Lifestyle factors, including diet, play an important role in the survival of cancer patients. However, the molecular mechanisms underlying pathogenic links between diet and particular oncogenic mutations in human cancers remain unclear. We recently reported that the ketone body acetoacetate selectively enhances BRAF V600E mutant-dependent MEK1 activation in human cancers. Here we show that a high-fat ketogenic diet increased serum levels of acetoacetate, leading to enhanced tumor growth potential of BRAF V600E-expressing human melanoma cells in xenograft mice. Treatment with hypolipidemic agents to lower circulating acetoacetate levels or an inhibitory homolog of acetoacetate, dehydroacetic acid, to antagonize acetoacetate-BRAF V600E binding attenuated BRAF V600E tumor growth. These findings reveal a signaling basis underlying a pathogenic role of dietary fat in BRAF V600E-expressing melanoma, providing insights into the design of conceptualized “precision diets” that may prevent or delay tumor progression based on an individual’s specific oncogenic mutation profile.

Moreover, our findings suggest that a ketogenic diet would likely worsen the disease burden in BRAF V600E-positive cancer patients. Since acetoacetate is cell permeable (), herein we tested a hypothesis that, in addition to the increased intracellular acetoacetate levels induced by BRAF V600E, dietary-fat-fueled ketogenesis may promote BRAF V600E melanoma growth in vivo through increased circulating and consequently intracellular levels of acetoacetate.

We recently performed a systematic, RNAi-based screen and identified metabolic vulnerabilities specifically required by the oncogenic BRAF V600E mutant, but not other oncogenes such as NRAS Q61R/K, in human melanoma (). More than 50% of melanomas express BRAF V600E mutant, which represents a therapeutic target due to its pathogenic role (). Moreover, BRAF V600E mutation is also found in 10% of colorectal cancer (), 100% of hairy cell leukemia (HCL) (), and 5% of multiple myeloma () cases. We found that HMG-CoA lyase (HMGCL), a key enzyme in ketogenesis-producing ketone bodies, is selectively essential in melanoma cells expressing BRAF V600E (). Ketogenesis mainly occurs in the mitochondria of liver cells, which normally produce ketone bodies as a result of fatty acid breakdown to generate energy when glucose levels in the blood are low (). HMGCL converts HMG-CoA to acetyl-coA and a ketone body, acetoacetate, which can be further converted to two other ketone bodies, including D-β-hydroxybutyrate (3HB) and acetone (). We found that oncogenic BRAF V600E upregulates HMGCL gene expression in cancer cells. HMGCL in turn selectively promotes BRAF V600E-dependent phosphorylation and activation of MEK1 by controlling intracellular levels of its product acetoacetate, which specifically promotes BRAF V600E (but not BRAF WT) binding to MEK1 (). These results support an emerging “metabolic rewiring” concept in which distinct oncogenes may require different metabolic alterations for tumor growth.

Understanding the molecular details of metabolic alterations in cancer may suggest connections between cancer risk and certain diets that fuel cancer-related metabolic changes that are informative to the development and design of diets that can lower cancer risk and improve treatment. For example, increasing evidence suggests that different human cancers may share common metabolic properties such as the Warburg effect, in which cancer cells avidly consume glucose (), suggesting that cancer may thrive on sugar; thus, a sugar-depleted “diet therapy” may lower cancer risk. In fact, a ketogenic diet (high fat, adequate protein, and low carbohydrate) has been evaluated for cancer prevention and treatment purposes to attenuate tumor development by limiting carbohydrate supply (). However, the clinical efficacy of such diet therapies may vary due to the fact that distinct oncogenic backgrounds in different cancer types require different metabolic properties for tumor development.

