Malignant melanoma is the most aggressive and therapy resistant type of cancer leading to malignant transformation of pigment skin cells, melanocytes.
Progression and metastasis of melanoma tumours strongly correlates with dynamic regulation of cadherin expression. In many cases, cell motility is physically and biochemically stimulated by N-cadherin – mediated cell interactions and signalling pathways. Our recent study showed that silencing of N-cadherin in melanoma cells significantly impaired signalling through AKT and ERK pathway [1].
The object of this study is recognition of the possible role of N-cadherin and selected downstream protein kinases: PI3K, ERK1/2, and mTOR in cell invasion. It will also examine their possible influence on metalloproteinases (MMPs) activity in human melanoma cells: WM793 (VGP), WM115 (VGP) from the primary tumour site, as well as Lu1205 (lung) and WM266-4 (skin) from metastatic sites.
Melanoma cells were transfected with the small interfering RNA (siRNA) that targets human N–cadherin gene (CDH2). siRNA sequences were generated based on in vitro transcription system (Silencer siRNA Construction Kit- Ambion). Inhibitors LY294002 (PI3K), U0126 (ERK1/2), Everolimus (mTOR) were used to inhibits selected kinases of signalling pathways. In vitro cell migration was studied using Matrigel and analysis of matrix metalloproteinases MMP-2 and MMP-9 activity by gelatinase zymogram assay.
Silencing of N-cadherin gene expression by siRNA, and treatment of melanoma cells with kinases’ inhibitors significantly reduced activity of MMP-2 and MMP-9, as well as cell migration and invasion. The most significant decrease in metalloproteinasse MMP-2 and MMP-9 activity was observed when using N-cadherin siRNA with U0126 or Everolimus (24h), as well as N-cadherin siRNA with LY294002 (48h). Comparable decreases in MMP activity were observed when the following combination of inhibitors was used: Everolimus with U0126 or LY294002, and LY294002 with U0126. Knockdown of N-cadherin reduced melanoma cell migration in range of 20-25%, whereas combination treatment of cells with siRNA for N-cadherin and Everolimus or U126 reduced cell migration by 38%.
Both of the current and the former results [1] suggest that activation of PI3/AKT, mTOR and ERK kinase following N-cadherin expression contributes not only to increased proliferation, but also invasive potential of melanoma cells. The results also indicate that N-cadherin, as well as the studied kinases, should be considered as a potential target in melanoma therapy.
Introduction
It is becoming ever clearer that integration of signals triggered by cell-cell interactions, cell-matrix signalling and growth factor signalling, plays a crucial role in cellular behaviour. Cadherin’s have been implicated in a number of signalling pathways that stimulate cellular behaviour. They are also of key importance for embryo compaction [2], cell intercalation [3, 4] and cell sorting [5]. Furthermore, cadherin’s with associated protein-mediated cell–cell adhesion promote a unique cytoskeletal structure that supplies adhesive strength [6-10]. Interest in cadherin’s has grown since the late 1980s, when they were found to be linked with cancer progression and other diseases.
Human melanocytes express both E-cadherin and P-cadherin. E-cadherin is primarily responsible for adhesion of melanocytes to keratinocytes. Melanocytes embedded in the basal layer of epidermis interact with keratinocytes through E-cadherin, which in effect regulates their growth [11]. Pathologic changes leading to growth of malignant melanoma disturb the delicate homeostatic balance between melanocytes and keratinocytes and can lead to altered cell-cell adhesion and cell-cell connections. In majority of epithelial cancers, loss of E-cadherin (CDH1), P-cadherin (CDH3) or both during tumour progression results in an increased expression of the mesenchymal cadherin, i.e. N-cadherin, with a significant change in the adhesive properties of cancer cells, as they lose their affinity for epithelial neighbours in favour of stromal cells [6, 12-15].
One of the first and most important steps in the metastatic cascade is the loss of cell-cell and cell-matrix interactions. N-cadherin, a crucial mediator of homotypic and heterotypic cell-cell interactions might play a central role in the metastasis.
It was shown that in various cancer cells, the expression of N-cadherin is associated with cell migration as it induces cells’ motility in many types of cancers such as breast, prostate, and gastric cancer. Interestingly, overexpression of N-cadherin enhances cell motility and invasion without decreasing E-cadherin levels, suggesting that increased cell motility is caused by expression of N-cadherin rather than lack of E-cadherin [16]. Therefore, strict regulation of N-cadherin expression is essential in normal epithelial cell function [16].
