Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling

Neutral endopeptidase 24.11 (NEP, CD10) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides im plicated in the growth of androgen-independent prostate cancer (PC). NEP su bstrates such as bombesin and endothelin-1 induce cell migration. We invest igated the mechanisms of NEP regulation of cell migration in PC cells, incl uding regulation of phosphorylation on tyrosine of focal adhesion kinase (F AK). Western analyses and cell migration assays revealed an inverse correla tion between NEP expression and the levels of FAK phosphorylation and cell migration in PC cell lines. Constitutively expressed NEP, recombinant NEP, and induced NEP expression using a tetracycline-repressive expression syste m inhibited bombesin- and endothelin-1-stimulated FAK phosphorylation and c ell migration. This results from NEP-induced inhibition of neuropeptide-sti mulated association of FAK with cSrc protein. Expression of a mutated catal ytically inactive NEP protein also resulted in partial inhibition of FAK ph osphorylation and cell migration. Coimmunoprecipitation experiments show th at NEP associates with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in an N EP-Lyn-PI3-K protein complex. This complex competitively blocks FAK-PI3-K i nteraction, suggesting that NEP protein inhibits cell migration via a prote in-protein interaction independent of its catalytic function. These experim ents demonstrate that NEP can inhibit FAK phosphorylation on tyrosine and P C cell migration through multiple pathways and suggest that cell migration which contributes to invasion and metastases in PC cells can be regulated b y NEP.