Overview of redox regulation by Keap1–Nrf2 system in toxicology and cancer

Accepted Manuscript Overview of redox regulation by Keap1-Nrf2 system in toxicology and cancer Mikiko Suzuki, Akihito Otsuki, Nadine Keleku-Lukwete, Masayuki Yamamoto PII:

S2468-2020(16)30006-7

DOI:

10.1016/j.cotox.2016.10.001

Reference:

COTOX 9

To appear in:

Current Opinion in Toxicology

Received Date: 18 July 2016 Revised Date:

2 October 2016

Accepted Date: 3 October 2016

Please cite this article as: M. Suzuki, A. Otsuki, N. Keleku-Lukwete, M. Yamamoto, Overview of redox regulation by Keap1-Nrf2 system in toxicology and cancer, Current Opinion in Toxicology (2016), doi: 10.1016/j.cotox.2016.10.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Suzuki et al.

Overview of redox regulation by Keap1-Nrf2 system

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in toxicology and cancer

Mikiko Suzuki1, 2 Akihito Otsuki2, Nadine Keleku-Lukwete2 and Masayuki Yamamoto1, 2

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Center for Radioisotope Sciences, Tohoku University Graduate School of Medicine, Sendai

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980-8575, Japan

Department of Medical Biochemistry, Tohoku University Graduate School of Medicine,

Sendai 980-8575, Japan

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To whom correspondence should be addressed: Masayuki Yamamoto Department of Medical Biochemistry Tohoku University Graduate School of Medicine

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Key words: Nrf2, Keap1

2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan Phone +81-22-717-8084; Fax +81-22-717-8090 E-mail: [email protected]

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Abstract The Keap1-Nrf2 pathway is a cellular defense system against oxidative and xenobiotic stresses derived from reactive oxygen species (ROS) and electrophiles, respectively. Nrf2 is a

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key transcription factor that activates a set of cytoprotective genes, including those encoding antioxidative and detoxifying enzymes. Keap1 is an adaptor protein of Cullin3-based E3 ligase, which regulates Nrf2 activity in response to these stresses. Under unstressed

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conditions, Keap1 constitutively degrades Nrf2 via the proteasome pathway. Keap1 interacts with Nrf2 through DLGex and ETGE sites in Nrf2 Neh2 domain, which is critical for

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regulation of Nrf2 degradation. ROS and electrophiles modify cysteine residues of Keap1 to inactivate the ubiquitin E3 ligase activity of Keap1, so that Nrf2 escapes from the Keap1-mediated repression, migrates into the nucleus, and activates expression of its target genes. As oxidative stresses give rise to many diseases, Nrf2 inducers that interact with Keap1 cysteine residues or Keap1-Nrf2 binding surface are expected as drugs against these diseases.

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On the other hand, several lines of evidence have showed that cancer cells hijack the Keap1-Nrf2 system to obtain resistance of chemo- and radiotherapies. Somatic mutations in human KEAP1 and NRF2 genes are observed in a number of cancers, resulting in constitutive

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activation of NRF2 and poor prognosis. In this review, we describe molecular basis underlying

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the Keap1-Nrf2 function and drug discovery.

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Keap1-Nrf2 system is a key defense system against oxidative and electrophilic stresses Nrf2 (NF-E2-related factor like-2) is a master regulatory transcription factor and induces genes that play critical roles in cytoprotection against oxidative and xenobiotic stresses [1].

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Nrf2 is constitutively degraded under unstressed conditions, whereas Nrf2 is rapidly activated in response to the stresses [2]. Keap1 (Kelch-like ECH-associated protein 1) serves as a stress sensor of Nrf2. In unstressed and quiescent conditions, Nrf2 is ubiquitinated by

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Keap1-Cullin3 ubiquitin E3 ligase complex in cytosol, and constantly degraded through the proteasome pathway [2,3] (Figure 1). This mechanism contributes to maintain cellular Nrf2

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levels low with half-time of degradation approximately 18 minutes. On the contrary, when cells were exposed to ROS (reactive oxygen species) or toxic xenobiotics (often electrophiles), the Nrf2 degradation is stopped and as a consequence, Nrf2 is stabilized and rapidly accumulates in nucleus, which induces the expression of target genes by forming heterodimers with sMaf (small Maf) family proteins [4-6]. In addition to Keap1-dependent

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degradation pathway, GSK-3 (glycogen synthase kinase-3) and β-TrCP (β-transducin repeat containing protein) pathway also inhibit accumulation of Nrf2. The GSK-3 catalyzes phosphorylation of Nrf2 leading to recruitment of β-TrCP by which Nrf2 proteins are

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ubiquitinated and degraded in nucleus [7-9].

