Gene Ther Mol Biol Vol 13, 26-35, 2009
FAK as a target for cancer therapy
Steven N. Hochwald*, Vita M. Golubovskaya
Department of Surgery, University of Florida College of Medicine, Gainesville, Florida
*Correspondence: Steven N. Hochwald MD, Department of Surgery, University of Florida College of Medicine, 1600 SW Archer Road, P.O. Box 100109, Gainesville, FL 32609, USA; Tel: 352-265-0761, Fax: 352-265-0262, e-mail: Steven.Hochwald@surgery.ufl.edu
Key words: ocal Adhesion Kinase; malignancy; cancer; Y15
Abbreviations: FAK, (Focal Adhesion Kinase); FERM, (Focal Adhesion Kinase Ezrin/Radixin/Moesin; FRNK, (FAK-related non kinase)
This work was supported by the following NIH grant: CA113766 (S.N.H.)
We have learned that malignant cells are similar to normal cells in the signaling pathways that they use. However, cancer cells acquire aberrations that favor their growth in the complex environments of living tissues. This includes their ability to invade and metastasize and their ability to grow and divide indefinitely. 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. One of the main tyrosine kinases that are linked to this malignant phenotype is the Focal Adhesion Kinase (FAK). FAK is overexpressed in many types of tumors and recently has been proposed to be a target for anti-cancer therapy. In this review, we will review the FAK structure, its role in signaling, and FAK targeted therapy approaches in malignancy.
Despite recent advances in surgery, chemotherapy and radiation treatment, survival of patients with advanced malignancy remains suboptimal. Fortunately, our understanding of the origins of cancer has changed dramatically over the last twenty-five years, owing in large part to the revolution in molecular biology that has changed all biomedical research. Powerful experimental tools are available to cancer biologists and have made it possible to uncover and dissect the complex molecular machinery operating inside normal and malignant cells. In addition, these tools have allowed researchers to pinpoint the defects that cause cancer cells to signal and proliferate abnormally.
Focal Adhesion Kinase (FAK) was discovered about 15 years ago as a tyrosine phosphorylated protein kinase. Investigations in several laboratories have shown that this protein plays a critical role in intracellular processes of cell adhesion, motility, survival, and cell cycle progression. The FAK gene encodes a non-receptor 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). Subsequently, the FAK gene was identified in human tumors, and FAK mRNA has been shown to be up-regulated in invasive and metastatic human breast and colon cancer samples as compared to normal tissues (Weiner et al, 1993). This was the first evidence that FAK might be regulated at the level of gene transcription. Subsequently, up-regulation of FAK has been demonstrated at the protein level in a wide variety of human tumors, including breast cancer, colon cancer, ovarian cancer, thyroid cancer, melanoma, and sarcoma (Owens et al, 1995, 1996; Judson et al, 1999; Cance et al, 2000). Recently, the regulatory promoter region of the FAK gene was cloned and confirmed transcriptional up-regulation in cancer cell lines (Golubovskaya et al, 2004).
II. Molecular structure of focal adhesion kinase
The human FAK (also known as PTK2a) gene has been mapped to chromosome 8 (Fiedorek, Jr. and Kay, 1995; Agochiya et al, 1999), and there appears to be a high degree of homology between vertebrate species. Human complete FAK mRNA sequence (NCBI Accession number: L13616) is 3791 bases long and includes a 5Ő-untranslated 233 base pair region (Whitney et al, 1993). Human FAK cDNA was first isolated from primary sarcoma tissue and increased FAKmRNA was seen 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 on human chromosome 8, locus p21.1, named PYK2 (RAFTK (related adhesion focal tyrosine kinase), CADTK (calcium-dependent tyrosine kinase), CAK (cell adhesion kinase) b, PTK 2b (protein tyrosine kinase 2b) (Avraham et al, 1995; Lev et al, 1995; Sasaki et al, 1995).
The gene coding FAK contains 34 exons (NCBI Gene ID: 5747), and genomic sequence spans 230 kb (Corsi et al, 2006). The FAK gene contains four 5Ő non-coding exons and 34 coding exons and has been shown to have multiple alternatively spliced forms. Comparison of the mouse and human FAK genes detected conservative and non-conservative 5Ő-untranslated exons that suggests a complex regulation of FAK expression. Exons (Sasaki et al, 1995; Burgaya and Girault, 1996; Fujimoto et al, 1999; Golubovskaya et al, 2002) 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 different exon inclusion, but can also occur from intron retention or 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, β-catenin, Crk) or angiogenesis (VEGFR-2, VEGFR-3 (Flt-4)). Thus, 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.).
The human FAK promoter regulating FAK expression 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). Interestingly, two transcription binding sites for p53 have been identified in the FAK promoter, and p53 can block FAK promoter activity (Golubovskaya et al, 2004). Recently, the mouse promoter has been cloned and found to be highly homologous to the human promoter and contains 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 Parsons group (Hayasaka et al, 2005), regulating expression of autonomously expressed FRNK protein.
