Focal Adhesion Kinase and p53 Signaling in Cancer Cells

C H A P T E R

T H R E E

Focal Adhesion Kinase and p53 Signaling in Cancer Cells Vita M. Golubovskaya*,† and William G. Cance*,†,‡ Contents 104 105 105 111 114 122 122 128 129 130 132 132 133 133 134 136 136 136 137 137 138 138 138

1. Introduction 2. Structure and Function of Focal Adhesion Kinase Protein 2.1. FAK structure 2.2. FAK functioning in cells 2.3. FAK-protein binding partners 3. FAK in Tumorigenesis 3.1. Overexpression in tumors 3.2. FAK and stem cells 4. FAK and p53 Association 4.1. Structure and function of the p53 protein 4.2. p53 mutations in cancer cells 4.3. p53 binds the FAK promoter 4.4. Direct FAK and p53 protein binding 4.5. Cytoplasmic–nuclear protein shuttling 4.6. Feedback model of FAK–p53 protein interaction 5. FAK and p53 Targeted Therapy 5.1. Downregulation of FAK 5.2. FAK inhibitors 5.3. Targeting protein–protein interactions 5.4. p53 therapy 6. Summary Acknowledgments References

Abstract The progression of human cancer is characterized by a process of tumor cell motility, invasion, and metastasis to distant sites, requiring the cancer cells to be able to survive the apoptotic pressures of anchorage-independent conditions. * { {

Department of Surgery, University of Florida School of Medicine, University of Florida, Gainesville, Florida 32610 UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610

International Review of Cytology, Volume 263 ISSN 0074-7696, DOI: 10.1016/S0074-7696(07)63003-4

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2007 Elsevier Inc. All rights reserved.

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One of the critical tyrosine kinases linked to these processes of tumor invasion and survival is the focal adhesion kinase (FAK). FAK was first isolated from human tumors, and FAK mRNA was found to be upregulated in invasive and metastatic human breast and colon cancer samples. Recently, the FAK promoter was cloned, and it has been found to contain p53-binding sites. p53 inhibits FAK transcription, and recent data show direct binding of FAK and p53 proteins in vitro and in vivo. The structure of FAK and p53, proteins interacting with FAK, and the role of FAK in tumorigenesis and FAK-p53-related therapy are reviewed. This review focuses on FAK signal transduction pathways, particularly on FAK and p53 signaling, revealing a new paradigm in cell biology, linking signaling from the extracellular matrix to the nucleus. Key words: Focal adhesion kinase, Cancer, p53, Apoptosis, Tumorigenesis. ß 2007 Elsevier Inc.

1. Introduction Focal adhesion kinase (FAK) was discovered about 15 years ago as a tyrosine phosphorylated protein kinase. Since then it has become clear that this protein plays a critical role in intracellular processes of cell adhesion, motility, survival, and cell cycle progression. Cancer is often characterized by defects of these processes. One of the critical tyrosine kinases that are linked to the processes of tumor invasion and survival is the FAK. The FAK gene encodes a nonreceptor tyrosine kinase that localizes at contact points of cells with extracellular matrix and is activated by integrin (cell surface receptor) signaling. The FAK gene was first isolated from chicken embryo fibroblasts transformed by v-src (Schaller et al., 1992). We were the first to isolate the FAK gene from human tumors, and in our initial report, we demonstrated that FAK mRNA was upregulated in invasive and metastatic human breast and colon cancer samples (Weiner et al., 1993). At the same time, matched samples of normal colon and breast tissue from the same patients had almost no detectable FAK expression. This was the first evidence that FAK might be regulated at the level of gene transcription, as well as by other mechanisms. Subsequently, we have demonstrated upregulation of FAK at the protein level in a wide variety of human tumors, including breast cancer, colon cancer, ovarian cancer, thyroid cancer, melanoma, and sarcoma (Cance et al., 2000; Judson et al., 1999; Owens et al., 1995, 1996). Recently, we cloned the regulatory promoter region of the FAK gene and confirmed transcriptional upregulation in cancer cell lines (Golubovskaya et al., 2004). We have found that the FAK promoter contains p53 binding sites and that p53 inhibits FAK transcription. In addition, our recent data show direct binding of FAK and p53 proteins in vitro and in vivo

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(Golubovskaya et al., 2005). Thus, this review will focus on FAK intracellular signaling in cancer, especially on the novel FAK and p53 signaling, linking signaling from the extracellular matrix to the nucleus. We will focus on the role of FAK expression, localization, transport, activity, protein interaction, and survival signaling in the development of cancer. We will discuss the structure, function, binding partners, and localization of FAK. Then we will discuss the novel cross-link of FAK and p53 signaling and its interaction, which opens a new paradigm in cell biology, and will pay attention to novel therapeutic approaches to target this interaction.

2. Structure and Function of Focal Adhesion Kinase Protein 2.1. FAK structure 2.1.1. FAK gene cDNA First, FAK cDNA encoding a 125-kDa protein was isolated from chicken embryo cells (Schaller et al., 1992). Then mouse FAK cDNA, encoding a 119-kDa FAK protein, was identified (Hanks et al., 1992). The human FAK (also known as PTK2a) gene has been mapped to chromosome 8 (Agochiya et al., 1999; Fiedorek and Kay, 1995), and there appears to be a high degree of homology between species. The human complete FAK mRNA sequence (NCBI Accession number: L13616) is a 3791-base long sequence that includes the 50 -untranslated 233-base pair region (Whitney et al., 1993). We were the first group to isolate human FAK cDNA from primary sarcoma tissue and found increased FAK mRNA in tumor samples compared with normal tissue samples (Weiner et al., 1993). Subsequently, Xenopus laevis FAK cDNA (Zhang et al., 1995) and rat FAK cDNA (Burgaya and Girault, 1996) were identified. Recently, Drosophila FAK cDNA (Dfak56) was isolated (Fujimoto et al., 1999). FAK cDNA is closely related to the homologous proline-rich calcium-dependent tyrosine kinase (45% amino acid identity) that is also located in humans at chromosome 8, locus p21.1, named PYK2 (RAFTK [related adhesion focal tyrosine kinase]), CADTK (calcium-dependent tyrosine kinase), CAK (cell adhesion kinase) b, and PTK 2b (protein tyrosine kinase 2b) (Avraham et al., 1995; Lev et al., 1995; Sasaki et al., 1995). Genomic structure Recently, the genomic structure of FAK has been characterized (Corsi et al., 2006). The gene coding sequence contains 34 exons (NCBI Gene ID: 5747), and the genomic sequence spans 230 kb (Corsi et al., 2006). The FAK gene contains four 50 noncoding exons and 34 coding exons, and it was shown to have multiple alternatively spliced forms. Comparison of the mouse and human FAK genes detected conservative

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and nonconservative 50 -untranslated exons, which suggests a complex regulation of FAK expression. Exons (13, 14, 16, and 31) are highly conserved among vertebrate species, suggesting their critical function in gene regulation (Corsi et al., 2006). It is known that alternative splicing often occurs and plays an important role in cancer (Caballero et al., 2001; Venables, 2006). Alternative splicing most often results from a different exon inclusion, but can also occur from intron retention or an alternative choice between two splice sites leading to changes in protein localization, structure, removal of phosphorylation sites, or proteasomal degradation (Venables, 2006). There were several cases of alternatively spliced genes that are involved in invasion and metastasis (Rac 1, b-catenin, Crk) or angiogenesis (VEGFR-2, VEGFR-3 [Flt-4]). Thus, a detailed study of alternatively spliced forms of FAK that are overexpressed in pre- and metastatic cancers will be critical for understanding mechanisms and regulation of FAK expression in carcinogenesis, either by changes in mRNA, by changes in the coding sequence (exon inclusion/exclusion), or by changes in protein levels (stability, etc.). FAK promoter We were the first group to clone the human FAK promoter regulating FAK expression (Golubovskaya et al., 2004). The core promoter contains 600 base pairs and includes many transcription binding sites, such as AP-1, AP-2, SP-1, PU.1, GCF, TCF-1, EGR-1, NF-kB, and p53 (Golubovskaya et al., 2004). We have demonstrated that NF-kB binds to the FAK promoter and upregulates its activity, as it was increased by an NFkB-inducing agent, tumor necrosis factor (TNF)-a, and blocked by an NFkB inhibitor, a superrepressor of NF-kB (Golubovskaya et al., 2004). Interestingly, we found two transcription binding sites for p53 in the FAK promoter and found that p53 can block FAK promoter activity (Golubovskaya et al., 2004). Recently, a mouse promoter has been cloned that was highly homologous to the human promoter and contained the same binding sites (Corsi et al., 2006). In addition, the FAK gene has an internal FRNK promoter or C-terminal, FAK-CD promoter that has been recently cloned by the Parsons group (Hayasaka et al., 2005), regulating expression of autonomously expressed FRNK protein.

2.1.2. FAK protein domains The FAK protein is a 125-kDa tyrosine kinase (p125FAK) with a large amino-N-terminal domain, exhibiting homology with a FERM (protein 4.1, ezrin, radixin, and moesin) domain with an autophosphorylation site (Y-397), a central catalytic domain, and a large carboxy-C-terminal domain that contains a number of potential protein interacting sites, including two proline-rich domains and an FAT domain (Hanks and Polte, 1997; Schaller and Parsons, 1994; Schaller et al., 1994) (Fig. 3.1).

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c-Met VEGFR-3

EGFR

Integrins

PDGFR

FAK

p53 N-terminus (1−415) Bmx/ Etk

FERM K152

Ezrin

p130Cas

Kinase domain (416−676) Pro

ASAP1

GRAF

C-terminus (677−1052) Pro

FRNK

Pro

Talin

FAT

Trio

K454 Y397 Y407 Y567 Y577

Y861

Y925

Paxillin

PIAS-1 ATP

JSAP1

Grb-7 RIP Shc

Src

S722 S732 S843 S910

Grb-2 PI-3K Nck-2

FIP200

RhoGEF

Hic-5

Figure 3.1 Focal adhesion kinase (FAK) structure. FAK has the N-terminal, kinase domain and the C-terminal domains.The N-terminal domain has theY-397-Y-autophosphorylation site. The kinase domain has the Y576/577 tyrosines, important for catalytic activity of FAK. The C-terminal part of FAK has Y861 and Y925 tyrosines. Different proteins bind to these domains and are involved in motility and survival signaling. The N-terminal domain (205^422 aa) of FAK is involved in interaction with Src, RIP, p53, PI3K, PIAS-1, Grb-7, EGFR/PDGFR, Ezrin, Bmx, Trio, and others. The kinase domain is involved in binding with FIP200 protein. ASAP, p130Cas, Grb-2, paxillin, talin, RhoGEFp190, and others bind the C-terminal domain of FAK. Interactions of FAK and other proteins demonstrated by group are shown in italics.

