EGFR/uPAR interaction as druggable target to overcome vemurafenib acquired resistance in melanoma cells

Abstract

Background: BRAF inhibitor (BRAF-I) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms behind BRAF-I responsiveness and acquired resistance is therefore an important issue. Here we assessed the role of urokinase type plasminogen activator receptor (uPAR) as a potentially valuable biomarker in the acquisition of BRAF-I resistance in V600E mutant melanoma cells.

Methods: We examined uPAR and EGFR levels by real time PCR and western blot analysis. uPAR loss of function was realized by knocking down uPAR by RNAi or using M25, a peptide that uncouples uPAR-integrin interaction. We investigated uPAR-β1integrin-EGFR association by co-immunoprecipitation and confocal immuno-fluorescence analysis. Acquired resistance to BRAF-I was generated by chronic exposure of cells to vemurafenib.

Findings: We proved that uPAR knockdown in combination with vemurafenib inhibits melanoma cell proliferation to greater extent than either treatment alone causing a decrease in AKT and ERK1/2 phosphorylation. Conversely, we demonstrated that uPAR enforced over-expression results in reduced sensitivity to BRAF inhibition. Moreover, by targeting uPAR and EGFR interaction with an integrin antagonist peptide we restored vemurafenib responsiveness in melanoma resistant cells. Furthermore, we found significant detectable uPAR and EGFR levels in tumor biopsies of 4 relapsed patients.

Interpretation: We disclosed an unpredicted mechanism of reduced sensitiveness to BRAF inhibition, driven by elevated levels of uPAR and identified a potential therapeutic strategy to overcome acquired resistance.

Funds: Associazione Italiana Ricerca sul Cancro (AIRC); Ente Cassa di Risparmio di Firenze.

Keywords: Acquired resistance; EGFR; Melanoma; Vemurafenib; uPAR.

Fig. 1

uPAR, EGFR expression level association in melanoma cells. a. List of melanoma cell lines evaluated for uPAR, EGFR and pERK1/2 protein levels. b. Western blot analysis of EGFR, uPAR and phosphorylation ERK1/2 levels with related densitometric quantification normalized to the internal control. Numbers on the right refer to molecular weights expressed in kDa. c, d. Real time PCR analysis was performed in M6 cells transfected with specific uPAR or EGFR siRNAs and no targeting siRNA as Control, to evaluate the expression levels of uPAR and EGFR.Results of three independent experiments performed in triplicates are expressed as fold changeaccording to 2^−ΔΔCT method, using 18S as calibrator. Statistical analysis was performed using unpaired Student's t-test, Error bars: mean ± SD; *p < .05 compared to Control. e, f. Cellular extracts of M6 before and after gene silencing were immunoblotted with antibodies against uPAR and EGFR. GAPDH was included as a loading control. Relative protein levels of uPAR and EGFR were quantified by densitometry and reported as values normalized to the GAPDH. Statistical analysis was performed using unpaired Student's t-test, Error bars: mean ± SD; *p < .05 compared to Control. g. Cellular extracts of A375 before and after enforced expression with pQ2-uPAR plasmid were analyzed with antibodies against uPAR and EGFR. GAPDH used as a loading control. Densitometric analysis of the levels of the same proteins relative to GAPDH expression. Error bars indicate mean ± SD; n = 3 experiments; Statistical analysis was performed using unpaired Student's t-test, Error bars: mean ± SD; *p < .05 compared to Mock Control.

