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Cadherin mechanotransduction in leader-follower cell specification during collective migration
Experimental Cell Research 376 (2019) 86–91
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Cadherin mechanotransduction in leader-follower cell specification during collective migration
T
Antoine A. Khalil, Johan de Rooij
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Dept. Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Stratenum 3.231, Universiteitsweg 100, 3584 CG, Utrecht, the Netherlands
ARTICLE INFO
ABSTRACT
Keywords: Cadherin Mechanotransduction Cell polarity Collective cell migration Collective invasion
Collective invasion drives the spread of multicellular cancer groups, into the normal tissue surrounding several epithelial tumors. Collective invasion recapitulates various aspects of the multicellular organization and collective migration that take place during normal development and repair. Collective migration starts with the specification of leader cells in which a polarized, migratory phenotype is established. Leader cells initiate and organize the migration of follower cells, to allow the group of cells to move as a cohesive and polarized unit. Leader-follower specification is essential for coordinated and directional collective movement. Forces exerted by cohesive cells represent key signals that dictate multicellular coordination and directionality. Physical forces originate from the contraction of the actomyosin cytoskeleton, which is linked between cells via cadherin-based cell-cell junctions. The cadherin complex senses and transduces fluctuations in forces into biochemical signals that regulate processes like cell proliferation, motility and polarity. With cadherin junctions being maintained in most collective movements the cadherin complex is ideally positioned to integrate mechanical information into the organization of collective cell migration. Here we discuss the potential roles of cadherin mechanotransduction in the diverse aspects of leader versus follower cell specification during collective migration and neoplastic invasion.
1. Introduction Collective migration is a principal mechanism of cell movement, whereby group of cells maintain cell-cell adhesions and move in a directional and coordinated manner. Collective migration drives the formation and regeneration of several tissue types during morphogenesis and wound healing and repair [1]. Collective migration also drives multicellular invasion of many epithelial cancer cells into the peritumor stroma [2,3]. Collective migration depends on the establishment of a polarized group of cells, with a front-rear asymmetry. A key step in setting up this polarization is the specification of cells into one of two morphologically and functionally distinct cell fates, leader or follower cells. Leader cells extend cytoplasmic protrusions, which probe the extracellular environment for soluble factors and engage with the extracellular matrix (ECM) through integrin-based adhesions (Fig. 1). This results in actomyosin contraction of the cell body, which generates traction forces on the ECM that allow the movement of leader cells to the front. Follower cells can be dragged by the leader cell, but are also actively engaged in directional movement by extending directional cytoskeletal protrusions underneath the cells that are in front of them
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(termed cryptic lamellipodia) [4]. Nevertheless, in general, follower cells show less pronounced cytoplasmic protrusions, and lower degrees of cell-ECM interactions and traction forces [5]. Depending on the tissue context, individual cells within a multicellular group show distinct (predetermined) or similar abilities to become leader cells [6]. For instance, during trunk neural crest cell migration, leader and follower cell fates are predetermined prior to migration through an unknown mechanism [7]. Predetermined leader cells are also apparent in the assistance of tumor invasion by stromal cells, where cancer-associated fibroblasts (CAFs) initiate and lead the invasion of epithelial tumor cells [8]. In wound healing and cancer invasion models, a subset of epithelial cells that express myoepithelial/basal cell markers (incl. basal keratins) appears to be exclusively capable to form the leader cells [9,10]. For example, during collective invasion in mouse models of invasive ductal carcinoma, groups of basal-like epithelial cells initiate and guide the movement of multicellular invasive strands, whereas luminal cells remain as followers and fail to invade in the absence of basal-like cells [11]. Since, not every basal-like cell in these models, shows leader cell behavior, follower cells within such invasive strands consist as well of basal-like cells. The switch of quiescent basal-like cells into invasive leader cells requires signals from
Corresponding author. E-mail address: [email protected] (J. de Rooij).
https://doi.org/10.1016/j.yexcr.2019.01.006 Received 15 October 2018; Received in revised form 3 January 2019; Accepted 7 January 2019 Available online 08 January 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.
Experimental Cell Research 376 (2019) 86–91
A.A. Khalil, J. de Rooij
Fig. 1. Cadherin-mechanics in leader cell specification during collective cancer invasion. a) Initial tension on free-edged cells and b) transition into a specified leader cell. (a) A subset of free-edged cells with basal/mesenchymal characteristics (purple) are connected to the cells at the back by a mixture of homotypic (E-E) and heterotypic (E-P or E-N) cadherin junctions. Junctions are subjected to tension generated from actomyosin contractions. The presence of cadherin junction to one side renders junctional tension anisotropic. Anisotropic junctional tension initiates the specification of basal/mesenchymal cells into leader cells. (b) Specification into leader cell involves the acquisition of a front-rear asymmetry elicited by the establishment of subcellular polarized structures such as nuclear positioning to the rear of the cell, actin-rich protrusions and enhanced integrin ECM adhesion to the front. Cell-ECM adhesion promotes contraction of the cell body, which generates traction forces on the ECM (depicted and further forces on the junctions that orient the cryptic lamellipodia of the follower cells that are close to the leading edge. Table 1 Summary of major leader cell specification signals and cell dynamics during the collective migration of various cell types. ECM: Extracellular matrix; LC: Leader cell; FC: Follower cell; ND: Not determined; Y/N: Yes/No; VEGF: Vascular endothelial growth factor; FGF: Fibroblast growth factor; EGF: Epidermal growth factor; TGFβ: Transforming growth factor beta; SDF-1: Stromal derived factor-1; PVF: Platelet-derived/vascular endothelium-derived growth factor homologue; LMN: Laminin; FN: Fibronectin, ColI: Collagen I. Collective migration
Extracellular stimulus associated with LC specification
model
Soluble factor
ECM
Cell-cell
interchange (Y or N)
Trunk neural crest cell migration Angiogenesis Tracheal branching Mesendoderm Epithelial sheet migration Lateral line primordium Astrocyte migration Border cell migration CAFs-guided cancer invasion Collective cancer invasion
ND VEGF FGF ND EGF, TGFβ SDF-1 ND PVF, EGF ND ND
ND ND ND ND ND ND LMN ND FN ColI
ND ND ND C-cadherin E-cadherin ND N-cadherin E-cadherin E-N cadherin E- and P-cadherin
N Y Y ND ND ND ND Y ND Y
the extracellular environment (Table 1). For example, the presence of collagen-I in the ECM promotes the induction of collective invasion in breast cancer [12]. In other contexts, such as during endothelial sprouting and epithelial branching, cells with similar potential to become leaders can be specified based on stochastic events such as competition for sensing soluble growth factors present in the extracellular environment [13]. Once specified, the new leader cells subsequently suppress leader cell behavior in the followers by lateral inhibition such as through delta-notch signaling [14]. Soluble factors may also promote follower phenotype among trailing cells as exemplified by the FGF-mediated follower cell specification during the migration of the lateral line [15]. Specified leader cells from different tissue contexts acquire similar topology, characterized by membrane protrusions towards the front and cell-cell contact at the back (Fig. 1b). However, the dynamics of leader cell specification varies. Whereas during trunk neural crest cell migration, leader cells remain at the front for a long time without leader to follower interchange, leader cells transiently appear and can be replaced by followers that overtake them during endothelial sprouting, epithelial tracheal branching and breast cancer invasion (Table 1).