A. FAK protein structure
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 FAT domain (Schaller and Parsons, 1994; Schaller et al, 1994; Hanks and Polte, 1997) (Figure 1).
B. The kinase domain
The central kinase domain of FAK (amino acids 424-676) contains the Y576 and Y577, major phosphorylation sites, and also K454, which is the ATP binding site (Figure 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 maximal FAK enzymatic activity (Calalb et al, 1995). The crystal structure of the FAK kinase domain reveals an open conformation similar to other kinases (Nowakowski et al, 2002). The FAK kinase domain structure has an unusual bisulphite bond between the conserved cysteines 456 and 459, 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, although the design and specificity of these inhibitors can be complicated by structural similarities between kinase domains. Thus, finding small structural differences between the ATP binding site of kinases is crucial in the design of small molecule kinase inhibitors. For example, the side chain of glutamic acid, E506 forms a bifurcated hydrogen bond to the 2Ő and 3Ő 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. The N-terminal domain
The first function of the N-terminal, homologous to FERM domain was linked to the binding of integrins, via their β subunits (Schaller et al, 1995). The N-terminal domain of FAK protein contains the major autophosphorylation site Y397-tyrosine, that in its 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 leads to tyrosine phosphorylation of other tyrosine phosphorylation sites of FAK: Y407; Y576,Y577- major phosphorylation sites in the catalytic domain of FAK; Y861 and Y925 (Hanks and Polte, 1997; McLean et al, 2005), and to phosphorylation of FAK binding proteins, such as paxillin and Cas (Schaller et al, 1999). This 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, motility and metastatic, invasive phenotype in cancer cells (Figures 1 and 2). Phosphorylated Y397 FAK is able to recruit important signaling molecules, p85 PI3-kinase (phosphoinositide 3-kinase), growth factor receptor bound protein Grb 7,
phospholipase Cγ (PLCγ) and others. Crystal structure of the N-terminal domain of avian FAK, containing the FERM domain, has been recently reported (Ceccarelli et al, 2006). Of note, negative regulation of FAK function by FERM domain was revealed (Cooper et al, 2003), where the N-terminal domain had an auto-inhibitory effect through interaction with the kinase domain of FAK.
Recently, several novel binding partners in cancer cells of the FAK N-terminus, such as EGFR ( Sieg et al, 2000; Golubovskaya et al, 2002), RIP (Kurenova et al, 2004) and p53 (Golubovskaya et al, 2005) have been reported (Figure 1). 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 (Lobo and Zachary, 2000; Jones et al, 2001; Stewart et al, 2002; Jones and Stewart, 2004). Thus, the N-terminal domain of FAK binds to the extracellular matrix receptors, integrins, growth factor receptors, and important cytoplasmic, cytoskeletal and nuclear proteins, mediating signaling from the extracellular matrix to the cytoplasm and nucleus and controlling cytoskeletal changes, survival, motility, and invasion.
Figure 1. Structure of FAK molecule with multiple interacting partners. FAK has multiple important functions including an impact on cell survival pathways and apoptosis.
Figure 2. FAK expression in human pancreatic cancers. (A, top) Immunohistochemical staining of FAK in human pancreatic adenocarcinomas. Intensity of FAK staining is higher in metastases than in primary tumor. (Mean±SE: 3.5±0.2 vs 4±0, p=0.001). (B, bottom) FAK staining in pancreatic cancer. Representative example demonstrating staining of FAK in primary and metastatic pancreatic cancer.
D. The C-terminal domain
Different proteins can bind to the C-terminal domain of FAK (amino acids 677-1052), including paxillin, p130cas, PI3-kinase, and GTP-ase-activating protein Graf, leading to changes in the cytoskeleton and to activation of the Ras-MAP kinase pathway (Schaller and Parsons, 1994; Windham et al, 2002; Hanks et al, 2003; Parsons, 2003). 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, FAK-related non-kinase (FRNK) (Richardson and Parsons, 1995). The crystal structure of the C-terminal domain of FAK, FAT, has been determined recently by several groups (Hayashi et al, 2002; Prutzman et al, 2004) and structure analysis demonstrates that it can exist as a dimer or monomer, allowing binding of several binding partners.
E. Post-translational protein modifications
FAK function is altered by post-translational modifications including phosphorylation of tyrosines or serines. FAK has numerous tyrosine phosphorylated sites: Y397, Y407, Y576/Y577, Y861 and Y925. Phosphorylation of Y397, creates 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 (bu-Ghazaleh et al, 2001). FAT domain mediates signaling through Grb-2 binding to Y925 site of FAK (Arold et al, 2002). Inhibition of FAK that resulted in decreased Y925 phosphorylation of FAK 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 have been reported to play a major role in FAK function, such as serines 722, 732, 843 and 910. The role of serine phosphorylation is less described than phosphorylation of tyrosines but was suggested to play a role in binding/stability of proteins (Parsons, 2003).
In addition, recent 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). The authors suggested that coordinated phosphorylation of FAK by tyrosine and serine/threonine-specific kinases may be critical a 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 conserved 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 total of 30 sites of phosphorylation of FAK, including those reported before, requiring detailed analysis of their biological functioning in vivo.
