The Promoter-Associated Noncoding RNA <i>pncCCND1_B</i> Assembles a Protein-RNA Complex to Regulate Cyclin D1 Transcription in Ewing Sarcoma.

Abstract

Most Ewing sarcomas are characterized by the in-frame chromosomal translocation t(11;22) generating the EWS-FLI1 oncogene. EWS-FLI1 protein interacts with the RNA helicase DHX9 and affects transcription and processing of genes involved in neoplastic transformation, including <i>CCND1</i> (the cyclin D1 gene), which contributes to cell-cycle dysregulation in cancer. In this study, we found that <i>CCND1</i> expression is significantly higher in patients with Ewing sarcoma compared with other sarcomas and that the <i>pncCCND1_B</i> RNA, a previously uncharacterized <i>CCND1</i> promoter-associated noncoding (pnc) transcript, is expressed in Ewing sarcoma cells. <i>PncCCND1_B</i> interacted with the RNA-binding protein Sam68 and repressed <i>CCND1</i> expression. Notably, knockdown of Sam68 affected <i>pncCCND1_B</i> subcellular localization and cyclin D1 expression. Pharmacologic impairment of DHX9/EWS-FLI1 interaction promoted RNA-dependent association of Sam68 with DHX9 and recruitment of Sam68 to the <i>CCND1</i> promoter, thus repressing it. Conversely, mitogenic stimulation of Ewing sarcoma cells with IGF1 impaired Sam68/DHX9 interaction and positively regulated <i>CCND1</i> expression. These studies uncover a fine-tuned modulation of the proto-oncogene <i>CCND1</i> in Ewing sarcoma cells via alternative complexes formed by DHX9 with either EWS-FLI1 or <i>pncCCND1_B</i>-Sam68. SIGNIFICANCE: A pncRNA-based mechanism represses expression of <i>CCND1</i> through the formation of a protein-RNA complex and provides new therapeutic opportunities for patients with Ewing sarcoma.<b>Graphical Abstract:</b> http://cancerres.aacrjournals.org/content/canres/79/14/3570/F1.large.jpg.