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Anoikis mechanisms Steven M Frisch* and Robert A Screaton

Anoikis is defined as apoptosis that is induced by inadequate or inappropriate cellmatrix interactions. It is involved in a wide diversity of tissue-homeostatic, developmental and oncogenic processes. The central problem of anoikis is to understand how integrin-mediated cell adhesion signals control the apoptotic machinery. In particular, the initiation of the caspase cascade in anoikis remains to be explained.
Addresses The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA *Correspondance: e-mail: sfrisch@burnham-inst.org Current Opinion in Cell Biology 2001, 13:555562 0955-0674/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. Abbreviations DLC1 dynein light chain 1 EGF epidermal growth factor EGFR EGF receptor ERK extracellular signal-regulated kinase FADD fas-associated death domain FAK focal adhesion kinase IGFRs insulin-like growth factor receptors IAP inhibitor of apoptosis ILK integrin-linked kinase JNKs Jun N-terminal kinases PAK p21-activated kinase PDGF platelet-derived growth factor PKA cAMP-dependent protein kinase A PI3K phosphoinositide-3 kinase SGKs serum- and glucorticoid-inducible kinases

attachment, facilitating eventual reattachment and colonization of secondary sites. Interestingly, a second selective advantage has been revealed by two recent studies [6,7]. In A431 epidermal carcinoma cells [6] and MDA-MB468 mammary carcinoma cells [7], stimulation with epidermal growth factor (EGF) induced either cell cycle progression or cell rounding, which triggered anoikis. This observation suggests that excessively stimulated growth factor signaling pathways, which commonly occur in tumor cells, may cause cytoskeletal perturbations capable of initiating anoikis. This phenomenon would provide a strong and immediate selective pressure for cells to become resistant to anoikis, perhaps by oncogene activation. Moreover, it may explain why several individual oncogenes simultaneously activate survival and cytoskeleton-altering pathways (e.g. ras.) These specific examples also underline the importance of coupling integrin- and growth-factor-signaling pathways for producing the appropriate cellular response. Surprisingly, resistance to anoikis also appears to be involved in the evolution of certain non-epithelial cancers such as melanoma [8,9] and neuroblastoma [10], where de-differentiation confers resistance. These results suggest that the circumvention of anoikis may be involved not only in carcinomas but also in other commonly occurring human tumors, although the mechanisms leading to aberrant survival may vary greatly among these cell types.

Introduction
Normal cell and tissue homeostasis reflects a dynamic balance of cell proliferation, differentiation and apoptosis. Anoikis the subset of apoptosis triggered by inadequate or inappropriate cellmatrix contacts maintains the correct cell number of high-turnover epithelial tissues. The clearest evidence for this is that the breakdown of anoikis contributes to neoplasia. Accordingly, this process is discussed first.

Role of protein kinase signaling pathways in anoikis

Numerous kinase/phosphatase signaling molecules have been implicated in anoikis as central regulators. Because ras activation prevents anoikis [1] and integrins can stimulate various aspects of the ras pathway, anoikis research has focused on the two major ras effectors, the kinases PI3K (phosphoinositide-3 kinase) and raf. Rather than cataloguing the signaling molecules that we may infer to control anoikis, the next section focuses on some recent findings regarding selected signaling pathways; it is not intended to be comprehensive.
Phosphoinositide-3 kinase-related signaling

Recent evidence for a role of anoikis resistance in malignancy

Anoikis was first documented in both epithelial cells the precursors of most human cancers and endothelial cells. In these early reports [1,2], the expression of certain oncogenes was shown to render normal epithelial cells resistant to anoikis. More recent reports confirm that the breakdown of anoikis contributes prominently to the malignancy of mammary and colon cancers [35]; a similar role has recently been reported for lung carcinomas (see Now in press). The breakdown of anoikis is expected to confer a selective advantage upon pre-cancerous epithelial cells, affording them an increased survival time in the absence of matrix

