Interplay between EGFR, E-cadherin, and PTP1B in epidermal homeostasis

ABSTRACT

Maintaining epithelial homeostasis is crucial to allow embryo development but also the protective barrier which is ensured by the epidermis. This homeostasis is regulated through the expression of several molecules among which EGFR and E-cadherin which are of major importance. Indeed, defects in the regulation of these proteins lead to abnormalities in cell adhesion, proliferation, differentiation, and migration. Hence, regulation of these two proteins is of the utmost importance as they are involved in numerous skin pathologies and cancers. In the last decades it has been described several pathways of regulation of these two proteins and notably several mechanisms of cross-regulation between these partners. In this review, we aimed to describe the current understanding of the regulation of EGFR and interactions between EGFR and E-cadherin and, in particular, the implication of these cross-regulations in epithelium homeostasis. We pay particular attention to PTP1B, a phosphatase involved in the regulation of EGFR.

KEYWORDS:

• Epithelium
• EGFR
• E-cadherin
• differentiation
• PTP1B

Introduction

Epithelia are specific tissues that support the structure of embryos and many organs but also act as a protective barrier against environmental insults and pathogens. Their structure is of graded complexities from monolayers as in the gut to multilayers as in the epidermis which is a stratified squamous epithelium composed of several cell types but mainly of keratinocytes. It is organized into discrete layers of basal, replicative cells, and suprabasal layers that undergo a stringent regulated program of terminal differentiation as cells move outward to the stratum corneum.¹ Epidermal keratinocytes hence exhibit growth control mechanisms that coordinate proliferation and differentiation. In human epidermis it takes about 4 weeks for cells to exit the basal layer and be extruded from the skin surface as squames. Indeed, pluristratified epithelia are polarized: basal keratinocytes are those that renew the tissue population while apical keratinocytes are highly differentiated maintaining a squamous cornified layer of dead keratinocytes endowed with highly resistant properties in contact to environmental media. The epidermis hence replenishes itself through a process during which the number of cell divisions in the basal layer compensates for the squamous dead cell lost.² Various proteins control epidermis’ homeostasis by affecting regenerative capacity and differentiation such as EGFR (Epidermal Growth Factor Receptor). The main cohesive key is the intercellular junctions that are built from cadherin as keystones of the epithelial structure. Epidermal adherens junctions are structured from transmembrane E-cadherin (Figure 1a). E-cadherin is of major importance for the maintenance of skin organization as it has been shown that deletion of E-cadherin causes hyperproliferation, defective differentiation and impaired barrier formation.^3–5

Figure 1. A Schematic representation of human epidermis. At the outermost side of the skin, the epidermis is composed of several layers of cells: basal, replicative cells, and suprabasal layers that undergo a regulated program of terminal differentiation as cells move outward to the stratum corneum (dead cells). On the dermis interface, in the basal layer of keratinocytes, EGFR signaling is highly activated and cells are proliferative. From the base and up in the epidermis, cells reduce their proliferation and start to differentiate and form more cell-cell junctions, such as tight, adherens and gap junctions; a precise regulation and balance between EGFR and E-Cadherin (E-Cad) are keystone in maintaining structure and integrity of the epidermis. B Regulation of EGFR intracellular trafficking and activation. EGFR activated in the presence of various concentrations of EGF is driven in two distinct endocytic pathways. 1: At low concentrations of EGF, the receptor undergoes the “recycling pathway”, through early and recycling endosomes, before being recycled back to the plasma membrane. 2: At high concentrations of EGF, it undergoes the “degradation pathway”. Signaling is down-regulated by two major mechanisms: PTP1B-dependent dephosphorylation at the RE membrane contact, and degradation inside the endolysosomes upon control by ubiquitination. C Overview of EGFR, E-Cadherin and PTP1B interactions, and their roles in the maintenance of AJ and intracellular signaling. 1: By dephosphorylating beta-catenin, PTP1B promotes the proper assembly of AJ. Their integrity is also conserved by the direct interaction of PTP1B with E-Cadherin (still at AJ location). 2: When PTP1B is not phosphorylated, it cannot dephosphorylate beta-catenin, and fails to maintain AJ integrity. 3: EGFR promotes proliferation, differentiation, cell growth and survival by activating several signaling pathways such as MAPK, STAT, PI3K/Akt and Erk. The latters are also regulated by E-Cadherin. EGFR interaction with E-Cadherin through galectin-7 partially impairs EGFR phosphorylation, contributing to the E-Cadherin/EGFR balance. 4: p120 direct interaction with EGFR enhances Akt and Erk signaling. 5: PTP1B inhibits EGFR by dephosphorylation, both at the plasma membrane and near the RE (Figure 1b), therefore down-regulating the corresponding signaling pathways. 6: Under tension, vinculin binds to unfolded alpha-catenin, after phosphorylation by Abl. This complex binds myosin II and F-actin, forming an organized contractile actin network.

