Malignant melanoma is an aggressive type of malignant skin lesions with worldwide increasing incidence 1, which is resistant to common cytotoxic therapies. As malignant melanomas have a potential to form organ metastases in the very early phase of primary growth, a better understanding of their progression is urgently needed. In the past few years, paradigm shifts have occurred in treatment modalities: in contrast to the previous practice when major survival benefit could only be achieved with early detection and complete surgical removal, recently target-based options have appeared on the horizon. Overall, 50–60% of malignant melanomas carry mutation in the BRAF oncogene, and vemurafenib and dabrafenib show activity against the V600E-mutant BRAF 2,3. Nevertheless, application of BRAF inhibitors can induce cutaneous squamous cell carcinoma owing to paradoxical activation of RAF signaling in cells carrying wild-type BRAF 4. Moreover, a number of BRAF-mutant melanomas show limited response owing to intrinsic resistance, and initially responding patients often relapse because of acquired resistance 5. Novel combinations are in development that may prevent resistance mechanism. In the past few years, clinical trials have proved that the addition of MEK-inhibitors to V600E-selective BRAF-inhibitors was associated with significant improvement in progression-free survival among patients of BRAF-mutated metastatic melanoma 6,7.
Hyperactivation of the epidermal growth factor receptor (EGFR) signaling components commonly presents in human melanoma (e.g. NRAS-BRAF-MAPK and PI3K-AKT), which suggests the potential role of EGFR itself as well. EGFR (Her-1 or c-erbB-1), a member of the c-erbB receptor tyrosine kinase (TK) family, is a glycoprotein (170 kDa) composed of an extracellular binding domain, transmembrane lipophilic segment and an intracellular protein TK domain with a regulatory carboxyl terminal segment. EGFR becomes activated by homodimerization, a mechanism that could be promoted by ligand binding as well as high receptor density due to overexpression. Receptor activation normally leads to the recruitment and phosphorylation of several intracellular substrates, regulating various cellular activities such as differentiation, increased proliferation, survival and migration 8.
Aberrant activation of EGFR has been shown to correlate with poor prognosis in a wide range of malignant tumors, for example, urinary bladder, cervix, esophagus, ovarian cancers, and tumors of the head and neck region 9. Therapeutic inhibition of TK activity by small-molecule substrates is a possible approach to interfere with such an aberrant activation of TK-type receptors: small-molecule tyrosine kinase inhibitors (TKIs) bind to the ATP cleft of the TK receptor and selectively block growth factor-stimulated signal activation via dimerization and autophosphorylation 10. Inhibition of phosphorylation leads to depletion of the activated downstream effectors, resulting in attenuation of tumor progression.
Previously a number of experimental studies suggested the importance of EGFR function in malignant melanoma cells 11–14. Furthermore, according to clinicopathological data, EGFR gene copy number alterations in primary cutaneous malignant melanomas were associated with poor prognosis 15. Several genomic and proteomic analysis confirmed the potential role of EGFR in the progression of malignant melanoma 16–18; therefore, EGFR-TKI strategy could serve as a potential antimelanoma approach. Recent studies demonstrated in BRAF-mutant colorectal cells that selective inhibition led to feedback activation of EGFR 19. Similar mechanism was proved in the development of adaptive resistance to vemurafenib in the case of BRAF-mutant malignant melanoma as well 20; therefore, simultaneous application of BRAF and EGFR inhibitors could be a potential novel combination.
In our preclinical study, we first examined the EGFR-TK-status at protein level in six human melanoma cell lines representing the major oncogenic driver mutations (mutant BRAF, mutant NRAS, and double wild-type cells). Moreover, we studied the potential effect of specific EGFR-TKIs in combination with vemurafenib on proliferation, apoptosis and migration of human melanoma cells in-vitro as well as on in-vivo growth and colonization of human melanoma xenografts.
Cell lines and culture conditions
BRAF-mutant A2058 cell line was provided by L.A. Liotta (NCI, Bethesda, Maryland, USA), HT168-M1 human melanoma was the derivative of A2058 with high metastatic potential 21, HT199 melanoma line was established by our group 22, and WM983B melanoma cell line was a gift from M. Herlyn (Wistar Institute, Philadelphia, Pennsylvania, USA). NRAS-mutant M24met melanoma line was kindly provided by B.M. Mueller (Scripps Research Institute, La Jolla, California, USA). Double wild-type MEWO and A431 squamous carcinoma cells (which served as a positive control for EGFR) are available from ATCC. Human melanoma cell lines were grown in RPMI-1640 medium, whereas A431 were cultured in DMEM containing 4500 mg/l glucose (Sigma; Sigma Chemical Co.); both were supplemented with 5% fetal bovine serum (Sigma; Sigma Chemical Co.) and 1% penicillin–streptomycin (Sigma; Sigma Chemical Co.), at 37°C in a humidified atmosphere of 5% CO2.