N-terminal domain The first function of the N-terminal domain, homologous to the FERM domain, was linked to the binding of integrins, via their b subunits (Schaller et al., 1995). The N-terminal domain of the FAK protein contains the major autophosphorylation site Y397-tyrosine, which in phosphorylated form becomes a binding site of the SH-2 domain of Src, leading to its conformational changes and activation (Hanks and Polte, 1997). Tyrosine phosphorylation of FAK and binding of Src lead to tyrosine phosphorylation of other tyrosine phosphorylation sites of FAK: Y407; Y576, Y577—the major phosphorylation sites in the catalytic domain of FAK; and Y861 and Y925 (Hanks and Polte, 1997; McLean et al., 2005)— to phosphorylation of FAK-binding proteins, such as paxillin and Cas (Schaller et al., 1999). That leads to subsequent cytoskeletal changes and activation of RAS-MAPK (mitogen-activated protein kinase) signaling pathways (Hanks et al., 2003; McLean et al., 2005). Thus, the FAK-Src signaling complex activates many signaling proteins involved in survival and

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Growth factors

Integrins

FAK VEGFR-3

RIP

p53

Paxillin

Talin N-terminus

C-terminus

FERM domain

Kinase domain

Y 397

Cytoplasm

F.A.T

Pro-1 Pro-2

P

Src-family PTKs

p130Cas

PI-3K

Angiogenesis ?

AKT

ERK/MAP Cell migration Lymphogenesis Cell invasion

FAK

p53

Survival signals Cell proliferation

p53 FAK Nucleus

Apoptosis P21, Bax

Figure 3.2 Focal adhesion kinase (FAK) functions in cells. Focal adhesion kinase integrates signals from growth factor receptors (EGFR,VEGFR) and integrins to control motility, survival, metastasis, lymphogenesis, and angiogenesis. Numerous binding partners of FAK mediate this signaling. The FAK and p53 complex is involved in apoptotic/survival signaling.

motility and the metastatic, invasive phenotype in cancer cells (Figs. 3.1 and 3.2). Phosphorylated Y397 FAK is able to recruit important signaling proteins: p85 phosphoinositide 3-kinase (PI3K), growth factor receptor bound protein Grb 7, phospholipase Cg (PLCg), and others. The crystal structure of the N-terminal domain of avian FAK, containing the FERM domain, has recently been reported (Ceccarelli et al., 2006). An interesting negative regulation of FAK function by the FERM domain was revealed by Cooper et al. (2003), where the N-terminal domain had an autoinhibitory effect through interaction with the kinase domain of FAK. Recently, our group discovered several novel binding partners of the N-terminal FAK domain in cancer cells, such as the epidermal growth factor receptor (EGFR) (Golubovskaya et al., 2002; Sieg et al., 2000), receptorinteracting protein (RIP) (Kurenova et al., 2004), and p53 (Golubovskaya et al., 2005) (Fig. 3.1, shown in italics). The N-terminal domain of FAK has been shown to cause apoptosis in breast cancer cells (Beviglia et al., 2003) and can be localized to the nucleus ( Jones and Stewart, 2004; Jones et al., 2001;

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Lobo and Zachary, 2000; Stewart et al., 2002). Thus, the N-terminal domain of FAK binds to the extracellular matrix receptors, integrins, growth factor receptors, or important signaling cytoplasmic, cytoskeletal, and nuclear proteins, mediating signaling from the extracellular matrix to the cytoplasm and nucleus and controlling cytoskeletal changes, survival, motility, and invasion. Kinase domain The central kinase (catalytic) domain of FAK (424–676 amino acids) is the most conserved domain in vertebrate and nonvertebrate organisms (Corsi et al., 2006). The central catalytic domain of FAK contains Y576 and Y577, major phosphorylation sites, and also K454, the ATPbinding site (Fig. 3.1). Phosphorylation of FAK by Src on Y576 and Y577 is an important step in the formation of an active signaling complex and is required for maximum enzymatic activity of FAK (Calalb et al., 1995). The crystal structure of the FAK kinase domain revealed an open conformation similar to the fibroblast growth factor receptor-1 (FGFR-1) and vascular endothelial growth factor receptor (VEGFR) (Nowakowski et al., 2002). The FAK kinase domain structure has an unusual bisulfite bond between the conserved cysteines 456 and C459, suggesting a possible role in protein– protein interactions and kinase function (Nowakowski et al., 2002). The ATP-binding site of protein kinases is the most common target for the small-molecule inhibitors design, although the design of these inhibitors can be complicated by structural similarities between kinase domains. Thus, finding small structural differences is a key in such a design. For example, the side chain of glutamic acid, E506, forms a bifurcated hydrogen bond to the 20 and 30 hydroxyl groups of the ribose (Nowakowski et al., 2002). The corresponding side chains in EphA2 and Aurora-A kinases are smaller and do not contact with sugar (Nowakowski et al., 2002). C-terminal domain Different proteins can bind to the C-terminal domain of p125FAK (677–1052 amino acids), including paxillin, p130cas, PI3K, and GTPase-activating protein Graf, leading to changes in the cytoskeleton and to activation of the Ras-MAPK pathway (Hanks et al., 2003; Parsons, 2003; Schaller and Parsons, 1994; Windham et al., 2002). The carboxy-terminal domain of FAK contains sequences responsible for its targeting to focal adhesions, also known as the FAT domain. Alternative splicing of FAK results in autonomous expression of the C-terminal part of FAK, FAKrelated nonkinase (FRNK) (Richardson and Parsons, 1995). The crystal structure of the C-terminal domain of FAK, the focal adhesion targeting domain (FAT), has been determined recently by several groups (Hayashi et al., 2002; Prutzman et al., 2004) and can exist as a dimer or monomer, allowing binding of several binding partners.

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2.1.3. Posttranslational modifications The main posttranslational modifications of FAK are phosphorylation of tyrosines and serines, and recently sumoylation of FAK has been reported (Kadare et al., 2003). Phosphorylation of tyrosines and serines FAK has numerous tyrosine phosphorylated sites: Y397, Y407, Y576/Y577, Y861, and Y925 (Fig. 3.1). Phosphorylation of Y397, as mentioned previously, created a binding site for Src, PI3K, PLC-g, Grb-7, and Grb-2-SOS. Phosphorylation of tyrosine 407, as well as Y397, correlated with differentiation and with the level of gastrin-releasing peptide and its receptor in colon cancer cells (Matkowskyj et al., 2003). Phosphorylation of Y576 and Y577 correlated with maximal activity of FAK (Calalb et al., 1995). Src-dependent phosphorylation of Y861 was induced by VEGFR in HUVEC endothelial cells (AbuGhazaleh et al., 2001). The FAT domain mediates signaling through Grb2 binding to the Y925 site of FAK (Arold et al., 2002). Inhibition of FAK, which resulted in decreased Y925 phosphorylation, resulted in decreased FAK-Grb2-MAPK signaling and VEGFR-induced tumor growth of 4T1 breast carcinoma cells (Mitra et al., 2006). In addition to tyrosine phosphorylation, several serine phosphorylation sites were reported to play a major role in FAK function, such as serines 722, 732, 843, and 910 (Fig. 3.1). The role of serine phosphorylation has been investigated less than the phosphorylation of tyrosines, but it was suggested that it plays a role in the binding/stability of proteins (Parsons, 2003). Some studies described serine phosphorylation of FAK. For example, phosphorylation of serine 722 was induced by Rho-dependent kinase in endothelial cells in response to VEGF (Le Boeuf et al., 2006). Serine 732 was reported to be phosphorylated by serine-threonine kinase Cdk5, which was important for microtubule organization, nuclear movement, and neuronal migration in cultured neuron cells (Xie et al., 2003). G-protein-coupled receptor activation induced phosphorylation of serine 843 (Fan et al., 2005). Recently, FAK phosphorylation at tyrosine 397 has been shown to be reverse correlated with phosphorylation at serine 843 ( Jacamo et al., 2007). While platelet-derived growth factor (PDGF) induced serine 910 phosphorylation of FAK in Swiss 3T3 cells, that differed in kinetics and in response to MAPK and PI3K inhibitors from Y397 tyrosine phosphorylation, suggesting activation of different signaling pathways (Hunger-Glaser et al., 2004). In addition, bombesin, lysophosphatic acid, and epidermal growth factor activated Ser-910 phosphorylation of FAK in Swiss 3T3 cells via an extracellular signalregulated kinase (ERK)-dependent pathway (Hunger-Glaser et al., 2003). In addition, recently mass spectrometry analysis of chicken FAK revealed 19 new sites of phosphorylation with some sites reported before: 15 serine, 5 threonine, and 5 tyrosine residues (Grigera et al., 2005). It was

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suggested that coordinated phosphorylation of FAK by tyrosine and serine/ threonine-specific kinases may be a critical step in regulation of FAK function (Grigera et al., 2005). Some of the sites were present only in chicken FAK, such as S386, T388, and T393, but several chicken phosphorylation sites were conservative in human, mouse, and frog species, such as S29, Y155, S390, S392, T394, Y397, T406, Y407, Y570, T700, S708, S722, S725, S726, S732, S766, S845 (S843 in human), S894, Y899, and S911 (S910 in human and mouse) (Grigera et al., 2005). Thus, now there are a total of 30 sites of phosphorylation of FAK, including those reported before, requiring detailed analysis of their biological functioning in vivo. Sumoylation Recently, the N-terminal domain of FAK was reported to contain lysine 152, which is sumoylated by a small ubiquitin-like modifier protein, SUMO, in the presence of PIAS1 ligase (Kadare et al., 2003). PIAS1 is a protein that initially was cloned as a protein inhibitor of activated STAT1 (signal transducer and activator of transcription) (Liu et al., 1998), which has been shown to interact with SUMO-1 and caused sumoylation of several proteins, such as p53 (Schmidt and Muller, 2002, 2003), regulating its transcriptional activity. Sumoylation of FAK at lysine 152 caused its nuclear localization and increased its autophosphorylation activity (Kadare et al., 2003). Since sumoylation occurs in the nucleus, and FAK was reported to be localized in the nucleus (Golubovskaya et al., 2005; Jones and Stewart, 2004; Lobo and Zachary, 2000), mechanisms of nucleocytoplasmic shuttling of FAK and its function between membrane, focal adhesions, and nucleus remain to be discovered.