Degradation of basement membranes and stromal extracellular matrix (ECM) is crucial for invasion and metastasis of malignant cells. Degradation of ECM is initiated by proteinases secreted by different cell types participating in tumour cell invasion, and increased expression or activity of every known class of proteinases (metallo-, serine-, aspartic-, and cysteine) has been linked to malignancy and invasion of tumour cells [17]. Gelatinase A (MMP-2, 72 kDa gelatinase) is expressed in a variety of normal and transformed cells, including fibroblasts, keratinocytes, endothelial cells and chondrocytes. Gelatinase B (MMP-9, 92kDa gelatinase) is produced by keratinocytes, monocytes, macrophages and many malignant cells. High levels of MMP-9 were detected in breast, colorectal and gastric cancers. The possibility of using MMP-9 as a marker of skin melanoma vertical growth phase has been suggested. [18].
In addition to induction of degradation of intracellular matrix components, MMP-9 is involved in regulation of the basic biological processes of: apoptosis, proliferation, and differentiation [19-20].
The role of N-cadherin in cell differentiation, transformation and invasion has also been documented [21,12] with some of these functions, most probably depending on the activation of intracellular signal transduction cascades [22]. However, neither the mechanism by which N-cadherin regulates entry into the cell cycle, nor its migratory characteristics have yet been fully elucidated.
Therefore following our earlier report on the role of N-cadherin on melanoma proliferation [1], we sought to look at the possible initiative role of this adhesion molecule on cancer cell migration and invasive potential.
Materials and Methods
Cell culture
Human melanoma cell lines: WM793 (VGP) – Lu1205 (metastatic; biopsy taken from the lung; selection in mice; a culture from the primary site (sternum area) from the same donor is WM793). WM115 (VGP) – WM266-4 (metastatic, skin) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics: penicillin, streptomycin. Cells were treated with inhibitors: PI3K – LY294002 (Cell Signalling TM) – 20μM concentration, ERK1/2 – U0126 (Cell Signalling TM) – 10 μM concentration, and mTOR– Everolimus (Selleck) in 5 nM concentration. The incubation time of melanoma cells with inhibitors were 24 and 48 hours. Cells were obtained from the ESTDAB Melanoma Cell Bank (Tubingen, Germany).
siRNA transfection of melanoma cells
Melanoma cells were grown until 60% confluence was reached, and then transfected using Oligofectamine reagent (Invitrogen), in keeping with the manufacturer’s protocol, with two different, previously described [1] 21bp double-stranded siRNA molecules specifically targeting for the N-cadherin: siRNA CDH2 (target sequence 5’-AAAGTGGCAAGTGGCAGTAAA-3’ -nucleotides 798-818; NM001792) – generated in vitro transcription (SilencerTM siRNA Construction Kit (Ambion)) and commercially available (Ambion ID#S27773) or a control non-silencing sequence (Ambion) [1]. The effects that RNA interference had on expression of CDH2 mRNA and protein were determined by reverse transcription-PCR (RT-PCR) and Western immunoblotting respectively. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to monitor efficiency of RT-PCR.
WM793, WM115, WM266-4 cells were transfected with 50 nM, and Lu1205 cells with 100 nM siRNA. Medium was replaced 24h later with a fresh one, and cells were grown for an additional 24h to 48h period (48h or 72h post-tansfection) prior to further analysis [1].
Cell Migration and Invasion Assay
Cell invasion assays were performed using conventional Boyden transwell methods in keeping with the manufacturer’s protocol (BD BioCoatTM FluoroBlok Invasion System No. 354166). Melanoma cell migration was studied with the use of Matrigel fluoro-chamber. Quantitation of invasion was achieved by post invasion cell labelling with Calcein AM (Fluka), and measuring the fluorescence of invading cell samples at excitation/emission wavelengths of 485/530 nm sensitivity 100 on a plate reader (BIO-TEK).
Zymography
Melanoma cells from primary (WM793, WM115) and metastatic sites (Lu1205, WM266-4) were grown as monoculture for various periods of time (24, 48 hours). Serum-free conditioned media were collected and analysed for presence and activity of the metalloproteinase: MMP-2 and MMP-9 using gelatin zymography.
Gels were prepared in the presence of 0.1% gelatin (Sigma) in non-reducing conditions. Proteins were loaded per well and separated with the use 4.5% stacking and 10% separation gel in a 4h run. Following electrophoresis, the gel was washed two times for 30 min in 2.5% TritonX-100.
The gel was intubated at 37°C for 48h in buffer (50 mM Tris pH 7.5; 10 mM CaCl2; 0.15 mM NaCl).
