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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Sara A Byron, David C Loch, Candice L Wellens, Andreas Wortmann, Jiayi Wu, John Wang, Kenichi Nomoto, Pamela M Pollock
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-75) contains supplementary material, which is available to authorized users.

Competing interests

This study was supported in part by a grant and drug from Eisai Inc., Andover, Massachusetts, USA. JWu and KN are both employees of Eisai Inc. JWa is an employee of H3 Biomedicine Inc. H3 Biomedicine Inc. is a subsidiary of Eisai Inc.

Authors’ contributions

SB carried out many of the in vitro experiments, including proliferation assays, cell cycle and cell death analyses, E6201/LY294002 combination studies and Western blots. SB also helped to draft the manuscript. DL performed some of the Western blots and helped to draft the manuscript. CW performed proliferation assays, cell cycle and cell death analyses, E6201/LY294002 and E6201/Rapamycin combination studies and Western blots. AW performed some of the Western blots and helped to draft the manuscript. JWu, JWa and KN carried out the xenograft experiments, participated in the study’s design and helped to draft the manuscript. PP conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines.

Results

The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1.

Conclusions

Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.
Zusatzmaterial
Additional file 1: Figure S1. Efficacy of MEK1/2 inhibition with E6201. Western blots demonstrating phosphorylated ERK1/2 levels in our panel of melanoma cell lines following treatment with either vehicle (0.05% DMSO) or 500 nM E6201. Briefly, 500,000 cells from each cell line were plated in duplicate in a 6-well plate on day 0. The next day cells were washed twice with PBS and serum-starved in DMEM containing 0.2% FBS. Sixteen hours after serum starvation, cells were treated with either 0.05% DMSO or 500 nM E6201. After 6 hours of treatment protein lysates were collected. 30 μg of total protein were analysed on a 12% SDS-PAGE gel. Phosphorylated ERK1/2 protein was probed for with a phospho-specific antibody from Cell Signaling Technology (Beverly, MA). Immunoblots were then stripped and re-probed for total ERK1/2 (Cell Signaling Technology, Beverly, MA) and tubulin (Sigma Aldrich, St Louis, MO). (PDF 195 KB)
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Additional file 2: Figure S2. Efficacy of PI3K inhibition with LY294002. Western blots demonstrating phosphorylated AKT (serine 473) levels in UACC647, UACC558, UACC903, MM622, WM35 and MM540 cell lines following treatment with either vehicle (0.15% DMSO) or 20 μM LY294002. Briefly, 500,000 cells from each cell line were plated in duplicate in a 6-well plate on day 0. The next day cells were washed twice with PBS and serum-starved in DMEM containing 0.2% FBS. Sixteen hours after serum starvation, cells were treated with either 0.15% DMSO or 20 μM LY294002. After 6 hours of treatment, protein lysates were collected. 30 μg of total protein were analysed on a 12% SDS-PAGE gel. Phosphorylated AKT protein was probed for with a phospho-specific antibody from Cell Signaling Technology (Beverly, MA). Immunoblots were then stripped and re-probed for total AKT (Cell Signaling Technology, Beverly, MA) and GAPDH (Abcam, Cambridge, MA). (PDF 119 KB)
12943_2012_1072_MOESM2_ESM.pdf
Additional file 3: Figure S3. LY294002 Single Agent Concentration Response Curves. Concentration response curves of UACC647, UACC558, UACC903, MM622, WM35 and MM540 melanoma cell lines to the PI3K inhibitor LY294002. The IC50 of LY294002 for each cell line is provided in the legend. (PDF 274 KB)
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Additional file 4: Figure S4. Concentration response curves for E6201 and LY294002 combinations normalized to DMSO. Concentration response curves of UACC647, UACC558, UACC903, MM622, WM35 and MM540 melanoma cell lines to increasing concentrations of E6201 (3 nM to 3 μM) in combination with LY294002 (1 μM, 5 μM, 10 μM, 20 μM and 30 μM) treatment. (PDF 409 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 5
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