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Succination of Keap1 and Activation of Nrf2-Dependent Antioxidant Pathways in FH-Deficient Papillary Renal Cell Carcinoma Type 2

2011; Cell Press; Volume: 20; Issue: 4 Linguagem: Inglês

10.1016/j.ccr.2011.10.005

1878-3686

Lisa N. Kinch, Nick V. Grishin, James Brugarolas,

Pancreatic and Hepatic Oncology Research

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. 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 type 2 (pRCC-2) (Tomlinson et al., 2002Tomlinson I.P. Alam N.A. Rowan A.J. Barclay E. Jaeger E.E. Kelsell D. Leigh I. Gorman P. Lamlum H. Rahman S. et al.Multiple Leiomyoma ConsortiumNat. Genet. 2002; 30: 406-410Crossref PubMed Scopus (1185) Google Scholar). In tumors, the wild-type FH allele is lost, and FH function is abrogated. pRCC-2 tumors in patients with HLRCC 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 proteins (PHD) 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., 2010Hayes J.D. McMahon M. Chowdhry S. Dinkova-Kostova A.T. Antioxid. Redox Signal. 2010; 13: 1713-1748Crossref PubMed Scopus (426) Google Scholar, Taguchi et al., 2011Taguchi K. Motohashi H. Yamamoto M. Genes Cells. 2011; 16: 123-140Crossref PubMed Scopus (1073) Google Scholar). 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., 2010Hayes J.D. McMahon M. Chowdhry S. Dinkova-Kostova A.T. Antioxid. Redox Signal. 2010; 13: 1713-1748Crossref PubMed Scopus (426) Google Scholar). In this issue of Cancer Cell, Ooi et al., 2011Ooi A. Wong J.-C. Petillo D. Roossien D. Perrier-Trudova V. Whitten D. Wong Hui Min B. Tan M.-H. Zhang Z. Yang X.J. et al.Cancer Cell. 2011; 20 (this issue): 511-523Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar and Adam et al., 2011Adam J. Hatipoglu E. O'Flaherty L. Ternette N. Sahgal N. Lockstone H. Baban D. Nye E. Stamp G.W. Wolhuter K. et al.Cancer Cell. 2011; 20 (this issue): 524-537Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar show that FH loss results in Keap1 inactivation and Nrf2-dependent activation of antioxidant pathways. 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., 2011Adam J. Hatipoglu E. O'Flaherty L. Ternette N. Sahgal N. Lockstone H. Baban D. Nye E. Stamp G.W. Wolhuter K. et al.Cancer Cell. 2011; 20 (this issue): 524-537Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, Ooi et al., 2011Ooi A. Wong J.-C. Petillo D. Roossien D. Perrier-Trudova V. Whitten D. Wong Hui Min B. Tan M.-H. Zhang Z. Yang X.J. et al.Cancer Cell. 2011; 20 (this issue): 511-523Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Complementarily, a membrane-permeable fumarate ester was sufficient to induce Nrf2 (Ooi et al., 2011Ooi A. Wong J.-C. Petillo D. Roossien D. Perrier-Trudova V. Whitten D. Wong Hui Min B. Tan M.-H. Zhang Z. Yang X.J. et al.Cancer Cell. 2011; 20 (this issue): 511-523Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Thus, Nrf2 regulation appeared to be linked to fumarate levels. Pollard and colleagues (Bardella et al., 2011Bardella C. El-Bahrawy M. Frizzell N. Adam J. Ternette N. Hatipoglu E. Howarth K. O'Flaherty L. Roberts I. Turner G. et al.J. Pathol. 2011; 225: 4-11Crossref PubMed Scopus (186) Google Scholar) 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, 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., 2011Adam J. Hatipoglu E. O'Flaherty L. Ternette N. Sahgal N. Lockstone H. Baban D. Nye E. Stamp G.W. Wolhuter K. et al.Cancer Cell. 2011; 20 (this issue): 524-537Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). MS/MS analyses revealed succination at several Cys previously shown to be electrophile targets, including Cys151 and Cys288 (Adam et al., 2011Adam J. Hatipoglu E. O'Flaherty L. Ternette N. Sahgal N. Lockstone H. Baban D. Nye E. Stamp G.W. Wolhuter K. et al.Cancer Cell. 2011; 20 (this issue): 524-537Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, Ooi et al., 2011Ooi A. Wong J.-C. Petillo D. Roossien D. Perrier-Trudova V. Whitten D. Wong Hui Min B. Tan M.-H. Zhang Z. Yang X.J. et al.Cancer Cell. 2011; 20 (this issue): 511-523Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). How Cys151 and Cys288 modification affects Keap1 remains an unresolved question (Hayes et al., 2010Hayes J.D. McMahon M. Chowdhry S. Dinkova-Kostova A.T. Antioxid. Redox Signal. 2010; 13: 1713-1748Crossref PubMed Scopus (426) Google Scholar). Notably, whereas Ser substitution of Cys288 abolishes Keap1 function and stabilizes Nrf2, Ser substitution of Cys151 does not affect Nrf2 degradation. However, substitution of Cys151 by amino acids with progressively bulkier side chains causes progressive loss of Keap1 function, suggesting that the effect at this site is steric (Hayes et al., 2010Hayes J.D. McMahon M. Chowdhry S. Dinkova-Kostova A.T. Antioxid. Redox Signal. 