The link between diet and cancer prevention is well supported by a large amount of data derived from diverse epidemiological studies and from preclinical studies using experimental animals. Overall, despite a poor understanding of the complex interactions of diverse dietary components, these epidemiological studies consistently support inverse relationships between cancer risk and intake of “healthy” food such as vegetables, fruits, whole grains, and dietary fiber and direct relationships between cancer risk and dietary fat as well as other “calorie-dense” food such as deep-fried food and processed meat (reviewed inand). However, these epidemiological studies are more descriptive than mechanistic in nature, and many questions remain to be resolved. It is unclear, for example, which specific dietary factors are predominantly important in providing a cancer-prevention effect and how they may function alone and/or interact with other dietary factors to control cancer risk in all or in specific cancer types. Most importantly, although epidemiological data suggest a general connection between diet and cancer risk, pathogenic links between diet and particular oncogenic mutations in certain cancer types remain unknown.

Consistent with our findings presented above, we found that treatment with a high-fat diet promoted—while DHAA alone inhibited—tumor growth rates, sizes, and masses in nude mice with BRAF V600E-expressing A2058 or A375 cell xenografts; cotreatment with DHAA effectively reversed the enhanced tumor growth potential of A2058 or A375 cells in xenograft mice fed with a high-fat diet ( Figures 7 A and S7 A). A high-fat diet in the presence or absence of DHAA treatment did not affect body weight ( Figure S7 B). Although DHAA treatment had no effect on serum levels of acetoacetate, 3HB, cholesterol, or glucose in mice fed with high-fat or normal foods ( Figures 7 B, 7C, S7 C, and S7D, respectively), DHAA significantly attenuated the high-fat-diet-dependent enhancement of phosphorylation of MEK1 and ERK1/2 ( Figure 7 D), BRAF V600E-MEK1 binding ( Figure 7 E), and cell proliferation rates, as assessed by IHC staining of Ki67 ( Figures 7 F and S7 E) in tumors derived from A2058 and A375 cells. These results suggest that dietary fat likely promotes BRAF V600E tumor growth through regulation of serum levels of acetoacetate in vivo.

(F) Summarized results of IHC staining assay detecting Ki67-positive cells in tumor tissue samples from xenograft mice. Data are expressed as mean ± SD; p values were obtained by a two-tailed Student’s t test.

(B and C) AA (B) and 3HB (C) levels in serum harvested from xenograft mice. Data are expressed as mean ± SD; n = 3. p values were obtained by a two-tailed Student’s t test.

(A) Tumor growth (left) and weight (right) of xenograft nude mice injected with BRAF V600E-positive human melanoma A375 (upper) and A2058 (lower) cells that were fed with normal or high-fat diets followed by intraperitoneal injection with DHAA. Data are expressed as mean ± SEM for tumor growth and mean ± SD for tumor weight; p values were obtained by a two-way ANOVA test.

Further studies revealed that the inhibitory effect of DHAA treatment on tumor growth potential of A375 cells in xenograft mice was not reversed by intraperitoneal injection with acetoacetate ( Figure 6 G) despite increased serum levels of acetoacetate in DHAA-treated mice that received acetoacetate injection ( Figure 6 H). DHAA treatment did not affect serum levels of 3HB, cholesterol, or glucose in mice in either the presence or the absence of acetoacetate injection ( Figures 6 I, 6J, and S6 G, respectively). Consistently, acetoacetate injection did not reverse the inhibitory effects of DHAA on phosphorylation of MEK1 and ERK1/2 ( Figure 6 K), BRAF V600E-MEK1 binding ( Figure 6 L), or cell proliferation rates assessed by IHC staining of Ki67 ( Figures 6 M and S6 H) in tumors derived from A375 cells in mice. These data are consistent with previous results ( Figures 5 D–5E, 5I, and S5 C) showing that acetoacetate was insufficient to reverse the effect of DHAA on BRAF V600E-expressing cells.

