ELL2 Regulates DNA Double-strand Break Repair In Prostate Cancer Cells And Its Mechanism

Aim: Incorrect repair of DSBs may induce cell apoptosis or senescence, or createmutations and chromosomal translocations that lead to abnormal cell growth and ultimately cancer. The role of DNA damage response and repair in the development and progression of prostate cancer ispoorly understood. DNA damage mightfacilitate or accelerate prostate tumorigenesis. ELL2 is an androgen-regulated tumor suppressor frequently down-regulated inprostate cancer, however the mechanisms involved remain to be fully elucidated.Thus, elucidating the mechanisms involved in DNAdamage response in the prostate will provide critical understanding of tumor development, progressionand treatment.Methods: 1. Prostate cancer cells were treated with siRNA of ELL2, then treated with ?-irradiation or doxorubicin andcollected for detecting the level of DNA damage marker ?H2ax and performing neutral comet assay.2. Plasmid of I-Sce I was transfectioninto H1299 dA3-1#1 cells(for NHEJ) or HeLa pDRGFPcells(for HR) pretransfected with siRNA of ELL2. ForFACS analysis, cells were harvestedand EGFP-positive cells were counted.3. C4-2 cells were transiently transfected with GFP or GFP-ELL2, and RFP or RFP-Ku70, RFP-Ku80 expression vectors, and treated with laser microirradiation to induce DSBs after transfection before and after irradiation. Accumulation of the transfected proteins was indicated by GFP(green) or RFP(red) fluorescence at laser-irradiated sites.4. Using HEK293 cells transfectedwith various combinations of GFP-Ku70, GFP-K80 and Flag-ELL2 plasmids, and then performing Co-IP. C4-2 cells were treated with or without Dx prior to harvest and nuclear extracts preparation, and the co-immunoprecipitation of endogenous ELL2 and Ku70/80.5. Foci intensity of GFP-Ku70/80 accumulation at sites of lasermicroirradiation in C4-2 cells treated withsiELL2 followed by transfectionwith GFP-Ku70/80. Mean foci intensity was measured every 20 seconds for 8 minutes and subtracted from background intensity.6. C4-2 cells were treated with siELL2 and then transfected with GFP-Ku70/80 aloneor combination of siELL2-resistant Flag-ELL2 plasmids. Kinetics of GFP-Ku70/80 accumulation at laser-irradiated sites was measured 24 hours later.Results: 1. Knockdown of ELL2 sensitizes prostate cancer cells to DNA damage, and Overexpression of ELL2 protects prostate cancer cells from DNA damage.2. Knockdown of ELL2 reduced the percentage of GFPpositivecells compared with cells treated with siRNA control in NHEJ assay. Using the Hela p DR-GFP-based assay for HR repair, no significantdifference was detected in the percentage of GFP-positive cells between siELL2-treated and siRNAcontrol groups.3. GFP-ELL2, RFP-Ku70 and RFP-Ku80 proteins accumulated at sites of DNA damage induced by 405 nm laser microirradiation, and GFP-ELL2 co-accumulated at DSB sites with RFP-Ku70 and RFP-Ku80.4. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. Nuclear extracts prepared from C4-2 cells with Dxwere also immunoprecipitated with ELL2, which showed endogenous Ku70 and Ku80 co-precipitation with endogenous ELL2 protein, while without Dx can not.5. Knockdown of EAF1 and/or EAF2 inhibited Ku70 and Ku80 accumulation and retention at DSB sites dramatically in C4-2 cells. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2 in C4-2 cells.Conclusion: 1.ELL2 protects prostate cancer cells from doxorubicin and ?-irradiation-induced DNA damage.2. Knockdown of ELL2 impairs nonhomologous end-joining(NHEJ) repair.3. ELL2 binds to and co-accumulates with Ku70/Ku80 proteins at sites of DNA damage.4. Knockdown of EAF family proteins impairs recruitment of Ku70/Ku80 at sites of DSB.