PKMYT1 Promotes The Progression Of Castration-resistant Prostate Cancer By Activating The AR Signaling Pathway

Prostate cancer is the most common malignancy of the male reproductive system.According to the 2020 global cancer statistics,prostate cancer has become the fourth largest tumor in the world.In 2020,the number of new cases of prostate cancer in the world was 1.41 million,accounting for 7.3%of the total number of new cancer cases.The number of deaths was 370000,making it the second most common malignancy in men.Although the incidence of prostate cancer in China is lower than the European and American countries,with the development of economy,ageing population,obesity,and prostate specific antigen（PSA）screening application gradually,the incidence of prostate cancer in our country is rising year by year,annual growth rate of 8.92%,annual mortality of 13.37%.Therefore,how to accurately diagnose and treat prostate cancer are a major medical problem to be solved.Prostate cancer is highly heterogeneous and hormone-sensitive.Currently,androgen deprivation therapy is still the preferred treatment for advanced prostate cancer,but after 14 to 30 months of treatment,almost all patients eventually develop into castration-resistant prostate cancer（CRPC）with poor prognosis.The results of our research and other research groups show that the occurrence mechanism of CRPC is androgen receptor（AR）dependent.The main mechanisms of AR dependent type include amplification and/or mutation of AR gene.production of splice variant,abnormality of co-regulating factors,endogenous androgen synthesis,abnormal expression of AR posttranslational modification,and non-liganddependent activation of AR.Due to the complex molecular mechanism of the occurrence and development of CRPC,it is of great significance to effectively identify and target the key oncogenes and related signaling pathways that promote the progression of CRPC,and to further explore their biological phenotypes and mechanisms of action in CRPC for the establishment of intervention strategies to overcome CRPC.Part Ⅰ PKMYT1 is overexpressed in CRPC and promotes development and progression of CRPCProstate cancer is highly heterogeneous and hormone-sensitive.Androgen promotes the proliferation,migration and invasion of prostate cancer cells and anti-apoptosis by activating the AR signaling pathway.After castration,although the level of androgen in the systemic circulation has decreased,AR and AR mediate functions in the body continue to remain active.It has been reported in the literature that some oncogenes and signaling pathways play a crucial role in maintaining the phenotype of prostate cancer cells and in the process of malignant transformation of prostate cancer cells.As an oncogenic gene,PKMYT1 promotes tumor cell proliferation and inhibits apoptosis in gastric cancer.In ovarian cancer and esophageal squamous cell carcinoma,it promotes cell proliferation,migration and invasion,changes the cycle distribution of esophageal cancer cells and inhibits apoptosis.In this part,clinical specimens of prostate cancer and prostate cancer cell lines are used as research objects to study and analyze the role of PKMYT1 at the clinicopathological and cellular levels respectively,and the following results are obtained:1.PKMYT1 is highly expressed in CRPC tissues and is associated with poor prognosis,tumor biochemical recurrence and aggressiveness in prostate cancer patients:We first used GEO（Gene Expression Omnibus）database to investigate the expression of PKMYT1 in mRNA benign proliferative prostate tissues,primary localized prostate cancer,and CRPC.Data analysis results showed that PKMYT1 expression was significantly increased in CRPC tissues compared with localized prostate cancer.In this study,we collected 30 samples of localized prostate cancer and 24 samples of CRPC from the Department of Pathology of Qilu Hospital of Shandong University from 2013 to 2018 for immunohistochemistry（IHC）staining.The statistical results showed that compared with localized prostate cancer,PKMYT1 was more highly expressed in CRPC tissues.Then we used the TCGA（The Cancer Genome Atlas）database,and the analysis results showed that the high expression of PKMYT1 was positively correlated with Gleason score,lymph node metastasis,clinical stage and pathological stage,and negatively correlated with survival prognosis.2.PKMYT1 promotes the proliferation,migration and invasion of prostate cancer cells:In order to select a suitable cell line,the protein expression