In nucleus, Nrf2-sMaf heterodimer recognizes DNA sequences referred to as ARE

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(antioxidant response element [10]) or EpRE (electrophile response element [11]), or collectively named as CsMBE (CNC-sMaf binding element [12]). Target genes of Nrf2-sMaf have been expanding and a ChIP-seq (chromatin immunoprecipitation- sequencing) has identified several classes of Nrf2-target genes, including detoxifying enzyme genes, antioxidative stress genes, glutathione biosynthesis enzymes, ABC transporters and pentose phosphate pathway enzymes [13-17]. In fact, Nrf2-sMaf induces expression of the genes involved in detoxification such as Nqo1 (NAD(P)H: quinone oxidoreductase 1), Gsts 3

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(glutathione S-transferases), Gclc (glutamate-cysteine ligase catalytic subunit) and Gclm (glutamate-cysteine ligase modifier subunit) , ROS elimination such as Hmox1 (heme oxygenase 1) and Prdx1 (Peroxyredoxin 1). In addition, ABC transporters known as Mrp2

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(multi-drug resistance-associated proteins 2) and Mrp4 are Nrf2 target genes.

Physiological significance of cytoprotective Nrf2 functions has been demonstrated by utilizing various Nrf2 knockout and Keap1 knockdown lines of animals. While Nrf2 knockout

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mice grow normally under unstressed conditions, they were fragile to oxidative and xenobiotic stresses [1]. We recently generated Nrf2 knockout rats using genome-editing technology [18].

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Nrf2 knockout rats exhibit sensitivity to aflatoxin B1-induced toxicity. As rats have a set of detoxifying enzymes that mimic the detoxication metabolism of humans, Nrf2 knockout rats are suitable surrogates for human in toxicology.

In contrast, Keap1 knockdown mice in which Nrf2 is constitutively activated are more resistant to toxicity of xenobiotics (e.g., acetaminophen) than wild-type mice are [19,20].

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Mice harboring a straightforward Keap1 knockout allele homozygously die at weaning stage of malnutrition derived from hyperkeratosis of upper digestive tract [21]. However, this adverse effect has elicited by severe Nrf2 overexpression caused by genetic modifications, and

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available lines of evidence have shown that mild and transient activation of Nrf2 by chemical

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Nrf2 inducers have beneficial effects rather than harmful effect.

Keap1 serves as a stress-sensor and regulates Nrf2 protein stability Keap1 regulates Nrf2 activity by serving as an adaptor protein connecting Nrf2 and Cullin3 -based E3 ubiquitin ligase. Keap1 contains three functional domains: N-terminal BTB (Broad complex, Tramtrack and Bric-a-brac) domain, IVR (intervening region), and DC domains that consist of a DGR (double glycine repeat) domain and a CTR (carboxyl-terminal region) [2] (Figure 2A). The BTB domain contributes to homo-dimerization of Keap1, and two molecules 4

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of Keap1 actually capture one Nrf2 molecule through the two DC domains. Keap1 possesses multiple cysteine (Cys) residues that act as sensors for various environmental stresses. These Cys residues contain thiol (-SH) that is highly reactive with

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electrophiles. Various chemicals serving as Nrf2 inducers interact with these Cys residues specifically through their electrophilic nature [22] (Figure 2B). Cys151 is particularly important for sensing tBHQ (tert-butylhydroquinone), DEM (diethylmaleate), DMF

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(dimethylfumarate), SFN (sulforaphane), CDDO-Im (2-cyano-3, 12 dioxooleana-1, 9 diene-28-imidazolide) and NO [23,24] (Figure 2B). In contrast, Cys288 is required for sensing

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15d-PGJ2 (15-deoxy-∆12,14-prostaglandin J2) [24]. On the other hand, Cys151, Cys273 and Cys288 are required for recognition of 9-OA-NO2 (9-nitro-octadec-9-enoic acid), 4-HNE (4-hydroxynonenal) and NaAsO2. Intriguingly, PGA2 (prostaglandin A2), CdCl2, ZnCl2, Dex-Mes (dexamethasone 21-mesylate) and H2O2 are independent of Cys151, Cys273 and Cys288, suggesting that the other cysteine residues are required for sensing of these inducers.