III. FAK functioning in cells
Attachment to the underlying extracellular matrix provides cells with both a means of anchorage needed for traction during migration via a link to the actin cytoskeleton and also with intracellular structures that house membrane-associated signaling proteins. This leads to the transmission of biochemical signals into the cell interior to induce multiple biological responses. Loss of regulation of the process of adhesion formation or turnover, or of downstream signaling is likely to contribute to primary tumor development and/or tumor dissemination. Signaling via adhesion-associated kinases controls the changes that are necessary for cell migration including regulation of cell-matrix adhesion turnover and coordination of remodeling of the actin cytoskeleton network (Cance et al, 2000). FAK has numerous functions in cell survival, motility, metastasis, invasion, and angiogenesis. FAK has also been shown to be important for cell motility (Hauck et al, 2001; Schaller, 2001; Hanks et al, 2003; Schlaepfer and Mitra, 2004). FAK-null embryos exhibit decreased motility in vitro (Ilic et al, 1995). Furthermore, forced 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 has been shown to be required for the organization of the leading edge in migrating cells by coordinating integrin signaling in order to direct the correct activation of membrane protrusion (Tilghman et al, 2005). SH2 domain of Src, targeting Src to focal adhesions and Y397 activity has been shown to be important for motility (Yeo et al, 2006). PI3 kinase has been also shown to be critical for FAK-mediated motility in Chinese hamster ovary (CHO) cells (Reiske et al, 1999). 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, 1999). Overexpression of FAK reversed the inhibitory effect of PTEN on cell migration (Tamura et al, 1998).
Activation of FAK is linked to invasion and metastasis signaling pathways. FAK was important in Erb-2/Erb3-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 an aggressive tumor phenotype (Lark et al, 2005). Subsequently, we analyzed FAK expression in pre-invasive ductal carcinoma in situ, DCIS tumors and detected protein overexpression in preinvasive tumors (Lightfoot, Jr. et al, 2004), suggesting that FAK survival function occurs as an early event in breast tumorigenesis.
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 et al, 1996; Frisch and Ruoslahti, 1997; Frisch, 1999; Frisch and Screaton, 2001; Windham et al, 2002). Similarly, both FAK antisense oligonucleotides (Xu et al, 1996; Smith et al, 2005), as well as dominant-negative FAK protein (FAK-CD), caused cell detachment and apoptosis in tumor cells (Xu et al, 1996, 1998, 2000; van de et al, 2001; Golubovskaya et al, 2002, 2003; Beviglia et al, 2003; Gabarra-Niecko et al, 2003; Park et al, 2004b). The anti-apoptotic 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 vector-transfected cells (Sonoda et al, 2000; Kasahara et al, 2002). 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 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, 2004a,b). 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.
Vascular endothelial growth factor (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 that 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 cell (HBMEC) (Avraham et al, 2003). Furthermore, inhibition of FAK with the dominant-negative inhibitor FRNK (FAK-related non-kinase) or the C-terminal FAK (FAK-CD) significantly decreased HBMEC spreading and migration (Avraham et al, 2003, 2004). In addition, angiogenic inhibitor endostatin blocked VEGF-induced activation of FAK (Kim et al, 2002). Recently, we have shown that FAK binds to 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 in the future.
IV. FAK as a target for therapy
Recently, several reports describe the properties of FAK inhibitors in vitro and in vivo. FAK has been proposed to be a new therapeutic target (McLean et al, 2005). Initial studies which evaluated the effects of FAK inhibition in preclinical models focused on dominant negative mutants of FAK, antisense oligonucleotides and siRNAs (Parsons et al, 2008). More recently, scientists at Novartis Pharmaceuticals designed and synthesized a series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines to inhibit FAK using molecular modeling in conjunction with a co-crystal structure (Choi et al, 2006). Chemistry was developed to introduce functionality onto the 9-aryl ring, which resulted in the identification of potent FAK inhibitors. We and others have published reports on the use of such FAK inhibitors that have targeted the ATP binding site in the kinase domain. In human pancreatic cancer, we have shown widespread expression of FAK in primary pancreatic adenocarcinoma. In addition, we have shown significant upregulation of FAK protein expression in metastatic lesions (Figure 2, unpublished data). In human pancreatic cancer cells, we have identified that the FAK kinase inhibitor, TAE226, decreases viability, increases cell detachment and increases apoptosis (Liu et al, 2008). Other studies have shown that TAE226 readily induced apoptosis in human breast cancer cells with overexpressed Src or EGFR. Of note, these cells were resistant to adenoviral FAK dominant negative treatment, indicating that kinase inhibition was important for downregulation of FAK function and the observed phenotypic changes (Golubovskaya et al, 2008b).