2.2. FAK functioning in cells FAK has numerous functions in cell survival, motility, metastasis, invasion, and angiogenesis (Fig. 3.2). 2.2.1. Motility FAK has also been shown to be important for cell motility (Hanks et al., 2003; Hauck et al., 2001; Schaller, 2001; Schlaepfer and Mitra, 2004). FAKnull embryos exhibit decreased motility in vitro (Ilic et al., 1995). Furthermore, enforced expression of FAK stimulated cell migration (Hildebrand et al., 1993; Sieg et al., 1999). Cell migration is initiated by protrusion at the leading edge of the cell, by the formation of peripheral adhesions, exertion of force on these adhesions, and then the release of the adhesions at the rear of the cell (Tilghman et al., 2005). FAK is involved in the regulation of migration, although the precise mechanism of this FAK-regulated migration is unclear. FAK has been shown to be required for the organization of the leading edge in migrating cells by coordinating integrin signaling in

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order to direct the correct activation of membrane protrusion (Tilghman et al., 2005). The SH2 domain of Src, targeting Src to focal adhesions and Y397 activity, has been shown to be important for motility (Yeo et al., 2006). PI3K has also been shown to be critical for FAK-mediated motility in Chinese hamster ovary (CHO) cells (Reiske et al., 1999). The tumor suppressor gene PTEN, encoding phosphatase, has been shown to interact with FAK, causing its dephosphorylation and blocked motility (Tamura et al., 1998). Moreover, Y397FAK was important for PTEN interaction with FAK (Tamura et al., 1999a). Overexpression of FAK reversed the inhibitory effect of PTEN on cell migration (Tamura et al., 1998). 2.2.2. Invasion and metastasis Activation of FAK is linked to invasion and metastasis signaling pathways. FAK was important in Erb-2/Erb-3-induced oncogenic transformation and invasion (Benlimame et al., 2005). Inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis processes (Benlimame et al., 2005). In addition, FAK has been shown to be activated in invading fibrosarcoma and regulated metastasis (Hanada et al., 2005). Inhibition of FAK with dominant-negative FAK-CD disrupted invasion of cancer cells (Hauck et al., 2001). We have also shown high FAK expression in breast cancers associated with tumor aggressive phenotype (Lark et al., 2005). Subsequently, we analyzed FAK expression in preinvasive ductal carcinoma in situ (DCIS) tumors and detected protein overexpression in preinvasive tumors (Lightfoot et al., 2004), suggesting that the survival function of FAK occurs as an early event in breast tumorigenesis. 2.2.3. Survival FAK plays a major role in survival signaling and has been linked to detachment-induced apoptosis or anoikis (Frisch et al., 1996). It has been shown that constitutively activated forms of FAK rescued epithelial cells from anoikis, suggesting that FAK can regulate this process (Frisch, 1999; Frisch and Ruoslahti, 1997; Frisch and Screaton, 2001; Frisch et al., 1996; Windham et al., 2002). Similarly, both FAK antisense oligonucleotides (Smith et al., 2005; Xu et al., 1996), as well as dominant-negative FAK protein (FAK-CD), caused cell detachment and apoptosis in tumor cells (Beviglia et al., 2003; Gabarra-Niecko et al., 2003; Golubovskaya et al., 2002, 2003; Park et al., 2004; van De Water et al., 2001; Xu et al., 1996, 1998, 2000). The antiapoptotic role of FAK was also demonstrated in FAK-transfected FAK/ HL60 cells that were highly resistant to apoptosis induced with etoposide and hydrogen peroxide compared with the parental HL-60 cells or the vectortransfected cells (Kasahara et al., 2002; Sonoda et al., 2000). HL-60/FAK cells activated the AKT pathway and NF-kB survival pathways with the induction of inhibitor-of-apoptosis proteins (IAPs) (Sonoda et al., 2000). We have

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demonstrated that EGFR and Src signaling cooperate with FAK survival signaling in colon and breast cancer cells (Golubovskaya et al., 2002, 2003; Park et al., 1999, 2004). We have also demonstrated that simultaneous inhibition of Src and FAK or EGFR and FAK pathways was able to increase apoptosis in cancer cells (Golubovskaya et al., 2002, 2003). Thus, cancer cells use the cooperative function of kinases and growth factor receptor signaling to increase survival. 2.2.4. Angiogenesis VEGF is one of the known angiogenic growth factors, stimulating formation of new blood vessels or angiogenesis. FAK has been shown to play a major role in vasculogenesis. It has been shown that VEGF induced tyrosine phosphorylation of FAK in human umbilical vein endothelial cells (HUVEC) and other endothelial cell lines (Abedi and Zachary, 1997). VEGF-induced stimulation of FAK phosphorylation was also demonstrated in cultured rat cardiac myocytes, which was accompanied by subcellular translocation of FAK from perinuclear sites to the focal adhesions and increased association with the adaptor proteins Shc, Grb-2, and c-Src (Takahashi et al., 1999). VEGF-induced phosphorylation of FAK was inhibited by the tyrosine kinase inhibitors tyrphostin and genistein (Takahashi et al., 1999). VEGF-induced phosphorylation of FAK was induced in human brain microvascular endothelial cells (HBMEC) (Avraham et al., 2003). Furthermore, inhibition of FAK with the dominant-negative inhibitor FRNK or the C-terminal FAK (FAK-CD) significantly decreased HBMEC spreading and migration (Avraham et al., 2003, 2004). In addition, the angiogenic inhibitor endostatin blocked VEGF-induced activation of FAK (Kim et al., 2002). Recently, we have shown that FAK binds to the VEGFR-3 (Flt-4) protein in cancer cell lines (Garces et al., 2006), suggesting an important role of FAK in lymphogenesis in addition to angiogenesis. We have shown that the C-terminal domain of FAK binds to VEGFR-3. Disruption of this binding with VEGFR peptides caused apoptosis in breast cancer cells, allowing novel therapeutic approaches in breast tumors (Garces et al., 2006). The detailed interaction of FAK and VEGFR signaling and its mechanisms remain to be discovered. Overexpression of FAK in vascular endothelial cells promoted angiogenesis in transgenic mice (Peng et al., 2004). Overexpession of FAK induced human retinal endothelial cell (HREC) migration and in vivo angiogenesis (Kornberg et al., 2004). FAK activity and phosphorylation of the Y925 site of FAK promoted an angiogenic switch during tumor progression (Mitra et al., 2006). FAK-Grb2-MAPK signaling has been shown to be important for promoting angiogenesis. Furthermore, inhibition of FAK resulted in disruption of angiogenesis (Mitra et al., 2006). FAK and Src catalytic activities are important in promoting VEGF-dependent angiogenesis (Mitra and

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Schlaepfer, 2006). Thus, FAK is involved in angiogenesis and plays a major role in tumorigenesis. The link between FAK and VEGFR-3 signaling opens a new area of angionegensis/lymphogenesis linkage that seems to be critical in tumorigenesis. A correlation analysis between FAK and VEGFR on a population-based series of tumor samples is needed to define this mechanism.

2.3. FAK-protein binding partners FAK has numerous binding partners in the N-terminal, central, and C-terminal domains (Fig. 3.1). The N-terminal domain of FAK contains one proline-rich domain, and the C-terminal domain of FAK contains another two proline-rich domains that are sites of binding proteins, containing SH3 domains. The C-terminal part of the C-terminal domain of FAK (853–1012 aa) is called FAT, a domain that is necessary for the targeting of FAK to focal adhesion complexes through binding with different proteins (paxillin, talin, Rho, etc.). We will focus on more than 20 known protein-binding partners, including four recent novel ones, found recently by our group (EGFR, RIP, p53, and VEGFR), classified by the main binding domain. 2.3.1. Proteins binding to the N-terminal domain of FAK Src Src is a well-known binding partner of FAK (Schaller et al., 1994). C-Src is a member of related tyrosine kinases, including Fyn, Yes, Lck, Blk, Hck, Lyn, Fgr, and Yrk. Src proteins are located in the cytoplasm at cellular sites of integrin clustering. Following integrin binding, p125FAK becomes tyrosine phosphorylated at the Y-397 site (Hanks and Polte, 1997). This leads to efficient binding of Src via its SH2 domain to FAK, generating an FAK–Src complex (Calalb et al., 1995; Schaller and Parsons, 1994; Xing et al., 1994). The SH3 domain of Src can also bind the proline-rich domain of the N-terminal FAK, regulating downstream cell signaling (Thomas et al., 1998). Src can be autophosphorylated at tyrosine Y-416, while tyrosine Y-527 is critical for a negative regulation of Src activity ( Jones et al., 2000). Recently, Src has been shown to regulate anoikis in human colon tumors (Windham et al., 2002). Similarly, we have shown that increased Src activity led to additional survival signals in suppressing apoptosis in colon cancer cell lines, overexpressing FAK (Golubovskaya et al., 2003). The same result was obtained in a breast cancer model with stably expressing activated Src (Park et al., 2004). Shc The adapter protein Shc was identified as an SH2-containing protooncogene, involved in growth factor signaling (Ravichandran, 2001). Three Shc proteins are known: Shc A, Shc B, and Shc C. The Shc A protein has three isoforms that are expressed from the same messenger RNA in cells: 46-, 52-, and 66-kDa proteins (Ravichandran, 2001). The 66-kDa form of

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Shc A is expressed in all cells, while Shc B and C are mostly expressed in neuronal cells (Ravichandran, 2001). Shc contains two domains: one binds phosphotyrosine (SH2 and PTB), and another, central domain, contains phosphorylation sites. Importantly, knockout of Shc A caused embryonic lethality in mice, suggesting its important role in vivo (Ravichandran, 2001). Activation of Shc leads to mitogen-activated protein kinase (MAPK) activation, which has been well described (Ravichandran, 2001). Activation of FAK autophosphorylation at Y397 leads to binding with the Shc adaptor, its phosphorylation, and activation of downstream MAPK pathway signaling (Hanks and Polte, 1997). Also, Shc has been reported to be hyperphosphorylated in many types of tumors (Bonsi et al., 2005; Finlayson et al., 2003). JSAP1 The N-terminal domain of FAK binds to the c-Jun N-terminal kinase ( JNK)/stress-activated protein kinase-associated protein 1 ( JSAP1) (also known as JIP3, JNK-interacting protein 3), which was originally identified as a scaffolding protein for the JNK cascade by binding to JNK, SEK1, and MEKK1 proteins (Takino et al., 2002). The complex between JSAP and FAK was stimulated by c-Src, leading to JSAP tyrosine phosphorylation (Takino et al., 2002). JSAP mediated the association of FAK and JNK and is involved in cell migration (Takino et al., 2005). JSAP was suggested to play an important role in brain tumorigenesis (Takino et al., 2005). Cell spreading and adhesion in mouse embryonic fibroblasts with knockout of JSAP1 were slower than in the wild-type cells, suggesting a role of JSAP-1 in adhesion and cell spreading (Chae et al., 2006). BMX/Etk Etk/BMX is a member of the Btk family of tyrosine kinases, which is highly expressed in endothelial cells and metastatic carcinoma cells (Chen et al., 2001). The ETK interacts with the N-terminal domain of FAK, playing a critical role in motility (Chen et al., 2001). Importantly, physically Etk also associates with p53 through its Src homology 3 domain and the proline-rich domain of p53 ( Jiang et al., 2004). Thus, Etk interacts with p53, which has been shown to be a binding partner of FAK (see the following) in the cytoplasm, leading to bidirectional inhibition of the activities of both proteins ( Jiang et al., 2004). Ezrin Ezrin belongs to a ERM (ezrin/radixin/moesin) family of proteins, present in actin-rich cell structures, such as membrane ruffles, filopodia, and microvilli, linking the plasma membrane and the actin-based cytoskeleton (Poullet et al., 2001). ERM proteins belong to members of the 4.1 superfamily with a conserved N-terminal domain (FERM domain). ERM proteins are targeted to the plasma membrane by interacting with phosphoinositides and proteins, such as CD44, intercellular adhesion molecules (ICAMs), or EBP-50 (Poullet et al., 2001). The C-terminal end of

