Role And Mechanism Of PLXNA1 In Prostate Cancer Progression And Its Clinical Application

Background:Prostate cancer is currently the number one malignant tumor in adult males worldwide,and the mortality rate is also ranked second in some areas,but its incidence and morbidity characteristics have shown significant differences in many countries in recent years.In Europe and the United States,the incidence rate is higher,while the incidence rate in Asian population is lower.However,in recent years,including Asian countries such as China,the incidence of prostate cancer has been rising quickly.In China it has rushed to the top ten of male malignant tumors.In recent years,people have increasingly realized that traditional clinical and pathological diagnostic indicators can no longer be fully used to predict prostate cancer prognosis of patients,and more were need to analyze prostate cancer from a deeper molecular aspect.Therefore,facing the rapidly increased incidence of prostate cancer and the significant differences between the characteristics of the prostate cancer population in the East and West,exploring the key factors affecting the pathogenesis of prostate cancer from the molecular level,looking for molecular markers that can effectively predict the prognosis of prostate cancer patients,and exploring the signaling pathway that affect the development of prostate cancer Access is especially important.Purpose:This current study is based on the results of the previous multi-omics sequencing data from a Chinese prostate cancer study group,to explore the signaling pathway closely related to the malignant progression of prostate cancer,to find genes closely related to the malignant progression of prostate cancer,and to explore the role of PLXNA1 in prostate cancer proliferation,invasion and migration.The prognostic ability of PLXNA1 in prostate cancer was analyzed in two independent postoperative radical prostatectomy groups from a Chinese population and a Caucasian population.Through molecular bioinformatics analyses,cell biology experiments and other methods,the molecular mechanism of PLXNA1 in promoting the malignant progression of prostate cancer and its related regulation pathway in prostate cancer were studied.Methods:Through the sequencing data of prostate cancer sequenced in this study,we conducted research on genomic changes(including somatic mutations,copy number and structural variation)in which high frequency changes occurred,and comprehensively analyzed the new possible effects on prostate cancer.The signaling pathway for malignant progression and screening for an analysis that may play an important role in the malignant progression of prostate cancer.Through screening of the possible targets,bioinformatics analysis of data from multiple published databases were applied to explore its amplification in prostate cancer samples.We also explore the relationship between the expression level and the patient's Gleason score,tumor stage,etc.,to explore the difference in expression between prostate cancer and adjacent normal tissues,and the difference in expression between metastatic and nonmetastatic tumors.Through overexpressing of this gene by transfecting plasmid,and knock down the expression of the gene by shRNA and siRNA in prostate cancer,its effects on proliferation,invasion and migration ability of prostate cancer tumors are explored.By establishing a subcutaneous tumor model in nude mice,the effects on the proliferation of subcutaneous tumor on nude mice were explored.The effect of its effect on the expression of key molecules affecting the malignant progression of prostate cancer in subcutaneous tumors of nude mice was explored by immunohistochemical staining.We enrolled two independent post radical prostatectomy patient group from a Chinese population and a Caucasian population.We collected the clinical information,follow-up data on the prostate cancer biochemical recurrence,tumor metastasis and overall survival after radical prostatectomy.The expression in prostate cancer after radical prostatectomy was studied by immunohistochemical staining.The Kaplan-Meier method(also known as the product-limited method,KM method)was used to analyze the protein expression level and patient prognosis,including the relationship between biochemical recurrence,tumor metastasis and overall survival time of patients.The statistical difference of prognosis in patients with high expression and low expression was analyzed by log-rank test.Through Cox proportional hazard regression model,we enrolled the factors that might influenced the prognosis of prostate including Gleason score,preoperative PSA value,AJCC tumor T stage,postoperative margin by univariate analyses and multivariate analyses.We also calculated the relative risk ratio to judge whether it can be used as an important factor in monitoring postoperative tumor recurrence,progression and affecting the survival of patients.Thus,we hope to provide a better monitor and early clinical intervention of high-risk patients,which could further lay the foundation for further prospective clinical trials in the future.Through bioinformatics and protein immunoprecipitation(IP)experiments,we conducted a comprehensive analysis of the proteins interacting with them,and analyzed the possible downstream signaling pathway changes.Through overexpression and knocking down this gene,through protein immunization Western blotting(WB),IP experiments,lactic acid production experiments,we found and verified to influence the changes