Mol. Cells

NADH-Reductive Stress in Cancer Cell Proliferation

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Cancer is a metabolic process reprogrammed to accept excessive bioenergetic and biosynthetic substances that promote tumorigenesis and cell proliferation. To survive and proliferate, cancer cells need to regulate tightly reactive oxygen species (ROS) produced by oxidative metabolism, which is achieved via redox balance (Harris and DeNicola, 2020). ROS levels are abnormally increased by the rapid proliferation of cancer cells during their metabolic processes. Moreover, to remove the oxidative stress induced by ROS accumulation, cancer cells need to overexpress genes involved in antioxidant activities, causing redox imbalance (Ghanbari Movahed et al., 2019). Although the redox imbalance in cancer cells has been mainly associated with oxidative stress, it has recently been shown that it is involved in reductive stress due to the accumulation of reducing equivalents (e.g., NADH, NADPH, and GSH) and overexpression of genes with antioxidant effects (Xiao and Loscalzo, 2020). However, the mechanism of reductive stress in cancer is relatively unknown. In this study, the authors examined the possible metabolic vulnerability of cancer cells associated with NADH-dependent reductive stress via NRF2-mediated ALDH3A1 activation (Weiss-Sadan et al., 2023).

In most cancers, cell proliferation is induced by NRF2-induced Keap1 inhibition (Wu et al., 2019). The authors hypothesized that the wild-type Keap1 variant may also contribute to cancer cell proliferation via NRF2 activation in non-small cell lung cancers (NSCLCs). To examine this hypothesis, the authors used KI696, a specific Keap1 inhibitor, to block wild-type Keap1 in various NSCLC cell lines. Interestingly, 13% of NSCLC cell lines exhibited reduced cell proliferation in Keap1-blocked cells by both KI696 and Keap1-specific shRNA.

To identify critical genes that are sensitive or resistant to NRF2 activation, the authors performed a genome-wide CRISPR screen in the KEAP1-dependent CALU6 cell line after treatment with KI696. The authors identified KI696-resistant and -sensitive genes to NRF2 activation in the CALU6 cell line. Depletion of both MYC and members of the mediator complex (i.e., TAF5L and TADA2B) induced resistance to NRF2 activation. On the one hand, sensitive genes to NRF2 activation, including NAGS, NDUFB1, and LIAS, were found to be involved in the mitochondrial metabolic pathway. When the authors analyzed cellular pathways and gene ontology categories, they found that metabolism was commonly categorized as a major group for resistance and sensitive gene sets to NRF2 activation. Subsequently, the authors performed a metabolism-focused CRISPR screen. After treatment with KI696, they identified gene subsets associated with different metabolic processes in CALU6 (Keap1-dependent) and H1975 (Keap1-independent) cells. Keap1-independent cells were sensitized to NRF2 activation by depletion of glycolytic gene subsets, whereas Keap1-dependent cells were sensitized by inactivation of OXPHOS (oxidative phosphorylation)-associated NRF2 activation genes. To clarify this phenomenon, the authors analyzed the ratio of extracellular acidification rate (ECAR) to oxygen consumption rate (OCR) of Keap1-dependent and -independent cells after treatment with KI696. The OCR/ECAR ratio was high in Keap1-dependent cells and low in Keap1-independent cells. This suggests that glycolytic rates are negatively correlated with NRF2 sensitivity upon activation. In Keap1-dependent cells, the proliferation was restored by overexpression of the PFK (phosphofructokinase) glycolytic gene or by hypoxic conditions. The proliferation of Keap1-independent cells decreased due to the activity of glycolytic pathway inhibitors such as galactose and oxamate. Based on these results, the authors suggested that the glycolytic pathway is required to regulate NRF2 sensitivity.

The authors observed that the maximal respiratory capacity and TCA (tricarboxylic acid) metabolite levels significantly decreased in Keap1-dependent cells treated with KI696. OCR was rescued by NRF2 knockdown, suggesting that NRF2 activation plays an important role in regulating mitochondria function in Keap1-dependent cells. OCR has a prominent role in the electron transport chain (ETC). To clarify the differences between the sensitivity of ETC complexes, each type of complex was inhibited specifically in Keap1-dependent and -independent cells. Interestingly, only the inhibition of the ETC Complex I induced hypersensitivity to NRF2 activation in Keap1-dependent cells. These results indicate that NRF2 activation is involved in the mitochondrial metabolism of Keap1-dependent cells.

