Overexpression of FAK promotes Ras activity through the formation of a FAK/p120RasGAP complex in malignant astrocytoma cells.

Focal adhesion kinase (FAK) signaling may be mediated through the modulation of Ras activity. We have shown previously that grade III malignant astrocytoma biopsy samples exhibit elevated levels of FAK, and that overexpression of FAK in U-251MG malignant astrocytoma cells promotes the phosphorylation of Shc, a potential upstream mediator of Ras activity. Here, we report that overexpression of FAK promotes Ras activity in U-251MG malignant astrocytoma cells cultured in aggregate suspension or as monolayers adherent to vitronectin. The overexpression of FAK also promoted the association of FAK with p120RasGAP, which is a negative regulator of Ras activity, in the U-251MG cells cultured in aggregate suspension, with this association being abrogated upon plating of the cells onto vitronectin. An association of FAK with p120RasGAP also was observed in malignant astrocytoma biopsy samples, but not in normal brain samples. As overexpression of FAK in U-251MG cells in aggregate suspension culture reduced the amount of p120RasGAP complexed with active Ras, we hypothesize that the association of FAK with p120 RasGAP may facilitate Ras activity. The overexpression of a mutated FAK in which the Y397 had been mutated to F did not result in the formation of the FAK/p120RasGAP complex and did not promote Ras activity, indicating that the Y397 residue of FAK plays a role in the formation of this complex and in the activation of Ras. Moreover, the overexpression of mutated FAK (397F) was found to inhibit anchorage-independent growth. These data provide the basis for a previously undescribed mechanism in which the elevated expression of FAK can promote Ras activity through its competitive recruitment of p120RasGAP, thereby diminishing the association of p120RasGAP with active Ras.

Focal adhesion kinase is expressed in the angiogenic blood vessels of malignant astrocytic tumors in vivo and promotes capillary tube formation of brain microvascular endothelial cells.

PURPOSE: Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that has been shown to promote proliferation, migration, and invasion of several cell types in vitro, and we have shown recently that FAK promotes proliferation of malignant astrocytoma cells in vivo. To determine the role of FAK in angiogenesis in malignant astrocytic tumors, we investigated the expression and function of FAK in brain endothelial cells. EXPERIMENTAL DESIGN: We characterized the expression of FAK and activated FAK in endothelial cells by immunohistochemistry. We also determined the function of FAK in brain microvascular endothelial cells by transfecting these cells with a dominant interfering form of FAK [FAK-related nonkinase (FRNK)] or a mutant FRNK (Leu-1034 to Ser) and assessed the effect on capillary tube formation and cell migration. RESULTS: We found that FAK was expressed in the endothelial cells of grade III (4 of 9 samples) and IV (9 of 10 samples) astrocytoma biopsies but not in the endothelial cells of normal brain (0 of 9 samples) and not in grade I (0 of 5 samples) or II (0 of 4 samples) astrocytoma biopsies. Furthermore, we found that both FAK and activated FAK were expressed in the endothelial cells in malignant astrocytoma tumors propagated intracerebrally in the severe combined immunodeficient mouse brain. As expected, immunofluorescence analysis showed FRNK protein to localize to focal adhesions, whereas mutant FRNK protein did not. FRNK-transfected endothelial cells showed a 55% reduction in branched tube formation and a 40% reduction in tube length when propagated in three-dimensional collagen gels, compared with cells transfected with the mutant FRNK construct. Furthermore, FRNK-transfected cells showed a 35-50% reduction in haptotactic migration toward fibronectin and collagen, compared with mutant FRNK-transfected cells. CONCLUSIONS: These data suggest that FAK promotes angiogenesis and that this occurs, at least in part, through the promotion of endothelial cell migration.

FAK signaling in anaplastic astrocytoma and glioblastoma tumors.

Focal adhesion kinase (FAK) is a non-receptor cytoplasmic-tyrosine kinase that is activated by several different cell surface receptors shown to be upregulated on glioblastoma cells (integrins alpha(v)beta3 and alpha(v)beta5, and the epidermal growth factor receptor). Activated FAK can signal through several different signaling pathways, which are reviewed here. Published data are summarized that have demonstrated 1) elevated FAK expression in anaplastic astrocytoma and glioblastoma tumor biopsy samples, 2) a role for FAK in the promotion of glioblastoma cell proliferation, survival and migration in vitro, and 3) a role for FAK in the promotion of glioblastoma cell proliferation in vivo in an animal model. The available data suggests that increased levels of FAK protein and activity may contribute to an increased ERK activity and cell proliferation in vivo in these tumors.

Focal adhesion kinase in cancer.

Focal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that transmits signals important in modulating several cell functions, including proliferation, migration, and survival. Several different types of malignant tumors have been reported to express elevated levels of FAK protein in vivo, potentially pointing to a role for FAK in either the progression of tumor cells to malignancy or the pathogenesis of cancer. Considerable knowledge has been gained regarding FAK signaling in non-neoplastic cells, such as fibroblasts, while much less is known regarding FAK signaling in malignant cells. Several studies to date suggest that the regulation of FAK activity and signaling may be different in malignant cells. In this review, we summarize what is known regarding the function and signaling of FAK in cancer cells, and highlight areas that need further study. Evidence is emerging that aberrant FAK expression, activity, and signaling can potentially promote the progression of several types of tumors in vivo through its effects on cell function.

Focal adhesion kinase enhances signaling through the Shc/extracellular signal-regulated kinase pathway in anaplastic astrocytoma tumor biopsy samples.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that on activation generates signals that can modulate crucial cell functions, including cell proliferation, migration, and survival. In vitro, overexpression of FAK has been shown to promote cell proliferation by signaling through the Ras/mitogen-activated protein kinase cascade in several cell types. We have shown previously that overexpression of exogenous FAK lacking alternative splicing in malignant astrocytoma clones injected intracerebrally into SCID mouse brains promotes tumor cell proliferation. Here, we show that in anaplastic astrocytoma biopsy samples, FAK is expressed as an unspliced variant and migrates with a faster mobility similar to that observed in embryonic brain. Compared with nonneoplastic adult brain biopsies, the levels of FAK protein are elevated as are its levels of activation as assessed by autophosphorylation and overall tyrosine phosphorylation. The activity of Src kinase in these tumors is also elevated, as well as the activity of Src kinase associated with FAK; the latter may result in enhanced Src kinase phosphorylation of FAK. Phosphorylated Shc is associated with FAK in the anaplastic astrocytoma biopsy samples and in astrocytoma cells overexpressing FAK in vitro but not in nonneoplastic brain biopsy samples. Elevated extracellular signal-regulated kinase-2 activation and elevated expression of cyclins D and E are also found in anaplastic astrocytoma biopsy samples. These data provide evidence that the increased FAK activity in these tumors contributes to phosphorylation of Shc and likely to the promotion of Ras activity, extracellular signal-regulated kinase-2 activation, and cell proliferation in vivo.