Bioenergetic modulation with dichloroacetate reduces the growth of melanoma cells and potentiates their response to BRAFV600E inhibition

Abstract

Background: Advances in melanoma treatment through targeted inhibition of oncogenic BRAF are limited owing to the development of acquired resistance. The involvement of BRAFV600E in metabolic reprogramming of melanoma cells provides a rationale for co-targeting metabolism as a therapeutic approach.

Methods: We examined the effects of dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, on the growth and metabolic activity of human melanoma cell lines. The combined effect of DCA and the BRAF inhibitor vemurafenib was investigated in BRAFV600E -mutated melanoma cell lines. Vemurafenib-resistant cell lines were established in vitro and their sensitivity to DCA was tested.

Results: DCA induced a reduction in glycolytic activity and intracellular ATP levels, and inhibited cellular growth. Co-treatment of BRAFV600E-mutant melanoma cells with DCA and vemurafenib induced a greater reduction in intracellular ATP levels and cellular growth than either compound alone. In addition, melanoma cells with in vitro acquired resistance to vemurafenib retained their sensitivity to DCA.

Conclusions: These results suggest that DCA potentiates the effect of vemurafenib through a cooperative attenuation of energy production. Furthermore, the demonstration of retained sensitivity to DCA in melanoma cells with acquired resistance to vemurafenib could have implications for melanoma treatment.

Figure 1

Metabolic characterization of melanoma cell lines and primary melanocytes. A, Metabolic profiles of two melanoma cell lines (ED-013 and ED-179) and human epidermal melanocytes (HEMn-LP), based on Seahorse XF96 measurements. Depicted are the ECAR (left panel) and the OCR (right panel) measurements during successive addition of oligomycin (1 μM), FCCP (1 μM), rotenone/antimycin (1 μM/1 μM) and 2-DG (100 mM). Data constitute 6 parallel measurements, and are representative of three independent experiments. B, Basal and maximal glycolytic ECAR values for melanoma cell lines and human primary melanocytes (HEMn-LP). The ECAR measured after addition of 2-DG (non-glycolytic ECAR) was subtracted from all values. The dashed line indicates the basal ECAR of HEMn-LP. C, Ratios between basal mitochondrial OCR and basal glycolytic ECAR. D, ATP coupling representing the fraction of the basal OCR used for ATP production (fraction inhibited by oligomycin). E, The respiratory control ratio, denoting the ratio between the maximal mitochondrial OCR and the proton leak (OCR after addition of oligomycin). (B – E), Values are the means of three independent measurements ± standard deviation. Students t-test was used to determine differences between HEMn-LP and melanoma cell lines (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 2

Effects of DCA on metabolism and ATP levels. A, Relative changes in basal mitochondrial OCR and glycolytic ECAR induced by treatment with 10 mM DCA for 2 hours. B, Relative ATP levels after treatment with DCA (10 or 20 mM) for 24 hours. One-way matched-samples ANOVA was used for statistical analysis and Tukey’s HSD test was used to determine statistical significance (*p < 0.05; **p < 0.01).

Figure 3

Effects of DCA on growth, proliferation and apoptosis. A, Representative selection of crystal violet stains of four melanoma cell lines (ED-007, ED-070, ED-179 and ED-196) treated with increasing concentrations of DCA (1–50 mM) for 96 hours. B, Proliferation determined by the incorporation of BrdU after treatment with DCA (1 and 10 mM) for 96 hours. One-way matched-samples ANOVA was used for statistical analysis and Tukey’s HSD test was used to determine statistical significance (*p < 0.05; **p < 0.01). C, Annexin V and PI based flow cytometric detection of apoptosis in ED-179 and ED-196 cells after treatment with DCA at concentrations below (10 mM) and above (100 mM) the IC₅₀ for 96 hours.

Figure 4

Effects of combined treatment with DCA and vemurafenib. A, B, Four melanoma cell lines (ED-013, ED-071, ED-117 and ED-196) were treated with DCA (1 mM), vemurafenib (50 nM for ED-071 and ED-117; 100 nM for ED-013 and ED-196) or the combination for 2 weeks. A, Crystal violet staining results representative of three independent experiments. B, Quantification of the data exemplified in A. C, Relative ATP levels in cells treated with DCA (IC₅₀), vemurafenib (IC₅₀) or the combination for 24 hours. B, C, Data represent means ± standard deviation of three independent measurements. One-way matched-samples ANOVA was used for statistical analysis and Tukey’s HSD test was used to determine statistical significance (*p < 0.05; **p < 0.01).

Figure 5

Response of vemurafenib-resistant melanoma cell lines to DCA. A, Metabolic profiles of two vemurafenib-resistant melanoma cell lines (ED-013-R1 and ED-196-R) compared with their parental cell lines (ED-013 and ED-196). The panels indicate the basal OCR and the changes during successive addition of oligomycin (1 μM), FCCP (1 μM), rotenone/antimycin (1 μM/1 μM) and 2-DG (100 mM). Data constitute 6 parallel measurements and are representative of three independent experiments. B, Relative cell growth of vemurafenib sensitive cell lines (ED-013, ED-071 and SK-MEL-28) and their vemurafenib resistant sub-cultures (denoted R1 and R2) after treatment with DCA (10 mM), vemurafenib (1 μM) or the combination for 96 hours. C, Relative ATP levels after treatment with DCA (IC₅₀), vemurafenib (IC₅₀) or the combination for 24 hours.