Then the gel was stained in a solution containing 1% Coomasie blue R250 in 50% methanol and 10% acetic acid for 1h. Gelatinolytic activity was observed as clear areas in the gel.
Densitometry analysis
Densitometry analyses of gelatinolytic activities of metalloproteinase MMP-2 and MMP-9 were performed on raw volume (sum of intensities of bound) using SynGene Gene Tools version 4.03.0 (Synoptics Ltd Beacon House, Nuffield Road Cambridge, CB4 1TF, UK).
Cytotoxicity assay
Cytotoxicity of N-cadherin siRNA (100 nM), PI3K inhibitor – LY294002 (20μM) ERK1/2 inhibitor – U0126 (10 μM) and Everolimus (5nM) mTOR inhibitor assay was determined using – Cytotoxicity Detection Kit LDH, Roche, Germany. In all examined melanoma cell lines both N-cadherin siRNA and inhibitors: Everolimus, LY294002, U0126 showed no cytotoxicity effect tested in a culture medium at the time of 72h. LDH activity in the culture medium in no case exceeded 3%.
Statistics
Cell migration and invasion data were calculated from mean values of replicate experiments as indicated, using the Student’s t-test. Results were considered significant at p< 0.05.
Results
N-cadherin regulates cell invasion
To investigate if and how N-cadherin affects cell migration and invasion in studied melanoma cells, specific N-cadherin siRNA were used [1].
As was already shown, N-cadherin could be successfully silenced designed by in vitro transcription with each two siRNAs molecules specifically and effectively targeting for the N-cadherin [1].
The study on the role of N-cadherin in migration and invasion of melanoma cells was performed using conventional Boyden trans-well methods. All tested melanoma cell lines manifested the ability for cellular migration as a capacity to chemotaxis. Cells’ migration and invasion after 48 h knock-down of N-cadherin compared with control (non-specific siRNA) or non-treated cells was measured.
N-cadherin siRNA-transfected cells showed reduction of migration and invasion by 20-25% when compared with control cells (Fig. 1). The effect was observed for primary WM793 and WM115 as well as metastatic Lu1205 and WM266-4 cell lines. The most profound inhibition was, however, noticed in case of WM793 (VGP) and WM266-4, invasion of which was reduced after 24h by: 25% (p<0.00005) and 23% (p<0.00005) respectively (Fig. 1).
Separate use of inhibitors: U0126 (ERK1/2) or Everolimus (mTOR) reduced melanoma cell invasion by approximately 21-25% (p<0.005), whereas treatment with LY294002 (PI3K) reduced only about 15% (p<0.005). Treatment of melanoma cells with combination of U0126 (ERK1/2) and LY294002 (PI3K) inhibitors decreased it to about 25% (p<0.005). Applications of a combination of siRNA for N-cadherin and U126 (ERK1/2) inhibitor resulted in a reduction of invasiveness of cells by about 38% (p<0.00005) in Lu1205 cell line, and similar response was observed using a combination of siRNA for N-cadherin with Everolimus (mTOR) inhibitor (Fig.1).
N-cadherin regulates gelatinolytic activities of the metalloproteinase MMP-2 and MMP-9
The invasive ability of cancer cells can be regulated through the expression of zinc-dependent endopeptidases, MMPs.
Therefore, activities of MMP-2 and MMP-9 were studied both in the primary (WM793, WM115) and metastatic (Lu1205, WM266-4) cells lines. MMP-9 activity was always much lower than that of MMP-2, except for the Lu1205 cells. Both monomeric and dimeric forms of MMP-9 were observed in all tested cell lines except Lu1205 (Fig.2).
Zymography used to measure activity of metalloproteinases in melanoma cells showed a significant difference between MMP-2 and MMP-9 activities in N-cadherin knocked-down cells in all tested cell lines in comparison with untreated ones. The greatest decreases in MMP-2 activity were observed after 48 h treatment and reached in the Lu1205 cells (about 47% ) and WM793 line (about 40%), whereas the smallest in WM115 – 12%. In the case of MMP-9, decreases of activity reached 62% and 52% of their initial values in Lu1205 and WM793 cells respectively. Summary of the results of densitometry analysis of the activity of MMP-2 and MMP-9 after N-cadherin knockdown is presented in Fig. 2.
It was formerly shown that inhibitors of some signalling kinases as AKT and ERK1/2 used in combination with siRNA against N-cadherin led to more effective inhibition of melanoma cells proliferation [1]. Therefore, we sought to check if the decreases in MMPs activities caused by N-cadherin silencing may be strengthenen by additional inhibition treatment with selected signal kinases inhibitors.