DHAA is a synthetic organic compound that is used mostly as a fungicide and bactericide (); it shows little to no clinical toxicity or irritating potential and has been safely used in skin-care products. Consistently, chronic injection of DHAA to nude mice for ∼4 weeks revealed that 200 mg/kg/day administered intraperitoneally is a well-tolerated dose that did not cause notable differences in histopathological analyses and weights of diverse organs ( Figures S6 C and S6D, respectively). Moreover, chronic treatment with DHAA had no obvious effect on the mouse gut microbiome, as evidenced by an unaltered total-DNA amount extracted from bacteria in mouse feces; this suggested no change in total bacterial number in the mouse gut ( Figure S6 E), and by altered proportions but no loss of any components of the gut microbiota ( Figure S6 F). DHAA treatment did not alter complete blood counts (CBC) or hematopoietic properties in representative A375 xenograft mice compared to the water-treated group ( Table S1 ). These results together suggest that DHAA treatment does not cause obvious toxicity in vivo.

Consistent with these findings, DHAA treatment for ∼3.5 weeks effectively inhibited tumor growth rates, sizes, and masses in nude mice with BRAF V600E-expressing human melanoma A2058 and A375 cell xenografts, but not in mice carrying control xenografts derived from HMCB cells expressing NRAS Q61K ( Figures 6 A and S6 A). Notably, DHAA treatment did not affect acetoacetate or β-hydroxybutyrate levels in tumors harvested from xenograft mice ( Figures 6 B and 6C, respectively). In contrast, DHAA treatment selectively inhibited phosphorylation of MEK1 and ERK1/2 without affecting HMGCL expression ( Figure 6 D), reduced binding between BRAF V600E-MEK1 ( Figure 6 E), and reduced cell proliferation rates as assessed by IHC staining of Ki67 ( Figures 6 F and S6 B) in tumors derived from A2058 or A375 cells, but not control HMCB cells, compared to corresponding control xenograft mice treated with water.

(M) Summarized results of IHC staining assay detecting Ki67-positive cells in tumor tissue samples from xenograft mice shown in (G). Data are expressed as mean ± SD; p values were obtained by a two-tailed Student’s t test.

(H–J) AA (H), 3HB (I), and cholesterol (J) levels in tumor samples obtained from xenograft mice shown in (G). Data are expressed as mean ± SD; n = 3; p values were obtained by a two-tailed Student’s t test.

(G) Tumor growth (left) and weight (right) of xenograft nude mice injected with A375 and intraperitoneally injected with DHAA in the absence or presence of AA. Data are expressed as mean ± SEM for tumor growth and mean ± SD for tumor weight; p values were obtained by a two-way ANOVA test and a two-tailed Student’s t test.

(F) Summarized results of IHC staining assay detecting Ki67-positive cells in tumor tissue samples from xenograft mice. Data are expressed as mean ± SD; p values were obtained by a two-tailed Student’s t test.

(B and C) AA (B) and 3HB (C) levels in tumor samples obtained from xenograft mice. Data are expressed as mean ± SD; n = 3; p values were obtained by a two-tailed Student’s t test.

(A) Tumor growth (left) and weight (right) of xenograft nude mice injected with human melanoma BRAF V600E-positive A375 (upper) or A2058 (middle) and HMCB (NRAS Q61K; lower) cells intraperitoneally injected with DHAA. Data are expressed as mean ± SEM for tumor growth and mean ± SD for tumor weight; p values were obtained by a two-way ANOVA test and a two-tailed Student’s t test.

