In Prostate Cancer Cells, AR Regulates Cell Cycle And Metastasis Through CXCL13

The chemokine CXCL13 protein(CXC ligand 13),also known as BCA-1(B cell attracting chemokine 1),is a CXC sub-type member of the chemokine superfamily.CXCL13-CXCR5 axis is proved to be involved in regulating B lymphocyte homing migration and in promoting inflammation.In addition,CXCL13-CXCR5 signal pathway may participate in tumor development.It has been reported that CXCL13-CXCR5 axis acts as a new target for the detection and treatment of lymphoma.However,research on CXCL13 is in initiative stage,the molecular mechanisms of CXCL13-CXCR5 axis in the progression of PCa remain unclear.It is well known that AR(Androgen Receptor)plays a crucial role in the tumorigenesis and its development in prostate cancer.Therefore,by employing cellular biology and molecular biology approaches,we are making an attempt to investigate the relationship between CXCL13 and AR in PCa and to explore the role of CXCL13 in promoting cancer progression with the control of androgen receptor.This may provide people a novel clue to understand the etiology and development of prostate cancer.For exploring the regulating mechanisms of CXCL13-CXCR5 axis and AR,we designed the experiments as following described:First,we explored the expression of CXCL13 in mRNA and protein levels in normal cells and a set of PCa cell lines.An aberrant expression of CXCR13 was observed in all PCa cell lines,which was much higher than that of normal cell.Interestingly,we found that expression of CXCR13 in androgen dependent cell lines were all higher than that in androgen independent cell lines.It means that AR might relate to the expression of CXCL 13.Secondly,we found that expressions of PSA(a prostate epithelial cell specific and AR regulated gene)and CXCL 13 were up-regulated in LNCaP and CWR22Rvl cells after treating with the Mibolerone(Mib,a synthetic androgen)by comparing with non-treatment cells.Furthermore,we found that the increasing trend of CXCL 13 transcription level was consistent with the increased concentration of Mib.All these inferred that the expression of CXCL13 might be positively correlated with the activity of AR.Third,we employed normal LNCaP cells and LNCaP-AI cells(a kind of low androgen dependent LNCaP cell line),transfection with AR expression plasmid in LNCap-AI cells and knockout of AR gene expression with its siRNA in normal LNCaP cells.The experimental data showed that expression of CXCL13 was increased with the over-expression of AR and decreased with the knockout of AR.These results demonstrated that AR played key role and mediated the expression of CXCL13 gene.Fourth,by using JASPAR software on internet,we found some potential AR binding sites within the promoter area and enhancer area of CXCL13 gene.By combining with our data base of AR ChIP-seq(chromatin immunoprecipitation sequence),we found that CXCL13 enhancer exists two strong AR binding sites.These results made us more convince of that AR,as a transcription enhancer,mediated the expression of CXCL13 gene.Fifth,to study CXCL13 functions,we constructed expression plasmids of CXCL13 and CXCR5(receptor of CXCL13).After transfection of CXCL13 and CXCR5 in LNCaP cells,we find the expression of snail(associated with cell migration)and cyclinB1 were increased;and these effect were abolished by silencing the expression of CXCL13 and CXCR5 with their siRNAs,respectively.To further explore the function of secreted CXCL13,we collected the culture medium of LNCaP cells which over-expressed CXCL13 and used the medium to culture LNCaP cells.The results showed that the expression of snail and cyclinB 1 were also increased by comparing to normal medium.In addition,we identified the regulation of CXCL13 on snail and cyclinBl in androgen-independent PC3 cells.Furthermore,we did the transwell assay,scratches and FCM(flow cytometry)assays.The experimental results indicated that CXCL13 could promote PCa migration and invasion.Taken together,AR regulates the expression of CXCL13 by combining CXCL13 enhancer in prostate cancer cells.The expression of CXCL13 can up-regulate snail and cyclinB1,so as to promote the transfer of prostate tumors and regulate PCa cellular cycle.