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the ERM proteins interacts with F-actin, and its N-terminal domain interacts with the N-terminal FERM domain of FAK (Poullet et al., 2001). Overexpression of ezrin increased phosphorylation of FAK Tyr-397, creating a docking site for FAK signaling partners (Poullet et al., 2001). c-Met Recently, it has been shown that the N-terminal domain of FAK interacts with the c-Met oncogene (Chen and Chen, 2006). The c-Met oncogene encodes a c-MET receptor protein that is activated by hepatocyte growth factor (HGF), which is a mesenchymally derived factor, transducing multiple cellular responses, including proliferation, motility, survival, and morphogenesis in various types of cells. Upon HGF/SF binding, c-Met autophosphorylation occurs on two tyrosine residues (Tyr-1234 and Tyr1235) within the activation loop of the tyrosine kinase domain, regulating kinase activity (Peruzzi and Bottaro, 2006). Phosphorylation of two tyrosine residues near the COOH terminus (Tyr-1349 and -1356) forms a multifunctional docking site for intracellular adapters (Grb2, Gab1, PI3K, phospholipase C-g, Shc, Src, Shp2, and Shp1) via Src homology-2 domains and other binding motifs, leading to downstream signaling (Peruzzi and Bottaro, 2006). Mainly tyrosines Tyr-1349 and, less, Tyr-1356 of c-Met are required for its interaction with the FERM domain of FAK. A patch of basic residues (216KAKTLRK222) in the FERM domain of FAK is critical for this interaction (Chen and Chen, 2006). Furthermore, the Met-FAK interaction activates FAK and plays a critical role in hepatocyte growth factor-induced cell motility and cell invasion (Chen and Chen, 2006). It has been suggested that the Met–FAK interaction plays an important role in tumorigenesis and serves as a target for therapeutic purposes (Chen and Chen, 2006). PI3 kinase FAK has been shown to directly interact with the p85 subunit of PI3K (Chen and Guan, 1994). PI3K is a known cytoplasmic kinase that is involved in different cellular functions, such as survival, cytoskeletal changes, and carcinogenesis (Bianco et al., 2006). The autophosphorylation of Y397FAK increased its interaction with PI3K (Chen and Guan, 1994). It was suggested that PI3K can be a substrate for FAK (Chen and Guan, 1994). The Y397 site of FAK is the site of binding with PI3K (Guan, 1997). The association of PI3K with FAK was mediated primarily through the association with the SH2 domain of p85, while the SH3 domain of PI3K was also able to bind FAK (Bachelot et al., 1996). FAK has recently been shown to activate the PI3K pathway to suppress doxorubicin-induced apoptosis (van Nimwegen et al., 2006). PIAS-1 Recently, the N-terminal domain of FAK has been shown to interact with FAK and to cause sumoylation in the presence of SUMO-1 (Kadare et al., 2003). The closely related protein Pyk-2 (CADTK/RAFTK)

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was not sumoylated by these proteins. This posttranslational modification of FAK increased its autophosphorylation activity. Interestingly, this sumoylation occurred in the nucleus, providing a basis for a novel functioning of the protein in the nucleus and for nucleocytoplasmic shuttling (Kadare et al., 2003). PIAS-1 also promoted sumoylation of p53 and inhibited its transcriptional activity (Schmidt and Muller, 2002). Trio Recently, a novel binding partner with FAK has been identified and named Trio, interacting with the N-terminal domain of FAK (Medley et al., 2003). The Trio guanine nucleotide exchange factor is essential for embryonic development and fetal skeletal muscle formation and organization of neural tissue (Medley et al., 2003). Trio is a large multidomain protein containing two functional DH GEF domains, which activate Rho-type GTPases, a protein serine/threonine kinase domain, two SH3-like domains, an Ig-like domain, and multiple spectrin-like repeats (Debant et al., 1996). Trio protein functions to activate Rho GTPases, such as RhoA, Rac1, and Cdc42, by promoting the exchange of GDP for GTP. Once activated, Rho GTPases function in several cellular processes, such as actin cytoskeleton organization, MAPK cascade signaling, and gene transcription (Medley et al., 2003). FAK phosphorylates 2737 tyrosine in the kinase domain of Trio. Trio can activate autophosphorylation and the kinase function of FAK. Thus, this interaction is a novel bidirectional signaling complex, playing an important role in cell motility and regulation of focal adhesion and cytoskeleton changes (Medley et al., 2003). Grb-7 Grb-7 is an Src homology (SH) 2-containing and pleckstrin homology domain-containing adaptor protein, which associates with the N-terminal domain of FAK through Y-397 tyrosine (Han and Guan, 1999). The Grb7 family members have similar structures, containing an amino terminal proline-rich region; a central part called the GM region, which includes a pleckstrin homology (PH) domain; and a carboxy-terminal SH2 domain. Like other adaptor molecules, Grb7 family members have been shown to interact with the different cell surface receptors and other signaling proteins (Shen et al., 2002).The FAK–Grb7 complex plays a critical role in cell migration stimulated by integrin signaling through FAK (Shen et al., 2002). Nck-2 A recently identified adaptor protein Nck-2, also known as Nckß or Grb4, contains three N-terminal SH3 domains and one C-terminal SH2 domain. The Nck-2 SH2 domain has been shown to bind the N-terminal FAK at Y397 and to decrease cell motility (Goicoechea et al., 2002).

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EGFR/PDGFR p125FAK interacts with other tyrosine kinases to affect survival functions. In breast cancer cell lines that overexpressed epidermal growth factor receptor (EGFR), we have found an association between EGFR and FAK, and demonstrated that FAK suppressed apoptosis by activating AKT/ERK1/2 pathways (Golubovskaya et al., 2002). Dual inhibition of FAK and EGFR induced death-receptor-mediated apoptosis, involving activation of caspases –8 and –3 and cleavage of poly(ADP-ribose) polymerase (PARP) (Golubovskaya et al., 2002). The N-terminal domain of FAK interacts with EGFR (180 kDa) and PDGFR protein complexes, linking growth factor receptors and integrin signaling pathways (Sieg et al., 2000). RIP To study death-induced apoptosis, we performed an analysis of death receptor proteins that bind FAK and found a physical association between FAK and a 66-kDa apoptosis-related death domain RIP (Kurenova et al., 2004). We have shown that RIP provides proapoptotic signals that are suppressed by its binding to FAK (Kurenova et al., 2004). The RIP protein has dual survival and apoptotic functions: it can recruit CRADD (apoptosis) and NF-kB (survival/proliferation) (Thakar et al., 2006). p53 The first indirect link between FAK and p53 was provided by Ilic et al. (1998). It was shown that extracellular matrix survival signals mediated by FAK suppressed p53-directed apoptosis (Ilic et al., 1998). We have shown direct binding of FAK and p53 in different cancer cells (Golubovskaya et al., 2005). The N-terminal domain of p53 (1–92 aa) interacts with the 205–422 aa of the N-terminal domain of FAK (Golubovskaya et al., 2005). We have shown previously that p53 can bind the FAK promoter and inhibit its luciferase activity (Golubovskaya et al., 2005). Moreover, FAK can block p53 transcriptional activity of p21, BAX, and Mdm-2. Thus, there is a feedback loop mechanism of regulation for these two proteins.

2.3.2. Proteins binding the kinase domain of FAK: FIP200 FIP200 (focal adhesion interaction) protein is a novel 200-kDa binding partner of FAK. FIP200 binds to the kinase domain of FAK and inhibits its kinase activity (Abbi and Guan, 2002; Gan et al., 2005). This protein plays an important role in various cellular functions, such as cell adhesion, motility, and survival. FIP has been shown to inhibit the G1–S phase transition of the cell cycle by increasing p21 and decreasing cyclin D1 protein expression (Melkoumian et al., 2005). FIP200 has been shown to interact with p53, stabilizing the protein half-life (Melkoumian et al., 2005). Targeted deletion of FIP200 in the mouse led to embryonic death associated

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with heart failure and liver degeneration (Gan et al., 2006). FIP200 knockout embryos had increased apoptosis and reduced phosphorylation of c-Jun N-terminal kinase in response to TNF-a treatment (Gan et al., 2006). A competition model of association between FIP200-p53 and FIP200-FAK has recently been proposed by Mitra and Schlaepfer (2006). 2.3.3. Proteins binding with the C-terminal domain of FAK Paxillin Paxillin is a 68-kDa cytoplasmic multidomain protein that localizes to focal adhesions and is known to interact with FAK, PYK2, Src, vinculin, and Crk (Turner, 1998). It is a phosphorylation substrate of FAK that binds the C-terminal domain of FAK, FAT (Schlaepfer et al., 2004; Turner, 2000). Paxillin is localized to focal adhesions through its LIM domains. Its primary function is as a scaffold or adapter protein that provides multiple binding sites at the plasma membrane for a variety of signaling and structural proteins, causing changes in the organization of the actin cytoskeleton that are necessary for cell motility and tumor metastasis (Schaller, 2004; Turner, 2000). Paxillin contains the N-terminal leucine-rich LD motifs, binding directly to FAK, and three LIM (Lin-11, Isl-1, and Mec-3) domains in the C-terminus (Wade and Vande Pol, 2006). It was demonstrated that paxillin LIM domains 1, 2, and 3, but not the LIM 4 domain, were required for FAK tyrosine phosphorylation (Wade and Vande Pol, 2006). Cas Cas (Crc-associated substrate) is a 130-kDa protein that binds to and is phosphorylated by FAK (Hanks and Polte, 1997). Phosphorylation of FAK caused Src- and FAK-dependent phosphorylation of the Cas protein, leading to the recruitment of a Crk adaptor protein and activation of a small GTPase and JNK, promoting membrane protrusion and cell migration (Cox et al., 2006). The Cas proteins have numerous protein–protein interaction domains, including: (1) an SH3 domain, mediating interaction with proline-rich PxxxP proteins; (2) the substrate domain with tyrosine phosphorylation sites, mediating interaction with SH2 domain proteins, a proline-rich motif; (3) the serine-rich region; and (4) a C-terminal dimerization domain, mediating homo- and heterodimerization (O’Neill et al., 2000). The FAK-binding domain is the N-terminal SH3 domain of Cas that associates with the proline-rich domain of the C-terminal domain of FAK. Cas proteins serve as docking molecules for numerous interacting proteins (O’Neill et al., 2000). The extracellular matrix and growth factor receptor signals are transduced through the interaction of FAK, Src, and Cas proteins, stimulating cells to migration and actin rearrangements. In transformed cells, Cas proteins can be constitutively tyrosine phosphorylated and might play a role in the development of cancer (O’Neill et al., 2000).