2010; 13: 1713-1748Crossref PubMed Scopus (426) Google Scholar). Placing Cys151 and Cys288 residues in a structural context should help unravel their role. Our analysis of two Keap1-related structures (Zhuang et al., 2009Zhuang M. Calabrese M.F. Liu J. Waddell M.B. Nourse A. Hammel M. Miller D.J. Walden H. Duda D.M. Seyedin S.N. et al.Mol. Cell. 2009; 36: 39-50Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar; 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 Cul3 because it causes a change in the conformation of a Cul3-binding "3-box" within the IVR (Zhuang et al., 2009Zhuang M. Calabrese M.F. Liu J. Waddell M.B. Nourse A. Hammel M. Miller D.J. Walden H. Duda D.M. Seyedin S.N. et al.Mol. Cell. 2009; 36: 39-50Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar) (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., 2010Ogura T. Tong K.I. Mio K. Maruyama Y. Kurokawa H. Sato C. Yamamoto M. Proc. Natl. Acad. Sci. USA. 2010; 107: 2842-2847Crossref PubMed Scopus (165) Google Scholar). Interestingly, Cys288 was found to form part of a conserved surface patch that we postulate interacts with the DC domain and that 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 Cys613) abrogates this interaction (Figure 1A). Ooi et al., 2011Ooi A. Wong J.-C. Petillo D. Roossien D. Perrier-Trudova V. Whitten D. Wong Hui Min B. Tan M.-H. Zhang Z. Yang X.J. et al.Cancer Cell. 2011; 20 (this issue): 511-523Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar 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., 2011Bardella C. El-Bahrawy M. Frizzell N. Adam J. Ternette N. Hatipoglu E. Howarth K. O'Flaherty L. Roberts I. Turner G. et al.J. Pathol. 2011; 225: 4-11Crossref PubMed Scopus (186) Google Scholar). Thus, how antioxidant genes are upregulated in sporadic pRCC-2 remains unclear. The contribution of antioxidant genes to renal tumorigenesis in patients with HLRCC requires further exploration. However, there is precedent implicating Keap1 and Nrf2 in tumor development. Somatically acquired loss-of-function KEAP1 mutations have been found in tumors (Taguchi et al., 2011Taguchi K. Motohashi H. Yamamoto M. Genes Cells. 2011; 16: 123-140Crossref PubMed Scopus (1073) Google Scholar). Nrf2 is also stabilized in tumors by mutation in either of the two motifs involved in Keap1 binding that are necessary for its degradation (Taguchi et al., 2011Taguchi K. Motohashi H. Yamamoto M. Genes Cells. 2011; 16: 123-140Crossref PubMed Scopus (1073) Google Scholar). 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., 2011Taguchi K. Motohashi H. Yamamoto M. Genes Cells. 2011; 16: 123-140Crossref PubMed Scopus (1073) Google Scholar). Understanding the biology of HLRCC-associated pRCC-2 will hopefully lead to new therapies. Because 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., 2011Yamasaki T. Tran T.A. Oz O.K. Raj G.V. Schwarz R.E. Deberardinis R.J. Zhang X. Brugarolas J. Nat. Rev. Urol. 2011; 8: 165-171Crossref PubMed Scopus (37) Google Scholar). Although exploiting this dependency may be challenging (Yamasaki et al., 2011Yamasaki T. Tran T.A. Oz O.K. Raj G.V. Schwarz R.E. Deberardinis R.J. Zhang X. Brugarolas J. Nat. Rev. Urol. 2011; 8: 165-171Crossref PubMed Scopus (37) Google Scholar), 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., 2011Frezza C. Zheng L. Folger O. Rajagopalan K.N. MacKenzie E.D. Jerby L. Micaroni M. Chaneton B. Adam J. Hedley A. et al.Nature. 2011; 477: 225-228Crossref PubMed Scopus (370) Google Scholar). 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., 2011Frezza C. Zheng L. Folger O. Rajagopalan K.N. MacKenzie E.D. Jerby L. Micaroni M. Chaneton B. Adam J. Hedley A. et al.Nature. 2011; 477: 225-228Crossref PubMed Scopus (370) Google Scholar). Interestingly, inhibition of HMOX1 is synthetically lethal with FH deficiency. However, the selectivity of this approach in patients remains to be determined, particularly because HLRCC patients are heterozygous for FH, and mutant FH may be incorporated into FH homotetramers, markedly reducing FH activity in non-neoplastic cells. Nonetheless, this concept deserves to be studied further in primary xenografts and, subsequently, in patients. An Antioxidant Response Phenotype Shared between Hereditary and Sporadic Type 2 Papillary Renal Cell CarcinomaOoi et al.Cancer CellOctober 18, 2011In BriefFumarate hydratase (FH) mutation causes hereditary type 2 papillary renal cell carcinoma (PRCC2). The main effect of FH mutation is fumarate accumulation. The current paradigm posits that the main consequence of fumarate accumulation is HIF-α stabilization. Paradoxically, FH mutation differs from other HIF-α stabilizing mutations, such as VHL and SDH mutations, in its associated tumor types. We identified that fumarate can directly up-regulate antioxidant response element (ARE)–controlled genes. Full-Text PDF Open ArchiveRenal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 SignalingAdam et al.Cancer CellOctober 18, 2011In BriefThe Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Full-Text PDF Open Access