Role Of Integrins In Apoptosis

Normal cell growth (net sum of cell proliferation and cell death) is a consequence of signals generated by integrin-ligand interactions and soluble growth factors. Deprivation of growth factors and hormones leads to apoptosis. Similarly, disruption of cell-matrix interactions induces apoptosis, in this case termed anoikis (108). Anoikis can be viewed as a mechanism by which cells use positional information delivered from the extracellular matrix via integrin ligation in order to determine whether they are in an appropriate environment for growth while deprivation of normal cues results in programmed cell death. When cells metastasise they almost certainly encounter a foreign ECM milieu compared with the original site of growth and thus the metastatic process is likely to be at least partially due to a failure of the apoptotic pathway. For example the invading transformed melanocyte or epithelial cell must pass through their underlying dermis, which is composed of many different matrix proteins compared with the suprabasal environment, before intravasating into blood-vessels or lymph nodes and transferring to distant sites.

Subsequent extravasation and growth into a secondary tumour, particularly if it is in a different organ from the tissue of origin of the transformed cell, must occur in an environment of different ECM proteins and growth factors. Thus an understanding of how integrins control entry into anoikis or apoptosis may identify abnormalities and potentially therefore, novel targets for the therapy of cancer (Figure 3).

There are now many examples of matrix-suppressed apoptosis (38, 109). Zhang et al have shown that anoikis is suppressed in Chinese hamster ovary cells ligated to fibronectin via a5pi and that this was associated with upregulation of Bcl-2 expression. This response to fibronectin also was shown to be integrin specific since cells expressing an alternative fibronectin receptor, underwent apoptosis when plated on fibronectin (38). Montgomery et al reported that expression of avP3 protected melanoma cells from apoptosis when growing in collagen gels (110). These data may have been related to the observation that ligation of is associated with down-regulation of the cyclin-dependent kinase inhibitor p21WAF" and also p53 (111). Certainly it is consistent with the activation of the p53-p21waf-i/cip-i pathway following disruption of cell-matrix interactions (112). However, de novo or increases in integrin expression does not invariably lead to protection from apoptosis. For example, expression of integrins in colon carcinoma cells has been shown to induce expression ofp21WAF"1/CIIM leading to growth arrest and apoptosis (113). Such results underlie the fact that integrin-mediated regulation of anoikis is complicated and, at present, imperfectly understood. However signalling events involved in integrin-dependent protection against apoptosis are being unravelled slowly.

Phosphoinositol 3-kinase (PI3K) has been implicated in protecting cells from entering adhesion dependent apoptosis (anoikis). Thus when MDCK cells express constitutively active Ras, protection from apoptosis was due to PI3K dependent activation of Akt/Protein kinase B promoting downstream blockade of apoptosis (71). In normal MDCK cells, adhesion via integrins activates PI3K although whether any of the other known pathways for activation of PI3K are involved is not clear. For example Shc can be activated by ligation of a variety of integrins (see above) resulting in activation of Ras. PI3K is a substrate for Ras (114) thus perhaps Shc could regulate PI3K activation upon adhesion. Alternatively PI3K may be activated subsequent to binding to the phosphorylated Y397 residue of FAK which is phosphorylated when integrins cluster(see above). In contrast to PI3K-dependent protection against anoikis, activation of the JNK/SAPK (stress activated protein kinase) pathway may promote anoikis (reviewed in (115).

Several groups have shown an involvement of focal adhesion kinase (FAK) in regulating anoikis. Xu et al showed that attenuation of FAK expression in tumour cells led to apoptosis (116) whereas expression of constitutively active FAK in MDCK cells blocked entry in to apoptosis even though cells were deprived of integrin ligation by being placed in suspension (88). Additionally anoikis was induced when peptides mimicking the FAK binding site on the cytoplasmic tail were microinjected into fibroblasts (84). Recently Ilic et al have shown that survival signals from the ECM, which are transduced by integrins via FAK, suppress a p53-regulated apoptotic pathway in fibroblasts and endothelial cells (117). This pathway was blocked by inhibitors of p53 including Bcl-2 but not by inhibitors of PI3K(117).

Moreover the capacity of the ECM to inhibit anoikis varied not only between substrates but also between different cell types. Thus fibronectin was equally effective at preventing anoikis in rabbit synovial fibroblasts (RSF) and mouse endothelial cells whereas vitronectin and laminin, though very effective for endothelial cells were very poor at preventing anoikis in RSF while adhesion to collagen was not an effective protection against anoikis for either cell type (117). Since each of these substrates are recognised by different groups of integrins these data further show that protection against anoikis is dependent not only upon which specific proteins in the matrix are present but which integrins are expressed.

Small peptides containing the RGD binding motif have been used extensively in the study of the role of integrins in tumour progression and metastasis. Co-injection of small peptides, which include the RGD motif, can significantly impair the lung colonising ability of melanoma cells (118) as well as reduce the formation of spontaneous metastases (119). The principal mechanism for these results was believed to be as a consequence of the ability of RGD peptides to disrupt integrin-ligand interactions and therefore prevent integrin-dependent processes including adhesion and migration. Although these conclusions may have been true, recent data from Buckley et al have demanded that we may need to interpret data generated by use of RGD peptides a little more cautiously (120). It seems that RGD peptides can promote apoptosis by entering cells in an integrin-independent manner and directly activating pro-apoptotic enzyme caspase 3 (120). The authors propose that the procaspase has an RGD-dependent conformational restraint that prevents it from becoming cleaved into the active form of the enzyme. RGD peptides entering the cell disrupt this intramolecular interaction and allow activation to take place. These results do not exclude a role for integrins in apoptosis since anti-integrin antibodies that disrupt ligand interactions also promote apoptosis (121) and attachment to anti-integrin antibodies but not nonspecific antibodies, prevents apoptosis in human umbilical vein endothelial cells (122).

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