The role of osteoprotegerin and receptor activator of nuclear factor ᴋb in osteotropic prostate and breast cancers

Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor ᴋB (RANK) and RANK ligand (RANKL), are members of the tumour necrosis factor receptor superfamily (TNFRSF), signal transducers which have pleiotropic actions. Each family member has unique structural attributes shown to couple them directly to specific signalling pathways involved in cell proliferation, differentiation and survival. Previous studies have clinically correlated OPG, RANK and RANKL expression, at both transcript and protein levels, with increasing cancer tumour burden, metastatic bone involvement and androgen status, however the mechanisms by which these molecules exert their effects remain elusive. This study aimed to establish what influence targeting OPG, RANK and RANKL expression may have on osteotrophic prostate and breast cancer cells in vitro and to subsequently explore the effect(s) Hepatocyte Growth Factor (HGF) and Bone Matrix Extract (BME) might also exert on cancer cell behaviour following manipulation of these molecules. The current study utilised 2 prostate cancer cell lines with varying androgen status, metastatic potential and bone metastasis phenotypes. Initial screening showed that the more aggressive osteolytic PC-3 cells expressed OPG, whilst weakly metastatic mixed-osseous LNCaP cells had very low expression. Whilst RANK was present in both cell lines, RANKL expression was only detected in the LNCaP cells. Reduction of OPG expression in the PC-3 cells resulted in increased cell invasion in vitro, which was further enhanced when treated with BME. No other cellular traits were affected by targeting OPG directly, however, cell migration was enhanced when the manipulated cells were exposed to the representative bone microenvironment. In contrast the addition of a recombinant form of OPG to LNCaP cells resulted in decreased cell invasion, a trend which was reversed when combined with BME. Combination of OPG and BME treatment reduced the migratory response of LNCaP cells, whilst combination of OPG and HGF were pro-migratory. The targeting of RANK in PC-3 cells affected cell proliferation and matrix adhesion in vitro though the addition of HGF or BME appeared to have no further direct influence on these manipulated cells. Targeting of the RANKL expression with a neutralising monoclonal antibody had little effect on cancer cell behaviour; however combined exposure with HGF or BME resulted in similar behaviour patterns seen under the OPG treatments. In our breast cancer cohort, RANK and RANKL expression were correlated with bone metastases and survival rates. Though OPG did not appear to be associated with grading, data also implied a role in overall survival. In the aggressive osteolytic MDA-MB-231 breast cancer cells, reduced OPG expression resulted in increased motility and invasion, traits which were little affected upon exposure to HGF or BME. In contrast the targeting of RANK expression in MDA-MB-231 cells resulted in reductions in all the cancer cell behaviours studied, but again these appeared unaffected under the influence of HGF or BME. The complexity of the bone environment underpins the vast number of soluble factors, signalling pathways and transcription regulators which can influence osteotrophic cancer cells. As indicated by the licensing of Denosumab, one therapeutic approach is not suitable for all osteotrophic cancers. Therefore further elucidation into the intricacies of these interactions is needed