Abstract 5189: The transcriptional regulation of the long non-coding RNA HOTAIR in ovarian cancer.

Abstract The Polycomb Group (PcG) Repressive Complexes 1 and 2 (PRC1 and PRC2) maintain gene expression patterns by epigenetically modifying chromatin. PRC2 suppresses transcription by catalyzing the trimethylation of histone H3 at lysine 27 (H3K27me3), a hallmark of repressed chromatin. Although overexpression of PRC2 components has been implicated in cancer initiation, progression and metastasis how PRC2 is recruited to its target genes remains poorly understood. Recent work suggests the interaction of PRC2 with the long-non coding (lnc) RNA HOX transcript antisense intergenic RNA (HOTAIR) could serve as a potential guide to silence target genes, but the mechanism has yet to be defined. In this study, we performed whole transcriptome RNA-seq analysis of isogenic cisplatin- sensitive and -resistant A2780 ovarian cancer (OC) cell lines. Of the lncRNA examined, HOTAIR was markedly induced in the cisplatin resistant cells (5-fold upregulation compared to the cisplatin-sensitve A2780 parental line). shRNA depletion of HOTAIR resensitized the drug-resistant cells to cisplatin. Based on analysis of the HOTAIR promoter using luciferase assays, we identified two putative NF-κB binding sites and three E-box binding sites. To further investigate whether these sites contributed to HOTAIR expression, platinum-sensitive cells were treated with different growth factors and cytokines (0 to 48hrs) and analyzed for changes in HOTAIR expression. Treatment with TNF-α, a well-known inflammatory cytokine and established activator of NF-κB, resulted in 16 fold-induction of HOTAIR. In accord with these results, we observed significant enrichment of both NF-κB and cMYC on the HOTAIR promoter using ChIP assay. Pharmacological inhibition of cMYC and NF-κB reduced HOTAIR expression by 2-fold, and siRNA depletion of the NF-κB p65 subunit or cMYC similarly reduced (P&amp;lt;0.01) HOTAIR expression. Based on these results, we hypothesize a positive feedback loop for HOTAIR transcriptional regulation: HOTAIR inhibits Iκ-Bα (an inhibitor of NF-κB) allowing the lncRNA to enter the nucleus and activate not only its own expression but also cMYC expression to further drive HOTAIR expression as well as cMYC-inducible genes. We are currently testing this hypothesis. We believe this novel pathway, whose components have been implicated in numerous other cancers, may be involved in cisplatin-resistant OC. Citation Format: Ali R. Ozes, Dave Miller, Cong Guo, Anurag Bhattrai, Yunlong Liu, Kenneth P. Nephew. The transcriptional regulation of the long non-coding RNA HOTAIR in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5189. doi:10.1158/1538-7445.AM2013-5189</jats:p

Abstract 4188: Whole transcriptome analyses of platinum-resistant ovarian cancer cells

Abstract Women with advanced stage ovarian cancer have a five-year survival rate of less than 25%. Although most patients respond to platinum-based chemotherapy, relapses are common, leading to platinum-resistant ovarian cancer, which is uniformly fatal. Similar to other malignancies, ovarian cancer progression is associated with transcriptional deregulation and such changes contribute to ovarian cancer progression and development of resistance to chemotherapy. In an effort to better understand the causes of acquired platinum resistance in ovarian cancer, we performed whole transcriptome analysis of platinum sensitive and resistant ovarian cancer cell lines. We used RNA-sequencing to identify candidate genes and pathways that contribute to platinum-resistant ovarian cancer. We detected altered mRNA expression of transcription factors (DMRT3, DMRT1, GATA1, EMX2, LHX1, SRY, SOX9, FOXL2, LHX9) and signaling pathway genes (RXFP2, AR, WT1, FGF9, RSPO1, TUSC3, NROB1, WNT4). Additionally, significant up regulation (&amp;gt;20 fold increase) of the long non-coding RNAs (lncRNAs) HOTAIR and HOTTIP, which direct polycomb group and trithorax complexes to HOX promoters, was detected. We observed elevated HoxA gene expression (&amp;gt; 100 fold in some cases) associated with increased HOTTIP lncRNA expression in the platinum resistant cells. Altered expression of genes associated with DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L MECP2, MBD3L1, MBD6, MBD2, MBD3L2, MBD5, MBD1, MBD3, MBD4) was also observed. Furthermore, the splicing patterns of BRCA1 and BRCA2 genes in platinum resistant cells were altered, resulting in splice variants lacking functional coding regions. Altered BRCA1 and BRCA2 splicing patterns were correlated with reduced expression levels for numerous splicing factors (A2BP1, PTBP1, RBM9, SFRS3, ARID3A, ARID3B, SF1). DNA methylation analysis indicated epigenetic derepression consistent with increased expression for gonad developmental genes (HOTAIR, HOTTIP, HOXA genes). Our data suggest that genes associated ovarian development and differentiation contribute to the development of platinum resistant ovarian cancer. We are currently conducting integrated data and bioinformatics analyses to further investigate changes found in platinum resistance. 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 4188. doi:1538-7445.AM2012-4188</jats:p