- Email: [email protected]
Integrins and anoikis
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Integrins and anoikis Steven M Frisch* and Erkki Ruoslahtit The loss of integrin-mediated cell-matrix contact induces apoptosis ('anoikis') in certain cell types. Recently it has been shown that protein kinase signaling pathways control anoikis both positively and negatively. Focal adhesion kinase, when activated by integrins, can suppress anoikis. Phosphatidylinositol 3-kinase and the AKT oncoprotein may mediate the anoikis-suppressing effects of focal adhesion kinase. Conversely, the stress-activated protein kinase/Jun amino-terminal kinase pathway promotes anoikis. Latest results indicate that caspase-mediated cleavage of the first component of this latter pathway, MEKK-1, may trigger activation of this pathway in anoikis. In addition, certain integrins may regulate bcl-2 expression levels, possibly adjusting the threshold for anoikis.
Addresses The Burnham Institute, La Jolla Cancer Research Center, 10901 N Torrey Pines Road, La Jolla, CA 92037, USA *e-mail: [email protected] re-mail: [email protected] Current Opinion in Cell Biology 1997, 9:701-706
http://biomednet.com/elecref/O955067400900701 © Current Biology Ltd ISSN 0955-0674 Abbreviations ERK extracellularsignal regulated kinase FAK focal adhesion kinase JNK Jun amino-terminal kinase MAP mitogen-activatedprotein PI3K phosphoinositide3-kinase
Introduction
Fibroblasts dissociated from their extracellular matrix undergo reversible growth arrest, thereby revealing their anchorage dependence [1]. Certain other cell types were recently found to be anchorage-dependent for a different reason. When displaced from the extracellular matrix, epithelial [2] and endothelial [3] cells undergo apoptosis ('anoikis'; see [4,5] for earlier reviews). Anoikis in vivo may prevent detached cells from reattaching to new matrices and growing dysplastically; this could be an important safeguard for the organism. In neoplastic cells, alterations in cell-cell adhesion molecules, integrins, integrinassociated signaling molecules or apoptosis regulators can lead to anoikis resistance, facilitating anchorage-independent growth. In this review, we summarize the main features of the anchorage-dependent apoptosis program and the current understanding of its molecular mechanisms. Anoikis
Anchorage-related apoptosis was discovered in epithelial [2] and endothelial [3] cells that were experimentally
dissociated from their extracellular matrix. Cell/nuclear morphology, internucleosomal DNA cleavage and nuclear lamin cleavage all indicated apoptosis rather than necrosis. Also, overexpression of bcl-2 protein protected MDCK cells from this cell death [2]; this information later provided an important insight into the mechanism of cell death. This apoptosis could be induced by various dissociation methods and the cells' commitment to it could be reversed by re-plating them within a short time of suspension. T h e new term 'anoikis' - - the ancient Greek word for 'homelessness'--was coined [2] to denote the apoptosis that occurs in cells that are detached from matrix (or that are attached via the wrong molecules). T h e existence of anoikis implies that integrin signaling regulates critical components of the apoptotic machinery, because integrins are the major matrix receptors in vivo. Anoikis has also been documented in vivo. It occurs in normal skin [6], in colonic epithelial tissues [7], and in the involuting mammary gland [8]. Interestingly, it is also important developmentally in the first cavitation step of embryogenesis [9]. T h e significance of anoikis for epithelial and endothelial cells is fairly straightforward to understand; presumably anoikis prevents these cells from colonizing elsewhere when detached. However, other cell types have curiously been found to display anoikis as well, namely, skeletal muscle cells [10], certain melanoma cell variants of low tumorigenic potential [11] and embryonic fibroblasts [12]. T h e normal or pathological significance of anoikis in these cell types is more subtle. R o l e o f t h e i n t e g r i n t y p e in a n o i k i s
Attachment of endothelial cells to surfaces coated with fibronectin or anti-lSl-integrin antibodies--but not to surfaces coated with polylysine--rescued the cells from anoikis [3]; this implied a specific requirement for integrin signaling. However, even polylysine, or any other surface on which cell spreading occurred, rescued MDCK cells (S Frisch, unpublished data). This promiscuity appeared to result from the de novo synthesis of matrix molecules by the MDCK cells followed by integrin-mediated attachment, and does not imply that non-integrin-mediated attachment can rescue these cells. Indeed, in certain cell types, only specific ligated integrins suppress anoikis. For example, mammary epithelial cells die when attached to fibronectin or collagen matrices, but live on basement membrane [8]. The use of specific (x-subunit-blocking antibodies confirmed that only specific (x integrins suppressed anoikis, even though other integrins could mediate attachment [13]. Similar results were obtained from study of CHO cells,
