Combination therapy with vemurafenib (PLX4032/RG7204) and metformin in melanoma cell lines with distinct driver mutations

Abstract

Background: A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines.

Materials and methods: The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot.

Results: Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways.

Conclusions: The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

Figure 1

Effects of vemurafenib, metformin or the combination on cell proliferation and viability. a) Example of growth curves of BRAF^V600E and NRAS^Q61 mutant melanoma cell lines. BRAF^V600E cell lines included M263 (synergistic) and M308 (antagonistic). As representatives for NRAS^Q61 mutants M207 (synergistic) and SKMEL173 (antagonistic) are shown. The black line represents the data obtained with vemurafenib treatment, the blue line with metformin, and the red line with combination treatment. b) Combination index for the combination of 5 μM vemurafenib and 10 mM metformin of all melanoma cell lines tested. The green cell lines harbor BRAF^V600E, red shows NRAS^Q61 mutant cell lines, and black the wild type cell line M257.

Figure 2

Effects of single agent and combination therapy on cell cycle progression. Melanoma cells were treated with 5 μM vemurafenib, 10 mM metformin, or the combination for 24 hours. Afterwards cell cycle analysis was performed using DAPI staining and analyzed by flow cytometry. a) Cell cycle analysis of M263 treated with DMSO, vemurafenib, metformin, and the combination. b) Cell cycle changes after single agent vemurafenib. c) Cell cycle changes after single agent metformin. d) Cell cycle changes after combination of vemurafenib and metformin. Columns indicate a ratio of change over baseline (DMSO treatment). BRAF^V600E mutants are presented in green, NRAS^Q61 in red, and the wild type M257 in black.

Figure 3

Effects of single agent and combination therapy on apoptosis. To test the cytotoxic effects of the different treatments, melanoma cells were treated for 72 hours with 5 μM vemurafenib, 10 mM metformin, or the combination. Cells were stained with Annexin V and PI and were analyzed by flow cytometry. a) Annexin/PI staining of M263 as a representative example. Upper left corner: DMSO, upper right corner: vemurafenib, lower left corner: metformin, lower right corner: Combination of vemurafenib and metformin. b) Early and late apoptotic cells after 72-hour treatment with the indicated agents. Percentages are shown in relation to the control, DMSO treated cells.

Figure 4

Western blot analysis of different BRAF^V600E mutant cell lines. Cells were exposed for 24 hours to 5 μM vemurafenib, 10 mM metformin, or the combination. Phosphorylation was analyzed with antibodies against specific phosphor-proteins compared to their total proteins. Synergistic characteristics of the cell lines are indicated as + (slight synergy), ++ (significant synergy), - (slight antagonism), and - - (significant antagonism).

Figure 5

Western blot analysis of different BRAF^WT cell lines. Cells were exposed for 24 hours to 5 μM vemurafenib, 10 mM metformin, or the combination. Phosphorylation was analyzed with antibodies against specific phosphor-proteins compared to their total proteins. Synergistic characteristics of the cell lines are indicated as + (slight synergy), ++ (significant synergy), - (slight antagonism), and - - (significant antagonism).