Bioenergetic modulation with dichloroacetate reduces the growth of melanoma cells and potentiates their response to BRAF^V600E inhibition

A hallmark of cancer is the reprogramming of cellular metabolism towards aerobic glycolysis. This metabolic pattern is characterized by increased glucose uptake and highly up-regulated glycolytic activity with fermentation of glucose into lactic acid instead of complete aerobic decomposition in the mitochondria. Aerobic glycolysis, also referred to as the Warburg effect, resembles the anaerobic metabolism of normal cells, but occurs in the context of an adequate oxygen supply [1]. The reprogramming of metabolism in cancer cells is a highly complex and heterogeneous process, which is driven by a wide variety of genetic and non-genetic strategies to overcome energy restriction [2]-[4].

The BRAF ^V600E oncogene, present in more than 50% of melanomas [5], has been directly implicated in the reprogramming of cellular metabolism. The constitutive activity of mutant BRAF reduces the expression of oxidative enzymes and the number of mitochondria, while increasing the expression of glycolytic enzymes and lactic acid production [6],[7]. Furthermore, a molecular link was recognized between the RAS-RAF-MEK-ERK-MAPK pathway and the energetic-stress check-point mediated by the liver kinase B1 (LKB1)-AMP activated protein kinase (AMPK) pathway, suggesting a role of BRAF^V600E in mediating resistance to energetic stress [8],[9]. BRAF affects oxidative metabolism through microphthalmia-associated transcription factor (MITF)-dependent control of the mitochondrial master regulator PGC1α [7]. Previous studies have shown that melanomas expressing PGC1α have a more oxidative phenotype than PGC1α-negative melanomas [4],[7]. In addition, BRAF^V600E was shown to mediate oncogene-induced senescence through metabolic regulation. This mechanism involves an increase in pyruvate dehydrogenase (PDH) activity through the suppression of pyruvate dehydrogenase kinase (PDK) [10]. PDH controls the coupling between glycolysis and mitochondrial respiration by facilitating the influx of pyruvate into the mitochondria, promoting complete utilization of glucose. The PDK-PDH axis is often dysregulated in cancer, where PDK over-expression reduces the coupling between the two energy systems and thereby contributes to the Warburg effect [11],[12]. On the basis of these findings, targeted inhibition of PDK was proposed as a therapeutic option for melanoma, with a possible synergistic effect of chemical BRAF^V600E inhibitors, such as vemurafenib [10],[13].

Dichloroacetate (DCA) is an inhibitor of the four isoforms of PDK and was previously used for treatment of lactic acidosis [14],[15], with low toxicity at effective dose levels [16],[17]. Several studies have demonstrated that DCA reverses the Warburg effect in cancer cells and negatively affects their growth and survival [13],[18]-[21]. This effect was attributed to a normalization of the mitochondrial membrane potential from the hyperpolarized state that characterizes cancer cells. The changes in membrane potential result in the reopening of voltage-gated anion channels and were shown to introduce a re-sensitization to apoptosis, due to a regained ability to release pro-apoptotic mediators [18]. Here we have investigated the effect of DCA on melanoma cells. Specifically, we analyzed cellular responses with regards to metabolism, bioenergetics, growth, proliferation and cell death in melanoma cell lines, primary human melanocytes, and BRAF^V600E-mutant melanoma cells with acquired resistance to vemurafenib.

Metabolic targeted therapy for cancer has been primarily focused on targeting the energy supply through inhibition of glycolysis. However, the recognition that mitochondria may be active contributors to melanoma progression has increased the attention on oxidative metabolism as a potential therapeutic target [10],[13],[29]. DCA promotes PDK-dependent activation of PDH, reversing lactate production in favor of influx of pyruvate into the mitochondria [15],[18]. Through this mechanism, DCA improves the coupling between glycolysis and mitochondrial respiration, which will have a greater impact on cells with a deficient coupling, such as cancer cells [18]. All melanoma cell lines examined in our study responded to DCA with reduced lactate production and an increased OCR. This shift towards mitochondrial respiration was expected to optimize substrate utilization and lead to a more efficient energy yield, but instead led to a significant drop in ATP levels despite an unaffected or even increased mitochondrial ATP coupling. The observed reduction in ECAR in response to DCA suggests that inhibition of glycolysis could be a major contributor to energy deprivation. A glycolysis-inhibitory mechanism of DCA has not been previously described. However, it has been demonstrated that pyruvate kinase, the last ATP-producing site in the glycolytic pathway, is negatively regulated by acetyl coenzyme A (acetyl-CoA) [30]. As PDH activation directly increases the formation of acetyl-CoA [31], this could explain the DCA-mediated inhibition of glycolysis. The structural similarity between DCA and pyruvate [32] could also imply a direct inhibition of glycolysis by DCA, possibly through an allosteric feedback mechanism.

