The Roles And Mechanisms Of Deubiquitinase USP33 In Castration Resistant Prostate Cancer

Background and ObjectiveProstate cancer is the common tumor in men, the incidence of which is highest in men in western countries, specially. The incidence of prostate cancer increased rapidly in China in recent years, becoming a serious tumor and attracted people’s attention. The main treatments for prostate cancer include radical prostatectomy, radiotherapy, androgen deprivation therapy(ADT) and so on. For advanced and metastic prostate cancer, ADT was the first option. However, after response to ADT, the majority prostate cancer of patients will become castration resistant prostate cancer(CRPC). There is no effective therapeutic regimens for CRPC till now. So the treatment for CRPC was the difficulty of clinical treatment, and which has become the hot spot of prostate cancer research.Ubiquitin modification is one of the most important post-translational modification, which can modificate specific substrate protein with different type of ubiquitin chain and leading to the effect of degradation, activation and the change of intracellular localization of substrate. It plays an indispensable role in nomal physiological activity of cell. However,dysfuction of ubiquitin modification could affect the celluler fuction, which lead to cancer or other diseases. Numerous researches indicated that ubiquitin modification plays an important role in tumorigenesis and cancer progression. Deubiquitinases(DUBs) is a key enzymas can reverse the process of ubiquitin modification. It plays a role in the progression of cancer by different fuctions and mechanisms. Previous studies have been reported that the expression of USP33 in Ln Cap cell line was increased significantly when prostate cell line stimulated by androgen, which indicated USP33 may have essential role in prostate cancer. The objective of this research was to discuss the role and mechanisms of deubiquitinase USP33 in prostate cancer, especially in castration resistant prostate cancer.Materials and MethodsRWPE, Ln Cap, PC3, DU145, Ln Cap-AI, Lncap C4-2, 22RV1 cell lines were cultured, and the different specimens were collected for this research. The USP33 m RNA in these cell lines were mesured by RT-PCR. The cell growth, migration, invasion and apotosis for cell lines were analysed by CCK8, Transwell and flow cytometry separately.The specific substrate of USP33 was found by co-immunoprecipiation plus massspectrometry. The interaction between USP33 and its substrate was verified by western blotting. The mechanism were also analysed by co-immunoprecipiation and western blotting. Tumor bearing nude mice and intratumor injection modle were established.Statistical analysis was performed by SPSS software.ResultsThe expression levels of USP33 in prostate cancer cell lines were higher than in nomal cell line, especially in androgen independent cell lines. The expression level of USP33 in the tissue of prostate cancer was also higher than benign prostatic hyperplasia(BPH) samples. USP33 could promote the viability, migration and the invasion of prostate cancer cell lines. PC3 cell line was typical androgen independent prostate cancer cell line,so we choose PC3 cell line to be our focused cell line for the further research. The Flow cytometry results showed that USP33 can inhibit the apoptosis of PC3 cell line by using the Annexin V kit. Western blotting results showed that USP33 could inhibit the activation of JNK1/2 and P38 in PC3 cell line. Mass spectrometry assays show that the DUSP1 might be a potential substrate of USP33 and the interaction between them was confirmed in PC3 cell line. And USP33 could stabilize the protein level of DUSP1 in PC3 cell line by decrease the K48 poly-ubiquinatinated level of DUSP1. By doing the rescue experiments,we found that DUSP1 and the activity of P38 and JNK1/2 were required for the effect of USP33 in regulating the apoptosis of prostate cancer. At last, we found that knockdown of USP33 could inhibit tumor progression in tumor-bearing nude mice model of PC3 cell line.ConclusionUSP33 could inhibit the apoptosis and promote the viability of prostate cancer cell line by decrease the K48 poly-ubiquinatinated level of DUSP1 and in a DUSP1, P38 and JNK1/2 dependent way. USP33 was a potential therapeutic target of castration resistant prostate cancer.