Original Article | Published:

RSUME inhibits VHL and regulates its tumor suppressor function

Oncogene volume 34, pages 48554866 (10 September 2015) | Download Citation

Abstract

Somatic mutations or loss of von Hippel–Lindau (pVHL) happen in the majority of VHL disease tumors, which present a constitutively active Hypoxia Inducible Factor (HIF), essential for tumor growth. Recently described mechanisms for pVHL modulation shed light on the open question of the HIF/pVHL pathway regulation. The aim of the present study was to determine the molecular mechanism by which RSUME stabilizes HIFs, by studying RSUME effect on pVHL function and to determine the role of RSUME on pVHL-related tumor progression. We determined that RSUME sumoylates and physically interacts with pVHL and negatively regulates the assembly of the complex between pVHL, Elongins and Cullins (ECV), inhibiting HIF-1 and 2α ubiquitination and degradation. We found that RSUME is expressed in human VHL tumors (renal clear-cell carcinoma (RCC), pheochromocytoma and hemangioblastoma) and by overexpressing or silencing RSUME in a pVHL-HIF-oxygen-dependent degradation stability reporter assay, we determined that RSUME is necessary for the loss of function of type 2 pVHL mutants. The functional RSUME/pVHL interaction in VHL-related tumor progression was further confirmed using a xenograft assay in nude mice. RCC clones, in which RSUME was knocked down and express either pVHL wt or type 2 mutation, have an impaired tumor growth, as well as HIF-2α, vascular endothelial growth factor A and tumor vascularization diminution. This work shows a novel mechanism for VHL tumor progression and presents a new mechanism and factor for targeting tumor-related pathologies with pVHL/HIF altered function.

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References

  1. 1.

    , , , , , et al. RSUME, a small RWD-containing protein, enhances SUMO conjugation and stabilizes HIF-1alpha during hypoxia. Cell 2007; 131: 309–323.

  2. 2.

    , . Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol 2007; 8: 947–956.

  3. 3.

    . SUMO: a history of modification. Mol Cell 2005; 18: 1–12.

  4. 4.

    , , , , , et al. RSUME enhances glucocorticoid receptor SUMOylation and transcriptional activity. Mol Cell Biol 2013; 33: 2116–2127.

  5. 5.

    , , , , , et al. In silico structural and functional characterization of the RSUME splice variants. PLoS One 2013; 8: e57795.

  6. 6.

    , , , , , et al. Concurrent gene signatures for han chinese breast cancers. PLoS ONE 2013; 8: e76421.

  7. 7.

    , , , , , et al. Genetic associations with taxane-induced neuropathy by a genome-wide association study (GWAS) in E5103. J Clin Oncol 2011; 29: Supplementary Abstract 1000.

  8. 8.

    , , , , , et al. GWAS-based association between RWDD3 and TECTA variants and paclitaxel induced neuropathy could not be confirmed in Scandinavian ovarian cancer patients. Acta Oncol 2013; 52: 871–874.

  9. 9.

    , , , , . Expression profiling of genes modulated by minocycline in a rat model of neuropathic pain. Mol Pain 2014; 10: 47.

  10. 10.

    , , , , , . Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision. Mol Cell Neurosci 2008; 37: 845–856.

  11. 11.

    . Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 1999; 15: 551–578.

  12. 12.

    , . HIF at a glance. J Cell Sci 2009; 122: 1055–1057.

  13. 13.

    , . Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 2008; 30: 393–402.

  14. 14.

    , , . HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nat Rev Cancer 2012; 12: 9–22.

  15. 15.

    , , , , . Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit. J Biol Chem 1997; 272: 11205–11214.

  16. 16.

    , . The silencing approach of the hypoxia-signaling pathway. Methods Enzymol 2007; 435: 107–121.

  17. 17.

    , , , . The updated biology of hypoxia-inducible factor. EMBO J 2012; 31: 2448–2460.

  18. 18.

    . Molecular basis of the VHL hereditary cancer syndrome. Nat Rev Cancer 2002; 2: 673–682.

  19. 19.

    , , , . HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci USA 2001; 98: 9630–9635.

  20. 20.

    , . Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol 2009; 49: 73–96.

  21. 21.

    . Oxygen sensing, homeostasis, and disease. N Engl J Med 2011; 365: 537–547.

  22. 22.

    , . Deciphering the emerging role of SUMO conjugation in the hypoxia-signaling cascade. Biol Chem 2013; 394: 459–469.

  23. 23.

