*Vita Golubovskaya a, William Cance a,b a Department of Surgery, University of Florida, School of Medicine, Gainesville, FL. b Department Biochemistry and Molecular Biology, University of Florida, Gainesville, FL. ABSTRACT The progression of human cancer is characterized by a process of tumor invasion, cell motility, and metastasis to distant sites. Focal Adhesion Kinase (FAK) is a central molecule in the processes of tumor progression. FAK is a 125 kda protein tyrosine kinase and is localized to focal adhesions, which are contact points between a cell and its extracellular matrix. Focal Adhesion Kinase (FAK) is over expressed in human cancer and plays an important role in integrin signaling, cellular motility, and survival signaling pathways. Despite this progress in understanding the biology of FAK and its signaling complex, it is still not known how FAK interacts with its signaling partners to resist activation of the apoptotic cascade. Key words: Focal Adhesion kinase, apoptosis, survival, cancer REVIEW 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 critical tyrosine kinases that is linked to these processes of tumor invasion and survival is the Focal Adhesion Kinase (FAK). Focal Adhesion Kinase (FAK) is a non-receptor protein tyrosine kinase that is localized to contact points between cells and their extracellular matrix. 1-3 The FAK gene encodes a non receptor tyrosine kinase that localizes at contact points of cells with extracellular matrix, and that is activated by integrin signaling. 1 The FAK gene was first isolated from chick embryo fibroblasts transformed by v-src. 1 FAK was isolated from human tumors, and FAK mrna was upregulated in invasive and metastatic human breast and colon cancer samples. 4 At the same time, matched samples of normal colon and breast tissue from the same patients had almost no detectable FAK expression. 4 FAK is a point * Corresponding author. Phone: 352-392-7461, FAX: 352-392-0080, E-mail: golubvi@surgery.ufl.edu 215
Vita Golubovskaya of convergence of a number of signaling pathways associated with cell adhesion, invasion, motility, mitogenesis, angiogenesis and oncogenic transformation. FAK STRUCTURE AND FUNCTION The human FAK gene was mapped to chromosome 8, 5,6 and there appears to be a high degree of homology between species. The FAK 1protein is a 125 kda tyrosine kinase (p125 FAK ) with a large amino-terminal domain with an autophosphorylation site (Y-397), a central catalytic domain, and a large carboxy-terminal domain that contains a number of potential protein interacting sites, including two proline-rich domains. 2,7,8 Integrins, via their β subunits, appear to bind to FAK at its amino N-terminal domain. Following integrin binding, p125 FAK becomes tyrosine phosphorylated at Y-397 site. 2 This allows for efficient binding of Src via its SH2 domain to form a FAK- Src complex. 8-10 Src-FAK binding activates Src and enhance FAK phosphorylation at Y576/577 at carboxy terminal sites. Different proteins can bind the C-terminal domain of p125 FAK, including paxillin, p130cas, PI3- kinase, GTP-ase-activating protein Graf, leading to changes in the cytoskeleton and to activation of the Ras- MAP kinase pathway. 8,11-13 The carboxy-terminal domain of FAK contains sequences responsible for its targeting to focal adhesions, also known as the FAT domain (Figure1). Alternative splicing of FAK results in autonomous expression of C-terminal part of FAK, FAK-related nonkinase (FRNK). 14 FRNK or FAK-CD (FAK-C-terminal domain) was shown to cause apoptosis in different cancer cell lines. 15-20 FAK plays a major role in survival signaling, and has been linked to detachment-induced apoptosis, or anoikis. 21 It has been shown that constitutively activated forms of FAK rescued epithelial cells from anoikis, suggesting that FAK can regulate this process. 13,21-24 Similarly, both FAK antisense oligonucleotides 25 as well as dominant-negative FAK protein (FAK-CD) caused cell detachment and apoptosis in tumor cells. 15-18,25-28 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. 29,30 HL-60/FAK cells activated the AKT survival pathway and activated NF-kappa B pathway with induction of inhibitorof-apoptosis proteins, IAPs. 29 FAK has also been shown to be important for cell motility. 12,31-33 FAK null embryos exhibit decreased motility in vitro. 34 Furthermore, enforced expression of FAK stimulated cell migration. 