Over the last decade, numerous studies have shed light on the importance of the regulation of EGFR and interactions between EGFR and E-cadherin and, in particular, the implication of these cross-regulations in epithelium homeostasis.⁶ We propose through this review to point out the latest advances in this area.

EGFR

EGFR, a tyrosine kinase receptor, has been identified as one of the major actors of skin homeostasis. It belongs to the ErbB family of receptors, which consists of 4 closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/c-neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4).⁷ EGFR has been described to mediate cell proliferation, to modulate terminal differentiation and to control overall epidermal homeostasis.⁸ Overexpression of EGFR is correlated with progression of numerous human cancers and therefore EGFR is a major target in many cancer therapies.^9,10 EGFR is regulated by at least seven different activating ligands in humans: EGF itself, transforming growth factor α (TGF-α), amphiregulin (ARG), epiregulin (EPR), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), and epigen (EGN).¹¹ Ligand binding induces a conformational change in the extracellular domain of the receptor resulting in receptor asymmetrical dimerization and autophosphorylation.¹² Depending on the position and degree of its phosphorylation EGFR recruits specific signaling complexes and thus has the potential to initiate a wide variety of downstream signaling cascades associated with EGF-dependent responses.^10,13 EGFR signaling is involved in many embryonic cellular processes such as development, tissue homeostasis and wound healing. Indeed, it activates the MAPK pathway (cell proliferation and angiogenesis), as well as the STAT pathway (cell proliferation, migration, cell adhesion, and angiogenesis) or even the PI3K/Akt pathway (metabolism, cell survival, inhibition of apoptosis and proliferation).¹⁴ Moreover, EGFR overexpression and/or hyperactivation through genetic alterations have been linked to malignant transformation.¹⁵

Following EGF-induced dimerization, activated EGFRs are internalized. Signaling from the endocytosed EGFR is subject to down-regulation by at least two mechanisms: (a) ESCRT (endosomal sorting complex required for transport)-mediated sequestration into the ILVs (intraluminal vesicles) of endosomes before lysosomal delivery and subsequent degradation and (b) dephosphorylation by protein tyrosine phosphatases, such as PTP1B (protein tyrosine phosphatase 1B)¹⁶ (Figure 1b).

PTP1B

PTP1B is a ubiquitous and abundant intracellular protein Tyrosine Phosphatase (PTP) that interacts with numerous phosphotyrosine proteins present in the cytoplasm, plasma membrane, vesicles, nucleus and intercellular junctions.^16,17 Specialized membrane contact sites may be involved in some of these interactions.¹⁸ It is the first described PTP among a hundred of the protein tyrosine phosphatase (PTP) family.^19,20 The PTP activity of PTP1B relies on the presence of a conserved and nucleophilic catalytic cysteine residue in the P loop of the enzyme.

More generally, PTP1B is involved in the regulation of several cellular pathways in the cell. Indeed, it has been shown to impact other receptors, like the insulin receptor (IR) .^21,22 This receptor, being also a member of the tyrosine kinase family, is modulated by PTP1B-dependent dephosphorylation.