Flow cytometric measurement of epidermal growth factor receptor protein expression
Cells from monolayer cultures were detached with 0.02% EDTA (Sigma; Sigma Chemical Co.), then washed with PBS for 3× 5 min, and then fixed and permeabilized by methanol for 15 min. After blocking nonspecific binding sites with 3% BSA for 15 min, cells were labelled for 45 min at 37°C with a mouse monoclonal antibody against the intracellular amino acid region of EGFR between 1020 and 1046, purchased from Becton-Dickinson (1 : 20 in PBS; Sunnyvale, California, USA). After the washing period, RPE-conjugated goat polyclonal anti-mouse antibody (Dako; DakoCytomation, Glostrup, Denmark) was applied for 45 min at 37°C. Fluorescence was analyzed by flow cytometer (CyFlow SL-Green; Partec, Munster, Germany) using FlowMax software (Partec). Positive events from a total of 104 cells were counted. Negative controls were prepared by isotype-matched non-immune IgG (Sigma; Sigma Chemical Co.) primary antibody.
Small-molecule tyrosine kinase inhibitors
EGFR-specific TKI gefitinib (ZD1839, Iressa (AstraZeneca Pharmaceuticals, Cambridge, UK); [N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine]) was a kind gift from AstraZeneca. Erlotinib (OSI-774, Tarceva (AstraZeneca Pharmaceuticals); [N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine]) and irreversible inhibitor pelitinib (EKB-569; [N-(4-(3-chloro-4-fluorophenylamino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide]) were synthesized by Vichem Chemie Ltd (Budapest, Hungary). PD153035 [4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline] were purchased from Calbiochem (La Jolla, California, USA). V600E-selective BRAF-inhibitor vemurafenib (PLX4032, [N-(3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl-2,4-difluorophenyl)propane-1-sulfonamide] was provided by Selleck Chemicals (Munich, Germany). All TKIs were suspended in DMSO (Sigma; Sigma Chemical Co.) and used at 0.01–100 μmol/l concentrations in 0.5% DMSO-medium for in-vitro studies. For in-vivo metastasis assays, vaporized inhibitors were suspended in Tween-20 and diluted in physiologic saline to reach a final concentration of 1%. The final applied doses of gefitinib, pelitinib and vemurafenib were 2 and 20 mg/kg, 0.04 and 0.4 mg/kg, 12.5 and 25 mg/kg, respectively (applied in-vivo doses were based on in-vitro proliferation assays).
Melanoma cells of monolayer culture were fixed in paraformaldehyde for 10 min and then permeabilized with 0.1% Triton X-100 (Sigma; Sigma Chemical Co.) in PBS for 1 min. After washing in PBS for 3× 5 min and blocking with 1% bovine serum albumin (BSA; Sigma; Sigma Chemical Co.) and goat serum (9 : 1) for 30 min at room temperature, slides were incubated with primary phosphospecific rabbit anti-EGFR[pY1068] antibody (1 : 20 in PBS; Biosource, Nivelles, Belgium) for 45 min at 37°C. After washing, biotin-conjugated anti-rabbit IgG (Amersham, Buckinghamshire, UK) was applied for 40 min at 37°C (dilution 1 : 100). EGFR protein was visualized by streptavidin-FITC (dilution 1 : 100; Vector Laboratories, Burlingame, California, USA). Negative controls were prepared by isotype-matched non-immune IgG (Sigma; Sigma Chemical Co.) primary antibody. Cell nuclei were stained with propidium iodide (PI; Sigma; Sigma Chemical Co.). Slides were covered with Vectashield (Vector Laboratories) and examined with confocal microscopy (Eclipse C1 Plus; Nikon Optoteam, Vienna, Austria).
Kinexus Kinex Kinetworks protein kinase screen
WM983B control and treated cells with 25 µmol/l gefitinib for 5 or 30 min were prepared according to the recommendations of Kinexus Bioinformatics Corporation (Vancouver, British Columbia, Canada; www.kinexus.ca). 1×107 adherent cells were washed twice with PBS, and 200 μl ice-cold lysis buffer was added to each sample (20 mmol/l MOPS, pH 7.0; 2 mmol/l EGTA; 5 mmol/l EDTA; 30 mmol/l sodium fluoride; 60 mmol/l β-glycerophosphate, pH 7.2; 20 mmol/l sodium pyrophosphate; 1 mmol/l sodium orthovanadate; 1% Nonidet P-40 (Sigma; Sigma Chemical Co.); 1 mmol/l phenylmethylsulfonylfluoride; 3 mmol/l benzamidine; 5 μmol/l pepstatin; 10 μmol/l leupeptin; 1 mmol/l dithiothreitol; final pH of the homogenizing buffer was adjusted to 7.2). Scrapped cells were collected and sonicated four times for 10 s each time with 10–15 s intervals on ice, and each homogenate was centrifuged at 90 000g 30 min at 4°C in an ultracentrifuge. Supernatants were transferred to 1.5-ml microcentrifuge tubes, resuspended in SDS-PAGE sample buffer (31.25 mmol/l Tris-HCl, pH=6.8; 1% SDS; 12.5% glycerol; 0.02% bromophenol blue; 1.25% β-mercaptoethanol) to final protein concentration of 1.4 mg/ml, and shipped to Kinexus. Commercially available service of fluorescent labelling, hybridization onto KPCS-1.0 microarray with selected phospospecific antibodies (ERK1/2, MEK1/2, p38 MAPK), as well as scanning, imaging and quantitative analysis of the enhanced chemiluminescence signal of the detected proteins in the EGFR-pathway were performed by Kinexus. Images of immunoblots were provided by Kinexus, and our further conclusions were based on their evaluations.
