Research Article: High extracellular lactate causes reductive carboxylation in breast tissue cell lines grown under normoxic conditions

Date Published: June 10, 2019

Publisher: Public Library of Science

Author(s): Arthur Nathan Brodsky, Daniel C. Odenwelder, Sarah W. Harcum, Jeffrey Chalmers.


In cancer tumors, lactate accumulation was initially attributed to high glucose consumption associated with the Warburg Effect. Now it is evident that lactate can also serve as an energy source in cancer cell metabolism. Additionally, lactate has been shown to promote metastasis, generate gene expression patterns in cancer cells consistent with “cancer stem cell” phenotypes, and result in treatment resistant tumors. Therefore, the goal of this work was to quantify the impact of lactate on metabolism in three breast cell lines (one normal and two breast cancer cell lines—MCF 10A, MCF7, and MDA-MB-231), in order to better understand the role lactate may have in different disease cell types. Parallel labeling metabolic flux analysis (13C-MFA) was used to quantify the intracellular fluxes under normal and high extracellular lactate culture conditions. Additionally, high extracellular lactate cultures were labelled in parallel with [U-13C] lactate, which provided qualitative information regarding the lactate uptake and metabolism. The 13C-MFA model, which incorporated the measured extracellular fluxes and the parallel labeling mass isotopomer distributions (MIDs) for five glycolysis, four tricarboxylic acid cycle (TCA), and three intracellular amino acid metabolites, predicted lower glycolysis fluxes in the high lactate cultures. All three cell lines experienced reductive carboxylation of glutamine to citrate in the TCA cycle as a result of high extracellular lactate. Reductive carboxylation previously has been observed under hypoxia and other mitochondrial stresses, whereas these cultures were grown aerobically. In addition, this is the first study to investigate the intracellular metabolic responses of different stages of breast cancer progression to high lactate exposure. These results provide insight into the role lactate accumulation has on metabolic reaction distributions in the different disease cell types while the cells are still proliferating in lactate concentrations that do not significantly decrease exponential growth rates.

Partial Text

Since the 1920s, many types of cancers have been shown to rely heavily on glycolysis and lactate fermentation to produce energy rather than the more energy efficient complete oxidation of glucose in the mitochondria, even in the presence of sufficient oxygen. This metabolic state is called the Warburg Effect [1–4]. In addition, lactate can be utilized by cancer cells in the presence of glucose, a process known as the Reverse Warburg Effect [5–9]. Not only does this capability to use lactate provide cancer cells a metabolic advantage in vivo, it seems to favor cancer progression. For example, when lactate was injected into mice with xenografts of the human breast cancer cell line MDA-MB-231, metastasis increased ten-fold [10]. When the human breast cancer cell line MCF7 was exposed to lactate in vitro, genes associated with “stemness” were upregulated and gene expression patterns consistent with the “cancer stem cell” phenotype were observed [7]. In several other types of cancers, intratumoral lactate levels—which can rise to as high as 40 mM—correlated with treatment resistance as well as poor patient prognosis [5]. Further, it has been shown in xenotransplants and mouse cancer models that inhibiting the ability of cancer cells to utilize lactate can force the cells to become glycolytic and retard tumor growth through glucose starvation, while rendering the remaining cells more susceptible to radiation treatments [9]. Since lactate accumulation and its subsequent utilization by surrounding cancer cells appears to negatively affect cancer patient outcomes, deciphering the role of lactate at the metabolic level within central carbon metabolism is crucial.

In this study, the metabolic responses of three breast cell lines—MCF 10A, MCF7, and MDA-MB-231—to high-lactate were examined using 13C-MFA. MCF 10A is a non-tumorous epithelial breast cell line, MCF7 is a tumorigenic, luminal breast cancer cell line, and MDA-MB-231 is a metastatic, basal breast cancer cell line. 13C-glucose, 13C-glutamine, and 13C-lactate tracers were used in parallel experiments to determine the relative contribution of each substrate to intracellular metabolism. [1,2-13C] glucose and [U-13C] glutamine were selected as tracers, since both have previously been shown to be suitable for characterizing fluxes through glycolysis, the PPP, and TCA cycle pathways for mammalian cells [18, 21, 41, 42]. In addition, the [U-13C] lactate tracer was used to determine the metabolic contribution of lactate to breast cancer cell metabolism. For metabolic flux analysis, a detailed metabolic network model was constructed for breast cancer cells based a CHO cell metabolic network model [21]. The intracellular MIDs from parallel labeling experiments and extracellular flux measurements were paired with a mammalian metabolic network model to develop intracellular metabolic flux maps for each cell line under the control and high-lactate conditions.




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