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Talin The cytoskeletal protein talin plays a key role in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton (Critchley, 2005). Talin contains the N-terminal 47-kDa globular head domain and a 190-kDa C-terminal domain. Talin binds to integrins via the 4.1 band of the FERM domain (Ratnikov et al., 2005). Talin binds to the FAT domain in the C-terminal domain of FAK (Schlaepfer et al., 2004), linking FAK indirectly to integrins (Chen et al., 1995) (Fig. 3.1). It has been shown that phosphorylation of type Ig phosphatidylinositol phosphate kinase (PIPKIg661) on tyrosine 644 (Y644) is critical for the interaction of PIPKIg with talin and its localization to focal adhesions (Ling et al., 2003). PIPKIg661 is specifically phosphorylated on Y644 by Src, which is regulated by FAK (Ling et al., 2003). Grb-2 Grb-2 is an adaptor protein (
24 kDa) that binds to the Y925 site of the C-terminal domain of FAK through the Grb-2 SH-2 domain (Schlaepfer and Hunter, 1996; Schlaepfer et al., 1994). FN-stimulated Grb2 binding to FAK can activate ERK2-MAPK intracellular signaling (Schlaepfer et al., 1997). It has been shown that the Src-dependent association of FAK with both Grb2 and p130(Cas) is required for the regulation of cell cycle progression by FAK (Reiske et al., 2000). RhoGEF RhoGEF (guanine nucleotide exchange factor; 190 kDa) is a RhoA-specific guanosine diphosphate/guanosine triphosphate (GDP/GTP) exchange factor protein that directly associates with the C-terminal domain of FAK, FAT (Zhai et al., 2003). Rho GTPases are members of the Ras GTPase superfamily that play an important role in a number of biological processes, such as actin cytoskeleton reorganization, cell motility, gene expression, and cell cycle progression. RhoGTPases are inactive when bound to GDP and active once bound to GTP (Zhai et al., 2003). Several regulatory proteins bind to RhoGTPases, such as RhoGEF, to promote conformation from the inactive to the active form. Binding of RhoGEF to FAK supports the role of FAK in the activation of the Rho pathway and plays an important role in different cellular functions. GRAF Another Rho pathway protein GAP, GTPase-activating protein termed GRAF (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42 (Hildebrand et al., 1996). GRAF colocalizes with actin stress fibers, cortical actin structures, and focal adhesions in Graf-transfected chicken embryo cells (Hildebrand et al., 1996). GRAF is expressed in embryonic brain and liver tissues (Hildebrand et al., 1996) and may function in mediating cross talk between FAK and the Rho family GTPase, controlling integrin-initiated signaling events. GRAF has been shown to be GAP for Rho in vivo in Swiss

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3T3 cells, and microinjection of GRAF cDNA changed actin cytoskeletal localization and cell shape (Taylor et al., 1999). The human GRAF gene is 90% homologous to the chicken GRAF gene, is located on the 5q31 chrosomosome, and functions as a GAP for RhoA in vivo (Borkhardt et al., 2000). This gene might be associated with hematological malignancies containing deletions of 5q (Borkhardt et al., 2000). ASAP1 ASAP1 (ADP ribosylation factor [ARF]-GTPase-activating protein [GAP] containing SH3, ANK repeats, and the PH domain) has been shown to bind Src and has been phosphorylated by Src (Brown et al., 1998). The second proline-rich domain of the C-terminal domain of FAK has been shown to bind the SH3 domain of ASAP-1 (Liu et al., 2002). Overexpression of wildtype ASAP1 prevented the organization of paxillin and FAK in focal adhesions during cell spreading, while it did not change vinculin localization and organization (Liu et al., 2002). Hic-5 Hic-5 (hydrogen peroxide-inducible clone), a senescence-related protein, binds the C-terminal domain of FAK (Fujita et al., 1998). hic-5 was initially identified as a gene induced by transforming growth factor b1 or by H2O2 (Fujita et al., 1998). The exogenous overexpression of Hic-5 induced growth arrest, senescence-like morphology, and the increased expression of p21/WAF1/Cip1/sdi1 and extracellular matrix-related proteins such as fibronectin (FN), collagen, and collagenase (Fujita et al., 1998). Structurally, Hic-5 contains four LIM domains with 62% homology to paxillin in the C-terminal half and LD domains in the N-terminal half. Hic-5 is localized to focal adhesions and is involved in integrin-mediated signal transduction. Hic-5 expression has been shown to be decreased in immortalized mouse fibroblasts (Ishino et al., 2000). Overexpression of Hic-5 decreased the colony-forming ability of MEF from FAK(þ/þ) mice but not of FAK (–/–) cells. These observations suggested the involvement of Hic-5 in the regulation of cellular proliferation (Ishino et al., 2000). VEGFR-3 We have performed a phage display assay to map protein– protein interactions with the C-terminal domain of FAK and demonstrated that VEGFR-3 binds to the C-terminal and FAT domains of FAK (Garces et al., 2006). We synthesized TAT-conjugated peptide, containing 12 amino acid binding sites of VEGFR-3, introduced it into the cancer cells, and caused cell detachment and displacement of FAK from focal adhesions (Garces et al., 2006). Thus, this approach can be used for specific targeting of protein–protein interactions and for future cancer therapy. A detailed study of FAK–VEGFR-3 association and downstream signaling is required for development of in vivo tumorigenesis models.

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3. FAK in Tumorigenesis 3.1. Overexpression in tumors Our laboratory was the first to isolate FAK from a primary human tissue and to link FAK to human tumorigenesis (Cance et al., 2000; Owens et al., 1995, 2001). FAK is elevated in a variety of human tumors, including colorectal cancer (Han et al., 1997), breast (Cance et al., 2000; Owens et al., 1995), sarcomas (Weiner et al., 1993), cervical carcinomas (McCormack et al., 1997), and prostatic carcinoma tumors and cancer cell lines (Tremblay et al., 1996). We identified and cloned tyrosine kinase fragments from primary tumors (Weiner et al., 1993, 1994) and identified FAK from a primary high-grade human sarcoma (Weiner et al., 1993). We analyzed FAK mRNA expression in normal, invasive, and metastatic human tissues. Northern blot analysis demonstrated low levels of FAK mRNA in normal tissues, while primary and metastatic tumors significantly overexpressed FAK (Fig. 3.3) (Weiner et al., 1993). Subsequently, we cloned the human FAK cDNA and generated anti-FAK antibodies that were used to demonstrate that FAK is overexpressed in different types of tumors (Owen et al., 1999; Owens et al., 1995, 1996; Weiner et al., 1993). In a recent study, we performed real-time PCR analysis on colorectal carcinoma and liver metastasis samples with matching normal tissues and demonstrated increased FAK mRNA levels in tumor and metastatic tissues versus normal tissues (Lark et al., 2003). To confirm this hypothesis, we first cloned the regulatory promoter region of FAK with potential transcription binding sites, identified the start of transcription of the FAK gene, and found that FAK mRNA was higher in cancer cells compared to normal fibroblast and monocyte cell lines (Golubovskaya et al., 2004). Although FAK gene amplification was demonstrated in tumors (Agochiya et al., 1999; Okamoto et al., 2003), it is not the only mechanism of FAK increased expression. Moreover, recently we analyzed breast and liver metastatic tumors with increased FAK mRNA from liver metastatic samples with increased FAK mRNA (Lark et al., 2003) by FISH and did not find gene amplification in these samples. Moreover, analysis of FAK expression in head and neck squamous cell carcinomaderived cells demonstrated that FAK protein expression correlated with mRNA levels, but not with FAK copy DNA levels (Canel et al., 2006). It was suggested the increase in FAK protein levels in head and neck tumors can be due to its upregulated transcription (Canel et al., 2006). Thus, defining the mechanisms of FAK upregulation in different types of tumors will be important in understanding the role of FAK in tumorigenesis. Next, we will describe FAK expression and signaling in different types of tumors.

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Breast cancer Normal breast tissue

B

Matched breast cancer tissue

Colon cancer Normal colon tissue with adjacent dysplasia

Invasive colon adenocarcinoma

Figure 3.3 Overexpression of focal adhesion kinase (FAK) in different types of tumors. Immunohistochemical staining of FAK in tumor samples. Breast cancer (A), colon cancer (B). FAK is overexpressed in these tumors.The arrow in colon normal tissues indicates it is adjacent to normal tissue dysplasia with overexpressed FAK.

3.1.1. Ovarian cancer We have shown that FAK expression was increased in ovarian cancers (26 cancer samples from patients with Stage I–IV ovarian carcinoma and two ovarian carcinoma cell lines) compared to normal samples ( Judson et al., 1999). It was suggested that FAK may be a potential target for therapeutic disruption of ovarian carcinoma progression ( Judson et al., 1999). Recently, FAK overexpression in ovarian cancer and its absence in normal ovarian epithelial cells were also demonstrated by several other groups (Gabriel et al., 2004; Grisaru-Granovsky et al., 2005; Sood et al., 2004). 3.1.2. Colon cancer We have shown that FAK was overexpressed in invasive and metastatic colon tumors on both protein and mRNA levels (Fig. 3.3) (Lark et al., 2003; Weiner et al., 1993). FAK protein overexpression was seen in 32 out of 80 cases of colon adenocarcinoma (Theocharis et al., 2003). An increase of tyrosine phosphorylation of FAK (Y-397) was detected in colorectal cancer

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cells (Yu et al., 2004). Phospho-FAK correlated with invasiveness and lymph node metastasis in colon cancers (Yu et al., 2006). We have demonstrated that simultaneous inhibition of FAK and Src pathways caused increased apoptosis in colon cancer cell lines, suggesting that multiple signaling is involved in tumorigenesis (Golubovskaya et al., 2003). 3.1.3. Prostate cancer Increased FAK protein and mRNA levels and phosphorylation were observed in metastatic prostate cancer cells that correlated with the progression and metastasis of tumors (Tremblay et al., 1996). It has been suggested that FAK signaling plays a critical role in the invasiveness and motility of prostate cancers (Zeng et al., 2006; Zheng et al., 1999). 3.1.4. Breast cancer We found that p125FAK was significantly elevated in 17 (100%) of 17 invasive and metastatic colonic lesions and in 22 (88%) of 25 invasive and metastatic breast tumors, suggesting that FAK can be a marker of invasive tumors (Owens et al., 1995). We have shown that dual inhibition of FAK and EGFR (which are both overexpressed in breast tumors) signaling pathways cooperatively enhanced apoptosis in breast cancers (Golubovskaya et al., 2002). We have also shown that overexpression of an activated form of the Src tyrosine kinase in breast cancer cells (BT474 and MCF-7) suppressed the loss of adhesion and apoptosis induced by dominant-negative adenoviral FAK-CD, indicating cooperative FAK and Src signaling in breast tumorigenesis (Madan et al., 2006; Mitra and Schlaepfer, 2006; Park et al., 2004). Overexpressed HER2 has been shown to be involved in the tumor malignancy and metastatic ability of breast cancer through FAK and Src signaling pathways (Schmitz et al., 2005). We analyzed FAK expression in DCIS breast tumors and demonstrated upregulation of FAK expression in DCIS is an early event in breast tumorigenesis (Lightfoot et al., 2004). Subsequently, we analyzed FAK expression in 629 formalin-fixed, paraffin-embedded tissue sections (Lark et al., 2005). High FAK expression was associated with poor prognostic indicators, including high mitotic index, nuclear grade 3, architectural grade 3, estrogen and progesterone receptor negative, and overexpression of HER-2/neu (Lark et al., 2005). The association of FAK and Her-2 in breast tumors indicated its important cooperative signaling to promote breast tumorigenesis (Lark et al., 2005). 3.1.5. Melanoma cancer Increased FAK constitutive phosphorylation was observed in human metastatic melanoma cells (Scott and Liang, 1995). Focal adhesion increased expression correlated with motility of human melanoma cells (Akasaka et al., 1995). FAK expression appeared to be important for tumor cell adhesion in melanoma (Maung et al., 1999). Constitutive activation of FAK was

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regulated by the cytoskeleton in melanoma cells important for aggressive tumor growth (Kahana et al., 2002). We treated melanoma cell lines with antisense FAK oligonucleotides, inhibiting FAK expression, and demonstrated increased cell apoptosis and sensitivity to the chemotherapy drug 5-fluorouracil (Smith et al., 2005). Treatment of melanoma cells with dominant-negative FAK-related FRNK increased the aggressive phenotype of the cells, demonstrated by decreased invasion and motility, and inhibited the Erk1/2-mediated signaling pathway (Hess and Hendrix, 2006). 3.1.6. Thyroid cancer We analyzed p125FAK expression in 30 human thyroid tissue samples that included paired normal and malignant specimens (Owens et al., 1996). The highest levels of p125FAK were seen in follicular carcinomas and tumors associated with distant metastatic foci (Owens et al., 1996). FAK was not expressed in normal tissue and nodular hyperplasia but was expressed in all of the follicular, papillary, medullary, and anaplastic thyroid carcinomas (Kim et al., 2004). This result indicates that the upregulation of FAK may play a role in the development of thyroid carcinogenesis. 3.1.7. Oral cancer FAK was found to be overexpressed in oral cancers (Kornberg, 1998). Overexpression of FAK in low-invading cells resulted in a 4.5-fold increase in the rate of invasion compared with control cell lines, suggesting that enhanced expression of FAK in oral carcinoma cells may lead to a selective growth advantage and increased invasive potential of the primary oral tumor (Schneider et al., 2002). In another study, it was suggested that activation of FAK by phosphorylation of c-Met could mediate the HGF/SF-induced motility of human oral squamous cell carcinoma cells and that Rho protein could regulate the tyrosine phosphorylation of FAK through translocation from the nucleus to the membrane (Kitajo et al., 2003). Increased FAK phosphorylation and expression were observed in oral carcinoma of the larynx (Aronsohn et al., 2003). 3.1.8. Head and neck cancer In a recent study, hepatocyte growth factor/scatter factor HFG/SF induced phosphorylation of FAK and Erk in cultured squamous cell carcinoma of the head and neck, indicating its role in tumorigenesis (Fleigel et al., 2002). Inhibition of FAK with a dominant-negative C-terminal FAK, Ad-FRNK, caused increased apoptosis and decreased cell motility in epithelial cells from head and neck carcinomas (SCCHN cells) (Kornberg, 2005). Analysis of FAK expression by immunohistochemistry in 211 head and neck squamous cell carcinoma (HNSCC) tissue samples, including 147 primary tumors, 56 lymph node metastases, 3 benign hyperplasias, and 5 dysplasias, demonstrated elevated FAK expression in 62% of tumor samples (Canel et al., 2006).

