Tracing the electron flow in redox metabolism: The appropriate distribution of electrons is essential to maintain redox balance in cancer cells.

Cancer cells are characterized by sustained proliferation, which requires a huge demand of fuels to support energy production and biosynthesis. Energy is produced by the oxidation of the fuels during catabolism, and biosynthesis is achieved by the reduction of smaller units or precursors. Therefore, the oxidation-reduction (redox) reactions in cancer cells are more active compared to those in the normal counterparts. The higher activity of redox metabolism also induces a more severe oxidative stress, raising the question of how cancer cells maintain the redox balance. In this review, we overview the redox metabolism of cancer cells in an electron-tracing view. The electrons are derived from the nutrients in the tumor microenvironment and released during catabolism. Most of the electrons are transferred to NAD(P) system and then directed to four destinations: energy production, ROS generation, reductive biosynthesis and antioxidant system. The appropriate distribution of these electrons achieved by the function of redox regulation network is essential to maintain redox homeostasis in cancer cells. Interfering with the electron distribution and disrupting redox balance by targeting the redox regulation network may provide therapeutic implications for cancer treatment.

Microenvironmental Factors Modulating Tumor Lipid Metabolism: Paving the Way to Better Antitumoral Therapy.

Metabolic reprogramming is one of the emerging hallmarks of cancer and is driven by both the oncogenic mutations and challenging microenvironment. To satisfy the demands of energy and biomass for rapid proliferation, the metabolism of various nutrients in tumor cells undergoes important changes, among which the aberrant lipid metabolism has gained increasing attention in facilitating tumor development and metastasis in the past few years. Obstacles emerged in the aspect of application of targeting lipid metabolism for tumor therapy, due to lacking of comprehensive understanding on its regulating mechanism. Tumor cells closely interact with stromal niche, which highly contributes to metabolic rewiring of critical nutrients in cancer cells. This fact makes the impact of microenvironment on tumor lipid metabolism a topic of renewed interest. Abundant evidence has shown that many factors existing in the tumor microenvironment can rewire multiple signaling pathways and proteins involved in lipid metabolic pathways of cancer cells. Hence in this review, we summarized the recent progress on the understanding of microenvironmental factors regulating tumor lipid metabolism, and discuss the potential of modulating lipid metabolism as an anticancer approach.

Lactate and glutamine support NADPH generation in cancer cells under glucose deprived conditions.

Although glucose, through pentose phosphate pathway (PPP), is the main source to generate NADPH, solid tumors are often deprived of glucose, hence alternative metabolic pathways to maintain NADPH homeostasis in cancer cells are required. Here, we report that lactate and glutamine support NADPH production via isocitrate dehydrogenase 1 (IDH1) and malic enzyme 1 (ME1), respectively, under glucose-deprived conditions. Isotopic tracing demonstrates that lactate participates in the formation of isocitrate. Malate derived from glutamine in mitochondria shuttles to cytosol to produce NADPH. In cells cultured in the absence of glucose, knockout of IDH1 and ME1 decreases NADPH/NADP+ and GSH/GSSG, increases ROS level and facilitates cell necrosis. In 4T1 murine breast tumors, knockout of ME1 retards tumor growth in vivo, with combined ME1/IDH1 knockout more strongly suppressing tumor growth. Our findings reveal two alternative NADPH-producing pathways that cancer cells use to resist glucose starvation, reflecting the metabolic plasticity and flexibility of cancer cells adapting to nutrition stress.

Mitochondrial malic enzyme 2 promotes breast cancer metastasis via stabilizing HIF-1α under hypoxia.

OBJECTIVE: α-ketoglutarate (α-KG) is the substrate to hydroxylate collagen and hypoxia-inducible factor-1α (HIF-1α), which are important for cancer metastasis. Previous studies have shown that the upregulation of collagen prolyl 4-hydroxylase in breast cancer cells stabilizes the expression of HIF-1α by depleting α-KG levels. We hypothesized that mitochondrial malic enzyme 2 (ME2) might also affect HIF-1α expression via modulating α-KG levels in breast cancer cells. METHODS: We evaluated ME2 protein expression in 100 breast cancer patients using immunohistochemistry and correlated with clinicopathological indicators. The effect of ME2 knockout on cancer metastasis was evaluated using an orthotopic breast cancer model. The effect of ME2 knockout or knockdown on the levels of α-KG and HIF-1α proteins in breast cancer cell lines was determined both in vitro and in vivo. RESULTS: ME2 was found to be upregulated in the human breast cancerous tissues compared with the matched precancerous tissues (P<0.001). The elevated expression of ME2 was associated with a poor prognosis (P=0.019). ME2 upregulation was also related to lymph node metastasis (P=0.016), pathological staging (P=0.033), and vascular cancer embolus (P=0.014). Also, ME2 knockout significantly inhibited lung metastasisin vivo. In the tumors formed by ME2 knockout cells, the levels of α-KG were significantly increased and collagen hydroxylation level did not change significantly but HIF-1α protein expression was significantly decreased, compared to the control samples. In cell culture, cells with ME2 knockout or knockdown demonstrated significantly higher α-KG levels but significantly lower HIF-1α protein expression than control cells under hypoxia. Exogenous malate and α-KG exerted similar effect on HIF-1α in breast cancer cells to ME2 knockout or knockdown. Additionally, treatment with malate significantly decreased 4T1 breast cancer lung metastasis. ME2 expression was associated with HIF-1α levels in human breast cancer samples (P=0.008). CONCLUSIONS: Our results provide evidence that upregulation of ME2 is associated with a poor prognosis of breast cancer patients and propose a mechanistic understanding of a link between ME2 and breast cancer metastasis.