Akt is involved in diverse survival signaling scenarios, including anoikis [11]. The three primary integrin signaling molecules that have been linked to cell survival are FAK (focal adhesion kinase), Shc and ILK (integrin-linked kinase) [12-14]. Each of these proteins may impinge upon the PI3K/Akt pathway, although recent evidence shows that they can also signal by distinct mechanisms. The evidence follows. ILK interacts with the cytoplasmic tails of 1 and 3 integrin subunits and is activated transiently by cellmatrix adhesion or growth factor stimulation (the latter presumably through a Nck- and ILK-interacting adaptor called Pinch.) When

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Figure 1 Cytoskeletal alterations potentially involved in anoikis. Diagrams illustrating the alterations in (a) attached cells and (b) suspended cells are shown. The various signaling molecules that are controlled by integrins and growth factor receptors have been omitted, so as to focus on cytoskeletal functions. (a) In the attached cells, the integrins are complexed with extracellular matrix and growth factor receptors, and the three intact cytoskeletal systems are linked through plectin. They sequester the BH3 domain proteins Bim and Bmf, the JNK pathway components JNK1, SEK1 and MLK2, and p150-Spir is able to stabilize actin polymers through Arp2/3; TNFR2 (not shown) may be kept inactive through interaction with intermediate filaments. Most of the caspase-8 is bound to mitochondria, as discussed in the text. (b) Upon suspension, the initiating signal for anoikis is not yet known. However, cytoskeleton of all types are presumably perturbed, which may cause the release of Bmf from actin, thus neutralizing bcl-2, and allowing cytochrome c release from mitochondria. Accompanying this, caspase-8 is activated, which promotes further cytochrome c release through Bid, plectin cleavage and activation of effector caspases. These in turn cleave filamin and cytokeratin intermediate filaments. The fate of actin polymers after detachment is not well understood but activation of the JNK pathway may inhibit Arp2/3 function through p150-Spir, leading to disassembly.

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Current Opinion in Cell Biology

overexpressed in cell lines, ILK activates Akt activity either direct-ly or indirectly. Inactivation of the phospholipid phosphatase PTEN, which occurs frequently in prostate cancer cell lines, constitutively activates both ILK and Akt (both kinases possess lipid-binding pleckstrin homology (PH) domains, which probably explains this activation). Interestingly, transfection of a dominant-negative ILK into PTEN-deficient prostate carcinoma cells or treatment of these cells with a specific chemical inhibitor of ILK decreases serine-473 phosphorylation of Akt and, in turn, its kinase activity [15]. This implicates ILK as an in vivo activator of Akt, which was recently confirmed biochemically [16]. Overexpression of ILK can also suppress aniokis in certain epithelial cell lines [17].

FAK may also activate Akt through direct PI3K activation as well as indirectly through a p130cas-crkII-DOCK180-rac pathway [18]. To date, only FAK has been demonstrated to regulate the expression of caspase inhibitors of the IAP (inhibitor of apoptosis) family [19], by a proposed mechanism involving PI3K/Akt activation of the NF-B pathway. Future experiments will determine whether or not IAPs are induced in response to the other integrin signaling molecules. Although Shc is thought to activate the MAP kinase pathway through grb2, recent results indicate that Shc is also a potent PI3K/Akt activator [20]. In hematopoietic cells, activated Shc recruits gab2 through grb2. Gab2 in turns

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recruits the regulatory subunit of PI3K, p85, leading to PI3K activation. Whether an analagous pathway pertains to integrin signaling wherein Shc is recruited to integrins via fyn and caveolin will be interesting to test.
Growth factor inputs into integrin-mediated phosphoinositide-3 kinase/Akt survival signaling