A critical role of PTP1B is its regulation of EGFR functions. Indeed, by dephosphorylating the receptor, PTP1B down regulates EGFR and several of its downstream signaling components²³ (Figure 1b). As mentioned above, EGFR signaling is involved in many cellular processes²³ and when dysregulated is involved in cancer development;²⁴ its precise regulation is therefore important in maintaining normal cellular function.⁷ However, PTP1B regulates several other cell components, among which some may antagonize EGFR signaling. It has therefore been shown that PTP1B can display both tumor promoting and suppressing effects depending on cellular conditions.^25,26

E-Cadherin

E-cadherin is a transmembrane glycoprotein that binds epithelial cells together via a homotypic calcium-dependent interaction.²⁷ E-cadherin contains five extracellular domains and a cytoplasmic tail that binds to several effectors to transduce physical and biochemical signals to the cell.²⁸ The five extracellular domains exhibit calcium-binding sites and are involved both in trans- and cis-homotypic interactions constituting multimolecular complexes seeding the intercellular adhesive function.²⁹ The intracellular domain of E-cadherin associates with many different cytoplasmic proteins, and notably the alpha, beta- and p120-catenins (Figure 1c). These E-cadherin binding intracellular partners mediate and regulate the activity of E-cadherin and in particular its association with the actin-myosin cytoskeleton, alongside with its intracellular trafficking and recycling.³⁰ E-cadherin hence forms the core of a mechano-transduction and signaling hub called the adherens junctions (AJ). Its function is critical for the induction and maintenance of tissue barriers by controlling cell adhesion, permeability, polarity, and differentiation in vitro and in vivo. Studies in mammalian cell cultures have identified a central role for E-cadherin in coordinating the establishment of apico–basolateral polarity with the formation of AJ but also desmosomes and tight junctions (TJ).³¹ The loss of E-cadherin alters the morphology of the skin but does not affect its integrity. Dysfunctional adherens junctions are linked to various pathologies but most relevant to cancer^32,33 and inflammatory³⁴ diseases. As such, several studies have described E-cadherin as a tumor suppressor even if recent studies have shown it may also promote cell migration, invasion and even tumor progression.³³ Indeed, the cytoplasmic domains of classic cadherins interact with various signaling-competent partners.³⁵ Thus, homophilic binding of E-cadherin can down-regulates expression levels of receptor tyrosine kinase (RTK) and tyrosine kinase Src and can suppress activation of Wnt/beta-catenin pathway and RTK/PI3K pathways.³⁶

Interaction between EGFR and E-Cadherin and their consequences

Molecular interactions

Various biochemical and microscopy evidence, such as immunoprecipitation and immunofluorescence studies, demonstrated that a fraction of EGFR is detected at sites of cell–cell contacts³⁷ and is present in its phosphorylated active form, thus strongly supporting the concept that E-cadherin can transactivate EGFR.^38,39

Qian et al. showed in MDCK cells that E-cadherin could inhibit cellular responses to EGFR stimulation documenting that mitogenic responsiveness to EGF decreased as cells grew to confluence.⁴⁰ This desensitization could be overcome by adding antibodies that block E-cadherin function. They also showed that reduced expression of E-cadherin increased both EGFR autophosphorylation and EGF-induced DNA synthesis. These results have been reinforced in head and neck cancer cells with the observation that downregulation of E-cadherin upregulated EGFR at the cellular membrane and at the mRNA level. This upregulation was due to the increase of EGFR mRNA stability, but not to changes in protein stability.²⁶ However, in epithelial ovarian cancer cells E-cadherin silencing was associated with a decrease of EGFR protein levels.³⁷ Notably in these cell lines EGF stimulation did not affect E-cadherin expression in control siRNA-treated and untreated cells.³⁷ Although it has not been shown for EGFR, it has been reported that the overexpression of another member of the family, ERBB2 in human mammary epithelial cells induces inhibition of the transcription of E-cadherin.⁴¹