Western blot analysis
Human malignant melanoma control cells and cells treated with 25 µmol/l gefitinib or pelitinib for 5 or 30 min were prepared. After treatment, the cells were washed with PBS, and then lysed with MLB (no. 20-168; Merck KGaA, Darmstadt, Germany) cell lysis solution containing 10 µg/ml aprotinin and 10 µg/ml leupeptin. We removed the cells from the flask surface using a cell scraper, and the samples were diluted 1 : 1 ratio by 2× Laemmli buffer (Sigma-Aldrich, St Louis, Michigan, USA), and stored at −80°C until analysis. The whole procedure was performed on ice to avoid protein degradation. Protein content of the samples was measured according to the method of Lowry 23. The proteins were separated in a 7.5% SDS-polyacrylamide gel, and then transferred to a PVDF membrane (Bio-Rad, Hercules, California, USA), using a wet electroblotting apparatus according to the conmanufacturer’s protocols. Blotted samples were primarily treated with rabbit monoclonal antibody (in dilution 1 : 500) against phospho-S6 ribosomal protein (pS6 clone D68F8; Cell Signaling Technologies, Danvers, Massachusetts, USA) and incubated overnight at 4°C. Horseradish-peroxidase jugated anti-rabbit IgG secondary antibody was purchased from Jackson Immuno Research Laboratories (West Grove, Pennsylvania, USA). Immunoblots were revealed by enhanced WesternBright chemiluminescence system (Advansta, Menlo Park, California, USA). Expression of pS6 was compared with the level of loading control anti-β-actin antibody (A1978, dilution 1 : 5000; Sigma; Sigma Chemical Co.).
Flow cytometric measurement of apoptosis
Melanoma cells of monolayer were previously treated for 48 h with different concentrations of EGFR-TKIs and/or vemurafenib, then detached with EDTA, washed with PBS, fixed with 70% ethanol (30 min, 4°C). Samples were washed twice with PBS and incubated with PI and RNAse (CyStain PI Absolute T; Partec) for 4 h at room temperature. Fragmented DNA in cells was measured by flow cytometer (CyFlow; Partec). The percentage of the apoptotic cells (sub-G1 fraction) was analyzed by FlowMax software. The applied gate provided that measurement included only cells with intact membrane.
Cell proliferation assay
Cell suspensions containing 5×104 viable cells/ml were plated in 96-well dishes (Greiner, Frickenhausen, Germany), incubated for 24 h and treated with gefitinib, PD153035, erlotinib, and pelitinib at concentrations of 0.1–100 and/or 5 μmol/l of PLX4032 in 200 μl serum-free or serum-containing medium for 48 h. At the end of incubation, cell monolayers were fixed with 10% trichloroacetic acid and stained for 15 min with sulforhodamine B. Wells were repeatedly washed with 1% acetic acid to remove excess dye. Protein-bound dye was dissolved in 10 mmol/l Tris, and absorbance was measured at 570 nm using microplate reader (Bio-Rad). The 50% inhibitory concentrations (IC50) were calculated by dose-effect analysis with Microcomputers software (Elsevier-Biosoft, Cambridge, UK).
Modified Boyden-chamber migration assay
Cell migration was assayed by a method reported previously 24. Human melanoma cells were treated with different concentrations of gefitinib, pelitinib or vemurafenib for 24 h at 37°C, then harvested with 0.02% EDTA, washed twice with serum-free medium, and resuspended at a density of 106 viable cells/ml in medium contained 0.1% BSA. Viability was controlled by previous trypan blue staining (Sigma; Sigma Chemical Co.). Overall, 20 μl of the cell suspension was placed on top of the 96-well CXF8 plates (polycarbonate filter with 8 μm pore size; Neuroprobe Inc., Cabin John, Maryland, USA), and the lower compartment was filled with 30 μl of fibronectin in RPMI (100 μg/ml; Sigma; Sigma Chemical Co.). Cells were allowed to migrate for 6 h (except M24met, where 24 h incubation period was applied) at 37°C in a humidified atmosphere of 5% CO2. The cells on the upper surface of the filter were then removed mechanically, and the membranes were stained with toluidine blue (Sigma; Sigma Chemical Co.). Six independent parallel samples were applied. Migrated cells were counted under a light microscope in three high-power fields per sample.
Animal experiments for liver colonization
All animal-model protocols were carried out in accordance with the Guidelines for Animal Experiments and were approved by the Institutional Ethics Committee at the National Institute of Oncology, Budapest, Hungary (permission number: 22.1/722/3/2010).
SCID mice (C.B-17/lcr-Prkdcscid/lcrlcoCrl) were bred and maintained in our specific pathogen-free colony and housed 10 to a cage. WM983B and HT168-M1 human melanoma cells from monolayer culture were prepared for one-cell suspension and inoculated into the spleen of SCID mice with a number of 106 or 5×104 cells/animal, respectively. Fourteen days after intrasplenic injection, animals were randomized (10 animals per group) and treated intraperitoneally daily for 21 days. Reversible EGFR-specific TKI gefitinib, irreversible pelitinib and V600E-selective BRAF-inhibitor vemurafenib were suspended in physiologic saline containing 1% Tween-20. After termination, the weight of the primary tumors was measured. Livers were fixed in 10% neutral buffered formalin for 48 h, and the number of liver colonies was counted under a stereomicroscope.
