Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells

Abstract Epidermal and hepatocyte growth factors can stimulate invasive abilities of melanoma cells, while treatment with combination of their receptors' (EGFR and MET, respectively) inhibitors reduces viability of these cells, as we have previously shown. Proposed therapy has potential; however, used drugs block more than one goal effectively, what raises the question about the real target of analysed inhibitors. For this reason, we analysed direct involvement of these receptors in the invasion of melanoma cells inducing EGFR and MET up‐ and down‐regulations in examined cells. Results were acquired with assays evaluating cell migration and invasion (scratch wound assay, Transwell filter‐based method and single‐cell tracking). We revealed that cells' motile abilities are increased after EGFR overexpression and decreased following EGFR and MET silencing. This outcome correlates with elevated (EGFR up‐regulation) or reduced (EGFR/MET down‐regulation) number of formed invadopodia, visualized with immunofluorescence, and their rate of proteolytic abilities, evaluated by fluorescent gelatin degradation assay, and gelatin zymography, compared to control cells. Above‐mentioned data indicate that both—EGFR and MET signalling is directly connected with melanoma cells invasion, what establishes these receptors as promising targets for anti‐cancer treatment.


| INTRODUC TI ON
Melanoma is a heterogeneous tumour with a very low cure rate in the case of metastasis in which conventional therapies fail to improve overall survival. Although many genes important for melanoma induction, transformation and metastasis have been identified, the established targeted therapies are often inefficient in the final outcome. This phenomenon may be related to the incomplete knowledge of the process of melanoma progression including possible mechanisms leading to development of drug resistance.
Understanding the acquisition of invasive behaviour by melanoma cells is therefore crucial. The research focused on the molecules and pathways involved in its progression is also needed. 1 Metastasis, the main cause of cancer patients' mortality, is a multi-step process, where cancer cells spread from primary tumour into the distant tissues moving through the surrounding extracellular matrix (ECM). Cell invasion is an essential stage of cancer spreading involving ECM degradation and remodelling. 2 In recent years, actinrich protrusions known as invadopodia have been shown to be critical for migration through the ECM. 3 These structures consist of an actin core surrounded by a number of protein components, including cytoskeletal modulators, adhesion proteins, scaffolding proteins and signalling molecules. Their main role is secretion of proteases digesting elements of the ECM, what enables cancer cells to migrate through surrounding microenvironment. 4,5 Previously, we showed that chemoattractants-epidermal growth factor (EGF) and hepatocyte growth factor (HGF) stimulate invadopodia formation, and extracellular matrix degradation, what correlates with higher invasive abilities of melanoma cells. 6 EGF receptor (EGFR) is up-regulated in many types of cancer.
In the case of melanoma, the gene expression data are inconsistent 7 ; however, some researches postulate that overexpression of EGFR often occurs in advanced stages of melanoma. 8 Following ligand binding EGFR undergoes dimerization what induces its autophosphorylation and is essential for downstream signalling pathways activation, with the most significant represented by PI3K/ AKT (Phosphoinositide 3-kinase/Protein kinase B) and MAPK (mitogen-activated protein kinase). These cascades participate in the regulation of several cellular processes, including cell proliferation, prevention of apoptosis and promotion of cell invasion. 9 Therefore, any aberrations in EGFR expression level or activity might be linked to the higher ability of cancer cells to invade and form metastasis. 10 The level of HGF receptor (MET) also seems to be related to the stage of malignancy in melanoma. 11 Its activation, mediated by HGF binding, promotes several processes involved in oncogenesis including tumour cell proliferation, migration, invasion and metastasis, through several intracellular signalling pathways such as PI3K/AKT, Src, STAT3 (Signal transducer and activator of transcription) and MAPK. 12 Moreover, MET localizes to invadopodia along with cortactin, one of the main components of migratory protrusions, and regulates its activation. 13 Interestingly, it was shown that both-EGFR and MET signalling regulates invadopodia formation and degradation of ECM by breast cancer cells. 13,14 Both receptors-EGFR and MET seem to be a promising target in anti-metastatic therapy, since our previous studies indicated that chemical inhibition of their activity results in synergistic cytotoxic effect on the viability and proliferation of melanoma cell lines derived from primary tumour and metastasis. 15 Additionally, we observed the reduction in number of formed invadopodia and decline of migration, and invasion capacity of breast cancer cells treated with EGFR, and MET inhibitors. 16 Despite the fact that use of chemical inhibitors appears to be a good strategy in the anti-melanoma therapy, there appears to be a problem with low specificity of these compounds which may block activity of several receptors. This in turn may generate some ambiguities related to the targets, against which the therapies should be directed. Therefore, in this work we focused on the analysis of direct involvement of EGFR or MET in the regulation of invasiveness of melanoma cells derived from primary tumour and metastasis.

