The Role And Mechanism Of MYSM1 In Castration-resistant Prostate Cancer

【Backgrounds】Prostate cancer is one of the most common causes of cancer death in the world.Many patients with localized prostate cancer have undergone cancer recurrence and progression after radical prostatectomy and local radiotherapy.At the molecular level,androgen activates the androgen receptor,which in turn acts as a nuclear receptor transcription factor and involves in the expression of specific tumorigenic genes.Therefore,the treatment for advanced prostate cancer mainly focuses on inhibition of androgen synthesis and androgen receptor antagonist on the basis of androgen deprivation therapy.However,most cancers become drug-resistant and subsequently get castration-resistant.In most cases,CRPC still relies on AR signaling pathways.CRPC is so fatal that the improvement of treatment for prostate cancer is particularly urgent.How AR regulates CRPC growth is not fully elucidated but it is certain that AR is able to be activated at castration level of androgen through unknown mechanisms.Histone ubiquitination is one important forms of epigenetic modification,which is involved in transcription regulation,cell cycle and DNA repairment and other biological events.MYSM1 is a histone H2A-specific deubiquitinase.Its JAMM domain in N-terminal is a common domain of metallases that possesses the hydrolytic activity of metalloproteases and removes ubiquitin molecules.MYSM1 molecular structure determines its ability to remove ubiquitin of histones in the nucleus.Data available and our preliminary work confirmed that MYSM1 is very important in the development and function of hematopoietic stem cells and a variety of immune cells.Although histone ubiquitination is closely related with tumorgenesis,the role of MYSM1 in the tumor has not been reported.MYSM1 is validated to be involved in the maintenance of genomic stability and cell proliferation process,which suggests that MYSM1 may play an important role in tumorigenesis.To investigate the role of MYSM1 in tumors and its mechanism,this study intends to verify the correlation between MYSM1 and prostate cancer,identify its role in the development of prostate cancer,identify the target genes of MYSM1 and explore the mechanisms through which MYSM1 regulates gene transcription.This will provide us with potential targets and markers for the diagnosis and prognosis of prostate cancer.【Objective】To explore the biological function and mechanism of MYSM1 in castration-resistant prostate cancer.【Methods】(1)The expression of MYSM1 in prostate cancer was clarified by making use of the public data published in The Human Protein Atlas.(2)MYSM1 was down-regulated by RNAi and the expression of MYSM1 was examined by qRT-PCR and Western blot.(3)The effect of MYSM1 on the proliferation of castration-resistant prostate cancer cells was examined by MTT assay.(4)The effect of MYSM1 on the cell cycle and apoptosis of castration-resistant prostate cancer cells was examined by flow cytometry.(5)The effect of MYSM1 on the migration ability of castration-resistant prostate cancer cells was detected by wound healing test.(6)The effect of MYSM1 on tumorigenesis of castration-resistant prostate cancer cells was examined by subcutaneous tumorigenesis in nude mice.(7)The interaction between MYSM1 and AR was confirmed by Co-IP.The effect of MYSM1 on transcription was further verified by ChIP.(8)The effect of MYSM1 on Akt/GSK-3β/C-Raf signaling pathway was detected by Western blot.【Results】 1.The expression of MYSM1 in prostate cancer decreased.The expression of MYSM1 in prostate cancer tissues was validated by the figures found in the Human Protein Atlas.It was found that MYSM1 expression decreased in prostate cancer in comparison with normal prostate tissue.Besides,its expression was negatively correlated with the malignant degree of prostate cancer.2.MYSM1-low cell lines and control cell lines were established.The castration-resistant prostate cancer cell lines were infected by sh-lenti-MYSM1 and control lentivirus respectively and then were treated with puromycin for one week.All of the cells were green fluorescent under the fluorescence microscope.The mRNA and protein levels of MYSM1 in infected cells were detected by qRT-PCR and Western blot respectively.As a result,the mRNA and protein expression of MYSM1 were significantly down-regulated by lentivirus.3.MYSM1 can significantly inhibit the proliferation of castration-resistant prostate cancer cells.The expression of MYSM1 in castration-resistant prostate cancer cells was down-regulated by lentivirus.A series of functional experiments were performed in vitro and in vivo.MTT assay confirmed that down-regulation of MYSM1 significantly promoted the growth of prostate cancer cells.The results of flow cytometry showed that down-regulation of MYSM1 could decrease the number of G1 phase cells and promote cell cycle progression.Down-regulation of MYSM1 could significantly promote the tumorigenic ability of prostate cancer cells in nude mice.Detection of apoptosis and wound healing test showed that MYSM1 had no significant effect on the apoptosis and migration of prostate cancer cells.4.MYSM1 can interact with AR and mediated prostate cancer growth through AR signaling pathway.Studies have confirmed that MYSM1 can coordinate histone acetylation and deubiquitination and then participate in gene transcription.By analyzing the TCGA database,we found that MYSM1 was positively correlated with AR expression.But we have demonstrated that down-regulation of MYSM1 did not affect AR protein expression at cell level.Down-regulation of MYSM1 promoted the transcription of AR-target genes.The co-immunoprecipitation(Co-IP)assay confirmed that MYSM1 interacted with AR.Chromatin immunoprecipitation(ChIP)further confirmed that MYSM1 could regulate the chromatin structure of the androgen response element region,which affected the transcription of AR signaling pathway.5.MYSM1 can inactivate Akt/GSK-3β/C-Raf signaling pathway and inhibit the growth of prostate cancer.Androgen can not only function as a transcription factor through the canonical androgen receptor pathway to regulate expression of androgen response elements in the nuclear;it can also participate in tumor growth through AR-independent pathways by activating PI3K/Akt signaling pathway.We found that in addition to interacting with AR,MYSM1 has an impact on noncanonical PI3K/Akt signaling pathway.Down-regulation of MYSM1 could promote the phosphorylation of Akt,GSK-3β and C-Raf.Meanwhile,the expression of p53 decreased and the expression of c-Myc increased.These indicated that down-regulation of MYSM1 activated Akt/GSK-3β/C-Raf signaling pathway to accelerate the growth of prostate cancer.【Conclusion】MYSM1,as an important epigenetic molecule,plays an important role in the development and progression of prostate cancer.Its effect on proliferation may depend on its interaction with AR and its epigenetic regulation of AR signaling pathway.Besides,the activation of Akt/GSK-3β/C-Raf signaling pathway can make sense to the effect mediated by MYSM1 as well.