Subsequent studies have studied the in vivo effects of TAE226. The expression status of FAK in BarrettŐs esophageal adenocarcinoma has been recently reported. FAK expression was studied in frank adenocarcinoma, areas of BarrettŐs epithelia, squamous epithelia, and gastric epithelia. FAK expression was increased in cancerous parts compared to non-cancerous areas and strong expression (>50% positive staining cells per area) were observed in 94% of BarrettŐs esophageal adenocarcinoma compared with 18% of BarrettŐs epithelia. In a subcutaneous model of human esophageal cancer, TAE226 given orally at 30 mg/kg significantly decreased tumor volume and weight compared with placebo (Watanabe et al, 2008). Similar results from in vivo studies have confirmed the ability of TAE226 to decrease the growth of ovarian and glioma xenografts (Shi et al, 2007).
While initial results with kinase inhibition of FAK has shown anti-neoplastic effects, TAE226 has been shown to also inhibit the activity of IGF-1R at nanomolar concentrations (Liu et al, 2007). Therefore, the activities against multiple tumor types likely reflect its dual inhibition of adhesion and growth promoting pathways. Recently, Pfizer pharmaceuticals have published results on an ATP competitive reversible inhibitor of FAK that has bioavailability suitable for preclinical animal and human studies. PF-562,271 was shown to exhibit >100 fold selectivity for FAK when assayed against a panel of unrelated kinases. Treatment of cancer cells lines showed a dose dependent decrease in FAK phosphorylation at the Y397 site. The IC50 for FAK phosphorylation was reported to be 5 nmol/L. Anti-tumor efficacy was observed in multiple human subcutaneous xenograft models with minimal weight loss or mortality (Parsons et al, 2008; Roberts et al, 2008).
PF-562,271 is currently in phase 2 clinical trials. Phase 1 study results with this drug in patients with advanced solid malignancy have been reported in abstract form (Siu ll et al, 2008). Studies have been performed in 2 centers in the United States and one center in Canada and Australia with oral dosing as a single agent. Thirty two patients received from 5 mg up to 105 mg twice a day. Adverse events possibly related to the drug in over 10% were nausea, vomiting, fatigue, anorexia, abdominal pain, diarrhea, headache, sensory neuropathy, rash, constipation, and dizziness. Adverse events were generally grade 1-2 and reversible. Doses over 15 mg twice a day produced steady state plasma concentrations exceeding target efficacious levels predicted from preclinical models. Prolonged disease stabilization was observed in several tumor types. Phase 1 results indicated good tolerability of this drug with favorable pharmacokinetics and pharmacodynamics (Siu ll et al, 2008). This drug represents the sole FAK inhibitor being tested in humans to date.
Another approach to inhibit FAK function can be to target protein-protein interactions between FAK and its binding partners such as p53, VEGFR-3 or EGFR or targeting sites of FAK phosphorylation (Golubovskaya et al, 2008a). Tyrosine 397 is an autophosphorylation site of FAK that is a critical component in downstream signaling, providing a high-affinity binding site for the SH2 domain of Src family kinases (Figure 3). Y397 is also a site of binding of PI3 kinase, growth factor receptor binding Grb-7, Shc and other proteins. Thus, the Y397 site is one of the main phosphorylation sites that can activate FAK signaling in cells. We recently demonstrated that computer modeling and screening can be performed to identify novel small molecules that inhibit protein-protein interactions at the Y397 site (Golubovskaya et al, 2008a).
Figure 3. The Y397
autophosphorylation site of FAK has several binding proteins and is critical for survival signaling.
Figure 4. (A, Left) Molecular modeling of Y15 compound in the Y397 pocket of FAK. Y15 is shown in purple and the FAK pocket in green. (B, right) Structure of Y15. Reproduced from Golubovskaya et al, 2008 with kind permission from Journal of Medicinal Chemistry.
Figure 5. Y15 significantly blocks tumor growth in vivo and its effects are synergistic with gemcitabine treatment. Mice (n=5/group) were subcutaneously injected with Panc-1 cells. The day after injection, mice were treated with daily intraperitoneal PBS, intraperitoneal Y15 (30mg/kg), intraperitoneal gemcitabine alone (30mg/kg) or Y15 (30mg/kg) + gemcitabine (30mg/kg). The combination of Y15 + gemcitabine significantly decreased tumor volume compared to Y15 or gemcitabine (Gen) alone. *p<0.05 vs. Y15 or gemcitabine alone.
In this approach, more than 140,000 small molecule compounds were docked into the N-terminal domain of the FAK crystal structure in 100 different orientations. Those compounds with the greatest energy of interaction based on van der Waals and electrostatic charges were identified as lead compounds. One compound, 1,2,4,5-benzenetetraamine tetrahydrocholoride (Y15) significantly decreased viability in most cancer cells and specifically and directly blocked phosphorylation of Y397-FAK in a dose and time dependent manner (Figure 4). Furthermore, it inhibited cell adhesion and effectively caused breast tumor regression in vivo (Golubovskaya et al, 2008a). Finally, we have shown that it inhibits pancreatic cancer growth in vivo both alone and in combination with gemcitabine chemotherapy (Figure 5, unpublished data).