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A number of reports show the association of oxidative stress with human diseases, including multiple sclerosis, COPD (chronic obstructive pulmonary disease), type II-diabetes and cancer. Our animal studies showed that Nrf2 activation improved diabetes mellitus [25,26]

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and sickle cell disease [27]. The Keap1-Nrf2 system is an attractive therapeutic target for these diseases. Indeed, DMF (Tecfidera®) has been approved by FDA as a drug for multiple

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sclerosis. Activation of Nrf2 using Nrf2 inducers targeting on multiple Cys residues of Keap1 or Keap1-Nrf2 interacting pockets is effective for the ROS-mediated disease.

Structure-function analyses of Nrf2 domains Structure-function analyses of Nrf2 domains have been conducted extensively. Nrf2 contains six domains referred to as Neh (Nrf2-ECH homology) 1 to 6 domains [2] (Figure 3A). Neh1 domain corresponds to a basic region-leucine zipper (bZIP) structure that allows Nrf2 5

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dimerization with sMaf and binding to DNA. Neh4 and 5 are transactivation domains that bind to CBP (CREB-binding protein) and other transcriptional co-activators [28]. Neh2 and Neh6 domains serve as degrons targeted by Keap1 and β-TrCP, respectively.

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Neh2 domain localizes in the N-terminal region of Nrf2 and responsible for the interaction with Keap1. One molecule of Nrf2 associates with two molecules of Keap1 (Keap1 homodimer) through two binding motifs within Neh2 domain; i.e., low affinity DLGex

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(extended DLG) and high affinity ETGE motifs [2,29] (Figure 3A and B). As lysine residues between the DLGex and ETGE motifs are targeted for ubiquitination, the two-site binding

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seems to be critical for Nrf2 degradation (Two-site substrate binding model for the Keap1-Nrf2 interaction). Upon exposure to oxidative or electrophilic stresses, the low affinity DLGex site dissociates from Keap1 by conformation changes of Keap1 homodimer, thus Nrf2 degradation is stopped [30-32]. As these ETGE and DLGex motifs behave as hinge and latch of doors, respectively, we have referred the sensing mechanism to as the two-site binding

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hinge-and-latch model for the Keap1-Nrf2 system. It turns out that the binding modes of ETGE and DLGex to Keap1 are quite different from each other. Our co-crystallization analyses of ETGE-Keap1 and DLGex-Keap1 revealed that

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DLGex motif exhibits a complicated triple-helix structure upon the co-crystallization [29], whereas ETGE motif possesses a simple β-hairpin conformation [31,33,34]. ETGE binding to

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Keap1 is achieved by a two-step reaction. The first step is characterized as a fast-on and fast-off mode, whereas the second step is characterized by slow-on and slow-off mode. The two-step binding reaction enables a stable binding between ETGE and Keap1. On the other hand, DLGex binding to Keap1 is achieved by a single-step reaction and is characterized as a fast-on and fast-off mode [29]. The difference of binding kinetics between DLGex and ETGE seems to be important to sense electrophilic and oxidative stress. Neh6 domain serves as another degron of Nrf2 that is independent of Keap1. Neh6 domain 6

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contains an interacting interface with β-TrCP, an adaptor protein of Cullin1-based ubiquitin ligase [7,8]. Recognition of Nrf2 by β-TrCP requires phosphorylation by GSK3 of 335th and 338th serine residues within Nrf2 Neh6 domain. The GSK3 activity is regulated by

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phosphorylation by PI3K (phosphatidyl inositol 3 kinase)-AKT signaling. Indeed, several studies have showed that PI3K-AKT signaling pathway activates Nrf2 [17,35]. Of note, Nrf2 activation by PI3K-AKT-GSK3 is observed especially in Keap1-null conditions, indicating

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that β-TrCP-dependent degradation targets Nrf2 that escapes from Keap1-dependent

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degradation [35].

Cancer cell hijacks the Keap1-Nrf2 system

While Nrf2 protects normal cells against oxidative stresses, Nrf2 activation in cancer cells confers resistance against chemo-/radiotherapy and accelerates tumor progression (Figure 4). Somatic mutations in NRF2 and KEAP1 genes, which result in constitutive activation of the

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NRF2 protein, have been identified in human cancer patients [34,36]. The patients bearing NRF2-activated tumors exhibit poor diagnosis [36-38]. Constitutively activated NRF2 induces expression of cytoprotective genes and confers chemo- and radio-resistance for cancer

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cells.