for anoikis in LNCap cells will require a direct assessment of the levels of IGF protein itself during cell detachment (because only mRNA levels were examined). As loss of membrane integrity frequently occurs late in or after the completion of apoptosis in most systems, the release of transfected luciferase protein from apoptotic LNCap cells may not accurately reflect the kinetics of anoikis. Furthermore, cellular transcription and translation are largely inhibited in suspended cells. Given that the kinetics of caspase activation (and the consequent pro-apoptotic effects) following cell detachment are generally more rapid than transcriptional or translational alterations, the impact of transcriptional changes on anoikis may be secondary. A precise determination of the role of Akt activity in anoikis is complicated by the fact that Akt itself is a target for activated caspases [29]. Thus the loss of Akt activity may be a consequence and not a cause of anoikis. Nevertheless, the role of soluble survival factors in anoikis warrants further examination. Akt phosphorylates regulators of apoptosis such as (human) caspase-9 and Bad (reviewed in [30]), as well as transcription factors whose target genes program the survival phenotype (e.g. Forkhead factors). However, Akt-mediated survival is likely to involve as yet unidentified novel Akt substrates as well as kinases possessing Akt-like activity. Notably, Akt is also required for the activation of p21-activated kinases (PAK) by ras [31], thus PAK itself or some PAK activator may be a critical new Akt substrate. Several anti-apoptotic effects of PAK which, like Akt, can phosphorylate BAD [32] have been reported (e.g. [33]; see section below.) Forkhead transcriptional regulators are also substrates for a set of Akt-related kinases known collectively as serum- and glucorticoid-inducible kinases (SGKs); both Akt and SGK1 can inhibit Forkhead function by promoting cytoplasmic accumulation of the phosphorylated Forkhead product [34]. Whether the other Akt substrates are also SGK substrates, and the relevance of SGKs to anoikis, remains to be determined.
RafERK-related signaling

As insulin and IGF receptors are major activators of Akt activity and their ligands are critical survival factors for numerous cell types, several laboratories have addressed the role of these factors in anoikis. Two studies demonstrated that although IGFs protected serum-starved mouse embryo fibroblasts [21] and LNCap prostate carcinoma cells [22] from anoikis, they did not inhibit anoikis in primary mammary epithelial cells even though the mammary cells normally require insulin for survival when attached to the extracellular matrix [23]. When mammary cells attached to collagen (a non-permissive matrix required for their survival) were stimulated with insulin, the insulin receptor was still capable of autophosphorylation. Further signaling, however, was blocked: the receptor was not able to phosphorylate and recruit the adapter IRS-1 or activate downstream PI3K/Akt survival signaling [23]. Thus, in this cell system, integrin ligation is a prerequisite for insulin signaling. Whether or not similar requirements exist in animal models remains to be confirmed. Nevertheless, the interdependence of integrins and certain growth factor receptors (insulin receptor, PDGF (platelet-derived growth factor) receptor, and the EGF receptor) warrants further examination of the possibility, in some systems at least, that anoikis is partly due to an inhibition of growth factor signaling. Interestingly, EGFR and integrins form complexes in certain cell types in which extensive cross-activation of the two receptor types is observed [24,25]; uncoupling cooperative signaling by growth factor and matrix receptors could dysregulate cell survival by dissociating growth factor signaling from cytoskeletal alterations. It is in this context that the prevalence of FAK overexpression in human tumor cells [26] which prevents anoikis [27] may be appreciated. In the case of the PDGF receptor, the interaction between the receptor and integrin is extracellular, raising the possibility of a direct collaboration or competition between matrix molecules and the growth factor for receptor binding [28]. In the LNCap system, IGF levels are reported to dissipate slowly over the course of 16 hours of cell detachment, which is immediately followed by anoikis [22]. The authors attribute the observed anoikis in LNCap cells to this decrease in IGF. Moreover, the absence of IGFs was proposed to initiate a decline in Akt activity and the following cascade of events: activation of Gsk-3, phosphorylation-driven degradation of cyclin D, Rb hyperphosphorylation and consequent repression of the IGF promoter. With the IGF promoter repressed, the cells are committed stably to anoikis. Confirmation of such a model