One potential advantage of this approach utilized to identify small molecules through in silico screening is increased target specificity. Y15 did not affect phosphorylation of the FAK homologue, Pyk-2, which can be explained by only 43% amino acid identity between N-terminal domains of FAK and Pyk-2. Other kinase inhibitors of FAK have shown inhibition of Pyk-2 autophosphorylation and likely are less specific for inhibition of FAK function.
FAK is an emerging target for therapy. A FAK inhibitor is currently in Phase II clinical trials in cancer patients. Novel approaches to FAK inhibition are needed and offer directed molecular therapy. This work was supported by NIH grant number CA113766.
Abedi H, 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.
Agochiya M, Brunton VG, Owens DW, Parkinson EK, Paraskeva C, Keith WN, Frame MC (1999) Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells. Oncogene 18, 5646-5653.
Arold ST, Hoellerer MK, Noble ME (2002) The structural basis of localization and signaling by the focal adhesion targeting domain. Structure 10, 319-327.
Avraham HK, Jiang S, Lee TH, Prakash O, Avraham S (2004) HIV-1 Tat-mediated effects on focal adhesion assembly and permeability in brain microvascular endothelial cells. J Immunol 173, 6228-6233.
Avraham HK, Lee TH, Koh Y, Kim TA, Jiang S, Sussman M, Samarel AM, Avraham S (2003) Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase. J Biol Chem 278, 36661-36668.
Avraham S, London R, Fu Y, Ota S, Hiregowdara D, Li J, Jiang S, Pasztor LM, White RA, Groopman JE (1995) Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J Biol Chem 270, 27742-27751.
Benlimame N, He Q, Jie S, Xiao D, Xu YJ, Loignon M, Schlaepfer DD, aoui-Jamali MA (2005) FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion. J Cell Biol 171, 505-516.
Beviglia L, Golubovskaya V, Xu L, Yang X, Craven RJ, Cance WG (2003) Focal adhesion kinase N-terminus in breast carcinoma cells induces rounding, detachment and apoptosis. Biochem J 373, 201-210.
bu-Ghazaleh R, Kabir J, Jia H, Lobo M, Zachary I (2001) Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells. Biochem J 360, 255-264.
Burgaya F, Girault JA (1996) Cloning of focal adhesion kinase, pp125FAK, from rat brain reveals multiple transcripts with different patterns of expression. Brain Res Mol Brain Res 37, 63-73.
Caballero OL, de Souza SJ, Brentani RR, Simpson AJ (2001) Alternative spliced transcripts as cancer markers. Dis Markers 17, 67-75.
Calalb MB, Polte TR, Hanks SK (1995) Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol 15, 954-963.
Cance WG, Harris JE, Iacocca MV, Roche E, Yang X, Chang J, Simkins S, Xu L (2000) Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes. Clin Cancer Res 6, 2417-2423.
Ceccarelli DF, Song HK, Poy F, Schaller MD, Eck MJ (2006) Crystal structure of the FERM domain of focal adhesion kinase. J Biol Chem 281, 252-259.
Choi HS, Wang Z, Richmond W, He X, Yang K, Jiang T, Karanewsky D, Gu XJ, Zhou V, Liu Y, Che J, Lee CC, Caldwell J, Kanazawa T, Umemura I, Matsuura N, Ohmori O, Honda T, Gray N, He Y (2006) Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 2. Bioorg Med Chem Lett 16, 2689-2692.
Cooper LA, Shen TL, Guan JL (2003) Regulation of focal adhesion kinase by its amino-terminal domain through an autoinhibitory interaction. Mol Cell Biol 23, 8030-8041.
Corsi JM, Rouer E, Girault JA, Enslen H (2006) Organization and post-transcriptional processing of focal adhesion kinase gene. BMC Genomics 7, 198.
Fiedorek FT, Jr., Kay ES (1995) Mapping of the focal adhesion kinase (Fadk) gene to mouse chromosome 15 and human chromosome 8. Mamm Genome 6, 123-126.
Frisch SM (1999) Evidence for a function of death-receptor-related, death-domain-containing proteins in anoikis. Curr Biol 9, 1047-1049.
Frisch SM, Ruoslahti E (1997) Integrins and anoikis. Curr Opin Cell Biol 9, 701-706.
Frisch SM, Screaton RA (2001) Anoikis mechanisms. Curr Opin Cell Biol 13, 555-562.
Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY (1996) Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 134, 793-799.
Fujimoto J, Sawamoto K, Okabe M, Takagi Y, Tezuka T, Yoshikawa S, Ryo H, Okano H, Yamamoto T (1999) Cloning and characterization of Dfak56, a homolog of focal adhesion kinase, in Drosophila melanogaster. J Biol Chem 274, 29196-29201.
Gabarra-Niecko V, Schaller MD, Dunty JM (2003) FAK regulates biological processes important for the pathogenesis of cancer. Cancer Metastasis Rev 22, 359-374.
Garces CA, Kurenova EV, Golubovskaya VM, Cance WG (2006) Vascular endothelial growth factor receptor-3 and focal adhesion kinase bind and suppress apoptosis in breast cancer cells. Cancer Res 66, 1446-1454.
