Modulation of NRF2 signaling pathway by nuclear receptors: Implications for cancer

Nuclear factor-erythroid 2 p45-related factor 2 (NRF2, also known as Nfe2l2) plays a critical role in regulating cellular defense against electrophilic and oxidative stress by activating the expression of an array of antioxidant response element-dependent genes. On one hand, NRF2 activators have been used in clinical trials for cancer prevention and the treatment of diseases associated with oxidative stress; on the other hand, constitutive activation of NRF2 in many types of tumors contributes to the survival and growth of cancer cells, as well as resistance to anticancer therapy. In this review, we provide an overview of the NRF2 signaling pathway and discuss its role in carcinogenesis. We also introduce the inhibition of NRF2 by nuclear receptors. Further, we address the biological significance of regulation of the NRF2 signaling pathway by nuclear receptors in health and disease. Finally, we discuss the possible impact of NRF2 inhibition by nuclear receptors on cancer therapy.     

The Nuclear Factor (Erythroid-derived 2)-like 2 and Proteasome Maturation Protein Axis Mediate Bortezomib Resistance in Multiple Myeloma

Resistance to the proteasome inhibitor bortezomib is an emerging clinical problem whose mechanisms have not been fully elucidated. We considered the possibility that this could be associated with enhanced proteasome activity in part through the action of the proteasome maturation protein (POMP). Bortezomib-resistant myeloma models were used to examine the correlation between POMP expression and bortezomib sensitivity. POMP expression was then modulated using genetic and pharmacologic approaches to determine the effects on proteasome inhibitor sensitivity in cell lines and in vivo models. Resistant cell lines were found to overexpress POMP, and while its suppression in cell lines enhanced bortezomib sensitivity, POMP overexpression in drug-naive cells conferred resistance. Overexpression of POMP was associated with increased levels of nuclear factor (erythroid-derived 2)-like (NRF2), and NRF2 was found to bind to and activate the POMP promoter. Knockdown of NRF2 in bortezomib-resistant cells reduced POMP levels and proteasome activity, whereas its overexpression in drug-naive cells increased POMP and proteasome activity. The NRF2 inhibitor all-trans-retinoic acid reduced cellular NRF2 levels and increased the anti-proliferative and pro-apoptotic activities of bortezomib in resistant cells, while decreasing proteasome capacity. Finally, the combination of all-trans-retinoic acid with bortezomib showed enhanced activity against primary patient samples and in a murine model of bortezomib-resistant myeloma. Taken together, these studies validate a role for the NRF2/POMP axis in bortezomib resistance and identify NRF2 and POMP as potentially attractive targets for chemosensitization to this proteasome inhibitor.     

SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD⁺-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.     

DNA damage and oxidative stress in human cells infected by Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2–related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.     

Selenoprotein H Suppresses Cellular Senescence through Genome Maintenance and Redox Regulation

Oxidative stress and persistent DNA damage response contribute to cellular senescence, a degeneration process critically involving ataxia telangiectasia-mutated (ATM) and p53. Selenoprotein H (SelH), a nuclear selenoprotein, is proposed to carry redox and transactivation domains. To determine the role of SelH in genome maintenance, shRNA knockdown was employed in human normal and immortalized cell lines. SelH shRNA MRC-5 diploid fibroblasts under ambient O₂ displayed a distinct profile of senescence including β-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation. Such senescence phenotypes were alleviated in the presence of ATM kinase inhibitors, by p53 shRNA knockdown, or by maintaining the cells under 3% O₂. During the course of 5-day recovery, the induction of phospho-ATM on Ser-1981 and γH2AX by H₂O₂ treatment (20 μm) subsided in scrambled shRNA but exacerbated in SelH shRNA MRC-5 cells. Results from clonogenic assays demonstrated hypersensitivity of SelH shRNA HeLa cells to paraquat and H₂O₂, but not to hydroxyurea, neocarzinostatin, or camptothecin. While SelH mRNA expression was induced by H₂O₂ treatment, SelH-GFP did not mobilize to sites of oxidative DNA damage. The glutathione level was lower in SelH shRNA than scrambled shRNA HeLa cells, and the H₂O₂-induced cell death was rescued in the presence of N-acetylcysteine, a glutathione precursor. Altogether, SelH protects against cellular senescence to oxidative stress through a genome maintenance pathway involving ATM and p53.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-α and -β, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

Screening active components of modified Xiaoyao powder as NRF2 agonists

Nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) regulates antioxidant enzymes and phase II detoxifying enzymes, such as NAD(P)H: quinone oxidoreductase 1 (NQO1). Modified Xiaoyao powder (MXP) is most frequently used in the prevention and treatment of breast cancer in China. This study aimed to screen active components of MXP for antioxidant stress and chemoprevention, which depend on NRF2‐NQO1 signalling pathway. A total of 25 monomeric compounds contained in MXP were screened using an antioxidant response element–luciferase reporter. The most potent antioxidant response element–luciferase inducers were chosen to further examine their effects on NRF2 and NQO1 in MCF‐7 cells. These results were then confirmed by determining the oxidative stress levels and chemopreventive effect on inhibiting carcinogenesis transformation in NRF2 knockdown (NRF2^KD) and NRF2 wild‐type MCF‐10A cells. We found that quercetin, kaempferol, and atractylenolide II in MXP were potent NRF2 inducers, which could up‐regulate the expression of NRF2 and its downstream enzymes NQO1. In addition, these components could decrease reduced oxidative stress and inhibit carcinogenesis transformation, which depended on NRF2‐NQO1 pathway. In conclusion, NRF2‐NQO1 pathway plays an essential role in mediating the activity of MXP and its active components, at least in part; some beneficial effects of MXP may be applicable to breast cancer chemoprevention. Our study firstly found MXP active components including quercetin, kaempferol, and atractylenolide II. Our results firstly demonstrate that NRF2‐NQO1 pathway plays an essential role in mediating the activity of MXP and its active components in breast cancer chemoprevention. Our study firstly found that atractylenolide II is a novel NRF2 inducer.