Anoikis, from the Greek word ‘homelessness’, is a particular apoptotic death due to loss or inappropriate cell adhesion 1. A correct adhesion to extracellular matrix (ECM) proteins is necessary to determine whether a cell is currently in the correct location. Displaced cells are efficiently removed by anoikis, thereby preventing their reattachment to new matrices and their dysplastic growth. Integrins regulate cell viability through their interaction with the ECM, sensing mechanical forces arising from contacts and converting them into intracellular signals 2. Hence, anoikis is a physiologically relevant process for development and tissue homeostasis and is often deregulated in several diseases 3, 4. Anoikis has been described in several cell types, although it appears that cells of different tissue origin activate dissimilar pathways leading to anoikis.
In spite of its unique definition, anoikis is essentially an apoptotic process. The initiation and execution of anoikis could be driven by different pathways that terminally converge into the activation of caspases and DNA fragmentation. In keeping with classical apoptosis, anoikis could follow either the intrinsic pathway, due to the perturbation of mitochondria, or the extrinsic pathway triggered by cell surface death receptors (Figure 1) 4, 5. Proteins of the Bcl-2 family are key players of both intrinsic and extrinsic pathways 6. They include anti-apoptotic proteins, such as Bcl-2, Bcl-XL and myeloid cell leukaemia sequence 1 (Mcl-1), or pro-apoptotic proteins, such as Bax, Bak and Bok (multi-BH3 domain proteins), or Bid, Bik, Bmf, Noxa, Bad, Bim and Puma (BH3-only proteins) 6.
In the intrinsic pathway, caspase activation occurs as a consequence of mitochondrial permeabilization due to formation of oligomers in the outer mitochondrial membrane (OMM) by the pro-apoptotic proteins of the Bcl-2 family, thereby creating channels and causing membrane permeabilization 7, 8. An adjuvant role of the voltage-dependent anion channels in membrane permeabilization has also been reported 9, 10. Disruption of OMM leads to the release of cytochrome c, which forms the ‘apoptosome’ with caspase-9 and the apoptosis protease activating factor (APAF). The final event is the activation of the effector caspase-3 and execution of the apoptotic process 11–13. Anoikis due to the intrinsic pathway is mainly initiated by Bim, although a role has been proposed also for Bid 14. Bim is activated following detachment of cells from the ECM and rapidly promotes the assembly of Bax–Bak oligomers within the OMM 15, 16. Bim is sequestered in the dynein complex and actin filaments until the loss of integrin engagement induces its release and translocation to the mitochondria, where it interacts with Bcl-XL, neutralizing its pro-survival function 17. Loss of integrin engagement inhibits both extracellular signal-regulated kinase (ERK) and phosphoinositide-3-kinase (PI3K)/Akt-mediated phosphorylation of Bim, thus avoiding Bim proteasomal degradation and determining its accumulation 18, 19. In addition, loss of ECM contact, leading to disruption of Bim sequestration by dynein cytoskeleton complexes, strongly increases Bim cytoplasmic accumulation and increases Bad availability 17. This leads to execution of apoptotic process through promotion of Bax/Bad oligomerization and OMM permeabilization.
Beside Bim and Bid, commonly called ‘apoptosis activators’, some other members of the BH3-only family, such as Bad, Bik, Bmf, Noxa, Puma and Hrk, behave as ‘apoptotic sensitizers’ 20, 21. Indeed, they are unable to directly activate Bax and Bak oligomerization, but contribute to cell death by competing for the binding to Bcl-2 of apoptotic activators 22, 23. Bcl-2 is the master anti-apoptotic member of the family, and prevents apoptosis by maintaining mitochondrial membrane integrity 3, 16, 24, 25. Bcl-2 inhibits apoptosis by heterodimerization with Bad/Bax apoptotic members and preventing their clustering into pores, or sequestering Bim apoptotic activators 21, 26.
Compelling evidence indicates the involvement of other members of the BH3-only family in anoikis execution of different cell histotypes. For example, Noxa and Puma are transcriptionally regulated by p53 and have been implicated in fibroblast anoikis 27, 28. Furthermore, in epithelial cells the Bcl-2 modifying factor (Bmf) behaves as sentinel able to register damage at the cytoskeleton and to convey a death signal. Indeed, upon cell detachment, Bmf is released from its previous interaction with the myosin V motor complex 29 and accumulates in the mitochondria, where it neutralizes Bcl-2, leading to cytochrome c release and anoikis execution 30.