Golubovskaya V, Beviglia L, Xu LH, Earp HS, III, Craven R, Cance W (2002) Dual inhibition of focal adhesion kinase and epidermal growth factor receptor pathways cooperatively induces death receptor-mediated apoptosis in human breast cancer cells. J Biol Chem 277, 38978-38987.
Golubovskaya V, Kaur A, Cance W (2004) Cloning and characterization of the promoter region of human focal adhesion kinase gene: nuclear factor kB and p53 binding sites. Biochim Biophys Acta 1678, 111-125.
Golubovskaya VM, Finch R, Cance WG (2005) Direct interaction of the N-terminal domain of focal adhesion kinase with the N-terminal transactivation domain of p53. J Biol Chem 280, 25008-25021.
Golubovskaya VM, Gross S, Kaur AS, Wilson RI, Xu LH, Yang XH, Cance WG (2003) Simultaneous inhibition of focal adhesion kinase and SRC enhances detachment and apoptosis in colon cancer cell lines. Mol Cancer Res 1, 755-764.
Golubovskaya VM, Nyberg C, Zheng M, Kweh F, Magis A, Ostrov D, Cance WG (2008a) A small molecule inhibitor, 1,2,4,5-benzenetetraamine tetrahydrochloride, targeting the y397 site of focal adhesion kinase decreases tumor growth. J Med Chem 51, 7405-7416.
Golubovskaya VM, Virnig C, Cance WG (2008b) TAE226-induced apoptosis in breast cancer cells with overexpressed Src or EGFR. Mol Carcinog 47, 222-234.
Grigera PR, Jeffery ED, Martin KH, Shabanowitz J, Hunt DF, Parsons JT (2005) FAK phosphorylation sites mapped by mass spectrometry. J Cell Sci 118, 4931-4935.
Hanada M, Tanaka K, Matsumoto Y, Nakatani F, Sakimura R, Matsunobu T, Li X, Okada T, Nakamura T, Takasaki M, Iwamoto Y (2005) Focal adhesion kinase is activated in invading fibrosarcoma cells and regulates metastasis. Clin Exp Metastasis 22, 485-494.
Hanks SK, Polte TR (1997) Signaling through focal adhesion kinase. Bioessays 19, 137-145.
Hanks SK, Ryzhova L, Shin NY, Brabek J (2003) Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front Biosci 8, d982-d996.
Hauck CR, Sieg DJ, Hsia DA, Loftus JC, Gaarde WA, Monia BP, Schlaepfer DD (2001) Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells. Cancer Res 61, 7079-7090.
Hayasaka H, Simon K, Hershey ED, Masumoto KH, Parsons JT (2005) FRNK, the autonomously expressed C-terminal region of focal adhesion kinase, is uniquely regulated in vascular smooth muscle: analysis of expression in transgenic mice. J Cell Biochem 95, 1248-1263.
Hayashi I, Vuori K, Liddington RC (2002) The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin. Nat Struct Biol 9, 101-106.
Hildebrand JD, Schaller MD, Parsons JT (1993) Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions. J Cell Biol 123, 993-1005.
Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, Yamamoto T (1995) Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377, 539-544.
Jones G, Machado J, Jr., Merlo A (2001) Loss of focal adhesion kinase (FAK) inhibits epidermal growth factor receptor-dependent migration and induces aggregation of nh(2)-terminal FAK in the nuclei of apoptotic glioblastoma cells. Cancer Res 61, 4978-4981.
Jones G, Stewart G (2004) Nuclear import of N-terminal FAK by activation of the FcepsilonRI receptor in RBL-2H3 cells. Biochem Biophys Res Commun 314, 39-45.
Judson PL, He X, Cance WG, Van LL (1999) Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma. Cancer 86, 1551-1556.
Kasahara T, Koguchi E, Funakoshi M, izu-Yokota E, Sonoda Y (2002) Antiapoptotic action of focal adhesion kinase (FAK) against ionizing radiation. Antioxid Redox Signal 4, 491-499.
Kim YM, Hwang S, Kim YM, Pyun BJ, Kim TY, Lee ST, Gho YS, Kwon YG (2002) Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1. J Biol Chem 277, 27872-27879.
Kurenova E, Xu LH, Yang X, Baldwin AS, Jr., Craven RJ, Hanks SK, Liu ZG, Cance WG (2004) Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein. Mol Cell Biol 24, 4361-4371.
Lark AL, Livasy CA, Dressler L, Moore DT, Millikan RC, Geradts J, Iacocca M, Cowan D, Little D, Craven RJ, Cance W (2005) High focal adhesion kinase expression in invasive breast carcinomas is associated with an aggressive phenotype. Mod Pathol 18, 1289-1294.
Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, Schlessinger J (1995) Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature 376, 737-745.
Lightfoot HM, Jr., Lark A, Livasy CA, Moore DT, Cowan D, Dressler L, Craven RJ, Cance WG (2004) Upregulation of focal adhesion kinase (FAK) expression in ductal carcinoma in situ (DCIS) is an early event in breast tumorigenesis. Breast Cancer Res Treat 88, 109-116.
