Abstract LB-345: Acetylenic tricyclic bis-(cyano enone) interacts with cysteine residues of actin and inhibits non-small cell lung cancer cell migration

During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their cell-cell junctions, apical-basal polarity, and reorganize their actin cytoskeleton. These changes, among others, increase the motility of individual cells and promote a highly invasive phenotype. The actin cytoskeleton is also key in cell migration and invasion, as the polymerization of actin at the leading edge provides the necessary driving force for translocation and protrusions. Therefore, the actin cytoskeleton is a potential target for inhibiting tumour progression. In this study we characterized a novel tri-cyclic synthetic triterpenoid derivative, TBE-31, in its ability to associate with actin and inhibit cell migration. Using a pull-down approach, we demonstrate that TBE-31 binds directly to actin. Furthermore, TBE-31 inhibited branched and linear actin polymerization in vitro, and stress fibre formation in cultured cells. Actin cytoskeleton reorganization and stress fiber formation was also inhibited in TGFβ-dependent EMT of non-small cell lung cancer cells. We next used SwissDock software to investigate the potential molecular interaction between TBE-31 and actin. Swissdock predicted that TBE-31 would have a high affinity for binding to the same cleft as Cytochalasin D, a bona-fide actin polymerization inhibitor. As triterpenoids associate with cysteine residues in target proteins, we generated cysteine to alanine mutants of actin and verified TBE-31 binding using pull-down assays. We identified that cysteine 374 is important for TBE-31 interaction with actin. Finally, we observed that TBE-31 inhibited fibroblast and non-small cell lung tumour cell migration with an IC50 of 1.0 and 2.5 μM, respectively. Taken together, our results suggest that TBE-31 targets actin polymerization to alter cell morphology and inhibit cell migration. Citation Format: Eddie Chan, Akira Saito, Tadashi Honda, John Di Guglielmo. Acetylenic tricyclic bis-(cyano enone) interacts with cysteine residues of actin and inhibits non-small cell lung cancer cell migration. [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 LB-345.