FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

Directional motility is a complex process requiring the spatiotemporal integration of signals that regulate cytoskeletal changes, and the establishment of an anteroposterior or polarized cell axis. Focal adhesion kinase (FAK) promotes cell migration, but a molecular role for FAK in promoting cell polarity remains undefined. Here, using wound healing and Golgi-reorientation analyses, we show that fibroblast, endothelial and carcinoma polarity during cell migration requires FAK and is associated with a complex between FAK, p120RasGAP and p190RhoGAP (p190A), leading to p190A tyrosine phosphorylation. Fibronectin-integrin-mediated FAK activation and phosphorylation promote SH2-mediated binding of p120RasGAP to FAK and FAK-mediated p190A tyrosine phosphorylation. The association of p120RasGAP with FAK facilitates the formation of a FAK-p120RasGAP-p190A complex targeted to leading-edge focal adhesions by FAK. Knockdown of p120RasGAP, mutation of FAK Y397 or inhibition of FAK activity prevent the association of FAK with p190A and subsequent tyrosine phosphorylation of p190A, and result in the loss of cell polarity. Because reconstitution of FAK-null fibroblasts with FAK or a Pyk2-FAK chimera restore the normal decrease in RhoA GTP binding upon cell spreading on fibronectin, our studies support a model whereby FAK activity facilitates the recruitment and stabilization of a p120RasGAP-p190A complex at leading-edge focal adhesions connected to the transient inhibition of RhoA activity and the regulation of cell polarity.

Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that a4 and a5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either a4b1-or a5b1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that a5b1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for a5b1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, a4b1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatasea overexpression inhibited a4b1-stimulated NB motility and Src activation consistent with a4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In a4 shRNA-expressing NB cells, a4b1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated a4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that a4b1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during a5b1-mediated NB migration and support the evaluation of inhibitors to a4, Src and FAK in the control of NB tumor progression.

Pyk2 is a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK).FERM expression in human fibroblasts upon FAK knockdown prevented cisplatin-mediated apoptosis. Our studies demonstrate that nuclear Pyk2 functions to limit p53 levels, thus facilitating cell growth and survival in a kinase-independent manner.

Moreover, analyses of Src؊/؊ , Yes ؊/؊ , and Fyn ؊/؊ fibroblasts showed that Src expression was inhibitory to TNF␣-stimulated IL-6 production. These studies provide evidence for a novel Srcindependent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression. Focal adhesion kinase (FAK)2 is best known for its role as an integrin-stimulated protein-tyrosine kinase (1). FAK is recruited to sites of integrin clustering via interactions of its C-terminal domain with integrin-associated proteins such as talin and paxillin. FAK contains a central kinase domain, C-terminal proline-rich regions that serve as binding sites for Src homology 3 (SH3) domain-containing proteins, and an N-terminal band 4.1, ezrin, radixin, moesin homology (FERM) domain that acts to regulate FAK kinase activity through an auto-inhibitory mechanism (2, 3). Integrin clustering promotes FAK activation, results in FAK phosphorylation at Tyr-397 (Tyr(P)-397), promotes Src family protein-tyrosine kinase binding to the FAK Tyr(P)-397 site, and facilitates the formation of the FAK-Src signaling complex that results in the secondary phosphorylation of FAK at Tyr-861 and Tyr-925 (4, 5).

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that canonically promotes signaling downstream of integrin receptors. FAK expression and activity are elevated during breast cancer progression and is associated with a poor clinical prognosis. FAK inhibitors are in clinical trials and we found that oral administration of a small molecule FAK inhibitor blocks orthotopic breast and ovarian tumor growth in mice without general toxicity (Walsh et al. and Tanjoni et al., in press). Our studies are the first to show that FAK inhibition can suppress spontaneous breast to lung metastasis using a syngenic mouse model system. FAK inhibition also prevented the metastasis of MDA-MB-231 human breast carcinoma containing activating mutations in KRAS and BRAF. Surprisingly, we found that FAK inhibition did not limit cell proliferation or promote cell death in breast or ovarian carcinoma cells in adherent two-dimensional culture. However, when the same cells were grown under three-dimensional (3D) conditions as spheroids, low concentrations of FAK inhibitors promoted caspase-3 activation and cell death. Spheroid-mediated FAK inhibition was associated with dephosphorylation of the FAK substrate p130Cas and the inhibition of the small GTPase Rap1. Spheroid-mediated cell death upon FAK inhibition was associated with increased entosis, a process of cell-cell engulfment. Integrin activation can counter-act entosis and we evaluated cells expressing wildtype (WT) or kinase-dead (KD) FAK for effects on β1 integrin activation via fibronectin binding as analyzed by flow cytometry. Whereas cells expressing WT FAK showed high levels of fibronectin binding, KD FAK-expressing cells showed 10-fold reduced levels of fibronectin binding with equal surface β1 integrin expression. Taken together, our results support the hypothesis that loss of integrin activation associated with FAK-p130Cas-Rap1 inhibition triggers entosis within spheroids. Ongoing studies are focused on determining whether FAK-mediated signaling promotes β1 integrin activation that affects the lodging, clearance rate, or survival of tumor cells upon metastasis to the lungs. There is a great need for drugs that might prevent tumor progression and metastasis. Culturing breast carcinoma cells as spheroids better mimics the growth and survival conditions of tumor cells dissociated from a primary tumor and undergoing metastasis. Unfortunately, tumor spheroids usually show increased resistance to various chemo-therapeutic drugs and this may be one reason limiting drug efficacy in vivo. In contrast, FAK inhibitors selectively effect spheroid survival and also prevent tumor growth and metastasis in vivo. Our studies may have uncovered a potential “Achilles heel” of breast carcinoma tumor growth in relying on FAK signaling to provide a non-canonical survival pathway through inside-out integrin activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1204.

No abstract