The regulation of cell-cell adhesion during epithelial-mesenchymal transition, motility and tumor progression

Adherens junctions (AJs) are essential for the maintenance of epithelial homeostasis and a key factor in the regulation of cell migration and tumor progression. AJs maintain cell-cell adhesion by linking transmembrane proteins to the actin cytoskeleton. Additionally, they participate in recruitment of signaling receptors and cytoplasmic proteins to the membrane. During cellular invasion or migration, AJs are dynamically regulated and their composition modified to initiate changes in signaling pathways and cytoskeleton organization involved in cellular motility. Loss of E-cadherin, a key component of AJs, is characteristic of epithelial-mesenchymal-transition (EMT) and is associated with tumor cell invasion. We will review recent findings describing novel mechanisms involved in E-cadherin transcription regulation, endocytosis of E-cadherin and signaling associated with loss of AJs as well as reorganization of the AJ during EMT.


Introduction
Epithelial cell-cell adhesion is maintained by three basic structures connecting adjacent cells: tight junctions (zonula occludens), adherens junctions (zonula adherens) and desmosomes (macula adherens). AJs are established via calcium-dependent homophilic binding of the extracellular cadherin domains. The cytoplasmic domains of these cadherins recruit the catenins, β-catenin and p120 catenin, which in turn connect the AJs to the actin cytoskeleton. Linking catenins with the cytoskeleton may be mediated by the clustering of cadherin/catenin complexes to recruit high levels of a-catenin 1-3 or by other cytoplasmic factors, such as epithelial protein lost in neoplasm (EPLIN or LIMA1). 4 EPLIN, a mechanosensitive biomarker, directly binds a-catenin and is absent in many cancer cell lines. 5,6 Other major components of AJs are the nectins and afadins. Unlike cadherins, nectins are capable of establishing adhesive contacts via either heterophilic or homophilic binding. 7 The nectin-like protein NECL-5, better known as the poliovirus receptor (CD155), plays a role in cell motility, associating with integrin avβ3 and plateletderived growth factor receptor (PDGFR) at the leading edge of migrating cells. 8 Other adhesion molecules have been shown to have dual function as a cell adhesion and motility promoter. In this special focus, Kiefer et al. show that L1 cell adhesion molecule (L1CAM), which plays a major role in the development of the nervous system, also functions to promote the malignancy of human tumors. While it acts as a glue between cells, L1CAM can also drive motility during neural development and supports metastasis of human cancers. Important factors that contribute to the switch in the functional mode of L1CAM are cleavage from the cell surface by membrane proximal proteolysis and the ability to change binding partners and engage in L1CAM-integrin binding. Cleavage of E-cadherin is also a mechanism by which the function of L1CAM can be disrupted.
One of the key steps in tumor progression is the loss of cell-cell adhesion. As single cells invade into the underlying stroma, they express mesenchymal markers and proteins involved in matrix degradation and motility. The induction of the EMT phenotype during cancer is reminiscent of the EMT observed during development and neural cell crest migration. EMT involves the suppression of E-cadherin and a switch to the expression of mesenchymal cadherins such as N-cadherin 9 or cadherin 11. 10 At the center of this process is the inhibition of E-cadherin, the major mediator of cell adhesion in AJs. 11,12 The regulation of E-cadherin involves both transcriptional and post-translational mechanisms. Whether by direct gene repression or as a consequence of weaker bonds between cells, loss of AJs is concomitant to the loss of the other junction complexes during EMT. 13,14 Although E-cadherin is the most studied target of EMT inducers, the transcription factor SNAIL (SNAI1) is also known to inhibit tight junction components. 15,16 In this review we will consider new aspects of the regulation of AJs, particularly cadherins, focusing on their role in EMT and tumor progression.

Transcriptional Regulation and Epigenetic Silencing of E-Cadherin (CDH1)
Regulation of E-cadherin transcription during EMT is dependent upon many known transcription factors capable of binding the Ebox sequence in the promoter region of CDH1. Two of these transcription factors, SNAI1 and the basic helix-loop-helix family member TWIST1, have been considered to be master regulators of EMT. 17 Table 1) to regulate stemness. These data have allowed for a much better understanding of how CDH1 is silenced in cancer cells.