Liu TJ, LaFortune T, Honda T, Ohmori O, Hatakeyama S, Meyer T, Jackson D, de GJ, Yung WK (2007) Inhibition of both focal adhesion kinase and insulin-like growth factor-I receptor kinase suppresses glioma proliferation in vitro and in vivo. Mol Cancer Ther 6, 1357-1367.
Liu W, Bloom DA, Cance WG, Kurenova EV, Golubovskaya VM, Hochwald SN (2008) FAK and IGF-IR interact to provide survival signals in human pancreatic adenocarcinoma cells. Carcinogenesis 29, 1096-1107.
Lobo M, Zachary I (2000) Nuclear localization and apoptotic regulation of an amino-terminal domain focal adhesion kinase fragment in endothelial cells. Biochem Biophys Res Commun 276, 1068-1074.
Matkowskyj KA, Keller K, Glover S, Kornberg L, Tran-Son-Tay R, Benya RV (2003) Expression of GRP and its receptor in well-differentiated colon cancer cells correlates with the presence of focal adhesion kinase phosphorylated at tyrosines 397 and 407. J Histochem Cytochem 51, 1041-1048.
McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC (2005) The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nat Rev Cancer 5, 505-515.
Mitra SK, Mikolon D, Molina JE, Hsia DA, Hanson DA, Chi A, Lim ST, Bernard-Trifilo JA, Ilic D, Stupack DG, Cheresh DA, Schlaepfer DD (2006) Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors. Oncogene 25, 5969-5984.
Nowakowski J, Cronin CN, McRee DE, Knuth MW, Nelson CG, Pavletich NP, Rogers J, Sang BC, Scheibe DN, Swanson RV, Thompson DA (2002) Structures of the cancer-related Aurora-A, FAK, and EphA2 protein kinases from nanovolume crystallography. Structure 10, 1659-1667.
Owens LV, Xu L, Craven RJ, Dent GA, Weiner TM, Kornberg L, Liu ET, Cance WG (1995) Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 55, 2752-2755.
Owens LV, Xu L, Dent GA, Yang X, Sturge GC, Craven RJ, Cance WG (1996) Focal adhesion kinase as a marker of invasive potential in differentiated human thyroid cancer. Ann Surg Oncol 3, 100-105.
Park HB, Golubovskaya V, Xu L, Yang X, Lee JW, Scully S, Craven RJ, Cance WG (2004b) Activated Src increases adhesion, survival and alpha2-integrin expression in human breast cancer cells. Biochem J 378, 559-567.
Park HB, Golubovskaya V, Xu L, Yang X, Lee JW, Scully S, Craven RJ, Cance WG (2004a) Activated Src increases adhesion, survival and alpha2-integrin expression in human breast cancer cells. Biochem J 378, 559-567.
Parsons JT (2003) Focal adhesion kinase: the first ten years. J Cell Sci 116, 1409-1416.
Parsons JT, Slack-Davis J, Tilghman R, Roberts WG (2008) Focal adhesion kinase: targeting adhesion signaling pathways for therapeutic intervention. Clin Cancer Res 14, 627-632.
Prutzman KC, Gao G, King ML, Iyer VV, Mueller GA, Schaller MD, Campbell SL (2004) The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation. Structure 12, 881-891.
Reiske HR, Kao SC, Cary LA, Guan JL, Lai JF, Chen HC (1999) Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration. J Biol Chem 274, 12361-12366.
Richardson A, Parsons JT (1995) Signal transduction through integrins: a central role for focal adhesion kinase? Bioessays 17, 229-236.
Roberts WG, Ung E, Whalen P, Cooper B, Hulford C, Autry C, Richter D, Emerson E, Lin J, Kath J, Coleman K, Yao L, Martinez-Alsina L, Lorenzen M, Berliner M, Luzzio M, Patel N, Schmitt E, LaGreca S, Jani J, Wessel M, Marr E, Griffor M, Vajdos F (2008) Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer Res 68, 1935-1944.
Sasaki H, Nagura K, Ishino M, Tobioka H, Kotani K, Sasaki T (1995) Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J Biol Chem 270, 21206-21219.
Schaller MD (2001) Biochemical signals and biological responses elicited by the focal adhesion kinase. Biochim Biophys Acta 1540, 1-21.
Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT (1992) pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A 89, 5192-5196.
Schaller MD, Hildebrand JD, Parsons JT (1999) Complex formation with focal adhesion kinase: A mechanism to regulate activity and subcellular localization of Src kinases. Mol Biol Cell 10, 3489-3505.
Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines RR, Parsons JT (1994) Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol 14, 1680-1688.
Schaller MD, Otey CA, Hildebrand JD, Parsons JT (1995) Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains. J Cell Biol 130, 1181-1187.
Schaller MD, Parsons JT (1994) Focal adhesion kinase and associated proteins. Curr Opin Cell Biol 6, 705-710.
Schlaepfer DD, Mitra SK (2004) Multiple connections link FAK to cell motility and invasion. Curr Opin Genet Dev 14, 92-101.
