The Cell Cycle Regulator CCDC6 Is a Key Target of RNA-Binding Protein EWS

<div><p>Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma.</p></div

Regulation of targets by EWS in vivo.

<p><b>A</b>. Protein domain organization of EWS and FLI1. The black vertical arrows indicate common breakpoints in Ewing sarcoma. Numbers correspond to exons and a typical EWS-FLI1 fusion protein is also shown. Note that the RNA-binding domain of EWS is lost in the process of translocation. <b>B</b>. Pie diagram showing the distribution of PAR-CLIP clusters across 3’UTR, 5’UTR, intronic and coding regions of Refseq RNAs. The three diagrams give the cluster distribution of all sequenced EWS PAR-CLIP targets, all targets regulated by EWS and the four targets we validated (FGF9, MDM2, CBFB, CCDC6). <b>C</b>. Relative mRNA levels of targets genes FGF9, MDM2, CBFB, CCDC6 and EWS in HEK293T cells following EWS knockdown assayed by qRT-PCR (mock: only transfection reagent used; scrambled: AllStars Negative Control siRNA; EWS: siRNA targeting EWS). Relative mRNA levels were normalized to beta actin and quantified relative to the mock and scrambled control levels. Results are shown as mean SEM values (*P < 0.05; n = 3 per group). <b>D</b>. Amount of CCDC6 mRNA transcript percentage is measured upon knocking down of EWS as compared to control. The level of transcript was measured by qRT-PCR after knocking down for 24 hours followed by treatment with actinomycin D. The linear regression and slopes were calculated and the data is presented as Mean and SEM on a linear scale. <b>E</b>. Luciferase activity of CCDC6 upon EWS transfection (normalized to empty psiCHECK-2 plasmid). Data is shown as the fold increase in luciferase activity (RLU units) relative to control. Results are shown as the mean SEM values (*P<0.05; n = 3 per group). <b>F</b>. Relative mRNA levels of CCDC6 and EWS in mock, control and EWS knockdown in MHH-ES-1 cells. Knockdown of EWS decreased the expression of CCDC6. The mRNA levels were normalized to beta actin. Data is represented as mean SEM values (*P<0.05; n = 3 per group). <b>G</b>. Western blot showing the downregulation of CCDC6 upon EWS knockdown in MHH-ES-1 cells. Antibodies are indicated.</p

EWS downregulation affects apoptosis, cell cycle and proliferation.

<p><b>A</b>. Decrease in the relative mRNA levels of FBXW7 upon knocking down EWS. <b>B</b>. Relative mRNA levels of FUS, EWS and TAF15 (FET family proteins) upon EWS knock down. <b>C</b>. Total percentage of living, necrotic and apoptotic cells after EWS and scrambled siRNA knockdown are represented on the bars. Apoptotic cells are defined by the sum of population of cells in early apoptosis and late apoptosis. Mock, scrambled and EWS KD had 10%, 11.8% and 20% apoptotic cells and 17%, 22% and 30% of necrotic cells respectively. The P values refer to the apoptotic cell population. <b>D</b>. Rescue effect upon co-transfection of EWS siRNA and CCDC6 overexpression. Bars represent total percentage of living, apoptotic and necrotic cells. 50nM siRNA, 100ng of empty vector and 100ng of CCDC6 expression vector were transfected. Mock and scrambled had 15% and 18.6% of apoptotic cells, EWS KD and EWS KD+ Empty vector had 39% and 40.5% apoptotic cells respectively and EWS KD+ CCDC6 vector had only 24% apoptotic cells. The P values refer to the apoptotic cell population. <b>E</b>. Proliferation rates on three consecutive days using CCK8 assay was calculated by measuring the absorbance which is proportional to the amount of living cells. <b>F</b>. Quantification of cell cycle distribution. The bars indicate the % of cells in each cell cycle phase subG1, G0/G1, S and G2/M phase. Mock has 11.3%, 44.7%, 16%, 28% of cells in subG1, G0/G1, S and G2/M phase respectively. 16%, 42%, 15%, 27% of cells for scrambled and 37%, 44%, 5%, 14% of cells for EWS KD in each phase respectively. The calculated P values refer to the cells in S phase. Data in all the above figures (A-F) is presented as mean SEM values (*P<0.05; where n = 3 per group).</p