Abstract PO-090: TGF-β induced EMT gene expression is associated with promoter demethylation in pancreatic cancer

Abstract

Abstract INTRODUCTION: Transforming growth factor-β (TGF-β), a ubiquitous molecule in pancreatic ductal adenocarcinoma (PDA) tumors, acts as a potent inducer of tumor invasion by regulating proteins involved in metastasis and driving epithelial-mesenchymal transition (EMT). We hypothesized that TGF-β signaling-induced EMT is regulated by DNA methylation. To evaluate this, we investigated the association between EMT gene promoter methylation status and gene expression in the TCGA PDA dataset. We also investigated vimentin gene (a mesenchymal marker)-specific changes in DNA methylation due to TGF-β treatment in PDA cells. METHODS: The methylation status of EMT-related genes (ZEB2, CDH2, Vimentin) was interrogated in the TCGA PDA data set. β-values (proportion of methylated gene probes in a location) were compared to gene expression levels. Next, a PDA cell line (Panc-1) was treated with TGF-β (10ng/ml), vehicle control, DNA methyltransferase inhibitor (5-azacytidine, AZA, 10mM), or TGF-β plus AZA to evaluate the effects on the DNA methylation status of the vimentin gene with methylation-specific PCR (MSP) against the promoter region of vimentin. Unmethylation and methylation levels at the vimentin promoter region were then compared. RESULTS: We found that β values of promotor regions of ZEB2, CDH2, and Vimentin were moderately negatively correlated with gene expression (Pearson r varies from 0.45 to 0.63) in the PDA TCGA data set. In Panc-1 cells, vimentin gene expression was increased following treatment with AZA compared to controls suggesting that DNA demethylation increases vimentin gene expression. MSP demonstrated that TGF-β treatment increased unmethylated vimentin gene levels compared to vehicle control treatment. TGF-β treatment increased unmethylated vimentin gene levels more so than methylated gene levels. CONCLUSIONS: These results demonstrate that gene expression of EMT-related genes may be at least partially regulated by DNA methylation. We demonstrate that TGF-β treatment leads to increased Vimentin promotor demethylation (lower β value) and increased gene expression in Panc-1 cells. This observation is consistent with our findings in the TCGA data set that lower β-values are associated with increased expression of mesenchymal genes, indicative of EMT. Further studies are underway to investigate the relationship between DNA methylation and TGF-β-induced EMT in general. Citation Format: Manjul Rana, Abul Elahi, Abidemi O. Ajidahun, Rita G. Kansal, Anders E. Berglund, David Shibata, Evan S. Glazer. TGF-β induced EMT gene expression is associated with promoter demethylation in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-090.