Promoter methylation. The silencing of E-cadherin expression by hypermethylation is a common event in cancer. 27,28 DNMTs target cytosine residues in CpG dinucleotides for methylation and have recently been identified in the repression of E-cadherin in normal 29-32 and pathological contexts, 33-36 such as colorectal cancer, gastric cancer and hepatocellular carcinomas. [37][38][39][40][41][42] Multiple signaling pathways involved in EMT and tumorigenesis activate DNMTs, e.g., ras 43 and TGF-β. 44,45 DNMTs bind several histone remodeling enzymes, such as MPP8, 46 Sirtuin 1 47 and G9a 48 ( Table 1). However, SNAI1 has been shown to be linked to DNMT1, 49 notably in association with G9a and Suv39H1 50,51 (see also Table 2). As polycomb proteins act as a platform to recruit DNMT, the two epigenetic mechanisms could intersect. 52 Cooperation between Polycomb proteins and EMT-inducing transcription factors. The polycomb proteins are part of repressor complexes that inhibit gene expression through chromatin remodeling. Polycomb-mediated gene expression is essential for the maintenance of embryonic stem cells and is involved in development as well as tumor suppression. 53,54 The polycomb repressive complex 2 (PRC2) recruits PRC1 after chromatin methylation at H3K27 through enhancer of Zeste Homolog 2 (EZH2), a histone H3 lysine-27-specific methyltransferase. 55 Both, PRC1 and PRC2 have been shown to interact with SNAI1 and TWIST1 to promote EMT. SNAI1 is stabilized through its interaction with the PRC1 component BMI-1 56,57 and interacts with Suppressor of Zeste 12 (Suz12) and EZH2 to repress CDH1 expression. 58,59 Interestingly, EZH2 also participates in transforming growth factor β 1 (TGF-β1) signaling, 60 a potent inducer of EMT. BMI-1 can also interact with TWIST to induce EMT. 61 The intricate interactions of EMT-inducing transcription factors and chromatin remodeling complexes PRC1 and PRC2 may offer novel approaches to control EMT and thus cell adhesion in cancer cells via a plethora of new drug, such as HDACs and DNMT inhibitors.

Endocytosis of AJ Components
AJs are highly dynamic structures. 62 Remodeling of AJs and associated proteins occurs through endocytosis and recycling of the complex components. 63 Endocytic pathways are often misregulated in cancers and a shift in the balance between recycling and degradation 64 can result, for example, in the degradation of E-cadherin and increased cell migration. Endocytic signaling pathways enclose a vast number of mechanisms and associated proteins some of them recently described such as CD2AP. 65,66 Others such as Rab, Rap and Rho GTPases, endocytosis proteins like dynamin and other associated proteins like Bar proteins etc have been extensively reviewed. 64,[67][68][69] Here we will here focus on some recent findings involving E-cadherin endocytosis, particularly during EMT.
Src is a known oncogene and activates different signaling pathways that can ultimately inhibit E-cadherin to promote tumor progression. 75 The proto-oncogenes epidermal growth factor receptor (EGFR) 76 and hepatocyte growth factor receptor (HGFR) target Src but also physically interact with Ecadherin. [77][78][79][80]  Protein tyrosine phosphatases (PTPs) have been considered tumor suppressors as they are the functional opposites of tyrosine kinases. PTPs (PTP-m, DEP1, PTP-LAR, PTP1B, PTB-PEST and others) activate kinases by removing inhibitory phosphates and are involved in the regulation of cell-cell junction (for a review see refs. 81 and 82). Rather than acting as pleiotropic suppressors of tyrosine kinases, PTPs recently have been described to act as specific regulators of signaling pathways. 83 In mammary epithelial cells, loss of the PTPs PTPRG, PTPRR and PTPN23 was associated with enhanced cell motility. However only PTPN23 was been shown to directly induce E-cadherin internalization and was associated with increased invasion. Src, E-cadherin and βcatenin appear to be direct substrates of PTPN23. An increase in Src activity with the subsequent loss of PTPN23 was responsible for the expression of mesenchymal markers and the observed increased invasion in mammary cell lines (Fig. 1).
Non-clathrin-mediated endocytic pathways. The suppression of PTPN23 is also associated with caveolin-1 mediated internalization and blocking of early endosome vesicle trafficking. 83 Caveolin-1 plays an important role in EGFR signaling 84,85 as downregulation of caveolin-1 in response to EGF results in the expression of SNAI1, EMT and loss of E-cadherin. 86 In cancer cells, EGF can induce caveolin-1-mediated degradation of Ecadherin and tumor cell dissociation. 87,88 Reggie/flotillins are microdomain scaffolding proteins (lipid raft components) that play an important role in Wnt and Shh signaling 89 and are involved in regulating membrane trafficking and turnover. 90,91 In association with prion protein (PrP), reggies/ flotillins control activation of Src kinases and the presence of receptor tyrosine kinases such as EGFR at the membrane. 92 Reggies contribute to the internalization and turnover of Ecadherin. Together with PrP they target the recycling of E-cadherin to the AJs and its binding to catenins for the maintenance of cell adhesion (Fig. 2). 93 In the context of EGFRdependent loss of cell adhesion, reggies are necessary for EGFR endocytosis to prevent sustained activity and concurrently cell migration.
