Cytotoxic Necrotizing Factor 1 Promotes Prostate Cancer Progression Through Activating The Cdc42–PAK1 Axis

Objective:Uropathogenic Escherichia coli(UPEC)is the leading cause of urinary tract infections(UTIs).UTIs caused by UPEC can induce cystitis,pyelonephritis,and prostatitis.UPEC plays a role in prostate cancer(PCa)progression.However,the mechanisms through which UPEC promotes PCa development are unclear.Cytotoxic necrotizing factor 1(CNF1)is one of the most important UPEC toxins and its role in PCa progression has never been studied.Monitoring and treatment of UPEC strains carrying cnf1 has important clinical significance in controlling PCa progression.This paper intends to solve the following three problems: firstly,to study the effects of CNF1 on PCa cells in vitro;secondly,to study the effects of CNF1 on PCa in vivo;lastly,to explore the molecule mechanism of CNF1 on PCa.Methods:1.The UPEC11 derived CNF1 was purified by nickle column.We detected the effects of CNF1 on the motility of PCa cells in vitro using wound healing,Transwell migraton and invasion assays.2.PC3 cell lines stably expressing CNF1 functional domain(CNF1c)and luciferase were selected.We constructed a tail vein injection mouse model.And luciferase stably transduced 22Rv1 cells treated with CNF1 were injected into the prostates of NOD/SCID mice.Bioluminescence imaging was performed to monitor the metastasis in vivo,which was verified by H&E staining and immunohistochemistry.3.We performed immunofluorescence and Western blotting(WB)assays to determine whether CNF1 can enter PCa cells and Pull-down assay to examine whether CNF1 can influence the activation levels of RhoA,Cdc42,and Rac1.4.The effects of inhibitors and siRNAs of RhoA,Cdc42,and Rac1 on CNF1 induced motility of PC3 cells were detected.The effects of Cdc42 on the movement of PCa cells were further analyzed after transfection of the wild type,constitutively active and dominant negative Cdc42 expression plasmids.5.The effects of PAK1 inhibitor,a Cdc42 downstream molecule,on CNF1 induced the motility of PC3 cells were detected.WB was performed to detect the phosphorylation of PAK1 at Thr423 after treatment with CNF1.The effects of PAK1 on the motility of PCa cells were further analyzed after transfection of the wild type,constitutively active and dominant negative PAK1 expression plasmids.6.Western blotting was performed to detect the expression of MMP2 and MMP9 in CNF1 treated PC3 and 22Rv1 cells.7.We performed IHC of pulmonary metastases from the mice injected by PC3 with CNF1 c expression to detect the MMP9 and phosphorylated PAK1 expression.8.The correlation of the phosphorylated PAK1 expression with histological grade of the PCa was analysed by IHC using a human tissue array.9.Other UPEC derived CNF1 was purified by nickel column.We used Transwell migration and invasion and WB assays to compare the effects of different UPEC derived CNF1 on PC3 cells.Results:1.CNF1 derived from the UPEC strain 11 generally promoted the migration and invasion of PCa cells in vitro.2.Mice intravenously receiving the PC3-CNF1 c had a higher level of lung metastasis,and mice orthotopically receiving 22Rv1-luci cells treated with CNF1 had a higher level of metastasis in various tissues and organs compared with the control group.CNF1 significantly promoted the in vivo metastasis of PC3 and 22Rv1.3.There was accumulation of CNF1 protein in the cytoplasm of PC3.RhoA,Cdc42,and Rac1 were activated in PC3 cells treated by CNF1,and CNF1's effect was stronger on Cdc42 and Rac1 than on RhoA.4.The enhancement of migration and invasion by CNF1 was attenuated by inhibiting Cdc42,but not Rac1.Inhibition of RhoA also significantly reduced PC3 migration and invasion in the control groups.The migration and invasion abilities of PCa cells transfected with constitutively active Cdc42 were significantly increased.5.Exposure of CNF1-treated PC3 cells to the PAK1 inhibitor decreased their migration and invasion.We found elevated phosphorylation of PAK1 at Thr423 after treatment with CNF1.The migration and invasion abilities of PCa cells transfected with constitutively active PAK1 were significantly increased.6.CNF1 up-regulated MMP9,but had no effect on MMP2 expression in PC3 cells.7.MMP9 and phosphorylated PAK1 expression were significantly enhanced in the pulmonary metastases of mice injected by PC3 cells with CNF1 c expression.8.In clinical samples,with the increase of histological grade,the percentage of samples with phosphorylated PAK1 staining and the levels of phosphorylated PAK1 were elevated.9.The different UPEC strain sources of CNF1 did not affect its effect on PCa cells,both significantly promoted the migration and invasion of PCa cells through the same mechanism.Conclusions:1.CNF1 can promote the migration and invasion of prostate cancer cells in vitro.2.CNF1 can promote the metastasis of prostate cancer in vivo.3.CNF1 can enter into the prostate cancer cells and activate RhoA,Cdc42,and Rac1.4.CNF1 promotes the migration and invasion of PCa cells by activating Cdc42.5.CNF1 can drive the migration,invasion and the metastasis of PCa cells through the Cdc42–PAK1–MMP9 pathway both in vitro and in vivo.6.Phosphorylation of PAK1 is positively correlated with the histological grade of the advanced human PCa.7.CNF1 derived from different UPECs has similar pro-migratory and pro-invasive effects on PCa cells.