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Focal Adhesion Kinase, a Downstream Mediator of Raf-1 Signaling, Suppresses Cellular Adhesion, Migration, and Neuroendocrine Markers in BON Carcinoid Cells

We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1–activated BON cells by targeted siRNA led to reversal of the Raf-1–mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAKTyr407, but not FAKTyr397, in these Raf-1–activated cells, indicating that FAKTyr407 may be associated with changes in the neuroendocrine phenotype. Overexpression of constitutively active FAK plasmids (wild-type FAK or FAKTyr397 mutant) into BON cells reduced neuroendocrine markers, whereas the FAKTyr407 mutant plasmid did not show any decrease in the levels of neuroendocrine markers, indicating that phosphorylation of FAK at the Tyr407 residue may be important for these effects. Our results showed for the first time that FAK is an essential downstream effector of the Raf-1/MEK1/2/ERK1/2 signaling cascade and negatively regulated the neuroendocrine and metastatic phenotype in BON cells. Mol Cancer Res; 8(5); 775–82. ©2010 AACR.