Beyond these crosstalks physical interactions between EGFR and E-cadherin have also been demonstrated as well as with some components of AJ complex such as catenin. This has been confirmed in several cell lines such as for example A431 (human epidermoid carcinoma cells),⁴² MDCK³⁹ (kidney), EOC (epithelial ovarian carcinoma)³⁷ or HaCaT (human keratinocyte cells).⁴³

The crosstalk between both molecules has firstly been described through interactions via intracellular partners belonging to the AJ complex. As early as the 1990s, it was demonstrated using recombinant proteins that beta-catenin directly interacts with EGFR. This interaction seems to be mediated by the central region of beta-catenin. This region having about 80% identity at the amino acid level with plakoglobin and armadillo protein suggests possible interactions of EGFR with these molecules.⁴² More recently, it has been shown that EGFR also interacts with another catenin of the AJ complex: δ-catenin. This direct interaction has been demonstrated in bosc23 (human kidney cell line) and CWR22Rv-1 cells (Human Prostate cancer cell line) cells. Moreover, the authors noted that this interaction was decreased in the presence of EGF without affecting the localization of δ-catenin.

Beyond the catenins, other partners of the AJ complex have been shown to be crucial in the interaction between EGFR and E-cadherin. PLEKHA7, a recently demonstrated new component of the AJ, is known to stabilize the molecular complex of E-cadherin by linking it to the minus ends of non-centrosomal microtubules.²⁹ However, it also appears that this molecule obliterates the interaction between EGFR and E-cadherin since upon PLEKHA7 overexpression in epithelial ovarian cancer, the association of E-cadherin with EGFR is lost and subsequent EGFR activation inhibited.^37,44 This type of interaction has also been illustrated for the neurofibromatosis type-2 (NF2) tumor suppressor and Merlin (also known as schwannomin). Merlin has been described to be recruited at the cadherin/catenin complex and to regulate cell adhesion.^45,46 A few years later it has been demonstrated that it could also binds to and regulates EGFR trafficking through NHE-FR1⁴⁷ and hence by this interaction with both E-cadherin and EGFR coordinate membrane receptor signaling and adhesion in mammalian cells.⁴⁸

However, there are other possible interactions through post-translational modifications such as glycosylation. Indeed, three canonical N-glycosylation sites in its extracellular domain were reported on EGFR in the UniProt database and several evidence that EGFR functions are linked to the N-glycosylation status of EGFR have been published.⁴⁴ These results and the importance of EGFR glycosylation and E-cadherin glycosylation have been nicely illustrated recently by Manwar-Hussain and colleagues.⁴⁹ Interestingly, we recently identified a galectin as a new partner of EGFR. Galectins are a family of proteins characterized by a common affinity for β-galactoside containing carbohydrates and an evolutionary conserved Carbohydrate Recognition Domain (CRD).³⁹ Among them, galectin-7, which is restricted to the pluristratified epithelia, has newly been shown to impair EGFR phosphorylation through a direct interaction with the carbohydrates of EGFR extracellular domain.⁴³ Moreover, galectin-7 was already known to regulate cell adhesion through the direct regulation of E-cadherin.³⁸ Evidence has now been established by protein recombinant experiments and in silico data that this molecule establishes a bridge between E-cadherin and EGFR regulating the proliferation/differentiation balance notably in the skin⁴³ (Figure 1c).