LC-FC
Ref.
[7] [13] [17] [18] [19–21] [22] [23,24] [25,26] [8,27] [9,12]
Besides soluble growth factors and chemokines, a key role is emerging for mechanical forces in determining leader-follower specifications. Mechanical forces originating from the actomyosin-dependent traction applied on the ECM are transmitted from the leader to the follower cells. Such mechanical coordination is essential for front-rear polarization and efficient collective migration [16]. Most of the insights come from studies in 2D grown cells such as the MDCK cell line. Typical experiments include scratch wounding or stencil release to induce collective migration and traction force microscopy (TFM) to measure cell-ECM forces and infer cell-cell forces (Box 1). Such experiments have shown that forces are coordinated at multicellular length scales and that such coordination determines the emergence and positioning of leader cells and the direction of migration of leader and follower cells [5,28–30]. Classical cadherins may have a central role in integrating multiple regulatory inputs in leader-follower specification and function: Classical cadherin-based junctions are a key component of migrating cell collectives; The cadherin complex connects the cytoskeletons of neighboring cells, is dynamic in nature and can respond to growth factor/chemokine signals, to changes in mechanical forces and to changes in cell fate such as EMT. Conversely, alterations in cadherin 87
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Box 1 . Scratch wound assay. Cells are seeded on ECM-coated surfaces to reach confluency. A scratch or wound is introduced by a pipette tip allowing the cells at the wound edge to polarize and migrate into the wounded region. The movement of most epithelial cells occurs as collective sheets with cell junction retained. Stencil release assay. To exclude the effect of the damage introduced by physical monolayer scratching, stencil release assay is used. Here, cells are seeded on a layer of PDMS that is on top of an ECM-coated surface. The PDMS layer contains PDMS-free regions with defined geometries. In those regions the ECM is available to the cells and allow adhesion and growth in a defined space. When confluency is reached, the PDMS stencil is removed that allow cells to move to free space. The movement away from the compact regions subject the cellcell junctions to forces Traction force microscopy (TFM). TFM uses the displacement of the ECM to compute the traction forces exerted by cells on their substrata. ECM displacement is detected by the net movement of the beads (placed within or under the cells) or by the displacement of ECM such collagen I fibers.
complex composition and stability affect many intracellular processes and cell behaviors that underlie leader-follower specification. Thus, integration of cell-intrinsic properties with extracellular signals from soluble mediators, cell-ECM and cell-cell contacts dictates leader cell specification (Table 1). The importance of leader-follower interplay in tumor invasion is becoming clear from several recent publications [9,11,31,32] and understanding the molecular mechanisms underlying their establishment and maintenance could provide targets for therapeutic intervention. Here we describe the signals that are elicited by forces on the cadherin complex and their potential involvement in controlling leader-follower cell specification during collective migration and invasion.
