Integrins contribute to progression in many cancers, including breast cancer. For example, the interaction of ␣ 5 ␤ 1 with plasma fibronectin causes the constitutive invasiveness of human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as invasion substrates. Immunoassays were used to compare the roles of

IntroductionRapid induction of keratinocyte and fibroblast migration into wounds is necessary for tissue repair. Reepithelialization begins soon after injury as epidermal cells become migratory (1, 2). Because plasma fibronectin (pFn) circulates in the blood at 0.3-0.5 mg/mL (reviewed in ref.3) and binds to fibrin, it is a significant component of clot provisional matrix. If the basement membrane is destroyed, epidermal cells migrate over the pFnrich provisional matrix (4-6). The fibronectin of the provisional matrix is also known to promote the movement of fibroblasts, macrophages, and blood vessels into the wound space (1, 7). Keratinocytes, other epithelial cell types such as mammary and prostate epithelial cells, and fibroblasts express the α5β1 integrin fibronectin receptor (8-11). Although keratinocytes express less α5β1 than α3β1, α5β1 is more important for migration (8). Also, expression of α5β1 (12, 13) and specific metalloproteinases (14) are increased in motile keratinocytes at the leading edge of the new epithelium. Keratinocyte and fibroblast cell movement is supported by pFn (15,16). Fragments of pFn containing the cell-binding domain, which are found in wounds due to localized proteinase activity (17, 18), stimulate monocyte (7) and fibroblast chemotaxis through the dermis (19-21), as well as matrix metalloproteinase expression by fibroblasts (11). This interaction also stimulates cellular invasion of the provisional matrix and wound stroma (16; reviewed in ref. 22). The pFn cell-binding domain also induces monocytes to extravasate from proximal vessels, enabling them to enter the wound and differentiate into inflammatory macrophages (23), and can stimulate normal human endothelial cell motility in vitro (24). Thus, the invasive/migratory phenotype expressed by epithelial cells and fibroblasts after wounding may result from pFn fragmentation and α5β1 integrin-mediated signaling. Furthermore, this interaction promotes the immigration or motility of a number of cell types involved in healing through a variety of extracellular matrices, including the stroma as well as vascular basement membranes (23,24).To define the invasion-stimulatory sequences of pFn, the naturally serum-free basement membranes and associated extracellular matrices of sea urchin embryos (SU-ECM) were used as in vitro invasion substrates (25). We report that the PHSRN sequence of pFn stimulates SU-ECM invasion by keratinocytes and fibroblasts. Acetylated and amidated PHSRN peptide (Ac-PHSRN-NH 2 ) is greatly increased in activity, whereas a randomized sequence peptide, Ac-HSPNR-NH 2 , is inactive. In derivatives of the PHSRN peptide, substitution of arginine by alanine or glutamic acid also results in inactivity. The PHSRN sequence of the plasma fibronectin (pFn) cell-binding domain induces human keratinocytes and fibroblasts to invade the naturally serum-free extracellular matricies of sea urchin embryos. The potency of acetylated, amidated PHSRN (Ac-PHSRN-NH 2 ) is significantly increased, making it more active on a molar basis ...

A 5 B 1 Integrin interacts with the PHSRN sequence of plasma fibronectin, causing constitutive invasion by human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as in vitro invasion substrates. Immunoassays were employed to dissect the roles of focal adhesion kinase (FAK), phosphatidylinositol 3 ¶-kinase (PI3K), and protein kinase CD (PKCD) in A 5 B 1 -mediated, matrix metalloproteinase-1 (MMP-1)-dependent invasion by metastatic human DU 145 prostate cancer cells. We found that a peptide composed of the PHSRN sequence induced rapid FAK phosphorylation at Tyr 397 (Y397), a site whose phosphorylation is associated with kinase activation. The technique of RNA silencing [small interfering RNA (siRNA)] confirmed the role of FAK in PHSRN-induced invasion. PHSRN also induced the association of the p85-regulatory subunit of PI3K with FAK at a time corresponding to FAK phosphorylation and activation, and maximal PI3K activity occurred at this same time. The necessity of PI3K activity in both PHSRN-induced invasion and MMP-1 expression was confirmed by using specific PI3K inhibitors. By employing a specific inhibitor, Rottlerin, and by using siRNA, we also found that PKCD, a PI3K substrate found in focal adhesions, functions in PHSRN-induced invasion. In addition, the induction of MMP-1 in PHSRN-treated DU 145 cells was shown by immunoblotting, and the role of MMP-1 in PHSRNinduced invasion was confirmed by the use of blocking anti-MMP-1 monoclonal antibody. Finally, a close temporal correspondence was observed between PHSRN-induced invasion and PHSRN-induced MMP-1 activity in DU 145 cells.