Abstract mda-9/Syntenin, a tandem PDZ-domain containing adaptor protein, functions as a positive regulator of cancer cell progression in several human cancers. Protein kinase Cα (PKCα) is a key intermediate between integrins and focal adhesion kinase (FAK) signaling in response to fibronectin (FN), but the molecular mechanism by which PKCα regulates FN-induced FAK activation remains unclear. Here, we report that mda-9/syntenin links FN-induced activation of FAK and PKCα in human breast cancer and melanoma cells. FN induced mda-9/syntenin expression and PKCα activation in a similar manner prior to FAK activation. PKCα inhibition resulted in the suppression of the endogenous and FN-induced mda-9/syntenin expression, as well as mda-9/syntenin-induced cell migration and invasion toward FN. mda-9/Syntenin expression and PKCα activation was interdependently regulated; e.g., inhibition of mda-9/syntenin suppressed both FN-induced and endogenous phosphorylation of PKCα at Thr638/641, and inhibition of PKCα suppressed both FN-induced and endogenous expression of mda-9/syntenin. Moreover, inhibition of either mda-9/syntenin or PKCα suppressed the FN-induced association of integrin- 1/FAK/c-Src signaling complexes. Consistently, suppression of either mda-9/syntenin or PKCα significantly inhibited FN-induced phosphorylation of FAK at Tyr397 and c-Src at Tyr416, activation of downstream signaling molecules such p38 and ERK mitogen-activated protein kinases (MAPKs), Cdc42 GTPase, and nuclear factor-κB (NF-κB). These findings demonstrate that PKC -dependent mda-9/syntenin up regulation could play a critical role in FN-induced activation of FAK, and mda-9/syntenin may act as a molecular adaptor that links PKCα and FAK during adhesion to FN in human breast cancer and melanoma cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-372.