Extracellular Matrix Survival Signals Transduced by Focal Adhesion Kinase Suppress p53-mediated Apoptosis

In many malignant cells, both the anchorage requirement for survival and the function of the p53 tumor suppressor gene are subverted. These effects are consistent with the hypothesis that survival signals from extracellular matrix (ECM) suppress a p53-regulated cell death pathway. We report that survival signals from fibronectin are transduced by the focal adhesion kinase (FAK). If FAK or the correct ECM is absent, cells enter apoptosis through a p53-dependent pathway activated by protein kinase C λ/ι and cytosolic phospholipase A₂. This pathway is suppressible by dominant-negative p53 and Bcl2 but not CrmA. Upon inactivation of p53, cells survive even if they lack matrix signals or FAK. This is the first report that p53 monitors survival signals from ECM/FAK in anchorage- dependent cells.     

Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation

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.     

Inhibition of urokinase-type plasminogen activator receptor induces apoptosis in melanoma cells by activation of p53

The urokinase-type plasminogen activator receptor (uPAR) is involved in several biological processes, including proteolysis, adhesion, migration and inflammation. Increased expression of uPAR is associated with metastasis in several tumor types. We studied the biological role of uPAR in melanoma and found that inhibition of uPAR via RNA interference induced massive death in three different metastatic cell lines. Annexin-V staining and caspase activation analysis revealed induction of the mitochondrial apoptotic pathway. The expression of members of the Bcl-2 family (Bax, Bcl-2, Bak and Bcl-x(L)) was changed in a pro-apoptotic manner. uPAR inhibition induced the expression of the tumor suppressor p53 and of its downstream target gene p21. Inhibition of p53 rescued cells from apoptosis indicating that p53 was critical for apoptosis induction. Apoptosis was observed in melanoma cells carrying activating BRAF mutations and occurred in the presence of extracellular signal-regulated kinase (ERK) phosphorylation. uPAR can activate focal adhesion kinase (FAK), which is implicated in adhesion-dependent tumor cell survival. However, inhibition of FAK did not induce apoptosis. Our data suggest a new function of uPAR acting as a survival factor for melanoma by downregulating p53. Inhibition of uPAR induces a pro-apoptotic signalling pathway via p53 that is independent of ERK or FAK signalling. These findings may offer new treatment strategies for metastatic melanoma.     

Squamous cell carcinoma cell aggregates escape suspension-induced, p53-mediated anoikis: fibronectin and integrin alphav mediate survival signals through focal adhesion kinase

Resistance to anoikis, or apoptosis triggered by detachment from the extracellular matrix (ECM), lengthens the survival of malignant cells, facilitating reattachment and colonization of secondary sites. To examine the molecular mechanisms underlying resistance to anoikis in human oral squamous cell carcinoma (SCC) cells, we cultured human squamous carcinoma (HSC-3) cells in suspension on plates coated with poly-2-hydroxyethyl methacrylate, which blocks access to the ECM. Cells in suspension that formed multicellular aggregates had significantly lower levels of apoptosis than single cells. Aggregates, but not single cells, had high levels of fibronectin. Preincubation with a cyclic arginine-glycine-aspartic acid peptide or fibronectin-blocking antibody significantly increased anoikis. Single cells had markedly lower expression of the integrin alpha(v) receptor than aggregates. Blocking alpha(v) function with a blocking antibody or by transfection with an antisense oligonucleotide increased apoptosis and inhibited aggregation. In single cells but not aggregates, phosphorylation of the integrin-associated focal adhesion kinase (FAK) at tyrosine 397 was reduced, and p53 levels were increased. Apoptosis was increased by blocking FAK with an antisense oligonucleotide and reduced by blocking p53. These findings show that SCC cells escape suspension-induced anoikis by forming multicellular aggregates that avail themselves of fibronectin survival signals mediated by integrin alpha(v). Single cells in suspension that do not form aggregates undergo anoikis because of decreased FAK phosphorylation and increased p53 levels. Thus, SCC cells appear to use neighboring cells and the ECM molecule FN to promote the metastatic phenotype.     