the re-organization of the cortex [39,41]. In addition, cell-cell junctions inhibit proximal integrin-mediated cell-ECM adhesion, which also depends on actomyosin organization and could be limited by cortical stiffening. Migratory adhesions and protrusions are not simply inhibited in follower cells, as exemplified by the presence of cryptic lamellipodia that extend underneath neighbors in the direction of migration. This is steered by E- and P-cadherin junctions in MDCK and myoblast cells [42,43]. Because junctional tension is anisotropic in follower cells, being higher at their front and back than at their sides, it is conceivable that force-activated α-catenin is involved in orienting cryptic lamellipodia, perhaps by causing asymmetry in the cortical cytoskeleton. Mutations in α-catenin that would perturb such asymmetry, by either inhibiting or enhancing vinculin interaction (ΔVBS [34] or Δmod [36] mutations, respectively), indeed disrupt the coordinated behavior of cell collectives and perturb directionality of collective migration. This has been observed in tissue culture models as well as in lateral mesoderm migration in zebrafish embryos [35,36,44]. Anisotropy in junctional tension may also explain why cell divisions among follower cells tend to align with the direction of migration as tension-dependent recruitment of LGN to E-cadherin was found to orient the spindle in the direction of force in stretched monolayers of epithelial cells [45]. The largest anisotropy in junctional tension obviously occurs in leader cells that have no junctions at their front. Applying tension on cadherins on one side of the cell results in polarization in the opposite direction (Fig. 1) in multiple systems [18,46]. This may depend on the mechanical regulation of the α-catenin-vinculin interaction as was shown in CAF-lead tumor invasion, where perturbation of this interaction resulted in a loss of polarization of the leading CAF [8,35,36]. In collective cell migration of astrocytes, N-cadherin-based focal adherens junctions undergo a rearward flow along the lateral contact between 2 leader cells from the leading edge to the back of the cell [23]. This flow is dependent on their contact to transvers actomyosin bundles that also flow backwards. The punctate appearance of the adhesion complexes, and their direct contact to radial actomyosin indicates a tensile state of the cadherin complex, but a role for a-catenin or vinculin at this junction was not investigated. This polarized cadherin junction flow was not observed among follower cells and was needed for efficient collective migration. Thus the actomyosin-dependent dynamics of complete cadherin adhesion domains that is differentially regulated between leaders and followers, is also important in leader and follower cell behavior [23]. A recent addition to the repertoire of force-induced cadherin-proximal processes is the recruitment of BAR domain-containing proteins to cadherin junctions between follower and leader cells in endothelial collectives. The higher contractility and stress fiber presence in leader vs follower cells results in the formation (or lingering) of finger-like protrusions that stick into follower cells and are stabilized by mechanically activated VE-cadherin junctions [47,48] These finger-like junctions are associated with highly curved membranes and this results
2. Proximal effects of cadherin mechanotransduction in early leader and follower specification The core of a classical cadherin adhesion complex is formed by the cadherin itself with β-catenin and α-catenin. Together with a mirror image in a neighboring cell, this complex forms an intercellular forcechain that connects 2 actomyosin cytoskeletons. In the simple configuration of the complex (Fig. 2a), changes in actomyosin contractility result in changes in tension across the cadherin complex. This may have a number of direct molecular effects that lead to the conversion of mechanical force into biochemical response (the definition of mechanotransduction). The best established among these is the opening of a vinculin binding site on α-catenin that results in enhanced vinculin residence and strengthening of the cadherin-based junction under increased tension [33,34]. Other α-catenin-interacting proteins like formins and affadin may also be recruited to cell-cell junctions in a tension-dependent manner, although a molecular explanation for the tension sensitivity of this is absent [8,35,36]. In addition, multiple catch-bonds within the protein complex will strengthen with increased tension [37] and a number of actin-regulating proteins like Vasp, Zyxin and TES are recruited [38]. Together these force responses influence the organization of the actomyosin cytoskeleton in the vicinity of a junction, which has been measured as cortical stiffening in magnetic twisting cytometry (MTC) experiments using cadherin-coated beads and observed in multiple studies by microscopy analysis of junctionassociated F-actin [37]. Although not directly shown, junction-proximal actomyosin adaptations may contribute to leader and follower cell acquisition in a number of ways. First of all cortical stiffening may control the extent and orientation of membrane protrusions in junction proximal regions [39–41]. Cytoskeletal protrusions are inhibited near the posterior cellcell junction of a cell with a free edge, whereas as the free edge allows active protrusion. This is in contrast to the cells that lack free edges, where protrusion-inhibition takes place along the complete junctioncontaining cortex. Cadherin junctions also regulate Rac and ARP2/3 activity to inhibit membrane protrusions, which likely interplays with 88
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Fig. 2. Molecular effects of tension on the cadherin junction and downstream cellular effects. a) Force-mediated molecular changes in the cadherin junction complex and b) downstream distant effects. (a) Actomyosin contractions subject cadherin junctions to tensions, which induces changes in α-catenin conformation (closed to open conformation), increasing its protein-binding sites availability. The open conformation of α-catenin allows its interactions with vinculin which binds to F-actin to strengthen the junction. Several of the interactions are reinforced in response to tension, such catch bonds (*) form between the engaged cadherin extracellular domains, between α-catenin – F-actin and Vinculin-F-actin. Several actin-regulators and -binding proteins are recruited to the force-activated junctional complex including TES, Vasp, Zyxin, afadin and formin. Tension on cadherins induces β-catenin phosphorylation. (b) Several signaling cascades are activated downstream of the tension-induced molecular changes. PI3K and Rho-GTPases mediate cytoskeletal rearrangement and integrin adhesion that modulate cellular stiffness and promote polarized actin-rich protrusions towards the cell-free edge. Increased contractility applies additional forces on the cadherin junctions. Additional tension on the junctions further increases nuclear translocation of β-catenin and YAP. In the nucleus, TCF- and TEAD-dependent gene transcription promotes mesenchymal and basal fate molecular programs that potentiate cell front-rear polarity and motility.
in the specific recruitment of N- and F-BAR domain proteins to the follower cell side of the junction. This leads to differential VE-cadherin turnover between leader and follower cells and directs the formation of cryptic lamellipodia in the follower cell in the direction of migration [47,48], both of which are essential for efficient collective migration in endothelial cell systems. Possible causes and consequences of cadherin mechanotransduction on leader cell specification mentioned in this section are summarized (Fig. 1) in a hypothetical cell that combines insights from multiple biological model systems. In conclusion, multiple mechanically controlled molecular responses in the proximity of the cadherin complex maybe involved in the control of leader and follower cell specifications and it is conceivable that this is involved in the rapid changes between leader-follower behaviors in dynamic modes of collective migration and in the early steps of leader cell specification in more stable modes.