level of PKMYT1 in prostate cancer cell lines are firstly detected.As CRPC cells,C42B with the highest expression level of PKMYT1 and CRPC cell 22RV1 with a slightly higher expression level were selected according to the results of Western blot.LNCaP,as a low hormone sensitivity expression was used as the cells in this study as well.Before the phenotypic study,we detected the transfection efficiency of the small interference and the plasmid of PKMYT1.Western blot results showed that the small interference of PKMYT1 transfected in C42B and 22RV1 resulted in a decrease in the protein level of PKMYT1,while the transfected plasmid of PKMYT1 resulted in an increase in the protein level.In order to analyzed the effect of PKMYT1 on prostate cancer cells,two experimental methods,MTS and EdU were used to detect the cell proliferation efficiency of PKMYT1 in the presence of FBS and CSS.The results of MTS and EdU experiments showed that PKMYT1 knockdown could inhibit cell proliferation under both FBS and CSS conditions,and PKMYT1 high expression could promote cell proliferation.Transwell detected the metastatic and invasive ability of PKMYT1 against prostate cancer cells.The results showed that PKMYT1 knockdown decreased the migration and invasion ability of prostate cancer cells in both FBS and CSS states.The high expression of PKMYT1 enhanced the migration and invasion of prostate cancer cells.3.PKMYT1 regulates the cycle distribution of prostate cancer cells and inhibits the occurrence of apoptosis:Long et al.reported that PKMYT1 could change the cycle distribution of lung adenocarcinoma,and Sun et al.reported that inhibition of PKMYT1 could promote apoptosis in non-small cell lung cancer.Therefore,in order to analyze the effect of PKMYT1 on the change of cycle distribution and apoptosis of prostate cancer cells,flow cytometry was used for detection and analysis.The results showed that compared with the control group,the ratio of G0/G1 phase in the cell cycle was decreased,the ratio of G2/M phase was increased and the apoptosis rate was increased after PKMYT1 knockdown.This study confirmed the high expression of PKMYT1 in CRPC at both clinical samples and cell lines.In the study of cell phenotypes,PKMYT1 could promote the proliferation.migration and invasion of prostate cancer cells under both FBS and CSS states.In addition,PKMYT1 could also affect the periodic distribution of prostate cancer cells and inhibit the occurrence of apoptosis.Part Ⅱ Molecular characterization of PKMYT1 in promoting CRPC via activation of AR signaling pathwayCRPC is the final stage of prostate cancer progression.Although its growth is not dependent on androgen,AR signaling pathway still plays an important role in the progression of CRPC.Post-translational modifications of AR,especially phosphorylation,have been reported to regulate subcellular localization of AR and transcription of downstream target genes.This provides a new idea for the mechanism of promoting the progression of prostate cancer.PKMYT1 can activate a variety of oncogenic signaling pathways in tumors,including MAPK signaling pathway,Notch signaling pathway,AKT/mTOR signaling pathway,etc.By analyzed the cell sequencing datas,we found that PKMYT1 was related to AR and MAPK signaling pathways in prostate cancer cells.It has been reported that the phosphorylation state of MAPK pathway increases the sensitivity of AR to DHT,and promotes AR entry into the nucleus to play a transcriptional regulatory role.In this study,the prostate cancer cell lines were taken as the research object to systematically explore the mechanism of PKMYT1 in the progression of CRPC,and the following results were obtained:1.PKMYT1 is an androgen responsive gene:In order to study the changes in the expression level of PKMYT1 under CSS,hormone-sensitive LNCaP was selected.Western blot results showed that,compared with FBS,the expression of PKMYT1 in LNCaP cells under CSS increased in a time-dependent manner.Then,LNCaP was cultured in CSS for 3d.treated with hormones and set different time and concentration gradients.The results of qRT-PCR and Western blot showed that the expression of PKMYT1 in mRNA and protein levels decrease in a time-dependent and dose-dependent manner with the increase of hormone treatment time and dose.Finally,C42B cells were treated with enzalutamide（ENZ）,the antagonist of AR,after the addition of hormones.QRT-PCR and Western blot results showed that the mRNA and protein levels of PKMYT1 decreased after the addition of androgen.The