Next we found that DHAA treatment selectively inhibited cell proliferation of A375, A2058, and SK-MEL-5 cells expressing BRAF V600E and WM-266-4 cells expressing BRAF V600D ( Figures 5 A and S5 B), but it did not inhibit control PMWK, CHL-1, and MeWo cells expressing BRAF WT or HMCB or SK-MEL-2 cells expressing active NRAS mutants ( Figures 5 A and S5 B). Consistent with these findings, DHAA treatment selectively inhibited phosphorylation of MEK1 and ERK1/2 ( Figure 5 B) and BRAF V600E-MEK1 association ( Figure 5 C) only in BRAF V600E-expressing A375 and A2058 cells, but not in control PMWK or HMCB cells. The inhibitory effect of DHAA on diverse BRAF V600E-expressing cells could not be reversed by acetoacetate treatment in terms of reduced cell proliferation ( Figures 5 D and S5 C) or decreased MEK-ERK activation and BRAF V600E-MEK1 binding ( Figures 5 E and S5 D). Similar results were obtained using immortalized melanocyte Mel-ST cells overexpressing BRAF WT, V600E, or tBRAF; DHAA treatment selectively inhibited cell proliferation, MEK-ERK activation, and BRAF V600E-MEK1 binding in BRAF V600E-expressing cells, but not in parental or control cells expressing BRAF WT or tBRAF ( Figures 5 F–5H, respectively). In addition, acetoacetate treatment did not reverse the inhibitory effect of DHAA on BRAF V600E-expressing Mel-ST cell proliferation ( Figure 5 I).

(I) The effect of DHAA with or without AA rescue treatment on cell proliferation rates of Mel-ST cells stably expressing BRAF V600E. Data are expressed as mean ± SD; p values were obtained by a two-tailed Student’s t test.

(D and E) Effect of DHAA with or without AA treatment on cell proliferation rates (D), MEK1 and ERK1/2 phosphorylation, and BRAF-MEK1 binding (E) of melanoma BRAF-V600E positive A2058 and A375 cells. Data are expressed as mean ± SD; p values were obtained by a two-tailed Student’s t test.

(A–C) Effect of DHAA treatment on cell proliferation rates (A), MEK1 and ERK1/2 phosphorylation (B), and BRAF-MEK1 binding (C) of melanoma PMWK, HMCB, A2058, and A375 cells. Data are expressed as mean ± SD; p values were obtained by a two-tailed Student’s t test.

We next sought to determine whether DHAA inhibits BRAF V600E directly. Previously, we demonstrated that acetoacetate binding resulted in increased Vmax and slightly decreased Km of BRAF V600E using MEK1 as a substrate (). Interestingly, we found that treatment with increasing concentrations of DHAA alone did not affect BRAF V600E kinase activity with unaltered Vmax and Km ( Figure 4 H, left). In contrast, DHAA treatment effectively reversed the activating effect of acetoacetate on BRAF V600E in terms of increased Vmax and decreased Km of BRAF V600E in the presence of acetoacetate (300 μM) when using MEK1 as a substrate ( Figure 4 H, right). Further mechanistic studies revealed that DHAA treatment effectively inhibited acetoacetate-enhanced MEK1 binding to BRAF V600E and consequent phosphorylation of V600E-bound MEK1 in a cell-free, in-vitro-coupled protein-protein binding and kinase assay using purified recombinant BRAF V600E pretreated with acetoacetate (300 μM) and incubated with recombinant purified MEK1 as a substrate ( Figure 4 I, right). In contrast, DHAA had no effect on BRAF WT-MEK1 binding or MEK1 phosphorylation in a control experiment using purified BRAF WT incubated with MEK1 in the presence of acetoacetate ( Figure 4 I, left). Notably, DHAA at 200 μM was sufficient to compete with acetoacetate at 300 μM for BRAF V600E binding ( Figures 4 E, 4F, and 4H) and inhibit BRAF V600E-MEK1 binding and phosphorylation of MEK1 enhanced by acetoacetate at 300 μM ( Figure 4 I). This is physiologically consistent with the acetoacetate levels determined as approximately 300 μM in stable HMGCL-knockdown A375 and A2058 cells (). These results together are consistent with our hypothesis that mutation at V600 is the predominant mechanism underlying acetoacetate binding to BRAF protein and that DHAA primarily functions by competing with acetoacetate for mutant BRAF binding.