There is much evidence to implicate ERK (extracellular signal-regulated kinase) activation in both cell cycle progression and survival (e.g. [35]). It is well established that raf-1 function is critical for ERK activation; a possible alternative function and novel activation mechanism for raf-1 are considered here. The Bcl-2-interacting protein BAG-1 activates raf-1 kinase activity and targets it to mitochondria, where its survival effect is apparently enhanced [36]. Interestingly, BAG-1 also interacts with the p53-inducible inhibitor of the rasrafMAP-kinase pathway, Siah-1, and prevents Siah-1 from inducing growth arrest [37]. Whether this phenotypic effect of Siah-1 is dependent on association with raf-1 through BAG-1 is not yet clear. In any event, the existence of critical mitochondrial raf-1 substrates is strongly implied by these results.

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Although the precise mechanism of raf-1 kinase activation remains unclear, it is known that the PAKs, whose role in integrin signaling is well recognized, can activate raf-1 by direct phosphorylation of serine 338 [38]. PAK activity is controlled both by phosphoinositides and PI3K, as the former interact with the PH domains of the rac/cdc42 GEFs, and the latter may directly activate PAK through the adaptor protein PIX [39]. Integrin ligation to fibronectin can stimulate raf phosphorylation at serine 338 by this pathway, which provides an interesting new mechanism for the control of raf-1 activity by integrin stimulation of PI3K activity. It may also explain the critical involvement of PAKs in the stimulation of ERK activity by integrins. Recently, an indirect mechanism for raf-mediated survival was indicated: the induction of transforming growth factor- (TGF-) expression [40]. In MCF10a epithelial cells, a significant increase in TGF- was noted in response to expression of a tamoxifen-inducible raf-1 protein. Exogenous addition of this cytokine sufficed for anoikis rescue, and inhibition of the TGF- receptor with AG1478 abrogated rescue by raf or TGF-. Anoikis rescue by EGF has also been demonstrated in keratinocytes [41]. As raf-1 can in turn be activated by EGFRs, this result implicates a positive feedback loop that promotes cell survival involving raf and autocrine growth stimulation. Finally, the ERK pathway is also affected by the state of the actin cytoskeleton. In 3T3 cells, both detachment and treatment of attached cells with cytochalasin D is sufficient to prevent ERK translocation into the nucleus and the consequent phosphorylation of its target, elk-1 in response to mitogenic stimulation [42]. It is difficult to reconcile the adhesion requirement for ERK translocation with the ability of EGF to rescue cells in suspension from anoikis, although cell-type differences between these studies may contribute. Nevetheless, this highlights the important challenge of elucidating how integrins control ERK nuclear import or export through cytoskeletal regulation.
Jun N-terminal kinases

response to attachment to, rather than detachment from, the matrix (fibronectin). In these experiments, as reported previously, JNK activation was stimulated by cotransfection of FAK, accompanied by enhanced survival. Thus, a survival pathway from integrins through FAK to JNKs was proposed. There is evidence to suggest that JNKs are in fact involved in normal cell cycle progression in fibroblasts: adherent 3T3 cells show a spike of JNK activity in the G1 phase of the cell cycle, and transfection of a dominantnegative version of MKK4 a positive upstream regulator of JNK activity causes 3T3 cells to growth-arrest in G1 [47]. In this study, JNK activity was stimulated both by attachment to the matrix (in HUVEC, 3T3 and 293 cells) and by FAK (in 293 cells), further implicating JNKs in adhesion-dependent cell cycle progression and survival. JNK was assayed immediately after detachment (time zero) versus various time points after reattachment. However, because JNK activity is stimulated rapidly by detachment [43] and cells reattach and spread slowly, it is possible that the stimulation of JNK observed in these [47] and other studies [48] was due to detachment indicating a proapoptotic effect of JNKs rather than reattachment. Clearly, JNK regulation is significantly different in fibroblasts versus epithelial cells: in fibroblasts but not in epithelial cells, ras stimulates JNK activity, and cell cycle progression by dominant-negative MKK4 is inhibited. In fact, anoikis is likely to proceed by different mechanisms in these two cell types altogether, given that fibroblasts must be deprived of growth factors in order to respond to matrix detachment. This might reflect the different circumstances under which fibroblasts and epithelial cells have evolved to undergo anoikis in vivo. Differences amongst epithelial cell lines may exist as well.