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Positive immunostaining was also detected in benign hyperplasias and preinvasive dysplastic lesions, indicating that FAK overexpression is an early event during tumorigenesis. FAK DNA copy number ratios were higher in 39% of the tumors compared with normal matched samples. However, not all cases of FAK overexpression contained gene amplification, indicating additional mechanisms of FAK expression regulation (Canel et al., 2006). Inhibiting of FAK by adenoviral Ad-FRNK and introduction of exogenous p53 with Ad-p53 cause increased apoptosis of the carcinoma cells and an improved cytotoxic effect of chemotherapy drugs (Kornberg, 2006). Photodynamic therapy, effective in the treatment of head and neck invasive cancers, decreased cell motility and FAK phosphorylation and downstream survival signaling (Yang et al., 2006). FAK overexpression correlated with the invasiveness and lymph node metastasis of 91 thoratic esophageal squamous cell carcinoma tumor samples (Miyazaki et al., 2003). Patient survival with FAK-overexpressing tumors was significantly lower than survival without FAK-overexpression tumor ( p ¼ 0.006). The 5-year survival rate of patients without FAK overexpression was 69%, whereas that of patients with FAK overexpression was 38% (Miyazaki et al., 2003). This suggests that FAK can be a prognostic factor in these tumors. 3.1.9. Hepatocellular carcinoma Analysis of FAK expression in 60 cases of hepatocellular carcinoma demonstrated overexpression of FAK mRNA and protein in tumor samples compared to matched nontumor liver samples (Fujii et al., 2004). The fact that FAK overexpression correlated significantly with tumor size ( p ¼ 0.034) and serum AFP level ( p ¼ 0.030) led to the suggestion that FAK can be a prognostic therapeutic factor in these tumors (Fujii et al., 2004). Downregulation of FAK with the dominant-negative FRNK caused metastatic adhesion of carcinoma cells within liver sinusoids (von Sengbusch et al., 2005). The hepatitis B virus (HBV) involved in hepatic cell transformation activated FAK, suggesting the importance of FAK signaling in HBV-associated hepatocellular carcinogenesis (Bouchard et al., 2006). 3.1.10. Brain tumors Anaplastic astrocytoma brain tumors expressed higher levels of FAK and autophosphorylation compared to nonneoplastic adult normal brain tissues (Hecker et al., 2002). Overexpression of FAK promoted Ras activity through the formation of the FAK/p120RasGAP complex in malignant astrocytoma cells cultured in aggregate suspension or as monolayers adherent to vitronectin (Hecker et al., 2004). Overexpression of FAK in serumstarved glioblastoma cells plated on recombinant (rec)-osteopontin resulted in enhancement of basal migration and in a more significant increase of PDGF-BB-stimulated migration. Both expression of mutant FAK(397F) and FAK downregulation with small interfering siRNA inhibited basal and

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PDGF-stimulated migration (Natarajan et al., 2006). Recently, a novel kinase inhibitor of FAK (TAE226) has been shown to increase apoptosis in brain tumors (Shi et al., 2007). 3.1.11. Lung cancer In lung surgical specimens, phosphorylated FAK has been shown to be the major component among 100- to 130-kDa phosphorylated proteins correlating with poor patient prognosis (Nishimura et al., 1996). In addition, increased phosphorylation of FAK has been demonstrated in lung cancer samples and its absence in normal tissues (Imaizumi et al., 1997). The increased phosphorylation of FAK was closely correlated with the nodal involvement of cancer and disease-free survival time (Imaizumi et al., 1997). Expression of laminin 5, regulating cell adhesion, and anoikis and increased phosphorylation of FAK in lung adenocarcinoma cells suggested the importance of the laminin–integrin–FAK pathway in tumorigenesis (Kodama et al., 2005). Stimulation of small-cell lung cancer cells with hepatocyte growth factor (HGF) activated c-Met and increased phosphorylation of Y397 FAK, suggesting that this pathway is a therapeutic target in lung tumorigenesis (Maulik et al., 2002). FAK signaling has been demonstrated to be important in the early stages of mammary adenocarcinoma lung metastasis (van Nimwegen et al., 2005). In an experimental model of mammary metastasis, formation of lung metastasis was prevented when the dominant-negative FAK inhibitor, FRNK, was expressed 1 day before tumor cell injection, whereas at 11 days after injection expression of FRNK had no effect (van Nimwegen et al., 2005). Immunohistochemical staining of FAK in 60 formalin-fixed and paraffin-embedded non-small-cell lung cancer (NSCLC) tumors demonstrated increased FAK levels compared with nonneoplastic tissues (Carelli et al., 2006). Moreover, Western blotting and real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed a statistically significant correlation between FAK upregulation and higher disease stages (I þ II versus III þ IV, p ¼ 0.019 and 0.028, respectively), indicating FAK involvement in tumorigenesis (Carelli et al., 2006). 3.1.12. Kidney cancer Comparative analysis of FAK expression in metastatic and nonmetastatic renal carcinoma cells revealed that FAK and paxillin mRNA expression were upregulated 2.0- to 2.5-fold in the metastatic Caki-1 cells over normal renal cortex epithelial cells (RCEC), suggesting its potential role as a marker of metastasis ( Jenq et al., 1996). Expression of FAK and HGF synergistically increased transformation of Madin–Darby canine kidney epithelial cells (Chan et al., 2002).

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3.1.13. Neuroblastoma Treatment of neuroblastoma cells with okadaic acid (OA), a serine phosphatase inhibitor, increasing serine/threonine phosphorylation and inhibiting tyrosine phosphorylation, induced focal adhesion loss, actin cytoskeleton disorganization, and cellular detachment, which corresponded to a loss of FAK Tyr-397 (Kim et al., 2003). It was suggested that drugs causing FAK dephosphorylation may be potentially therapeutic in neuroblastoma cells. A detailed study of FAK expression and phosphorylation in neuroblastoma will be critical in understanding FAK regulation in these cells. 3.1.14. Pancreatic cancer Inhibition of FAK with FAK siRNA potentiated gemcitabine-induced cytotoxicity in pancreatic cells. FAK siRNA treatment downregulated AKT activity (Duxbury et al., 2003). In addition, downregulation of FAK with siRNA caused cell increased anoikis in pancreatic adenocarcinoma PANC1, BxPC3, and MiaPaCa2 cell lines (Duxbury et al., 2004). FAK siRNA also inhibited metastasis in a nude mouse model (Duxbury et al., 2004). Furthermore, downregulating FAK with siRNA decreased cell motility and invasiveness in pancreatic cells (Huang et al., 2005). Analysis of FAK expression in 50 patients with pancreatic invasive ductal carcinoma by immunohistochemistry showed its detection in 24 samples (48%) (Furuyama et al., 2006). There was a statistically significant correlation between FAK expression and tumor size ( p ¼ 0.004), although FAK expression did not significantly correlate with other factors such as tumor histological grade, lymph node metastasis, distant metastasis, histological stage, and overall survival. It was concluded that FAK expression may not be a prognostic marker for pancreatic cancer patients (Furuyama et al., 2006). The more extended study of pancreatic cancer needed to be analyzed for FAK expression, as it correlated with tumor size. 3.1.15. Osteosarcoma Expression analysis of 16 osteosarcoma tumors, 5 osteosarcoma cell lines, and 6 normal tissues demonstrated FAK overexpression in high-grade osteosarcomas (Schroder et al., 2002). FAK expression analysis performed in our group in 13 high-grade sarcomas showed high levels in all tumor samples compared to benign, noninvasive mesenchymal specimens (Owens et al., 1995). This analysis shows that FAK can be a potential target in these tumors, although a more detailed study with correlation analysis of other tumor markers will be critical.

3.2. FAK and stem cells We would like to review several studies on FAK functioning in stem cells, as these cells are now considered precursors of cancer cells. Stem cell studies generate an important and promising field in cancer research. FAK has been

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shown to be upregulated differently by various cytokines in primary murine bone marrow (BM) cells (Kume et al., 1997). In unstimulated BM cells, FAK mRNA was detected in myeloid and lymphoid cells, but not in erythroid cells. FAK mRNA and protein levels have been shown to be upregulated by granulocyte macrophage colony-stimulating factor (GM-CSF), but not by interleukin-3 ( IL-3) (Kume et al., 1997). FAK increased levels correlated with motility and morphology cell polarization, suggesting the involvement of FAK in the maturity of GM-CSF-stimulated monocyte–macrophage lineages. In the developing brain, precursor cells give origin to cells that mature into microglia and neurons (astrocytes and oligodendrocytes). Oligodendrocytic progenitors are motile cells and are stimulated for proliferation by a number of growth factors: neurotrophin-3, fibroblast growth factor-2, and PDGF form isoforms. By RT-PCR and immunohistochemical analysis of 17 different protein kinases, FAK and c-Met have been shown to be expressed by cultured rat oligodendrocytes, suggesting their role in cell motility (Kilpatrick et al., 2000). In embryonic mouse stem cells, a decrease of mobility has been observed in FAK-deficient endodermal cells (Ilic et al., 1996). FAK has been shown to play a role in laminin-5 and extracellular matrix contact-induced differentiation of human mesenchymal stem cells into osteoblasts (Salasznyk et al., 2007a,b). Thus, these reports suggest that FAK functioning is important during stem cell maturation and motility. Future studies will reveal the role of FAK and stem cell signaling in tumorigenesis.

