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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Cell. Author manuscript; available in PMC 2012 October 18.
Published in final edited form as:
PMCID: PMC3226726
NIHMSID: NIHMS331566

Succination of Keap1 and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type-2

Abstract

Fumarate hydratase (FH) is a tumor suppressor, but how it acts is unclear. Two reports in this issue of Cancer Cell reveal that FH-deficiency leads to succination of Keap1, stabilization of Nrf2, and induction of stress-response genes including HMOX1, which is important for the survival of FH-deficient cells.

The fumarate hydratase gene (FH) encodes a TCA cycle enzyme and functions as a tumor suppressor gene. Heterozygous germline FH mutations result in hereditary leiomyomatosis and renal cell cancer (HLRCC), a syndrome characterized by smooth muscle tumors and papillary renal cell carcinoma-2 (pRCC-2) (Tomlinson et al., 2002). In tumors, the wild-type FH allele is lost and FH function is abrogated. pRCC-2 tumors in HLRCC patients tend to metastasize early and currently there is no therapy.

How FH suppresses tumor formation is unknown. FH loss causes fumarate accumulation in tumor cells and fumarate is a competitive inhibitor of 2-oxoglutarate-dependent prolyl hydroxylase domain-containing (PHD) proteins that hydroxylate HIFα. When hydroxylated, HIFα is recognized by the pVHL E3 ubiquitin ligase complex and is degraded. Because VHL is frequently mutated in renal cancer leading to HIF stabilization, a model whereby HIF is upregulated in pRCC-2 as a consequence of PHD inhibition by fumarate is attractive. However, the importance of PHD inhibition and HIF stabilization in the development of FH-deficient tumors remains unknown.

Keap1 is the substrate recognition subunit of a Cul3-based E3 ubiquitin ligase complex that regulates Nrf2, a pivotal transcription factor in the antioxidant response. Keap1 proteins dimerize through an N-terminal BTB domain, which through an intervening region (IVR) is linked to a C-terminal DC domain that contains a β-propeller made up largely of kelch repeats (Figure 1A). Two β-propellers in a Keap1 dimer interact with an Nrf2 monomer. Nrf2 contains two different Keap1 interacting motifs. Binding through both motifs is required for Nrf2 degradation, which led to a ‘hinge and latch’ model (Hayes et al., 2010; Taguchi et al., 2011). Under normal conditions, Keap1 promotes Nrf2 ubiquitylation and proteosomal-mediated degradation. However, in the presence of electrophiles or reactive oxygen species, Keap1 is modified at several reactive Cys residues resulting in Nrf2 stabilization and the activation of a protective gene expression program that includes HMOX1, an archetypal stress response gene (Hayes et al., 2010).

Figure 1
A proposed update of the Keap1 ‘Hinge and Latch’ model of Nrf2 degradation

In this issue of Cancer Cell, Ooi et al., and Adam et al., show that FH loss results in Keap1 inactivation and Nrf2-dependent activation of antioxidant pathways (Adam et al., 2011; Ooi et al., 2011). Through gene expression analyses, both groups discovered that FH deficiency was associated with increased expression of antioxidant genes and this was accompanied by the accumulation of Nrf2. Reconstitution of FH-deficient cells with wild-type FH or an extramitochondrial FH decreased fumarate levels and restored Nrf2 regulation (Adam et al., 2011; Ooi et al., 2011). Complementarily, a membrane permeable fumarate ester was sufficient to induce Nrf2 (Ooi et al., 2011). Thus, Nrf2 regulation appeared to be linked to fumarate levels.

Pollard and colleagues had previously shown that in FH-deficient tumor cells fumarate spontaneously reacts with Cys thiol groups in proteins to form S-(2-succinyl) Cys (2SC) adducts (Bardella et al., 2011) and both groups asked whether Keap1 was succinated. Using an anti-2SC antibody, ectopically expressed Keap1 was found to be succinated in FH-deficient, but not wild-type cells (Adam et al., 2011). MS/MS analyses revealed succination at several Cys previously shown to be electrophile targets including Cys151 and Cys288 (Adam et al., 2011; Ooi et al., 2011).