    , . Role of VHL gene mutation in human cancer. J Clin Oncol 2004; 22: 4991–5004.

  24. 24.

    , . von Hippel-Lindau disease. Medicine (Baltimore) 1997; 76: 381–391.

  25. 25.

    , , . The molecular biology of renal cell carcinoma. Semin Oncol 2013; 40: 421–428.

  26. 26.

    , , , , , et al. State of the science: an update on renal cell carcinoma. Mol Cancer Res 2012; 10: 859–880.

  27. 27.

    , , , , , et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol 2000; 2: 423–427.

  28. 28.

    . The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat Rev Cancer 2008; 8: 865–873.

  29. 29.

    , , , , , et al. Sumoylation increases HIF-1alpha stability and its transcriptional activity. Biochem Biophys Res Commun 2004; 324: 394–400.

  30. 30.

    , , , . SUMO-specific protease 1 is essential for stabilization of HIF1alpha during hypoxia. Cell 2007; 131: 584–595.

  31. 31.

    , , , , , et al. Failure to prolyl hydroxylate hypoxia-inducible factor alpha phenocopies VHL inactivation in vivo. EMBO J 2006; 25: 4650–4662.

  32. 32.

    , , , , . Hypoxia inactivates the VHL tumor suppressor through PIASy-mediated SUMO modification. PLoS ONE 2010; 5: e9720.

  33. 33.

    , , , , , et al. PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells. Br J Cancer 2013; 109: 1795–1804.

  34. 34.

    , , , , , . In vitro and in vivo models analyzing von Hippel-Lindau disease-specific mutations. Cancer Res 2004; 64: 8595–8603.

  35. 35.

    , , , , , . VHL gene mutations and their effects on hypoxia inducible factor HIFalpha: identification of potential driver and passenger mutations. Cancer Res 2011; 71: 5500–5511.

  36. 36.

    , , . VHL type 2B mutations retain VBC complex form and function. PLoS ONE 2008; 3: e3801.

  37. 37.

    , , , , , et al. Endothelial HIF-2alpha regulates murine pathological angiogenesis and revascularization processes. J Clin Invest 2012; 122: 1427–1443.

  38. 38.

    , , , , , et al. PIASy stimulates HIF1alpha SUMOylation and negatively regulates HIF1alpha activity in response to hypoxia. Oncogene 2010; 29: 5568–5578.

  39. 39.

    , , , . RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2alpha. Nucleic Acids Res 2010; 38: 1922–1931.

  40. 40.

    , , , , . Increased protein SUMOylation following focal cerebral ischemia. Neuropharmacology 2008; 54: 280–289.

  41. 41.

    , , , . Transient focal cerebral ischemia induces a dramatic activation of small ubiquitin-like modifier conjugation. J Cereb Blood Flow Metab 2008; 28: 892–896.

  42. 42.

    , , , , , et al. SENP3 is responsible for HIF-1 transactivation under mild oxidative stress via p300 de-SUMOylation. EMBO J 2009; 28: 2748–2762.

  43. 43.

    , , , , , et al. RSUME is implicated in HIF-1-induced VEGF-A production in pituitary tumour cells. Endocr Relat Cancer 2012; 19: 13–27.

  44. 44.

    , , , , , et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012; 2: 401–404.

  45. 45.

    , , , , , et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 2013; 6: pl1.

  46. 46.

    Cancer Genome Atlas Research Network. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature 2013; 499: 43–49.

  47. 47.

    , , , , , . von Hippel-Lindau protein mutants linked to type 2C VHL disease preserve the ability to downregulate HIF. Hum Mol Genet 2001; 10: 1019–1027.

  48. 48.

    , , , . Renal cell carcinoma risk in type 2 von Hippel-Lindau disease correlates with defects in pVHL stability and HIF-1alpha interactions. Oncogene 2006; 25: 370–377.

  49. 49.

    . HIF-1 mediates metabolic responses to intratumoral hypoxia and oncogenic mutations. J Clin Invest 2013; 123: 3664–3671.

  50. 50.

    , , , , . VHL mutations linked to type 2C von Hippel-Lindau disease cause extensive structural perturbations in pVHL. J Biol Chem 2009; 284: 10514–10522.

  51. 51.

    , . Recent updates in renal cell carcinoma. Curr Opin Oncol 2010; 22: 250–256.

  52. 52.

    , . VHL gene mutations in renal cell carcinoma: role as a biomarker of disease outcome and drug efficacy. Curr Oncol Rep 2009; 11: 94–101.

