Abstract 3551: Non-coding RNA HOTAIR connects DNA damage signaling to NF-κB activation in cisplatin resistant ovarian cancer

Abstract

The Polycomb Repressive Complex 2 (PRC2) has been implicated in cancer and a role for PRC2 during DNA damage response (DDR) has recently been reported. To examine changes in gene expression with DDR in cisplatin (cddp)-resistant ovarian cancer (OC), we performed whole transcriptome RNA-seq analysis of isogenic cddp-sensitive and -resistant OC cell lines (A2780 and A2780-cp). Differential expression of PRC components (SUZ12, EZH1, SIRT1, PHC1 &2) (P<0.05), NF-κB pathway members (NCAM, ABCB9, RAGE, IL4R, IL6R, BCL2L11) (P<0.05), and long non-coding RNAs (lncRNAs) known to associate with PRC2 (P<0.05), including Hox transcript antisense intergenic RNA or HOTAIR, was observed. In tumors from patients with high grade serous OC at diagnosis, basal expression of HOTAIR was greater (P<0.01) compared to normal ovarian surface epithelium and marked overexpression of HOTAIR was observed in tumors obtained from patients who had developed platinum-resistant OC. Ablation of HOTAIR using dsiRNA resensitized A2780-cp to cddp, while ectopic over-expression of HOTAIR increased (P<0.05) A2780 cell survival after cddp treatment (3-fold vs. control). To further examine HOTAIR regulation, we conducted a promoter analysis using bioinformatics tools and luciferase assays. We identified a putative p65-NF-κB binding site (906-GGGACACCCC-915) 906 bp upstream of the HOTAIR transcription start site. Treatment of A2780 cells with the NF-κB activator TNF-α induced HOTAIR (16-fold vs. control) and NF-κB enrichment (3-fold assessed by ChIP assays) at the HOTAIR promoter. Furthermore, in A2780-cp compared to A2780, total Iκ-Bα levels were reduced (P<0.05) and nuclear p65 levels were increased, indicating that endogenous activation of NF-κB contributes to cddp resistance and DDR. Consistent with this observation, cddp treatment (20μM for 0-24hrs) of A2780 cells increased (P<0.05) HOTAIR expression by 5- and 16-fold at 8 and 24 hrs and decreased (P<0.05) Iκ-Bα protein levels at similar time points. Furthermore, inhibiting NF-κB by either gliotoxin (5μM) or Bay-11 (3μM) completely abolished cddp-induced HOTAIR expression in A2780 cells, demonstrating that NF-κB is a HOTAIR transcriptional activator during cddp-induced DDR. Importantly, EZH2 and histone H3 lysine-27 trimethylation (H3K27me3) levels were enriched (6- and 17-fold) in the Iκ-Bα promoter at 24 and 48 hours post cddp treatment, and HOTAIR depletion using dsiRNA reduced the observed EZH2-H3K27me3 enrichment at the Iκ-Bα promoter, demonstrating that HOTAIR recruits PRC2 complex to the Iκ-Bα promoter to prolong NF-κB activation during cddp-induced genotoxic stress. Mouse xenograft studies with A2780 cells overexpressing HOTAIR are ongoing. The results of this study support a role for HOTAIR as a positive regulator of the NF-κB pathway and PRC2 during cisplatin-induced DNA damage. We further suggest that HOTAIR may serve as a therapeutic target in cisplatin-resistant OC. Citation Format: Ali R. Ozes, Dave F. Miller, Yunlong Liu, Kenneth P. Nephew. Non-coding RNA HOTAIR connects DNA damage signaling to NF-κB activation in cisplatin resistant ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3551. doi:10.1158/1538-7445.AM2014-3551