The extrinsic apoptosis pathway also leads to anoikis execution (Figure 1). This is initiated by ligation of extracellular death ligands, such as Fas Ligand (FasL) or tumour necrosis factor-α (TNF-α), to their transmembrane receptors, Fas and TNF-α receptor (TNFR), resulting in the assembly of a death-inducing signalling complex (DISC). DISC, through the adaptor protein Fas-associated death domain (FADD), engages and aggregates several molecules of caspase-8. Autoactivated caspase-8 then proteolytically activates caspase-3 and -7, culminating in substrate proteolysis and cell death 16, 31. Caspase-8 can now independently activate effector caspases (type I extrinsic anoikis) or cleave the BH3-only protein Bid (type II extrinsic anoikis) (Figure 1) 7. t-Bid (truncated-Bid) promote mitochondrial cytochrome c release and assembly of the apoptosome 32–36.
Loss of anchorage to the ECM leads to increase in Fas expression and Fas-L expression and to a decreased expression of FLIP, an endogenous inhibitor of Fas-mediated signalling 37. Furthermore, also changes in cell shape during detachment (cell rounding) can induce extrinsic anoikis 38, mainly through membrane relocalization and activation of Fas 39. Finally, activation of the death receptor pathway could be secondary to mitochondrial damage, thereby creating a crosstalk between intrinsic and extrinsic pathways 40.
Activation of the caspase-3 is the common event of both intrinsic and extrinsic anoikis. This initiates a downstream proteolytic cascade affecting signalling molecules like focal adhesion-kinase (FAK), Cas and paxillin 6, 41–43. Cleavage of FAK shuts down its survival signal and disrupts focal adhesion architecture 44. p130Cas cleavage produces a carboxy-terminal fragment, which regulates transcription of p21 cyclin kinase inhibitor, thus contributing to anoikis execution by blocking the cell cycle 45. Detached cells undergoing caspase-3 cleavage of FAK and p130Cas then experience the final steps of the anoikis programme.
Physiological protection from anoikis
Anoikis prevents that epithelial cells, shed from their original location, colonize elsewhere. Epithelial cells are protected by anoikis in several circumstances, including continuous ECM contacts or transient release from the ECM happening in both mobile and dividing cells. Hence, due to their high mobility, mesenchymal cells are usually more resistant to anoikis 46, 47. In addition, professionally non-adherent cells, such as haematopoietic mature cells and leukocytes, are protected from anoikis 48.
Primarily, cells are prevented to enter anoikis while adherent on permissive ECM proteins. The role of ECM as anoikis suppressor is well established 1, 49 and several integrins (α1β1, α2β1, α3β1, α5β1, α6β1, α6β4, αvβ3) have a profound impact on cell survival 2, 44, 50 in both normal or neoplastic cells 2, 51, 52. Key players in integrin-mediated signal transduction leading to anoikis protection are FAK, integrin-linked kinase (ILK), Src tyrosine kinase, PI3K, ERK and the adaptor protein Shc (Figure 2). Upon integrin ligation by proper ECM proteins, FAK and ILK recruit and activate PI3K/Akt, ERK and the Jun-kinase (JNK) pathway 53, 54. PKB/Akt is an essential element of cell survival signalling as integrin-, growth factor- and cell–cell anchorage-mediated signals converge to its activation to grant survival of cells. Activation of PKB/Akt inhibits at multiple levels of the anoikis programme, such as inactivation of caspase-9 55 and phosphorylation of the pro-apoptotic protein Bad 56, activation of nuclear factor-κB (NF-κB) 57, as well as inhibition of Fork-head transcription factors 58. ILK conveys integrin-mediated survival signals independently of FAK, as indicated by the failure of dominant-negative FAK to revert to the ILK-mediated protection from anoikis 59, 60. In addition, the adaptor protein Shc may also transduce adhesion signals to ERKs independently from FAK or ILK 61, 62. Both ERKs and PI3K have been reported to negatively regulate Bim through its phosphorylation, which commits the BH3-only protein to degradation. This prevents Bim from antagonizing Bcl-2 function or stimulating Bax activation and consequently grant for the block of anoikis.