Role of the cytoskeleton

The readily apparent differences between the cytoskeletal structures of attached versus suspended cells suggest that survival signaling in anoikis is likely to be extensively regulated by cytoskeleton. Such regulation may be affected by the multiple cytoskeletal changes apparent in transformed cells [49]. Indeed, substantial evidence now exists that both signaling molecules and apoptosis regulators are associated with the cytoskeleton, and as such may together regulate anoikis by serving as sensors of cytoskeletal integrity. For example, several interesting connections exist between the cytoskeleton and the JNK pathway. First, MKK4/SEK1 interacts directly with the integrin-associated cytoskeletal protein ABP280/filamin [50]. Filamin-deficient melanoma cells are refractory to JNK stimulation, and reconstitution of filamin rescued JNK activity, an observation that implicates the cytoskeleton in the control of JNK activation. Moreover, filamin is a caspase substrate, and cleavage of filamin may play a role in the regulation of JNKs [51]. Second, JNKs may control cytoskeletal arrangement by direct association with a novel WASP family member (a family of effectors for rholike GTPases that regulate actin cytoskeleton), p150Spir

Numerous reports have suggested both pro- and anti-apoptotic roles for Jun N-terminal kinases (JNKs), creating some degree of complexity in the field. Early reports indicated that detachment of epithelial cells from matrix strongly and rapidly induces the activation of JNKs, which may contribute to anoikis [43]. Recent evidence supporting a pro-apoptotic role for JNKs comes from the observation that mouse embryo fibroblasts lacking both JNKs 1 and 2 (due to a compound knockout) were resistant to UV-, anisomycinand DNA damage-induced apoptosis [44]. These effects correlated with a failure to release cytochrome c from mitochondria or activate Bid. Interestingly, anoikis also involves mitochondrial cytochrome c release [45]. Perhaps determining the effect, if any, of the JNK1 and 2 gene knockouts on cellular response to matrix detachment will clarify this issue. In contrast to previous reports, Almeida and co-workers [46] observed that serum-starved fibroblasts activated JNKs in

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[52], with which activated JNK collaborated in transfection experiments to cause cytoskeletal rearrangements. Third, MLK2, a MAP kinase kinase kinase that activates JNK, colocalized with JNK along microtubules and associated with the kinesin superfamily motor KIF3 [53]. Another interesting cytoskeletal connection involves two potently proapoptotic Bcl-2-family proteins of the BH3only class, Bim and Bmf [54,55,56]. In untreated cells, Bim protein is apparently sequestered by microtubule-associated dynein light chain 1 (DLC1). When released from this sequestration upon treatment with microtubule-disrupting agent taxol, Bim interacts directly with Bcl-2 and stimulates release of cytochrome c from mitochondria. Analogously, Bmf which was recently isolated by virtue of its interaction with the Bcl-2 homolog Mcl-1 is sequestered by the actin/myosin-associated dynein light chain-2 (DLC-2) in MCF7 cells. Cell suspension, cytochalasin treatment or UV-irradiation all induce the release of Bmf from DLC-2, which allows Bmf to complex with and presumably neutralize Bcl-2. Interestingly, release of both Bim and Bmf occurred independently of caspase activity. Thus, these proteins may directly regulate the initial stages of apoptosis, and perhaps anoikis, by serving as sensors for microtubule and actin cytoskeleton integrity, respectively. Future functional and genetic studies will help to establish the precise role of these proteins in anoikis. Another conceptually attractive connection between the actin cytoskeleton and apoptosis is provided by the actin severing/capping protein gelsolin [57]. Recent in vitro experiments with isolated mitochondria have indicated that full-length gelsolin prevents the Bax-stimulated release of cytochrome c, whereas caspase-cleaved gelsolin promotes apoptosis by an as yet unknown mechanism. As will be discussed in the next section, there is emerging evidence that some components of the anoikis pathway may overlap with those of death receptor pathways. In this context, it is interesting to note that mouse embryonic fibroblasts isolated from keratin-8/ mice are about 100-fold more sensitive to killing by tumor necrosis factor (TNF) than normal cells [58]. An association between TNF receptor 2 and cytokeratins has been implicated in this effect, and these results suggest that keratin intermediate filaments can serve to attenuate certain death receptor responses. Preliminary results (S Frisch, R Oshima, unpublished data) also indicate that these keratin 8-knockout cells are also sensitized to anoikis. A final connection is provided by the cytoskeletal protein plectin, which forms links with all three major cytoskeletal systems (microtubules, intermediate filaments and actin). Recently, the majority of procaspase-8 (the inactive holoenzyme) was found associated with mitochondria in unstimulated MCF-7 cells [59]. Remarkably, stimulation of apoptosis with FAS ligand caused immediate processing of mitochondrial procaspase-8, after which the active subunits of the enzyme translocated to and cleaved plectin.