How Cys151 and Cys288 modification affects Keap1 remains an unresolved question (Hayes et al., 2010). Notably, while Ser substitution of Cys288 abolishes Keap1 function and stabilizes Nrf2, Ser substitution of Cys151 does not affect Nrf2 degradation. However, substitution of Cys151 by residues with progressively bulkier side chains causes progressive loss of Keap1 function suggesting that the effect at this site is steric (Hayes et al., 2010).

Placing Cys151 and Cys288 residues in a structural context should help unravel their role. Our analysis of two Keap1 related structures (Zhuang et al., 2009; see also Figure 1 legend) suggests that the IVR rotates with respect to the BTB domain giving rise to either a linear or a V-shape dimer (Figure 1A). The rotation may affect the interaction with Cucl3 as it causes a change in the conformation of a Cul3-binding ‘3-box’ within the IVR (Zhuang et al., 2009; see also Figure 1B). Interestingly, Cys151 is at the bending point and we propose that succination induces the linear state and releases Cul3 resulting in Nrf2 stabilization (Figure 1C). Although the structural context of Cys288 is less clear, the IVR, where Cys288 is located, closely associated with the DC domain in a low resolution electron microscopy model of Keap1 (Ogura et al., 2010). Interestingly, Cys288 was found to form part of a conserved surface patch that we postulate interacts with the DC domain, and which placed Cys288 in close proximity with Cys613. We propose that these two Cys stabilize the interaction between the IVR and DC domains either through a disulfide bond or metal coordination (together with Glu289 and His246) and that modification of Cys288 (and possibly Cys613) abrogates this interaction (Figure 1A).

Ooi et al., report that Nrf2 target genes are induced also in sporadic pRCC-2. However, in contrast to familial pRCC-2, mutations in FH have not been detected in sporadic pRCC-2, and sporadic pRCC-2 tumors do not accumulate succinated proteins (Bardella et al., 2011). Thus, how antioxidant genes are upregulated in sporadic pRCC-2 remains unclear.

The contribution of antioxidant genes to renal tumorigenesis and HLRCC requires further exploration. There is precedent, however, implicating Keap1 and Nrf2 in tumor development. Somatically acquired loss of function KEAP1 mutations have been found in tumors (Taguchi et al., 2011). Nrf2 is also stabilized in tumors by mutations in the two motifs involved in Keap1 binding that are necessary for its degradation (Taguchi et al., 2011). Consistent with the notion that these two proteins function in concert, mutations in KEAP1 and NRF2 tend not to be observed together in the same tumor (Taguchi et al., 2011).

Understanding the biology of HLRCC-associated pRCC-2 will hopefully lead to new therapies. Since FH is lost in tumor cells and this truncates the TCA cycle, these tumors may be exquisitely dependent on glycolysis for energy generation. As determined by 2-deoxy-2-(18F)fluoro-D-glucose (FDG) positron emission tomography (PET), FH-deficient tumors take up large amounts of glucose (Yamasaki et al., 2011). This may offer an opportunity for therapeutic intervention.

Recently, HMOX1, which is required for heme synthesis and is upregulated in FH-deficient tumors, was proposed as a therapeutic target (Frezza et al., 2011). FH-deficient cells maintain segmental TCA cycle function and NADH generation by using glutamine and disposing of excess carbon through the synthesis of heme and its excretion from the cell as bilirubin (Frezza et al., 2011). Interestingly, inhibition of HMOX1 is synthetic lethal with FH deficiency. The selectivity of this approach in patients remains to be determined however, particularly since HLRCC patients are heterozygous for FH and mutant FH can be incorporated into FH homotetramers markedly reducing FH activity in non-neoplastic cells. Nonetheless, this concept deserves to be studied further in primary tumorgrafts and subsequently in patients.

Footnotes

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