Adherent cells can exploit a functional cross-talk between integrins ligated to their ECM proteins and several growth factor receptors, which are activated in a ligand-independent manner through such interactions. Epidermal growth factor receptor (EGFR) 63, hepatocyte growth factor receptor (HGFR) 64 vascular endothelial growth factor receptor (VEGFR) 65 and platelet-derived growth factor receptor (PDGFR) 66 are the main growth factor receptors recruited in integrin platforms. In epithelial cells, either ligand-dependent or integrin-mediated ligand-independent EGFR activation stimulates ERK and PI3K signalling, thereby suppressing the activity of apoptotic factors. In detached cells disengagement of β1 integrins leads to down-regulation of EGFR expression culminating in upregulation of Bim death signalling 67. EGFR expression is further reduced by prolonged suspension, thereby leading to attenuation of its survival signalling, but the re-establishment of ECM contact rescues EGFR expression and its pro-survival spur 68.
The ligand-independent phosphorylation of EGF receptor in response to integrin ligation is strictly dependent by its association with the adaptor protein p130Cas and the c-Src tyrosine kinase 63, 68. Src kinase is activated in response to integrin ligation due to its redox sensitivity. Indeed, integrin engagement leads to activation of the small GTPase Rac-1 and generation of reactive oxygen species (ROS), which in turn oxidize and activate Src kinase. In turn, the ligand-independent trans-phosphorylation of EGFR is carried out by activated Src, recruited at the integrin/EGFR platform, and switches both ERK and PKB–Akt pathways. Final event are the degradation of Bim and protection from anoikis 69.
Last, a coordination between autophagy and anoikis resistance of epithelial cells is recently emerging 70, 71. Indeed, the RNA activated protein kinase-like endoplasmic reticulum kinase (PERK) facilitates the survival of ECM-detached cells by concomitantly promoting autophagy and ATP production 72. ECM detachment activates the canonical autophagic pathway through ATG6 and ATG8, sustains ATP levels and delays anoikis. The functional players of such integration are Beclin-1, an autophagic protein acknowledged to modulate the anti-apototic role of Bcl-2 and Bcl-XL 73, ROS and ERKs 72. Autophagy allows epithelial cells to survive given that they re-adhere onto the ECM in a timely fashion, and it is likely a previously unrecognized tool used by circulating cancer cells to survive anoikis, thereby facilitating tumour cell dormancy though nutrient recovery, as well as dissemination of metastases 74.
Pathophysiological deregulation of anoikis
Anoikis resistance in cancer
Cancer cells, in harsh contrast to normal epithelial cells, are insensitive to anoikis and do not require adhesion to the ECM to survive and proliferate. This ability has important implications in the metastatic process where cancer cells must survive and migrate in the absence of proper ECM contacts 75, 76. In this scenario, anoikis resistance is a natural molecular prerequisite for the aggressive metastatic spread of cancer (Figure 3).
Different strategies have been described to achieve anoikis resistance (Figure 4). A functional approach involves the modulation of the expression of cell adhesion molecules, such as integrins, which stabilize the normal cell–matrix contacts 77, 78. Indeed, a change in the pattern of integrin expression may influence the downstream signalling cascades to enhance cell survival. In keeping, many cancer cells switch their integrins expression, in order to express the correct integrins that allow to survive in a different microenvironment, thereby preventing the induction of death signalling. A switch in integrin expression has often been described in epithelial cells. Indeed, while normal epithelial cells express collagen receptor α2β1 and laminin receptors α3β1 and α6β1 79, 80, hyperproliferating epithelial cells over-express αvβ5 and αvβ6 integrins 81, 82 and the anoikis-resistant squamous carcinoma cells over-express αvβ6 with respect to αvβ5 83, 84. This gradual change in integrin pattern expression allows cells to acquire enhanced invasiveness and anoikis resistance. Another example of integrin switch is during melanoma progression. Melanoma cells express αVβ3 integrin, which binds to fibronectin, vitronectin, collagen, laminin and integrin αVβ1, which selectively binds to fibronectin 85. Indeed, in melanocytes or melanoma at early stages, the expression of the β3 integrin is undetectable, since β3 integrin has been shown to correlate with malignancy 86–88. It is of note that αVβ3 is important for the adhesion of melanoma cells to dermal collagen and for the suppression of anoikis 89, mainly shifting the Bcl2:Bax ratio 90. This change in integrin expression allows only transformed melanocytes to survive in the new tissue environment during melanoma invasion.
