The focal adhesion kinase (FAK) is a mediator of cellextracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125FAK , and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125 FAK . These results indicate that FAKmediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway.As normal epithelial cells become transformed and develop the capacity for invasion and metastasis, they must acquire the property of anchorage-independent growth. This is essential for tumor cells to survive the apoptotic stimuli associated with the loss of adhesion, proteolysis, and migration through their extracellular matrix (ECM) 1 (1, 2). Oncogenic transformation has been shown to suppress apoptosis as a means of enhancing tumor cell survival (3-6). Cells from human tumors have been shown to be more resistant to apoptotic stimuli than normal cells (7). Normal cells undergo apoptosis when they lose ECM adhesion, and this phenomenon has been termed "anoikis" (8, 9). Tumor cells have been thought to be resistant to anoikis, thus allowing them to grow in an anchorage-independent fashion. One of the critical signaling molecules involved in both cell-ECM interactions and anoikis is the focal adhesion kinase (FAK) (10), a tyrosine kinase that localizes to focal adhesions (11,12). Previous studies have shown that FAK is overexpressed in breast, colon, and thyroid cancers (13-17), whereas normal tissues express little detectable FAK. The overexpression of FAK in tumors is likely to affect three functions as follows: motility, adhesion, and survival. FAK is thought to play a role in adhesion-mediated survival because overexpression of a constitutively activated form of FAK in Madin-Darby canine kidney cells has been shown to confer resistance to apoptosis following loss of adherence (10). FAK overexpression in Chinese hamster ovary (CHO) cells caused an increase in migration (18), suggesting that FAK may play a role in motility of CHO cells. Although these experiments were performed in normal cells, they raise the possibility that tumor cells upregulate FAK expression in ord...

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays a key role in maintaining focal adhesion function and cell survival and is implicated in cell migration, adhesion, and cell cycle control (9,13,18,20,33,44). Overexpression of FAK is a common event in numerous tumor systems, including breast, colon, and thyroid carcinomas (2,24,32,41), and occurs at early stages of tumorigenesis, before a tumor has developed the capacity for invasion and metastasis (2). Importantly, FAK has been shown to be one of the critical factors protecting cells from apoptosis, but the exact mechanism is unknown (8,9,12,19,37,43). Attenuation of FAK expression by antisense oligonucleotides led to apoptosis in tumor cells (42), and the treatment of cells with anti-FAK antibody (18, 26) or overexpression of the focal adhesion targeting (FAT) domain of FAK led to cell rounding, detachment, and apoptosis (19,21,40). We have created a model system for the attenuation of FAK function by adenoviral gene transduction of the carboxy-terminal domain of FAK (FAK-CD) and have demonstrated a loss of adhesion and apoptosis in breast cancer cells with this treatment (43). Both anchoragedependent and anchorage-independent apoptotic signaling required Fas-associated death domain protein (FADD) and caspase 8, suggesting an important role for FAK in inhibiting death receptor-related apoptosis (43). This finding provided additional evidence that a death receptor-mediated apoptotic pathway or death receptor-related death domain proteins are involved in the apoptotic process triggered by the expression of FAK-CD.The loss of adhesion and induction of apoptosis upon attenuation of FAK function by the expression of FAK-CD is similar to the phenomenon of anoikis (7-9). Intriguingly, there is evidence for the involvement of death receptor-related, death domain-containing proteins in anoikis (7, 35), whereby the silencer of death domain (SODD) and dominant-negative FADD efficiently inhibited anoikis in Madin-Darby canine kidney (MDCK) cells and in a number of untransformed epithelial cell lines. In these studies, it was also shown that cell matrix detachment activated caspase 8. However, the linkage of the signaling pathways to the death receptors remains unknown.RIP is a serine/threonine kinase that contains a death domain (17, 38) and is named for its association with the death receptor complex. RIP interacts with the death domains of cell surface receptors of the tumor necrosis factor (TNF) superfamily and death domain adaptor proteins (3,5,17) and plays an indispensable role in 39). Recently, it was shown that TNF alpha-mediated activation of NF-B depends on the association of RIP and FAK (11). TNF-induced NF-B DNA binding activity and activation of IB kinases were markedly impaired in FAK Ϫ/Ϫ cells (11). However, it has been well established that RIP has a dual function and is capable of either inducing apoptosis or activating cellular survival signals (14,17,23,27,38,39). Similarly, it has been proposed that RIP is one of the switch...

Overexpression of FAK may be part of a mechanism for invasion and metastasis of thyroid cancer. Furthermore, the levels of p125FAK may serve as a marker of biologic behavior in this disease.

The focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) are protein-tyrosine kinases that are overexpressed and activated in human breast cancer. To determine the role of EGFR and FAK survival signaling in breast cancer, EGFR was stably overexpressed in BT474 breast cancer cells, and each signaling pathway was specifically targeted for inhibition. FAK and EGFR constitutively co-immunoprecipitated in EGFR-overexpressing BT474 cells. In low EGFR-expressing BT474-pcDNA3 vector control cells, inhibition of FAK by the FAK C-terminal domain caused detachment and apoptosis via pathways involving activation of caspase-3 and -8, cleavage of poly(ADP-ribose) polymerase, and caspase-3-dependent degradation of AKT. This apoptosis could be rescued by the dominantnegative Fas-associated death domain, indicating involvement of the death receptor pathway. EGFR overexpression did not inhibit detachment induced by the FAK C-terminal domain, but did suppress apoptosis, activating AKT and ERK1/2 survival pathways and inhibiting cleavage of FAK, caspase-3 and -8, and poly(ADPribose) polymerase. Furthermore, this protective effect of EGFR signaling was reversed by EGFR kinase inhibition with AG1478. In addition, inhibition of FAK and EGFR in another breast cancer cell line (BT20) endogenously overexpressing these kinases also induced apoptosis via the same mechanism as in the EGFR-overexpressing BT474 cells. The results of this study indicate that dual inhibition of FAK and EGFR signaling pathways can cooperatively enhance apoptosis in breast cancers.

Emerging data suggest that urolithins, gut microbiota metabolites of ellagitannins, contribute toward multiple health benefits attributed to ellagitannin-rich foods, including walnuts, red raspberry, strawberry, and pomegranate. However, there is limited data on whether the potential neuroprotective effects of these ellagitannin-rich foods are mediated by urolithins. Herein, we evaluated the potential mechanisms of antineuroinflammatory effects of urolithins (urolithins A, B, and C; 8-methyl-O-urolithin A; and 8,9-dimethyl-O-urolithin C) in BV2 murine microglia in vitro. Nitrite analysis and qRT-PCR suggested that urolithins A and B reduced NO levels and suppressed mRNA levels of pro-inflammatory genes of TNF-α, IL-6, IL-1β, iNOS, and COX-2 in LPS-treated microglia. Western blot revealed that urolithins A and B decreased phosphorylation levels of Erk1/2, p38 MAPK, and Akt, prevented IκB-α phosphorylation and degradation, and inhibited NF-κB p65 subunit phosphorylation and nuclear translocation in LPS-stimulated microglia. Our results indicated that urolithins A and B attenuated LPS-induced inflammation in BV2 microglia, which may be mediated by inhibiting NF-κB, MAPKs (p38 and Erk1/2), and Akt signaling pathway activation. The antineuroinflammatory activities of urolithins support their role in the potential neuroprotective effects reported for ellagitannin-rich foods warranting further in vivo studies on these ellagitannin gut microbial derived metabolites.