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where the ectopic expression of ¢x51~1 integrin, but not av~l integrin, suppressed anoikis of fibronectin-bound cells under serum-free conditions [14]; a privileged role for ot51~l integrin was also shown in endothelial cells [15°]. MG-63 human osteosarcoma cells were similarly protected on fibronectin (by oc5131 integrin) but not on vitronectin (the vitronectin receptor is ave3 integrin) [14]. Serum-starved HT29 carcinoma cells readily underwent apoptosis, which was partially suppressed by transfection of the ¢x5 integrin subunit [16]. Angiogenic endothelial cells are particularly dependent on the ¢xvl33 integrin in vivo and can be induced to undergo apoptosis by treatment with antagonists of this integrin [17]. The same antagonists can also induce apoptosis in melanoma cells [11]. Likewise, the importance of a particular integrin is also implied in myotubes, where anoikis is suppressed by merosin, but not by the related laminin-1 [10]. Finally, an unusual role of the cx61M integrin - - unique in that it interacts with cytokeratin intermediate filaments rather than actin microfilaments - - has begun to emerge. Transfection of the full-length 134 molecule, but not a truncated form lacking the cytoplasmic domain, into the cell line RKO induced apoptosis [18]. This raises the possibility that this particular integrin uses unique signaling pathways. T h e differing abilities of specific integrins to rescue cells from anoikis suggests that they utilize distinct signaling pathways. Some distinctions have become apparent recently. For example, cxv~3 integrin uniquely associates with insulin receptor substrate-1 (IRS-1), linking it to the growth-regulated pathways that are mediated by insulin, insulin-like growth factor (IGF) and plateletderived growth factor (PDGF) [19,20]. Interestingly, the ~5131, cxvl~3 and ocll~l integrins, but not some other integrins, activate tyrosine phosphorylation of the adaptor protein Shc and through this pathway possibly activate mitogen-activated protein (MAP) kinases [15"]. All of the above experiments were conducted with only limited cell types. T h e results leave open the possibility that the same integrin might connect to different pathways in different cell types and that a given integrin could, therefore, provide a survival signal in one cell type and an anoikis signal in another. What does seem clear is that integrins differ in their ability to suppress anoikis in a single cell type. Perhaps tumor cells often express aberrant integrin types (reviewed in [21]) to evade anoikis. Clearly, much more work needs to be done to understand integrin-specific signaling and its role in anoikis. R o l e of cell s h a p e in a n o i k i s Cell detachment or attachment to a different kind of matrix causes a profound change in cell shape. Therefore, does the correct cytoskeletal organization--as opposed to integrin signaling per s e - - suppress anoikis? This question can be resolved easily by positing that cytoskeletal organization is a component of integrin signaling.
For example, endothelial cells that were placed in suspension underwent anoikis [22]. Interestingly, the addition of microbeads coated with RGD peptides (single-letter code for amino acids; the RGD sequence is found in the integrin-binding site within many matrix proteins [23]) that were too small to permit cell spreading did not rescue these cells. Larger-than-cell-size plastic surfaces coated with RGD peptides, however, rescued efficiently [22]. These data indicate that the mere attachment of cells to the m a t r i x - - e v e n through integrins--is insufficient for rescue from anoikis, and that cell shape changes are required. However, cytochalasin treatment, which prevents cytoskeletal reorganization, blocked certain integrin-mediated signaling events, such as autophosphorylation of focal adhesion kinase [24]. T h e role of cell shape in anoikis was recently explored in a system that allows the total surface area available for cell attachment to remain constant, while forcing the cell either to remain round or to spread out [25°°]. This was accomplished by the use of microfabricated surfaces composed of islands of adhesive surface interspersed at varying distances from one another. When endothelial cells were cultured on these surfaces, round cells were found to be susceptible to apoptosis, whereas cells forced to stretch over several widely dispersed small islands survived and proliferated [25°°]. Thus, rescue from anoikis involves the cooperation of integrin signaling molecules and the cytoskeleton. It is seemingly more difficult to explain the integrin specificity of anoikis on the basis of cytoskeletal changes, because there are no noticeable differences in the shapes of the cells destined to die through apoptosis and those that will survive in the CHO system described above [14]. However, a thorough comparison of these cytoskeletons might, in principle, reveal important differences in, for example, microtubules (which can control apoptosis [26°]). Resolution of the specific role of cell shape, and of how tumor cells circumvent it to become anoikis-resistant, obviously remains a challenge. Role of cell-cell interactions in a n o i k i s In the MDCK system, cells engaged in cell--cell interactions are sensitive to anoikis, whereas cells in sparse cultures are not, suggesting that cell--cell interactions sensitize cells toward anoikis [2]. Thus, the breakdown of these interactions might contribute to neoplasia (reviewed in [27]) by conferring anoikis resistance. Identifying which cell adhesion molecules are involved and how they signal will add an important new dimension to the study of anoikis. It is tempting to speculate that cadherin-catenin complexes might be involved, given the role of ~-catenin in signaling pathways involving the wnt-1 oncoprotein and the adenomatous polyposis coil tumor suppressor protein (reviewed in [28]). In other cell systems, cell-cell adhesion was suggested to play a different role. Treatment of LIM 1863 colon
Integrins and anoikls Frisch and Ruoslahti
carcinoma cells with antibodies against the integrin a v subunit inhibits cell--cell aggregation and induces apoptosis [29]. T h e authors propose that cell-cell interactions are mediated by a v integrin in this system, and conclude that the breakdown of these interactions triggers apoptosis, a conclusion that is seemingly at odds with the results from M D C K cells cited above. However, it is not clear whether the interactions in the LIM 1863 cell culture represent physiological cell--cell interactions or an artifactual aggregation phenomenon in which a v integrin bridges neighboring cells by binding to small matrix fragments. Thus, an alternative interpretation is simply that the antibodies directly induced anoikis in this system.