Recently, a fascinating role of the ECM itself to ensure survival signals has been revealed. In fact, murine lung carcinoma cells, over-expressing type IV collagen, acquire a selective advantage in their liver-metastasizing potential. The advantage is due to sustained stimulation of the α2 integrin–FAK–PI3K pathway, granting anoikis protection 91. Hence, these cancer cells, lacking their proper ECM, bring their own soil (collagen) to assure survival.
Another strategy used by tumours to avoid anoikis is the exploitment of oxidative stress, a common feature of several cancers. Indeed, ROS are key molecules that elicit pro-survival signals. Giannoni et al.48 observed that in cancer cells the oxidative intracellular milieu strongly correlates with induction of anoikis resistance; ROS production protects tumour cells from anoikis through the oxidation/activation of the tyrosine kinase Src, thus granting the activation of pro-survival pathways through a Src-dependent and ligand-independent phosphorylation of EGFR, which leads to Bim degradation. Furthermore, in human lung adenocarcinoma cells, Src activation compensates for the loss of cell-survival signals caused by disruption of cell-ECM interactions and contributes to anoikis resistance 92. A redox-mediated activation of Src has been recently evidentiated also in several different tumour types over-expressing angiopoietin-like 4 protein (ANGPTL4). Indeed, ANGPTL4 is linked to tumour progression and its suppression impairs tumour growth associated with enhanced apoptosis. ANGPTL4 interacts with integrins, thus stimulating NADPH oxidase-dependent production of O2−. This leads to Src activation, therefore activating downstream PI3K/Akt and ERK pro-survival pathways. Furthermore the 14–3–3 adaptor protein sequesters the pro-apoptotic Bad protein from mitochondria, conferring resistance to anoikis and favouring tumour survival and growth 93. Recently, PTEN has also been involved in protection from anoikis through a redox control. PTEN oxidation determines its inactivation with consequent phosphorylation/activation of Akt, leading to a pro-survival signal 94.
More recently, an intriguing connection between ROS and caveolin1 (Cav1) in regulating anoikis has also been evidentiated 95. In human lung carcinoma cells H2O2 is an upstream mediator of Cav1-dependent resistance to anoikis. Following detachment, H2O2 production stabilizes the Cav1 protein level and prevents anoikis. In contrast, scavenging H2O2 decreases Cav1 levels through an increase of its proteosomal degradation. A possible explanation of these phenomena is due to the constitutive activation of PI3K/Akt signalling caused by ROS-mediated increased level of Cav1 95.
During tumour progression, epithelial–mesenchymal transition (EMT) is a key event to provide epithelial-derived cells with a motile and highly invasive phenotype, ultimately facilitating metastatic spread. EMT is an essential process for the acquisition of anoikis resistance, too. During EMT, several genes, such as Snail, Twist, HGF-R/cMet and NF-κB, are induced and play a crucial role to evade anoikis by constitutively activating specific pro-survival signals 46. Snail is a zinc-finger transcriptional factor over-expressed in high-grade primary human breast carcinomas and lymph node-positive breast tumours 96. Snail inhibits the transcription of the epithelial marker E-cadherin and confers anoikis resistance by activating survival genes, such as the PI3K/Akt cascade 97. Twist, a helix–loop–helix transcriptional factor, up-regulated in several malignancies promotes EMT, inducing mesenchymal markers such as fibronectin and N-cadherin. Twist, regulates anoikis by increasing the Bcl-2:Bax ratio 98. Indeed, loss of Twist expression provides the cancer cells to become more sensitive to anoikis induction. An hallmark of EMT is the loss of E-cadherin expression correlated with tumour grade and stage 99. Increasing evidence indicates that loss of E-cadherin is associated with anoikis resistance. E-cadherin is a type I cadherin that forms homophilic interactions with neighbouring cells, regulating cell–cell contacts. In mammary tumourigenesis models, loss of E-cadherin grants for anoikis resistance and increased angiogenesis, thus contributing to efficient metastatic spread 100, 101. Furthermore, loss of E-cadherin in gastric cancers leads to a decrease in cell–cell contacts, followed by a release of β-catenin from adherens junctions and translocation to the nucleus where it activates tumour-promoting genes 102. Besides integrins, cadherins are also subjected to a switch of class during cancer progression; many carcinoma cells decrease their E-cadherin expression and increase N-cadherin. N-cadherin recruits PI3K which in turn activates Akt and promotes anoikis resistance 98, 103.