The metabolic response to DCA was accompanied by reduced proliferation of melanoma cells, independent of the genetic driver status and metabolic profiles of these cells. Several previous studies have demonstrated an apoptotic effect of DCA on cancer cells [13],[18],[19],[32]-[34]. However, in accordance with our results, the apoptotic response was only triggered at concentrations too high to be clinically relevant [32]. To further explore the clinical relevance of DCA to melanoma treatment, we examined the efficacy of this agent in combination with the BRAF inhibitor vemurafenib. These experiments demonstrated a potentiating effect of DCA on the growth inhibition of BRAF^V600E-mutant melanoma cells. At low concentrations of DCA that alone had no effect on cell growth, the combination with low concentrations of vemurafenib had a significantly stronger growth-reducing effect than vemurafenib alone. This potentiating effect of DCA was also reflected in the reduction of ATP levels. Biochemical analysis has demonstrated the ability of BRAF^V600E to uncouple the LKB1-AMPK energy sensing pathway, promoting resistance to energy deprivation and preventing an apoptotic response [8],[9]. Treatment with BRAF inhibitors restores this pathway [35] and may, therefore, potentiate the response to compounds that reduce the generation of ATP. Both DCA and vemurafenib suppress glycolytic activity in melanoma cells and thus render them more dependent on mitochondrial respiration [6]. As glycolysis accounts for a large fraction of the total energy production in these cells, inhibition of this process will place a high demand on the oxidative system for ATP production. The lower performance of the mitochondria in melanoma cells could explain the inability of these cells to sustain ATP levels in the presence of DCA and vemurafenib. The cooperative effect of these compounds in lowering ATP levels suggests that the energetic threshold promoting growth arrest or cell death in melanoma cells can be reached with lower concentrations of vemurafenib in the presence of DCA.

Previous studies have investigated the ability of metabolic modulators to improve the therapeutic effect of BRAF inhibitors for treatment of melanoma. The combination of PLX4720 (a vemurafenib analogue) with either of the two anti-diabetic biguanides, metformin and phenformin, showed synergistic inhibition of melanoma cell viability [35],[36]. Both agents impair ATP synthesis through inhibition of the mitochondrial complex I activity, leading to a reduction in the ATP to ADP ratio and activation of the LKB1-AMPK pathway to suppress growth [35],[36]. Unlike DCA, metformin and phenformin both stimulate glycolysis and lactic acid production [37],[38], which could explain the growth-stimulating effects of metformin on some melanoma cell lines when used as a single agent. In addition, the concentrations at which metformin was effective were above a therapeutically relevant level [35]. Phenformin was significantly more potent than metformin [36], but has been associated with a high risk of lactic acidosis [39], and was taken off the market for treatment of type 2 diabetes in many countries. DCA, on the other hand, was here demonstrated to potentiate the effect of vemurafenib at concentrations down to 1 mM, and was previously shown to have few adverse effects when administered to patients [17],[19],[40]. These findings allude to a therapeutic potential of DCA as a co-drug for vemurafenib treatment of BRAF^V600E-mutant melanoma. This was reinforced by the demonstration that sensitivity to DCA was retained in melanoma cell lines with acquired resistance to vemurafenib. Although resistant cells showed an altered metabolic profile with significantly increased maximal mitochondrial respiration, as also shown by Corazao-Rozas et al. [41], they were as sensitive to DCA as the parental vemurafenib-sensitive cells. Therefore, DCA could possibly provide a strategy to prevent the appearance of vemurafenib-tolerant subpopulations during initial treatment and thereby postpone or prevent the development of resistance.

We here provide a more elaborate understanding of the effects of DCA on the metabolism and growth of melanoma cells. The ability of DCA to lower ATP levels and melanoma growth appears to potentiate the effect of vemurafenib, a drug already used in the clinic for treatment of BRAF^V600E-mutant metastatic melanomas. Importantly, melanoma cells with acquired resistance to vemurafenib retained their sensitivity to DCA. These findings should encourage further investigation of this drug combination and the in vivo application of DCA.