Routinely fixed xenograft tumors were dehydrated in a graded series of ethanol, infiltrated with xylene and embedded into paraffin at a temperature not exceeding 60°C. Three-to-four-micron thick sections were mounted on Superfrost slides (Thermo Shandon, Runcorn, UK) and were manually deparaffinized. Endogenous peroxidase activity was blocked by 3% H2O2 in methanol for 5 min at room temperature. Slides were immersed in 0.05-mmol/l citrate buffer (pH=9) and exposed to 93°C for 10 min (MFX-800-3 automatic microwave, Meditest, Budapest, Hungary).
Slides were primarily treated with rabbit antibody (in dilution 1 : 100) against phospho-S6 ribosomal protein (pS6; Cell Signaling Technologies) and incubated overnight at 4°C. After washing, secondary antibody Biotinylated Link (Dako; DakoCytomation, Glostrup, Denmark) was used and incubated for 10 min at room temperature. For visualization, a standard avidin–biotin peroxidase technique (ABC system; Dako; DakoCytomation) was used with aminoethyl carbazole as chromogen.
To determine statistical differences between different strata, analysis of variance were used with the post-hoc Scheffé-test where parametric methods were available. For the animal experiments, we used nonparametric Kruskal–Wallis test with post-hoc analysis. Statistical significance was determined when P values were under 0.05. Statistical analysis was performed by statistica 11.0 software (StatSoft, Tulsa, Oklahoma, USA).
Epidermal growth factor receptor-signalization in human melanoma cells
Fixed and permeabilized cells were labeled with antibodies specific for the intracellular domain of the EGF receptor, and the ratio of positive cells was evaluated by flow cytometry. Expression of EGFR detected by antibody against the intracellular domain showed 52–88% positivity in the studied human melanoma cell lines (Fig. 1a and b).
In our previously published work, TK activation and inactivation were demonstrated by immunofluorescence microscopy using a phosphospecific antibody 25. By the application of EGFR-pY1068-specific antibody, we detected constitutively phosphorylation of EGFR without exogenous EGF stimulation in HT168-M1 and WM983B human melanoma cell lines (Fig. 2a and c). Furthermore, the EGFR signal could be inhibited by the EGFR-specific TKI, gefitinib (Fig. 2b and d).
Kinexus Kinex Kinetworks (Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada) phosphoprotein assay confirmed that EGFR-specific inhibition by gefitinib affected elements of the EGFR pathway in WM983B cells: activation of MEK1/2 and Erk1 was blocked at both end points, whereas Erk2 and p38a MAPK were blocked at 5 min, albeit the inhibitory effect was weakened at 30 min (Fig. 3a and b). Of note, although p38a MAPK is involved in the EGFR signaling via RAC1, the major inducing stimuli are hypoxia and stress 26.
Ribosomal S6 protein is one of the key downstream effectors of EGFR signal, which becomes activated by phosphorylation through mTOR or alternatively MAPK pathway 27. Our western blot experiments confirmed that the level of pS6 was reduced by EGFR-TKIs gefitinib and pelitinib in BRAF-mutant malignant melanoma cells HT168-M1 and WM983B (Fig. 3c). In addition, NRAS-mutant M24met cells show relative resistance against gefitinib. In contrast, irreversible inhibitor pelitinib proved to be more effective in this aspect.
Effect of epidermal growth factor receptor-tyrosine kinase inhibitors on the in-vitro proliferation of human melanoma cells
The inhibitory potential of gefitinib on the phosphorylation of EGFR suggested that EGFR-TKIs may have an effect on malignant melanoma at cellular level (Fig. 2). The inhibition of EGFR significantly decreased in-vitro proliferative capacity of the human melanoma cells in serum-free and serum-containing media (Table 1). The most potent inhibitor was irreversible EGFR-TKI pelitinib (IC50 values were in the range: 0.27–2.16 μmol/l). In the case of gefitinib, IC50 values were between 0.25 and 17.2 μmol/l; wild-type EGFR receptor expressing NRAS-mutant M24met cell line 11 and double wild-type (NRAS/BRAF) MEWO showed relative resistance to such inhibitor treatment. Compared with the effect of gefitinib, in BRAF-mutant lines, response to PD153035 was relatively weaker, whereas M24met and MEWO showed extensive resistance. Regardless of oncogenic mutational status, all studied human melanoma cell lines proved to be resistant to erlotinib, whereas the proliferation of the reference nonmelanoma cell line, A431 was inhibited successfully. Generally IC50 values of human melanoma cells were higher than that of EGFR-amplified A431 human squamous cell line. Furthermore, in BRAF-mutant melanoma cells, vemurafenib enhanced the inhibitory effect of gefitinib, whereas it failed to affect that in wild-type BRAF-carrying cells in serum-free and serum-containing media either (Table 1).
