TAK-1 inhibition prevents cytokine-mediated CXCL12 production in a bone cancer cell line

The chemokine, CXCL12, (SDF-1), has a profound effect on cancer progression due to its effects on cells within the tumour microenvironment. A number of inhibitors for the CXCL12 receptor, CXCR4, have been developed for treatment of CXCR4-expressing tumours, including hematopoietic cancers [1]. However, these drugs have limited clinical success and issues with unknown long-term effects and target engagement remain [2]. In this study, we examined the potential to prevent CXCL12 induction in response to cytokines such as IL-1β and TNFα through pharmacological inhibition of the signalling pathways regulating this pathway. Studies were carried out in the osteosarcoma line U2OS stably transfected with a luciferase coupled CXCL12 promoter, with CXCL12 induction measured by luciferase reporter activity. Cell signalling pathways were studied by Western blotting using phospho- and total antibodies. Each experiment was performed a minimum of three times and data quantified and statistical analysis conducted using Graph pad prism 6. We found that; IL-1β stimulated a time-dependent induction of CXCL12 reporter activity (Fold stim at 6 h IL-1β = 3.18 ± 0.23, ***P<0.001). Pre-treatment with the MAP kinase pathway inhibitors SP600125 (JNK), SB203580 (p38 MAP kinase), UO126 (ERK) or an inhibitor of the canonical NFκB cascade (IKK2-X1) had no significant inhibitory effect. In contrast, inhibition of transforming growth factor β-activated kinase-1(TAK-1) with 5Z-7-oxozeanenol (5Z-7-oxo) caused a concentration dependent inhibition of CXCL12 activity (IC50 value of 4.89 µM). TNFα-dependent CXCL12 reporter activity was also inhibited with 10 μM 5Z-7-0xo. IL-1β stimulated JNK phosphorylation, and activated canonical NFκB pathway markers, cellular IκBα degradation and phosphorylation of p65 NFκB. Pretreatment with 5Z-7-oxo inhibited all three responses. Additionally, stimulation with IL-1β significantly increased the phosphorylation of p100 NFκB, a component of the non-canonical NFκB pathway (Fold stim for IL-1β – 13.30 ± 1.8, ***P<0.001). Phosphorylated p100 was significantly inhibited with 5Z7-oxo, (Fold stim for IL-1β + 10 µM 5Z-7-oxo – 1.28 ± 0.2, ***P<0.001). Taken together, these results exclude a role for the MAP kinase pathways and the canonical NFκB pathway in the regulation of CXCL12 production in response to IL-1β. This points to the potential involvement of a novel non-canonical NFκB pathway downstream of the MAP3 Kinase TAK-1.