Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion.

Current therapies for pancreatic ductal adenocarcinoma (PDA) target individual tumor cells. Focal adhesion kinase (FAK) is activated in PDA and levels are inversely associated with survival. We investigated the effects of PF-562,271 (a small molecule inhibitor of FAK/PYK2) on a) in vitro migration, invasion and proliferation, b) tumor proliferation, invasion and metastasis in a murine model, and c) stromal cell composition in the PDA microenvironment. Migration assays were performed to assess tumor and stromal cell migration in response to cellular factors, collagen and the effects of PF-562,271. An orthotopic murine model was used to assess the effects of PF-562,271 on tumor growth, invasion and metastasis. Proliferation assays measured PF-562,271 effects on in vitro growth. Immunohistochemistry was used to examine the effects of FAK inhibition on the cellular composition of the tumor microenvironment. FAK and PYK2 are activated and expressed in patient-derived PDA tumors, stromal components and human PDA cell lines. PF-562,271 blocked phosphorylation of FAK Y397 in a dose-dependent manner. PF-562,271 inhibited migration of tumor cells, cancer associated fibroblasts, and macrophages. Treatment of mice with PF-562,271 resulted in reduced tumor growth, invasion, and metastases. PF-562,271 had no effect on tumor necrosis, angiogenesis or apoptosis, but did decrease tumor cell proliferation and resulted in fewer tumor-associated macrophages and fibroblasts compared to control or gemcitabine. These data support a role for FAK in PDA and suggest that inhibitors of FAK may contribute to efficacious treatment of patients with PDA.

Antiestrogens such as tamoxifen are widely used in the clinic to treat estrogen receptor-positive breast tumors. Resistance to tamoxifen can occur either de novo or develop over time in a large proportion of these tumors. Additionally, resistance is associated with enhanced motility and invasiveness in vitro. One molecule that has been implicated in tamoxifen resistance, breast cancer antiestrogen resistance-3 (BCAR3), has also been shown to regulate migration of fibroblasts. In this study, we investigated the role of BCAR3 in breast cancer cell migration and invasion. We found that BCAR3 was highly expressed in multiple breast cancer cell lines, where it associated with another protein, p130Cas (also known as breast cancer antiestrogen resistance-1; BCAR1), that plays a role in both tamoxifen resistance and cell motility. In cells with relatively low migratory potential, BCAR3 overexpression resulted in enhanced migration and colocalization with p130Cas at the cell membrane. Conversely, BCAR3 depletion from more aggressive breast cancer cell lines inhibited migration and invasion. This coincided with a relocalization of p130Cas away from the cell membrane and an attenuated response to epidermal growth factor stimulation that was characterized by a loss of membrane ruffles, decreased migration toward EGF, and disruption of p130Cas /Crk complexes. Based on these data, we propose that the spatial and temporal regulation of BCAR3/p130Cas interactions within the cell is important for controlling breast cancer cell motility. [Cancer Res 2007;67(13):6174-82]

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.