| Transfection procedure
Cells were transfected with 29-mer shRNA constructs directed against human EGFR or MET or 29-mer non-targeting shRNA (shCTRL), which were purchased from OriGene. For EGFR overexpression, pcDNA3 plasmid (Invitrogen) with cloned cDNA encoding human EGFR was ap-

| qRT-PCR analysis of gene expression
To measure the expression level of EGFR and MET in obtained cell lines, total RNA was isolated using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) following the manufacturer's protocol.
After DNase I (Sigma-Aldrich) treatment, reverse transcription reaction was performed using 0.5 μg of RNA and the High Capacity

| Western blotting analysis
To detect the protein level of EGFR and MET, cell lysates were prepared from examined cells by harvesting them in urea buffer (50 mmol/L TRIS-HCl pH 7.4, 5% SDS, 8.6% sucrose, 1 mmol/L DTT, 0.45% urea), supplemented with protease inhibitors cocktail (Sigma-Aldrich). Protein concentration was determined by the Bradford procedure, 17 and an identical amount of proteins were separated by SDS-PAGE electrophoresis 18 and transferred to nitrocellulose sheets. 19 Then, membranes were incubated with suitable primary antibodies directed against EGFR, MET or GAPDH (Santa Cruz Biotechnologies). Next, goat anti-mouse or goat anti-rabbit antibodies conjugated with horseradish peroxidase (Cell Signaling Technologies) were applied. Immunoblots were developed using the Clarity Western ECL Substrate (Bio-Rad), scanned with ChemiDoc (Bio-Rad) and analysed with ImageLab software (ver. 6.0, Bio-Rad). At least three independent experiments were performed in each case. For the evaluation of migration distances and cell trajectories, cells were seeded in low density, and images were analysed using ImageJ software with Manual Tracking plugin. 20 The distance covered by every cell was measured as the total distance based on the cumulative track lengths. The experiments were performed three times, and each time 40 cells were analysed.

| Transwell migration and invasion assay
Cell migration and invasion tests were performed using Transwell filters with 8 µm pore size (BD Biosciences) placed in 24-well plates.
Prior to the experiment, cells were starved for 16 hours in serumfree DMEM medium. Cells were seeded in medium deprived of

| Fluorescent staining
The subcellular distribution of actin filaments and cortactin was examined by immunofluorescence in cells seeded on 1 mg/mL Matrigel-coated coverslips in 24-well plates. After 24 hours, cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Coverslips were then blocked with 1% bovine serum albumin in PBS. Anti-cortactin antibodies (Santa Cruz Quantitative analysis of the number of invadopodia per cell was performed using ImageJ software. 20 Only invadopodia positive for F-actin and cortactin were scored, and at least 40 cells were analysed per condition.

| Fluorescent gelatin degradation assay
The experiments were done as previously described. 21

| Gelatin zymography
The MMP-9 activity was determined in serum-free media col-

| Statistical analysis
All data are presented as mean ± standard deviation (SD), and their significance was determined using Student's t test. The significance test was set at P ≤ .05 (*), P ≤ .01 (**) or P ≤ .001 (***).

| Characterization of the generated cell lines
In our studies, we used two melanoma cell lines: one isolated from primary amelanotic tumour (A375) and the second derived from lymph node metastasis (WM9). Previously, we demonstrated that both cell lines express HGF receptor at high level, whereas they differ in EGFR expression, which was detected at lower level in A375 in comparison with WM9 cells. 15

| The level of EGFR and MET regulates migration and invasion abilities of melanoma cells
Firstly, the influence of EGFR and MET expression level on spontaneous migration, where cells were seeded sparsely, and there was no factor inducing directional migration, was verified (Figure 2A,B). We noticed that A375 EGFR cells were able to cover much longer distances To analyse whether modified protein levels of MET and EGFR are able to impact melanoma cell migration in three-dimensional (3D) conditions, the invasion assays were subsequently performed ( Figure 3A). We observed that tested cells invade through Matrigel layer in a similar way as they migrate in 2D conditions. Overexpression of EGFR stimulated the invasion of A375 EGFR cells, whereas in the case of silenced EGFR and MET expression decreased invasion capacity was noticed in both cell lines.

| Influence of EGFR and MET level on invadopodia formation
As a result of our observation that EGFR and MET may regulate the invasion of primary and metastatic melanoma, we decided to put our attention to the invadopodia. They are actin-rich protrusions crucial for cell movement through the ECM. 3 Previously, we demonstrated, that tested A375 and WM9 cells are able to form these structures. 6 Therefore, to evaluate the influence of differential expression level of EGFR and MET receptors on invadopodia formation, cortactin (a marker of these protrusions) and filamentous actin (F-actin) were stained using immunocytochemistry ( Figure 3B). Invadopodia were visible as dots in the cell nuclei proximity, where F-actin and cortactin colocalized (which is indicated by white arrows in merge pictures, Figure 3B). Analysis of fluorescently labelled proteins showed increased number of invadopodia in A375 cells overexpressing EGFR and a contrary results were detected in WM9 shEGFR cells ( Figure 3C).
Moreover, decreased expression of MET also led to lowered number of invadopodia in examined cells.