Apoptosis induction by epidermal growth factor receptor-tyrosine kinase inhibitors in human melanoma cell lines
To investigate the effect of EGFR inactivation on cell survival/apoptosis, human melanoma cells were treated by small-molecule TKIs for 48 h, and after PI staining, fragmentation of DNA was analyzed by flow cytometry. As erlotinib proved to be ineffective to interfere with cell viability, we have not tested its potential for apoptosis induction. Measurement of sub-G1 fractions (Fig. 4a–c) revealed that significant induction of apoptosis has not occurred in the range of the IC50 values for proliferation inhibition in most of the studied cell lines. After treatment with higher concentrations of gefitinib (25 and 50 μmol/l), a strong, dose-dependent induction of apoptosis was shown in BRAF-mutant melanoma cells (Fig. 4a). PD153035 showed the weakest proapoptotic effect (Fig. 4b). Irreversible inhibitor pelitinib was already effective at lower concentrations, 5 μmol/l led to 23–30% of sub-G1 fraction (Fig. 4c). Similarly to the proliferation assay, reversible EGFR-TKIs were less capable to induce intense apoptosis in mutant NRAS-expressing M24met cell line, whereas intermediate response was detected in double wild-type MEWO cells. However, irreversible inhibitor pelitinib showed activity in both cell lines with wild-type BRAF. Of note, vemurafenib has not induced significant apoptosis in any of the studied cell lines. Its inhibitory effect was rather realized through the blockade of cell cycle in G1-phase, and this effect was detectable only in cell lines harboring mutant BRAF (data not shown).
Effects of epidermal growth factor receptor-tyrosine kinase inhibitors on the in-vitro migration of human melanoma cells
To investigate the effect of gefitinib and pelitinib on cell migration, modified Boyden-chamber assay was applied. Similarly to vemurafenib, pretreatment with the EGFR-specific TKIs significantly reduced 6 h migration of the BRAF-mutant human melanoma cells (Fig. 4d–f). The inhibitory capacity of gefitinib and pelitinib proved to be dose dependent. Pretreatment with 10-μmol/l concentration of pelitinib completely abolished viability of HT168-M1 cells; therefore, migration assay could not be performed.
Epidermal growth factor receptor-tyrosine kinase inhibitor strategy inhibited liver colonization of BRAF-mutant WM983B and HT168-M1 xenografts
Based on our in-vitro results, we examined the in-vivo effect of gefitinib in combination with vemurafenib and pelitinib alone on the liver colonization of WM983B and HT168-M1 human melanoma cells in SCID mice. Fourteen days after intrasplenic inoculation of WM983B or HT168-M1 cells, mice were treated intraperitoneally with gefitinib or pelitinib daily for 3 weeks, at doses of 2, 20 or 0.04, 0.4 mg/kg, respectively. Based on the in-vitro IC50 values, we applied equivalent in-vivo dose, 2 mg/kg of gefitinib or 0.4 mg/kg of pelitinib, and we administered 10-fold higher or lower concentrations, respectively. Vemurafenib was applied at clinically relevant doses (12.5 or 25 mg/kg). The weight of the primary tumors was measured during the autopsy and the number of liver colonies was determined under stereomicroscope after formaldehyde fixation. Contrary to the in-vitro results in the case of HT168-M1 cells, gefitinib did not inhibit primary tumor and liver colonization (data not shown) as compared with the irreversible inhibitor pelitinib, which reduced liver colonies at a dose of 0.4 mg/kg (Fig. 5a). In the case of the other BRAF-mutant WM983B melanoma cells, gefitinib significantly (P<0.05) inhibited liver colonization at the dose of 2 mg/kg as well as 20 mg/kg in a dose-dependent manner (Fig. 5b). Compared with the control group, vemurafenib significantly affected liver colonization; however, synergistic effect of the combination did not reach significance, and only a statistical trend appeared. In the TKI-treated groups, primary tumor sizes did not differ significantly from that of solvent-treated control; however, a tendency of decrease was observed: 18 and 27% in the case of 2 and 20 mg/kg of gefitinib and 9 and 14% in the case of 0.04, 0.4 mg/kg of pelitinib, respectively. Interestingly, vemurafenib has not shown any effect on primary tumors either alone or in combination (data not shown).
Ribosomal S6 protein is the part of the translational machinery, one of the key effectors of EGFR pathway; therefore, its phosphorylation status is highly dependent of signaling activity. By immunohistochemical examination of the primary WM983B xenograft tumors, we confirmed in-vivo inhibitory effect of gefitinib, because compared with solvent control, gefitinib-treated cells showed lower pS6-positivity (Fig. 5c–d).
Aberrant activation of the TK EGFR was demonstrated in several common solid tumors, resulting in increased proliferation, survival, invasiveness and metastasis. EGFR-inhibitory strategy has already been approved in cancers of the head and neck region, colon cancers and non-small cell lung cancer 28. Nevertheless, the effectiveness of EGFR inhibition may be influenced by oncogenic mutations in the downstream signaling pathway, for instance by the V600E-mutant BRAF or by mutant NRAS, which are the two most common driver mutations in malignant melanoma cases 29.
