Integrins: roles in cancer development and as treatment targets

The integrin family of cell adhesion proteins promotes the attachment and migration of cells on the surrounding extracellular matrix (ECM). Through signals transduced upon integrin ligation by ECM proteins or immunoglobulin superfamily molecules, this family of proteins plays key roles in regulating tumour growth and metastasis as well as tumour angiogenesis. Several integrins play key roles in promoting tumour angiogenesis and tumour metastasis. Antagonists of several integrins (α5β1, αvβ3 and αvβ5) are now under evaluation in clinical trials to determine their potential as therapeutics for cancer and other diseases.

During the last 10 years, novel insights into the mechanisms that regulate cell survival as well as cell migration and invasion have led to the development of novel integrin-based therapeutics for the treatment of cancer. Several integrins play important roles in promoting cell proliferation, migration and survival in vitro and in vivo. Antagonists of these integrins suppress cell migration and invasion of primary and transformed cells and also induce apoptosis of primary cells. Integrin antagonists also block tumour angiogenesis and tumour metastasis. Currently, humanised antibody antagonists of integrins a5b1 and avb3 as well as peptide inhibitors of integrins avb3/avb5 are under evaluation as angiogenesis-inhibiting therapeutics in cancer clinical trials.

INTEGRINS REGULATE CELL SURVIVAL AND MIGRATION
The invasion and survival of cells in vivo controls embryonic development, angiogenesis, tumour metastasis and other physiological processes (Aplin et al, 1998;Carmeliet and Jain, 2000;Hood and Cheresh, 2002). Cell surface receptors for the extracellular matrix (ECM), such as the integrins, play key roles in the regulation of normal and tumour cell migration and survival. The integrin family of cell adhesion proteins controls cell attachment to the ECM (Figure 1). While some integrins selectively recognise primarily a single ECM protein ligand (e.g., a5b1 recognises primarily fibronectin), others can bind several ligands (e.g., integrin avb3 binds vitronectin, fibronectin, fibrinogen, denatured or proteolysed collagen, and other matrix proteins). Several integrins recognise the tripeptide Arg -Gly -Asp (e.g., avb3, a5b1, aIIbb3), whereas others recognise alternative short peptide sequences (e.g., integrin a4b1 recognises EILDV and REDV in alternatively spliced CS-1 fibronectin). Inhibitors of integrin function include function-blocking monoclonal antibodies, peptide antagonists and small molecule peptide mimetics matrix (reviewed in Hynes, 1992;Cheresh, 1993).
Although integrins mediate cellular adhesion to ECM proteins found in intercellular spaces and basement membranes, they also transduce intracellular signals that promote cell migration (reviewed in Aplin et al, 1998;Schwartz and Shattil, 2000) as well as cell survival (Meredith et al, 1993;Stromblad and Cheresh, 1996). However, unlike growth factor receptors, integrins have no intrinsic enzymatic activity but activate signalling pathways strictly by coclustering with kinases and adaptor proteins in focal adhesion complexes. The association of integrins with polyvalent or crosslinked ECM proteins clusters integrins and their associated cofactors, thus activating integrin-regulated signalling pathways. For example, integrin ligation suppresses apoptosis by activating suppressors of apoptosis (Pankov et al, 2003) and by inhibiting caspase activation (Stupack et al, 2001;Kim et al, 2002). Integrins also stimulate cell migration by activating Rho and Rac GTPases (Ren et al, 1999) and by anchoring actin filaments to the membrane. These adhesion proteins promote cell cycle entry by stimulating expression of cyclins (Assoian and Schwartz, 2001). Integrin ligation, therefore, supports signal transduction cascades that promote cell proliferation, cell survival and cell migration. In contrast, inhibition of cell integrin -ligand interaction inhibits cell migration (Kim et al, 2000a, b;Bakre et al, 2002) and proliferation and induces apoptosis (Meredith et al, 1993;Boudreau et al, 1996;Stupack et al, 2001;Bakre et al, 2002;Kim et al, 2002).