sheets [52]. Similarly, during collective migration of myoblasts, Pcadherin mediates the polarized and dynamic focal contacts at the leading edge [43]. Rho-GTPases are also involved in these responses to tension at cadherin junctions. Rho-GTPases are the central regulators of actomyosin contractility and actin rich protrusions (lamellipodia, pseudopodia, filopodia) [53]. Rho-GTPases have been found important in the collective migration of several cell types including mesendoderm, border, neural crest, myoblasts and epithelial cells [18,25,28,43,54]. In border cell migration, Rac1 activation in leader cells is proposed to be dependent on the increase in E-cadherin tension in leader cells as compared to follower cells [25]. In MDCK sheets, Rac1 activity is controlled by the tight junction adhesion complex, which releases Merlin in a tension-dependent manner into the cytoplasm of follower cells, resulting in a front-rear gradient of Rac activity that promotes migration in the direction of the leader cell [55]. Activation of RhoA, and CDC42, both inducers of actomyosin contractility, downstream of cadherin adhesion has been documented in multiple models, but the exact molecular link and the possible regulation by tension has remained elusive, despite a number of Rho-family GEFs being found at the cadherin junction [56,57]. Interestingly, different classical cadherins appear to have different capacities to regulate Rho-GTPases and cell mechanics, even though they all assemble the same core mechanotransduction complex and can all sense forces through the α-catenin-vinculin interaction [37]. For instance, in MCF10A mammary epithelial cells that express both E- and P-cadherin, E-cadherin determines the rate of monolayer stiffening upon a stencil release, whereas P-cadherin determines the amount of tissue tension that develops [58]. In another cell type (C2C12 myoblasts) increased P-cadherin, but not E-cadherin expression led to increased contractility through activation of CDC42 [43]. Strikingly, Pcadherin is expressed in basal-like epithelial cells that lead collective breast cancer invasion, but not in the luminal cells that are limited to
3. Global effects of cadherin mechanotransduction on cell mechanics in collective migration The subcellular responses to cadherin tension that maybe involved in leader-follower specification likely extend farther than the vicinity of cell-cell junctions (Fig. 2b). For instance, posterior cadherin junctions induce basic features of leader cells namely the extension of actin-rich protrusions towards the free edge [18,20,25,41,46,49]. Through the cytoskeletal protrusions leader cells engage with the ECM via integrinmediated adhesion and generate traction forces that allow leader cells to initiate migration. Recently it was found that tension on E-cadherin drives these processes as it activates PI3K in an EGFR-dependent mechanism to promote the formation of new integrin adhesions and ROCK-dependent cell contractility [50]. In line with this, E-cadherinbased junctions are required for directed traction force generation at the periphery of keratinocyte colonies [51] and migrating epithelial 89
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follower behavior [9]. Such breast cancer leader cells indeed show enhanced ECM traction force generation and collagen-I fiber alignment [59,60]. In general, positive feedback systems appear to exist where increasing tension on cadherin-based junctions drives Rho-GTPase signaling to increase global cellular contractility leading to a further increase in junctional tension (Fig. 2b). The specific types of cadherins expressed and GTPases activated may determine the details of this feedback. The arising of differences in cadherin tension between early leader and follower cells may thus be propagated to be a driving force in stabilization of leader-follower behavior. Most of these potential links between cadherin mechanotransduction and leader-follower behavior are currently speculative. Unraveling the molecular details of the interactions between cadherin junctions, Rho-GTPase signaling and actomyosin contractility is needed to really understand how cadherin junctions and cellular mechanics interplay to regulate collective cell behavior.
stabilize leader and follower cell behavior. Finally, cadherin-dependent transcriptional programs including YAP/TEAD and β-catenin/TCF may provide longer term stabilization of leader and follower cell fates in migrating collectives. Only certain aspects of this model may apply in particular biological systems. For instance, stochastic leader cell selection occurs in dynamic models such as the neural crest and endothelial cell collectives, whereas transcriptional distinction already exists in CAF-lead tumor invasion, but leader behavior still is induced and stabilized by the cadherin contact between CAF and tumor cell. CAFs express a different cadherin than epithelial cells and specific cadherin expression profiles may indeed feed into this model as well. This may occur in invasive ductal carcinoma models, where a leader is stochastically selected among the P-cadherin expressing basal-like cells. The presence of P-cadherin may enlarge the effects on global contractility in these leader cells. In conclusion, multiple roles of cadherin mechanotransduction in various aspects of collective cell migration can be envisioned and molded into a sequential model. This underscores the central position of classical cadherins in interpreting mechanical forces in collective cell behavior.
4. Cadherin mechanotransduction and transcriptional regulation of leader-cell specification In addition to the mechanoresponses that regulate proximal and global cell mechanics, tension on cadherin junctions also affect transcriptional programs involved in leader-follower cell specification. For instance, tension on E-cadherin results in the increased phosphorylation of β-catenin at Y654 (Fig. 2b), which lowers its stability at E-cadherinbased junctions and increases its nuclear activity to drive TCF-dependent gene transcription and cell proliferation [61,62]. Notably, another recent study reports the opposite, where loss of tension on E-cadherin promotes transcriptional activities of β-catenin independent of phosphorylation of Y654 [63]. Similar to β-catenin, the transcriptional cofactor YAP is activated by tension at the cadherin complex [61]. This may involve sequestration of components of YAP-phosphorylation pathways at junctions [64] or release of sequestration of YAP itself [65]. Different YAP-regulating mechanisms have been assigned to Eand VE-cadherin and may point to differences among classical cadherins. As none of the described intermediates in these pathways actually interact with the mechanically regulated parts of the cadherin complex, we still lack a solid molecular explanation of YAP regulation by force on cadherins. Besides by phosphorylation-dependent mechanisms, YAP activation is also more directly regulated by cellular mechanics. Cell stiffening, a global response to cadherin tension as described above, is one of the key drivers of YAP activation [66] and may thus further enhance transcriptional regulation downstream of cadherin tension. Even though the exact mechanisms remain unclear, it is clear that mechanical forces at cadherin junctions influence YAP and β-catenin transcriptional programs, both of which are implicated in the EMT-like processes that occur in normal and neoplastic leader cells [31,67]. Thus, together with the proximal and global regulation of cell mechanics, regulation of transcriptional pathways may contribute to the effect of cadherin mechanotransduction on leader-follower cell specification.
