Functional And Mechanistic Studies Of Histone Methyltransferase KMT2D Loss Induced Oxidative DNA Damage In Prostate Cancer

Background and objectiveProstate cancer(PCa)is the second malignancies in men worldwide and is considered to threaten the health of elder men.PCa is a heterogenous disease and evidence shows that the incidence,prognosis and treatment response are various across different regions,indicating the molecular heterogeneity.However,we notice most of gene sequencing studies and the clinical correlation analysis are restricted in Western populations,and rare studies are performed for Asian patients.Moreover,although lots of genetic alterations have been identified by the sequencing studies,most of them still need to be functionally analyzed for a better understanding of PCa.Histone lysine(K)methylation transferase KMT2D is an epigenetic enzyme that catalyze the formation of H3K4me1.The H3K4me1 is found to accmulate at enhancers and considered as a principal marker of active enhancers.In PCa,our previous study showed that KMT2D sustained prostate carcinogenesis and metastasis via epigenetically activating LIFR and KLF4.However,given the divergent functions of KMT2D in other cancers,the pathophysiological function of KMT2D in PCa is likely to be complicated and needs to be comprehensively investigated.Results and conclusion1.Integrative molecular characterization of Chinese prostate cancer specimensIn this study,we applied an integrative genetic/transcriptomic assay that combines targeted next-generation sequencing and quantitative real-time PCR(qRT-PCR)on samples from 46 Chinese patients with PCa.KMT2D,ZFHX3,AKAP9 and GLI1 were frequently mutated in our cohort.Moreover,a clinicopathological analysis showed that RB1 deletion was common in patients with a high risk of disease progression.Remarkably,four genomic events,MYC amplification,RBI deletion,APC mutation or deletion,and CDK12 mutation,were correlated with poor disease-free survival.In addition,a close link between KMT2D expression and the AR signaling pathway was observed both in our cohort.In summary,our results demonstrate the feasibility and benefits of integrative molecular characterization of PCa samples in disease pathology research and personalized medicine.2.Loss of KMT2D induces prostate cancer ROS-mediated DNA damage bysuppressing the enhancer activity and DNA binding of FOXO3In current study,we found that knockdown of KMT2D sensitized cells to DNA damage via the comet assay,immunofluorescence staining and flow cytometry.Meantime,through the CellROX intracellular eactive oxygen species(ROS)analysis,we found the level of ROS was also increased in the KMT2D-loss cells,while treating the cells with antioxidant NAC could reverse the DNA damage induced by KMT2D depletion.The apoptosis,cell cycle and cell senescence assay reveled that the ROS-mediated DNA damage led to cell apoptosis and senescence.Mechanically,after KMT2D knockdown,the antioxidative gene expression was significantly decreased.Interestingly,ChIP-PCR results showed the DNA binding of FOXO3,a critical mediator of the cellular response to oxidative stress,was simultaneously attenuated in KMT2D deficiency cells.Finally,we concluded that in PCa,the loss of KMT2D reduced the abundance of enhancer activity markers H3K4mel and H3K27ac,which blocked the DNA binding of FOXO3 and suppressed antioxidative gene transcription.Moreover,KMT2D deletion in PCa cells also increased their sensitivity to genotoxic anticancer drugs and a PARP inhibitor,which suggested that lower levels of KMT2D may mediate the response of PCa to particular treatments.These findings further highlighted the important role of KMT2D in PCa progression and suggested that targeting KMT2D might be therapeutically beneficial for advanced PCa treatment.