35,36 Figure 1. Structure of Focal Adhesion Kinase, FAK. FAK has N-terminal, Kinase domain and C-terminal domains. N-terminal domain has Y-397Y-autophosphorylation site. Kinase domain has Y576/577 tyrosines important for catalytic activity of FAK. C-terminal part of FAK has Y861 and Y925 tyrosines. Different proteins bind to these domains and involved in motility and survival signaling, Integrins, Shc; PI-3K(p85); Src, Cas, p130; Graf; Talin; Paxillin; Grb2. Taken together, these results show that FAK has numerous functions in cell survival and motility. FAK IS UP-REGULATED IN EARLY STAGES OF TUMORIGENESIS FAK was isolated from a primary human tissue and linked to human tumorigenesis. 37,38 FAK is elevated in a variety of human tumors, including colorectal cancer, 39 breast, 38 sarcomas, 4 cervical carcinomas, 40 prostatic carcinoma tumors and cell lines, 41 FAK was identified and cloned from primary tumors. 4,42 Northern blot analysis demonstrated low or undetectable levels of FAK mrna 216
in normal tissues, while primary and metastatic tumors significantly overexpressed FAK. 4 Subsequently, human FAK cdna has been cloned and anti-fak antibodies were generated and used to demonstrate that FAK is overexpressed in different types of tumors. 4,38,43,44 ELEVATED FAK EXPRESSION IN HUMAN TUMORS SUPPRESSES APOPTOSIS FAK expression was linked to apoptosis by treating FAK positive tumor cell lines with different antisense oligonucleotides to FAK that specifically inhibited p125fak expression. 25,45 The cells treated with antisense oligonucleotides lost their attachment and underwent apoptosis. 25 In addition, p125 FAK interacts with other tyrosine kinases to affect survival functions. Recently, Src was shown to regulate anoikis in colon human tumors; 13 similarly, we have shown that increased Src activity led to additional survival signals in suppressing apoptosis in colon cancer cell lines overexpressing FAK. 18 The same result was obtained in a breast cancer model with stably expressing activated Src. 19 In breast cancer cell lines that overexpressed EGFR, we have found that association of EGFR and FAK suppressed apoptosis by activating AKT/ERK1/2 pathways. 17 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. 17 To study death-induced apoptosis we performed analysis of death receptor proteins to bind FAK and found physical association of FAK and an apoptosis-related receptor interacting protein, RIP. 46 RIP provided pro-apoptotic signals that were suppressed by its binding to FAK (Figure 2). 46 In a recent study, real-time PCR analysis on colorectal carcinoma and liver metastasis samples with matching normal tissues demonstrated increased FAK mrna levels in tumor and metastatic tissues versus normal tissues. 47 However, the structure of the regulatory promoter region of FAK was unknown. We have cloned the FAK promoter region and characterized transcription factors that bind to this region. 48 In addition, there have been several reports on the localization of the N-terminal part of FAK in the nucleus. 49, 26, 50, 26, 49, 51 Furthermore, the N-terminus of FAK was shown to cause apoptosis in breast cancer cell lines 26 and its nuclear localization was regulated by caspase inhibitors in endothelial cells. 51 Thus, deregulated FAK could affect the ability of cancer cells to die in response to killing drugs. Figure 2. FAK-mediated survival signalling. EGFR was shown to bind N-terminal domain of FAK and caused activation of ERK and AKT pathways. Src and FAK binding leads to activation of AKT survival pathway. Recently, RIP (Receptor interacting protein) was shown to bind N-terminus of FAK to suppress apoptosis. CONCLUSION/FUTURE PERSPECTIVES Thus, FAK can be a central molecule in tumor cell survival, and disruption of critical signaling complexes in the amino terminus and carboxy terminus can induce apoptosis in tumor cells. Characterization of the functional role of FAK and its binding partners in cancer cells may help to understand survival signaling in tumors with overexpressed FAK. Studies to reveal FAK-mediated intracellular signaling from cell contacts with extracellular matrix to the nucleus and to the cytoplasm will be important for the development of new cancer therapy approaches and cancer treatment program. REFERENCE 1. Schaller, M.D., Borgman, C.A., Cobb, B.S., Vines, R.R., Reynolds, A.B. and Parsons, J.T. (1992) pp125fak a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proceedings of the National Academy of Sciences of the United States of America, 89, 5192-6. 2. Hanks, S.K. and Polte, T.R. (1997) Signaling through focal 217
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