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5. Conclusion Classical cadherins are ideally positioned to regulate the behaviors of cell collectives in response to forces that drive their migration and invasion. In the first step of a sequential model, cadherin proximal mechanotransduction, dependent on α-catenin, vinculin and local actomyosin organization, provides a fast mode of cell-cell communication. This may drive the initial polarization of the cortices of individual cells to adapt to their emerging distinct roles as leaders or followers. In the next step, cadherin-induced regulation of actomyosin contractility in more distant parts of the cell, through PI3K-dependent integrin adhesion and through modulation of Rho-GTPase signaling, affects global cell mechanics and may further polarize the migration machinery to 90
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Cadherin mechanotransduction in leader-follower cell specification during collective migration
T
Antoine A. Khalil, Johan de Rooij
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Dept. Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Stratenum 3.231, Universiteitsweg 100, 3584 CG, Utrecht, the Netherlands
ARTICLE INFO
ABSTRACT
Keywords: Cadherin Mechanotransduction Cell polarity Collective cell migration Collective invasion
Collective invasion drives the spread of multicellular cancer groups, into the normal tissue surrounding several epithelial tumors. Collective invasion recapitulates various aspects of the multicellular organization and collective migration that take place during normal development and repair. Collective migration starts with the specification of leader cells in which a polarized, migratory phenotype is established. Leader cells initiate and organize the migration of follower cells, to allow the group of cells to move as a cohesive and polarized unit. Leader-follower specification is essential for coordinated and directional collective movement. Forces exerted by cohesive cells represent key signals that dictate multicellular coordination and directionality. Physical forces originate from the contraction of the actomyosin cytoskeleton, which is linked between cells via cadherin-based cell-cell junctions. The cadherin complex senses and transduces fluctuations in forces into biochemical signals that regulate processes like cell proliferation, motility and polarity. With cadherin junctions being maintained in most collective movements the cadherin complex is ideally positioned to integrate mechanical information into the organization of collective cell migration. Here we discuss the potential roles of cadherin mechanotransduction in the diverse aspects of leader versus follower cell specification during collective migration and neoplastic invasion.
1. Introduction Collective migration is a principal mechanism of cell movement, whereby group of cells maintain cell-cell adhesions and move in a directional and coordinated manner. Collective migration drives the formation and regeneration of several tissue types during morphogenesis and wound healing and repair [1]. Collective migration also drives multicellular invasion of many epithelial cancer cells into the peritumor stroma [2,3]. Collective migration depends on the establishment of a polarized group of cells, with a front-rear asymmetry. A key step in setting up this polarization is the specification of cells into one of two morphologically and functionally distinct cell fates, leader or follower cells. Leader cells extend cytoplasmic protrusions, which probe the extracellular environment for soluble factors and engage with the extracellular matrix (ECM) through integrin-based adhesions (Fig. 1). This results in actomyosin contraction of the cell body, which generates traction forces on the ECM that allow the movement of leader cells to the front. Follower cells can be dragged by the leader cell, but are also actively engaged in directional movement by extending directional cytoskeletal protrusions underneath the cells that are in front of them
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(termed cryptic lamellipodia) [4]. Nevertheless, in general, follower cells show less pronounced cytoplasmic protrusions, and lower degrees of cell-ECM interactions and traction forces [5]. Depending on the tissue context, individual cells within a multicellular group show distinct (predetermined) or similar abilities to become leader cells [6]. For instance, during trunk neural crest cell migration, leader and follower cell fates are predetermined prior to migration through an unknown mechanism [7]. Predetermined leader cells are also apparent in the assistance of tumor invasion by stromal cells, where cancer-associated fibroblasts (CAFs) initiate and lead the invasion of epithelial tumor cells [8]. In wound healing and cancer invasion models, a subset of epithelial cells that express myoepithelial/basal cell markers (incl. basal keratins) appears to be exclusively capable to form the leader cells [9,10]. For example, during collective invasion in mouse models of invasive ductal carcinoma, groups of basal-like epithelial cells initiate and guide the movement of multicellular invasive strands, whereas luminal cells remain as followers and fail to invade in the absence of basal-like cells [11]. Since, not every basal-like cell in these models, shows leader cell behavior, follower cells within such invasive strands consist as well of basal-like cells. The switch of quiescent basal-like cells into invasive leader cells requires signals from
Corresponding author. E-mail address: [email protected] (J. de Rooij).
https://doi.org/10.1016/j.yexcr.2019.01.006 Received 15 October 2018; Received in revised form 3 January 2019; Accepted 7 January 2019 Available online 08 January 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.