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Figure 1
Role of focal adhesion kinase in anoikis Focal adhesion kinase (FAK) is a good candidate for a mediator of protection from anoikis. FAK becomes activated by autophosphorylation upon integrin-mediated cell attachment and is thought to initiate a signaling cascade that, in fibroblasts, leads to the activation of the MAP kinases extracellular signal regulated kinase (ERK)-I and ERK-2 [30]. Development of a constitutively activated form of FAK made it possible to test the role of FAK in anoikis. Fusion of FAK to the ectodomain and transmembrane domain of the CD2 antigen targeted it to the plasma membrane, resuiting in constitutive activation of FAK [31]. Specifically, FAK's autophosphorylation and src-interaction functions, which are normally active in only matrix-attached cells, were found to remain active in the CD2-FAK chimeric proteins expressed in suspended cells [31,32,33°°]. T h e C D 2 - F A K chimeras substantially protected M D C K cells from anoikis [33"°], demonstrating the importance of FAK in regulating adhesion-dependent cell survival; this conclusion is supported by peptide microinjection experiments where the inactivation of FAK caused apoptosis [12]. Interestingly, the M D C K cells that expressed C D 2 - F A K also became somewhat anchorage-independent and tumorigenic [33"']. This transformation appeared to result primarily from rescue from anoikis rather than from activation of growth factor response pathways. Thus, C D 2 - F A K produces a transformed phenotype that is quite commonly found in actual human carcinoma cells: the cells display normal morphology and growth rate, but are tumorigenic. This underscores the importance of anoikis resistance as a parameter in human carcinogenesis. There may be many other oncoproteins that, like activated FAK, transform human epithelial cells by activating integrin signal transduction. This might be exploited to control cancer, as has been accomplished in rhabdomyosarcoma cells, where downregulation of FAK by antisense oligonucleotides induced apoptosis [34].
Role of AKT and phosphoinositide 3-kinase in anoikis Events downstream of FAK that lead to rescue from anoikis were less clear until a recent study implicated
P_xtracellular matrix © 1997CurrentOpinionin CellBiology Pathways implicated in anoikis, with arrows indicating information flow but not necessarily stimulation. At the left is shown a pathway in which FAK becomes activated in response to cell-matrix interaction. This pathway is initiated by integrin-mediated cell attachment to the extracellular matrix. Upon cell attachment, FAK becomes autophosphorylated. This leads to the activation of PI3K and then of the kinase AKT, suppressing anoikis by unknown mechanisms. An apparently separate (S Frisch, unpublished data) pathway is shown at the right. The dissociation of cells from the extracellular matrix leads to the activation of initiator and effector caspases, which cleave and thereby activate MEKK-1 (MAP kinase/ERK kinass kinase-1). MEKK-1 may then activate .INKs and perhaps other substrates, leading via unknown effectors to anoikis. A positive feedback loop in this pathway may exist, whereby MEKK-1 activates caspases which activate MEKK-1. Bcl-2 has been shown to inhibit anoikis, and it may achieve this by suppressing the activation of caspases.
the involvement of the FAK- and ras-interaction partner phosphoinositide 3-kinase (PI3K [35"']) (see Figure 1). Activation of ras or adhesion of M D C K cells was shown to activate this enzyme, and the resulting lipid products activated the kinase AKT. Interestingly, introduction of activated forms of the PI3K p l l 0 subunit, or of AKT, rescued the M D C K cells from anoikis. This was consistent with the ability of non-raf-interacting ras mutants, but not non-PI3K-interacting mutants, to rescue cells. It was also consistent with AKT's anti-apoptotic function in a different system, serum-starved fibroblasts that overexpress c-myc [36]. Activated ras was also shown to promote the interaction of FAK with PI3K but not the phosphorylation of FAK [35"']. T a k e n together, these data strongly suggest that ras or FAK controls anoikis through
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PI3K, which in turn regulates kinases such as AKT. It will be interesting to identify the relevant substrates for AKT.
