TEM 4-18), followed by monensin (IC50 457nM for PC3E vs 11nM for TEM 4-18) and salinomycin (IC50 1687nM for PC3E vs 278nM for TEM 4-18). Monensin and nigericin induced apoptosis, a cell cycle arrest, and an increase in ROS production in TEM 4-18 without apparent mitochondrial disruption. In addition, monensin rapidly induced swelling of golgi apparatus likely resulting in a blockage of intracellular protein trafficking and consequently cell death. To extend our findings, we evaluated the toxicity of these three ionophores in 15 cancer cell lines from different origins and classified them as resistant or sensitive. We analyzed publically available gene expression data for these cell lines and performed a Gene Set Enrichment Analysis to identify the gene sets differentially represented in the 2 groups. Supporting our hypothesis, the gene set involving EMT was the top gene sets enriched in the sensitive group. Conclusion: In this study we identified three chemically-related compounds (monensin, salinomycin and nigericin) exhibiting relatively greater toxicity against EMT-like cells. These compounds may serve as chemical probes for exploring the biology of EMT-like cells and point to strategies for selectively targeting these cells to disrupt metastasis and/or therapeutic resistance. Citation Format: Marion Vanneste, Qin Huang, Meng Wu, Michael D. Henry. Identification of EMT-selective cytotoxic compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1589.