These data together suggest that DHAA binds to BRAF V600E with a higher affinity that enables DHAA to compete with acetoacetate for V600E binding. Consistently, we found thatC-labeled acetoacetate bound only to BRAF proteins harboring different substitutions of V600, including clinically reported V600E, V600D, and V600R with Kd values determined as approximately 92 μM, 93 μM, and 113 μM, respectively; it did not bind to a negative control mutant V600A or to other clinically reported BRAF mutants, including L597Q and K601E ( Figure 4 G, left). Moreover, DHAA effectively competed for AA binding with BRAF V600E, V600D and V600R in the presence of 300 μMC-labeled acetoacetate with Ki values determined as approximately 88 μM, 88 μM and 91 μM, respectively ( Figure 4 G, right). These findings are also consistent with results from a binding assay usingC-labeled acetoacetate incubated with diverse BRAF mutants: acetoacetate bound to V600E, V600D, and V600R but did not bind to control V600A mutant or other clinically reported BRAF mutants, including K507E, N581S, D594N, L597Q, K601E, and S616F ( Figure S5 A, upper), and acetoacetate promoted MEK1 binding to BRAF V600E, V600D, and V600R with increased MEK1 phosphorylation, but not to other BRAF mutants ( Figure S5 A, lower).

We next sought to determine whether functional inhibition of acetoacetate would attenuate BRAF V600E tumor growth. We examined a group of commercially available acetoacetate analogs and found that dehydroacetic acid (DHAA) ( Figure 4 A ) is an inhibitory homolog of acetoacetate. Similarly to acetoacetate (), DHAA directly binds to BRAF V600E, but not BRAF WT, in a thermal melt-shift assay using purified recombinant BRAF WT or V600E incubated with increasing concentrations of DHAA ( Figure 4 B). Moreover, in a cellular thermal-shift assay using cell lysates from 293T cells transfected with FLAG-tagged BRAF WT or V600E, both acetoacetate (400 μM) and DHAA (400 μM) bound only to BRAF V600E, but not WT, and DHAA bound to BRAF V600E with higher affinity than acetoacetate ( Figure 4 C). In addition, we performed a series of radiometric metabolite-protein interaction analyses usingC-labeled acetoacetate incubated with purified BRAF variants in the presence and absence of DHAA. As shown in Figure 4 D,C-labeled acetoacetate specifically bound to BRAF V600E and a V600E mutant of an active, truncated C-terminal domain of BRAF (tBRAF, 416-766 aa) () but did not bind to control proteins including BRAF WT, tBRAF WT, and a truncated N-terminal domain of BRAF (BRAF-N, 1-415 aa), whereas treatment with DHAA resulted in a significant decrease in the binding ability of BRAF V600E mutant forms to acetoacetate ( Figure 4 D). Additionally, DHAA competed with acetoacetate for BRAF V600E binding in a dose-dependent manner in a binding assay where purified BRAF V600E mutant pretreated withC-labeled acetoacetate was incubated with increasing concentrations of DHAA ( Figure 4 E). Furthermore, pretreatment of purified BRAF V600E mutant with DHAA (200 μM) was sufficient to block acetoacetate binding to recombinant BRAF V600E incubated with increasing concentrations ofC-labeled acetoacetate up to 400 μM ( Figure 4 F).

(H) Vmax and Km of BRAF V600E were measured using purified BRAF V600E protein incubated with increasing concentrations of ATP in the presence and absence of increasing concentration of DHAA (left) or increasing concentration of DHAA with 300 μM AA (right) using excessive amount of purified MEK1 as substrates. Data are expressed as mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test.

(G) Kd values (left) were determined by 14 C-labeled acetoacetate binding assay. BRAF WT and mutant proteins were incubated with increasing concentrations of 14 C-labeled acetoacetate. Effect of increasing concentrations of DHAA on 14 C-labeled acetoacetate binding to BRAF mutant proteins (right).

(E and F) Radiometric metabolite-protein interaction analysis using purified recombinant BRAF V600E (rBRAF V600E) pretreated with either 14 C-labeled acetoacetate incubated with increasing concentrations of DHAA (E) or rBRAF V600E pretreated with DHAA incubated with increasing concentrations of 14 C-labeled acetoacetate (F). Data are expressed as mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test.