Role of bcl-2 and related proteins The expression levels of bcl-2, bax and related proteins may determine the sensitivity of cells to apoptosis (reviewed in [37]). Consistent with this, bcl-2 levels were found to decrease to undetectable levels when MDCK became confluent (i.e. sensitive to anoikis [32]), suggesting that the bcl-2 gene may be regulated by cell--cell interactions. Surprisingly, individual integrins can also regulate bcl-2 levels in CHO cells stably transfected with integrins [14]. Cells attached through (x5131 integrin (and thus capable of survival) expressed bcl-2 at detectable levels, whereas cells that were attached through other integrins and were destined for anoikis did not. Moreover, transfection of bcl-2 into the cells lacking (x5131 integrin prevented the anoikis response [14]. Similarly, the blockage of endothelial cell (xv~3 integrin with antibodies specifically caused the activation of p53 and the downregulation of bcl-2 [38]; the subsequent decrease in the bcl-2 : bax ratio was proposed to induce apoptosis. It will be interesting to identify the integrin signal transduction events that regulate the bcl-2 promoter. Recently, anticancer drugs such as taxol that perturb microtubules were shown to dramatically stimulate the phosphorylation of bcl-2, thus inhibiting its ability to neutralize bax and suppress apoptosis [26°]. This raises the unexplored possibility that integrins might control microtubule structure or dynamics so as to regulate bcl-2 function and anoikis. Jun a m i n o - t e r m i n a l k i n a s e s a n d t h e bcl-2/caspase connection The Jun amino-terminal kinases (JNKs) are a family of MAP kinase related serine/threonine kinases that respond to a variety of 'stress-inducing' stimuli, such as UV or y irradiation, ceramide, tumor necrosis factor-(x and interleukin-1 (reviewed in [39,40]). The apoptosis-inducing activity of JNK is counteracted by ERK, and the balance between the activation of these pathways has been proposed to control cell survival versus apoptosis [41].
Recently, the dissociation of epithelial cells from the extracellular matrix was found to rapidly and dramatically induce the activity of the JNKs as well [32] (see Figure 1). Furthermore, the blockage of the JNK pathway by a dominant-negative form of JNK kinase partially inhibited anoikis. Thus, the JNK pathway also plays a critical role in anoikis. These results provide a possible mechanism for the suppression of anoikis by bcl-2. The overexpression of bcl-2--which had previously been shown to suppress anoikis--suppresses the activation of caspases involved
in anoikis [32] and other apoptotic systems (e.g. [42"]). Caspase activity is required for the activation of the JNK pathway [32] (see Figure 1); this may result from the requirement of the upstream kinase MAP kinase/ERK kinase kinase-1 (MEKK-1) to be activated by caspase-mediated cleavage [43"]. This suggests that bcl-2 may suppress anoikis in part by suppressing JNK activation. Thus, the signals regulating anoikis appear to flow from the integrins through bcl-2-related proteins to caspases, then to MEKK-1 and JNK. T h e MEKK/JNK pathway and caspases also communicate in a positive feedback loop, because blockage of MEKK/JNK inhibits caspases and blockage of caspases inhibits MEKK/JNK [43"]. The MEKK substrates or JNK substrates that mediate anoikis remain to be identified, but c-jun itself should be considered, because it can induce apoptosis when overexpressed in at least one system [44"]; activation of the I~B(x kinase complex by MEKK-1 [45] might also be important. An alternative way for bcl-2 to block anoikis has been proposed [46"]. In the prostate carcinoma cell line LNCaP, bcl-2 overexpression suppressed the accumulation of the cyclin dependent kinase inhibitors p21 and p27. Presumably because of this, the retinoblastoma protein (Rb) remained in the inactive, hyperphosphorylated state even in suspended cells. In the absence of active Rb, LNCaP cells were resistant to anoikis. Thus, bcl-2 might directly prevent the Rb-mediated cell cycle arrest that is required for the induction of anoikis in this system.
Future prospects Clearly, a substantial amount of experimental work will be needed to understand how integrin signaling and apoptotic signaling are integrated. New methodology might be required to analyze the possible scaffolding of both classes of transducer by the cytoskeleton, which may play a key role in anoikis. In the immediate future, there are several particularly compelling candidate integrating molecules to explore, including the following: first, integrin-linked kinase, a protein kinase that binds to the integrin 1~1 subunit [47]; second, PYK-2, a FAK-related molecule that regulates the JNK pathway [48]; third, rho, rac and cdc42, ras-superfamily members ~J~at are involved in cytoskeletal organization [49]; and fourth, death-domain-containing receptors [50], whose functions may somehow be regulated by integrin signaling. It will also be interesting to systematically compare the effects of the various integrin types in anoikis, in the hope of understanding how tumor cells might evade anoikis by expressing certain integrins. Finally, many observations cited above suggest that a cell must have a fairly extensive epithelial transcription program in order to be sensitive to anoikis. This program is
Integrins and anoikis Frisch and Ruoslahti
apparently reinforced by cell-cell interactions and the loss of the program may contribute to carcinoma progression. In this light, it will be important to analyze the transcriptional mechanisms by which the epithelial transcription program is established in order to understand anoikis more fully.
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Acknowledgements This work was partially supported by the following grants from the National Institutes of Health: GM 51452 (to SM Frisch), and CA 62042, CA 28896, and CA 74238 (to E Ruoslahti). Our work was also supported by Cancer Center Support Grant CA30199.
