The results showed that macrophage CM increased the migration of MCF7 cells significantly, whereas knockdown of YAP rescued this phenomenon (Figures 1a and b). consists of heterogeneous components including extracellular matrix, tumor-associated stromal cells and a myriad of signaling molecules,1 which can significantly influence tumor growth and metastasis.2 Macrophages in tumor microenvironment have a key role in promoting tumor metastasis.3 TNF, mainly derived from activated macrophages, is a well-known cytokine that regulates the inflammatory processes in tumor development. High level of tumor necrosis factor (TNF ) is usually associated with an aggressive behavior and a poor prognosis in many malignant cancers, including breast cancers.4 Studies reported that TNF induces epithelialCmesenchymal transition and further facilitates metastasis in breast malignancy and prostate malignancy.5 The signaling mechanisms underlying the pro-invasive activity of TNF are still largely unknown. In tumor cells, TNF activates IB kinases (IKKs), c-Jun N-terminal kinase and mitogen-activated protein kinase signaling to stimulate the nuclear translocation of transcription factors including activator protein-1 (AP-1) and nuclear factor kappa B (NF-B) via TNF receptor 1 (TNFR1).6 TNF promotes the expression of genes involved in tumor invasion and PRKDC metastasis such as interleukin-8 (IL-8), monocyte chemotactic protein-1 and matrix metalloproteinase, thus enhancing tumor progression.6, 7 The Hippo pathway is a highly conserved signaling that controls organ size and is tightly involved in tumorigenesis. The core components of the Hippo pathway constitute a kinase cascade. In complex with Sav1, Mst1/2 phosphorylates and activates Lats1/2. Lats1/2 phosphorylates yes-associated protein (YAP)/TAZ and promotes the binding of YAP/TAZ to 14?3?3, which leads to cytoplasmic retention of YAP/TAZ. YAP/TAZ, in conjunction with TEA domain name family members (TEAD1C4), mediates the major physiological functions of the Hippo pathway.8, 9 The functions of YAP in oncogenesis, including the promotion of cell proliferation, the inhibition of apoptosis and the induction of the epithelialCmesenchymal transition, have been elucidated.9, 10, 11, 12 Many upstream signaling contributes to tumorigenesis have been found to trigger YAP. For example, hypoxia stimulates YAP though SIAH2-mediated degradation of LATS2.13 Recently, it was reported that intestinal IL-6-gp130 signaling triggers activation of YAP that dependent on Src-mediated phosphorylation to maintain epithelial cell proliferation, providing the evidence that YAP is responsive to the inflammatory microenvironment.14 However, whether YAP also has an essential role in inflammation-associated tumor progression is still largely unknown. In our study, we found that TNF triggers IKK-mediated YAP phosphorylation and activation in breast malignancy cells. We found that conditioned medium (CM) from macrophage or TNF treatment stabilizes YAP protein and increases the conversation between YAP and p65. Further, YAP/TEAD/p65 triplet synergistically upregulates hexokinase 2 (HK2) transcription, which promotes breast malignancy cell migration. Thus, our results uncovered a non-autonomously regulatory mechanism of YAP in malignancy cells by environmental cues and provided a molecular basis for targeting TNF-IKK-YAP/p65-HK2 pathway to effectively treat breast malignancy cell metastasis. Results Macrophage CM treatment promotes the transactivation of YAP YAP is usually overexpressed in various cancers and closely related to breast malignancy tumorigenesis.15, 16, 17, 18, 19, 20, 21 YAP could promote cancer cell migration, and we hypothesized that YAP might be involved in macrophage-mediated and inflammation-induced cancer cell metastasis. First, we established MCF7 PIM447 (LGH447) breast malignancy cells stably expressing YAP short hairpin RNAs (shRNA) via lentiviral contamination. PIM447 (LGH447) Then, the stable cell lines were exposed to CM from cultured human THP-1 macrophages. The ability of cell migration was PIM447 (LGH447) measured by transwell assay. The results showed that macrophage CM significantly increased the migration of MCF7 cells, whereas knockdown of YAP rescued this phenomenon (Figures 1a and b). This evidence prompted us to investigate whether macrophage CM stimulated the activity of YAP. As expected, we found the protein level of YAP increased upon macrophage CM treatment (Physique 1c) and the mRNA level of YAP is not changed (Physique 1d). Open in a separate windows Physique 1 Macrophage CM promotes cell migration and YAP activation. (a) Control or shYAP stably transfected MCF7 cells were cultured with control medium or macrophage CM in the transwells for 24?h. The migratory ability was determined by transwell assay. The right panel showed the YAP knockdown efficiency. (b) The transwell membranes were washed by dimethylsulfoxide (DMSO) and the OD values were calculated at the absorbance of 570?nm. Data were collected from three impartial experiments. (c, d) MCF7 cells were cultured in control medium or macrophage CM for 24?h, YAP expression was detected by western blot (c) and real-time PCR (d). (e) Control or shYAP stably transfected MCF7 cell lines were cultured with macrophage CM for 24?h and then the CYR61 mRNA level was assessed real-time PCR..