(D) Radiometric metabolite-protein interaction analysis using 14 C-labeled acetoacetate incubated with purified BRAF variants, followed by treatment with DHAA. Data are expressed as mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test.

(C) Intracellular thermal melt-shift assay was performed to examine the protein (BRAF WT or BRAF V600E; left and right, respectively) and ligand (AA or DHAA) interaction.

(B) Thermal melt-shift assay was performed to examine the protein (BRAF WT or BRAF V600E; left and right, respectively) and ligand (DHAA) interaction. Arrows indicate melting temperatures at 0 μM (left) and 400 μM (right).

We found that fluvastatin and niacin treatment effectively attenuated tumor growth potential of BRAF V600E-expressing A375 cells in xenograft mice; this could be reversed by intraperitoneal injection with acetoacetate ( Figures 3 A , top, and S4 A, left). Similarly, treatment with fenofibrate attenuated tumor growth potential of BRAF V600E-expressing A2058 melanoma cells in xenograft nude mice ( Figures 3 A, middle, and S4 A, upper right) but not in control mice injected with NRAS Q61K-expressing HMCB cells ( Figures 3 A, bottom, and S4 A, lower right). Intraperitoneal acetoacetate injection effectively rescued the decreased tumor growth of A2058 cells in mice treated with fenofibrate but had no effect on tumor growth potential of HMCB cells in xenograft mice. Consistent with these findings, treatment with fluvastatin, niacin, or fenofibrate resulted in reduced serum levels of acetoacetate, but not β-hydroxybutyrate, in mice ( Figures 3 B and 3C, respectively), while acetoacetate injection rescued the decreased serum acetoacetate levels but did not affect 3HB levels. Although these three drugs did not affect serum glucose levels ( Figure S4 B) or body weight ( Figure S4 C) of mice, and only niacin treatment resulted in marginally decreased serum cholesterol levels that were not affected by acetoacetate injection ( Figure 3 D), all three hypolipidemic agents effectively reduced serum levels of triglyceride in mice, despite acetoacetate injection ( Figure 3 E). Consistently, fluvastatin, niacin, or fenofibrate treatment resulted in decreased phosphorylation of MEK1 and ERK1/2 ( Figure 3 F), decreased BRAF V600E-MEK1 association ( Figure 3 G), and reduced cell proliferation rates as evidenced by decreased Ki67 IHC staining ( Figures 3 H, S4 D, and S4E). This was the case only in tumors derived from BRAF V600E-expressing A375 and A2058 cells, not in control HMCB cells, and these inhibitory effects were effectively reversed by injection with acetoacetate. Similar results were obtained in fluvastatin or niacin-treated xenograft nude mice injected with A2058 cells ( Figures S4 F–S4H).

(H) Summarized results of IHC staining assay detecting Ki67-positive cells in tumor tissue samples from xenograft mice. Data are expressed as mean ± SD; n = 3; p values were obtained by a two-tailed Student’s t test.

(B–E) AA(B), 3HB (C), cholesterol (D), and triglyceride (E) levels in serum harvested from A375, A2058, and HMCB xenograft mouse tissue. Data are expressed as mean ± SD; n = 3; p values were obtained by a two-tailed Student’s t test.

(A) Tumor growth (left) and weight (right) of xenograft nude mice injected with human melanoma BRAF V600E-positive A375 cells (upper) that were orally treated with one of two different lipid-lowering agents, Niacin and Fluvastatin, alone or in combination with intraperitoneal injection with acetoacetate (AA); tumor growth (left) and weight (right) of xenograft nude mice injected with human melanoma BRAF V600E-positive A2058 (middle) cells or HMCB (NRAS Q61K; lower) cells that were orally treated with the lipid-lowering agent Fenofibrate alone or in combination with intraperitoneal injection with AA. Data are expressed as mean ± SEM for tumor growth and mean ± SD for tumor weight; p values were obtained by a two-way ANOVA test for tumor growth rates and a two-tailed Student’s t test for tumor masses.