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Integrins and anoikis Steven M Frisch* and Erkki Ruoslahtit The loss of integrin-mediated cell-matrix contact induces apoptosis ('anoikis') in certain cell types. Recently it has been shown that protein kinase signaling pathways control anoikis both positively and negatively. Focal adhesion kinase, when activated by integrins, can suppress anoikis. Phosphatidylinositol 3-kinase and the AKT oncoprotein may mediate the anoikis-suppressing effects of focal adhesion kinase. Conversely, the stress-activated protein kinase/Jun amino-terminal kinase pathway promotes anoikis. Latest results indicate that caspase-mediated cleavage of the first component of this latter pathway, MEKK-1, may trigger activation of this pathway in anoikis. In addition, certain integrins may regulate bcl-2 expression levels, possibly adjusting the threshold for anoikis.
Addresses The Burnham Institute, La Jolla Cancer Research Center, 10901 N Torrey Pines Road, La Jolla, CA 92037, USA *e-mail: [email protected] re-mail: [email protected] Current Opinion in Cell Biology 1997, 9:701-706
http://biomednet.com/elecref/O955067400900701 © Current Biology Ltd ISSN 0955-0674 Abbreviations ERK extracellularsignal regulated kinase FAK focal adhesion kinase JNK Jun amino-terminal kinase MAP mitogen-activatedprotein PI3K phosphoinositide3-kinase
Introduction
Fibroblasts dissociated from their extracellular matrix undergo reversible growth arrest, thereby revealing their anchorage dependence [1]. Certain other cell types were recently found to be anchorage-dependent for a different reason. When displaced from the extracellular matrix, epithelial [2] and endothelial [3] cells undergo apoptosis ('anoikis'; see [4,5] for earlier reviews). Anoikis in vivo may prevent detached cells from reattaching to new matrices and growing dysplastically; this could be an important safeguard for the organism. In neoplastic cells, alterations in cell-cell adhesion molecules, integrins, integrinassociated signaling molecules or apoptosis regulators can lead to anoikis resistance, facilitating anchorage-independent growth. In this review, we summarize the main features of the anchorage-dependent apoptosis program and the current understanding of its molecular mechanisms. Anoikis
Anchorage-related apoptosis was discovered in epithelial [2] and endothelial [3] cells that were experimentally
dissociated from their extracellular matrix. Cell/nuclear morphology, internucleosomal DNA cleavage and nuclear lamin cleavage all indicated apoptosis rather than necrosis. Also, overexpression of bcl-2 protein protected MDCK cells from this cell death [2]; this information later provided an important insight into the mechanism of cell death. This apoptosis could be induced by various dissociation methods and the cells' commitment to it could be reversed by re-plating them within a short time of suspension. T h e new term 'anoikis' - - the ancient Greek word for 'homelessness'--was coined [2] to denote the apoptosis that occurs in cells that are detached from matrix (or that are attached via the wrong molecules). T h e existence of anoikis implies that integrin signaling regulates critical components of the apoptotic machinery, because integrins are the major matrix receptors in vivo. Anoikis has also been documented in vivo. It occurs in normal skin [6], in colonic epithelial tissues [7], and in the involuting mammary gland [8]. Interestingly, it is also important developmentally in the first cavitation step of embryogenesis [9]. T h e significance of anoikis for epithelial and endothelial cells is fairly straightforward to understand; presumably anoikis prevents these cells from colonizing elsewhere when detached. However, other cell types have curiously been found to display anoikis as well, namely, skeletal muscle cells [10], certain melanoma cell variants of low tumorigenic potential [11] and embryonic fibroblasts [12]. T h e normal or pathological significance of anoikis in these cell types is more subtle. R o l e o f t h e i n t e g r i n t y p e in a n o i k i s
Attachment of endothelial cells to surfaces coated with fibronectin or anti-lSl-integrin antibodies--but not to surfaces coated with polylysine--rescued the cells from anoikis [3]; this implied a specific requirement for integrin signaling. However, even polylysine, or any other surface on which cell spreading occurred, rescued MDCK cells (S Frisch, unpublished data). This promiscuity appeared to result from the de novo synthesis of matrix molecules by the MDCK cells followed by integrin-mediated attachment, and does not imply that non-integrin-mediated attachment can rescue these cells. Indeed, in certain cell types, only specific ligated integrins suppress anoikis. For example, mammary epithelial cells die when attached to fibronectin or collagen matrices, but live on basement membrane [8]. The use of specific (x-subunit-blocking antibodies confirmed that only specific (x integrins suppressed anoikis, even though other integrins could mediate attachment [13]. Similar results were obtained from study of CHO cells,
