The Mechanisms Underlying KAT2A-mediated AR Translocation Into Nucleus In Castration-resistant Prostate Cancer Cell With Abiraterone-Resistance

Part Ⅰ KAT2 A is highly expressed in abiraterone-resistant CRPC cells and is associated with poor prognosis of prostate cancer Objective:To test the expression of KAT2 A in abiraterone resistant CPRC model and confirm the relationship between KAT2 A expression and clinical prognostic end-points.Methods:The concentration gradient increasing method was used to induce the establishment of C4-2 abiraterone resistant cell model(C4-2-Abi R)in vitro.Colony formation experiments were used to detect the proliferation level of drug-resistant cell lines and parental cell lines.The CCK-8 method was used to determine the abiraterone IC50 of the drug-resistant cell line C4-2-Abi R and the parental cell line(C4-2-P),and then the resistance index(RI)was calculated.The CCK-8 method was used to draw the growth curve of parental cells and drug-resistant cells.Flow cytometry was used to test cell cycle.Western blot was used to detect the expression of KAT2 A in parental cells and drug-resistant cells.Bioinformatics methods were used to evaluate the relationship between the expression level of KAT2 A and various clinical prognostic indicators.Results:1.The IC50 of C4-2-Abi R model established by the concentration gradient increasing method was 22.89μM,while that of C4-2-P was 3.66μM.RI =(IC50 of drug-resistant cells)/(IC50 of parental cells)= 6.25.2.Colony formation experiments showed that in the presence of abiraterone,the number of C4-2-Abi R clones was more than C4-2-P.3.The growth curve shows that the growth rate of C4-2-Abi R is slower than that of C4-2-P in the absence of abiraterone.4.Flow cytometry showed that cell cycle of C4-2-Abi R was blocked in the stage S compared with C4-2-P.5.Western blot results indicated that the expression level of KAT2 A in C4-2-Abi R was significantly higher than that of C4-2-P.6.Bioinformatics results determined that prostate cancer with high KAT2 A expression has shorter OS,DFS,PFS,and higher biochemical recurrence rate and Gleason score.Conclusion:The expression of KAT2 A increased in C4-2 abiraterone-resistant cells.Prostate cancer patients with higher KAT2 A expression have shorter survival intervals and a higher Gleason score.Part Ⅱ The effect of KAT2 A on the biological behavior of abiraterone-resistant prostate cancer cellsObjective:To explore the effect of KAT2 A on the biological behavior of abiraterone-resistant prostate cancer in vitro and in vivo experiments.Methods:By constructing KAT2 A overexpression and knockdown vectors,screen for stable KAT2 A overexpression/knockdown prostate cancer cell lines after transfection.The CCK-8 method was used to detect the growth activity of prostate cancer cell lines treated with abiraterone after regulating KAT2 A expression;the colony formation assay was used to detect the proliferation ability of prostate cancer cell lines treated with abiraterone after regulating KAT2 A expression.Nude mice were subcutaneously inoculated with C4-2-Abi R prostate cancer cells with stable knockdown of KAT2 A to establish a subcutaneous ectopic tumor transplantation model,and the effect of knockdown of KAT2 A on the ectopic tumor model was analyzed.Results:1.After transfection with KAT2 A knockdown plasmids and subsequently stable selection,the expression level of KAT2 A in C4-2-Abi R cell line was significantly reduced;after infection with KAT2 A overexpression lentivirus and subsequently stable selection,the expression level of KAT2 A in C4-2-P and parental LNCa P cell lines increased significantly.2.The growth and proliferation ability of the C4-2-Abi R cell line with stably knocked-down KAT2 A was significantly inhibited in the complete medium in the presence of abiraterone,while those of the C4-2-Abi R cell line that was stably transfected with vector was basically unaffected in the presence of abiraterone.3.The growth and proliferation ability of the C4-2-P cell line stably overexpressing KAT2 A in the presence of abiraterone is basically not affected,while that of the C4-2-P cell line stably transfected with vector was significantly inhibited in the presence of abiraterone.4.The growth and proliferation ability of parental LNCa P cell line stably overexpressing KAT2 A in the presence of abiraterone is basically not affected,while that of parental LNCa P cell line stably transfected with vector was significantly inhibited in the presence of abiraterone.5.The animal xenograft tumor model showed that the growth of tumors with stable knockdown of KAT2 A was obviously inhibited by abiraterone,while the growth of tumors transfected with vector was not affected by abiraterone.6.IHC staining of the transplanted tumor showed that the expression of Ki-67 was significantly reduced after the tumor with stable knockdown of KAT2 A was treated with abiraterone,while the expression of Ki-67 was not affected after the transplanted tumor with vector was treated with abiraterone.Conclusion:Inhibiting the expression of KAT2 A in vitro can enhance the sensitivity of CRPC to abiraterone.Inhibiting the expression of KAT2 A in vivo can increase the sensitivity of prostate cancer tumor tissues to abiraterone.Part Ⅲ KAT2 A mediates AR acetylation and promotes its nuclear translocation,leading to abiraterone resistance in CRPC cellsObjective:To explore the interaction between KAT2 A and AR signaling pathway in abiraterone-resistant CRPC cells.Methods:Regulating KAT2 A to detect the changes in m RNA level and protein level expression of related molecules in the AR signaling pathway.Immunofluorescence and nucleoplasmic protein separation experiments were used to detect the nuclear translocation of AR after the regulation of KAT2 A.The dual-luciferase reporter gene experiment was used to detect the binding of AR and androgen response element of the target gene.Immunofluorescence and co-immunoprecipitation experiments were used to detect the binding of AR and KAT2 A.Immunoprecipitation and Western blot were used to detect the level of AR acetylation.CCK-8 test was used to test cell viability.The dual luciferase reporter gene experiment was used to detect the combination of AR and KAT2 A androgen response element.Results:1.KAT2 A knockdown in abiraterone-resistant cell line can reduce the mRNA level and protein level of the AR target gene PSA,while the expression level of AR is not affected;overexpression of KAT2 A in the parent cell line can increase the m RNA level and protein level of PSA level,but the expression level of AR is not affected.2.Overexpression of KAT2 A can enhance the combination of AR and PSA androgen response element.3.Overexpression of KAT2 A in parental cell line can promote AR nuclear translocation,and knockdown of KAT2 A in abiraterone-resistant cell line can inhibit AR nuclear translocation.4.KAT2 A can acetylate the K630 position of AR.5.AR can enhance transcription level of KAT2 A by binding to the androgen response element of KAT2 A.Conclusion:KAT2A can acetylate the K630 site of AR to promote AR nuclear translocation.KAT2 A promotes abiraterone resistance in prostate cancer cells by acetylating AR.