The largest decreases in the activities of metalloproteinases, particularly in MMP-2, was observed in the case of 48h treatment of cells with combination of siRNA for N-cadherin and PI3K inhibitor – LY294002 (Fig. 3). With shorter (24h) incubation time best results were achieved with application of combination of siRNA for N-cadherin and inhibitor U0126 (ERK1/2). High efficiency of MMPs inhibition was also reached for the combination of siRNA for N-cadherin and inhibitor Everolimus (mTOR), regardless of incubation time. The use of single inhibitor gave similar results to N-cadherin gene silencing but much less promising than in the case of combinations of inhibitors and siRNA for N-cadherin (Fig 3).
The activity of MMP-9 was much lower than that of MMP-2 with the exception of Lu1205.
As in the case of MMP-2, the greatest decreases in MMP-9 activity were observed when using a combination of siRNA for N-cadherin and PI3K inhibitor – LY294002. Similar results were observed for both metalloproteinases: MMP-2 and MMP-9 after application of mTOR inhibitors – Everolimus.
Melanoma cells react differently to MEK kinase inhibitor. Observed small increase in the activity of metalloproteinases in the two cell lines: WM115 (VGP) and WM 266-4 (from the same source). Like the results obtained by Ferguson et al. 2013 [22] where a rise in MMP-2, WM266-4 after application of MEK kinase inhibitors: PD184352 or selumetinib.
(Fig 3) illustrates the results of densitometry comparison of MMP-2 and MMP-9 activity after knockdown of N-cadherin and treating with U0126 (ERK1/2), LY294002 (PI3K) and Everolimus (mTOR) inhibitors.
Discussion
It has been previously demonstrated that N-cadherin had significant effect on cell signalling, cell cycle regulation and, in consequence, proliferation of melanoma cells, as the use of siRNA against N-cadherin led to significant reduction of number of such treated cancer cells [1].
The current study confirmed that melanoma cells transfected with N-cadherin specific siRNA successfully and transiently decreased its expression at mRNA and protein levels. This resulted in inhibition of metalloproteinase 2 and 9 activities and reduced cell invasion.
In various cancer cells abnormal expression of N-cadherin correlates with induction of cell motility. Expression of N-cadherin induces cell migration in e.g. breast cancer, melanoma, prostate cancer, and gastric cancer [21, 24, 25]. Furthermore, the E- to N-cadherin switch is often observed in aggressive cancers [26]. The up-regulation of N-cadherin in aggressive carcinomas suggests that the level of its expression is a critical parameter for cancer cell invasion.
Melanoma cell invasion.
The results of the study on the effects of N-cadherin knock down on cell invasion confirm the role of this adhesion molecule in the process of invasion of melanoma cells. In all tested melanoma cell lines a statistically significant decrease number of invading cells in the range between 20-25% was observed. Furthermore, impact of protein kinase inhibitors: LY294002, U126, Everolimus, and combination of siRNA for N-cadherin and protein kinase inhibitors on the process of melanoma cell invasion reported here suggests involvement of all three kinases in N-cadherin initiated signalling. Simultaneous administration of any of protein kinase inhibitors and siRNA for N-cadherin resulted in a decrease of approximately 30%, whereas with the use of a combination of siRNA for N-cadherin and ERK MAPK pathway inhibitors-U126 diminished cells invasion up to 38%.
MMPs in tumour progression
MMPs play an important role in many biological and pathological processes. Uncontrolled, activity of metalloproteinases may well lead to development of many diseases such as arthritis, atherosclerosis, aneurysms, nephritis, tissue ulcers or fibrosis and cancer [27]. The MMPs have been present in the discussion on cancer for more than 40 years, being overexpressed in a wide range of malignant tumours in response to oncogenic cellular transformation, cytokines and several growth and angiogenic factors [28].
Initial observations on the role of MMPs in the cancer biology have suggested that the ability of tumour cells to metastasize correlates with increased levels of metalloproteinase activity. Elevated levels of gelatinases, MMP-2 and MMP-9 are often observed in malignant cancers. Among human melanoma cells, MMP-2 and -9 have attracted attention in the recent years, especially with regard to cutaneous, eye and oral melanomas [28]. Expression of MMP-2 has frequently been associated with malignant progression and poor prognosis [29]. Particularly high levels of MMP-2 were observed in WM793 melanoma cell line from the primary vertical growth phase (VGF).
