Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Sep 9;105(36):13568-73.
doi: 10.1073/pnas.0806268105. Epub 2008 Aug 29.

Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy

Tatsuhiro Shibata  1 , Tsutomu Ohta, Kit I Tong, Akiko Kokubu, Reiko Odogawa, Koji Tsuta, Hisao Asamura, Masayuki Yamamoto, Setsuo Hirohashi
Affiliations

Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy

Tatsuhiro Shibata et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10393

Abstract

The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
NRF2 mutations in human cancer. (A) Functional domains of Nrf2 protein showing distribution and types of NRF2 mutations. SQ, lung squamous cell carcinoma; AD, adenocarcinoma; LCNEC, large cell neuroendocrine carcinoma; HN, head and neck cancer; cell, lung cancer cell lines. (B) Representative DNA chromatograph of NRF2 mutations in human cancer cell lines with corresponding normal sequences in the bottom panel. (C) Copy number alteration map [shown by log2 ratio (tumor/normal)] of chromosome 2 in the tumors with mutated NRF2 and having lost the wild-type allele, detected by array-based comparative genomic hybridization. The arrow indicates the chromosomal location of NRF2. (D) Kaplan–Meier plot showing disease-free survival (DFS) of squamous cell lung carcinoma patients segregated according to the presence or absence of NRF2 mutations.
Fig. 2.
Fig. 2.
NRF2 mutations impair the two-site substrate recognition mechanism of Keap1. ITC titration profiles of Keap1-DC with Neh2[Δ1–33,D77V] (A), Neh2[Δ1–33,E79Q] (B), Neh2[Δ1–33,T80K] (C), and of Keap1 with Neh2 (D), Neh2[W24C] (E), Neh2[L30F] (F), raw thermograms (upper graph) and fitted binding isotherms (lower graph). Stoichiometry (n) and association constant (Ka) are as indicated. (G–I), binding site of the human Keap1-DC and wild-type ETGE peptide structure (G) and modeled structures of Keap1-DC with E79Q (H) or E82D (I) mutant ETGE peptide. Stick representation showing side chains of interacting residues from Keap1 (aqua) and ETGE peptide (pink). Dotted lines indicate predicted hydrogen bonds. Figures were drawn with PyMOL (www.pymol.org).
Fig. 3.
Fig. 3.
NRF2 mutations disturb proper Nrf2-Keap1 binding, inhibit Keap1-mediated degradation, promote transcriptional activity, and enhance nuclear localization of Nrf2 in vivo. (A) Expression level of the transfected Flag-tagged Keap1 and Myc-tagged wild-type or mutant Nrf2 (E79K, T80K, and L30F) in 293T cells. (B) Keap1-Nrf2 association was evaluated by immunoprecipitation of transfected 293T cells using anti-Flag antibody and immunoblotted by anti-Myc antibody. (C) Ubiquitination efficiency of wild-type or mutant Myc-tagged Nrf2 in vivo. (D) Immunoblot (left) showing degradation rate of Nrf2 protein from 15 to 60 min after cycloheximide chase. Intensities (%) relative to time 0 are plotted (right). Data represent mean ± SD of three independent runs. Dotted line indicates 50% expression of control. (E) Transactivation activities of Nrf2 mutants. Data represent mean ± SD. of three independent runs (*, P < 0.001). (F) Rescue analysis in KEAP1- or NRF2-mutated cancer cells. H1650, A549, LK2, and EBC-1 cells bearing both wild-type KEAP1 and NRF2 or mutations in one of the genes are indicated. Relative expressions of Nrf2 target genes peroxiredoxin1 (PRDX1), multidrug resistance protein 3 (MRP3), and NAD(P)H quinine oxidoreductase-1 (NQO1) to β-ACTIN with or without cotransfected KEAP1 plasmid are shown. (G) Nuclear accumulation of mutant Nrf2 proteins when coexpressed with Keap1 (left). Ratio of cytoplasmic/nuclear localization of Nrf2 proteins was shown as bar chart (right).
Fig. 4.
Fig. 4.
Down-regulation of NRF2 restores sensitivity to oxidative stress and chemotherapeutic agent. (A) Down-regulation of Nrf2 expression by siRNA. Nonsilencing control or NRF2 siRNA was transfected into cancer cells with either KEAP1 (A549) or NRF2 (EBC-1) mutation. Cell lysates were electrophoresed and immunoblotted with anti-Nrf2 or anti-β-actin (loading control) antibody (top). Relative expressions of Nrf2 target genes (PRDX1, MRP3, and NQO1) to β-ACTIN are shown in the histogram (bottom). (B) Cell viability of siRNA treated A549, EBC-1, and H1650 cells exposed to oxidative stress. Data present percentage of cell viability after a 5-h treatment with H2O2 relative to nontreated control (mean ± SD of three independent runs) (C) A549 and EBC-1 cells were treated with control or NRF2 siRNA and various concentrations of cisplatin. Data present percentage of cell viability after 48 h relative to vehicle (DMSO)-treated control (mean ± SD of three independent runs). (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
See this image and copyright information in PMC

References

    1. Kasai H, Kawai K. Oxidative DNA damage: Mechanisms and significance in health and disease. Antioxid Redox Signal. 2006;8:981–983. - PubMed
    1. Motohashi H, Yamamoto M. Carcinogenesis and transcriptional regulation through Maf recognition elements. Cancer Sci. 2007;98:135–139. - PMC - PubMed
    1. Fahey JW, Kensler TW. Role of Dietary Supplements/Nutraceuticals in Chemoprevention through Induction of Cytoprotective Enzymes. Chem Res Toxicol. 2007;20:572–576. - PMC - PubMed
    1. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 2007;47:89–116. - PubMed
    1. Ramos-Gomez M, et al. Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci USA. 2001;98:3410–3415. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources