ABSTRACT
NRF2 (nuclear factor erythroid 2-related factor 2) is a transcription factor which plays a major role in oxidative stress responses by regulating antioxidant gene expression. We have recently identified the ARF tumor suppressor as a key regulator of NRF2. ARF can significantly inhibit NRF2 transcriptional activities, and the ARF-NRF2 interaction may function as a novel checkpoint for oxidative stress responses.
KEYWORDS: Tumor suppressor, ARF, NRF2, oxidative stress
ARF, an alternative reading frame product of the CDKN2A locus, can suppress tumors, mainly by inhibiting MDM2 and ARF-BP1 (HUWE1), E3-ubiquitin ligases and negative regulators of TP53.1 Interestingly, ARF also has p53-independent tumor suppressor activities, although these function are still not well understood. NRF2 (nuclear factor erythroid 2-related factor 2) is regarded as one of the most important regulators of the cellular pathway to defend oxidative stress.2 Under normal conditions, NRF2 protein is kept at low levels in the cytoplasm by the E3 ubiquitin ligase KEAP1 (Kelch ECH-associated protein 1). However, under oxidative conditions, cysteine residues in KEAP1 become oxidized leading to a dissociation with NRF2, allowing both NRF2 protein stabilization and re-localization to the nucleus.2 NRF2 then binds to antioxidative response element (ARE) which causes transactivation of key antioxidant genes, such as NQO1 (quinine oxidoreductase 1), HO-1 (heme oxygenase 1), GSR (Glutathione-Disulfide reductase), PRDX1 (peroxiredoxin 1) and SLC7A11.3 Moreover, acetylation of NRF2 by CBP/p300 has been shown as another mechanism to control NRF2 function.4 SLC7A11, a key component of the cysteine/glutamate antiporter, is highly expressed and induced by NRF2 in human cancers. Downregulation or inhibition of SLC7A11, results in intracellular cysteine depletion, increasing Reactive oxygen species (ROS) levels to activate a non-apoptotic form of cell death termed ferroptosis.5 In a recent study, we purified the NRF2-complex, and identified ARF as a novel binding partner both in vitro and in vivo.6,7 ARF does not modulate NRF2 protein levels by interfering with KEAP1-mediated ubiquitination, however, we found that ARF inhibits CBP-dependent NRF2 acetylation and therefore decreases NRF2 binding to promoters, significantly repressing NRF2 transcriptional activity. As a consequence, SLC7A11 expression is dramatically suppressed when ARF is activated, in an NRF2-dependent manner (Figure 1). SLC7A11 suppression induces ferroptotic cell death in cancer cells upon induction with oxidative stress. Overexpression of NRF2 can abrogate ARF-mediated tumor suppression. In contrast, depletion of ARF augments NRF2 activity and reduces cancer cell death upon oxidative stress.
Figure 1.
ARF-NRF2: a new checkpoint for oxidative stress responses. ARF tumor suppressor can significantly inhibit NRF2 (nuclear factor erythroid 2-related factor 2) transcriptional activities, including SLC7A11. ARF-NRF2-SLC7A11 axis drives ferroptosis under oxidative stress and tumor suppression in a p53-independent manner. ARF also potentially affect other NRF2 target genes. DEM ((Diethyl maleate); tBHQ (tert-Butylhydroquinone).
As oxidative stress responses play emerging roles in metabolism, ferroptosis, and tumor suppression, the research on NRF2, a key regulator of oxidative stress, becomes increasingly important. NRF2 has been found to regulate about 500 target genes, which can be functionally divided into several major groups: synthesis and conjugation of glutathione, antioxidant, drug metabolizing enzymes and transporters, metabolic enzymes, heme and iron metabolism, and transcriptional factors.3 Therefore, it will be interesting to decipher whether ARF can regulate those other NRF2 target genes, besides SLC7A11, NQO-1, HO-1, GSR and PRDX1.6,7 Anti-oxidative reagents, such as DEM (Diethyl maleate) and tBHQ (tert-Butylhydroquinone), are well-known inducers of NRF2 to activate AREs.8 Besides DEM and tBHQ, a variety of other chemicals, including phytochemicals and derivatives (CDDO; sulforaphane), chemotherapeutic drugs (oltipraz; auranofin), environmental agents (paraquat; arsenic), and endogenous chemicals (nitric oxide; 15-Deoxy-Delta-12,14-prostaglandin J2; nitro-fatty acids; and 4-hydroxynonenal), induce NRF2 transcriptional activities.8 It will be worth to test whether ARF modulation will affect those chemical-modulated NRF2 activities to regulate cancer cell death in the future. The protective nature of NRF2 has also been suggested for cancer cells to create a prosurvival microenvironment for tumor growth and drug resistance. Hence, NRF2 has been suggested to play important roles in Cisplatin resistance.9 Methylation of the INK4 locus has been found as the frequent epigenetic change in cancer.1 Therefore, drugs that inhibit or interfere with DNA methylation should be used to reactivate and induce silenced ARF to re-express in those malignancies. Interesting, azacitidine, a demethylating agent, could be used to sensitize certain cisplatin-resistant cancers, at least partially through modulation of the ARF-NRF2 interplay. Coincidently, the National Institutes of Health started clinical trials for combination of azacitidine and Cisplatin. Although more mechanistic research is needed, it is tempting to speculate that activating ARF levels could enhance cancer cell death. Therefore, it is worth detecting the status of ARF in these patients receiving treatment; ARF mutation therefore would lead to a dampened response to chemotherapeutic drugs, particularly in those cancers with simultaneous upregulation of SLC7A11.10
Funding Statement
HHS | NIH | National Cancer Institute (NCI) This work was supported by the National Cancer Institute of the National Institutes of Health under Award 5RO1CA190477, 5RO1CA224227, 5RO1CA216884 and 5RO1CA085533 to W.G. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Competing financial interests
The authors declare no competing financial interests.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
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