Cross-talk between EGFR and T-cadherin: EGFR activation promotes T-cadherin localization to intercellular contacts

Abstract

Reciprocal cross-talk between receptor tyrosine kinases (RTKs) and classical cadherins (e.g. EGFR/E-cadherin, VEGFR/VE-cadherin) has gained appreciation as a combinatorial molecular mechanism enabling diversification of the signalling environment and according differential cellular responses. Atypical glycosylphosphatidylinositol (GPI)-anchored T-cadherin (T-cad) was recently demonstrated to function as a negative auxiliary regulator of EGFR pathway activation in A431 squamous cell carcinoma (SCC) cells. Here we investigate the reciprocal impact of EGFR activation on T-cad. In resting A431 T-cad was distributed globally over the cell body. Following EGF stimulation T-cad was redistributed to the sites of cell–cell contact where it colocalized with phosphorylated EGFR^Tyr1068. T-cad redistribution was not affected by endomembrane protein trafficking inhibitor brefeldin A or de novo protein synthesis inhibitor cycloheximide, supporting mobilization of plasma membrane associated T-cad. EGF-induced relocalization of T-cad to cell–cell contacts could be abrogated by specific inhibitors of EGFR tyrosine kinase activity (gefitinib or lapatinib), lipid raft integrity (filipin), actin microfilament polymerization (cytochalasin D or cytochalasin B), p38MAPK (SB203580) or Rac1 (compound4). Erk1/2 inhibitor PD98059 increased phospho-EGFR^tyr1068 levels and not only amplified effects of EGF but also per se promoted some relocalization of T-cad to cell–cell contacts. Rac1 activation by EGF was inhibited by gefitinib, lapatinib or SB203580 but amplified by PD98059. Taken together our data suggest that T-cad translocation to cell–cell contacts is sensitive to the activity status of EGFR, requires lipid raft domain integrity and actin filament polymerization, and crucial intracellular signalling mediators include Rac1 and p38MAPK. The study has revealed a novel aspect of reciprocal cross-talk between EGFR and T-cad.

Introduction

Cadherins are a major class of transmembrane cell–cell adhesion proteins that play fundamental roles in orchestrating morphogenetic and differentiation processes during development and in maintaining tissue integrity and homeostasis. The classical (or Type I) cadherins, such as E-, N-, P- or VE-cadherin, function as membrane spanning macromolecular complexes. They maintain stable cell–cell cohesion via homophilic ectodomain binding and cytoplasmic tail interactions with α/β-catenins and the actin cytoskeleton [1]. They can also behave as adhesion-activated signalling receptors via direct or indirect interactions with a variety of cytoplasmic and membrane proteins that participate in fundamental intracellular processes such as signal transduction, protein trafficking, cytoskeletal arrangement, inter alia [1]. Dysfunction of classical cadherins is a major contributor to cancer progression; perturbed cadherin-mediated cell–cell adhesion and intracellular signalling pathways result in the loss of contact inhibition and aberrant cell migration, metastasis, invasion and proliferation [2].

T-cadherin (T-cad) is an atypical cadherin family member which lacks transmembrane and cytoplasmic domains and is instead anchored to the membrane by a glycosylphosphatidylinositol (GPI) moiety. T-cad expression is frequently downregulated through allelic loss or hypermethylation of the T-cad gene promoter region in many different cancers [3]. Ectopic re-expression/upregulation of T-cad in tumor cells can reduce cell proliferation, migration and invasion while silencing of T-cad facilitates these processes [4], [5], [6], [7]. Therefore T-cad is considered a putative tumor suppressor, although this action may not primarily depend on the maintenance of stable cell–cell cohesion. T-cad is capable of homophilic binding [8] but its structural peculiarities engender relatively weak adhesive interactions [9]. Available evidence would indicate that the control of tissue integrity by T-cad depends rather on its actions as a signalling receptor participating in recognition of extracellular cues, dynamic cellular adhesion–deadhesion and guidance [10]. T-cad can interact directly or indirectly with a number of membrane-associated adaptor molecules to activate signal transduction pathways that impact cell–matrix adhesion, proliferation, survival and migration [11], [12], [13], [14], [15].

An alternative aspect of cadherin-regulated cell behavior concerns reciprocal cross-talk between cadherins and receptor tyrosine kinases [16]. Of particular interest in the context of tumor progression is bi-directional crosstalk between classical E-cadherin and the epidermal growth factor receptor (EGFR) which is overexpressed in many epithelial tumors. E-cadherin-mediated cell–cell adhesion reduces ligand-dependent activation of EGFR [17]. Tumor-derived E-cadherin mutations increase EGF-induced Ras activation by stabilizing the EGFR in the cell surface [18] and downregulation of E-cadherin upregulates EGFR expression [19]. Conversely, EGFR activation counteracts E-cadherin-mediated adhesion by disrupting interactions between E-cadherin and the cytoskeleton [20], [21]. Recently we reported that EGFR pathway activity can also be regulated by T-cad; in A431 squamous cell carcinoma (SCC) T-cad upregulation promoted retention of EGFR in lipid rafts and inhibited EGFR signalling and functional effects, whereas T-cad silencing released EGFR from this compartment, rendering EGFR more accessible to ligand stimulation [11]. We proposed a function for T-cad as a negative auxiliary regulator of EGFR. In this study we explore the possible converse scenario of effects of EGFR activation on T-cad.