HGF/Met, another key molecule for EMT, has been linked to anoikis escape typical of tumour cells. Indeed, in uterine endometrial cancer cells, HGF induces anoikis resistance through a PI3K–Akt pathway-dependent up-regulation of cyclooxygenase-2 (COX-2) expression. Similarly, Met receptor induces anoikis resistance in head and neck squamous cell carcinoma cells by activation of ERK and PI3K/Akt signalling 104. Moreover, recently it has been reported that breast cancer cells undergoing EMT acquire many of the properties of cancer stem cells 105, 106. Stemness is indeed a device to survive in a hostile microenvironment, once the primary cancer has grown. So the acquisition of stem cell features, linked to both EMT 107, 108 and ROS production 109, may contribute to the survival and subsequent proliferation of cancer cells.
In addition to EMT in some cancers, another type of cell plasticity, mesenchymal–amoeboid transition (MAT), has been highlighted. One of the main inducers of MAT is the EphA2 receptor, which stimulates RhoA and matrix metalloproteinase (MMP)-independent cell invasion 110, 111. In analogy with the acquisition of anoikis resistance induced by EMT, EphA2, which is over-expressed in many types of cancer 112, also confers resistance to anoikis through the action of the guanine nucleotide exchange factor Ephexin4, PI3K and Akt 113.
Another strategy adopted by neoplastic cells to evade anoikis is through constitutive activation of pathways responsible for cell survival, such as activation of Src family kinases and the PKB–Akt and ERK pathways. Really, activation of Src has been reported in various cancers characterized by anoikis resistance 48, 69, 114, 115. Actually, a crucial role is played by the modulation of PI3K–Akt signal pathway, regulated by PTEN-mediated dephosphorylation 116. Loss of tumour suppressor PTEN is often found in prostate cancers resistant to treatment and poorly differentiated, in which the constitutive activation of the PI3K–Akt pathway leads to anoikis resistance 117, 118. Also, the proto-oncogene Ras, which is aberrantly activated in many types of cancer, confers resistance to anoikis through PI3K–Akt signalling 1, 119. Furthermore, oncogenic Ras can interact with the Bcl-2 family of proteins, leading to down-regulation of the pro-apoptotic protein Bak and allowing anoikis resistance 120, 121. Moreover, it has been recently found that tumour cells displaying aggressive metastatic behaviour lack both p66Shc and retinoblastoma (pRB), thereby bypassing anoikis. The ability of re-expression of p66Shc to restore anoikis and to protect from metastatic dissemination of these Ras-dependent tumour cells underscores a novel role of p66Shc in promoting anoikis and suppressing metastases 122.
Other examples of acquisition of constitutive activity of pro-survival pathways to evade anoikis have been described: for instance, an autocrine bFGF loop is a critical component in melanoma progression, leading to cell survival and proliferation 123. Similar circuitries have been shown for growth factors, such as HGF, interleukin-8 and PDGF-AA, to support the proliferation, survival and migration of melanoma cells 124.