of downstream signaling pathways.Through bioinformatics experiments,in vivo ubiquitination experiments,drug treatment and protein half-life detection experiments,proteins that interacted with it and affect its protein levels were discovered.Their effects on downstream signaling pathways were also analyzed.Results:In this study,we analyzed the data of from a multi-omics sequencing data to find that there were some similarities between genomic mutations in Chinese male prostate cancer and TCGA(mainly males in a Caucasian population),but there are also significant differences in genetic changes and gene expression level.Furthermore,through a comprehensive analysis of multiple pathways,we found that high-frequency changes in the Axon Guidance signaling pathway in prostate cancer are closely related to the malignant progression of prostate cancer.Further,we found that high-frequency changes of PLXNA1 in the Axon Guidance signaling pathway,and comprehensive analysis of data from multiple databases showed that the expression of PLXNA1 was closely related to the degree of malignancy of prostate cancer in patients,and promotes the malignant progression of prostate cancer.By over-expressing and knocking down PLXNA1 in the prostate cancer cells,we found that knockdown of PLXNA1 significantly reduced the ability of prostate cancer cells to proliferate,invade and migrate,while overexpression of PLXNA1 promoted prostate cancer cells to proliferate,invade and migrate.PLXNA1 affected changes in prostate cancer cell proliferation,epithelial-mesenchymal transition,and neuroendocrine differentiation.Further animal experiments have also confirmed these results.Furthermore we enrolled a Chinese population study group(419 patients,median follow-up time of 52 months)and a Caucasian population(213 patients,median follow-up time 16.4 years)from two independent post radical prostatectomy study groups.The expression of PLXNA1 protein level was explored by immunohistochemistry.Through verification of this gene in this two large study groups,it was found that PLXNA1 is an important factor in identifying the malignant degree of prostate cancer.We also found that PLXNA1 is an independent risk factor of biochemical recurrence,tumor metastasis and overall survival after radical prostatectomy in patients.It can be used as a high-risk factor for the identification of postoperative tumor recurrence and metastasis in radical prostatectomy,and it is helpful to provide close monitor and early clinical intervention for high-risk patients.In a further mechanism study,we found that PLXNA1 interacted with OS-9 protein.When PLXNA1 was overexpressed,the interaction between HIF-1? and OS-9 is reduced.OS-9 promotes the degradation of HIF-1?.In vivo experiments showed that elevated PLXNA1 significantly promoted the stability of HIF-1? protein and increased its protein level.Knockdown of PLXNA1 expression significantly reduced the protein level of HIF-1?,and further caused the change of a number of key enzymes such as Hexokinase I(HXK I),GLUT-1,PDK-1,PKM,LDH,GLUT-1,VEGF in the downstream aerobic glycolysis and related regulatory signaling pathways of HIF-1?.These changes could further promote aerobic glycolysis process,facilitated tumor nutrition and tumor energy metabolism changes.We verified the interaction between PLXNA1 and Smurf2 by protein immunoprecipitation experiments.Further,we found that Smurf2 is an E3 ubiquitin ligase that interacted with PLXNA1.In vivo ubiquitination assay,and specific drug treatments confirmed that Smurf2 can promote the degradation of PLXNA1 through its ubiquitination,and further regulated tumor metabolism through the changes of PLXNA1-HIF-1? axis and several key enzymes in the downstream of aerobic glycolytic signaling pathway.Conclusion:In this study,we have analyzed the prostate cancer sequencing data in a Chinese population,and identified that PLXNA1 from Axon Guidance signaling pathway is an important regulator of malignant progression of prostate cancer.It can promote the proliferation,invasion and migration of prostate cancer.Bioinformatics analysis also shows the expression of PLXNA1 is closely related to the degree of malignancy of prostate cancer in patients.Data from two independent prostate cancer surgery groups of east and west showed that the expression of PLXNA1 in tumor tissues was an independent risk factor for biochemical recurrence,tumor metastasis and overall survival after radical prostatectomy.It can be used as a risk factor for the identification of tumor recurrence and metastasis after radical prostatectomy.Further mechanistic studies indicate that the protein level of PLXNA1 is regulated by the HECT family E3 ubiquitin ligase Smurf2,which promotes the ubiquitination degradation of PLXNA1,whereas PLXNA1 can bind to OS-9 protein and repressed OS-9 degradation of HIF-1?,thereby stabilizing HIF-1? at the protein level,and regulating tumor metabolism through promoting changes in key enzymes in the downstream aerobic glycolytic signaling pathway.In summary,we have studied the role and mechanism of PLXNA1 in promoting the malignant progression of prostate cancer from multiple aspects,and studied its regulatory mechanism in prostate cancer,and its role in survival and prognosis of patients.The current study provided the foundation for its further research and clinical translational applications.It also provided a reliable basis for further exploration of new therapeutic targets for prostate cancer and the provision of new clinical diagnosis and treatment.