In addition to chemo-/radio-resistance, Nrf2 activates enzyme genes in the pentose

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phosphate pathway and NADPH (nicotinamide adenine dinucleotide phosphate) production, such

as

G6PD

(glucose-6-phosphate

dehydrogenase),

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(phosphogluconate

dehydrogenase), TALDO1 (transaldolase 1), TKT (transketoklase), and ME1 (malic enzyme 1). Furthermore, Nrf2 strongly promotes purine nucleotides synthesis and glutamine consumption, which are advantageous for cancer cell proliferation [17]. Of note, all somatic mutations of NRF2 gene observed in human lung, head and neck, gallbladder cancer cells are found to reside within DLGex and ETGE motifs [8,36,39,40]. This 7

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phenomenon strongly supports our two-site substrate-binding model. Moreover, in hepatocellular carcinoma (HCC), accumulation of phosphorylated p62 is found to induce Nrf2 activation [41,42]. Autophagy chaperone protein p62 has Keap1-interacting region (KIR;

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amino acids 346-358) that harbors STGE motif, which will become similar upon serine phosphorylation (pSTGE), to the ETGE motif of Nrf2. Indeed, it has been shown that phosphorylation of 351st serine residue of p62 confers high affinity for p62 upon binding to

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Keap1 and pSTGE show β-hairpin structure similar to ETGE upon co-crystallization with the Keap1 DC domain.

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An intriguing observation is that the phosphorylated p62 inhibits interaction of KEAP1 and NRF2, resulting in constitutive NRF2 activation [42,43]. Thus, in addition to the somatic mutations NRF2 activation can be brought to cancer cells by the accumulation of “disrupter” proteins. In addition, covalent modification of KEAP1 by fumarate, one of the oncometabolites [44,45], provokes inactivation of KEAP1 and accumulation of NRF2 through

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cysteine residue modification. Furthermore, transcriptional upregulation of NRF2 gene by K-Ras signaling [46] and transcriptional down-regulation of KEAP1 by DNA methylation of

(Figure 4).

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the KEAP1 promoter [47] have also been identified, which eventually elicit NRF2 activation

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For therapies against chemo-/radio-resistant cancers with Nrf2 constitutive activation, Nrf2 inhibitors seem to be effective. In fact, a chemical compound Brusatol was found to serve as an Nrf2 inhibitor [48]. Brusatol decreases NRF2 protein levels as well as other short half-life proteins by suppressing protein synthesis [49]. Brusatol enhances chemo-sensitivity of lung cancer cell line. Recently we identified a chemical compound K67 that binds to the pocket of KEAP1 DC domain [50]. While K67 disturbs the interaction between KEAP1 and p62, K67 maintains the interaction between KEAP1 and NRF2. Therefore, K67 promotes NRF2 degradation by inhibiting interference by p62 in HCC cells. Importantly, treatment of 8

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HCC cells with K67 enhances chemo-sensitivity.

Nrf2 promotes immune system against cancer by suppressing MDSC

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Chronic inflammation promotes onset and development of tumors. Myeloid-derived suppressor cells (MDSC) are a heterogeneous myeloid population containing macrophages, dendritic cells and neutrophils [51]. MDSC supports tumor development and metastasis by

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inhibiting innate and adaptive immunity. MDSC are accumulated in peripheral blood of cancer patients and tumor-bearing mouse models. ROS is an important activator of

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immunosuppression by MDSC.

It should be noted that Nrf2 activation in myeloid cells inhibits the immunosuppressive function of MDSC (Figure 4). Germline and myeloid cell-specific Nrf2-deficient mice exhibit an increased susceptibility to pulmonary metastasis of the mouse Lewis lung carcinoma cells [52,53]. The Nrf2-null MDSC in tumor-bearing mice showed a high ROS accumulation,

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supporting the notion that Nrf2 represses the immunosuppressive function of MDSC by reducing the ROS levels [53].

In addition, we recently found that Nrf2 suppresses expression of proinflammatory cytokine

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genes, such as IL-6 and IL-1β, in a ROS-independent manner in myeloid cells [54]. As these proinflammatory cytokines play a critical role in sustaining the MDSC function, Nrf2 might

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suppress MDSC not only by ROS elimination but also by suppressing proinflammatory cytokines. Administration of an Nrf2-inducer CDDO-Im suppresses cancer cell metastasis in normal mice [53], indicating that Nrf2 activation in host myeloid cells has a beneficial effect on cancer therapies.

As mentioned above, constitutive activation of Nrf2 in cancer cells gives rise to advantages, such as chemo- and radiotherapy resistance and proliferation. On the other hand, Nrf2 activation in host promotes defense against cancer cells [55]. Thus, Nrf2 behaves as a 9

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double-edged sword in cancer therapies: we should consider Nrf2 activation levels both in cancer cells and host environment including MDSC.