In addition, the tropomyosin-related kinase B (TrkB), whose over-expression has been documented in several tumours 125–127, is a potent and specific suppressor of anoikis 128. Transfection of TrkB in highly anoikis-sensitive rat intestinal epithelial cells confers anoikis resistance through the activation of the PI3K–Akt pathway 128. Furthermore, TrkB over-expression results in EMT via the induction of Twist and Snail 129. Snail is able to induce also ZEB1, another transcription factor which suppresses E-cadherin 130. An intriguing feedback loop between ZEB transcription factors and members of the miR200 family 131 in controlling EMT, anoikis resistance and metastasis has been recently elucidated 132. The expression of ZEB1 is strictly correlated to the miR200 family 131, although they control opposite phenomena, ZEB1 being a crucial EMT activator, whereas miR200 members induce the reverse process, ie epithelial differentiation. Actually, ZEB factors and miR200 reciprocally control their expression in a strict feedback loop 133. Of note, ZEB1 is a key EMT regulator in many human cancers including prostate, colon, breast and pancreatic 132, 134–136, while miR200 family members are often down-regulated in cancer cells 131. Actually, the regulation of EMT by miRNAs is a very general phenomenon not limited to TrkB signalling 137. Evidence shows that re-expression of miR200 family members suppresses anoikis resistance in breast cancer cells 131, 138, 139. In addition, Penna et al140 recently showed that during melanoma progression miR214 is up-regulated and positively enhances cell movement and survival to anoikis in vitro as well as metastasis formation in vivo.
Hypoxia is likely another factor affecting anoikis resistance. This hypothesis is supported by the ability of hypoxia to induce EMT in a redox-dependent manner 141. Furthermore, hypoxia has been linked to mitochondrial oxidative stress, leading to HIF1α stabilization 141, 142. Owing to the acknowledged pro-survival role of both the EMT programme and oxidative stress, we therefore speculate that an hypoxic environment may act on tumour cells, stimulating their resistance to anoikis cell death. In keeping with a central role of hypoxia in the induction of EMT, low oxygen tension is able to promote the cadherin switch, characterized by loss of E-cadherin and acquisition of N-cadherin 143. Actually, it is possible hypothesize that the expression of N-cadherin induced by hypoxia may be responsible for conferring anoikis resistance. Indeed, in melanoma cells, it has been demonstrated that the gain of expression of N-cadherin promotes cell survival by activating PKB–Akt signalling and subsequently increasing β-catenin and inactivating the pro-apoptotic factor Bad 144. In addition, it has been suggested that the progression of early breast cancer lesions may be characterized by anoikis resistance caused by hypoxia. Indeed, hypoxic conditions block the expression of the pro-apoptotic Bim as well as increasing ERK pathway activation 145, thereby identifying the molecular players of hypoxia-induced anoikis resistance.
Anoikis induction in cardiovascular diseases and diabetes
Enhancement of anoikis, not compensated by cell healing or overcompensated by a dysfunctional healing process, as observed during fibrosclerosis, is probably responsible for cardiovascular degenerative pathologies, such as cardiac myocyte detachment in heart failure, plaque rupture in atherosclerosis, smooth muscle cell disappearance in aneurysms and varicose veins and extensive loss of vascular cells during cardiovascular infections 146–148. Proteases able to degrade ECM proteins induce anoikis of endothelial cells or other components of the cardiovascular system, such as smooth muscle cells, fibroblasts and cardiac myocytes. Active proteases can either be secreted directly by inflammatory cells, such as elastase and cathepsin G by polymorphonucleated leukocytes, chymase and tryptase by mast cells, and granzymes by lymphocytes, or produced by the activation of circulating zymogens at the cell surface. This is the case for the pericellular conversion of plasminogen to plasmin, which degrades fibronectin and induces anoikis of endothelial cells and smooth muscle cells. These pericellular proteolytic events are claimed to play a predominant role in the initiation of atherosclerosis and during the consequent athero-thrombotic complications 149, 150. A similar proteolytic cascade involving pericellular plasminogen activation and fibronectin degradation is instrumental in the disappearance of smooth muscle cells during aneurysm and varicose vein development 151, 152. In the latter case, release of proteases by polymorphonucleated leukocytes, recruited and activated in response to blood stasis and hypoxia, is also involved in endothelial cell detachment and disappearance 152, 153. The absence of cell adhesion and growth resulting from cleavage of adhesive proteins also represents a major obstacle to cellular healing, including the absence of cell recolonization of proteolytically injured tissues and the low efficacy of cell transplantation 154.