Fig. 2

uPAR levels and responsiveness to BRAF inhibitor in A375 and M6 melanoma cells. a. Schematic representation of M6 generation. b. Immunoblotting for uPAR and EGFR in A375 and M6, αTubulin used as internal control.c. M6 and A375 were treated for 96 h with increasing concentrations of vemurafenib. DMSO-treated cells were set as the control vehicle. Pictures of treated cultures were taken with a phase-contrast microscopy (on the left), viable cells (trypan blu-negative) were counted with the aid of a Burker (on the right). Scale bar = 50 μm. Statistical analysis was performed using unpaired Student's t-test, Error bars: mean ± SD; *p < .05 indicate significant differencefrom DMSO. d. Clonogenic assay of M6 and A375 cellsafter a 10 d-treatment with the indicated doses of BRAF-I. Colonies were stained with May Grunwald and then counted. Statistical analysis was performed using student t-test. Representative data of three independent experiments is shown (mean ± SD). Asterisks indicate significant differences (P < .05.) from DMSO-treated cells. e. Cells were treated with 0.5 μM or 1 μM vemurafenib ERK1/2 phosphorylated and unphosphorylated levels were monitored by immunoblotting and quantified by densitometric analysis; αTubulin was also examined to ensure equal loading of samples in each lane. f. mRNA levels of Cyclin D1 were determined by qRT-PCR analysis. g. Western blot analysis of cyclin d1. The Student's t-test was used to analyze the data. Error bars indicate mean ± SD; n = 3 experiments; *P < .05 indicates significant difference from DMSO treated cells. All the experiments were performed independently at least three times.

Fig. 3

uPAR overexpression prevents the sensitivity of melanoma cells to vemurafenib. a. M6 cells transfected with no targeting siRNA (siRNA Control) and a specific siRNA for uPAR. After 24 h the cells were seeded at the same density (800 cells/ml) and cultured in the presence of vemurafenib at indicated concentration for 10 days. The colonies were fixed, stained, photographed and counted. The Student's t-test was used to analyze the data. Error bars indicate mean ± SD; n = 3 experiments; *p < .05 indicates significant difference from DMSO treated cells b.M6 were transfected with PLAUR siRNA and negative control siRNA and incubated for 48 h in presence of 1 μM vemurafenib. Cells were harvested for immunoblotting with the indicated antibodies. αTubulin was included as a loading control. Densitometric analysis of the expression of the same proteins normalized to the internal control is reported on the right. Analysis of variance followed by Newman-Keuls post test was performed for comparing means of multiple groups. Error bars indicate mean ± SD; n = 3 experiments; *P < .05 indicate significant difference of vemurafenib treated cell from DMSO treated control, and of combo treatment (vemurafenib + siRNA PLAUR) versus either DMSO or vemurafenib treated cells. All the experiments were performed independently at least three times. c. A375 were transfected either with Mock or uPAR overexpressing plasmid pQ2-uPAR. After 24 h were seeded at the same density (800 cells/ml) and cultured in the presence of vemurafenib at indicated concentrations for 10 days. The colonies were fixed, stained, photographed and counted. Significance was assessed by Student's t-test. Error bars indicate mean ± SD; n = 3 experiments. *p < .05 indicates significant difference from DMSO treated cells. d. A375 Mock or uPAR transfected cells were harvested for Western blot analysis for the indicated antibodies. αTubulin was included as a loading control. Densitometric analysis of the expression of the same proteins normalized to the internal control is reported on the right. Significance was assessed by one-way ANOVA test followed by Newman-Keuls post test.Error bars indicate mean ± SD; Asterisks (*p < .05) indicate significant differences of vemurafenib treated cells, uPAR overexpressing cells or combo (vemurafenib + pQ2-uPAR) from Mock treated cells. All the experiments were performed independently at least three times.

Fig. 4

Analysis of the biological and molecular features of naïve cells and its BRAF-resistant counterpart a. Schematic representation of the induced vemurafenib resistance protocol in M6 cells. b. M6 vem sensitive (M6P) cells and c, M6 vem resistant cells (M6R), were treated for 24 h with DMSO or the indicated doses ofvemurafenib,stained with propidium iodide, and analyzed for cell cycle progression by flow cytometry. Representative images from one of three independent experiments are shown. The percentage of cells in the different phases of the cell cycle was calculated by the ModFit program and depicted in each panel. d. M6P and M6R cells (800 cells/ml) were exposed to graded concentrations of vemurafenib for ten days. Colonies were stained with May Grunwald and the counts reported in the related table. Representative data of three independent experiments is shown (mean ± SD). e. Immunoblot analysis for AKT, ERK1/2 and mTOR activity in M6R and M6P untreated or treated for 24 h with 2 μM vemurafenib. GAPDH was used as loading control. f. Images of M6P and M6R spheroids on agar coated plates following treatment with DMSO or μM vemurafenib. Spheroid area is reported on the right. Scale bar = 200 μm.