INTEGRIN ROLES IN CELL SURVIVAL
Studies from several groups showed that cell attachment is required for the survival of normal cells (Meredith et al, 1993;Stupack et al, 2001;Bakre et al, 2002). Complete loss of cell contact with the substratum (e.g., suspension culture) or adhesion to a nonspecific substratum such as poly-L-lysine induces apoptosis ('anoikis') of primary cells such as fibroblasts (Meredith et al, 1993), endothelial cells Kim et al, 2002) and epithelial cells (Frisch and Francis, 1994;Boudreau et al, 1996;Stupack et al, 2001). In contrast, loss of contact with the substratum does not necessarily kill tumour cells. Anchorageindependent tumour cells survive loss of contact with the substrate because they accumulate mutational changes in survival factors, such as upregulation of Bcl 2 expression and/or loss of p53 activity, which render the cells independent of integrin-mediated survival signals.
Recent studies have shown that cell death can also occur when a subset of integrins in a cell fail to bind their ECM ligands (Stupack et al, 2001;Kim et al, 2002). For example, expression of avb3 or a5b1 can inhibit cell survival in cells attached to the matrix through other integrins (Stupack et al, 2001;Kim et al, 2002). The expression of avb3 inhibits cell survival in cells attached to native collagen through integrin a2b1 (Stupack et al, 2001). As integrin avb3 does not bind native collagen, these results indicate that the unligated integrin avb3 induces cell death. In a similar manner, inhibition of integrin a5b1 activity with antibody antagonists induces apoptosis of endothelial cells that are attached to vitronectin through av integrins (Kim et al, 2002). In addition, expression of dominant negative integrins (e.g., Tac-b3, the IL-2 receptor fused with the integrin beta 3 subunit cytoplasmic tail) also inhibits survival by impairing normal integrin-mediated survival signalling (Stupack et al, 2001). Integrin ligation suppresses caspase 8 activation, while unligated integrins facilitate caspase 8 activation in a stress response and death receptor independent manner (Stupack et al, 2001;Kim et al, 2002). Additional studies suggest that unligated integrins activate membrane-associated protein kinase A (PKA), which itself can activate caspase 8 in endothelial cells (Kim et al, 2002). Thus, in normal cells, some integrins regulate survival when ligated and induce apoptosis when unligated.

INTEGRIN ROLES IN CELL MIGRATION
While integrin ligation by the ECM positively regulates migration, antagonising integrins inhibits cell migration. Although blocking integrin ligation can prevent cell attachment to the ECM and thus inhibit migration, recent studies show that antagonised integrins actively inhibit signal transduction leading to cell migration (Kim et al, 2000b). For example, the inhibition of integrin a5b1 negatively regulates fibroblast, endothelial cell and tumour cell migration even when other integrin receptors for provisional matrix proteins are ligated (Kim et al, 2000b). Antagonists of integrin a5b1 suppress cell migration on vitronectin, but not cell attachment to vitronectin, indicating that these antagonists affect the migration machinery rather than integrin receptors for vitronectin (Kim et al, 2000b). In fact, a5b1 antagonists activate PKA, which then inhibits cell migration by disrupting the formation of stress fibres (Kim et al, 2000b). Direct activation of PKA by forskolin or by overexpression of the catalytic, active subunit of PKA also inhibits cell migration Kim et al, 2000b). Thus, integrins regulate cell migration by making contact with the substratum and by promoting signal transduction cascades that support migration.