Recent studies have also described a role of enzymes of the fibrinolytic system in aortic valvular tissue remodelling 155. In particular, plasminogen activation and plasmin generation occurs on the surface of valvular myofibroblasts that express the urokinase-type plasminogen activator (uPA). In aortic valves, inflammatory cells such as macrophages, recruited towards the stenotic valves, can secrete uPA, like myofibroblasts themselves 156. The plasmin generated in situ leads to the disruption of cell–ECM interactions, triggering an extensive anoikis of valvular myofibroblasts 155. The occurrence of an extensive anoikis of myofibroblasts within the valvular tissues likely plays a fundamental role during degenerative aortic stenosis, although this evidence still needs further clarification.
A similar proteinase-dependent aberrant tissue remodelling also contributes to the evolution of cardiovascular infectious disorders, such as infective endocarditis (IE). Beaufort and collegues have recently reported that the haematotropic pathogen Pseudomonas aeroginosa induce an intense anoikis of cardiomyocytes and valvular myofibroblasts, thus contributing to host tissue destruction 146. In particular, the uncontrolled pericellular proteolysis responsible for extensive cell detachment mainly depends on the metalloproteinase LasB, one of the well-known proteolytic enzymes secreted by P. aeruginosa. LasB causes a massive degradation of ECM-associated fibronectin and vitronectin, which precedes cell de-adherence. Moreover, LasB produces a rapid endoproteolysis of the cell-associated urokinase receptor uPAR, leaving a truncated receptor that is unable to support cell adhesion and survival via interactions with vitronectin and integrins 146. The LasB-dependent proteolysis of ECM components and loss of functional cell receptors affect most vascular tissues, both in the acute infectious context and likely during chronic vascular disease, including atherosclerosis and aneurysm.
Chronic vascular diseases are the major causes of mortality in diabetes 157. These disorders are associated with increased detachment and premature death of endothelial cells by anoikis. Indeed, in diabetic patients the early stages of microangiopathy is characterized by the development of acellular capillaries and decreased angiogenesis with consequent ischaemia 158. Interestingly, Dobler reported that methylglyoxal, a dicarbonyl glycating agent whose formation is improved in hyperglycaemia, provokes strong modifications in integrin-binding sites of type IV collagen within the vascular basement membrane, causing detachment and anoikis of endothelial cells 159, 160. Thus, increased formation of methylglyoxal and ECM glycation in hyperglycaemia impairs endothelial cell survival and vasculature structure and inhibits angiogenesis, likely contributing to vascular dysfunction in diabetes 159. Emerging therapeutic interventions aimed at scavenging methylglyoxal prevent the loss of endothelial cells and the impairment of angiogenesis, ultimately inhibiting the vascular complications of diabetes.
Diabetic retinopathy (DR) is another common complication in diabetes. Loss of retinal pericytes is one of the distinctive features of DR, which is characterized by retinal capillary obliteration. Because of their tissue localization outside the blood–retinal barrier, retinal pericytes rely entirely on survival signals derived from their surroundings ECM 161. However, diabetes-induced hyperglycaemia affects both the composition and function of the ECM, leading to accelerated death of retinal pericytes and endothelial cells. The cystein-rich protein 61 (Cyr61) and connective tissue growth factor (CTGF) are two ECM proteins whose expression is largely up-regulated in the retinal vasculature from the early stages of DR 162. An interesting study identifies Cyr61 and CTGF as anti-adhesive molecules initiating retinal pericytes anoikis, since these proteins compete with the constitutively expressed ECM proteins for integrin binding 163. In particular, Cyr61- and CTGF-induced cell detachment and consequent anoikis is mediated by the proteolytic activity of MMP-2. Of note, type IV collagen, the major structural element of the basement membrane in which pericytes are embedded in retinal capillaries, is the main target of MMP-2 activity. The MMP-2 gene has also been shown to be transcriptionally activated by p53, suggesting an important role of p53 as a mediator of Cyr61 and CTGF activity in pericytes 163.