Fig. 5

Effect of the combination of Vemurafenib and M25 on the growth, invasion, PI3K and MAPK signaling pathways of M6R. a. Schematic representation of integrin, EGFR and uPAR interaction. b. Clonogenic assay of M6R and M6P treated with vemurafenib, M25 or the combo (vemurafenib + M25) for 10 days. Colonies were stained, and then counted. One-way ANOVA test followed by Newman-Keuls post test was used to determine the significance of combination treatments versus control and single treatments. Error bars indicate mean ± SD; n = 3 experiments; Asterisks indicate significant differences (P < .05.) of the combo versus untreated, vemurafenib or M25 treated cells.c.M6R and M6P treated for 24 h with M25 or vemurafenib, either administered alone or in combination were harvested for immunoblotting with the indicated antibodies. GAPDH was included as a loading control. Relative protein phosphorylation levels of AKT, ERK1/2 and mTOR were quantified by densitometry and reported as values normalized to the GAPDH. Significance was assessed by one-way ANOVA test followed by Newman-Keuls post test.Error bars indicate mean ± SD; n = 3 experiments; Asterisks indicate significant differences (P < .05.) of the combo treatment from DMSO-treated, while number signs indicate significant differences (P < .05) of either M25 or Vemurafenib alone from DMSO-treated cells. d. Representative images and invasive area quantification of M6P and M6R spheroids embedded into the matrigel substrate in presence of either vemurafenib or M25 administered alone or in combination. Cell sprouting and invasion cells are depicted in yellow. Scale bar = 200 μm.Measures of invasive areas (invasive area = total area-spheroid area) are reported in graphs on the right panel. On the left, comparative pictures of M6P and M6R invasive spheroids with the relative invasive area quantification. e. Representative images of spheroids on agar-coated plates of M6R in presence of either vemurafenib or M25 administered alone or in combination. M6R cells were pre-incubated with Vemurafenib, M25 or the combo for 24 h. Scale bar = 1 mm. f. Immunoprecipitation of α5β1-integrin. Input: Western blotting of aliquots (30 μg of proteins) of cell lysates before immunoprecipitation, used as a reference loading control. IP uPAR: immunoprecipitate (500 μg of proteins) obtained with anti-uPAR R5 antibody; alpha5-beta1 lane: immunoblotting with anti-α5β1 antibody; uPAR lane: immunoblotting with anti-uPAR antibody; EGFR lane: immunoblotting with EGFR antibody: IgG a lysate that was incubated with non-specific IgG instead of the antibody and used as negative control.Densitometric quantification of the immunoblots normalized to the relative input is reported on the right. Significance was assessed by one-way ANOVA test followed by Newman-Keuls post test.Error bars indicate mean ± SD; Asterisks (p < .05) indicate significant differences of either M25 or the combo treatment from DMSO-treated. Experiments have been performed three times in triplicate with similar results. g. Representative images of confocal microscopy of companion M6R treated cultures stained with specific anti-uPAR (red), α5β1 (green) and DAPI (blue). Experiments have been performed three times in triplicate with analogous results. The co-localization score is quantified by image J andreported within each picture as Manders' coefficient (MC). The shown pictures are representative of 20 different pictures for each experimental condition. Scale bar = 20 μm.

Fig. 6

uPAR and EGFR expression levels on tumor biopsies of metastatic melanoma patients before vemurafenib treatment: predictive values to BRAF-I responsiveness? A, Biological and clinical data of the six metastatic melanoma patients treated with vemurafenib as monotherapy. B, Level of uPAR and EGFR expression on isolated micrometastatic cells in 6 patients. Relative EGFR and uPAR expression levels compared to mRNA 18S and GAPDH on formalin-fixed paraffin embedded (FFPE) melanoma tissue sections from BRAF(V600E) mutant melanoma patients. C, Scatter plots depicted direct correlation between uPAR and EGFR in relapsed patients Experiments have been performed three times in triplicate with similar results.