Ketone bodies and two-compartment tumor metabolism: stromal ketone production fuels mitochondrial biogenesis in epithelial cancer cells

Abstract

We have previously suggested that ketone body metabolism is critical for tumor progression and metastasis. Here, using a co-culture system employing human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts, we provide new evidence to directly support this hypothesis. More specifically, we show that the enzymes required for ketone body production are highly upregulated within cancer-associated fibroblasts. This appears to be mechanistically controlled by the stromal expression of caveolin-1 (Cav-1) and/or serum starvation. In addition, treatment with ketone bodies (such as 3-hydroxy-butyrate, and/or butanediol) is sufficient to drive mitochondrial biogenesis in human breast cancer cells. This observation was also validated by unbiased proteomic analysis. Interestingly, an MCT1 inhibitor was sufficient to block the onset of mitochondrial biogenesis in human breast cancer cells, suggesting a possible avenue for anticancer therapy. Finally, using human breast cancer tumor samples, we directly confirmed that the enzymes associated with ketone body production (HMGCS2, HMGCL and BDH1) were preferentially expressed in the tumor stroma. Conversely, enzymes associated with ketone re-utilization (ACAT1) and mitochondrial biogenesis (HSP60) were selectively associated with the epithelial tumor cell compartment. Our current findings are consistent with the "two-compartment tumor metabolism" model. Furthermore, they suggest that we should target ketone body metabolism as a new area for drug discovery, for the prevention and treatment of human cancers.

Figure 1. Co-culture with MCF7 cells induces HMGCL expression in fibroblasts and ACAT1 downregulation in fibroblasts. hTERT-fibroblast-MCF7 co-cultures were maintained for 5 d. Then, cells were fixed and immunostained with anti-HMGCL (Fig. 1A) or anti-ACAT1 (Fig. 1B) antibodies. MCF7 cells were identified using anti-K8–18 (green) antibodies. Nuclei were stained with DAPI (blue). (A) HMGCL staining (red) and DAPI (blue) is shown in the top panels to better appreciate the co-culture-induced HMGCL upregulation in fibroblasts. (B) ACAT1 staining (red) and DAPI (blue) is shown in the top panels to better appreciate the co-culture-induced ACAT1 upregulation in MCF7 cells and ACAT1 downregulation in fibroblasts. Original magnification, 40x.

Figure 2. Serum starvation or Cav-1 downregulation induces the expression of ketogenic enzymes in fibroblasts. (A) Serum starvation induces the expression of enzymes for ketone body synthesis. hTERT-fibroblasts were cultured in 10% NuSerum or 0.2% BSA (serum starvation) for 72 h. Cells were then lysed and subjected to western blot analysis with antibodies directed against BDH1 and HMGCS1. Note that when fibroblasts were serum starved, BDH1 and HMGCS1 were upregulated. Equal loading was assessed by β-tubulin immunoblotting. (B) Cav-1 knock-down leads to increased expression of enzymes involved in ketone body synthesis. hTERT-fibroblasts were treated with Cav-1 siRNA or control siRNA for 48 h. Cells were then lysed and subjected to western blot analysis with antibodies directed against Cav-1, HMGCS1, HMGCS2 and BDH1. Note that when Cav-1 levels were knocked-down, HMGCS1, HMGCS2 and BDH1 were upregulated. Equal loading was assessed by β-actin immunoblotting.

Figure 3. Lactate or ketone bodies induces mitochondrial mass and metabolism in MCF7 cells. MCF7 cells were cultured in the presence of 10 mM L-lactate, 10 mM β-hydroxybutyrate, 10 mM butanediol, or vehicle alone for 2 d. (A) MCF7-treated cells were fixed and immunostained with anti-intact mitochondrial membrane antibody. Mitochondrial staining (red) is shown in the top panels to better appreciate that lactate and ketone body increase mitochondrial mass. Mitochondrial staining (red) and DAPI (blue) is shown in the bottom panels. Original magnification, 40×. (B) MCF7-treated cells were lysed and subjected to western blot analysis with antibodies directed against the intact mitochondrial membrane. Equal loading was assessed by β-actin immunoblotting. (C) MCF7-treated cells were lysed and subjected to western blot analysis with antibodies directed against multiple functional subunits of PDH complex. Equal loading was assessed by β-actin immunoblotting. Note that lactate, β-hydroxy-butyrate and butanediol increase the expression of subunits of the PDH enzymatic complex.

