P-cadherin counteracts myosin II-B function: implications in melanoma progression

Cutaneous melanoma, an aggressive cancer type originating from melanocytes in the human skin, is characterized as an invasive and commonly metastasizing tumor which is the major cause of death of melanoma patients [1, 2] Normal cutaneous melanocytes form cell-cell contacts with adjacent keratinocytes, providing a molecular anchor by which melanocytes participate in the normal function and architecture of the human skin. Malignant transformation of melanocytes is featured by downregulation of cell-cell adhesion molecules like E- and P-cadherin, resulting in the loss of keratinocyte-mediated growth and motility control [3, 4]. Concomitant with these changes, melanoma cells often undergo a phenomenon, referred to as epithelial-to-mesenchymal transition (EMT), and obtain a migratory and protease-producing phenotype, leading to invasion and the formation of distant metastasis [5, 6]. P-cadherin is a calcium-dependent cell-cell adhesion molecule belonging to the cadherin superfamily which comprises transmembrane proteins grouped by the presence of one or more cadherin repeats in their extracellular domains. The classical cadherin family consists of E(pithelial)-, N(euronal)-, V(ascular)E(ndothelial)- and P(lacental)-cadherin, named after the tissue they were first identified in [7, 8]. Classical cadherins exert cohesive and organising functions that are required for tissue development and integrity. In contrast to the universal expression of E-cadherin in epithelia, P-cadherin is only expressed in the basal layer of squamous epithelia [9, 10]. In epithelial-derived cancer, cadherins are often downregulated resulting in decreased cellular cohesion, increased invasion and formation of metastasis [11]. Our group showed that stable introduction of P-cadherin in BLM melanoma cells inhibits invasive capacities, stimulates homo- and heterotypic adhesion and induces an epithelioid phenotype [12]. Other experimental work elucidating the functions of P-cadherin pointed out that this molecule can have an opposite role depending on the cellular context [9, 12‐14]. Non-muscle myosin II belongs to the myosin superfamily and is an ATP-dependent molecular motor protein that can interact with and contract filamentous actin (F-actin) [15]. In vertebrates, three isoforms of the non-muscle myosin II heavy chain have been described: II-A, II-B and II-C. Each isoform is encoded by a specific gene and despite considerable homology between the different isoforms, differences in subcellular localization, enzymatic properties, filament assembly-disassembly regulation and tissue expression patterns have been described [16]. Nonmuscle myosin II heavy chain B (myosin II-B) plays a major role in the retraction phase of the migratory cycle in contrast to the bipolar shape- and substrate adhesion-related protrusion functions of nonmuscle myosin II heavy chain A (myosin II-A) [17‐19]. In migrating endothelial cells, it has been shown that myosin II-A is more abundant near the leading edge and myosin II-B in trailing ends. Moreover, myosin II-A moves with a higher velocity into new protrusions whereas myosin II-B is confined much longer to the retraction site of the migrating cell [20]. Myosin II-B filament assembly and ATPase activity are regulated by phosphorylation via two main kinases, myosin light chain kinase and Rho kinase, which phosphorylate Ser19 at the regulatory light chain [21‐23].

We show here that the anti-migratory and anti-invasive capacity of P-cadherin in BLM melanoma cells can be related to P-cadherin-dependent downregulation and organization of the myosin II-B isoform, implicating a coordinated cross-talk between adhesion molecules and cellular migration-related proteins.