NADH oxidation is regulated in ETC and glycolysis (Martínez-Reyes and Chandel, 2020). To interrogate whether NRF2 activation affects the NADH/NAD++ ratio, the authors used an NSCLC cell line continuously expressing SoNar, a genetically encoded NADH/NAD++ reporter. After treatment with KI696, the NADH/NAD++ ratio maintained a high level in Keap1-dependent cells but not in Keap1-independent cells. When the NAD+ precursor β-nicotinamide mononucleotide was added to Keap1-dependent cells, the NADH/NAD++ ratio and cell proliferation were restored to normal levels upon NRF2 activation. Therefore, the authors indicated that NRF2 induces NADH-reductive stress in Keap1-dependent cells, thus decreasing cell proliferation. Although most NADH oxidation in keap1-dependent and -independent cells occurs via lactate dehydrogenase, this study showed that NADH oxidation via ETC may contribute to Keap1 dependency and sensitivity to Complex I blockade.

The authors observed that the activation of NRF2 leads to an increase in NADH levels by inducing the expression of ALDH3A1 in Keap1-dependent cells. ALDH3A1 upregulation increases NAD+ consumption and changes the redox status. Therefore, ALDH3A1 could be regulated by NRF2 to control the NADH/NAD++ ratio in Keap1-dependent cells. A high MYC level is well known to increase transcriptional amplification in tumor cells (Lin et al., 2012). In this study, the authors showed that the expression of ALDH3A1 and the NADH/NAD++ ratio decreased by MYC depletion in Keap1-dependent cells following NRF2 activation. Consequently, ALDH3A1 expression and a high NADH/NAD++ ratio inhibited cell proliferation in Keap1-dependent cells upon NRF2 activation.

Based on their results, the authors hypothesized that NRF2 activation negatively affects cell proliferation by inducing NADH/NAD++ imbalance. To test this hypothesis, Keap1-mutant cells were treated with IACS-010759, a potent Complex I inhibitor. Then, the authors measured NADH/NAD++ ratio levels and cell proliferation rates. After treatment with IACS-010759, the NADH/NAD++ ratio increased, and cell proliferation decreased in Keap1-mutant cells. To confirm these results in vivo, the authors developed Keap1-mutant and –wild-type patient-derived xenograft (PDX) mice models. The treatment with IACS-010759 strongly inhibited tumor growth in the Keap1-mutant PDX mouse model. These results suggest that a high NADH/NAD++ ratio induces reductive stress and suppresses cell proliferation in Keap1-mutant cells.

In this study, the authors suggested that proper control of the NADH/NAD++ ratio can be a promising therapeutic option in cancer cells carrying mutations in the Keap1 gene and with a non-glycolytic metabolism susceptible to reductive stress induced by NRF2. Currently, attention is being focused on targeting the NRF2 activation as a therapeutic strategy for treating mitochondrial diseases (Holmström et al., 2016) and Leigh syndrome (Quintana et al., 2010), but the NADH/NAD++ ratio should also be considered since it is critical for mitochondrial respiration.

This study unraveled a suppressive mechanism for tumor proliferation via the alteration of NADH-reductive stress by NRF2-mediated ALDH3A1 upregulation. From this work, the authors suggested a direct relationship between NADH-reductive stress and cancer cell proliferation.

Article information

Mol. Cells.Aug 31, 2023; 46(8): 473-475.
Published online 2023-08-4. doi:  10.14348/molcells.2023.0094
Division of Life Sciences, Korea University, Seoul 02841, Korea
Received June 1, 2023; Accepted July 3, 2023.
Articles from Mol. Cells are provided here courtesy of Mol. Cells


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Figure 1

NRF2-mediated ALDH3A1 upregulation via pharmacologic inhibition or KEAP1 mutation induces NADH-reductive stress. The disruption of mitochondrial Complex I activity leads to metabolic vulnerability in Keap1-dependent and mutant cell lines, increasing NADH levels. The induction of NADH-reductive stress selectively blocks the proliferation of cancer cell lines. NRF2, nuclear factor erythroid-derived 2-related factor 2; KEAP1, Kelch-like ECH-associated protein 1; ALDH3A1, aldehyde dehydrogenase 3 family member A1; NADH, nicotinamide adenine dinucleotide hydride; TCA, tricarboxylic acid; OXPHOS, oxidative phosphorylation.