Abstract 2593: HDAC inhibition reverses EMT through modulation of ZEB1

Abstract

Abstract Histone deacetylase (HDAC) inhibition is known to inhibit the Epithelial-to-Mesenchymal Transition (EMT) in various cancer models by suppressing the acetylation of histone H3 and H4 of E-Cadherin promoter. However, the exact mechanism of EMT inhibition by HDAC inhibition is not known. In the present study, using various HDAC inhibitors such as Vorinostat (SAHA), Tricostatin A (TSA) and Panobinostat (LBH-589), we explored the molecular mechanisms of EMT inhibition in pancreatic tumor cells in vitro and in vivo. The expression of epithelial markers such as E-Cadherin and cytokeratin 18 were significantly up regulated inPanC-1, BxPC-3 and AsPC-1 cells treated with HDAC inhibitors, whereas the expression of mesenchymal marker vimentin decreased. Morphology of the cells also changed indicating that HDAC inhibitors suppressed EMT in pancreatic cancer cells. To determine whether HDAC inhibition correlates with the down-regulation of key transcriptional suppressors of E-Cadherin such as SNAIL1, SNAIL2, TWIST and ZEB, cells were treated with panobinostat. To our surprise, SNAIL1 expression was drastically increased, whereas expression of SNAIL2, TWIST and ZEB were decreased by HDAC inhibition in all the cell lines. Since, it has been previously shown that HDAC activity is required to suppress E-Cadherin expression by its repressors, we wanted to see whether SNAIL1 also requires HDAC activity to repress E-Cadherin expression in our model. HDAC1/2 was co-immunoprecipitated with SNAIL1. Our results revealed that this interaction was more pronounced in control cells as compared to HDAC inhibitors treated cells. These results suggest that although SNAIL1 expression was significantly increased by HDAC inhibition, it remained ineffective in E-Cadherin promoter. To evaluate other repressors, PanC-1 cells were transfected with SNAIL2 and ZEB1 and treated with HDAC inhibitors. Interestingly, our results show that SNAIL2 overexpression was unable to abrogate increase in E-Cadherin by HDAC inhibition. However, ZEB1 overexpression completely blocked the induction of HDAC-mediated E-Cadherin expression. To validate in vitro observations in an in vivo model, BxPC-3 cells were subcutaneously implanted in athymic nude mice. When the tumors reached about 70mm3, 20mg/Kg panobinostat was administered intraperitonealy three times a week while control group received vehicle alone. After 4 weeks of treatment, the growth of pancreatic tumors was significantly suppressed in panabinostat treated mice. Moreover, the tumors from panabinostat treated mice showed an increase in E-Cadherin and decrease in ZEB expression. The expression of other E-Cadherin repressors SNAIL1 and SNAIL2 were also down regulated. In conclusion, our in vitro and in vivo results clearly show that increase in E-Cadherin by HDAC inhibition was due to the down regulation of ZEB1. [Supported in part by R01 grants CA129038 and CA106953 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2593. doi:1538-7445.AM2012-2593