The interactions between these two molecules can be even more indirect involving additional partners in the regulation of their membrane stability or intracellular trafficking. Hence, reggie proteins (also known as flotillins) favor EGFR internalization by enhancing its phosphorylation and stabilize cell adhesion and AJ formation and dynamics. The defect in AJ formation and dynamics which results in the case of the down-regulation of reggie proteins is caused by an imbalanced rate of the macropinocytic uptake and redelivery of E-cadherin to AJ.⁵⁰ Thus, it appears that the regulation between E-cadherin and EGFR can also involve many other proteins, particularly at the level of intracellular trafficking and signaling. It is to note that in spite it is not the main subject of this review E-cadherin and EGFR can also impact tight junctions (TJ) through the regulation of claudins, a family of 24 proteins in humans.⁵¹ For example, it has been shown that EGF induces expression of claudin −1, −3 and −4 cells but also claudin-2 and −18 in several cell types and cancers^52–56

Interactions between PTP1B and cadherins have also been described. The enzyme interacts directly with cadherins leading to a PTP1B/cadherins/catenins complex.^57,58 PTP1B direct association with the N-cadherin cytoplasmic domain, especially in neural cells, has been well described.⁵⁸ More recently, its direct interaction with E-cadherin was also reported in intestinal epithelial cells (CaCo2).⁵⁹ This binding has been shown to be essential for AJ integrity and cell–cell adhesion in general. Indeed, dephosphorylation of beta-catenin tyrosine residues by PTP1B is needed for its binding to cadherins⁵⁸ (Figure 1c). It has been shown that the absence or inhibition of PTP1B prevents those bindings and disrupts adhesion.⁶⁰ PTP1B is therefore an up-regulator of cell–cell adhesion in several cell types. However, this mechanism also depends on PTP1B phosphorylated state. Indeed, the enzyme is only capable of binding cadherins and dephosphorylating catenins when itself phosphorylated on a tyrosine residue.⁶⁰ This underscores the importance of PTP1B regulation itself and suggests the presence of a more complex mechanism involving kinases as well as phosphatases.

Intracellular trafficking and signaling

Several findings suggest that E-cadherin may exert an extrinsic control on outside-in signal transducing pathways through interaction with tyrosine kinase receptors such as EGFR. Indeed, during the formation of AJ in keratinocytes, clustering of E-cadherins can induce a ligand-independent activation of the EGFR kinase which seems to involve the formation of higher order molecular complexes between E-cadherin and EGFR.³⁹ This aggregation of E-cadherin into assembling AJ has also been demonstrated in mammary epithelial cells where it induces tyrosine phosphorylation of specific substrates within sites of cell-cell contact.⁶¹ Furthermore Fedor-Chaiken and colleagues have demonstrated that EGFR phosphorylation requires the extracellular domain of E-cadherin. However, and interestingly this activation does not impact mammary epithelial cell motility governed by E-cadherin.⁶² Perrais et al. further demonstrated that E-cadherin homophilic ligation inhibited serum-stimulated cell proliferation by preventing E-cadherin binding to beta-catenin.⁶³

At the same time, it was observed that downregulation of E-cadherin upregulated EGFR expression compared with control siRNA-transfected cells after 72 hours. Cellular membrane localization of EGFR was also increased and downstream activated molecules of the EGFR signaling pathway, p-AKT, and p-ERK, were thus increased.⁶⁴ Current knowledge of E‐cadherin/catenin–EGFR cross‐talk hence suggests that loss of E‐cadherin function activates PI3K, mTOR, EGFR, or FAK signaling.⁶⁵

Furthermore, E-cadherin promotes EGFR-mediated cell differentiation of cultured human airway epithelial cells via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.⁶⁶

In a recent study Sehgal P. et al. revealed a key role for EGFR in E-cadherin force sensitive mechanism in A431 (human epidermoid carcinoma cells) and MCF7 (human epithelial breast carcinoma).⁶⁷ The tension dependent junctional E-cadherin response was shown to involve EGFR mediated Abl activated vinculin via stressed E-cadherin activation of integrin (Figure 1c). This complex cascade of activation further recruits vinculin to junctional domain linking cytoskeletal reorganization and force-modified E-cadherin.

