Disruption of prostate cancer cell—macrophage—osteoclast crosstalk by a novel TRAF6 inhibitor

Tumour necrosis factor receptor-associated factor 6 (TRAF6) is a key regulator of the pro-inflammatory NFkB pathway and plays a key role in immunity and osteoclast formation through the regulation of RANK/CD40-mediated NFkB signalling. TRAF/NFkB signalling is implicated in cancer, but its role in the prostate cancer—immune cell crosstalk remains unknown. Here, we report that TRAF6 is highly expressed in a panel of androgen-independent, bone-seeking cell lines including C42B4, PC3 and DU145 cells compared to the androgensensitive LNCaP. Stable knockdown and the pharmacological inhibition of TRAF6 using the verified TRAF6 inhibitor 6877002 and its novel and more potent congener FSAS3 reduced PC3 cell viability (50% reduction, p.05), migration (23% reduction, p.05) and invasion (35% reduction, p.05). Conditioned medium from M2 macrophage—but not M0 and M1 phenotype—enhanced human PC3 proliferation, migration and invasion and these effects were significantly inhibited by treatment with FSAS3 (0.3-1lL—proliferation, 22% reduction; migration, 35% reduction; invasion, 79% reduction) (p.005). 6877002 and FSAS3 reduced the ability of M0 macrophage to form osteoclasts in the presence of RANKL and PC3 cells and/or their derived factors. Interestingly, FSAS has no effect on the ability of M1 macrophage to inhibit the proliferation of PC3 cells. Mechanistically, FSAS3 (1lL) reduced IjB phosphorylation induced by RANKL and macrophage-derived factors in PC3 cells, indicative of NFjB inhibition. We conclude that inhibition of TRAF6 in prostate cancer cells reduces their ability to influence the differentiation of bone marrow macrophage into osteoclast and tumour associated macrophage in vitro. In vivo studies to test the effects of FSAS, alone and in combination with chemotherapeutic agents, on mouse models of prostate cancer metastasis are ongoing. .