Mechanistic Study Of Exosomal PGAM1 Targeting Combined With ACTG1 To Promote Prostate Cancer Metastasis Through Induction Of Angiogenesis

Background and purposeProstate cancer(PCa)is one of the most prevalent malignancies affecting men’s health,and angiogenesis is a key step in tumorigenesis and metastasis.In the tumor microenvironment,exosome-mediated complex signaling networks and material transport between tumor and non-tumor cells are involved in essentially every stage of tumorigenesis and metastasis.Non-metabolic pathways are an important aspect of studying metabolic enzymes in tumor progression.Phosphoglycerate mutase 1(PGAM1)is a key glycolytic enzyme,and studies have shown that PGAM1 is involved in the progression of a variety of tumors,and recent studies have found that PGAM1 promotes tumor metastasis largely dependent on non-metabolic pathways.Our previous study found that PGAM1 expression was increased in PCa and was present in exosomes secreted by PCa cells.However,the molecular mechanism of exosomal PGAM1 in PCa metastasis has not been clarified.The aim of this study was to elucidate the novel mechanism by which exosomal PGAM1 promotes PCa angiogenesis.MethodsThe TCGA database was applied to verify the expression of PGAM1 and its clinical significance;exosomes secreted by normal human,PCa patient sera and normal prostate epithelial cells,PCa cells were extracted and identified using exosome extraction technique;the expression of PGAM1 and CD31 in PCa patients and paraneoplastic tissues were detected using immunohistochemistry(IHC)and correlation with clinicopathological parameters was analyzed;stable knockdown PGAM1 cell lines were constructed and exosomes from each group of cells were collected and co-cultured with vascular endothelial cells(HUVEC).The cloning assay was applied to detect the proliferation ability of HUVEC;the Transwell migration assay and wound healing assay were applied to detect the migration ability of HUVEC;the tubule formation assay and chick embryo chorioallantoic membrane vascular assay(CAMVA)were applied to detect the angiogenic ability of HUVEC.In HUVEC,GST-pulldown,Co-IP assay and bioinformatics were applied to find downstream molecules interacting with PGAM1;immunofluorescence technique(IF),gelatin degradation assay and animal experiments were applied to verify how PGAM1 promotes angiogenesis while participating in the metastasis of PCa.ResultsPCa patients who developed metastasis were accompanied by high expression of PGAM1.The upregulation of exosomal PGAM1 was found to be positively correlated with PCa metastasis progression.IHC showed that CD31 expression was positively correlated with Gleason score in PCa patients.western blot demonstrated that PGAM1 content in exosomes was consistent with PGAM1 content in PCa cells.Uptake experiments demonstrated that PCa exosomal PGAM1 had enhanced proliferation,migration,and angiogenesis of itself after ingestion by HUVEC.GST-pulldown,Co-IP experiments demonstrated that PGAM1 and ACTG1 interact and that they co-localize in the cytoplasm.IF found increased podosomes production and neovascular sprouting after HUVEC uptake of PCa exosomal PGAM1.On the other hand PGAM1 in PCa cells also promoted increased invadopodia production leading to PCa metastasis.Animal experiments further corroborate that exosomal PGAM1 can promote PCa metastasis by inducing angiogenesis.ConclusionThe expression level of PGAM1 in PCa exosomes is positively correlated with poor prognosis and malignancy of patients.after the uptake of exosomal PGAM1 by HUVEC,PGAM1 and ACTG1 act in combination with each other,triggering increased podosomes and neovascularization in HUVEC;meanwhile,PGAM1 in PCa cells also binds to ACTG1 leading to increased PCa cell invadopodia formation increases,provoking a cascade response of tumor metastasis.In conclusion,this study illustrates that exosomal PGAM1 regulates the metastatic progression of PCa by targeting binding to ACTG1 to promote angiogenesis,which is expected to provide a new theoretical basis and molecular target for the diagnosis and prognosis of PCa patients.