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Fig. 1. Cadherin-mechanics in leader cell specification during collective cancer invasion. a) Initial tension on free-edged cells and b) transition into a specified leader cell. (a) A subset of free-edged cells with basal/mesenchymal characteristics (purple) are connected to the cells at the back by a mixture of homotypic (E-E) and heterotypic (E-P or E-N) cadherin junctions. Junctions are subjected to tension generated from actomyosin contractions. The presence of cadherin junction to one side renders junctional tension anisotropic. Anisotropic junctional tension initiates the specification of basal/mesenchymal cells into leader cells. (b) Specification into leader cell involves the acquisition of a front-rear asymmetry elicited by the establishment of subcellular polarized structures such as nuclear positioning to the rear of the cell, actin-rich protrusions and enhanced integrin ECM adhesion to the front. Cell-ECM adhesion promotes contraction of the cell body, which generates traction forces on the ECM (depicted and further forces on the junctions that orient the cryptic lamellipodia of the follower cells that are close to the leading edge. Table 1 Summary of major leader cell specification signals and cell dynamics during the collective migration of various cell types. ECM: Extracellular matrix; LC: Leader cell; FC: Follower cell; ND: Not determined; Y/N: Yes/No; VEGF: Vascular endothelial growth factor; FGF: Fibroblast growth factor; EGF: Epidermal growth factor; TGFβ: Transforming growth factor beta; SDF-1: Stromal derived factor-1; PVF: Platelet-derived/vascular endothelium-derived growth factor homologue; LMN: Laminin; FN: Fibronectin, ColI: Collagen I. Collective migration
Extracellular stimulus associated with LC specification
model
Soluble factor
ECM
Cell-cell
interchange (Y or N)
Trunk neural crest cell migration Angiogenesis Tracheal branching Mesendoderm Epithelial sheet migration Lateral line primordium Astrocyte migration Border cell migration CAFs-guided cancer invasion Collective cancer invasion
ND VEGF FGF ND EGF, TGFβ SDF-1 ND PVF, EGF ND ND
ND ND ND ND ND ND LMN ND FN ColI
ND ND ND C-cadherin E-cadherin ND N-cadherin E-cadherin E-N cadherin E- and P-cadherin
N Y Y ND ND ND ND Y ND Y
the extracellular environment (Table 1). For example, the presence of collagen-I in the ECM promotes the induction of collective invasion in breast cancer [12]. In other contexts, such as during endothelial sprouting and epithelial branching, cells with similar potential to become leaders can be specified based on stochastic events such as competition for sensing soluble growth factors present in the extracellular environment [13]. Once specified, the new leader cells subsequently suppress leader cell behavior in the followers by lateral inhibition such as through delta-notch signaling [14]. Soluble factors may also promote follower phenotype among trailing cells as exemplified by the FGF-mediated follower cell specification during the migration of the lateral line [15]. Specified leader cells from different tissue contexts acquire similar topology, characterized by membrane protrusions towards the front and cell-cell contact at the back (Fig. 1b). However, the dynamics of leader cell specification varies. Whereas during trunk neural crest cell migration, leader cells remain at the front for a long time without leader to follower interchange, leader cells transiently appear and can be replaced by followers that overtake them during endothelial sprouting, epithelial tracheal branching and breast cancer invasion (Table 1).
LC-FC
Ref.
[7] [13] [17] [18] [19–21] [22] [23,24] [25,26] [8,27] [9,12]
Besides soluble growth factors and chemokines, a key role is emerging for mechanical forces in determining leader-follower specifications. Mechanical forces originating from the actomyosin-dependent traction applied on the ECM are transmitted from the leader to the follower cells. Such mechanical coordination is essential for front-rear polarization and efficient collective migration [16]. Most of the insights come from studies in 2D grown cells such as the MDCK cell line. Typical experiments include scratch wounding or stencil release to induce collective migration and traction force microscopy (TFM) to measure cell-ECM forces and infer cell-cell forces (Box 1). Such experiments have shown that forces are coordinated at multicellular length scales and that such coordination determines the emergence and positioning of leader cells and the direction of migration of leader and follower cells [5,28–30]. Classical cadherins may have a central role in integrating multiple regulatory inputs in leader-follower specification and function: Classical cadherin-based junctions are a key component of migrating cell collectives; The cadherin complex connects the cytoskeletons of neighboring cells, is dynamic in nature and can respond to growth factor/chemokine signals, to changes in mechanical forces and to changes in cell fate such as EMT. Conversely, alterations in cadherin 87
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Box 1 . Scratch wound assay. Cells are seeded on ECM-coated surfaces to reach confluency. A scratch or wound is introduced by a pipette tip allowing the cells at the wound edge to polarize and migrate into the wounded region. The movement of most epithelial cells occurs as collective sheets with cell junction retained. Stencil release assay. To exclude the effect of the damage introduced by physical monolayer scratching, stencil release assay is used. Here, cells are seeded on a layer of PDMS that is on top of an ECM-coated surface. The PDMS layer contains PDMS-free regions with defined geometries. In those regions the ECM is available to the cells and allow adhesion and growth in a defined space. When confluency is reached, the PDMS stencil is removed that allow cells to move to free space. The movement away from the compact regions subject the cellcell junctions to forces Traction force microscopy (TFM). TFM uses the displacement of the ECM to compute the traction forces exerted by cells on their substrata. ECM displacement is detected by the net movement of the beads (placed within or under the cells) or by the displacement of ECM such collagen I fibers.
complex composition and stability affect many intracellular processes and cell behaviors that underlie leader-follower specification. Thus, integration of cell-intrinsic properties with extracellular signals from soluble mediators, cell-ECM and cell-cell contacts dictates leader cell specification (Table 1). The importance of leader-follower interplay in tumor invasion is becoming clear from several recent publications [9,11,31,32] and understanding the molecular mechanisms underlying their establishment and maintenance could provide targets for therapeutic intervention. Here we describe the signals that are elicited by forces on the cadherin complex and their potential involvement in controlling leader-follower cell specification during collective migration and invasion.
