Abstract 4082: Global miRNA expression changes induced by HDAC-42 and 5-azaCdr in lung cancer cells
Authors: Xin Wu, Fengting Yan, Wenrui Duan, Jianying Zhang, Li Gao, Melissa Crawford, Miguel A. Villalona, S. Patrick Nana-Sinkam, Gregory A. Otterson
Journal: Cancer Research (2012)

Abstract Introduction: Epigenetic alterations are universally seen in lung and other cancers. These include DNA methylation and histone modifications, both pharmacologically targetable abnormalities by DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors. Despite preclinical evidence, epigenetic modifiers have, to date, been ineffective in the treatment of most solid tumors. MicroRNAs are small non-coding RNAs that impact RNA and protein expression through imperfect base pairing in the 3′UTRs of transcribed genes. We investigated the ability of epigenetic drugs to affect the miRNA expression from four lung cancer cell lines, A549, H1299, H719, H520 (representing adeno, large cell, small cell and squamous carcinomas respectively). Materials and Methods: MTT was used to evaluate cell growth, while Western immunoblot and immunofluorescence was used to detect histone H3 acetylation. Annexin-V by flow cytometry was used for apoptosis detection. NanoString was used for the global profiling of miRNAs and corresponding statistical methods was performed by the Biostatistical Core of OSU-CCC. Results: We evaluated the impact of 6 HDAC inhibitors on cell growth of A549 and H1299 cell lines. HDAC-42 was among the most potent drugs and inhibited growth at nM concentrations. Both HDAC-42 and 5-azaCdr increased apoptosis in all 4 cell lines. The most significant synergy between HDAC-42 and 5-azaCdr was in H719 in which apoptosis increased from 4.9 to 38.0% at 500 nM. As expected, synergistic histone H3 acetylation was also seen. Following 72 hour treatment, RNA was harvested and global miRNA expression by NanoString showed that the expression patterns of miRNAs are different among the 4 cell lines after treatment with 200 nM HDAC-42 and 3 µM 5-azaCdr. 5-azaCdr caused more miRNA difference than HDAC-42 (19/3 differential expressed miRNAs in 5-azaCdr treated and HDAC-42 treated H520 cells, 21/6 in H719, 29/9 in H1299, 9/8 in A549). We found that some miRNA changes were common across cell lines in the HDAC-42 treated group, (increased miR129-3p in HDAC-42 treated H1299, H719 and H520 and increased miR148a in HDAC-42 treated H1299, A549 and H719 cells). We also found that 5-azaCdr induced changes in miRNA expression patterns were similar between H520, H719 and H1299 (16 out of 20 miRNAs in H520, 16 out of 21 in H719, 11 out of 29 in H1299). The synergistic activity of 5-azaCdr and HDAC-42 was observed with several individual miRNAs like miR-129-3p and miR-515-5p. Conclusion: Both HDAC-42 and 5-azaCdr could regulate the expression of certain miRNAs in lung cancer cells. The effect of HDAC-42 and 5-azaCdr on four different lung cancer cell lines showed distinct miRNA expression patterns. The role of several candidate miRNAs such as miR-129-3p, miR-515-5p, miR-148a and miR-200c related to acetylation and methylation requires further study. 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 4082. doi:1538-7445.AM2012-4082