Lactate dehydrogenases amplify reactive oxygen species in cancer cells in response to oxidative stimuli.

Previous studies demonstrated that superoxide could initiate and amplify LDH-catalyzed hydrogen peroxide production in aqueous phase, but its physiological relevance is unknown. Here we showed that LDHA and LDHB both exhibited hydrogen peroxide-producing activity, which was significantly enhanced by the superoxide generated from the isolated mitochondria from HeLa cells and patients' cholangiocarcinoma specimen. After LDHA or LDHB were knocked out, hydrogen peroxide produced by Hela or 4T1 cancer cells were significantly reduced. Re-expression of LDHA in LDHA-knockout HeLa cells partially restored hydrogen peroxide production. In HeLa and 4T1 cells, LDHA or LDHB knockout or LDH inhibitor FX11 significantly decreased ROS induction by modulators of the mitochondrial electron transfer chain (antimycin, oligomycin, rotenone), hypoxia, and pharmacological ROS inducers piperlogumine (PL) and phenethyl isothiocyanate (PEITC). Moreover, the tumors formed by LDHA or LDHB knockout HeLa or 4T1 cells exhibited a significantly less oxidative state than those formed by control cells. Collectively, we provide a mechanistic understanding of a link between LDH and cellular hydrogen peroxide production or oxidative stress in cancer cells in vitro and in vivo.

The quantitative relationship between isotopic and net contributions of lactate and glucose to the tricarboxylic acid (TCA) cycle.

Whether growing cancer cells prefer lactate as a fuel over glucose or vice versa is an important but controversial issue. Labeling of tricarboxylic acid (TCA) cycle intermediates with glucose or lactate isotope tracers is often used to report the relative contributions of these two metabolites to the TCA cycle. However, this approach may not yield accurate results, as isotopic labeling may not accurately reflect net contributions of each metabolite. This may be due to isotopic exchange occurring during the conversion between pyruvate and lactate. To evaluate this quantitatively, we used an equation (CG - CG' = CL' - CL) assessing the relationship between isotopic labeling and net consumption measurements in vitro. CG and CL refer to the contributions of glucose and lactate to the TCA cycle as measured by their net consumption, whereas CG' and CL' refer to glucose's and lactate's contributions determined with isotopic labeling. We found that the isotopic labeling data overestimate the net contribution of lactate to the TCA cycle and underestimate that of glucose. The overestimated amount is equal to the isotopic exchange amount between pyruvate and lactate. After excluding the interference of isotopic exchange, the major carbon contribution (i.e. acetyl-CoA) to the TCA cycle comes from glucose rather than lactate in vitro We propose that these relative contributions of glucose and lactate may also be present in cancer cells in vivo.

Quantification of lactate from various metabolic pathways and quantification issues of lactate isotopologues and isotopmers.

13C-labeled glucose combined with chromatography and mass spectrometry enables us to decipher the percentage of lactate generated from various metabolic pathways. We showed that lactate derived from glycolysis, pentose phosphate pathway, Krebs cycle, and other sources accounted for 82-90%, 6.0-11%, 0.67-1.8% and 1.5-7.9%, respectively, depending on different types of cells. When using glucose isotopomers ([1-13C]-, [3-13C]-, [4-13C]-, and [6-13C]glucose) or isotopologues ([1,2-13C2]- and [1,2,3-13C3]glucose) for tracing, the ratio of lactate derived from glucose carbon 1, 2, 3 over 4, 5, 6 via glycolysis varied significantly, ranging from 1.6 (traced with [1,2-13C2]glucose) to 0.85 (traced with [6-13C]glucose), but the theoretical ratio should be 1. The odd results might be caused by intramolecular 13C, which may significantly affect lactate fragmentation under tandem mass spectrometry condition, leading to erroneous quantification. Indeed, the fragmentation efficiency of [U-13C]lactate, [2,3-13C]lactate, and [3-13C]lactate were 1.4, 1.5 and 1.2 folds higher than lactate, respectively, but [1-13C]lactate was similar to lactate, suggesting that carbon-13 at different positions could differentially influence lactate fragmentation. This observed phenomenon was inconsistent with the data based on theoretical calculation, according to which activation energies for all lactate isotopomers and isotopologues are nearly identical. The inconsistency suggested a need for further investigation. Our study suggests that calibration is required for quantifying metabolite isotopolugues and isotopomers.

Lactic acidosis switches cancer cells from aerobic glycolysis back to dominant oxidative phosphorylation.

While transformation of normal cells to cancer cells is accompanied with a switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, it is interesting to ask if cancer cells can revert from Warburg effect to OXPHOS. Our previous works suggested that cancer cells reverted to OXPHOS, when they were exposed to lactic acidosis, a common factor in tumor environment. However, the conclusion cannot be drawn unless ATP output from glycolysis and OXPHOS is quantitatively determined. Here we quantitatively measured ATP generation from glycolysis and OXPHOS in 9 randomly selected cancer cell lines. Without lactic acidosis, glycolysis and OXPHOS generated 23.7% - 52.2 % and 47.8% - 76.3% of total ATP, respectively; with lactic acidosis (20 mM lactate with pH 6.7), glycolysis and OXPHOS provided 5.7% - 13.4% and 86.6% - 94.3% of total ATP. We concluded that cancer cells under lactic acidosis reverted from Warburg effect to OXPHOS phenotype.