On the contrary, activation of EGFR and vemurafenib resistance is linked to the signal of microphthalmia-associated transcription factor, which also suggests potential role of EGFR-signal in malignant melanoma 30. In addition, it was previously described that heregulin (ligand of ErbB3 and ErbB4 receptors) stimulated the proliferation of both melanocytes and malignant melanoma cells 12. As heregulin contains similar domains to EGF, the oncogenic effect of EGFR (ErbB1) could not be excluded in malignant tumor types that share neuroectodermal features, for example, malignant melanoma or glioblastoma multiforme. In the latter case, the role of EGFR seems to be more clear 31. Among numerous other TKs, Tworkoski et al. 18 detected the activity of EGFR in human malignant melanoma cell lines. A previous work using standardized ATP-based chemosensitivity assay showed significant response of human melanoma cells to gefitinib; however, the extracellular domain of EGFR could be detected only in a minority of tumor samples 14. These findings are in concordance with our results that all the studied human melanoma cell lines expressed the intracellular domain of the receptor that harbored the TK domain of EGFR. In mutant BRAF-carrying melanoma cell lines, EGFR-TKI treatment led to significant response in the signaling cascade and inhibited phosphorylation level of EGFR itself, which resulted in inactivation of the major elements in the downstream signal (e.g. MEK1/2, Erk1/2, p38a MAPK) in 30 min. These short-term alterations in the EGFR-signal may be the explanation of the detected long-term biological responses. Furthermore, we are the first to categorize the effectiveness of EGFR-TKIs in human melanoma according to the molecular pattern: treatment blocked proliferation activity of BRAF-mutant cells, whereas wild-type BRAF-carrying human melanoma cells showed relative insensitivity against gefitinib.
Inhibition of EGFR leads to the inactivation of PI3K/Akt survival signal, which results in increased apoptosis 32–34. Our results confirmed previous studies showing that albeit a minority of malignant melanoma cells expressing extracellular domain of EGFR, gefitinib still proved to be an apoptosis-inducing agent 13,35. This observation suggests the involvement of intracellular domain in the survival signal, whereas the presence of the extracellular region is not essential. Another novel statement of our current work is that irreversible inhibition of EGFR by pelitinib had a more potent effect on apoptosis as well as on proliferation than the already clinically administered reversible agents. Previously Djerf Severinsson et al. 36 showed that pan-ErbB TKI canertinib also had better antitumor activity in malignant melanoma. Additionally, our results served the first evidence that irreversible inhibition could work in NRAS-BRAF double wild-type as well as NRAS-mutant melanoma cells.
The relative resistance of NRAS-mutant M24met cell line to gefitinib could be explained by previous observations that confirmed the inactivity of EGFR in those cells 11, and the receptor was not capable to react to exogenous EGF stimulation despite the gene being amplified 15. It is probable that the relative resistance of double wild-type MEWO cells is caused by EGFR-independent signaling, because this line shows loss of NF1 function, which associates with NRAS activation 37. In our present study, we have not only confirmed the experimental work of Djerf et al. 35, but according to the driver oncogenic mutational status, we have systematically explored the potential of EGFR-TKI strategy in malignant melanoma. Moreover, based on previous theories in colorectal cancer that selective BRAF(V600E) inhibition led to feedback activation of EGFR 19, we have first shown that vemurafenib was capable of enhancing the efficacy of EGFR-TKIs.
EGFR plays a crucial role in the regulation of cell migration as well. Selective EGFR-TKI treatment resulted in inhibition of adhesion, migration and invasion in several tumor cell lines, such as cutaneous squamous cell carcinoma, carcinoma of the head and neck region, malignant mesothelioma, hepatocellular carcinoma and prostate cancer 38–42. Moreover, numerous data on animal experiments are available that show gefitinib has an in-vivo inhibitory effect on metastasis formation in mice using hepatocellular carcinoma, head and neck cancer, squamous cell carcinoma of the vulva and prostate carcinoma cells 43–47. We are the first to show that in BRAF-mutant melanoma cells, selective inhibition of EGFR prevented both in-vitro motility and in-vivo metastasis formation. Moreover, irreversible inhibitor pelitinib could open a new option for those cells which showed relative resistance against the reversible inhibitor gefitinib (e.g. NRAS-mutant, NRAS-BRAF double wild-type cells). At the same time, in contrast to previous findings 48, vemurafenib treatment only has affected migratory activity of BRAF-mutant human melanoma cells; however, we applied other cell lines and incubation period than the cited work.
Genetic analysis of tumors of patients with melanoma that relapsed on vemurafenib treatment revealed several acquired resistance mechanisms. These include among others overexpression of previously overseen growth factor pathways of melanoma involving EGFR 20, EGFR3 49, EGFR2/HER2, AXL and PDGFRβ receptors 50. Studies revealed also acquired genetic alterations such as ERBB4, besides FLT1, PTPRD, RET, TERT and RUNX1T1 50. These data all conclude to the same direction that in human melanoma cells inhibition of mutant BRAF frequently results in the (re-)activation of the EGFR receptor family signaling pathway. Moreover, recent clinical trials confirmed that in combination downstream elements of the TK-signal should be feasible targets: MEK-inhibitors improved the antitumor effect of mutant BRAF-specific inhibitors 6,7. Our data suggest that these pathways are already active in mutant BRAF-expressing human melanoma cells and themselves serve targets for therapeutic interventions which can further be exploited later upon vemurafenib resistance. A phase II study of gefitinib showed minimal clinical efficacy as a single agent in unselected patients with metastatic melanoma 50, which can be explained by the different EGFR activities in various molecular subgroups of human melanoma. Beside of others, our data also suggest revisiting the clinical application of EGFR-TKIs, as several new agents are now available.
Our study suggests that EGFR is a potential target in the therapy of BRAF-mutant malignant melanoma; however, more benefits could be expected from irreversible EGFR-TKIs and combined treatment settings.
The authors thank Katalin Derecskei for her excellent technical assistance and Andrea Ladányi for critical reviewing of the manuscript.