Shi Q, Hjelmeland AB, Keir ST, Song L, Wickman S, Jackson D, Ohmori O, Bigner DD, Friedman HS, Rich JN (2007) A novel low-molecular weight inhibitor of focal adhesion kinase, TAE226, inhibits glioma growth. Mol Carcinog 46, 488-496.
Sieg DJ, Hauck CR, Ilic D, Klingbeil CK, Schaefer E, Damsky CH, Schlaepfer DD (2000) FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2, 249-256.
Sieg DJ, Hauck CR, Schlaepfer DD (1999) Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J Cell Sci 112 ( Pt 16), 2677-2691.
Siu ll, Burris HA, Mileshkin L, Camidge DR, Rishchin D, Chen EX, Jones S, Tin D, Fingert H (2008) Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part 1. J Clin Oncol 25, 3527.
Smith CS, Golubovskaya VM, Peck E, Xu LH, Monia BP, Yang X, Cance WG (2005) Effect of focal adhesion kinase (FAK) downregulation with FAK antisense oligonucleotides and 5-fluorouracil on the viability of melanoma cell lines. Melanoma Res 15, 357-362.
Sonoda Y, Matsumoto Y, Funakoshi M, Yamamoto D, Hanks SK, Kasahara T (2000) Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60. J Biol Chem 275, 16309-16315.
Stewart A, Ham C, Zachary I (2002) The focal adhesion kinase amino-terminal domain localises to nuclei and intercellular junctions in HEK 293 and MDCK cells independently of tyrosine 397 and the carboxy-terminal domain. Biochem Biophys Res Commun 299, 62-73.
Takahashi N, Seko Y, Noiri E, Tobe K, Kadowaki T, Sabe H, Yazaki Y (1999) Vascular endothelial growth factor induces activation and subcellular translocation of focal adhesion kinase (p125FAK) in cultured rat cardiac myocytes. Circ Res 84, 1194-1202.
Tamura M, Gu J, Danen EH, Takino T, Miyamoto S, Yamada KM (1999) PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 274, 20693-20703.
Tamura M, Gu J, Matsumoto K, Aota S, Parsons R, Yamada KM (1998) Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science 280, 1614-1617.
Tilghman RW, Slack-Davis JK, Sergina N, Martin KH, Iwanicki M, Hershey ED, Beggs HE, Reichardt LF, Parsons JT (2005) Focal adhesion kinase is required for the spatial organization of the leading edge in migrating cells. J Cell Sci 118, 2613-2623.
van de WB, Houtepen F, Huigsloot M, Tijdens IB (2001) Suppression of chemically induced apoptosis but not necrosis of renal proximal tubular epithelial (LLC-PK1) cells by focal adhesion kinase (FAK). Role of FAK in maintaining focal adhesion organization after acute renal cell injury. J Biol Chem 276, 36183-36193.
Venables JP (2006) Unbalanced alternative splicing and its significance in cancer. Bioessays 28, 378-386.
Watanabe N, Takaoka M, Sakurama K, Tomono Y, Hatakeyama S, Ohmori O, Motoki T, Shirakawa Y, Yamatsuji T, Haisa M, Matsuoka J, Beer DG, Nagatsuka H, Tanaka N, Naomoto Y (2008) Dual tyrosine kinase inhibitor for focal adhesion kinase and insulin-like growth factor-I receptor exhibits anticancer effect in esophageal adenocarcinoma in vitro and in vivo. Clin Cancer Res 14, 4631-4639.
Weiner TM, Liu ET, Craven RJ, Cance WG (1993) Expression of focal adhesion kinase gene and invasive cancer. Lancet 342, 1024-1025.
Whitney GS, Chan PY, Blake J, Cosand WL, Neubauer MG, Aruffo A, Kanner SB (1993) Human T and B lymphocytes express a structurally conserved focal adhesion kinase, pp125FAK. DNA Cell Biol 12, 823-830.
Windham TC, Parikh NU, Siwak DR, Summy JM, McConkey DJ, Kraker AJ, Gallick GE (2002) Src activation regulates anoikis in human colon tumor cell lines. Oncogene 21, 7797-7807.
Xu LH, Owens LV, Sturge GC, Yang X, Liu ET, Craven RJ, Cance WG (1996) Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells. Cell Growth Differ 7, 413-418.
Xu LH, Yang X, Bradham CA, Brenner DA, Baldwin AS, Jr., Craven RJ, Cance WG (2000) The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Involvement of death receptor-related signaling pathways. J Biol Chem 275, 30597-30604.
Xu LH, Yang X, Craven RJ, Cance WG (1998) The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells. Cell Growth Differ 9, 999-1005.
Yeo MG, Partridge MA, Ezratty EJ, Shen Q, Gundersen GG, Marcantonio EE (2006) Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function. Mol Cell Biol 26, 4399-4409.
Zhang X, Wright CV, Hanks SK (1995) Cloning of a Xenopus laevis cDNA encoding focal adhesion kinase (FAK) and expression during early development. Gene 160, 219-222.
Steven N. Hochwald and Vita M. Golubovskaya