A good transcription factor gone rogue: the two faces of NRF2 in oxidative stress

Oxidative Stress
Author: Allison Bierly, PhD
Art Direction: Ken Mattiuz
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Highlights
  • Oncogenes activate Nrf2 independent of Keap1 mutations, via a Ras/MEK/ERK pathway
  • miR200a, silenced in breast cancer cells, is a negative regulator of KEAP1
  • New studies target NRF2 to counter COPD, multiple sclerosis, and chemoresistant cancer
Summary
Introduction: the NRF2 antioxidant pathway
Oncogenes lead to tumorigenesis through elevated Nrf2 and ROS detoxification
NRF2, KEAP1, and miR200a in breast cancer
Recent findings in NRF2 drug development
Conclusions
References
Back to top Summary
Recent studies point to novel mechanisms of NRF2 regulation, with implications for cellular detoxification and cancer. Reactive oxygen species (ROS) can contribute to cancer by damaging macromolecules like DNA and proteins (1), and the beneficial impact of antioxidant molecules in reducing cancer risk is well appreciated. However, constitutive activation of the antioxidant master regulator NRF2 via somatic mutations has also been implicated in carcinogenesis. New evidence has emerged pinpointing a role for oncogenes in activating the NRF2 antioxidant pathway independent of NFE2L2/KEAP1 mutations (2). Conversely, restoration or amplification of NRF2 activity continues to emerge as a potential treatment target for a wide variety of disease states. Therefore, the role of NRF2-initiated antioxidant activity in etiology, progression, and treatment of disease continues to increase in complexity.
Back to top Introduction: the NRF2 antioxidant pathway
NRF2 (nuclear factor-erythroid 2-related factor 2) binds to the antioxidant response elements (ARE) within promoters of antioxidant enzymes and detoxifying enzymes, such as glutathione S-transferase A2 (GSTA2) and NADPH quinone oxidoreductase 1 (NQO1) (3). Antioxidants counteract oxidative stress, employing a variety of techniques to diminish ROS levels in the intracellular environment (4). ROS are elevated during cancer and have been shown to activate signaling pathways involved in cell proliferation and migration, as well as cause DNA damage leading to mutations (1). As might be expected due to its role in induction of cytoprotective genes, Nrf2 knockout mice are more susceptible to injury and tumorigenesis in response to oxidative and chemical stresses (5-6).

KEAP1 (Kelch-like ECH-associated protein 1) represses NRF2 activity by binding to its Neh2 domain, which contains two binding sites for KEAP1's Kelch domain, the ETGE and DLG motifs. This binding promotes contact between NRF2 and the Cul3/Rbx1 ubiquitin ligase complex, leading to ubiquitination and degradation of NRF2 in the proteasome (7). Under normal conditions, this repression mechanism keeps antioxidant gene expression tightly regulated.
Back to top Oncogenes lead to tumorigenesis through elevated Nrf2 and ROS detoxification
While the NRF2 program is usually regarded as beneficial, recent work has undercut the notion that antioxidant activity is unambiguously protective against cancer. Excessive NRF2 activity resulting from mutations in NFE2L2 or KEAP1 is observed to contribute to chemotherapy resistance (7). Studies have suggested that the excessive induction of NRF2 genes in cancer cells, and therefore superior protection against cell stress, may confer a survival advantage (8).

Adding to this growing body of evidence, a recent study by DeNicola et al defines a new mechanism for Nrf2 activation in cancer. While ectopic expression of oncogenic K-Ras led to high ROS levels in fibroblasts, endogenous K-RasG12D expression caused ROS suppression, as did activation of endogenous MycERT2. Additionally, endogenous K-RasG12D raised mRNA and protein levels of Nrf2 and its target genes. Unlike in previous studies regarding upregulated Nrf2 and cancer, however, Keap1 regulation of Nrf2 was unchanged in these cells. Instead, K-RasG12Dappears to use the Raf/MEK/ERK pathway to amplify NRF2 expression, as a MEK inhibitor alleviated the oncogene's effects, increasing ROS levels and diminishing Nrf2 and antioxidant gene expression. Endogenous expression of oncogenic B-RafV619E led to the same trends in ROS and Nrf2 levels as did expression of K-RasG12D, in addition to ERK phosophorylation, lending further support to this pathway's involvement. The MAPK-related transcription factors Jun and Myc were also implicated.

Oncogenic induction of Nrf2 and its antioxidant targets also occurs in murine and human tumors in vivo. Murine pancreatic tumors from K-Ras mutants and lung adenomas from B-Raf mouse mutants showed elevated protein levels of a Nrf2 target antioxidant gene, Nqo1. Levels of 7,8-dihydro-8-oxo-29-deoxyguanosine or 8-oxo-dGuo, a DNA oxidation product commonly used as a marker of oxidative stress, were lowered in these tumors. Importantly, the same features were also observed in human pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma. Previous studies had indicated that Keap1 mutations and subsequent dysregulation of Nrf2 contributed to cancer, but the team sequenced more than 100 human pancreatic cancer samples and found KEAP1 and KRAS mutations together only one time. Therefore, in vivo as well as in vitro, NRF2 activity was independent of changes in KEAP1 regulation.

Emphasizing the importance of Nrf2 in detoxification under cancerous conditions in vivo, murine pancreatic tumor cells deficient in Nrf2 lacked expression of Nqo1, and showed no difference from non-cancerous cells with regard to the presence of oxidative stress markers. Cancer cell proliferation and survival were also affected by the absence of Nrf2, suggesting that the observed changes in oxidative damage and antioxidant induction can promote cancer development. PanIN from Nrf2-deficient mice showed less proliferation, and K-RasG12D MEFs underwent senescence after siRNA knockdown of Nrf2. However, adding an antioxidant to these cells, N-acetyl cysteine, reversed these effects.
Back to top NRF2, KEAP1, and miR200a in breast cancer
While NRF2 overactivation and excessive antioxidant activity promote tumors in some oncogene-induced cancers, Eades et al recently described a very different role for NRF2 in the human breast cancer cell lines MDA-MB-231 and Hs578T (9). Lowered NRF2 expression has been observed in breast cancer cells compared to normal cells (10). Through miRNA screening, Eades et al demonstrated that miR200a is silenced in breast cancer cells. Moreover, they showed that miR-200a can negatively regulate KEAP1 via mRNA destabilization, leading to higher levels of NRF2 and its antioxidant target gene NQO1.

Epigenetic therapy using a histone deacetylase inhibitor increased miR-200a and NRF2 levels, and led to a greater presence of NRF2 on the NQO1 promoter. Intriguingly, reactivation of NRF2 through HDAC therapy, as well as overexpression in breast cancer cells, prevented cells from forming colonies in soft agar. Additionally, mice treated with estrogen to induce epithelial transformation showed higher expression levels of Keap1 and lowered Nrf2 and miR-200a, while co-treating with the HDAC inhibitor raised Nrf2 and miR-200a levels and reduced Keap1. This role contrasts sharply with the role of NRF2 in promoting cancer cell survival, and emphasizes that, even among cancers, NRF2 can play substantially different roles.  
Back to top Recent findings in NRF2 drug development
Over the past year, studies have looked at the potential of NRF2 as a drug target in diseases ranging from COPD to multiple sclerosis, in addition to the anticipated role for anti-NRF2 drugs in enhancing cancer cell susceptibility to chemotherapy. Treatment with the NRF2 activator sulforaphane has recently shown two beneficial effects in COPD, rescuing impaired phagocytosis in alveolar macrophages and breaking down macrophage resistance to glucocorticosteroid treatment via HDAC2 denitrosylation (11-12). Linker et al. tested the effects of dimethylfumarate, a compound that had shown promise in clinical multiple sclerosis trials, in a mouse MS model (experimental autoimmune encephalomyelitis, or EAE). They found that this molecule attenuated some of the damaging effects of the disease in dependence on Nrf2. Treatment enhanced Nrf2 stabilization and activity, as well as transcription of Nqo1 (13).