This work was supported by the following grants: National Research, Development and Innovation Office (NKFIH) PD109580 (I.K.), K116295, K84173 (J.T.), K112371, K116151 (J.T.), National Development Agency-NFU KTIA AIK 12-1-2013-0041, INNO 08-3-2009-0248 (2010) (J.T.), NVKP-16-1-2016-0020 (J.T. and J.T.), NAPB KTIA-NAP-13-2-2014-0021, Hungarian Academy of Sciences-Med In Prot (J.T.). István Kenessey is a recipient of János Bolyai Research Scholarship of the Hungarian Academy of Sciences.
Conflicts of interest
There are no conflicts of interest.
1. Garbe C, Leiter U. Melanoma epidemiology and trends. Clin Dermatol 2009; 27:3–9.
2. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364:2507–2516.
3. Young K, Minchom A, Larkin J. BRIM-1, -2 and -3 trials: improved survival with vemurafenib in metastatic melanoma patients with a BRAF(V600E) mutation. Future Oncol 2012; 8:499–507.
4. Sharma A, Shah SR, Illum H, Dowell J. Vemurafenib: targeted inhibition of mutated BRAF for treatment of advanced melanoma and its potential in other malignancies. Drugs 2012; 72:2207–2222.
5. Girotti MR, Pedersen M, Sanchez-Laorden B, Viros A, Turajlic S, Niculescu-Duvaz D, et al. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. Cancer Discov 2013; 3:158–167.
6. Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A, Stroiakovski D, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med 2015; 372:30–39.
7. Larkin J, Ascierto PA, Dreno B, Atkinson V, Liszkay G, Maio M, et al. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N Engl J Med 2014; 371:1867–1876.
8. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001; 2:127–137.
9. Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001; 37 (Suppl 4):S9–S15.
10. Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 2000; 19:6550–6565.
11. Mueller BM, Romerdahl CA, Trent JM, Reisfeld RA. Suppression of spontaneous melanoma metastasis
in scid mice with an antibody to the epidermal growth factor receptor
. Cancer Res 1991; 51:2193–2198.
12. Stove C, Stove V, Derycke L, van Marck V, Mareel M, Bracke M. The heregulin/human epidermal growth factor receptor
as a new growth factor system in melanoma with multiple ways of deregulation. J Invest Dermatol 2003; 121:802–812.
13. Ivanov VN, Hei TK. Combined treatment with EGFR inhibitors and arsenite upregulated apoptosis in human EGFR-positive melanomas: a role of suppression of the PI3K-AKT pathway. Oncogene 2005; 24:616–626.
14. Knight LA, Di Nicolantonio F, Whitehouse P, Mercer S, Sharma S, Glaysher S, et al. The in vitro effect of gefitinib (Iressa) alone and in combination with cytotoxic chemotherapy on human solid tumours. BMC Cancer 2004; 4:83.
15. Rakosy Z, Vizkeleti L, Ecsedi S, Voko Z, Begany A, Barok M, et al. EGFR gene copy number alterations in primary cutaneous malignant melanomas are associated with poor prognosis. Int J Cancer 2007; 121:1729–1737.
16. Gast A, Scherer D, Chen B, Bloethner S, Melchert S, Sucker A, et al. Somatic alterations in the melanoma genome: a high-resolution array-based comparative genomic hybridization study. Genes Chromosomes Cancer 2010; 49:733–745.
17. Timar J, Gyorffy B, Raso E. Gene signature of the metastatic potential of cutaneous melanoma: too much for too little? Clin Exp Metastasis
18. Tworkoski K, Singhal G, Szpakowski S, Zito CI, Bacchiocchi A, Muthusamy V, et al. Phosphoproteomic screen identifies potential therapeutic targets in melanoma. Mol Cancer Res 2011; 9:801–812.
19. Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D, et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature 2012; 483:100–103.
20. Sun C, Wang L, Huang S, Heynen GJ, Prahallad A, Robert C, et al. Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma. Nature 2014; 508:118–122.
21. Ladanyi A, Timar J, Paku S, Molnar G, Lapis K. Selection and characterization of human melanoma lines with different liver-colonizing capacity. Int J Cancer 1990; 46:456–461.
22. Ladanyi A, Gallai M, Paku S, Nagy JO, Dudas J, Timar J, et al. Expression of a decorin-like molecule in human melanoma. Pathol Oncol Res 2001; 7:260–266.
23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265–275.
24. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 1987; 47:3239–3245.
25. Kenessey I, Keszthelyi M, Kramer Z, Berta J, Adam A, Dobos J, et al. Inhibition of c-Met with the specific small molecule tyrosine kinase inhibitor SU11274 decreases growth and metastasis
formation of experimental human melanoma. Curr Cancer Drug Targets 2010; 10:332–342.
26. Ichijo H. From receptors to stress-activated MAP kinases. Oncogene 1999; 18:6087–6093.
27. Roux PP, Shahbazian D, Vu H, Holz MK, Cohen MS, Taunton J, et al. RAS/ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation. J Biol Chem 2007; 282:14056–14064.
28. Scaltriti M, Baselga J. The epidermal growth factor receptor
pathway: a model for targeted therapy. Clin Cancer Res 2006; 12:5268–5272.
29. Lazar V, Ecsedi S, Szollosi AG, Toth R, Vizkeleti L, Rakosy Z, et al. Characterization of candidate gene copy number alterations in the 11q13 region along with BRAF and NRAS mutations in human melanoma. Mod Pathol 2009; 22:1367–1378.