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Concluding remarks

The Keap1-Nrf2 system is an attractive therapeutic target for diseases, including airway disorders, cancer and type II diabetes mellitus. Especially, previous works to delineate

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structural features of Keap1 and Nrf2 offer us further insight into drug discovery. Physiologically, Nrf2 is a cytoprotective molecule that buffers oxidative stress, detoxifies

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various xenobiotics, and contributes as a disease preventing or therapeutic agent. Therefore, activation of Nrf2 using inducers is effective for treatment of ROS-mediated diseases and cancer prevention. In contrast, in a part of cancer, Keap1-Nrf2 system is “hijacked” and the cancer cells acquire drug resistance and active proliferative capacity. Against the malignant tumor, Nrf2 inhibitors based on Keap1 independent mechanisms are required. Further studies

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the future of therapies.

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of novel Nrf2 function as well as anti-oxidative stress and detoxification will be important for

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Funding This work was supported in part by AMED-P-CREATE and AMED-CREST from Japan Agency for Medical and Development (to MY), JSPS (KAKENHI 26461395 to MS), the

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NAITO Foundation, Mitsubishi Life Science Foundation, and the Takeda Science Foundation

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(to MY). AO was a JSPS Research Fellow.

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Figure legends Figure 1. Schematic overview of the Keap1-Nrf2 pathway Under unstressed condition (left), Nrf2 is captured by Keap1 and constantly degraded through

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the ubiquitin (Ub)-proteasome pathway in cytosol. When cells are exposed to ROS or electrophiles (right), the negative regulation by Keap1 is stopped. Newly synthesized Nrf2 translocates into the nucleus and forms a heterodimer with sMaf to activate antioxidant and

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detoxification genes. It is noteworthy that the cysteine (Cys) residues of Keap1 act as a sensor for environmental stresses. In addition, the GSK-3 catalyzes phosphorylation of Nrf2 leading

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to recruitment of β-TrCP by which Nrf2 proteins are degraded in nucleus.

Figure 2. Domain structure of Keap1

(A) Domain structure of Keap1. Keap1 contains BTB, IVR, and DC (DGR plus CTR) domains. BTB domain is involved in homo-dimerization, while DC domain interacts with

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Nrf2. The 151st, 273rd and 288th cysteine residues are highly reactive residues, which are known as targets of several electrophiles that act as Nrf2 inducers. (B) Keap1 cysteine residues responding multiple drugs. Cys151 is important for sensing tBHQ, DEM, DMF, SFN,

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CDDO-Im and NO. Cys288 is required for sensing 15d-PGJ2. Cys151, Cys273 and Cys288 are required for recognition of 9-OA-NO2, 4-HNE and NaAsO2. PGA2, CdCl2, ZnCl2,

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Dex-Mes and H2O2 are independent of Cys151, Cys273 and Cys288.

Figure 3. Domain structure of Nrf2 (A) Domain structure of Nrf2. Nrf2 is composed of six functional domains referred to as Neh1 to Neh6. Neh2 domain containing DLGex and ETGE domains is responsible for Keap1 binding, which leads Nrf2 proteins to proteasomal degradation. Neh6 interacts with β-TrCP and acts as a secondary degron. Neh4 and Neh5 are transactivating domains that interact with 16

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several transcriptional co-activators. Neh1 binds to sMaf, involving to DNA recognition. (B) Binding model of Nrf2 ETGE and DLGex motifs. ETGE binding to Keap1 is achieved by a two-step reaction, which enables a stable binding between ETGE and Keap1. DLGex binding

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to Keap1 is achieved by a single-step reaction and is characterized as a fast-on and fast-off mode.

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Figure 4. Keap1-Nrf2 pathway and cancer

In cancer cells, Nrf2 is often constitutively activated due to somatic mutations of Keap1 and

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Nrf2 genes, accumulation of Keap1 disruptors such as p62 and fumarate, oncogene-induced Nrf2 transcription and suppression of Keap1 gene by DNA methylation. Constitutively activated Nrf2 induces its target genes, which confers chemo- and radiotherapy resistance of to cancer cells. On the other hand, Nrf2 activation suppresses MDSC and represses cancer cell

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progression by reducing ROS and proinflammatory cytokines.

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Highlights The Keap1-Nrf2 pathway is a cellular defense system against oxidative stresses.

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Nrf2 is a key transcription factor that activates a set of cytoprotective genes.

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Keap1 regulates Nrf2 activity in response to oxidative stresses.

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Nrf2 inducers are expected as drugs against oxidative stresses-related diseases.

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Cancer cells hijack the Keap1-Nrf2 system to obtain resistance of therapies.

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