Src kinase activation is mandatory for MDA-9/syntenin-mediated activation of nuclear factor-κB

The scaffolding postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain-containing protein melanoma differentiation associated gene-9 (MDA-9)/syntenin is a tandem PDZ protein overexpressed in human melanoma, and breast and gastric cancer cells. MDA-9/syntenin affects cancer cell motility and invasion through distinct biochemical and signaling pathways, including focal adhesion kinase and p38 mitogen-activated protein kinase (MAPK), resulting in activation of the nuclear factor (NF)-kappa B pathway. MDA-9/syntenin also promotes melanoma metastasis by activating c-Src, but how c-Src regulates NF-kappa B activation is unclear. Using a human melanoma model, we document that MDA-9/synteninc-Src interactions are positive regulators of NF-kappa B activation. Inhibition of c-Src by PP2 treatment, by blocking c-Src or mda-9/syntenin expression with small interfering RNA, or in c-Src (-/-) knockout cell lines, reduces NF-kappa B activation following overexpression of mda-9/syntenin or c-Src. Deletion or point mutations of the PDZ binding motif preventing MDA-9/syntenin association with c-Src reveals that both PDZ domains, with PDZ2 being the dominant module, are required for activating downstream signaling pathways, including p38 MAPK and NF-kappa B. We also document that MDA-9/syntenin-c-Src complexes functionally cooperate with NF-kappa B to promote anchorage-independent growth, motility and invasion of melanoma cells. These findings underscore PDZ domains of MDA-9/syntenin as promising potential therapeutic targets for intervening in a decisive component of cancer progression, namely, metastatic tumor spread. Oncogene (2010) 29, 3054-3066; doi: 10.1038/onc.2010.65; published online 15 March 2010