Ketones and lactate "fuel" tumor growth and metastasis: Evidence that epithelial cancer cells use oxidative mitochondrial metabolism

Abstract

Previously, we proposed a new model for understanding the "Warburg effect" in tumor metabolism. In this scheme, cancer-associated fibroblasts undergo aerobic glycolysis and the resulting energy-rich metabolites are then transferred to epithelial cancer cells, where they enter the TCA cycle, resulting in high ATP production via oxidative phosphorylation. We have termed this new paradigm "The Reverse Warburg Effect." Here, we directly evaluate whether the end-products of aerobic glycolysis (3-hydroxy-butyrate and L-lactate) can stimulate tumor growth and metastasis, using MDA-MB-231 breast cancer xenografts as a model system. More specifically, we show that administration of 3-hydroxy-butyrate (a ketone body) increases tumor growth by ∼2.5-fold, without any measurable increases in tumor vascularization/angiogenesis. Both 3-hydroxy-butyrate and L-lactate functioned as chemo-attractants, stimulating the migration of epithelial cancer cells. Although L-lactate did not increase primary tumor growth, it stimulated the formation of lung metastases by ∼10-fold. Thus, we conclude that ketones and lactate fuel tumor growth and metastasis, providing functional evidence to support the "Reverse Warburg Effect". Moreover, we discuss the possibility that it may be unwise to use lactate-containing i.v. solutions (such as Lactated Ringer's or Hartmann's solution) in cancer patients, given the dramatic metastasis-promoting properties of L-lactate. Also, we provide evidence for the up-regulation of oxidative mitochondrial metabolism and the TCA cycle in human breast cancer cells in vivo, via an informatics analysis of the existing raw transcriptional profiles of epithelial breast cancer cells and adjacent stromal cells. Lastly, our findings may explain why diabetic patients have an increased incidence of cancer, due to increased ketone production, and a tendency towards autophagy/mitophagy in their adipose tissue.

Figure 1

Ketones promote tumor growth. We used a xenograft model employing MDA-MB-231 breast cancer cells injected into the flanks of athymic nude mice to evaluate the potential tumor promoting properties of the products of aerobic glycolysis (such as 3-hydroxy-butyrate and L-lactate). Tumor growth was assessed by measuring tumor volume, at 3-weeks post tumor cell injection. During this time period, mice were administered either PBS alone or PBS containing 3-hydroxybutyrate (500 mg/kg) or L-lactate (500 mg/kg), via daily intra-peritoneal (i.p.) injections. Note that 3-hydroxy-butyrate is sufficient to promote an ∼2.5-fold increase in tumor growth, relative to the PBS-alone control. Under these conditions, L-lactate had no significant effect on tumor growth. *p < 0.05, PBS alone versus 3-hydroxybutyrate (Student's t-test). N = 8 tumors for the PBS group. N = 10 tumors each, for L lactate and 3-Hydroxy-butyrate groups

Figure 2

Ketones promote tumor growth without any increase in angiogenesis. Tumor angiogenesis could account for the tumor-promoting properties of 3-hydroxy-butyrate. Thus, we next evaluated the status of tumor vascularity using antibodies directed against CD31. However, the vascular density (number of vessels per field) was not increased by the administration of either 3-hydroxy-butyrate or L-lactate. Thus, other mechanisms, such as the “reverse Warburg effect” may be operating to increase tumor growth. (A) Quantitation; (B) Representative images of CD31 immuno-staining in primary tumor samples. n.s., not significant

Figure 3

Ketones and lactate function as chemo-attractants, stimulating cancer cell migration. We assessed whether 3-hydroxy-butyrate or L-lactate can function as chemo-attractants, using a modified “Boyden Chamber” assay, employing Transwell cell culture inserts. MDA-MB-231 cells were placed in the upper chambers and 3-hydroxy-butyrate (10 mM) or L-lactate (10 mM) were introduced into the lower chambers. Note that both 3-hydroxy-butyrate and L-lactate promoted cancer cell migration by nearly 2-fold. *p < 0.05, control (vehicle alone) versus 3-hydroxy-butyrate or L-lactate (10mM) (Student's t-test).

Figure 4

Lactate fuels lung metastasis. To examine the effect of 3-hydroxy-butyrate and L-lactate on cancer cell metastasis, we used a well-established lung colonization assay, where MDA-MB-231 cells are injected into the tail vein of athymic nude mice. After 7 weeks post-injection, the lungs were harvested and the metastases were visualized with India ink staining. Using this approach, the lung parenchyma stains black, while the tumor metastatic foci remain unstained and appear white. (A) Quantitation. The number of metastases per lung lobe was scored. Note that relative to PBS-alone, the administration of L-lactate stimulated the formation of metastatic foci by ∼10-fold. Under these conditions, 3-hydroxy-butyrate had no effect on metastasis formation. *p < 0.01, PBS alone versus L-lactate (Student's t-test and ANOVA); *p < 0.05, PBS alone versus L-lactate (Mann-Whitney test). N = 20 lung lobes counted for each group (B) Images of Lung Metastases. Representative examples of lung metastasis in PBS-alone controls and L-lactate-treated animals are shown. Note that the metastatic foci formed in L-lactate treated animals are more numerous, and are also larger in size.