Clathrin-mediated E-cadherin recycling. Endocytosis of Ecadherin can be mediated by clathrin-coated vesicles upon calcium depletion. 94 Several adaptor proteins in this pathway, notably Numb and Disabled-2 (Dab2), participate in the regulation of members of adherens junctions during EMT (Fig. 3).
Numb is often misregulated in cancers and plays many roles in the cell from endocytosis, to cell migration, cell adhesion, signaling pathways and asymmetric cell division. 95 However, Numb's function during tumorigenesis remains controversial: Much of the work done on Numb highlights its role as a tumor suppressor. Not only does it inhibit ubiquitination and subsequent destruction of p53, but it also has been shown to inhibit Rac1-GTP accumulation. Rac1 is initially increased by cell-cell contacts and E-cadherin 96 but is rapidly downregulated after the establishment of adherens junctions. 97,98 Indeed, Rac1-GTP accumulation is associated with loss of cell-cell adhesion and lamellipodia formation. 99 With respect to AJs more specifically, Numb knockdown was recently shown to sensitize cells to HGFmediated EMT. 100 The proposed mechanism for Numb's role in maintaining cell adhesion and apicobasal polarity involves its binding and sub-membranous localization, via its phosphotyrosine-binding domain (PTB), to both the PAR3-aPKC-PAR6 complex 101 and E-cadherin (Fig. 3). Upon phosphorylation by aPKC, Numb is endocytosed. Wang et al. propose that Numb binds the same NVYY motif that Hakai binds to target Ecadherin for degradation. Numb would therefore be an important regulator of cell adhesion and maintenance of cell polarity. In a similar fashion, signaling induced by TGF-β1 destabilizes PAR6 and affects both E-cadherin expression and localization of the PAR complex. 102,103 Because Numb establishes a dynamic link between AJs and tight junctions, loss of Numb upon HGF stimulation could affect both structures simultaneously during EMT.
By contrast, Numb expression at the cell membrane has been shown to promote the loss of cell-cell adhesion. It was suggested that Numb causes E-cadherin endocytosis by binding directly to p120 catenin, which itself prevents E-cadherin internalization. 104 Sato et al. propose that phosphorylation of Numb by the atypical protein kinase aPKC prevents Numb binding to p120 and helps maintain apicobasal polarity. Overall, more work is needed to elucidate the roles of Numb in the regulation of E-cadherin endocytosis.
Dab2 is an endocytotic adaptor protein 105 and is considered a tumor suppressor as Dab2 expression is frequently lost in different types of cancer. [106][107][108][109][110] Interestingly, Dab2 is a central node to TGF-β1 mediated EMT-signaling. As a downstream target of TGF-β1, 111,112 Dab2 gene expression is induced by Akt2, which phosphorylates hnRNP E1, thereby preventing suppression of the Dab2 promoter. 113,114 However, more recent studies have found that the epigenetic silencing of Dab2 is responsible for the switch of TGF-β1 signaling from a tumor suppressor to an oncogenic factor. 115,116 Furthermore, Dab2 participates in the endocytosis of TβRII and promotes signaling of TGFβ through canonical Smad signaling. Dab2 can decrease E-cadherin levels and upregulate the mesenchymal marker vimentin 117 demonstrating not only a role for Dab2 in EMT, but also in the regulation of E-cadherin. In addition, a study focusing on Dab2 function in embryogenesis, identified that Dab2 mediates directional trafficking and the polarized distribution of E-cadherin. 118 Taken together, it could be conceivable that Dab2 is affecting E-cadherin trafficking and degradation directly, but the mechanisms have yet to be identified.

The Fate of Cell-Cell Adhesion in Tumor Progression
In the absence of E-cadherin, individual tumor cells migrate by one of two modes: mesenchymal movement (directional migration with proteolysis at the leading edge) or amoeboid movement with lack of direction and of polarity. 119 In the presence of E-cadherin, tumor cells use collective migration. 119,120 Yang et al. propose that the induced mesenchymal mode of migration will be associated with local invasion but limited metastasis. Interestingly, in head-and-neck squamous cell carcinoma individual as well as collective migration can be observed. This prompts the question of whether cell-cell contacts could still be preserved after EMT.