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where the ectopic expression of ¢x51~1 integrin, but not av~l integrin, suppressed anoikis of fibronectin-bound cells under serum-free conditions [14]; a privileged role for ot51~l integrin was also shown in endothelial cells [15°]. MG-63 human osteosarcoma cells were similarly protected on fibronectin (by oc5131 integrin) but not on vitronectin (the vitronectin receptor is ave3 integrin) [14]. Serum-starved HT29 carcinoma cells readily underwent apoptosis, which was partially suppressed by transfection of the ¢x5 integrin subunit [16]. Angiogenic endothelial cells are particularly dependent on the ¢xvl33 integrin in vivo and can be induced to undergo apoptosis by treatment with antagonists of this integrin [17]. The same antagonists can also induce apoptosis in melanoma cells [11]. Likewise, the importance of a particular integrin is also implied in myotubes, where anoikis is suppressed by merosin, but not by the related laminin-1 [10]. Finally, an unusual role of the cx61M integrin - - unique in that it interacts with cytokeratin intermediate filaments rather than actin microfilaments - - has begun to emerge. Transfection of the full-length 134 molecule, but not a truncated form lacking the cytoplasmic domain, into the cell line RKO induced apoptosis [18]. This raises the possibility that this particular integrin uses unique signaling pathways. T h e differing abilities of specific integrins to rescue cells from anoikis suggests that they utilize distinct signaling pathways. Some distinctions have become apparent recently. For example, cxv~3 integrin uniquely associates with insulin receptor substrate-1 (IRS-1), linking it to the growth-regulated pathways that are mediated by insulin, insulin-like growth factor (IGF) and plateletderived growth factor (PDGF) [19,20]. Interestingly, the ~5131, cxvl~3 and ocll~l integrins, but not some other integrins, activate tyrosine phosphorylation of the adaptor protein Shc and through this pathway possibly activate mitogen-activated protein (MAP) kinases [15"]. All of the above experiments were conducted with only limited cell types. T h e results leave open the possibility that the same integrin might connect to different pathways in different cell types and that a given integrin could, therefore, provide a survival signal in one cell type and an anoikis signal in another. What does seem clear is that integrins differ in their ability to suppress anoikis in a single cell type. Perhaps tumor cells often express aberrant integrin types (reviewed in [21]) to evade anoikis. Clearly, much more work needs to be done to understand integrin-specific signaling and its role in anoikis. R o l e of cell s h a p e in a n o i k i s Cell detachment or attachment to a different kind of matrix causes a profound change in cell shape. Therefore, does the correct cytoskeletal organization--as opposed to integrin signaling per s e - - suppress anoikis? This question can be resolved easily by positing that cytoskeletal organization is a component of integrin signaling.
For example, endothelial cells that were placed in suspension underwent anoikis [22]. Interestingly, the addition of microbeads coated with RGD peptides (single-letter code for amino acids; the RGD sequence is found in the integrin-binding site within many matrix proteins [23]) that were too small to permit cell spreading did not rescue these cells. Larger-than-cell-size plastic surfaces coated with RGD peptides, however, rescued efficiently [22]. These data indicate that the mere attachment of cells to the m a t r i x - - e v e n through integrins--is insufficient for rescue from anoikis, and that cell shape changes are required. However, cytochalasin treatment, which prevents cytoskeletal reorganization, blocked certain integrin-mediated signaling events, such as autophosphorylation of focal adhesion kinase [24]. T h e role of cell shape in anoikis was recently explored in a system that allows the total surface area available for cell attachment to remain constant, while forcing the cell either to remain round or to spread out [25°°]. This was accomplished by the use of microfabricated surfaces composed of islands of adhesive surface interspersed at varying distances from one another. When endothelial cells were cultured on these surfaces, round cells were found to be susceptible to apoptosis, whereas cells forced to stretch over several widely dispersed small islands survived and proliferated [25°°]. Thus, rescue from anoikis involves the cooperation of integrin signaling molecules and the cytoskeleton. It is seemingly more difficult to explain the integrin specificity of anoikis on the basis of cytoskeletal changes, because there are no noticeable differences in the shapes of the cells destined to die through apoptosis and those that will survive in the CHO system described above [14]. However, a thorough comparison of these cytoskeletons might, in principle, reveal important differences in, for example, microtubules (which can control apoptosis [26°]). Resolution of the specific role of cell shape, and of how tumor cells circumvent it to become anoikis-resistant, obviously remains a challenge. Role of cell-cell interactions in a n o i k i s In the MDCK system, cells engaged in cell--cell interactions are sensitive to anoikis, whereas cells in sparse cultures are not, suggesting that cell--cell interactions sensitize cells toward anoikis [2]. Thus, the breakdown of these interactions might contribute to neoplasia (reviewed in [27]) by conferring anoikis resistance. Identifying which cell adhesion molecules are involved and how they signal will add an important new dimension to the study of anoikis. It is tempting to speculate that cadherin-catenin complexes might be involved, given the role of ~-catenin in signaling pathways involving the wnt-1 oncoprotein and the adenomatous polyposis coil tumor suppressor protein (reviewed in [28]). In other cell systems, cell-cell adhesion was suggested to play a different role. Treatment of LIM 1863 colon
Integrins and anoikls Frisch and Ruoslahti
carcinoma cells with antibodies against the integrin a v subunit inhibits cell--cell aggregation and induces apoptosis [29]. T h e authors propose that cell-cell interactions are mediated by a v integrin in this system, and conclude that the breakdown of these interactions triggers apoptosis, a conclusion that is seemingly at odds with the results from M D C K cells cited above. However, it is not clear whether the interactions in the LIM 1863 cell culture represent physiological cell--cell interactions or an artifactual aggregation phenomenon in which a v integrin bridges neighboring cells by binding to small matrix fragments. Thus, an alternative interpretation is simply that the antibodies directly induced anoikis in this system.
