Abstract Background: Epithelial-Mesenchymal-transition (EMT) is a highly conserved biologic process that is involved in organ development, tissue regeneration, tumor invasion and metastasis. EMT is phenotypically associated with the loss E-cadherin and a gain of mesenchymal markers (e.g., vimentin). Both promoter methylation by DNMT1 and histone H3- K27 trimethylation by the histone methyl transferase EZH2 have been shown to cause E-cadherin expression. Several transcription factors, including ZEB1, Snail, Slug and Twist, directly bind to E-cadherin promoter, recruit histone deacetylase (HDAC) activity and repress E-cadherin. We have previously shown that the pan-HDAC inhibitor panobinostat (PS, Novartis Pharmaceuticals) depletes EZH2 and DNMT1 levels, and co-treatment with PS and DNMT1 inhibitor decitabine (DAC) synergistically depletes EZH2 and DNMT1. Methods: Here, we determined the effects of PS (20 to 100 nM) and/or DAC (0.5 to 5.0 uM) on E-cadherin levels and other EMT-associated markers, as well as on invasiveness of human breast cancer MB-231 and SUM159PT cells, which have undetectable levels of E-cadherin. Results: Treatment with PS dose dependently inhibited the migration and invasion of the breast cancer cells (by Boyden chamber assay). This was associated with induction of mRNA and protein levels of E-cadherin and decline in vimentin levels. PS treatment inhibited HDAC activity and reduced DNMT1 and EZH2 levels. Additionally PS repressed ZEB1 mRNA and protein levels. Co-treatment with bortezomib partially restored ZEB1 levels, indicating that PS treatment promotes proteasomal degradation of ZEB1. Chromatin immunoprecipitation (ChIP) analyses demonstrated that PS treatment inhibited H3K27 chromatin repressive and increased H3K4 chromatin permissive mark associated with the E-cadherin promoter. PS treatment also diminished ZEB1 occupancy on the E-cadherin promoter. Pre-treatment of MB-231 and SUM 159PT cells with DAC or 5-azacytidine (AC, also a DNMT1 inhibitor) (0.5 to 5.0 uM) further enhanced PS-mediated induction of E-cadherin, which was associated with further decline in ZEB1 levels. Additionally, co-treatment with PS and DAC caused greater inhibition of in vitro invasion by the breast cancer cells. Discussion: Collectively, these findings indicate that combined epigenetic targeting with PS and DAC depletes ZEB1, induces E-cadherin and reverses EMT, thereby, attenuating invasiveness of human breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-03.