N-cadherin adherens junctions: collective migration after EMT. Recent work demonstrates that the transition between single cell migration and collective migration in breast cancer cell lines is reversible and depends upon TGF-β1 signaling. 121 In fact, alternating between single-cell and collective migration is conducive to a more metastatic disease phenotype. Collective cell migration requires mechanisms to maintain cell adhesion and is dependent on cell polarity.
One hallmark of EMT is the shift from E-cadherin to Ncadherin expression. 9 The "cadherin switch" does not necessarily imply that E-cadherin will always be replaced by N-cadherin. Rather, N-cadherin expression increases as cells undergo EMT. Ectopic expression of N-cadherin increases migration of breast cancer cell lines 122 as well as in the mouse mammary epithelial cell line NmuMG after treatment with TGF-β1. 123 Interestingly, in this last study N-cadherin upregulation preceded morphological changes induced by EMT. The precise mechanism controlling an increase in N-cadherin expression is unknown, although previous studies have suggested it to be TWIST1-dependent. 124,125 It is important to note that although N-cadherin AJs are weaker than their E-cadherin counterparts, 126 they may still maintain cellcell contacts during migration. In the collective migration of neural crest cells, N-cadherin inhibits formation of protrusions at cell-cell contacts helping the cells at the leading edge to progress. 127 The mechanism involved in the regulation of Rac1 activity to promote N-cadherin-mediated adhesion was also shown to be integrin-dependent. 128 N-cadherin can also play an essential role in collective migration. 129 Cells grown in a three-dimensional environment have been shown to establish focal adhesion differently compared with cells grown in a two-dimensional environment such as on plastic, which allows for the possibility that cell-cell adhesion could also be affected. 130,131 A three-dimensional environment, in which the cells were embedded in an extracellular matrix rather than grown on plastic, was required for the establishment of Ncadherin-mediated cell-cell adhesion after EMT hinting at matrixdependent cues, such as stiffness of the matrix, which have been demonstrated to affect cell migration and invasion (see below). Shih and Yamada also described a distinct role of the cytoplasmic domain of N-cadherin in promoting migration by an unknown mechanism. However, the potential role of AJs formed by Ncadherin with respect to the migration of epithelial cells undergoing EMT remains largely unexplored.
The role of the matrix in collective invasion. Matrix rigidity and orientation promotes collective cell migration. 119 Matrix stiffening was recently shown to sensitize cells to EGF. 132 Moreover, as the matrix stiffens, TGF-β1 loses its antiproliferative role to become pro-tumorigenic. This effect is not mediated by Smad signaling, but by the PI3K/Akt pathway. 133 Matrix stiffness is important in controlling control the strength of AJs in collective migration. 134 The composition of the matrix itself is regulated by the fibroblasts, which can be activated by paracrine factors secreted from the tumor. At the same time, secretion of growth factors from the activated fibroblasts contributes to tumor progression. 135 Carcinoma-associated fibroblasts can promote tumor invasion and generate a track in the matrix for the invading tumor cells to follow. 136 Interestingly, lysyl oxidase-like 2 (LOXL2), a tumor marker, increases collective cell migration. 137 LOXL2 interacts with and stabilizes SNAI1 at the invasion front in actively migrating cells. In addition, LOXL2 catalyzes the cross-linking of collagen fibrils and contributes to the matrix stiffening. Proteins such as LOXL2, which participate in mechanotransduction-mediated invasion are likely the regulators of temporal and spatial assembly and disassembly of cell junctions to facilitate single vs. collective migration.

Concluding Remarks
The regulation of cell adhesion is under control of multiple signaling pathways and mechanisms that allow fine-tuning of the process. Continuous formation and disassembly is necessary during embryogenesis and development. Some of the processes involved at this stage, such as EMT, are hijacked during carcinogenesis. While the structural importance of adherens junctions has been known for a long time, the impact of the complex components on cell signaling has been identified rather recently. The central role of E-cadherin is apparent by the many layers of regulation: promoter methylation, histone methylation, transcriptional repression and post-translational modifications leading to endocytosis. As our understanding of these machineries increases, we will discover novel players and, hopefully, therapeutics that could restore cell adhesion in the clinical setting.