Abstract B70: Histone deacetylase inhibitors reverse EMT phenotype and chemoresistance in pancreatic cancer cell lines

Abstract

Abstract Background: The epithelial-to-mesenchymal transition (EMT) is a cell development program charyyacterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell motility regulated by transcription factors and noncoding microRNAs. Recently we reported that EMT may contribute to gemcitabine resistance in pancreatic cancer. Histone deacetylase inhibitors (HDACi) are a new class of antineoplastic agents that have been shown to have a number of antitumor effects including enhanced differentiation of cancer cells. The aims of this study were to determine the potential role of HDACi in regulating EMT and in overcoming gemcitabine resistance in pancreatic cancer cell lines. Methods: SAHA (HDACi) and gemcitabine were tested against a panel of 7 human pancreatic cancer cell lines. Apoptotic cell death was measured by flow cytometry/propidium iodine staining (PIFACS). Cell lines were considered resistant if <50% cell death occurred. To elucidate the mechanism of drug responsiveness, transcriptome analysis was performed before and after drug treatment. Microarray findings were confirmed by western blot analysis and RT-PCR. MicroRNA 200c (miR200c) levels were analyzed by RTPCR. Resistant cell lines were treated with SAHA followed by gemcitabine. Cell death was evaluated by PIFACS. Results: 4 cell lines were identified as resistant. Unsupervised hierarchical analysis of all cell lines demonstrated accurate clustering of cell lines based on gemcitabine sensitivity. E-cadherin and Zeb-1 expression were highly correlated with gemcitabine sensitivity. E-cadherin was increased in chemosensitive and decreased in chemoresistant cell lines; zeb-1 was inversely correlated to E-cadherin. As a potential regulator of zeb-1 expression, miR 200c was evaluated and found to closely correlate inversely with Zeb-1 level and drug sensitivity. Silencing of Zeb-1 led to increased E-cadherin expression and increased sensitivity to gemcitabine. Silencing of E-cadherin in chemosensitive cells did not induce chemoresistance. Pre-treatment of resistant pancreatic cancer cell lines with SAHA led to decreased expression of Zeb-1, increased expression of E-cadherin and mir200c. Importantly it increased sensitivity to gemcitabine. Microarray analysis of resistant pancreatic cell lines after treatment with SAHA demonstrated that these lines now clustered with the sensitive pancreas cell lines. Conclusion: We identified a close association between the expression of miR200c, E-cadherin level, Zeb-1 level, epithelial phenotype and chemosensitivity to gemcitabine in pancreatic cancer cell lines. Interestingly, pre-treatment of resistant cells with SAHA increased miR200c and E-cadherin levels and increased sensitivity to gemcitabine. It appears that combined therapy with HDAC inhibitors and gemcitabine increases chemosensitivity in resistant cell lines and may be a target for overcoming chemoresistance in pancreatic cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B70.