Of note soluble E-Cadherin has been shown to act as a survival factor and to signal through EGFR to mediate its anti-apoptotic effects hence suggesting that soluble E-Cadherin present in patients’ sera might contribute to cancer progression by inhibiting apoptosis in cancer cells.⁶⁸

Conclusion

Current knowledge underscores that there is a strong link between adhesion molecules such as E-cadherin and growth factor receptors such as EGFR in the regulation of epithelial homeostasis. These interactions involve direct contact but also other partners, such as PTP1B, which regulate both activation pathways. Recent findings provide evidence that E-cadherin can differentially and concurrently regulate specific growth-related signaling pathways in a context-specific fashion, with direct functional consequences for cell proliferation and population growth. Several observations not only reveal a novel, complex role for E-cadherin in normal epithelial cell homeostasis and tissue regeneration, but also provide the basis for a more complete understanding of the consequences of E-cadherin loss on malignant transformation.⁶⁹ Furthermore, EGFR is also able to regulate differentiation by modulating the expression of adhesion molecules, notably E-cadherin which itself integrates the various concomitant signals.⁷⁰

Mutations of PTP1B have been documented in various types of cancer.^71,72 More precisely, frequent somatic mutations have been identified in primary mediastinal B cell lymphoma and Hodgkin lymphoma.^73,74 Most of them (i.e. 60%) are missense mutations located in the catalytic domain (i.e. 83%), inactivating the catalytic activity. Other frequent mutations affect glutamine residues (Q) of the first exon, leading to premature inactivation of the protein. In this case, but also in colon and thyroid cancers⁷⁵ and leukemia,⁷⁶ PTP1B mutations are associated with tumor development, as reduced phosphatase activity induces higher phosphorylation of several pathways, such as EGFR, JAK-STAT, Src or some downstream oncogenic targets.⁷⁷ Refined analysis of deletion mutants of PTP1B demonstrated enhanced proteolysis of WT enzyme linked to higher affinity dimer formation with the exon 6 deleted mutant.⁷⁸ On the other hand, mutants deleted from exon 2–4 did not impair coexpressed WT PTP1B suggesting additional mechanisms such as competition with ligands and substrates, interaction with novel molecular partners or interference with diverse signaling pathways.⁷⁹ Interestingly, combinatorial mutations in JAK/STAT effectors and PTP1B have been reported, adding further complexity.⁷³

However, PTP1B can also be a tumor promoter, as it has been described to activate the c-src pathway in colon and breast cancers, the enzyme being frequently overexpressed, alongside with increasing the formation of invadopodia.²⁶ This role has also been shown in prostate cancer cells, where an elevated PTP1B expression correlates with neuroendocrine differentiation.⁸⁰

Even if mutations have been described outside of the catalytic domain, for example, in the proline-rich motifs thus preventing the enzyme to interact with p130Cas;⁸¹ most of them are localized in the catalytic domain and have been identified in several cancers. However, its wide range of substrates and interactions broaden its implication in cellular processes.

PTP1B, by regulating E-cadherin and EGFR via its phosphatase activity, probably allows a very precise control of this balance which opens new therapeutic perspectives. Therefore, the development of PTP1B modulators is a very promising point that has been considered in recent years.⁸² Moreover, it has been demonstrated that environmental xenobiotics such as naphthoquinones are potent inhibitors of PTP1B and increase EGFR signaling. Furthermore, Li et al., showed that menadione can reduce the secondary effects of treatment with EGFR inhibitors in epithelial tumors. Indeed, the toxicity of EGFR inhibitors on the skin has long been reported, and, if not life-threatening, skin toxicity may cause significant physical and psycho-social discomfort. In their study the authors showed that the use of menadione topical cream can be efficient in the treatment or prevention of EGFR inhibitor-induced skin toxicity.⁸³ In parallel, using mice expressing HER2/Neu and lacking PTP1B specifically in the mammary epithelium, it was also shown that PTP1B delays HER2/Neu-induced mammary cancer.⁸⁴

In conclusion, the EGFR/E-cadherin balance is a major component of epithelial regulation and homeostasis and its regulation by the phosphatase PTP1B is a major and promising element.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Centre National de la Recherche Scientifique.