These events occurred well before the cleavage of well established initiator caspase targets could be detected, including PARP poly (ADP-ribose) polymerase, lamin or effector caspases. Moreover, embryonic fibroblasts from plectin-deficient mice showed attenuated cytoskeletal reorganization following FAS ligand treatment, indicating that plectin degradation is a watershed event. It will be interesting to test the plectin/ cells for anoikis sensitivity to elucidate a possible role for plectin.

Role of death receptors in anoikis

One of the major outstanding questions in anoikis research is how the caspase cascade is initially activated by simple detachment of cells from the matrix. One hypothesis is that death receptors somehow become activated, either through their propensity for self-association which may suffice for signaling or by an interaction with endogenous death ligands. Recent results indicate that the death receptor adaptor molecule FADD may be involved in anoikis, as a dominant-negative truncated FADD containing only the death domain inhibited anoikis [60,61]. This result does not prove, however, that death receptors themselves are involved in anoikis only that death domains are. Nevertheless, anoikis was also accompanied by an early activation of caspase-8, as would be expected for death receptor activation. However, extensive attempts to document the assembly of death-inducing signaling complexes (DISCs) in detached epithelial cells have so far proven disappointing (S Frisch, R Screaton, unpublished data). This suggests either that a specific FADD-requiring death receptor that has not yet been assayed is involved or that death receptors are not involved at all. This remains to be resolved. Recently in support of the death receptor hypothesis it has been reported that anoikis in HUVECs requires interactions between the endogenous death receptor FAS and its cognate ligand, FAS ligand [62]. In this study, anoikis was inhibited by FAS- or FAS-ligand-blocking antibodies, and FAS was upregulated and associated with FADD after detachment. However, our results (S Frisch, R Screaton, unpublished data) indicate that HUVECs are devoid of detectable FAS ligand protein. Moreover, anoikis of HUVECs could not be blocked by anti-FAS antibodies, and FAS is neither upregulated nor associated with FADD after detachment; the reason for this discrepancy is not yet clear. In summary, a role for death receptors in anoikis has yet to be established. Interestingly, in T cells FAS is tightly associated with the actin cytoskeletal protein ezrin, which is required for FAS ligand responses of the receptor [63]. If an analogous interaction occurs in other cell types, this may provide a novel mechanism for the cytoskeleton to control death receptor function positively (in detached cells) or negatively (in attached cells).

