Abstract 4788: Enhancer of zeste homolog 2 couples with HOTAIR to inhibit tumor suppressor miR-34a in human pancreatic ductal adenocarcinoma

Abstract

Abstract We attempt to study the role of EZH2 in suppressing (microRNA-34a) miR-34a, an important tumor suppressor miR-34a silenced in PDAC cells, and identify the silencing partner of EZH2. We have previously showed that enhancer of zeste homolog 2 (EZH2), an enzyme catalyzing trimethylation of H3K27, represses microRNA in pancreatic ductal adenocarcinoma (PDAC) by multiple mechanisms including H3K27 trimethylation, heterochromatin formation and DNA methylation. miR-34a is frequently downregulated in human cancers by inactivation of p53 or promoter DNA methylation, but in PDAC the major player that silences miR-34a is unknown. Methods: miR-34a levels were measured by qRT-PCR in PDAC cells treated with small molecule inhibitor of EZH2 3-deazaneplenocin A (DZP) and siRNAs targeting EZH2 and HOTAIR. miR-34a levels were also measured in human pancreatic ductal epithelial (HPDE) cells overexpressed with EZH2 and various EZH2-domain deleted mutants. We studied the effect of EZH2 to the promoter activity of miR-34a gene by luciferase assay. To identify the silencing partners of EZH2, we inhibited SUZ12, DNMT1, DNMT3A, DNMT3B and HOTAIR in PDAC cells and measured the change of miR-34a level. The effect of miR-34a to EZH2-induced cell growth and tumor formation were studied by MTT assay and in vivo subcutaneous tumor formation study respectively. Colony formation rate was also measured in HPDE cells overexpressed with wildtype and EZH2-binding domain deleted HOTAIR. Results: miR-34a was upregulated upon the inhibition of EZH2 and HOTAIR, while EZH2 overexpression in HPDE reduced miR-34a level. Consistent to previous studies, inhibition of DNMTs failed to upregulate miR-34a in PDAC cells. Inhibition of EZH2 by shRNAs increased the promoter activity of miR-34a in PDAC cells. We showed that EZH2 enhanced cell proliferation and colony formation of HPDE cells, and overexpression of miR-34a could attenuate this growth promoting effect. EZH2-mediated cell proliferation was attenuated by miR-34a mimics transfection and miR-34a precursor lentivirus transduction. Inhibition of miR-34a by miRZIP-34a lentivirus could decrease the tumor formation rate of PDAC cells expressing shEZH2. Moreover, overexpression of HOTAIR increased colony formation in HPDE cells, while deletion of EZH2-binding domain in HOTAIR attenuated this effect. Conclusion: Ectopic expression of EZH2 suppressed miR-34a in non-tumor human pancreatic duct cells and PDAC cells. In addition to PRC2 core member Suz12, inhibition of miR-34a by EZH2 is dependent on HOTAIR. miR-34a was the functional target silenced by EZH2 that contributed to EZH2-mediated cell proliferation and tumor growth in PDAC. We believed that HOTAIR guided EZH2 to exhibit silencing effect to the promoter of miR-34a. Note: This abstract was not presented at the meeting. Citation Format: Samson Li, Yangchao Chen. Enhancer of zeste homolog 2 couples with HOTAIR to inhibit tumor suppressor miR-34a in human pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4788. doi:10.1158/1538-7445.AM2015-4788