The formation of metastasis is the main cause of mortality of cancer patients. These processes require an initial, crucial step, namely invasion of tumor cells in surrounding stroma, where loss of intercellular adhesion and the gain of a migratory phenotype are of major importance. Differentiating basal cells migrate perpendicularly to the basal layer towards the surface of the skin, and various established cell-cell contacts maintain the epithelial structure and promote coordinated cellular migration. However, melanoma has been described as a strong invasive and metastasizing tumor, implicating loss of cellular cohesion and simultaneously acquirement of a migratory phenotype. Loss of cohesion has been evidenced by showing a cadherin shift in melanocytic tumors leading to decreased homo- and heterotypical intercellular adhesion and providing molecular anchors to interact with vascular endothelial cells and fibroblasts [24, 25]. The close relationship between cell-cell adhesion proteins with polarity- and migration-related proteins, both in normal tissue and tumor, implicates a coordinated cross-talk between cell-cell adhesion molecules and migration-related proteins. This interaction has been shown between E-cadherin and polarity- and migration-related proteins like Rho GTPases. However, in most cases signal transduction pathways are pointed out, in contrast to the functional effector proteins [26, 27]. In this paper, we describe that the expression of P-cadherin counteracts the expression and organization of nonmuscle myosin II-B which results in the repression of the migratory and invasive behaviour of melanoma cells. Due to the initial observation that introduction of P-cadherin in invasive BLM melanoma cells promoted cell-cell adhesion and counteracted invasion [12], we tried to determine molecular targets of P-cadherin that contribute to the anti-invasive and anti-migratory effect mediated by P-cadherin. Nonmuscle myosin II-B, implicated in cellular migration, cytokinesis and other cellular activities, could be identified as a functional effector protein that was downregulated in BLM P-cad cells. Myosin II-B staining of BLM LIE and BLM P-cad showed a stress fiber pattern in BLM LIE occurring at the trailing end of a polarized migrating cell, in contrast to BLM P-cad where no myosin II-B stress fibers, but a faint and diffuse, cytoplasmic expression pattern could be detected. Moreover, the localization of myosin II-B stress fibers relative to Golgi and the function of myosin II-B in cellular migration and invasion, demonstrates its contribution to these cellular activities by retracting the tail. Furthermore, the amount of phosphorylated regulatory light chain associated with myosin II-B is significantly higher in BLM LIE than in BLM P-cad. One of the morphological features of BLM P-cad cells is the formation of lamellopodia and filopodia but the tails of the cells are anchored by P-cadherin-mediated cell-cell contacts. Since the subcellular localization of both P-cadherin and myosin II-B occur at the same site of nucleus with reference to the Golgi apparatus, mutual and localized regulation can easily occur. All the tested melanoma cell lines showed no P-cadherin expression and high levels of myosin II-B. Interestingly, MeWo has been described as a non-invasive melanoma cell line and showed P-cadherin positivity. In order to confirm whether the P-cadherin switch in accordance with high myosin II-B expression effectively acts in melanoma patients, we evaluated nodular melanomas for P-cadherin and myosin II-B positivity, because of the deep infiltrative nature of this melanoma subtype. Normal epidermal layers showed a strong honeycomb-like P-cadherin staining, designating the cohesive nature of the normal skin. In contrast, the deeper tumor sites showed an aberrant or lack of expression of P-cadherin.

Cross-talk between adhesion molecules, e.g. E-cadherin, N-cadherin and αvβ3 integrin, and melanoma progression has been widely described. αvβ3 Integrin expression in human melanomas promotes invasion through physical interaction with MMP-2, and inducing transendothelial migration by association with the human neural cell adhesion molecule L1 expressed on endothelium [28]. For cadherins, loss of E-cadherin expression during melanoma progression has been shown to promote melanoma migration and, paralleled by N-cadherin upregulation, increased gap-junctional and N-cadherin-mediated communication with dermal fibroblasts and endothelial cells [29‐31]. In this study, we present an important effector molecule that can be related to P-cadherin expression in invasive melanoma.

The molecular mechanism underlying P-cadherin-mediated myosin II-B regulation concerning migration and invasion will be the major focus for our future research. Our data suggest that mainly MLCK activity contributes to cellular migration in BLM melanoma cells. This means in our proposed model that introduction of P-cadherin in melanoma cells results in spatial MLCK inhibition at sites of P-cadherin-mediated cell-cell contacts. Since melanocytes originate from segregation of cell lineages derived from the cephalic neural crest, the neural genetic and epigenetic background may not be overruled [32]. Therefore, we hypothesize that, as shown in neuronal cells, cadherin expression can modulate calcium channels and thereby alter transient calcium influxes and available intracellular calcium at sites of cadherin-mediated cell-cell contacts, as several ion channels and ion channel regulated proteins are presented in the microarray list (Additional file 1). Directional migration of a neuron is the succession of three major processes: protrusion of the leading edge, cell body translocation and retraction of the trailing end [33]. For each of these processes, a critical, regulating role of calcium waves has been pointed out [34]. Important, Shengyu Yang et al. recently showed that calcium-influxes, rather than calcium release of intracellular stores, are critical for growth-factor induced cell migration. Moreover, they describe that calcium channels facilitating calcium-influxes activate MLCK specifically at the trailing tail, without an effect on the phosphorylation status of myosin light chain at the lamellopodia, supporting our hypothesis [35]. An intensively studied calcium flux modulator is the NMDA receptor. Besides its presence in an 'industrial complex' associated with an enormous variety of cytoskeletal proteins (including myosin II-B), cellular adhesion proteins (including cadherins) and signaling proteins (including calmodulin kinase and tyrosine kinases) at the postsynaptic membrane for regulating calcium influx, it is a known regulator of cellular migration. Indeed, it has been shown that there is a positive correlation between the rate of cell movement and the amplitude and frequency of calcium fluctuations governed by NMDA receptors [36, 37]. Therefore, it would be interesting to explore further the molecular link between cadherins, calcium signaling and myosin II activity and their implications in melanoma progression.

In general, we can conclude that the inhibition of invasion and impaired migration of melanoma cells can, at least partially, be ascribed to a P-cadherin-mediated decreased myosin II-B expression and impaired organization. This interplay between adhesion and migration may be an important strategy of the melanoma cell to invade the deeper stroma and to form metastasis.