On the cancer front, Ren et al. used lung cancer cells to study the activity of brusatol, a compound found in a plant extract, in inhibiting the NRF2 pathway. Brusatol increases ubiquitination of NRF2 while leaving most other signaling pathways unaffected, and renders both cancer cell lines and xenografts more susceptible to the chemotherapeutic agent cisplatin (14). Conversely, Thangasamy et al discovered that NRF2 acts as a repressor for a kinase involved in cancer cell invasion and tamoxifen resistance, recepteur d' origine nantais (RON). The NRF2 activator sulforaphane, in addition to other NRF2-enhancing treatments, was able to block RON ligand-induced invasion by breast carcinoma cells in an in vitro matrigel model. (15).
Back to top Conclusions
Normal levels of NRF2 are needed for cytoprotection from oxidative stress, and NRF2 has been suggested as a therapeutic target for a host of disease states, from diabetic nephropathy to Parkinson's disease to malaria (16-18). Moreover, excessive NRF2 suppression is seen in breast cancer cell lines, and its restoration inhibits colony formation (2). Nevertheless, recent developments have revealed that a variety of mechanisms can enhance NRF2 levels in cancerous cells, reducing the intracellular environment and imparting survival advantages and drug resistance. While antioxidant activity is frequently seen as protective against cancer due to the mutagenic potential of ROS, a more nuanced view has emerged. When judging the benefit or harm of oxidative stress in cancer, as with so many biological processes, context is everything.

RT2 Profiler PCR Arrays profile the expression of genes involved in oxidative stress, antioxidant defense, cancer pathways, and many others. Additionally, miScript miRNA PCR Arrays permit profiling of miRNAs related to breast cancer, brain cancer, autoimmunity, development, and more. These tools are the most focused, extensively verified method available to get a thorough picture of the mRNA and miRNA expression changes occurring in your pathway of interest.
Back to top References
  1. Klaunig, J.E., Kamendulis, L.M. and Hocevar, B.A. (2010) Oxidative stress and oxidative damage in carcinogenesis. Toxicol. Pathol. 38, 96.
  2. DeNicola, G.M. et al. (2011) Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 475, 106.
  3. Nguyen, T., Nioi, P. and Pickett, C.B. (2009) The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J. Biol. Chem. 284, 13291.
  4. Lu, J-M., Lin, P.H., Yao, Q. and Chen, C. (2010) Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. J. Cell. Mol. Med. 14, 840.
  5. Hayes, J.D. and McMahon, M. (2006) The double-edged sword of Nrf2: subversion of redox homeostasis during the evolution of cancer. Mol. Cell 21, 732.
  6. Frohlich, D.A., McCabe, M.T., Arnold, R.S. and Day, M.L. (2008) The role of Nrf2 in increased reactive oxygen species and DNA damage in prostate tumorigenesis. Oncogene 27, 4353.
  7. Kensler, T.W. and Wakabayashi, N. (2010) Nrf2: friend or foe for chemoprevention? Carcinogenesis 31, 90.
  8. Lau, A., Villeneuve, N.F., Sun, Z., Wong, P.K. and Zhang, D.D. (2008) Dual roles of Nrf2 in cancer. 58, 262.
  9. Eades, G., Yang, M., Yao, Y., Zhang, Y. and Zhou, Q. (2011) miR-200a regulates Nrf2 activation by targeting Keap1 mRNA in breast cancer cells. J. Biol. Chem. Epub ahead of print.
  10. Loignon, M. et al. (2009) Cul3 overexpression depletes Nrf2 in breast cancer and is associated with sensitivity to carcinogens, to oxidative stress, and to chemotherapy. Mol. Cancer Ther. 8, 2432.
  11. Harvey, C.J. et al. (2011) Targeting Nrf2 signaling improves bacterial clearance by alveolar macrophages in patients with COPD and in a mouse model. Sci. Trans. Med. 3, 78ra32.
  12. Malhotra, D. et al. (2011) Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients. J. Clin. Invest. 121, 4289.
  13. Linker, R.A. et al. (2011) Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway. Brain 134, 678.
  14. Ren, D. et al. (2011) Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism. Proc. Natl. Acad. Sci. USA 108, 1433.
  15. Thangasamy, A., Rogge, J., Krishnegowda, N.K., Freeman, J.W. and Ammanamanchi, S. (2011) Novel function of transcription factor Nrf2 as an inhibitor of RON tyrosine kinase receptor-mediated cancer cell invasion. J. Biol. Chem. 286, 32115.
  16. Zheng, H. et al. (2011) Therapeutic potential of Nrf2 activators in streptozotocin-induced diabetic nephropathy. Diabetes 60, 3055.
  17. Cook, A.L et al. (2011) NRF2 activation restores disease-related metabolic deficiencies in olfactory neurosphere-derived cells from patients with sporadic Parkinson's disease. PLOS One 6, e21907.
  18. Olagnier, D. et al. (2011) Nrf2, a PPAR-gamma alternative pathway to promote CD36 expression on inflammatory macrophages: implication for malaria. PLoS Pathog. 7, e1002254.

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