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Conclusions
It is evident that much work remains to be done to establish mechanistic linkages between cell adhesion and apoptosis. Two new frontiers for research have recently emerged: the involvement of the cytoskeleton and the role of the gene expression program. The architectural state of the cytoskeleton is expected to affect the interactions of signaling molecules in threedimensional space. Currently one of the challenges of anoikis research is to connect cytoskeletal organization (as opposed to cytoskeletal content) with molecular signaling. For example, the small GTPase rac-1, whose major function is in cytoskeletal control, has recently been shown to confer anoikis resistance upon MDCK cells [64]. The complete inhibition of cellular PI3K activity restores sensitivity to detachment in MDCK(rac) cells, suggesting that rac protects cells by activating the PI3K/Akt pathway. However, other cytoskeletal effects of rac cannot be ruled out. For example, both rac and cdc42 are known to signal through PAK, and they have recently been shown to be regulated by cAMP-dependent protein kinase A (PKA; [65]). Recent insight into the significance of PAKs in anoikis of 3T3 cells has come from the observation that cell detachment transiently activates PKA. Activated PKA, in turn, is proposed to phosphorylate and inactivate PAKs, thus preventing the responsiveness of the MAPKs to growth factor stimulation. Interestingly, PKA, like FAS, also associates with ezrin [66], an association that provides some insight into how input from cell metabolic, cell death and cytoskeletal systems may be integrated. For the future, sorting out the precise manner by which integrin signal transduction events and architectural changes in the cytoskeleton are interrelated may require the development of new in vitro systems in which the cytoskeleton controls apoptosis. Given the rapid progress of in vitro actin-assembly, microtubule-assembly and apoptosis systems, an in vitro assembly system possessing at least some aspects of the desired linkage may soon be feasible. This would allow for a biochemical analysis of anoikis that may yield different and/or complementary results to those relying on protein overexpression or ablation in transfection experiments. Future research is also likely to focus on the importance of the epithelial gene expression program. The literature is currently replete with reports demonstrating that genes that cause an epithelial to mesenchymal transition (EMT) also often confer resistance to various forms of apoptosis, including anoikis. These genes include various signaling molecules (e.g. raf-1: [40]), transcription factors (e.g. snail/slug family repressors: reviewed [67,68]) and cellcell adhesion molecules [1,69,70]). For the co-repressor CtBP, functional knockout of the protein using adenovirus E1a protein induces a mesenchymal to epithelial phenotypic conversion and greatly enhances sensitivity to anoikis [70]. E1a-mediated disruption of cellular CtBP function also

provides an amenable system for investigation of the relationship between phenotypic conversion and sensitivity to anoikis. In particular, it will be of interest to determine whether epithelial cells display a characteristic pattern comprising expressed proapoptotic genes and repressed anti-apoptotic genes or, alternatively, whether the subnuclear compartmentalization of chromatin serves to modulate apoptosis sensitivity.

Update
Two additional reports relevant to this review were recently published [71,72]. The first relates to the phenomenon that fibronectin adhesion of CHO-based stable transfectants that express 5 1 integrin protects against apoptosis under serum-starved conditions but adhesion to vitronectin does not. This protection correlates with bcl-2 expression levels, and reference [71] reports that the bcl-2 promoter is regulated by signaling molecules such as FAK, Shc and Akt that may respond to specific integrins in this system. It will be interesting to identify transcription factors involved in this pathway and whether integrins regulate other survival gene transcription analogously. The second paper relates to the mechanism by which ras protects RIE-1 rat intestinal cells from anoikis [72]. In this paper, effector-domain mutants of ras were used to show that PI3K, raf or RalGDS activation were individually insufficient for anoikis rescue, indicating that multiple pathways had to be activated. Inhibitor studies and kinase assays indicated that ERK activation was required, although Akt was not required for anoikis-rescue, and, surprisingly, ras did not activate Akt substantially in this cell line; inhibition of the EGF receptor had no effect. The authors also point out the lack of ras activation of Akt in a second epithelial cell line and the lack of effect of ras-knockout on Akt activation in DLD1 carcinoma cells. These results, which differ dramatically from those published earlier in MDCK cells, underscore the importance of cell context in signaling pathways controlling anoikis.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

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Now in press
73. Wei L, Yang Y, Yu Q: Tyrosine kinase-dependent P13-kinase and mitogen-activated protein-kinase-independent signaling pathways prevent lung adenocarcinoma anoikis. Cancer Res 2001, 61:2439-2444.