30. Ji Z, Erin Chen Y, Kumar R, Taylor M, Jenny Njauw CN, Miao B, et al. MITF modulates therapeutic resistance through EGFR signaling. J Invest Dermatol 2015; 135:1863–1872.
31. Sangar V, Funk CC, Kusebauch U, Campbell DS, Moritz RL, Price ND. Quantitative proteomic analysis reveals effects of EGFR on invasion-promoting proteins secreted by glioblastoma cells. Mol Cell Proteomics 2014; 13:2618–2631.
32. Shen L, Li Z, Shen S, Niu X, Yu Y, Liao M, et al. The synergistic effect of EGFR tyrosine kinase inhibitor gefitinib in combination with aromatase inhibitor anastrozole in non-small cell lung cancer cell lines. Lung Cancer 2012; 78:193–200.
33. Feng Y, Dai X, Li X, Wang H, Liu J, Zhang J, et al. EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis. Cell Prolif 2012; 45:413–419.
34. Xu H, Stabile LP, Gubish CT, Gooding WE, Grandis JR, Siegfried JM. Dual blockade of EGFR and c-Met abrogates redundant signaling and proliferation in head and neck carcinoma cells. Clin Cancer Res 2012; 17:4425–4438.
35. Djerf EA, Trinks C, Abdiu A, Thunell LK, Hallbeck AL, Walz TM. ErbB receptor tyrosine kinases contribute to proliferation of malignant melanoma cells: inhibition by gefitinib (ZD1839). Melanoma Res 2009; 19:156–166.
36. Djerf Severinsson EA, Trinks C, Green H, Abdiu A, Hallbeck AL, Stal O, et al. The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo. Biochem Biophys Res Commun 2011; 414:563–568.
37. Nissan MH, Pratilas CA, Jones AM, Ramirez R, Won H, Liu C, et al. Loss of NF1 in cutaneous melanoma is associated with RAS activation and MEK dependence. Cancer Res 2014; 74:2340–2350.
38. Barnes CJ, Bagheri-Yarmand R, Mandal M, Yang Z, Clayman GL, Hong WK, et al. Suppression of epidermal growth factor receptor
, mitogen-activated protein kinase, and Pak1 pathways and invasiveness of human cutaneous squamous cancer cells by the tyrosine kinase inhibitor ZD1839 (Iressa). Mol Cancer Ther 2003; 2:345–351.
39. Abu-Ali S, Fotovati A, Shirasuna K. Tyrosine-kinase inhibition results in EGFR clustering at focal adhesions and consequent exocytosis in uPAR down-regulated cells of head and neck cancers. Mol Cancer 2008; 7:47.
40. Giannelli G, Sgarra C, Porcelli L, Azzariti A, Antonaci S, Paradiso A. EGFR and VEGFR as potential target for biological therapies in HCC cells. Cancer Lett 2008; 262:257–264.
41. Liu Z, Klominek J. Inhibition of proliferation, migration, and matrix metalloprotease production in malignant mesothelioma cells by tyrosine kinase inhibitors. Neoplasia 2004; 6:705–712.
42. Bonaccorsi L, Marchiani S, Muratori M, Carloni V, Forti G, Baldi E. Signaling mechanisms that mediate invasion in prostate cancer cells. Ann N Y Acad Sci 2004; 1028:283–288.
43. Matsuo M, Sakurai H, Saiki I. ZD1839, a selective epidermal growth factor receptor
tyrosine kinase inhibitor, shows antimetastatic activity using a hepatocellular carcinoma model. Mol Cancer Ther 2003; 2:557–561.
44. Shintani S, Li C, Mihara M, Nakashiro K, Hamakawa H. Gefitinib (Iressa), an epidermal growth factor receptor
tyrosine kinase inhibitor, mediates the inhibition of lymph node metastasis
in oral cancer cells. Cancer Lett 2003; 201:149–155.
45. Matar P, Rojo F, Cassia R, Moreno-Bueno G, Di Cosimo S, Tabernero J, et al. Combined epidermal growth factor receptor
targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting. Clin Cancer Res 2004; 10:6487–6501.
46. Angelucci A, Gravina GL, Rucci N, Millimaggi D, Festuccia C, Muzi P, et al. Suppression of EGF-R signaling reduces the incidence of prostate cancer metastasis
in nude mice. Endocr Relat Cancer 2006; 13:197–210.
47. Torrance CJ, Jackson PE, Montgomery E, Kinzler KW, Vogelstein B, Wissner A, et al. Combinatorial chemoprevention of intestinal neoplasia. Nat Med 2000; 6:1024–1028.
48. Halaban R, Zhang W, Bacchiocchi A, Cheng E, Parisi F, Ariyan S, et al. PLX4032, a selective BRAF(V600E) kinase inhibitor, activates the ERK pathway and enhances cell migration and proliferation of BRAF melanoma cells. Pigment Cell Melanoma Res 2010; 23:190–200.
49. Abel EV, Basile KJ, Kugel CH 3rd, Witkiewicz AK, Le K, Amaravadi RK, et al. Melanoma adapts to RAF/MEK inhibitors through FOXD3-mediated upregulation of ERBB3. J Clin Invest 2013; 123:2155–2168.
50. Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P, et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol 2011; 29:3085–3096.