Abstract NRF2 is a transcription activator that plays a key role in cytoprotection against oxidative stress. Although increased NRF2 activity is principally beneficial for our health, NRF2 activation in cancer cells is detrimental, as it drives their malignant progression. We previously found that CCAAT/enhancer-binding protein B (CEBPB) cooperates with NRF2 in NRF2-activated lung cancer and enhances tumour-initiating activity by promoting NOTCH3 expression. However, the general contribution of CEBPB in lung cancer is rather controversial, probably because the role of CEBPB depends on cooperating transcription factors in each cellular context. To understand how NRF2 shapes the function of CEBPB in NRF2-activated lung cancers and its biological consequence, we comprehensively explored NRF2-CEBPB–coregulated genes and found that genes involved in drug metabolism and detoxification were characteristically enriched. Indeed, CEBPB and NRF2 cooperatively contribute to the drug resistance. We also found that CEBPB is directly regulated by NRF2, which is likely to be advantageous for the coexpression and cooperative function of NRF2 and CEBPB. These results suggest that drug resistance of NRF2-activated lung cancers is achieved by the cooperative function of NRF2 and CEBPB.

Transcriptional dysregulation, which can be caused by genetic and epigenetic alterations, is a fundamental feature of many cancers. A key cytoprotective transcriptional activator, NRF2, is often aberrantly activated in non-small cell lung cancers (NSCLCs) and supports both aggressive tumorigenesis and therapeutic resistance. Herein, we find that persistently activated NRF2 in NSCLCs generates enhancers at gene loci that are not normally regulated by transiently activated NRF2 under physiological conditions. Elevated accumulation of CEBPB in NRF2-activated NSCLCs is found to be one of the prerequisites for establishment of the unique NRF2-dependent enhancers, among which the NOTCH3 enhancer is shown to be critical for promotion of tumor-initiating activity. Enhancer remodeling mediated by NRF2-CEBPB cooperativity promotes tumor-initiating activity and drives malignancy of NRF2-activated NSCLCs via establishment of the NRF2-NOTCH3 regulatory axis.

<title>Abstract</title>The KEAP1-NRF2 system is a sulfur-employing defense mechanism against oxidative and electrophilic stress. NRF2 is a potent transcription activator for genes mediating sulfur-involving redox reactions, and KEAP1 controls the NRF2 activity in response to the stimuli by utilizing reactivity of sulfur atoms. In many human cancer cells, the KEAP1-mediated regulation of NRF2 activity is abrogated, resulting in the persistent activation of NRF2. Persistently activated NRF2 drives malignant progression of cancers by increasing therapeutic resistance and promoting aggressive tumorigenesis, a state termed as NRF2 addiction. In NRF2-addicted cancer cell, NRF2 contributes to metabolic reprogramming in cooperation with other oncogenic pathways. In particular, NRF2 strongly activates cystine uptake coupled with glutamate excretion and glutathione synthesis, which increases consumption of intracellular glutamate. Decreased availability of glutamate limits anaplerosis of the TCA cycle, resulting in low mitochondrial respiration, and nitrogen source, resulting in the high dependency on exogenous non-essential amino acids. The highly enhanced glutathione synthesis is also likely to alter sulfur metabolism, which can contribute to the maintenance of the mitochondrial membrane potential in normal cells. The potent antioxidant and detoxification capacity supported by abundant production of glutathione is achieved at the expense of central carbon metabolism and requires skewed metabolic flow of sulfur. These metabolic features of NRF2 addiction status provide clues for novel therapeutic strategies to target NRF2-addicted cancer cells.

NRF2 (nuclear factor erythroid 2-related factor 2) is a key transcriptional activator that mediates the inducible expression of antioxidant genes. NRF2 is normally ubiquitinated by KEAP1 (Kelch-like ECH-associated protein 1) and subsequently degraded by proteasomes. Inactivation of KEAP1 by oxidative stress or electrophilic chemicals allows NRF2 to activate transcription through binding to antioxidant response elements (AREs) and recruiting histone acetyltransferase CBP (CREB-binding protein). Whereas KEAP1-dependent regulation is a major determinant of NRF2 activity, NRF2-mediated transcriptional activation varies from context to context, suggesting that other intracellular signaling cascades may impact NRF2 function. To identify a signaling pathway that modifies NRF2 activity, we immunoprecipitated endogenous NRF2 and its interacting proteins from mouse liver and identified glucocorticoid receptor (GR) as a novel NRF2-binding partner. We found that glucocorticoids, dexamethasone and betamethasone, antagonize diethyl maleate-induced activation of NRF2 target genes in a GR-dependent manner. Dexamethasone treatment enhanced GR recruitment to AREs without affecting chromatin binding of NRF2, resulting in the inhibition of CBP recruitment and histone acetylation at AREs. This repressive effect was canceled by the addition of histone deacetylase inhibitors. Thus, GR signaling decreases NRF2 transcriptional activation through reducing the NRF2-dependent histone acetylation. Consistent with these observations, GR signaling blocked NRF2-mediated cytoprotection from oxidative stress. This study suggests that an impaired antioxidant response by NRF2 and a resulting decrease in cellular antioxidant capacity account for the side effects of glucocorticoids, providing a novel viewpoint for the pathogenesis of hypercorticosteroidism.