Induction of integrin α 2 in a highly bone metastatic human prostate cancer cell line: roles of RANKL and AR under three-dimensional suspension culture

Background Prostate cancer (PCa) bone metastasis can be markedly enhanced by increased receptor activator of NF kappa-B ligand (RANKL) expression in PCa cells. Molecular mechanisms that account for the increased predilection of PCa for bone include increased bone turnover, promotion of PCa cell growth and survival in the bone environment, and recruitment of bystander dormant cells to participate in bone metastasis. The current study tests the hypothesis that PCa cells acquire high adhesion to bone matrix proteins, which controls PCa bone colonization, under the RANKL/RANK and AR axes. Methods We used a highly bone metastatic RANKL-overexpressing LNCaP PCa cell line, LNCaP RANKL , as a model to pursue the molecular mechanisms underlying the increased adhesion of PCa cells to collagens. A three-dimensional (3-D) suspension PCa organoid model was developed. The functions of integrin α 2 in cell adhesion and survival were evaluated by flow cytometry and western blot. AR expression and functionality were compared in 2-D monolayer versus 3-D suspension cultures using AR promoter- and PSA promoter-luciferase activity. AR role in cell adhesion was assessed using an adhesion assay. Results LNCaP RANKL cells were shown to adhere tightly to ColI matrix through increased α 2 integrin expression. This increased adhesion, concomitant with activation of the FAK and Akt pathways, was further enhanced by culturing LNCaP RANKL cells in 3-D suspension. Under the influence of 3-D suspension culture, AR was restored in LNCaP RANKL cells via downregulation of AP-4 transcription factor, and supported increased α 2 integrin expression and adhesion to ColI. Conclusion 3-D suspension culture and in vivo PCa tumor growth restore AR through downregulation of AP-4, enhancing integrin α 2 expression and adhesion to ColI which is rich in bone matrices. The interactions of PCa with ColI, mediated by integrin α 2 and AR expression, could be a key molecular event accounting for PCa bone metastasis.