| Impact of EGFR and MET signalling on proteolytic activity of examined melanoma cells
The main role of invadopodia is secretion of proteases digesting elements of the ECM, what enables cancer cells to invade through surrounding microenvironment and form metastasis. 4,5 Therefore, to estimate the proteolytic activity of tested cells, the gelatin-FITC degradation assay was performed. In this test, sites of gelatin digestion appeared as black spots present on a fluorescently labelled background (white arrows, Figure 4A). Obtained data confirmed that all tested cells were able to digest gelatin mainly because of the activity of invadopodia. Next, the digested area corresponding to the proteolytic activity of cells was quantified ( Figure 4B). The area was increased in A375 cells with up-regulated EGFR expression level and lowered in WM9 shEGFR cells in comparison with control cells. Cells with silenced MET (A375 shMET and WM9 shMET) also presented decreased proteolytic activity and digested lower area in comparison with appropriate controls.
Moreover, we performed gelatin zymography, which is another way to test proteolytic activity of cells. We noticed that activity of MMP-9 was lowered in cells with down-regulated EGFR and MET protein level ( Figure 4C), what was also confirmed by densytometric measurements ( Figure 4D). Surprisingly, similar effect on MMP-9 activity was induced by the overexpression of EGFR in A375 cells.
We suppose that the level of other protease(s) present in these cells is elevated, since the surface of gelatin digestion is higher in EGFR overexpressing than in the control cells. were associated with poor prognosis, 10 while overexpression of EGFR was often detected in advanced stage of melanoma. 8 MET was also demonstrated to be connected with malignant skin cancer development and the level of its expression seems to be related to the stage of malignancy in melanoma. 11,22 Moreover, based on our previous analysis we demonstrated that transcripts of both receptors are present in tumour tissue samples from patients suffering from melanoma (results for 114 primary and 155 metastatic melanoma samples from public database GEO). 15 Similar results were obtained in the melanoma tumour samples collected and analysed by our group (data not shown). Therefore, both of these receptors emerge as promising therapeutic targets.

| D ISCUSS I ON
Signal transduction activated by EGFR has an important role in cell motility in various types of cancer. [23][24][25] The crosstalk between EGFR and G-protein-coupled receptors modulates Rho GTPases activity and may contribute to the cell migration. 26,27 In cancer cells, various mechanisms may lead to permanent activation of EGFR, that is overexpression of ligands and receptors, EGFR gene amplification or activating mutations. MET also regulates tumour cell migration, invasion and metastasis. 25,28 Signalling molecules activated by MET promote tumour metastasis by changing the expression of proteins involved in cytoskeletal rearrangements (cadherins, Arp2/3, N-WASP) and cell adhesion (paxillin, integrins and focal adhesion kinase). [29][30][31] Majority of the studies carried out on cancer cells focused only on verification how chemical inhibition of EGFR or MET activity affects cell viability 32,33 or tumour growth, 34 what led to conclusions concerning involvement of these receptors in the regulation of tumour development. However, it is only part of the story since metastasis is the main cause of mortality among patients suffering from melanoma. Previously, we demonstrated that EGF and HGF stimulated invasiveness of melanoma cells. 6 In this work, we tested two mela- Cell migration is the multi-step process, where formation of actin-rich protrusions is needed. 42 We have previously shown that EGF and HGF stimulate invadopodia formation, and extracellular matrix degradation, what correlates with higher invasive abilities of melanoma cells. 6 Interestingly, both EGFR and MET signalling also regulate invadopodia formation in breast cancer cells. 13 and MMP-9-mediated degradation of E-cadherin. 49 Therefore, we also analysed proteolytic activity of generated variants of melanoma cells. We noticed that protein level of EGFR correlates with the ability to digest fluorescently labelled gelatin by melanoma cells. Further analysis revealed that activity of MMP-9 was lowered in cells with down-regulated EGFR. This is in line with observation of Zuo et al 49 F I G U R E 4 Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F-actin visible in red) detected using FITC-conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar-10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP-9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001 who showed that pharmacologic inhibition of EGFR activity reduced the production of MMP-9, as well as squamous carcinoma cell migration and invasion. Zhen et al 50  as well as their tissue inhibitors (TIMP-1, TIMP-2 and TIMP-3). 47,48 Therefore, it is possible that overexpression of EGFR led to up-regulation of other type of MMP, what in consequence is balanced by decreased activity of MMP-9; however, this hypothesis needs further studies. It was also found that MET signalling is essential for dendritic cell migration through the extracellular matrix, since both MMP-2 activity and MMP-9 activity were regulated by this receptor. 51 Our results indicate that expression of MET receptor is also crucial for In summary, our research presents the direct effect of EGFR and MET receptors protein level on the invasive abilities of melanoma cells. Obtained data indicate that both EGFR and MET signalling is strictly connected with migration and invasion abilities of melanoma cells, mostly because of the regulation of their proteolytic activity and the ability to form invadopodia. Therefore, these receptors seem to be good targets for anti-melanoma therapy, which aim will be the reduction of metastasis.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.