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 CCND1 (the cyclin D1 gene), which contributes to cell-cycle dysregulation in cancer. In this study, we found that CCND1 expression is significantly higher in patients with Ewing sarcoma compared with other sarcomas and that the pncCCND1_B RNA, a previously uncharacterized CCND1 promoter-associated noncoding (pnc) transcript, is expressed in Ewing sarcoma cells. PncCCND1_B interacted with the RNA-binding protein Sam68 and repressed CCND1 expression. Notably, knockdown of Sam68 affected pncCCND1_B 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 CCND1 promoter, thus repressing it. Conversely, mitogenic stimulation of Ewing sarcoma cells with IGF1 impaired Sam68/DHX9 interaction and positively regulated CCND1 expression. These studies uncover a fine-tuned modulation of the proto-oncogene CCND1 in Ewing sarcoma cells via alternative complexes formed by DHX9 with either EWS-FLI1 or pncCCND1_B-Sam68. SIGNIFICANCE: A pncRNA-based mechanism represses expression of CCND1 through the formation of a protein-RNA complex and provides new therapeutic opportunities for patients with Ewing sarcoma.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/14/3570/F1.large.jpg.
Highlights
We found that CCND1 expression is significantly higher in patients with Ewing sarcoma compared with other sarcomas and that the pncCCND1_B RNA, Sam68, DHX9, and pncCCND1_B form a multimolecular inhibitory complex on the CCND1 promoter, fostered by EWS-FLI1 dissociation and counteracted by mitogenic stimuli, such as Insulin growth factor 1 (IGF1) stimulation
CCND1 is upregulated in Ewing sarcoma By querying gene-expression datasets obtained from 137 sarcoma samples (30–33; dataset details are provided in the Supplementary Information), we found that CCND1 expression is significantly higher in Ewing sarcoma (28 patients) with respect to other sarcomas, such as alveolar rhabdomyosarcomas (ARMS; 23 patients), alveolar soft part sarcomas (ASPS; 12 patients), synovial sarcomas (SS; 46 patients) and desmoplastic small round cell tumors (DSRCT; 28 patients; Fig. 1A; statistical analysis is reported in the Supplementary Fig. S1A)
Expression of CCND1 in sarcomas was significantly upregulated in tumors bearing the most common EWS–ETS chromosomal translocations in Ewing sarcoma, such as EWS–ERG and EWS–FLI1 (Fig. 1B; statistical analysis is reported in Supplementary Fig. S1B)
Summary
Aberrant expression of cyclin D proteins plays pivotal roles in human tumors by contributing to the loss of regulatory circuitsNote: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).Ó2019 American Association for Cancer Research.governing cell-cycle progression [1]. Aberrant expression of cyclin D proteins plays pivotal roles in human tumors by contributing to the loss of regulatory circuits. In addition to its role in the regulation of the G1–S transition of the cell cycle, cyclin D1 influences gene expression programs by directly associating with several transcription factors and chromatin remodeling proteins [4], which likely contribute to its role in oncogenesis. One type of cancer that is sensitive to cyclin D1 levels is Ewing sarcoma, where increased cyclin D1 expression in early phase of the tumor correlates with poor prognosis [5]. Ewing sarcoma cells strictly require cyclin D1 and its cognate kinase CDK4 (cyclin-dependent kinase 4) for survival and anchorageindependent growth [8]. Pharmacologic inhibition of cyclin D1/CDK4 complex caused cytostatic effects and cell death of Ewing sarcoma cell lines [8]
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