PO-156 TNFα-induced cell fusion between MDA-MB435-pFDR.1 and M13SV1-Cre cells is suppressed by minocycline through inhibition of the NF-KB pathway

Introduction Cell fusion is involved in a wide range of physiological processes like fertilisation or wound healing. But also diseases could arise by cell fusion, such as cancer. In a cancer context, cell fusion generates hybrid cells, which often show a more malignant phenotype as their parental cells. Here, we have recently demonstrated that the fusion of M13SV1-Cre cells and MDA-MB435-pFDR.1 cells is induced by TNFα and that the TNFα induced fusion could be blocked by minocycline. Material and methods To quantify occurring cell fusion events, a Cre-LoxP recombination system was established, which allows the identification of hybrid cells originated from M13SV1-Cre breast epithelial cells and MDA-MB435-pFDR.1 breast cancer cells. The phosphorylation state of IKK, p38, Erk 1/2 and JNK was carried out by western blots. Transcriptional activity of NFκB was characterised by immunocytochemistry and by ChIP coupled with a downstream qPCR assay to measure the NF-κB yield of selected genes. Results and discussions The TNFα signal transduction pathway of both cell types was investigated to clarify the TNFα and minocycline effect in detail. We looked upstream and saw that TNFα bound to TNFR1 to recruit the adaptor TRAF2, which further resulted in activation of the IKK signalosome and activation of the MAP kinases JNK, Erk1/2 and p38. It was found that minocycline downregulate the TRAF2-TNFR1 recruitment and activation of IKK in both cell types, while the MAPK mediators p38, JNK and Erk1/2 were differently affected by minocycline treatment. Next, the activation of NFκB in the nucleus was examined and it was seen that TNFα increased the NFκB level in M13SV1-Cre, which in turn was blocked by minocycline. Addition of several specific inhibitors that block NFκB and MAPK activation confirmed that the TNFα induced cell fusion depends on activation of the NFκB nexus. A ChIP assay was used to detect putative cell fusion genes, which are TNFα up and minocycline down regulated. Several targets were picked, inhibited and analysed. Results implicated that the zymogen MMP9 seems to be fusion relevant in inhibiting the TNFα fusion process. Conclusion Our data show that TNFα is strong inductor of cell fusion by activation of the NFκB pathway. This TNFα-induced cell fusion effect was abrogated by minocycline, preventing the NFκB translocation into the nucleus, which affected the target gene expression of fusion-relevant factors, like MMP9.