Results of studies using tissue microarray, immunohistochemistry of melanoma biopsies of primary and metastatic lesions, as well as nevi, confirmed that MMP-2 is predictive of primary and metastatic stages [28]. High MMP-2 expression in the primary lesion contributes to the invasiveness of primary tumour cells, leading to metastases and poor survival outcomes [30].
Similarly to other publications [31], this study showed a high level of the MMP-2 activity in all tested melanoma cell lines.
The second gelatinase MMP-9 proved to be much less active than MMP-2, except for the Lu1205 from metastatic cell line, where activity of MMP-9 was at a high level. Here, monomeric and dimeric forms of MMP-9 were detected in all tested lines except for Lu1205. MMP-9 also exists as a monomeric and homodimeric molecule, in both its latent and active forms. Both monomeric and dimeric forms of MMP-9 have been identified in a variety of cells (normal and tumour cells alike) and in biological fluids and tissues, indicating that both forms are physiologically relevant. Dimerization or multimerization is mediated by the carboxyterminal domains of MMP-9, and occurs intracellularly [32]. The functional biological role of the MMP-9 dimer has not yet been elucidated, however, dimerization significantly decreases the activation rate of pro-MMP-9 by stromelysin (MMP-3) [32].
The herein reported here significant decrease of MMP-2 and MMP-9 activities that were observed after knockdown of N-cadherin remains in agreement with the view on the important role of this adhesion molecule in activation of matrix metalloproteinases, and in effect stimulation of invasion and metastasis. The largest decreases of MMP-2 activity were observed in the Lu1205 and line WM793 (by about 47% and 40% respectively). Concurrently, MMP-9 activity in the cell lines dropped by about 62% and 52% respectively.
Decreases of MMP-2 and MMP-9 activities were also observed upon using a combination of siRNA for N-cadherin and protein kinases inhibitors: LY294002 (PI3K), U126 (ERK1/2) and Everolimus (mTOR). The largest decrease in activity of metalloproteinase, MMP-2 in particular, was observed upon using a combination of siRNA for N-cadherin and PI3K inhibitor LY294002 after 48h treatment, while at shorter incubation time (24h) application of a combination of siRNA for N-cadherin and inhibitor U0126 produced best results. Using the combination of siRNA for N-cadherin and inhibitor of mTOR – Everolimus gave similar results regardless of the incubation time.
Hazan et al. 2004 [21] suggested that N-cadherin functionally interacts with the FGF receptor, causing sustained downstream signalling by PI3K, and through MAPK-ERK promotes cell survival, migration and invasion. Stabilization of FGF-1 receptor by N-cadherin followed by MAPK-ERK activation, may result in increased transcription of the extracellular matrix-degrading enzyme MMP-9 and hence, increased cellular invasiveness. The FGF-1 may interact with the fourth extracellular domain (EC4) of N-cadherin based on the fact that transfer of EC4 domain from N-cadherin onto E-cadherin reconstituted the invasive function of N-cadherin [21].
Current results show important role of N-cadherin in melanoma cell invasion. In light of our former studies, where we observed significant reduction of melanoma cell proliferation in effect of N-cadherin silencing the hypothesis that MMP-9 expression and cellular invasion are governed by at least two, but possibly more, distinct intracellular signalling pathways, when stimulated by N-cadherin-FGFR signalling [21], seems to be sound.
Melanoma cells react differently to the MEK kinase inhibitors, as Ferguson et al. 2013 [22] observed a slight increase in the activity of MMP-2 in the use of ERK1/2 inhibitor – U126. Preclinical data presented by Catalanotti et al. 2013 [33]
suggest that patients with B-RAF mutant melanomas and PI3K/AKT pathway activation are less sensitive to MEK inhibition. 50% of melanoma cases showed presence of B-RAF mutation. E.g. All studied cell lines exhibit B-RAF mutations: Lu1205 and WM793 – V600E, WM115 and WM266-4 V600D.
Particularly promising are the results with regard to the decrease of melanoma cells invasiveness after N-cadherin gene silencing and the use of Everolimus inhibitor of the mTOR pathway. Everolimus is a low toxicity drug in use for several years as an immunosuppressant in organ transplant patients, in particular in cancer-related cases [34]. In recent years, use of Everolimus and other inhibitors of the mTOR pathway for antitumor therapy has attracted some interest [35]. The fact that their inhibitory effect may be significantly enhanced by simultaneous use of siRNA for N-cadherin may be worth considering as a potentially new approach to effective treatment of melanoma.