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Figure 1
Role of focal adhesion kinase in anoikis Focal adhesion kinase (FAK) is a good candidate for a mediator of protection from anoikis. FAK becomes activated by autophosphorylation upon integrin-mediated cell attachment and is thought to initiate a signaling cascade that, in fibroblasts, leads to the activation of the MAP kinases extracellular signal regulated kinase (ERK)-I and ERK-2 [30]. Development of a constitutively activated form of FAK made it possible to test the role of FAK in anoikis. Fusion of FAK to the ectodomain and transmembrane domain of the CD2 antigen targeted it to the plasma membrane, resuiting in constitutive activation of FAK [31]. Specifically, FAK's autophosphorylation and src-interaction functions, which are normally active in only matrix-attached cells, were found to remain active in the CD2-FAK chimeric proteins expressed in suspended cells [31,32,33°°]. T h e C D 2 - F A K chimeras substantially protected M D C K cells from anoikis [33"°], demonstrating the importance of FAK in regulating adhesion-dependent cell survival; this conclusion is supported by peptide microinjection experiments where the inactivation of FAK caused apoptosis [12]. Interestingly, the M D C K cells that expressed C D 2 - F A K also became somewhat anchorage-independent and tumorigenic [33"']. This transformation appeared to result primarily from rescue from anoikis rather than from activation of growth factor response pathways. Thus, C D 2 - F A K produces a transformed phenotype that is quite commonly found in actual human carcinoma cells: the cells display normal morphology and growth rate, but are tumorigenic. This underscores the importance of anoikis resistance as a parameter in human carcinogenesis. There may be many other oncoproteins that, like activated FAK, transform human epithelial cells by activating integrin signal transduction. This might be exploited to control cancer, as has been accomplished in rhabdomyosarcoma cells, where downregulation of FAK by antisense oligonucleotides induced apoptosis [34].
Role of AKT and phosphoinositide 3-kinase in anoikis Events downstream of FAK that lead to rescue from anoikis were less clear until a recent study implicated
P_xtracellular matrix © 1997CurrentOpinionin CellBiology Pathways implicated in anoikis, with arrows indicating information flow but not necessarily stimulation. At the left is shown a pathway in which FAK becomes activated in response to cell-matrix interaction. This pathway is initiated by integrin-mediated cell attachment to the extracellular matrix. Upon cell attachment, FAK becomes autophosphorylated. This leads to the activation of PI3K and then of the kinase AKT, suppressing anoikis by unknown mechanisms. An apparently separate (S Frisch, unpublished data) pathway is shown at the right. The dissociation of cells from the extracellular matrix leads to the activation of initiator and effector caspases, which cleave and thereby activate MEKK-1 (MAP kinase/ERK kinass kinase-1). MEKK-1 may then activate .INKs and perhaps other substrates, leading via unknown effectors to anoikis. A positive feedback loop in this pathway may exist, whereby MEKK-1 activates caspases which activate MEKK-1. Bcl-2 has been shown to inhibit anoikis, and it may achieve this by suppressing the activation of caspases.
the involvement of the FAK- and ras-interaction partner phosphoinositide 3-kinase (PI3K [35"']) (see Figure 1). Activation of ras or adhesion of M D C K cells was shown to activate this enzyme, and the resulting lipid products activated the kinase AKT. Interestingly, introduction of activated forms of the PI3K p l l 0 subunit, or of AKT, rescued the M D C K cells from anoikis. This was consistent with the ability of non-raf-interacting ras mutants, but not non-PI3K-interacting mutants, to rescue cells. It was also consistent with AKT's anti-apoptotic function in a different system, serum-starved fibroblasts that overexpress c-myc [36]. Activated ras was also shown to promote the interaction of FAK with PI3K but not the phosphorylation of FAK [35"']. T a k e n together, these data strongly suggest that ras or FAK controls anoikis through
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PI3K, which in turn regulates kinases such as AKT. It will be interesting to identify the relevant substrates for AKT.
