The Interplay And Mechanism Study Of Spermine Metabolism And PRMT1 In Prostate Cancer

Prostate cancer is the leading cause of morbidity and mortality in men,and the mortality is second only to lung cancer.Hormone receptor AR is the main transcription factor in prostate tumor.The overactive AR signaling pathway is the main cause of prostate tumor development.At present,targeted inhibition of AR can only play an anti-tumor effect at an early stage.With the progression of tumor,there will be drug resistance,resulting in higher fatality rates.It is urgent to develop a new treatment method for prostate cancer.The development of tumor involves abnormal changes and interactions of epigenetics,cancer metabolism,tumor microenvironment and many other factors.Although the genomics and proteomics of prostate tumor have been extensively studied,little is known about their metabolomics changes and functions.Understanding the metabolic characteristics of tumor progression will help us understand the characteristics of prostate tumor more comprehensively and help to intervene their progression.The metabolites from clinical tissue samples of prostate tumor were tested and analyzed.A number of metabolite molecules,including creatine,citric acid and spermine,were found.The content of spermine is negatively correlated with the malignancy of prostate tumor.It can be used as the"biomarker"to evaluate cancer process.However,there is still a gap in the study of the biological function and mechanism of spermine in tumor.Therefore this paper mainly studies the function of spermine in prostate tumor.Firstly,in chapter 2 the effect of spermine on tumor growth was investigated.It was found that the growth of AR-positive prostate tumor cells and organoids could be inhibited more significantly with spermine treatment in 11 prostate tumor cells and organoids.At the animal level,spermine also inhibited the growth of 22RV1xenograft tumor,and had cooperative anti-tumor effect with enzalutamide.RNA-seq analysis with spermine treatment was carried out,it was found that compared with enzalutamide,spermine could both inhibit the AR signaling pathway and the AR-V7signaling pathway.The sequencing results were verified with q PCR and Western Blot experiments.Intervention in the metabolic enzymes of spermine allowed spermine to accumulate in the cells,which could also inhibit the AR signaling pathway.AR and AR-V7 signaling pathways in tumor tissues could also be downregulated at animal level.Chapter 3 mainly discusses the mechanism of spermine inhibiting AR and AR-V7 signaling pathway.Using computational method and biological experiments,we found that spermine could inhibit the enzymatic activity of type I protein arginine methyltransferase（PRMTs）at protein level and cellular level.The molecular level IC₅₀s were from 4μM to 40μM.PRMTs could regulate DNA transcription,RNA splicing and many other important biological processes,their expression level ofen increases when tumors develop.PRMTs could also affect prostate tumor growth,a lot of studies showed that PRMTs could enhance the AR signaling pathway.In order to further study the biological functions of spermine and type I PRMTs in prostate tumor,the compounds P7 and C26 were developed based on human PRMT1 and PRMT4 protein structures,through virtual screening,molecular docking,chemical synthesis and experimental verification at molecular and cellular levels,their activity and selectivity were both improved siginificantly.Compound P7 could inhibit PRMT1,PRMT6,PRMT8,with IC₅₀values from 0.5 n M to 2.5 n M,Compound C26could also inhibit PRMT4 apart from PRMT1,6,8,with IC₅₀values from 8 n M to30 n M.The mechanisms of spermine and type I PRMTs in prostate tumors was investigated more intensively in Chapter 4.Based on the expression levels of PRMTs in prostate tumor and their respective correlation with AR signaling pathway,it was further confirmed that spermine mainly interacted with PRMT1 in prostate tumor.It was confirmed that spermine could bind directly with PRMT1 through NMR experiment and molecular docking.Genetic intervention of PRMT1 at the cellular level or treatment with PRMT1 small molecule inhibitor could both inhibit tumor cells growth and proliferation,menawhile downregulate AR and AR-V7 signaling pathways.PRMT1 inhibitor also inhibited castration resistance model 22RV1xenograft tumor growth at animal level,and had cooperative anti-tumor effect with enzalutamide.The important function of PRMT1 in prostate tumors thus was confirmed.Spermine and PRMT1 small molecular inhibitor could both inhibit the transcriptional activity of AR in dual luciferase reporter assay.An ATAC experiment found that both spermine and PRMT1 inhibitor could inhibit the chromatin open state of genes in AR and AR-V7 signaling pathways,therefore inhibit their transcriptional expression.Compared with the wild type cells,spermine had weaker effect on AR,AR-V7 and their target genes expression in PRMT1-kockdown cells,indicating that spermine inhibited AR,AR-V7 signaling pathways through inhibiting PRMT1.This conclusion was further confirmed by H4R3me2a Ch IP-q PCR assay,in which H4R3me2a abundance at the AR promoter regions was reduced by spermine.H4R3me2a is an important transcription activating marker methylated by PRMT1.In this study,it was found that the metabolite spermine could selectively inhibit the growth of AR-positive prostate tumors at the cellular and animal levels.In the molecular mechanism,spermine could bind with and inhibit PRMT1,resulting the decreased level of H4R3me2a at AR promoter regions,and inhibit the chromatin open state of AR,AR-V7 and target genes,leading to less active AR and AR-V7signaling pathway.In this study,it was first clarified that spermine could inhibit prostate tumor growth through regulating epigentic modification.When compared with enzalutamide,spermine and type I PRMTs inhibitors could not only reduce AR signaling pathway,but also inhibit the expression of AR-V7 and target genes,overcoming the drug resistance of prostate tumors,and showed promising clinical application.