Study On The Mechanisms Of Androgen Deprivation Therapy Activating Siah2 Ubiquitin Ligase To Promote CPPC Formation

Objective:Androgen deprivation therapy(ADT)is always regarded as first-line treatment for metastatic prostate cancer(PCa).Although ADT initially effective,almost all patients eventually progress to castration-resistant prostate cancer(CRPC).The E3 ubiquitin ligase Siah2 is considered to contributes to foramation of CRPC,however,the mechanism is still unknown.The study focuses on the mechanisms of the regulation of ADT on Siah2 Ubiquitin Ligase Activity,which provides a new idea of the treatment of progressive PCa.Methods:Part I:1.LNCa P and 22RV1 prostate cancer cells were treated with dihydrotestosterone(DHT),Western Blot and Real-time PCR were used to detect the protein and m RNA expression levels of Siah2.PCa cells treated with or without DHT were then treated with protein synthesis inhibitor cycloheximide(CHX)at 50 μg/ml for various times.Western Blot was used to detect the protein degradation of Siah2.Use si RNA and Flutamide to inhibit AR and Western Blot was used to detect the protein expression level of Siah2.293 T wells transfected with different dose of AR plasmid and Siah2 plasmid and then Western Blot was used to detect the protein expression level of Siah2.2.Immunoprecipitation was used to detect the self-ubiquitination level of Siah2.LNCa P and 22RV1 prostate cancer cells were treated with dihydrotestosterone(DHT),Western Blot was used to detect the protein levels of Spry2 which is identified as the substrates of Siah2 and represent the activity of Siah2 ubiquitin ligase.Use si RNA and Flutamide to inhibit AR and Western Blot was used to detect the protein expression level of Spry2.293 T wells transfected with AR,Siah2 and Spry2 plasmids and then treated with CHX for various times.Western Blot was used to detect the protein degradation of Spry2.3.Phenol red-free medium supplied with 5% dextran coated charcoal-stripped fetal bovine serum(CS-FBS)is widely used to mimic castration condition and can be identified as ADT.Western Blot was used to detect Siah2 and Spry2 protein expression level in PCa cells after ADT.PCa cells were maintained in phenol red-free medium supplied with 5% dextran coated CS-FBS and then treated with CHX for various times.Western Blot was used to detect the protein degradation of Siah2 and Spry2.4.Immunohistochemistry was used to detect the Siah2 expression in human PCa samples including the same patient after transrectal prostate puncture and after radical prostatectomy with ADT.Part II1.Western Blot was used to detect the protein expression of Siah2 and Spry2 and CCK-8 was used to detect cell proliferation after treating PCa cells with vitamin K3(Vit K3).2.Use LNCa P to conduct xenograft model in nude mice.Nude mice were divided into the normal control group,castration group and castration combined with Vit K3 therapy group and tumor volumes were measured twice one week.Western Blot was used to detect AR and Siah2 expression level in Xenograft tumors.Results:Part I:1.Treating LNCaP and 22RV1 prostate cancer cells with DHT,we have observed a obvious increasing protein level of Siah2.However,there has no changes in the m RNA level.After treating PCa cells with cycloheximide,the degradation of Siah2 shows more slowly in the presence of DHT.Use si RNA and Flutamide to inhibit AR and we found a reduction in Siah2 protein level.2.We performed immunoprecipitation analysis in 293 T cells overexpressing FLAGSiah2,HA-ubiquitin and HA-AR.We found that Siah2 was more self-ubiquitinated in the absence of HA-AR compared with that in the presence of HA-AR.Western Blot was used to detect the protein levels of Spry2 and found decreasing Spry2 expression when adding 10 n M DHT to PCa cells.Furthermore,we monitored opposite changes in Spry2 expression when using si RNA and Flutamide to inhibit AR.293 T wells transfected with AR,Siah2 and Spry2 plasmids and then treated with CHX for various times.The degradation of Siah2 shows more slowly in the presence of AR plasmid.3.Western blotting results indicated that the Siah2 and Spry2 protein level is notably higher in cells maintained in growth media with CM-FBS compared to with CS-FBS.Cycloheximide chase experiment showed that Siah2 and SPRY2 degraded more quickly in CS-FBS growth media than in CM-FBS growth media.4.Immunohistochemistry was used to detect the Siah2 expression in human PCa samples after radical prostatectomy and ADT respectively and wo found that Siah2 expression was decreased after ADT.Part II1.Western Blot results showed that addition of Vit K3 can enhance the expression of Siah2 and Spry2,which means Vit K3 is able to inhibit Siah2 ligase activity in prostate cancer cells and attenuates ADT effect on Siah2.Furthermore,Vit K3 can inhibit the proliferation of AR positive PCa cells but not AR negative PCa cells.2.Animal experiments found that ADT combined with Vit K3 therapy could inhibit the growth of prostate xenograft tumor contrast to castration group and control group.We might conclude that ADT combined with Vit K3 therapy delays the development of PCa.Conclusion:1.Androgen stabilizes Siah2 protein expression through inhibiting Siah2 selfubiquitination2.ADT increases Siah2 ubiquitin ligase activity in PCa cells.3.Siah2 inhibitor vitamin K3 attenuates ADT effect on Siah2 and inhibit the proliferation of AR positive PCa cells4.Animal experiments found that ADT combined with Vit K3 therapy could significantly delay the formation of CRPC.