The 7-amino-acid site in the proline-rich region of the N-terminal domain of p53 is involved in the interaction with FAK and is critical for p53 functioning

It is known that p53 alterations are commonly found in tumour cells. Another marker of tumorigenesis is FAK (focal adhesion kinase), a non-receptor kinase that is overexpressed in many types of tumours. Previously we determined that the N-terminal domain of FAK physically interacted with the N-terminal domain of p53. In the present study, using phage display, sitedirected mutagenesis, pulldown and immunoprecipitation assays we localized the site of FAK binding to a 7-amino-acid region(amino acids 65-71) in the N-terminal proline-rich domain of human p53. Mutation of the binding site in p53 reversed the suppressive effect of FAK on p53-mediated transactivation ofp21, BAX (Bcl-2-associated X protein) and Mdm2 (murine double minute 2) promoters. In addition, to functionally test this p53 site, we conjugated p53 peptides [wild-type (containing the wild-type binding site) and mutant (with a mutated 7-aminoacid binding site)] to a TAT peptide sequence to penetrate the cells, and demonstrated that the wild-type p53 peptide disrupted binding of FAK and p53 proteins and significantly inhibited cell viability of HCT116 p53+/+ cells compared with the control mutant peptide and HCT116 p53-/- cells. Furthermore, the TAT-p53 peptide decreased the viability of MCF-7 cells, whereas the mutant peptide did not cause this effect. Normal fibroblast p53+/+ and p53-/- MEF (murine embryonic fibroblast) cells and breast MCF10A cells were not sensitive to p53 peptide. Thus, for the first time, we have identified the binding site of the p53 andFAK interaction and have demonstrated that mutating this site and targeting the site with peptides affects p53 functioning and viability in the cells.     

Pyk2 Inhibition of p53 as an Adaptive and Intrinsic Mechanism Facilitating Cell Proliferation and Survival

Pyk2 is a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK). Compensatory Pyk2 expression occurs upon FAK loss in mice. However, the impact of Pyk2 up-regulation remains unclear. Previous studies showed that nuclear-localized FAK promotes cell proliferation and survival through FAK FERM domain-enhanced p53 tumor suppressor degradation (Lim, S. T., Chen, X. L., Lim, Y., Hanson, D. A., Vo, T. T., Howerton, K., Larocque, N., Fisher, S. J., Schlaepfer, D. D., and Ilic, D. (2008) Mol. Cell 29, 9–22). Here, we show that FAK knockdown triggered p53 activation and G₁ cell cycle arrest in human umbilical vein endothelial cells after 4 days. However, by 7 days elevated Pyk2 expression occurred with a reduction in p53 levels and the release of the G₁ block under conditions of continued FAK knockdown. To determine whether Pyk2 regulates p53, experiments were performed in FAK^−/−p21^−/− mouse embryo fibroblasts expressing endogenous Pyk2 and in ID8 ovarian carcinoma cells expressing both Pyk2 and FAK. In both cell lines, Pyk2 knockdown increased p53 levels and inhibited cell proliferation associated with G₁ cell cycle arrest. Pyk2 FERM domain re-expression was sufficient to reduce p53 levels and promote increased BrdUrd incorporation. Pyk2 FERM promoted Mdm2-dependent p53 ubiquitination. Pyk2 FERM effects on p53 were blocked by proteasomal inhibition or mutational-inactivation of Pyk2 FERM nuclear localization. Staurosporine stress of ID8 cells promoted endogenous Pyk2 nuclear accumulation and enhanced Pyk2 binding to p53. Pyk2 knockdown potentiated ID8 cell death upon staurosporine addition. Moreover, Pyk2 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.     

Role of p53/FAK association and p53 phosphorylation in staurosporine-mediated apoptosis: Wild type versus mutant p53-R175H

A novel survival role of focal adhesion kinase (FAK) that involves its nuclear translocation and direct association with p53 has been demonstrated. Here we examined the relationship between the p53/FAK interaction and Ser46 phosphorylation of p53 (p-p53^Ser46) in the apoptotic regulation of human esophageal squamous cell carcinoma (HOSCC) cell lines, expressing either wild type (wt) p53 or mutant (mt) p53-R175H. In contrast to the wt p53 cell lines, the mt p53-R175H cell line was resistant to staurosporine (STS)-mediated detachment and caspase-3 activation. Furthermore, despite the resistance of mt p53-R175H to Ser46 phosphorylation, both wt and mt HOSCC cells translocate FAK into the nucleus and maintain the p53/FAK interaction post STS treatment. These findings provide unique insight into how tumor cells harboring the R175H mutant may resist chemotherapeutic intervention.