We next examined whether treatment with hypolipidemic agents may attenuate circulating acetoacetate levels and consequently BRAF V600E tumor growth potential in mice. We chose three drugs that are clinically used to treat hypercholesterolemia: fluvastatin, which belongs to a class of cholesterol-lowering statins that are HMG-CoA reductase inhibitors (); niacin (a.k.a. vitamin B3), which lowers triglycerides and is also clinically used to treat cardiovascular patients not taking a statin (); and fenofibrate, a fibric acid derivative that also lowers triglycerides ().

We chose to use the lithium-salt form of acetoacetate, which provides the anion form of acetoacetate that is approximately 55 times more stable, with a half-life of 130 hr, than the acid form (acetoacetic acid), which has a half-life of 140 min at 37°C in water (). To exclude potential effects of the lithium ion, we performed a series of experiments using a control salt lithium chloride. We found that lithium chloride did not bind to purified BRAF V600E in a thermal-shift assay ( Figure S2 A) or promote the association between purified BRAF V600E and MEK1 ( Figure S2 B) as acetoacetate does. Consistently, treatment with lithium chloride did not affect phosphorylation levels of MEK1 or ERK1/2, BRAF V600E-MEK1 binding, or cell proliferation rates in diverse BRAF V600E-positive or -negative human melanoma cells ( Figures S2 C–S2E, respectively). Moreover, lithium chloride treatment did not affect tumor growth potential of BRAF V600E-expressing A375 cells in xenograft mice in vivo ( Figure S2 F). It also did not affect serum levels of acetoacetate, 3HB, cholesterol, or glucose ( Figure S2 G); phosphorylation levels of MEK1 or ERK1/2; BRAF V600E-MEK1 binding; or cell proliferation potential in tumors derived from A375 cells in xenograft mice ( Figures S2 H–S2J, respectively). In addition, similar studies were performed to exclude possible effects from acetone, the potential degradation product of acetoacetate. We found that acetone did not bind to purified BRAF V600E in either a thermal-shift assay or a binding assay usingC-labeled acetone ( Figure S3 A, left and right, respectively), and did not affect the association between purified BRAF V600E and MEK1 ( Figure S3 B). Acetone also did not affect phosphorylation levels of MEK1 or ERK1/2, BRAF V600E-MEK1 binding, or cell proliferation rates in diverse BRAF V600E-positive or -negative human melanoma cells ( Figures S3 C–S3E, respectively). These results, together with our previous finding thatC-labeled acetoacetate binds to purified BRAF V600E (), suggest that the anion form of acetoacetate is the functional compound that binds to and regulates BRAF V600E.

We found that intraperitoneal injection with acetoacetate, but not 3HB, resulted in increased growth rates and masses of tumors derived from BRAF V600E-expressing A375 melanoma cells in xenograft nude mice ( Figure 2 A ; upper left and right, respectively). In contrast, injection with either acetoacetate or 3HB had no effect on growth rates or masses of tumor xenografts derived from control NRAS Q61K-expressing HMCB cells ( Figure 2 A; lower left and right, respectively).

(F) Summarized results of IHC staining assay detecting Ki67-positive cells in tumor tissue samples from xenograft mice. Data are expressed as mean ± SD; p values were obtained by a two-tailed Student’s t test.

(B and C) AA (B) and 3HB (C) levels in serum harvested from A375 and HMCB xenograft mice treated with AA or 3HB. Data are expressed as mean ± SD; n = 3; p values were obtained by a two-tailed Student’s t test.

(A) Tumor growth (left) and weight (right) of xenograft nude mice injected with human melanoma BRAF V600E-positive A375 (upper) or HMCB (NRAS Q61K; lower) cells that were intraperitoneally injected with AA or 3HB. Data are expressed as mean ± SEM for tumor growth and mean ± SD for tumor weight; p values were obtained by a two-way ANOVA test for tumor growth rates and a two-tailed Student’s t test for tumor masses.