expression of PKMYT1 mRNA and protein levels were reversed after the addition of androgen combined with ENZ treatment.2.PKMYT1 is an AR inhibitory gene:In this study,the correlation between PKMYT1 and AR expression was further detected.The results of qRT-PCR and Western-blot showed that after AR knockdown in LNCaP and C42B cells,the expression levels of PKMYT1 both protein and mRNA levels were increased,while after AR high expression in DU145 cells,the expression levels of PKMYT1 both protein and mRNA levels were decreased.As a transcriptional regulator,AR may bind to the promoter of PKMYT1 and played a regulatory role.Four PKMYT1 promote sequences that AR may bind were analyzed and predicted by JSPAR website,and the binding of AR to PKMYT1 promoter was detected by ChIP assay.The data showed that AR could bind to the promoter region of PKMYT1 and inhibit it.Compared with CSS status,AR was enriched in promoter region of PKMYT1 in androgen-treated LNCaP cells.3.PKMYT1 activates downstream target genes of AR:Based on the research content of the first part,we speculated that AR and AR signaling pathway may be involved in PKMYT1 to promote the occurrence and development of CRPC.Therefore,WE extracted RNA from 22RV1 NC and 22RV1 Si-PKMYT1 cells for RNA-seq sequencing.After screening differential genes（fold change>1.5.p<0.05）,GSEA prediction software was used for analysis,and it was found that androgen-induced gene sets were significantly enriched in the 22RV1 NC side（p<0.01）.qRT-PCR results showed that knockdown of PKMYT1 inhibited the transcription of AR downstream target genes,and overexpression of PKMYT1 promoted the transcription of AR downstream target genes.However,after knockdown or overexpression of PKMYT1 in C42B and LNCaP cells,qRT-PCR and Western blot results showed that the mRNA and protein expression levels of AR did not change significantly.4.PKMYT1 promotes translocation of AR into the nucleus by phosphorylation of AR:We analyzed the effect of PKMYT1 protein expression on AR subcellular localization using cytoplasmic separation assay.Western blot results showed that AR entry into C42B cells could be inhibited by knocking down PKMYT1 in FBS and CSS cultures.Overexpression of PKMYT1 in LNCaP cells could promote the entry of AR into the nucleus.Co-IP assay was used to detect the binding of PKMYT1 protein to AR protein.Western blot analysis showed that PKMYT1 did not bind to AR in LNCaP and C42B cells.PKMYT1 is known to regulate serine/threonine kinases.Western blot results showed that the protein expression level of AR phosphorylated ser81（AR-S81）decreased after the knockdown of PKMYT1 expression in C42B cells.After PKMYT1 was overexpressed in LNCaP cells,the protein expression level of AR-S81 increased.It has been reported that in prostate cancer,the catalytic "C" subunit PP2Ac of PP2A phosphatase can directly bind to AR-S81 and dephosphorylate it.The expression of pPP2Ac-Tyr307 protein was negatively correlated with the activity of PP2A phosphatase.So does PKMYT1 regulate the protein expression level of AR-S81 by affecting the activity of PP2A phosphatase?Western blot analysis showed that the expression of PP2Ac did not change after PKMYT1 was knocked down in C42B cells,but the expression of p-PP2Ac-Tyr307 was significantly decreased,which increased the activity of PP2A phosphatase and decreased the protein expression of AR-S81.5.PKMYT1 activates the MAPK signaling pathway:Based on RNA-seq sequencing data in "3" and the differential genes screened under the same conditions（fold change>1.5、p<0.05）,we found that k-RAS-induced gene sets were significantly enriched on the 22RV1 NC side by using GESA prediction software（p<0.0001）;The MAP2K1-induced gene set was also enriched in the 22RV1 NC side（p<0.05）;GSEA prediction software was also used to screen the differential genes（fold change>1.5、p<0.05）GO functional enrichment analysis showed that differential genes were significantly enriched in MAPK signaling pathway.These datas suggest that PKMYT1 may regulate MAPK signaling pathway.Western blot analysis showed that the phosphorylation level of key proteins in MAPK signaling pathway decreased after PKMYT1 expression was knocked down in 22RV1 cells.In conclusion,PKMYT1 inhibited PP2A phosphatase activity and then increased AR phosphorylation level and promoted AR entry into the nucleus,thus promoting CRPC occurrence and development.PKMYT1 mediates abnormal activation of MAPK signaling pathway,which also plays an important role in the progression of CRPC.