Structured summary

MINT-7294020: FAK (uniprotkb:Q05397) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by anti-bait coimmunoprecipitation (MI:0006)     

Focal adhesion kinase controls cellular levels of p27/Kip1 and p21/Cip1 through Skp2-dependent and -independent mechanisms

Endothelial cell proliferation is a critical step in angiogenesis and requires a coordinated response to soluble growth factors and the extracellular matrix. As focal adhesion kinase (FAK) integrates signals from both adhesion events and growth factor stimulation, we investigated its role in endothelial cell proliferation. Expression of a dominant-negative FAK protein, FAK-related nonkinase (FRNK), impaired phosphorylation of FAK and blocked DNA synthesis in response to multiple angiogenic stimuli. These results coincided with elevated cyclin-dependent kinase inhibitors (CDKIs) p21/Cip and p27/Kip, as a consequence of impaired degradation. FRNK inhibited the expression of Skp2, an F-box protein that targets CDKIs, by inhibiting mitogen-induced mRNA. The FAK-regulated degradation of p27/Kip was Skp2 dependent, while levels of p21/Cip were regulated independent of Skp2. Skp2 is required for endothelial cell proliferation as a consequence of degrading p27. Finally, knockdown of both p21 and p27 in FRNK-expressing cells completely restored mitogen-induced endothelial cell proliferation. These data demonstrate a critical role for FAK in the regulation of CDKIs through two independent mechanisms: Skp2 dependent and Skp2 independent. They also provide important insights into the requirement of focal adhesion kinase for normal vascular development and reveal novel regulatory control points for angiogenesis.     

FERM control of FAK function: Implications for cancer therapy

Integrins are transmembrane receptors that bind to extracellular matrix proteins and convey anchorage-dependent signals regulating normal cell proliferation. Integrin signals within the tumor micro-environment also impact cancer cell survival and invasion during tumor progression. These integrin-associated signaling events are transduced in part through the activation of non-receptor protein-tyrosine kinases. Focal adhesion kinase (FAK) is activated by β-subunit integrins in both normal and transformed cells. As genetic inactivation of β1 integrin or FAK yield early embryonic lethal phenotypes associated with decreased cell proliferation, and dominant-negative inhibition of FAK can cause increased cell apoptosis, there is a concern that FAK inhibition may have cytotoxic effects on cell growth or survival.However, FAK-specific small molecule inhibitors do not directly impact cell growth in culture, but yet show potent anti-tumor growth effects in vivo. Additionally, recent studies have shed new insight into the FAK kinase-independent regulation of cell proliferation and survival mediated by the FAK N-terminal FERM (band 4.1, ezrin, radixin, moesin homology) domain.Herein, we review the role of the FAK FERM domain in both the intrinsic regulation of FAKkinase activity and how FERM-mediated nuclear localization of FAK promotes enhanced cell survival through the inhibition of tumor suppressor p53 activation during development and under conditions of cellular stress. As we find that FAK FERM-mediated regulation of p53 occurs in human carcinoma cells, elevated FAK expression in tumors may promote both kinase-dependent and –independent survival mechanisms. We discuss how the pharmacological inhibition of FAK kinase activity may impact tumor progression through combined effects of blocking both tumor- and stromal-associated signaling regulating neovascularization.     

Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.     

Src-dependent autophagic degradation of Ret in FAK-signalling-defective cancer cells

We have recently described that autophagic targeting of Src maintains cancer cell viability when FAK signalling is defective. Here, we show that the Ret tyrosine kinase is also degraded by autophagy in cancer cells with altered/reduced FAK signalling, preventing its binding to FAK at integrin adhesions. Inhibition of autophagy restores Ret localization to focal adhesions. Importantly, Src kinase activity is required to target Ret to autophagosomes and enhance Ret degradation. Src is thus a general mediator of selective autophagic targeting of adhesion-linked kinases, and Ret a second FAK-binding tyrosine kinase degraded through autophagy in cancer cells under adhesion stress. Src-by controlling not only its own degradation but also that of other FAK-binding partners-allows cancer cell survival, suggesting a new therapeutic strategy.     

Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21(WAF1/Cip1) resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3sigma, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3sigma (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.     