Figure 4. The MCT1 inhibitor CHC inhibits mitochondrial biogenesis in MCF7 cells. Co-cultures of MCF7 and fibroblasts were maintained for 5 d. Forty-eight h prior to fixation, cells were incubated with 10 mM CHC or vehicle alone. Then, cells were fixed and immunostained with antibodies against the intact mitochondrial membrane (red). and K8–18 (green). Nuclei were stained with DAPI (blue). Mitochondrial membrane staining is shown in the top panels to better appreciate that the MCT1 inhibitor CHC blocks the co-culture-induced increase in mitochondrial mass. Original magnification, 40×.

Figure 5. Lactate or ketone bodies induces Smad2 activation in MCF7 cells. MCF7 cells were treated with 10 mM L-lactate (Fig. 5A) or 10 mM β-hydroxybutyrate (Fig. 5B) for the indicated times. Then, cells were lysed and subjected to western blot analysis with antibodies directed against phospho-Smad2 and total Smad2/3. Equal loading was assessed by β-actin immunoblotting. Note that treatment with lactate or β-hydroxybutyrate is sufficient to induce the activation of the Smad2 pathway, suggesting an EMT induction.

Figure 6. Co-culture with fibroblasts and β-hydroxybutyrate treatment induce mitochondrial anti-stress responses in MCF7 cells. (A) hTERT-fibroblast-MCF7 co-cultures were maintained for 5 d. Then, cells were fixed and immunostained with anti-HSP60 (red) and anti-K8–18 (green) antibodies. Nuclei were stained with DAPI (blue). HSP60 staining is shown in the top panels to better appreciate the co-culture-induced HSP60 upregulation in MCF7 cells. (B) MCF7 cells were cultured in the presence of 10 mM β-hydroxybutyrate or vehicle alone for 2 d. Then, cells were fixed and immunostained with anti-TRAP1 or SCO1 antibodies. Nuclei were stained with DAPI (blue). Note that β-hydroxybutyrate treatment induces the expression of TRAP1 and SCO1 in MCF7 cells, indicating an increased mitochondrial anti-stress responses. Original magnification, 40×.

Figure 7. Compartmentalization of ketone metabolism: Human breast cancers show increased expression of a key enzyme for ketone utilization in the epithelial tumor cells. Paraffin-embedded tissue sections from human breast cancer samples were immunostained with antibodies directed against ACAT1, a key enzyme for ketone utilization. Slides were counterstained with hematoxylin. Note that ACAT1 is highly overexpressed in the epithelial tumor cell compartment. Original magnification, 40× and 60×.

Figure 8. Compartmentalization of ketone metabolism: Human breast cancers show increased expression of key enzymes for ketone generation in the stromal compartment. Paraffin-embedded tissue sections from human breast cancer samples were immunostained with antibodies directed against HMGCS2, HMGCL and BDH1, key enzymes for ketone generation. Slides were counterstained with hematoxylin. Note that HMGCS2, HMGCL and BDH1 are highly overexpressed in the stromal compartment of human breast cancers. Original magnification, 40×.

Figure 9. HSP60 expression is increased in the epithelial tumor compartment of human breast cancers. Paraffin-embedded tissue sections from human breast cancer samples were immunostained with antibodies directed against HSP60, a protein involved in the folding and assembly of mitochondrial proteins. Slides were counterstained with hematoxylin. Note that HSP60 is highly overexpressed in the epithelial tumor compartment. Original magnification, 40× and 60×.