Cell-based therapies and anoikis
Cell transplantation has attracted great interest as a potential means of treating several kinds of disease, such as neurological diseases, heart failure and myophathies. In the attempt to restore the lost function, one of the main problems encountered by researchers is the limited viability of transplanted cells. Indeed, despite its potential benefits, cell transplantation is limited in its clinical applications. It has been established that only ∼5% of mesenchymal stem cells, which are one of the best candidates for cell therapy in heart diseases, can survive for 14 days in the infarcted porcine heart 164. In addition, the survival rate of transplanted human mesenchymal stem cells in a mouse heart reaches < 0.5% at 4 days after transplantation 165. These discouraging results were also reported using different cell types, such as skeletal myoblasts, smooth muscle cells and unfractionated bone marrow cells, which can survive only few days after transplantation 166–168. In vivo, bone marrow-derived circulating mesenchymal stem cells are able to repopulate damaged tissues, leading to reconstruction of their function, due to the secretion of soluble factors exerting proliferative and anti-apoptotic actions 169. Conversely, during ex vivo treatments for cell-based therapy, mesenchymal stem cells increase their sensitivity to anoikis, due to up-regulation of pro-apoptotic molecules. For example, plasminogen activator inhibitor 1 is secreted by transplanted cells, leading to a decrease of their ECM contacts, leading to anoikis. In keeping with this, the inhibition of plasminogen activator inhibitor 1 improves autograft survival of bone marrow-derived cells 170.
During ex vivo cell preparation and after injection, cells are deprived of integrin signalling generated by ECM contact. In this period, for many hours, cells lose important adhesion-related survival signals, which are reinitiated when the cells find themselves in the recipient tissue. The association of cells with the ECM is fundamental for all cell types used in cell-based therapy, such as cardiac myocytes, smooth muscle cells, skeletal muscle precursors and stem cells in general. To further increase the damage, ROS can increase the anoikis signals during transplantation of myocardial precursors, since they are dramatically increased in the ischaemic heart and hinder cell adhesion 171. Hence, co-injection of skeletal myoblasts with ROS scavengers increases cell survival by about two-fold after the engraftment 168. Therefore, knowledge of molecules able to enhance the viability of the transplanted cells is crucial. Various strategies have been carried out to potentiate cell viability after transplantation: (a) pretreatment of the cells with growth factors or cytokines, including bFGF, insulin-like growth factor and bone morphogenic protein-2 in myocardial precursors 172 or adiponectin 173 and nitric oxide in mesoangioblasts 174; (b) genetic modification to induce over-expression of pro-survival molecules, including Akt 175, 176, bFGF 177, haem oxygenase-1 178, VEGF 179 and Bcl-2 180. Hence, strategies that improve cell viability during transplantation, allowing cells to evade anoikis when circulating in the bloodstream until they reach the target organ, appear essential for the success of cell-based therapy.
Anoikis has now emerged as a leading theme in the literature, and this is confirmed by the number of relevant papers that have recently appeared. Although intensive research on anoikis is only little more than a decade old, significant advances have been made. The success of the topic is mainly due to the different ways exploited by cancer cells to elude the innate suicidal response that an ‘homeless’ cell holds. Unfortunately, the anchorage dependence of different cells employs different signal transduction pathways and often many of these signals are concomitantly affected in cells that should be committed to anoikis. The final decision is the sum of all these inputs, thereby creating serious difficulties to a selective pharmacological approach to anoikis resistance. The current focus on anoikis-sensitizing drugs should be on network-targeted combinatorial algorithms, and the aim is to maximize therapeutic efficacy while minimizing the activation of alternative signalling pathways, which will otherwise contribute to emergence of single-drug resistance. One of the most promising approaches is genetic modulation of miRNAs able to regulate EMT and anoikis resistance. Indeed, the expression of miR-200c restores anoikis sensitivity through repression of mesenchymal genes involved in cell motility and anoikis resistance 139. Efficient in vivo delivery of the miR-200 family could successfully inhibit anoikis resistance, as well as other EMT-related steps during tumour progression, such as motility, stemness and chemoresistance. An integrated analysis of potential anoikis-sensitizing drugs and genetic manipulation of the miR-200 family will provide a relevant rational framework for the therapy of anoikis-resistant aggressive tumours.
The authors contributed equally to the writing of this paper.