We found that high-fat diets in either solid or paste forms resulted in increased growth rates, masses and sizes of tumors without affecting body weight in nude mice with BRAF V600E-expressing human melanoma A375 cell xenografts ( Figures 1 A and S1 A; upper). In contrast, a high-fat diet did not affect tumor growth rates, masses, sizes, or body weight in mice with control SK-MEL-2 tumor xenografts expressing an active NRAS Q61R mutant ( Figures 1 A and S1 A; lower). The increased tumor growth in A375 xenograft mice fed a high-fat diet was not due to differences in food intake amounts ( Figure S1 B). In both A375 and SK-MEL-2 models, consumption of a high-fat diet did not significantly affect serum levels of D-β-hydroxybutyrate (3HB) ( Figure 1 C), but significantly increased serum cholesterol levels ( Figure 1 D) and reduced serum glucose levels ( Figure S1 C) compared to control mice fed with normal food. Serum levels of acetoacetate were also increased in both A375 and SK-MEL-2 xenograft mice fed with high-fat diets ( Figure 1 B). The increased serum levels of acetoacetate led to enhanced phosphorylation of MEK1 and ERK1/2 without affecting HMGCL expression ( Figure 1 E); they also led to increased binding between BRAF V600E-MEK1 ( Figure 1 F) in tumors derived from A375 cells, but not control SK-MEL-2 cells, compared to corresponding control xenograft mice fed with normal food. Consistent with these findings, consumption of a high-fat diet resulted in increased cell proliferation rates in tumors derived from A375 cells, but not control SK-MEL-2 cells—as assessed by increased immunohistochemistry (IHC) staining of Ki67—compared to corresponding control xenograft mice fed with normal food ( Figures 1 G and S1 D). Similar results were obtained in nude mice carrying BRAF V600E-expressing human-melanoma A2058 xenograft tumors compared to mice with control PMWK cell xenografts expressing BRAF wild-type (WT) or HMCB cell xenografts expressing an active NRAS Q61K mutant ( Figures S1 E–S1J).

(G) Summarized results of immunohistochemical (IHC) staining assay detecting Ki67-positive cells in tumor tissue samples from A375 and SK-MEL-2 xenograft mice. Data are expressed as mean ± SD; p values were obtained by a two-tailed Student’s t test.

(B–D) Acetoacetate (AA; B), β-hydroxybutyrate (3HB; C), and cholesterol (D) levels in serum harvested from A375 and SK-MEL-2 xenograft mice fed with normal or different high-fat diets. Data are expressed as mean ± SD; n = 3; p values were obtained by a two-tailed Student’s t test.

(A) Tumor growth (left), weight (middle), and body weight (right) of xenograft nude mice injected with human melanoma BRAF V600E-positive A375 (upper) or SK-MEL-2 cells (NRAS Q61R; lower) that were fed with normal diet or different high-fat diets. Data are expressed as mean ± SEM for tumor growth and mean ± SD for tumor weight; p values were obtained by a two-way ANOVA test for tumor growth rates and a two-tailed Student’s t test for tumor masses.

Discussion

Interestingly, changes of circulating acetoacetate at submillimolar levels are sufficient to alter BRAF V600E tumor growth in vivo, whereas acetoacetate treatment at a millimolar level is required to promote cell proliferation of cultured BRAF V600E tumor cells in vitro. Such response disparity might be due to the fact that in the in vitro cell proliferation assay, cells are cultured in complete media with various growth factors and abundant nutrients that provide an optimized culture condition for cells to achieve almost maximized cell proliferation potential, whereas cells in growing tumors likely are not provided with such optimized conditions. This might explain why the cultured cells are less sensitive to acetoacetate treatment in terms of enhanced cell proliferation. Moreover, tumor formation and growth in animals at the whole-organism level are complex processes not just involving cell proliferation potential, so it is possible that other related processes including, for example, tumorigenesis and angiogenesis might be more sensitive to the change of circulating acetoacetate level.