Abstract
Tumor hypoxia induces the upregulation of hypoxia-inducible factor 1α (Hif-1α), which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis. Interference with the Hif-1 pathway and neoangiogenesis is an attractive antitumor target. The hydroxylation of Hif-1α by prolyl-hydroxylase (PHD) proteins during normoxia serves as a recognition motif for its proteasomal degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves polyubiquitylated and degraded by Siah ubiquitin ligases. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a fragment derived from a Drosophila protein (phyllopod) interferes with the PHD degradation. Furthermore, cells stably expressing the phyllopod fragment display reduced upregulation of Hif-1α protein levels and Hif-1-mediated gene expression under hypoxia. In a syngeneic mouse model of breast cancer, the phyllopod fragment reduced tumor growth and neoangiogenesis and prolonged survival of the mice. In addition, levels of Hif-1α and its target Glut-1 are reduced in tumors expressing the phyllopod fragment. These data show, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for antitumor therapies.
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References
Bottger A, Bottger V, Garcia-Echeverria C, Chene P, Hochkeppel HK, Sampson W et al. (1997). Molecular characterization of the hdm2-p53 interaction. J Mol Biol 269: 744–756.
Boulton SJ, Brook A, Staehling-Hampton K, Heitzler P, Dyson N . (2000). A role for Ebi in neuronal cell cycle control. EMBO J 19: 5376–5386.
Casey AE, Laster Jr WR, Ross GL . (1951). Sustained enhanced growth of carcinoma EO771 in C57 black mice. Proc Soc Exp Biol Med 77: 358–362.
Chene P, Fuchs J, Bohn J, Garcia-Echeverria C, Furet P, Fabbro D . (2000). A small synthetic peptide, which inhibits the p53-hdm2 interaction, stimulates the p53 pathway in tumour cell lines. J Mol Biol 299: 245–253.
Della NG, Senior PV, Bowtell DD . (1993). Isolation and characterisation of murine homologues of the Drosophila seven in absentia gene (sina). Development 117: 1333–1343.
Depaux A, Regnier-Ricard F, Germani A, Varin-Blank N . (2006). Dimerization of hSiah proteins regulates their stability. Biochem Biophys Res Commun 348: 857–863.
Dickins RA, Frew IJ, House CM, O'Bryan MK, Holloway AJ, Haviv I et al. (2002). The ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice. Mol Cell Biol 22: 2294–2303.
Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR et al. (2001). C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107: 43–54.
Freedman SJ, Sun ZY, Kung AL, France DS, Wagner G, Eck MJ . (2003). Structural basis for negative regulation of hypoxia-inducible factor-1alpha by CITED2. Nat Struct Biol 10: 504–512.
Frew IJ, Hammond VE, Dickins RA, Quinn JM, Walkley CR, Sims NA et al. (2003). Generation and analysis of Siah2 mutant mice. Mol Cell Biol 23: 9150–9161.
Fukuba H, Yamashita H, Nagano Y, Jin HG, Hiji M, Ohtsuki T et al. (2007). Siah-1 facilitates ubiquitination and degradation of factor inhibiting HIF-1alpha (FIH). Biochem Biophys Res Commun 353: 324–329.
Germani A, Prabel A, Mourah S, Podgorniak MP, Di Carlo A, Ehrlich R et al. (2003). SIAH-1 interacts with CtIP and promotes its degradation by the proteasome pathway. Oncogene 22: 8845–8851.
Glickman MH, Ciechanover A . (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82: 373–428.
Hochstrasser M . (2000). Evolution and function of ubiquitin-like protein-conjugation systems. Nat Cell Biol 2: E153–E157.
Holinger EP, Chittenden T, Lutz RJ . (1999). Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases. J Biol Chem 274: 13298–13304.
House CM, Frew IJ, Huang HL, Wiche G, Traficante N, Nice E et al. (2003). A binding motif for Siah ubiquitin ligase. Proc Natl Acad Sci USA 100: 3101–3106.
House CM, Hancock NC, Moller A, Cromer BA, Fedorov V, Bowtell DD et al. (2006). Elucidation of the substrate binding site of Siah ubiquitin ligase. Structure 14: 695–701.