INTEGRINS REGULATE ANGIOGENESIS
Angiogenesis is the process by which new blood vessels develop from pre-existing vessels. The growth of new blood vessels promotes embryonic development, wound healing and the female reproductive cycle, and also plays a key role in the pathological development of solid tumour cancers, haemangiomas, diabetic retinopathy, age-related macular degeneration, psoriasis, gingivitis, rheumatoid arthritis and possibly osteoarthritis and inflammatory bowel disease (reviewed in Carmeliet and Jain, 2000). New advances in understanding the mechanisms regulating angiogenesis, such as those that promote cell migration and invasion, are leading to the development of novel therapeutics for cancer.
Growth factors released by hypoxic tissues or pathological tissues such as tumours stimulate new blood vessel growth. New vessels grow by sprouting from pre-existing vessels (reviewed in Carmeliet and Jain, 2000) or by recruitment of bone marrowderived endothelial progenitor cells (Asahara et al, 1997). While growth factors and their receptors play key roles in angiogenic sprouting, adhesion to the ECM also regulates angiogenesis ( Figure 2). Adhesion promotes endothelial cell survival (Kim et al, 2002;Stupack and Cheresh, 2002), as well as endothelial cell proliferation and motility (Kim et al, 2000a, b) during new blood vessel growth. One ECM protein in particular, fibronectin, is associated with vascular proliferation (Kim et al, 2000a, b); it is expressed in provisional vascular matrices and provides proliferative signals to vascular cells during wound healing, atherosclerosis and hypertension. Notably, fibronectin-null mice die early in development from a collection of defects, which include an   Figure 2 Integrin family. Integrin alpha beta heterodimers can be grouped into three subfamilies. Integrins a1b1, a2b1, a5b1, a4b1, avb3 and avb5 (highlighted in blue) have been shown to play important roles in regulating tumour angiogenesis.
improperly formed vasculature (George et al, 1993(George et al, , 1997. Recent experimental studies showed that fibronectin regulates angiogenesis, as antibody inhibitors of fibronectin block angiogenesis (Kim et al, 2000a). Studies in experimental angiogenesis models and in mutant mice indicate that several integrins play key roles in regulating angiogenesis. Embryonic deletion of integrin a5b1 induces early mesenchymal abnormalities, which include defects in the organisation of the emerging vasculature (Yang et al, 1993;Goh et al, 1997) and defects in the ability of endothelial cells to form vessellike structures ex vivo (Taverna and Hynes, 2001;Francis et al, 2002). Similarly, loss of integrin a4b1 leads to aorta, heart and other vascular malformations (Yang et al, 1995). Deletion of the av subunit causes 80% of embryos to die early in development from uncertain causes, while the few surviving embryos die a few hours after birth with significant defects in brain development, including failure of blood vessels to form properly (Bader et al, 1998). In contrast, individual loss of the b3orb5 subunit during embryogenesis does not cause noticeable defects in the formation of the cardiovascular system (Hodivala-Dilke et al, 1999;Huang et al, 2000). In fact, one study show that loss of the b3 or the b3 and b5 subunits promotes tumour angiogenesis (Reynolds et al, 2002). These studies led to the controversial conclusion that the avb3/ avb5 integrins are not required for angiogenesis, but instead may suppress angiogenesis. As many studies have shown that avb3 and avb5 inhibitors block angiogenesis by inducing apoptosis in proliferating endothelial cells (Brooks et al, 1994b), it is possible that loss of the integrin-mediated death mechanism can lead to enhanced angiogenesis (Cheresh and Stupack, 2002). In fact, loss of either the b3 or b5 subunits does block angiogenesis induced by the angiomatrix protein Del-1 (Zhong et al, 2003). Thus, integrins appear to have diverse roles in the establishment of the cardiovascular system, with integrin a5b1 clearly playing a major role during development of the vascular system.
Studies in experimental models of angiogenesis also indicate that several integrins can play important roles in regulating angiogenesis in normal animals. The expression of both integrins avb3 (Brooks et al, 1994a) and a5b1 (Kim et al, 2000a) are significantly upregulated on endothelium during angiogenesis. The expression of integrins avb3 and a5b1 partially controls angiogenesis; neither integrin is expressed by quiescent endothelium and both are expressed in response to angiogenic growth factors (Brooks et al, 1994a;Kim et al, 2000a, b). Their expression is controlled by the transcription factor Hox D3 (Boudreau et al, 1997;Boudreau and Varner, 2003;Zhong et al, 2003). Hox D3 is a Homeobox gene expressed by ECs that may regulate an angiogenic switch. When expressed in vivo, Hox D3 promotes a haemangioma-like proliferation of blood vessels (Boudreau et al, 1997;Zhong et al, 2003); this transcription factor promotes the expression of integrin avb3, a5b1 and uPA, molecules with established roles in angiogenesis. Thus, Hox D3 may provide a switch to activate a program of angiogenesis. Once integrins a5b1 and avb3 are expressed, angiogenesis depends on each integrin as antagonists of each can block angiogenesis in vivo (Brooks et al, 1994a, b;Kim et al, 2000a). Antibody and peptide antagonists of integrins avb3 and a5b1 inhibit growth factor as well as tumour angiogenesis, tumour growth and tumour metastasis (Brooks et al, 1994a, b;Carron et al, 1998;Kim et al, 2000a;Stoeltzing et al, 2003). These studies indicate that these integrins function in part by promoting survival in proliferating endothelial cells in vivo (Brooks et al, 1994b;Kim et al, 2002). Studies of the signals transduced when integrins are antagonised indicate that unligated integrins activate PKA, which then activates caspase 8 and induces apoptosis Kim et al, 2002).
In addition, other integrins have been shown to regulate angiogenesis. Integrin avb5 promotes VEGF-, but not bFGF-, mediated angiogenesis (Friedlander et al, 1995). Integrin receptors for laminin and collagen also play roles in regulating blood vessel formation as antagonists of a2b1 and a1b1 suppressed VEGFmediated angiogenesis (Senger et al, 1997). Thus, integrins play key roles in regulating tumour angiogenesis, and integrin antagonists hold promise as future therapeutics for cancer.

