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Focal Adhesion kinase (FAK) localized in focal adhesions influences the dynamic equilibrium of actin cytoskeleton (AC) regulating cell motility and invasion. We have recently identified a novel, specific signalling pathway regulating actin cytoskeleton dynamics and cell motility in malignant breast and prostate human epithelial cells. Incubation of MCF7 breast cancer cells with the opioid agonist αs1 casomorphin and LNCaP prostate cells with testosterone-BSA (testo-BSA) stimulated actin reorganization. This was associated with FAK phosphorylation and subsequent phosphatidylinositol-3 kinase (PI-3K) and Rac1 activation. In the present study we first investigated whether the FAK/PI-3K pathway operates in MCF-7 cells upon treatment with a different stimulus. Since membrane androgen receptors have also been identified in breast cancer cells we investigated the effect of testo-BSA on MCF7 cell line. Immunofluorescence microscopy with testo-BSA FITC confirmed the presence of membrane androgen receptors in MCF7 cells. Treatment with testo-BSA resulted in early actin reorganization associated with rapid FAK and PI3K activation. To test whether actin rearrangement affects cell motility wound healing assay was performed demonstrating a significant inhibition of wound closure. We next investigated the functional role of the FAK/PI-3K-actin signalling pathway in cells of different origin, the A375 melanoma cell line. Incubation with αs1 casomorphin for 15 min to up to 24 h resulted in early significant actin reorganization. In line with this, morphological analysis showed increased number of lamelipodia and stress fibers equally distributed to the cytoplasm. Casomorphin also induced significant early phosphorylation of FAK and PI-3K. FAK phosphorylation was still evident in cells pretreated with the PI-3K inhibitor wortmanin, indicating that FAK activation occurs upstream of PI-3K. Affinity precipitation with a GST-fusion protein corresponding to the p21-binding domain of PAK1 performed in cell lysates confirmed Rac-1 activation. Wound healing assay revealed that casomorphin significantly inhibited cell motility. In conclusion, actin reorganization through FAK and subsequent PI3-K/Rac-1 activation operates in various human cancer cell systems supporting a broader role for FAK in controlling cell motility and underlines the importance of FAK as a new therapeutic target in cancer.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA