An important aspect of multicellularity is that cells only grow and differentiate when in the correct context within a tissue, and remove themselves by apoptosis when they are not. Cells sense their location through specific interactions with the extracellular matrix (ECM) as well as neighbouring cells. Apoptosis in response to inappropriate cell/ECM interactions is termed anoikis, a name that in some way implies a special case of cell death initiated by signals not used in response to other proapoptotic insults.1 In practice, different cell types use diverse mechanisms to interpret signals from the ECM, all of which are found to regulate apoptosis in response to many other stimuli. The purpose of this review is to show that rather than anoikis being a specific stimulus of cell death, it is in fact a broad range of cellular responses to loss of adhesion that utilise diverse signalling and apoptotic pathways.
Anoikis – A Short Historical Perspective
Anoikis is apoptosis induced by lack of correct cell/ECM attachment, and many experimental systems study this by completely detaching cells from the ECM. Some thought is required regarding the significance of anoikis in vivo. Cells in tissues require very specific ECM attachments, and the wrong type of ECM can have the same consequences as no ECM at all. The importance of anoikis in vivo can readily be seen when alterations that perturb its normal control are seen to enhance tumour metastasis, a process which requires cells to survive in totally inappropriate ECM environments.2 Anoikis, therefore, should not be considered as an experimental system in vitro, but the mechanism by which cells in vivo use ECM-derived signals to maintain tissue integrity.
Although anchorage dependence of cells has been recognised for many years, particularly regarding proliferation,3 anoikis as we understand it was first described in the early 1990s. Almost simultaneous papers from the groups of Martin Schwartz and Steve Frisch showed that cells that were deprived of attachment to the ECM underwent classical apoptosis.1, 4 The significance of these papers was that signals from the ECM were recognised to be required to prevent cells from actively undergoing programmed cell death. These papers also demonstrated some fundamental aspects of anoikis. Firstly, ECM receptors of the integrin family are essential for cells to suppress anoikis. Thus, plating cells onto anti-integrin antibodies prevented anoikis, whereas attachment to antibodies recognising other cell surface proteins did not, suggesting that specific integrin-dependent signalling is required. Second, anoikis was inhibited by overexpression of Bcl-2, indicating a requirement for mitochondrial membrane permeabilisation (MMP) to occur. Thirdly, not all cell types are equally sensitive to anoikis. Epithelial and endothelial cells were found to be more sensitive than fibroblasts, the latter being able to survive in the absence of ECM if serum growth factors were present. Furthermore, epithelial cells can be switched between anoikis sensitive and insensitive by oncogenic transformation or treatment with scatter factor. Such processes not only induce cells to become migratory but also to become insensitive to anoikis. Transformation with H-Ras was reverted by adenovirus E1a, which restored sensitivity to anoikis. The sensitivity of cell to anoikis appears, therefore, to be associated with epithelial to mesenchymal transition, transformation and immortalisation.
Signals from the Matrix – Clarifying or Confusing?
Adhesion receptors not only provide a physical attachment to the ECM but they also create an adhesion-dependent signalling scaffold containing a number of adaptor proteins and kinases.5, 6 Thus, integrins function in an analogous way to growth factor receptors (GFRs), and indeed activate many of the same downstream pathways. Integrins also crosstalk directly with GFRs, and allow cells to respond optimally to soluble cytokines only when they are attached to the correct ECM.
Specificity for particular types of ECM occurs through the range of integrins expressed on cells.6 Humans have at least 24 different integrins, and although some are expressed on the same cells and even recognise the same ECM components, many have essential roles in specific tissues. Mammary epithelial cells adhere to a laminin-rich basement membrane via integrin α6β1. The stroma underlying the mammary ducts and alveoli contain collagen I, which is recognised by α2β1 on the mammary cells. However, although they express collagen-binding integrins, they do not support mammary cell survival and they eventually undergo apoptosis.7, 8 Melanocytes are similarly kept in the correct tissue compartment through integrin/ECM interactions. The underlying dermis is rich in collagen, and this fails to support melanocyte adhesion and survival, as unlike MEC they do not express suitable integrins. However, during melanoma invasion through the collagen-rich dermis, upregulation of αvβ3 on the melanoma cells allows them to receive antiapoptotic signals from a normally hostile ECM environment.9 Inhibition of αvβ3 function using blocking antibodies induced melanoma cell apoptosis.
So how does adhesion via integrins keep cells alive? Integrin-mediated adhesion regulates all the same signalling pathways that control apoptosis in growth factor-mediated survival, DNA damage responses and death receptor-mediated apoptosis, although to different extents. Which pathways regulate anoikis varies depending on cell type, with different integrins activating distinct signalling cascades (Figure 1). For example, integrins can activate PI3-kinase signalling, the classical ERK pathway, as well as stress-activated MAP kinases like c-Jun N-terminal kinase (Jnk).5 These can be activated in a number of ways specific to different integrins. Some integrins (α1β1, α5β1 and αvβ3) recruit the src family kinases Fyn and Yes via an interaction with caveolin 1, which can then activate the classical ERK pathways by recruiting Shc, Grb2 and Sos10 (Figure 1a). Many integrins recruit pp125FAK (focal adhesion kinase), a nonreceptor tyrosine kinase that is activated in response to adhesion11 (Figure 1b). Pp125FAK interacts with a range of signalling and adaptor molecules, including Src, PI3-kinase, paxillin and p130CAS, and has been linked to a number of signalling pathways controlling migration, proliferation and apoptosis. Integrin-linked kinase is also recruited to adhesion sites, and has been implicated in survival signalling.12, 13 There is considerable overlap between the downstream pathways activated by these alternative integrin signalling mechanisms. I will highlight a few examples that illustrate the diversity in anoikis signalling.
Pp125FAK has been shown to be required to suppress anoikis in a number of cell types, either through expression of dominant-negative forms, microinjection of anti-FAK antibodies or use of dominant active forms to suppress cell death.14, 15, 16 FAK can regulate PI3-kinase signalling, MAP kinase signalling, small GTPases and other tyrosine kinases such as those of the Src family, all of which can influence cell survival.11 In MDCK cells, detachment from ECM can activate Jnk, providing a proapoptotic signal.17 However, a different study also using MDCK cells found no correlation between Jnk and anoikis.18 Instead, activation of PI3-kinase in adherent MDCK cells was required to suppress anoikis, similar to that seen in mammary epithelial cells.14 Jnk can be either pro- or antiapoptotic, depending on its cellular context. A study in primary fibroblasts found that pp125FAK activation of Jnk in adherent cells was required to suppress anoikis.19 In fibroblasts, anoikis was also found to be dependent on p53.20 In serum-free conditions, FAK is required to suppress p53-dependent apoptosis. In the absence of FAK or adhesion, p53 is activated via phospholipase A2 and protein kinase Cγ. Anoikis was inhibited by either overexpression of Bcl-2 or by a dominant-negative form of p53, indicating that this p53-dependent mechanism still required mitochondrial permeabilisation. In the same study, growth factors provided a strong PI3-kinase-dependent survival signal and the fibroblasts were no longer sensitive to inhibition of pp125FAK.
Adhesion to the correct ECM alone is not sufficient to provide a survival signal. Cell spreading and shape can profoundly influence phenotype, and the role of the cytoskeleton in these aspects of adhesion signalling is critical. The degree of spreading of endothelial cells on micropatterned substrates caused a switch between proliferation, differentiation and apoptosis, independent of the type of ECM and integrin used for attachment.21, 22 Similarly, mammary epithelial cells require a specific three-dimensional (3-D) arrangement to suppress apoptosis.23 This regulation of apoptosis through the arrangement of cells within a 3-D architecture may contribute to mammary gland morphogenesis.24 Cell shape is controlled by the cytoskeleton and its connections with integrins at cell/ECM and cell/cell junctions. Changes in these mechanical forces can alter cellular signalling pathways associated with cell adhesion, thus influencing survival.25, 26 The ways in which cells sense mechanical forces associated with spreading and tissue architecture, and how these affect signalling, are reviewed in detail elsewhere.27, 28, 29
A further complication arises when we consider crosstalk between integrins and GFRs. Many GFRs are influenced by adhesion to ECM, allowing, for example, anchorage-dependent control of proliferation.30 Pp125FAK signalling can directly influence the ability of GFR to control ERK activation and G1–M transition. It is not surprising, therefore, that this crosstalk can influence anoikis (Figure 1c). In primary oligodendrocytes, integrins crosstalk with GFR, allowing target-dependent survival in conditions of limiting growth factors.31 At physiological levels of neuregulin, attachment of newly formed oligodendrocytes to laminin on axons, via α6β1, was required to fully activate survival signals. The laminin/α6β1 interaction allowed neuregulin to activate a strong ERK dependent survival signal. Epithelial cells also show regulation of anoikis through integrin GFR crosstalk. Epithelial cells are dependent on both growth factors and adhesion for survival, and the absence of either results in apoptosis. However, they are not necessarily working through distinct signals. Primary mammary epithelial cells depend upon adhesion to laminin along with the insulin-like growth factor 1 (IGF-1).32 Activation of the IGF-1 receptor suppresses apoptosis through a PI3-kinase-dependent pathway.33 Attachment to laminin appears to be a requirement for this IGF receptor signalling. Primary MEC grown on collagen do not efficiently activate PI3-kinase in response to IGF-1 and undergo apoptosis. The human MEC line MCF10A shows requirement for epidermal growth factor (EGF) receptor (EGFR) signalling, although in this case it appears to function through the classical ERK pathway.34 In the absence of adhesion, MCF10A cells rapidly lost cell surface expression of EGFR, leading to upregulation of the Bcl-2 protein Bim. A breast tumour cell line did not show this detachment-induced downregulation of EGFR, and overexpression of EGFR in MCF10A cells inhibited anoikis. Interestingly, different MEC lines appear to show differences in how integrins link with GFRs to control anoikis.35 Thus, the boundary between integrins and growth factors in apoptosis regulation appears blurred.
Anoikis is an essential mechanism for maintaining the correct position of cells within tissues. Induction of anoikis occurs when cells lose attachment to ECM, or adhere to an inappropriate type of ECM, the latter being the more relevant in vivo. However, this superficial similarity in how anoikis is initiated masks the many diverse ways in which cells signal via adhesion receptors to regulate apoptosis. How one cell responds to incorrect adhesion will be quite different from another type of cell, involving a distinct set of signalling enzymes and apoptosis regulatory proteins. Also, given the complexity of integrin/growth factor crosstalk, it also becomes difficult to determine the boundary between adhesion and cytokine dependent survival. As such, it is perhaps misleading to think that anoikis is regulated by, for example, specific BH3-only proteins. Instead, loss of adhesion can control signalling pathways that can activate most of, if not all, the BH3-only proteins, depending upon which cell type you examine. Choice of cell type for study has a further important implication. Cell lines have been selected for their ability to grow over many passages in culture. Such lines therefore show a marked loss in sensitivity to anoikis, as every passage is detached from ECM before being replated. Many studies on anoikis have therefore been based on culture models that are not truly anchorage dependent for survival. Mammary cell lines, such as FSK-7 and MCF10A, do not require adhesion to laminin to suppress apoptosis, unlike primary MEC that absolutely require laminin. Changes in cell lines most likely dampen the exquisite sensitivity of primary cells to anoikis. To truly understand the significance of anoikis, a greater understanding of how it operates in vivo is required.
I would like to thank Professor Charles Streuli for comments on the manuscript.
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Cell Death Discovery (2017)