Hu G, Chung YL, Glover T, Valentine V, Look AT, Fearon ER . (1997). Characterization of human homologs of the Drosophila seven in absentia (sina) gene. Genomics 46: 103–111.
Huang LE, Gu J, Schau M, Bunn HF . (1998). Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 95: 7987–7992.
Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, Wenger RH et al. (1998). Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 12: 149–162.
Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ et al. (2001). Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292: 468–472.
Johnsen SA, Subramaniam M, Monroe DG, Janknecht R, Spelsberg TC . (2002). Modulation of transforming growth factor beta (TGFbeta)/Smad transcriptional responses through targeted degradation of TGFbeta-inducible early gene-1 by human seven in absentia homologue. J Biol Chem 277: 30754–30759.
Kane RC, Bross PF, Farrell AT, Pazdur R . (2003). Velcade: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncologist 8: 508–513.
Khurana A, Nakayama K, Williams S, Davis RJ, Mustelin T, Ronai Z . (2006). Regulation of the ring finger E3 ligase Siah2 by p38 MAPK. J Biol Chem 281: 35316–35326.
Lando D, Peet DJ, Gorman JJ, Whelan DA, Whitelaw ML, Bruick RK . (2002). FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev 16: 1466–1471.
Laney JD, Hochstrasser M . (1999). Substrate targeting in the ubiquitin system. Cell 97: 427–430.
Li S, Li Y, Carthew RW, Lai ZC . (1997). Photoreceptor cell differentiation requires regulated proteolysis of the transcriptional repressor Tramtrack. Cell 90: 469–478.
Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME et al. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399: 271–275.
Nakayama K, Frew IJ, Hagensen M, Skals M, Habelhah H, Bhoumik A et al. (2004). Siah2 regulates stability of prolyl-hydroxylases, controls HIF1alpha abundance, and modulates physiological responses to hypoxia. Cell 117: 941–952.
Nakayama K, Gazdoiu S, Abraham R, Pan ZQ, Ronai Z . (2007). Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2. Biochem J 401: 217–226.
Polekhina G, House CM, Traficante N, Mackay JP, Relaix F, Sassoon DA et al. (2002). Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling. Nat Struct Biol 9: 68–75.
Salceda S, Caro J . (1997). Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 272: 22642–22647.
Schofield CJ, Ratcliffe PJ . (2004). Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 5: 343–354.
Semenza GL . (1999). Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 15: 551–578.
Semenza GL . (2000). Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochem Pharmacol 59: 47–53.
Tang AH, Neufeld TP, Kwan E, Rubin GM . (1997). PHYL acts to down-regulate TTK88, a transcriptional repressor of neuronal cell fates, by a SINA-dependent mechanism. Cell 90: 459–467.
Wang JL, Zhang ZJ, Choksi S, Shan S, Lu Z, Croce CM et al. (2000). Cell permeable Bcl-2 binding peptides: a chemical approach to apoptosis induction in tumor cells. Cancer Res 60: 1498–1502.
Wasylyk C, Salvi R, Argentini M, Dureuil C, Delumeau I, Abecassis J et al. (1999). p53 mediated death of cells overexpressing MDM2 by an inhibitor of MDM2 interaction with p53. Oncogene 18: 1921–1934.
Zhang J, Guenther MG, Carthew RW, Lazar MA . (1998). Proteasomal regulation of nuclear receptor corepressor-mediated repression. Genes Dev 12: 1775–1780.
Acknowledgements
The EO771 cells were a kind gift from Dr Robin Anderson, Peter MacCallum Cancer Centre. The expression plasmid for FLAG-TIEG-1 was provided by Steven Johnsen and Thomas Spelsburg (Mayo Clinic and Foundation, Rochester, NY, USA). The expression plasmid for Myc-FIH-1 was a kind gift from Daniel Peet (School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia). This study was funded by grants of the Australian National Health and Medical Research Council (NHMRC) to AM, CH and DB (no. 400321 and no. 509331).
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
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Möller, A., House, C., Wong, C. et al. Inhibition of Siah ubiquitin ligase function. Oncogene 28, 289–296 (2009). https://doi.org/10.1038/onc.2008.382
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DOI: https://doi.org/10.1038/onc.2008.382
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