Role of bcl-2 and related proteins The expression levels of bcl-2, bax and related proteins may determine the sensitivity of cells to apoptosis (reviewed in [37]). Consistent with this, bcl-2 levels were found to decrease to undetectable levels when MDCK became confluent (i.e. sensitive to anoikis [32]), suggesting that the bcl-2 gene may be regulated by cell--cell interactions. Surprisingly, individual integrins can also regulate bcl-2 levels in CHO cells stably transfected with integrins [14]. Cells attached through (x5131 integrin (and thus capable of survival) expressed bcl-2 at detectable levels, whereas cells that were attached through other integrins and were destined for anoikis did not. Moreover, transfection of bcl-2 into the cells lacking (x5131 integrin prevented the anoikis response [14]. Similarly, the blockage of endothelial cell (xv~3 integrin with antibodies specifically caused the activation of p53 and the downregulation of bcl-2 [38]; the subsequent decrease in the bcl-2 : bax ratio was proposed to induce apoptosis. It will be interesting to identify the integrin signal transduction events that regulate the bcl-2 promoter. Recently, anticancer drugs such as taxol that perturb microtubules were shown to dramatically stimulate the phosphorylation of bcl-2, thus inhibiting its ability to neutralize bax and suppress apoptosis [26°]. This raises the unexplored possibility that integrins might control microtubule structure or dynamics so as to regulate bcl-2 function and anoikis. Jun a m i n o - t e r m i n a l k i n a s e s a n d t h e bcl-2/caspase connection The Jun amino-terminal kinases (JNKs) are a family of MAP kinase related serine/threonine kinases that respond to a variety of 'stress-inducing' stimuli, such as UV or y irradiation, ceramide, tumor necrosis factor-(x and interleukin-1 (reviewed in [39,40]). The apoptosis-inducing activity of JNK is counteracted by ERK, and the balance between the activation of these pathways has been proposed to control cell survival versus apoptosis [41].
Recently, the dissociation of epithelial cells from the extracellular matrix was found to rapidly and dramatically induce the activity of the JNKs as well [32] (see Figure 1). Furthermore, the blockage of the JNK pathway by a dominant-negative form of JNK kinase partially inhibited anoikis. Thus, the JNK pathway also plays a critical role in anoikis. These results provide a possible mechanism for the suppression of anoikis by bcl-2. The overexpression of bcl-2--which had previously been shown to suppress anoikis--suppresses the activation of caspases involved
in anoikis [32] and other apoptotic systems (e.g. [42"]). Caspase activity is required for the activation of the JNK pathway [32] (see Figure 1); this may result from the requirement of the upstream kinase MAP kinase/ERK kinase kinase-1 (MEKK-1) to be activated by caspase-mediated cleavage [43"]. This suggests that bcl-2 may suppress anoikis in part by suppressing JNK activation. Thus, the signals regulating anoikis appear to flow from the integrins through bcl-2-related proteins to caspases, then to MEKK-1 and JNK. T h e MEKK/JNK pathway and caspases also communicate in a positive feedback loop, because blockage of MEKK/JNK inhibits caspases and blockage of caspases inhibits MEKK/JNK [43"]. The MEKK substrates or JNK substrates that mediate anoikis remain to be identified, but c-jun itself should be considered, because it can induce apoptosis when overexpressed in at least one system [44"]; activation of the I~B(x kinase complex by MEKK-1 [45] might also be important. An alternative way for bcl-2 to block anoikis has been proposed [46"]. In the prostate carcinoma cell line LNCaP, bcl-2 overexpression suppressed the accumulation of the cyclin dependent kinase inhibitors p21 and p27. Presumably because of this, the retinoblastoma protein (Rb) remained in the inactive, hyperphosphorylated state even in suspended cells. In the absence of active Rb, LNCaP cells were resistant to anoikis. Thus, bcl-2 might directly prevent the Rb-mediated cell cycle arrest that is required for the induction of anoikis in this system.
Future prospects Clearly, a substantial amount of experimental work will be needed to understand how integrin signaling and apoptotic signaling are integrated. New methodology might be required to analyze the possible scaffolding of both classes of transducer by the cytoskeleton, which may play a key role in anoikis. In the immediate future, there are several particularly compelling candidate integrating molecules to explore, including the following: first, integrin-linked kinase, a protein kinase that binds to the integrin 1~1 subunit [47]; second, PYK-2, a FAK-related molecule that regulates the JNK pathway [48]; third, rho, rac and cdc42, ras-superfamily members ~J~at are involved in cytoskeletal organization [49]; and fourth, death-domain-containing receptors [50], whose functions may somehow be regulated by integrin signaling. It will also be interesting to systematically compare the effects of the various integrin types in anoikis, in the hope of understanding how tumor cells might evade anoikis by expressing certain integrins. Finally, many observations cited above suggest that a cell must have a fairly extensive epithelial transcription program in order to be sensitive to anoikis. This program is
Integrins and anoikis Frisch and Ruoslahti
apparently reinforced by cell-cell interactions and the loss of the program may contribute to carcinoma progression. In this light, it will be important to analyze the transcriptional mechanisms by which the epithelial transcription program is established in order to understand anoikis more fully.
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Acknowledgements This work was partially supported by the following grants from the National Institutes of Health: GM 51452 (to SM Frisch), and CA 62042, CA 28896, and CA 74238 (to E Ruoslahti). Our work was also supported by Cancer Center Support Grant CA30199.
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