the re-organization of the cortex [39,41]. In addition, cell-cell junctions inhibit proximal integrin-mediated cell-ECM adhesion, which also depends on actomyosin organization and could be limited by cortical stiffening. Migratory adhesions and protrusions are not simply inhibited in follower cells, as exemplified by the presence of cryptic lamellipodia that extend underneath neighbors in the direction of migration. This is steered by E- and P-cadherin junctions in MDCK and myoblast cells [42,43]. Because junctional tension is anisotropic in follower cells, being higher at their front and back than at their sides, it is conceivable that force-activated α-catenin is involved in orienting cryptic lamellipodia, perhaps by causing asymmetry in the cortical cytoskeleton. Mutations in α-catenin that would perturb such asymmetry, by either inhibiting or enhancing vinculin interaction (ΔVBS [34] or Δmod [36] mutations, respectively), indeed disrupt the coordinated behavior of cell collectives and perturb directionality of collective migration. This has been observed in tissue culture models as well as in lateral mesoderm migration in zebrafish embryos [35,36,44]. Anisotropy in junctional tension may also explain why cell divisions among follower cells tend to align with the direction of migration as tension-dependent recruitment of LGN to E-cadherin was found to orient the spindle in the direction of force in stretched monolayers of epithelial cells [45]. The largest anisotropy in junctional tension obviously occurs in leader cells that have no junctions at their front. Applying tension on cadherins on one side of the cell results in polarization in the opposite direction (Fig. 1) in multiple systems [18,46]. This may depend on the mechanical regulation of the α-catenin-vinculin interaction as was shown in CAF-lead tumor invasion, where perturbation of this interaction resulted in a loss of polarization of the leading CAF [8,35,36]. In collective cell migration of astrocytes, N-cadherin-based focal adherens junctions undergo a rearward flow along the lateral contact between 2 leader cells from the leading edge to the back of the cell [23]. This flow is dependent on their contact to transvers actomyosin bundles that also flow backwards. The punctate appearance of the adhesion complexes, and their direct contact to radial actomyosin indicates a tensile state of the cadherin complex, but a role for a-catenin or vinculin at this junction was not investigated. This polarized cadherin junction flow was not observed among follower cells and was needed for efficient collective migration. Thus the actomyosin-dependent dynamics of complete cadherin adhesion domains that is differentially regulated between leaders and followers, is also important in leader and follower cell behavior [23]. A recent addition to the repertoire of force-induced cadherin-proximal processes is the recruitment of BAR domain-containing proteins to cadherin junctions between follower and leader cells in endothelial collectives. The higher contractility and stress fiber presence in leader vs follower cells results in the formation (or lingering) of finger-like protrusions that stick into follower cells and are stabilized by mechanically activated VE-cadherin junctions [47,48] These finger-like junctions are associated with highly curved membranes and this results
2. Proximal effects of cadherin mechanotransduction in early leader and follower specification The core of a classical cadherin adhesion complex is formed by the cadherin itself with β-catenin and α-catenin. Together with a mirror image in a neighboring cell, this complex forms an intercellular forcechain that connects 2 actomyosin cytoskeletons. In the simple configuration of the complex (Fig. 2a), changes in actomyosin contractility result in changes in tension across the cadherin complex. This may have a number of direct molecular effects that lead to the conversion of mechanical force into biochemical response (the definition of mechanotransduction). The best established among these is the opening of a vinculin binding site on α-catenin that results in enhanced vinculin residence and strengthening of the cadherin-based junction under increased tension [33,34]. Other α-catenin-interacting proteins like formins and affadin may also be recruited to cell-cell junctions in a tension-dependent manner, although a molecular explanation for the tension sensitivity of this is absent [8,35,36]. In addition, multiple catch-bonds within the protein complex will strengthen with increased tension [37] and a number of actin-regulating proteins like Vasp, Zyxin and TES are recruited [38]. Together these force responses influence the organization of the actomyosin cytoskeleton in the vicinity of a junction, which has been measured as cortical stiffening in magnetic twisting cytometry (MTC) experiments using cadherin-coated beads and observed in multiple studies by microscopy analysis of junctionassociated F-actin [37]. Although not directly shown, junction-proximal actomyosin adaptations may contribute to leader and follower cell acquisition in a number of ways. First of all cortical stiffening may control the extent and orientation of membrane protrusions in junction proximal regions [39–41]. Cytoskeletal protrusions are inhibited near the posterior cellcell junction of a cell with a free edge, whereas as the free edge allows active protrusion. This is in contrast to the cells that lack free edges, where protrusion-inhibition takes place along the complete junctioncontaining cortex. Cadherin junctions also regulate Rac and ARP2/3 activity to inhibit membrane protrusions, which likely interplays with 88
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Fig. 2. Molecular effects of tension on the cadherin junction and downstream cellular effects. a) Force-mediated molecular changes in the cadherin junction complex and b) downstream distant effects. (a) Actomyosin contractions subject cadherin junctions to tensions, which induces changes in α-catenin conformation (closed to open conformation), increasing its protein-binding sites availability. The open conformation of α-catenin allows its interactions with vinculin which binds to F-actin to strengthen the junction. Several of the interactions are reinforced in response to tension, such catch bonds (*) form between the engaged cadherin extracellular domains, between α-catenin – F-actin and Vinculin-F-actin. Several actin-regulators and -binding proteins are recruited to the force-activated junctional complex including TES, Vasp, Zyxin, afadin and formin. Tension on cadherins induces β-catenin phosphorylation. (b) Several signaling cascades are activated downstream of the tension-induced molecular changes. PI3K and Rho-GTPases mediate cytoskeletal rearrangement and integrin adhesion that modulate cellular stiffness and promote polarized actin-rich protrusions towards the cell-free edge. Increased contractility applies additional forces on the cadherin junctions. Additional tension on the junctions further increases nuclear translocation of β-catenin and YAP. In the nucleus, TCF- and TEAD-dependent gene transcription promotes mesenchymal and basal fate molecular programs that potentiate cell front-rear polarity and motility.
in the specific recruitment of N- and F-BAR domain proteins to the follower cell side of the junction. This leads to differential VE-cadherin turnover between leader and follower cells and directs the formation of cryptic lamellipodia in the follower cell in the direction of migration [47,48], both of which are essential for efficient collective migration in endothelial cell systems. Possible causes and consequences of cadherin mechanotransduction on leader cell specification mentioned in this section are summarized (Fig. 1) in a hypothetical cell that combines insights from multiple biological model systems. In conclusion, multiple mechanically controlled molecular responses in the proximity of the cadherin complex maybe involved in the control of leader and follower cell specifications and it is conceivable that this is involved in the rapid changes between leader-follower behaviors in dynamic modes of collective migration and in the early steps of leader cell specification in more stable modes.