INTEGRINS PLAY ROLES IN TUMOUR INVASION AND METASTASIS
Tumour metastasis promotes the spread of tumours to local and distant sites away from primary tumours. Metastasis is the leading cause of the morbidity and mortality associated with cancer. Tumour cells isolated from metastases are highly migratory and invasive. Therefore, understanding the mechanisms regulating cell migration may be helpful in developing new modes of therapy for metastatic cancer.
Increased levels of expression of integrins avb3 is closely associated with increased cell invasion and metastasis (Felding-Habermann et al, 2002). Notably, integrin avb3 is expressed on invasive melanoma but not benign nevi or normal melanocytes (Gehlsen et al, 1992). Additionally, increased avb3 expression levels correlate with increased rates of melanoma metastases (Nip et al, 1992).
Integrin a6 expression is also significantly upregulated in numerous carcinomas, including head and neck cancers and breast cancer (Garzino-Demo et al, 1998;Mercurio et al, 2001;Ramos et al, 2002). Integrin a6b4 expression enhances tumour cell invasiveness and metastasis, particularly in breast carcinomas (Mercurio et al, 2001;Ramos et al, 2002). Thus, antagonists of these integrins may be useful to prevent the spread of tumour cells.

INTEGRIN INHIBITORS AS THERAPEUTIC AGENTS FOR CANCER
Several integrin inhibitors are currently under investigation as therapeutics for cancer. Antibody and peptide inhibitors of integrins avb3 and avb5 (for review, see Kerr et al, 2002) and of a5b1 are currently in clinical trials for the inhibition of angiogenesis in cancer. A humanised anti-avb3 antibody, Vitaxin, is currently in Phase II trials for cancer (Gutheil et al, 2000;Patel et al, 2001;Posey et al, 2001;Mikecz, 2000), while a humanised anti-a5b1 antibody is in Phase I trials for cancer (Varner, personal communication; www.pdl.com). A cyclic peptide inhibitor of integrin avb3/avb5, Cilengitide, is in Phase I/II trials for glioblastoma and other cancers (Burke et al, 2002;Eskens et al, 2003;Smith, 2003). Other promising integrin a5b1and avb3-blocking peptides with antitumour angiogenesis and tumour metastasis activities are currently in preclinical development (Carron et al, 1998;Reinmuth et al, 2003;Stoeltzing et al, 2003). As Avastin, the antibody inhibitor of VEGF, has recently shown promise as a therapeutic for colon cancer in Phase III clinical trials (Fernando and Hurwitz, 2003), these integrin-based antiangiogenesis therapeutics hold great promise as powerful therapeutics for the treatment of cancer.

CONCLUSION
The studies reviewed here indicate that integrin promote cellular migration and survival in tumour and primary cells. Antagonists of integrins avb3, a5b1, avb5 and a6b4 show great promise as potential inhibitors of tumour growth and metastasis as well as tumour angiogenesis. Clinical trials are currently underway to evaluate inhibitors of integrin avb3, avb5 and a5b1 for their usefulness in the treatment of cancer.