4. FAK and p53 Association FAK activity and expression can be regulated not only by cooperation with oncogens, but also by association with tumor suppressor gene proteins. One of the known proteins encoded by a tumor suppressor gene that can regulate FAK activity is PTEN, which is able to bind and dephosphorylate FAK and thus negatively regulate motility and invasion (Tamura et al., 1999b). Another protein is the neurofibromatosis type 2 (NF2) gene product, Merlin, encoded by a tumor suppressor gene frequently inactivated in malignant mesothelioma that has been shown to inactivate FAK and also inhibit invasion and motility (Poulikakos et al., 2006). A recent report demonstrated that tumor suppressor neurobifromin, NF1, modulated AKT, RAS, and FAK signaling pathways and affected cytoskeletal organization (Boyanapalli et al., 2006). We have recently demonstrated a novel direct interaction of FAK and p53, where FAK blocked tumor suppressor

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apoptotic activity (Golubovskaya et al., 2005). These data link FAK with tumor suppressors, especially important in association with p53 signaling. It is known that the p53 tumor suppressor is the most frequent target for genetic alterations in human cancers and is mutated in almost 50% of all tumors (Baker et al., 1990; Hollstein et al., 1999; Sidransky et al., 1992; Wang et al., 2003; Willis and Chen, 2002). Inactivation of the p53 gene is a critical step in tumorigenesis (Fearon and Vogelstein, 1990). Following induction by a variety of cell stresses such as DNA damage, hypoxia, or the presence of activated oncogenes, p53 upregulates a set of genes that can promote cell death and growth arrest, such as p21, GADD45, cyclin G, and Bax, reviewed in Giaccia and Kastan (1998). Recently, it was shown that p53 can repress promoter activities of a number of antiapoptotic genes and cell-cycle genes: survivin (Hoffman et al., 2002), cyclin B1, cdc2, (Taylor and Stark, 2001; Wu et al., 2001), cdc25 c (Krause et al., 2000), stathmin ( Johnsen et al., 2000), Map4 (Murphy et al., 1996), and bcl-2 (Wu et al., 2001). Overexpression of p53 induced apoptosis (Oren, 2003; Yonish-Rouach et al., 1991) and p53 inactivation caused a decrease in radiation-induced apoptosis (Fridman and Lowe, 2003; Lowe et al., 1993, 1994). The first report that analyzed a role of FAK and p53 genes in apoptosis was performed by Ilic et al. (1998). They reported that FAK suppressed a p53-regulated apoptotic pathway in anchorage-dependent cells (fibroblasts and endothelial cells) (Ilic et al., 1998). They also demonstrated that in the absence of FAK function, p53-regulated apoptosis was activated by protein kinase C and phospholipase A2, and this process was suppressible by dominantnegative p53 and Bcl-2. (Ilic et al., 1998). They analyzed apoptosis in anchorage-dependent human fibroblasts and endothelial cells but did not study these processes in tumors and cancer cell lines (Ilic et al., 1998; Zhang et al., 2004). In addition, immunohistochemical analysis of 115 endometrial carcinoma samples by our group demonstrated a correlation between FAK and p53 overexpression (Livasy et al., 2004). Our group showed a direct association between FAK and p53. Thus, we will focus briefly on the structure and biology of p53 to understand its novel interaction.

4.1. Structure and function of the p53 protein 4.1.1. p53 gene structure p53 is a tumor suppressor gene that is located at the chromosome 17p13 region, spans 20 kb, and contains 11 exons (Levine, 1997). The p53 protein is a phosphoprotein transcription factor that binds to the 50 Pu-Pu-PuC-A/T-T/A-G-Py-Py-Py30 (Pu-purine; Py-pyrimidine) consensus DNA sequence in the promoters of the genes and activates their transcription (Farmer et al., 1992). The p53 gene encodes the 393 amino acid protein. The promoter of p53 lacks a TATA box and contains various binding sites

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for known transcription factors, such as NF-kB, Sp1, or c-Jun (Bouchet et al., 2006). 4.1.2. p53 protein structure The p53 protein contains three domains: an acidic N-terminal transcriptionalactivating domain (1– 92 aa), a central DNA-binding domain (102–292 aa), and a C-terminal (102–292 aa) tetramerization domain (325–393 aa) (Fig. 3.4). The p53 protein contains many sites for phosphorylation by different kinases: ATM, Chk2, ATR, JNK, MAPK, CKI, and CKII. N-terminal transactivation domain A DNA-binding domain crystal structure shows that this domain contains a scaffold of b-sheets, supporting flexible helixes and loops, involved in interaction with DNA. Mutations in this domain alter this structure and disrupt DNA–protein interactions. Codons 17–28 interact with the double minute-2 homologue (Mdm-2), which plays a major role in p53 inhibition and degradation via the ubiquitin–proteasome pathway (Levine, 1997). Mdm-2 regulates p53 via an autoregulatory feedback loop in which both proteins control its cellular expression. p53 activates Mdm-2 transcription and expression. In turn, Mdm-2 binds to p53 and negatively regulates its stability and activity. Codons 22–26 are involved in binding of histone acetyltransferase P300 (Lacroix et al., 2006). A proline-rich domain (codons 63–97) that contains five PXXP motifs is required for interaction with various proteins and regulates apoptosis (Lacroix et al., 2006). PXXP motifs have been shown to create a binding site for Src homology SH3 domains (Yu et al., 1994). A mutant deleted from the proline-rich domain in human p53 (D62–91) is not able to cause apoptosis, but maintains the ability for cell cycle arrest (Edwards et al., 2003). Interestingly, a polymorphism at codon 72 has been demonstrated with either proline or arginine that can lead to differences in binding of transcription factors, apoptosis/survival signaling, and chemotherapy responses (Bergamaschi et al., 2003).

FAK

Transactivation domain TAD 1−92 a.a.

p53 DNA-binding central domain DBD

102−292 a.a.

Tetramerization domain TD 325−393 a.a.

Figure 3.4 p53 structure. p53 contains three domains: the N-terminal, transactivation domain, the central DNA-binding domain, and the C-terminal tetramerization domain. Focal adhesion kinase (FAK) interacts with the N-terminal domain of p53 (1^92 aa). The FAK protein binds directly to the N-terminal transactivation domain of p53, which is marked by an arrow.

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Central domain, DNA-binding domain This region is highly conserved, homologous to other p53 family member (p63 and p73) regions. The DNA-binding domain binds to promoter binding sites 50 Pu-Pu-Pu-C-A/ T-T/A-G-Py-Py-Py30 (Levine, 1997). C-terminal tetramerization domain This domain is involved in protein tetramerization and regulation of p53 activity. It contains three nuclear localization signals that allow a p53 nuclear import tetramerization region (323–356 aa) and a negative regulatory region (363–393 aa) (Lacroix et al., 2006).

4.2. p53 mutations in cancer cells p53 is the most commonly mutated gene in human malignancies, with up to 75% mutations in invasive cancers. The majority of mutations are missense mutations with four out of five mutations in the DNA-binding domain; only 1% is found in the N-terminal domain and 4% in the tetramerization domain (Stoklsa and Golab, 2005). The latest version of the International Agency for Research on Cancer p53 mutation database (http://www-p53.iarc.fr/) from July 2005 reported 21,512 somatic mutations. The most frequent ‘‘hot-spot’’ mutations are in codons: 175 Arg!His (breaks the bond between the L2 and L3 loop), Arg!Gln (Trp) (breaks contact with DNA in the minor groove), 273 Arg!His (Cys) (breaks contact with DNA in the major groove), and 282 Arg!Trp (destabilizes the H2 helix and DNA binding in the major groove and breaks contact on the b-hairpin) (Stoklsa and Golab, 2005). Among reported mutations, 75% are missense mutations; 80% are located in the DNA-binding domain of p53 (Bouchet et al., 2006), and 30% are located in five hotspot codons: 175, 245, 248, 273, and 282. Arginine residues (248 and 273) involved in interaction of p53 with DNA and arginines (175 and 282) stabilize the DNA-binding sequence (Bouchet et al., 2006). Wild-type p53 binds to promoters differently; for example, p53 activates the p21 promoter with higher affinity than the Bax promoter (Bouchet et al., 2006). Some p53 mutants are able to transactivate different genes, such as EGFR, MDR1, c-Myc, PCNA, IGF-2, or VEGF, providing growth-promoting phenotypes and drug resistance (Bouchet et al., 2006).

4.3. p53 binds the FAK promoter Our group was the first to clone the human FAK promoter and to find two p53-binding sites in it (Golubovskaya et al., 2004). We have shown that p53 can bind the FAK promoter and inhibit its transcriptional activity (Golubovskaya et al., 2004). In addition, several other transcription factors, such as SP-1, AP-2, TCF-1, and NF-kB, were shown to be present in the

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FAK promoter. The NF-kB protein has been shown to be linked to the p53 pathway (Benoit et al., 2006). For example, activation of Cox-2 transcription required the cooperation of NF-kB and p53 (Benoit et al., 2006). Thus, regulation of the FAK promoter can also include the association of these two transcription factors, thus providing an additional indirect p53-regulated FAK expression mechanism. Moreover, while the wild-type inhibited FAK promoter activity, the mutant p53 did not. The recent global analysis of the p53 transcription factor-binding sites demonstrated that induction of HCT116 colon cancer cells with 5-fluorouracil transcriptionally downregulated FAK (Wei et al., 2006). Thus, it was suggested that p53 can suppress metastasis through downregulation of metastasis-related genes, such as FAK. Since p53 is often mutated in cancer, a population-based study is needed to identify which mutations of p53 will correlate with upregulation of FAK.

4.4. Direct FAK and p53 protein binding In a recent report, we demonstrated that the N-terminal transactivation domain (1–92 aa) of p53 is physically directly associated with the N-terminal part (205–422 amino-acids) of FAK (Golubovskaya et al., 2005). In addition, there have been several reports on the localization of the N-terminal part of FAK in the nucleus (Beviglia et al., 2003; Jones and Stewart, 2004; Lobo and Zachary, 2000; Stewart et al., 2002). Furthermore, the N-terminus of FAK was shown to cause apoptosis in breast cancer cell lines (Beviglia et al., 2003), and its nuclear localization was regulated by caspase inhibitors in endothelial cells (Lobo and Zachary, 2000). In addition, p53 has been reported to be localized in the cytoplasm (Chipuk and Green, 2004). p53 directly activated Bax and released proapoptotic molecules, activating multidomain proteins in the cytoplasm. This mechanism required 62–91 residues in the proline-rich N-terminal domain of p53 (Chipuk and Green, 2004). Thus, understanding of the mechanism and functions of FAK/p53 interaction may ultimately have important implications for targeted cancer therapy.