sheets [52]. Similarly, during collective migration of myoblasts, Pcadherin mediates the polarized and dynamic focal contacts at the leading edge [43]. Rho-GTPases are also involved in these responses to tension at cadherin junctions. Rho-GTPases are the central regulators of actomyosin contractility and actin rich protrusions (lamellipodia, pseudopodia, filopodia) [53]. Rho-GTPases have been found important in the collective migration of several cell types including mesendoderm, border, neural crest, myoblasts and epithelial cells [18,25,28,43,54]. In border cell migration, Rac1 activation in leader cells is proposed to be dependent on the increase in E-cadherin tension in leader cells as compared to follower cells [25]. In MDCK sheets, Rac1 activity is controlled by the tight junction adhesion complex, which releases Merlin in a tension-dependent manner into the cytoplasm of follower cells, resulting in a front-rear gradient of Rac activity that promotes migration in the direction of the leader cell [55]. Activation of RhoA, and CDC42, both inducers of actomyosin contractility, downstream of cadherin adhesion has been documented in multiple models, but the exact molecular link and the possible regulation by tension has remained elusive, despite a number of Rho-family GEFs being found at the cadherin junction [56,57]. Interestingly, different classical cadherins appear to have different capacities to regulate Rho-GTPases and cell mechanics, even though they all assemble the same core mechanotransduction complex and can all sense forces through the α-catenin-vinculin interaction [37]. For instance, in MCF10A mammary epithelial cells that express both E- and P-cadherin, E-cadherin determines the rate of monolayer stiffening upon a stencil release, whereas P-cadherin determines the amount of tissue tension that develops [58]. In another cell type (C2C12 myoblasts) increased P-cadherin, but not E-cadherin expression led to increased contractility through activation of CDC42 [43]. Strikingly, Pcadherin is expressed in basal-like epithelial cells that lead collective breast cancer invasion, but not in the luminal cells that are limited to
3. Global effects of cadherin mechanotransduction on cell mechanics in collective migration The subcellular responses to cadherin tension that maybe involved in leader-follower specification likely extend farther than the vicinity of cell-cell junctions (Fig. 2b). For instance, posterior cadherin junctions induce basic features of leader cells namely the extension of actin-rich protrusions towards the free edge [18,20,25,41,46,49]. Through the cytoskeletal protrusions leader cells engage with the ECM via integrinmediated adhesion and generate traction forces that allow leader cells to initiate migration. Recently it was found that tension on E-cadherin drives these processes as it activates PI3K in an EGFR-dependent mechanism to promote the formation of new integrin adhesions and ROCK-dependent cell contractility [50]. In line with this, E-cadherinbased junctions are required for directed traction force generation at the periphery of keratinocyte colonies [51] and migrating epithelial 89
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follower behavior [9]. Such breast cancer leader cells indeed show enhanced ECM traction force generation and collagen-I fiber alignment [59,60]. In general, positive feedback systems appear to exist where increasing tension on cadherin-based junctions drives Rho-GTPase signaling to increase global cellular contractility leading to a further increase in junctional tension (Fig. 2b). The specific types of cadherins expressed and GTPases activated may determine the details of this feedback. The arising of differences in cadherin tension between early leader and follower cells may thus be propagated to be a driving force in stabilization of leader-follower behavior. Most of these potential links between cadherin mechanotransduction and leader-follower behavior are currently speculative. Unraveling the molecular details of the interactions between cadherin junctions, Rho-GTPase signaling and actomyosin contractility is needed to really understand how cadherin junctions and cellular mechanics interplay to regulate collective cell behavior.
stabilize leader and follower cell behavior. Finally, cadherin-dependent transcriptional programs including YAP/TEAD and β-catenin/TCF may provide longer term stabilization of leader and follower cell fates in migrating collectives. Only certain aspects of this model may apply in particular biological systems. For instance, stochastic leader cell selection occurs in dynamic models such as the neural crest and endothelial cell collectives, whereas transcriptional distinction already exists in CAF-lead tumor invasion, but leader behavior still is induced and stabilized by the cadherin contact between CAF and tumor cell. CAFs express a different cadherin than epithelial cells and specific cadherin expression profiles may indeed feed into this model as well. This may occur in invasive ductal carcinoma models, where a leader is stochastically selected among the P-cadherin expressing basal-like cells. The presence of P-cadherin may enlarge the effects on global contractility in these leader cells. In conclusion, multiple roles of cadherin mechanotransduction in various aspects of collective cell migration can be envisioned and molded into a sequential model. This underscores the central position of classical cadherins in interpreting mechanical forces in collective cell behavior.
4. Cadherin mechanotransduction and transcriptional regulation of leader-cell specification In addition to the mechanoresponses that regulate proximal and global cell mechanics, tension on cadherin junctions also affect transcriptional programs involved in leader-follower cell specification. For instance, tension on E-cadherin results in the increased phosphorylation of β-catenin at Y654 (Fig. 2b), which lowers its stability at E-cadherinbased junctions and increases its nuclear activity to drive TCF-dependent gene transcription and cell proliferation [61,62]. Notably, another recent study reports the opposite, where loss of tension on E-cadherin promotes transcriptional activities of β-catenin independent of phosphorylation of Y654 [63]. Similar to β-catenin, the transcriptional cofactor YAP is activated by tension at the cadherin complex [61]. This may involve sequestration of components of YAP-phosphorylation pathways at junctions [64] or release of sequestration of YAP itself [65]. Different YAP-regulating mechanisms have been assigned to Eand VE-cadherin and may point to differences among classical cadherins. As none of the described intermediates in these pathways actually interact with the mechanically regulated parts of the cadherin complex, we still lack a solid molecular explanation of YAP regulation by force on cadherins. Besides by phosphorylation-dependent mechanisms, YAP activation is also more directly regulated by cellular mechanics. Cell stiffening, a global response to cadherin tension as described above, is one of the key drivers of YAP activation [66] and may thus further enhance transcriptional regulation downstream of cadherin tension. Even though the exact mechanisms remain unclear, it is clear that mechanical forces at cadherin junctions influence YAP and β-catenin transcriptional programs, both of which are implicated in the EMT-like processes that occur in normal and neoplastic leader cells [31,67]. Thus, together with the proximal and global regulation of cell mechanics, regulation of transcriptional pathways may contribute to the effect of cadherin mechanotransduction on leader-follower cell specification.
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