  53. 53.

    , , , , , et al. ClearCode34: A prognostic risk predictor for localized clear cell renal cell carcinoma. Eur Urol 2014; 66: 77–84.

  54. 54.

    , , , , , et al. Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene. Proc Natl Acad Sci USA 1996; 93: 10589–10594.

  55. 55.

    , , , . Tumour suppression by the human von Hippel-Lindau gene product. Nat Med 1995; 1: 822–826.

  56. 56.

    , , , . Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL-/- tumors. Mol Cancer Res 2004; 2: 89–95.

  57. 57.

    , , , , , . The contribution of VHL substrate binding and HIF1-alpha to the phenotype of VHL loss in renal cell carcinoma. Cancer Cell 2002; 1: 247–255.

  58. 58.

    , , . Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. J Biol Chem 2000; 275: 26765–26771.

  59. 59.

    , , , , , et al. Phosphorylation of c-Fos by members of the p38 MAPK family. Role in the AP-1 response to UV light. J Biol Chem 2005; 280: 18842–18852.

  60. 60.

    , . Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein. Mol Cell Biol 2002; 22: 5319–5336.

  61. 61.

    , , , , , et al. Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production. Proc Natl Acad Sci USA 2006; 103: 105–110.

  62. 62.

    , , , , . Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 2002; 1: 237–246.

  63. 63.

    , . Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells. Gene Ther 2001; 8: 1801–1807.

  64. 64.

    , , . Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products. Proc Natl Acad Sci USA 2000; 97: 8507–8512.

  65. 65.

    , , . SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol Cell 1998; 2: 233–239.

  66. 66.

    , , , , , et al. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J Biol Chem 2001; 276: 35368–35374.

  67. 67.

    , , . SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J Biol Chem 2001; 276: 12654–12659.

  68. 68.

    , , , , , et al. Combining the receptor tyrosine kinase inhibitor AEE788 and the mammalian target of rapamycin (mTOR) inhibitor RAD001 strongly inhibits adhesion and growth of renal cell carcinoma cells. BMC Cancer 2009; 9: 161.

  69. 69.

    , , , , , et al. Tumor suppression by the von Hippel-Lindau protein requires phosphorylation of the acidic domain. J Biol Chem 2005; 280: 22205–22211.

  70. 70.

    , , , , , et al. Reduced expression of the cytokine transducer gp130 inhibits hormone secretion, cell growth, and tumor development of pituitary lactosomatotrophic GH3 cells. Endocrinology 2003; 144: 693–700.

  71. 71.

    , , , , , et al. Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk. Proc Natl Acad Sci USA 2003; 100: 1034–1039.

  72. 72.

    , , , . Sequence determinants in hypoxia-inducible factor-1alpha for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3. J Biol Chem 2002; 277: 39792–39800.

  73. 73.

    , , , , , et al. Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. J Biol Chem 2000; 275: 25733–25741.

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Acknowledgements

We thank Sergio Senin, María Antunica Noguerol, Jimena Druker and Mariana Haedo for technical help. We also thank the Argentinian Instituto Nacional del Cáncer (INC) and Bunge&Born Foundation for financial support to Lucas Tedesco and Juan Jose Bonfiglio, respectively. This work was supported by grants from the Max Planck Society, Germany; the University of Buenos Aires; CONICET; the Agencia Nacional de Promoción Científica y Tecnológica, Argentina and FOCEM-Mercosur (COF 03/11).

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Author notes

    • J Gerez
    •  & L Tedesco

    These authors equally contributed to this work.

Affiliations

  1. Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)—CONICET—Partner Institute of the Max Planck Society, Buenos Aires, Argentina

    • J Gerez
    • , L Tedesco
    • , J J Bonfiglio
    • , M Fuertes
    • , S Silberstein
    •  & E Arzt
  2. Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

    • J Gerez
    • , J J Bonfiglio
    • , S Silberstein
    •  & E Arzt
  3. Center for Endocrinological Investigations (CEDIE), Hospital de Niños R. Gutiérrez, Buenos Aires, Argentina

    • M Barontini
  4. Department of Clinical Research, Max Planck Institute of Psychiatry, Munich, Germany

    • Y Wu
    • , U Renner
    • , M Páez-Pereda
    • , F Holsboer
    •  & G K Stalla

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The authors declare no conflict of interest.

Corresponding author

Correspondence to E Arzt.

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https://doi.org/10.1038/onc.2014.407

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)