4.5. Cytoplasmic–nuclear protein shuttling Moving proteins between cellular localizations is a tightly regulated process and provides an important mechanism for controlling protein functioning in the cell. FAK and p53 can both be localized under different conditions in either cytoplasm or nucleus. Although p53 was known mainly as a nuclear protein, recently evidence appeared to demonstrate that an extranuclear function of p53 in the cytoplasm caused apoptosis (Chipuk et al., 2004). p53 has been shown to directly bind the proapoptotic BAX protein, allowing for mitochondrial membrane permeabilization, cytochrome c release, and apoptosis (Chipuk et al., 2004). Thus, evidence was provided for the apoptotic

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transcription-independent function of p53 in the cytoplasm. Importantly, the proline-rich subdomain of the N-terminal p53 domain has been found to be critical for this p53 functioning (Chipuk et al., 2004). Cytoplasmic sequestration of p53 by the E1B 55-kDa protein played an important role in inhibiting p53 activities (Zhao et al., 2003). Different mechanisms of p53 nuclear export have been proposed: either through Mdm-2 binding in the nucleus and shuttling it to the cytoplasm, through p53 nuclear export signals (NES), or through Mdm-2-directed ubiquitination of p53 in the nucleus to promote its nuclear export (Zhang et al., 2001). It was found that a novel nuclear export signal in the N-terminal domain of p53 is required for the nuclear export of p53 (Zhang et al., 2001). p53 Arg-72 was critical for Bax binding with the p53 N-terminal proline-rich domain (Chipuk et al., 2004). The polymorphism with arginine instead of proline expressed more apoptotic functioning in mitochondria (Chipuk et al., 2004). These data suggest that the p53 N-terminal domain interaction with FAK can be critical for protein localization, transport, and shuttling from the nucleus to the cytoplasm. It was shown that p53 uses microtubules for nuclear localization (Vousden and Woude, 2000). FAK is mainly a cytoplasmic protein, but it was also recently reported to be localized in the nucleus ( Jones and Stewart, 2004; Jones et al., 2001; Lobo and Zachary, 2000). The N-terminal domain of FAK has been shown to be localized in the nucleus of endothelial cells (Lobo and Zachary, 2000). Loss of FAK from focal adhesions induced aggregation of the N-terminal domain of FAK in the nuclei in apoptotic glioblastoma cells, demonstrating FAK functioning not only in the cytoplasm and in the nucleus ( Jones et al., 2001). Treatment of cells with an inhibitor of nuclear transport, leptomycin B, resulted in the accumulation of the nuclear localization of FAK ( Jones and Stewart, 2004). Thus, FAK can shuttle between the nuclear and cytoplasmic compartments ( Jones and Stewart, 2004). In addition, recently a novel functioning of FAK was demonstrated in the nucleus, such as the PIAS-1-mediated sumoylation of FAK that occurred at lysine 152 in the N-terminal domain (Kadare et al., 2003). Thus, it is important to understand the mechanisms of protein shuttling between the cytoplasm and the nucleus that are critical for the regulation of their activity (Vousden and Woude, 2000).

4.6. Feedback model of FAK–p53 protein interaction We have shown that p53 can suppress FAK transcription (Golubovskaya et al., 2004). Recently, global characterization of 65,572 p53 ChIP DNA fragments was done in an HCT116 colorectal cancer cell line treated with 5-fluorouracil for 6 h, which activated p53 (Wei et al., 2006). Novel targets of p53 were identified that are involved in cell adhesion, migration, and metastasis, and PTK2 or FAK was one of these kinases (Wei et al., 2006).

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Interestingly, in HCT116 cells treated with 5-fluorouracil, which increases the p53 level, PTK2 (FAK) expression was also inhibited (Wei et al., 2006), which is consistent with our data (Golubovskaya et al., 2004). We have also shown that FAK can suppress the transcriptional activity of p53 through its interaction, as p53-mediated activation of p53 targets: p21, Mdm-2, and Bax were blocked by overexpression of FAK (Golubovskaya et al., 2005). Thus, p53 can regulate FAK, and in turn, FAK can regulate p53. Thus FAK and p53 can be regulated by comprising a feedback mechanism (Fig. 3.5). Mutations of p53 that are frequently found in cancers can lead to upregulation and overexpression of FAK (Fig. 3.3). This mechanism was recently discussed in Mitra and Schlaepfer (2006). Interestingly, the novel link between FAK and p53 was supported by a recent report on the novel FAK inhibitor FIP200, which binds to FAK directly and inhibits cellular functions, such as cell adhesion, spreading, and motility (Melkoumian et al., 2005). FIP200 overexpression resulted in increased levels of p21 and caused growth arrest (Melkoumian et al., 2005). It was found that FIP200 interacted with FAK and p53 (Melkoumian et al., 2005). An interesting model of the FAK–p53–FIP200 interaction has been proposed (Mitra and Schlaepfer, 2006). It was suggested that FIP200/FAK and FIP200/p53 bindings can compete, and when FAK has low expression, FIP200 can bind p53 and

p21

Bax Mdm-2 Apoptosis Wild-type p53 protein

p53

p53

FAK protein

Low expression of FAK protein FAK protein

FAK promoter

Mutant p53 protein

p53

Other factors (NF-kappa B) High expression of FAK protein p53

FAK FAK FAK protein protein protein

FAK promoter

Figure 3.5 The feedback model of focal adhesion kinase (FAK) and p53 interaction. The wild-type p53 binds to the FAK promoter and blocks its transcriptional activity, resulting in low FAK expression. Overexpressed FAK protein binds to p53 protein and blocks p53 transcription by repressing p53-induced apoptotic targets: p21, Mdm-2, and Bax, thus generating a feedback loop mechanism.

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increase its expression (Mitra and Schlaepfer, 2006). For the reverse, it was also suggested that FAK overexpression can limit binding of FIP200 to p53, enhancing cellular survival. Thus, novel mechanisms of FAK survival function remain to be discovered during carcinogenesis.

5. FAK and p53 Targeted Therapy 5.1. Downregulation of FAK It was recently proposed that FAK is a new therapeutic target (McLean et al., 2005). Several in vitro approaches have been used to downregulate FAKadenoviral FAK-CD (dominant-negative FAK) (Xu et al., 2000), antisense oligonucleotides (Smith et al., 2005), and siRNA for FAK (Han et al., 2004). We linked FAK expression to apoptosis by treating FAK-positive tumor cell lines with different antisense oligonucleotides to FAK that specifically inhibited p125 FAK expression ( Judson et al., 1999; Xu et al., 1996). The melanoma cells treated with antisense oligonucleotides lost their attachment and underwent apoptosis (Smith et al., 2005; Xu et al., 1996). The same effect was observed with Ad-FAK-CD in different cancer cells. While breast cancer cells underwent apoptosis by downregulation of FAK with FAKCD, normal MCF-10A and HMEM cells did not (Xu et al., 2000). These inhibitor applications are limited due to in vivo cell toxicity. Thus, developing small-molecule drugs is critical for the future of FAK-targeting therapy, involving kinase inhibitors and drugs, targeting FAK–protein interactions.

5.2. FAK inhibitors There were no pharmacological inhibitors reducing FAK kinase activity. Recently, Novartis Inc. developed novel FAK inhibitors, causing downregulation of its kinase activity (Choi et al., 2006). A series of 2-amino-9aryl-7H-pyrrolo[2,3-d]pyrimidines was designed and synthesized to inhibit FAK using molecular modeling in conjunction with a cocrystal structure. Chemistry was developed to introduce functionality onto the 9-aryl ring, which resulted in the identification of potent FAK inhibitors. The novel Novartis FAK inhibitor TAE226 was recently employed in brain cancer and effectively inhibited FAK signaling and caused apoptosis in these cells (Shi et al., 2007). We also used this inhibitor in breast, neuroblastoma, and pancreatic cancer cells and found that this inhibitor can effectively cause apoptosis, although it can also inhibit other signaling pathways in addition to FAK. Thus, the specificity of this drug remains to be discovered. Future detailed studies will be needed to address the specificity of these drugs.

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5.3. Targeting protein–protein interactions One of the approaches to inhibit FAK function can be targeting its protein– protein interaction with its binding partners such as p53. Small-molecule drugs can be found either through high-throughput screening or through database searches using protein crystal structures.

5.4. p53 therapy Experimental approaches that target p53 for cancer treatment include therapies to activate p53 as well as to inactivate p53. Anticancer therapies include: (1) a gene therapy approach to replace wild-type p53 with p53expressing retroviruses or adenoviruses, (2) killing of p53-deficient cells with modified adenoviruses, and (3) pharmacological activation by inhibition of Mdm-2 binding with nutlins or by direct activation with polyamines or reactivation of wild-type activity in mutant cells with PRIMA-1 (Bouchet et al., 2006). Retroviruses are attractive agents for cancer gene therapy, as in the stable form they integrate into the genome and require cell division for transduction. The second strategy is adenoviruses that are double-stranded DNA viruses. In contrast to retrovirus, their effect is not limited to high-proliferating cells, and there is no risk of insertional mutagenesis. One such drug is Ad5CMV-p53 (Advexin INTROGEN Therapeutics Inc.; Gendicine Shenzden SiBiono Gene Tch. Co. Ltd.), which is a recombinant E1-deleted serotype 5 adenoviral vector encoding p53. In vitro and in vivo studies confirmed the therapeutic affect of Ad5CMV-p53. This therapy approach was used effectively in clinical trials, Phase I and II, although some cases of tumor resistance were also demonstrated (Bouchet et al., 2006). The second major approach to kill p53-defective cells is the use of E1Bdefective adenoviruses. The delta 1520 virus (dl1520; ONYX-015) is an adenovirus that contains a deletion in the E1B region. ONYX-015 can be used as a selective drug against tumors with mutant p53. The same problems as with adenoviruses can occur in some types of tumors. Small molecule drug inhibitors are effectively used to target p53 protein–protein interactions, particularly with Mdm-2 protein (Vassilev, 2005). The first potent inhibitors targeting p53–Mdm-2 interaction have been identified by high-throughput screening followed by structure-based optimization (Vassilev, 2005). The screening identified nutlins that represent a class of cis-imidazole analogues that bind to the p53 pocket interacting with Mdm-2. The same strategy can be used to target interaction with FAK. Recently, we used the crystal structure of the N-terminus of FAK and screened 20,000 small molecules from the NCI bank for their ability to target this binding site and for their oral bioavailability using Lipinski rules. We identified 10 potential lead compounds and tested them on human

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breast, colon, and melanoma cell lines for their ability to disrupt p53 FAK binding and to induce cancer cell death. We found several potential compounds that were able to decrease FAK phosphorylation, decrease cell viability, and activate PARP, suggesting their potential role in future therapy.

6. Summary Thus, an understanding of FAK biology during tumorigenesis, the mechanisms of its upregulation in different tumors, its role in stem cell biology, angiogenesis, and motility, and especially the mechanisms of its direct physical interaction with the p53 protein and downstream signaling pathways will be critical in developing targeted therapeutics. Studies with peptide inhibitors already have indicated that blockade of specific protein–protein interactions has therapeutic promise for treating a variety of diseases, including cancer (Aarts et al., 2002; Akhter et al., 1998; Aramburu et al., 1999; Chen et al., 1999; May et al., 2000; van Rooij et al., 2002). Small-molecule drugs are particularly attractive as inhibitors of intracellular protein–protein interactions due to their ability to modify their structures to achieve optimal target binding. As we further define the mechanisms of FAK signaling in cancer cells, we will identify the optimal sites for targeting this protein and disrupting its signaling to cause apoptosis in human tumors. Thus, the interaction of FAK and p53, as well as complexes with other binding partners, can be important therapeutic targets in cancer treatment programs.

ACKNOWLEDGMENTS We apologize to those whose papers we have not cited because of space limitations. The research is supported by NIH RO1-CA065910 (W.G.C.) and by Susan G. Komen for the Cure grant (V.M.G.).

REFERENCES Aarts, M., Liu, Y., Liu, L., Besshoh, S., Arundine, M., Gurd, J. W., Wang, Y. T., Salter, M. W., and Tymianski, M. (2002). Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science 298, 846–850. Abbi, S., and Guan, J. L. (2002). Focal adhesion kinase: Protein interactions and cellular functions. Histol. Histopathol. 17, 1163–1171. Abedi, H., and Zachary, I. (1997). Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. J. Biol. Chem. 272, 15442–15451.

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