CXCL13 Participates In The Molecular Mechanism That AR Regulates The Migration, Invasion And Cycle Transition Of Prostate Cancer Cells

Prostate cancer(PCa)is the most commonly diagnosed malignancy and the predominant causes of cancer death among men in the western countries.Androgen have always been the chief driving factor for PCa which plays a significant role in the progression of prostate cancer.Although it is well established that androgen and AR promote prostate cancer cell metastasis,the molecular mechanisms of androgen-mediated cell metastasis remains poorly understood.Chemokines(chemotactic cytokines)are a family of low molecular weight cytokines,that have a crucial regulation effect in diverse immunoinflammatory responses by activation of specific leukocytes through interacting with their specific receptors.CXCL13 is demonstrated in regulating lymphocyte migration and promoting inflammation and acts as a new targets for the detection and treatment of lymphoma,and CXCL13 is overexpressed in breast cancer and colon cancer patients which is tightly related with the poor prognosis and overall survival of patients.In prostate cancer,that serum CXCL13 levels is positively correlated with prostate specific antigen and prostatic disease,and PCa cell lines selectively express certain matrix metalloproteinases(MMPs)in response to aberrant expression of CXCL13,which may support PCa cell migration and invasion by degrading extracellular matrix(ECM)components.However,the relationship between AR and CXCL13,and the effect of CXCL13 in AR mediated prostate cancer procession is still unknown,therefore we designed the experiments as follows:Firstly,by performing quantitative reverse transcription PCR and immunohistochemistry assays,CXCL13 had increased expression in the PCa tissues compared with the para-tumorous tissues at both mRNA and protein levels.Furthermore,we found that the levels of CXCL13 were abundant in androgen-responsive LNCaP and CWR22Rv1 cell lines compared to androgen-unresponsive DU 145 and PC3 cell lines at both mRNA and protein levels,and the expression of CXCL13 in normal prostate epithelial cell line WPMY-1 were the lowest.Thus,the results indicated that the high expression of CXCL13 was significantly correlated with prostate tumor in clinic,and the expression of CXCL13 might be regulated by Androgen-Androgen Receptor axis.Secondly,our results revealed that a synthetic potent anabolic androgen Mibolerone(Mib)has a dramatic effect on the induction of CXCL13 expression at both mRNA and protein levels after hormone-stripped treatment in LNCaP and CWR22Rvl cell lines.Next,by analyzing the AR ChIP-seq data in LNCaP cells,we found that there exist two obvious AR binding peaks located within CXCL13 intron Ⅰ.Corresponding to the two peaks of CHIP-seq results,there were two AR-binding sites(called as ARBS-1 and ARBS-2)in CXCL13 intron Ⅰ.By further using transcription factor binding sites prediction website JASPAR,four and one canonical androgen responsive elements(ARE)sequences were separately identified in ARBS-1 and ARBS-2 within CXCL13 intron Ⅰ.In addition,by performing luciferase reporter assays and chromatin immunoprecipitation(ChIP)assays,we demonstrated that Mib induced CXCL 13 increase is mediated by AR.Thirdly,LNCaP and CWR22Rv1 cells were cultured in 10%CSS medium immediately after endogenous CXCL13 were knocked down by siRNA for 48 hr,and then treated with or without 10 nM Mib for another 24 hr,results showed that CXCL 13 plays a key role in AR induced up-regulation of ETS-1,snail,and Cyclin B1 levels.By performing wound healing assay and transwell migration and invasion assay,knocking-down of endogenous CXCL13 by siRNA largely diminished Mib-induced LNCaP cells migration and invasion.Furthermore,by performing flow cytometry analysis in LNCaP and PC3 cells,our results suggest that CXCL13 plays an important role in promoting G2/M cell cycle progression in prostate cancer cell lines.In summary,our study demonstrated that CXCL 13 is a downstream target gene of AR,and plays an key role in androgen-induced migration and invasion in PCa cells.Moreover,our results revealed that CXCL 13 promotes cell cycle progression at G2/M checkpoint by induced up-regulation of Cyclin B1.These data suggest that CXCL 13 plays an critical role in the progression of prostate cancer.