An altered fibronectin matrix induces anoikis of human squamous cell carcinoma cells by suppressing integrin alpha v levels and phosphorylation of FAK and ERK

Fibronectin regulates many cellular processes, including migration, proliferation, differentiation, and survival. Previously, we showed that squamous cell carcinoma (SCC) cell aggregates escape suspension-induced, p53-mediated anoikis by engaging in fibronectin-mediated survival signals through focal adhesion kinase (FAK). Here we report that an altered matrix, consisting of a mutated, nonfunctional high-affinity heparin-binding domain and the V region of fibronectin (V⁺H⁻), induced anoikis in human SCC cells; this response was blocked by inhibitors of caspase-8 and caspase-3. Anoikis was mediated by downregulation of integrin alpha v in a panel of SCC cells and was shown to be proteasome-dependent. Overexpression of integrin alpha v or FAK inhibited the increase in caspase-3 activation and apoptosis, whereas suppression of alpha v or FAK triggered a further significant increase in apoptosis, indicating that the apoptosis was mediated by suppression of integrin alpha v levels and dephosphorylation of FAK. Treatment with V⁺H⁻ decreased the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, and direct activation of ERK by constitutively active MEK1, an ERK kinase, increased ERK1 and ERK2 phosphorylation and inhibited the increase in apoptosis induced by V⁺H⁻. ERK acted downstream from alpha v and FAK signals, since alpha v and FAK overexpression inhibited both the decrease in ERK phosphorylation and the increase in anoikis triggered by V⁺H⁻. These findings provide evidence that mutations in the high-affinity heparin-binding domain in association with the V region of fibronectin, or altered fibronectin matrices, induce anoikis in human SCC cells by modulating integrin alpha v-mediated phosphorylation of FAK and ERK.     

Direct interaction of focal adhesion kinase with p190RhoGEF

Focal adhesion kinase (FAK) is a protein-tyrosine kinase that associates with multiple cell surface receptors and signaling proteins through which it can modulate the activity of several intracellular signaling pathways. FAK activity can influence the formation of distinct actin cytoskeletal structures such as lamellipodia and stress fibers in part through effects on small Rho GTPases, although the molecular interconnections of these events are not well defined. Here, we report that FAK interacts with p190RhoGEF, a RhoA-specific GDP/GTP exchange factor, in neuronal cells and in brain tissue extracts by co-immunoprecipitation and co-localization analyses. Using a two-hybrid assay and deletion mutagenesis, the binding site of the FAK C-terminal focal adhesion targeting (FAT) domain was identified within the C-terminal coiled-coil domain of p190RhoGEF. Binding was independent of a LD-like binding motif within p190RhoGEF, yet FAK association was disrupted by a mutation (Leu-1034 to Ser) that weakens the helical bundle structure of the FAK FAT domain. Neuro-2a cell binding to laminin increased endogenous FAK and p190RhoGEF tyrosine phosphorylation, and co-transfection of a dominant-negative inhibitor of FAK activity, termed FRNK, inhibited lamininstimulated p190RhoGEF tyrosine phosphorylation and p21 RhoA GTP binding. Overexpression of FAK in Neuro-2a cells increased both endogenous p190RhoGEF tyrosine phosphorylation and RhoA activity, whereas these events were inhibited by FRNK co-expression. Because insulin-like growth factor 1 treatment of Neuro-2a cells increased FAK tyrosine phosphorylation and enhanced p190RhoGEF-mediated activation of RhoA, our results support the conclusion that FAK association with p190RhoGEF functions as a signaling pathway downstream of integrins and growth factor receptors to stimulate Rho activity.     

Understanding the Roles of FAK in Cancer: Inhibitors,Genetic Models,and New Insights

Focal adhesion kinase (FAK) is a protein tyrosine kinase that regulates cellular adhesion, motility, proliferation and survival in various types of cells. Interestingly, FAK is activated and/or overexpressed in advanced cancers, and promotes cancer progression and metastasis. For this reason, FAK became a potential therapeutic target in cancer, and small molecule FAK inhibitors have been developed and are being tested in clinical phase trials. These inhibitors have demonstrated to be effective by inducing tumor cell apoptosis in addition to reducing metastasis and angiogenesis. Furthermore, several genetic FAK mouse models have made advancements in understanding the specific role of FAK both in tumors and in the tumor environment. In this review, we discuss FAK inhibitors as well as genetic mouse models to provide mechanistic insights into FAK signaling and its potential in cancer therapy.