Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Endothelial deletion of Sag/Rbx2/Roc2 E3 ubiquitin ligase causes embryonic lethality and blocks tumor angiogenesis

Oncogene volume 33, pages 5211–5220 (2014)Cite this article

Subjects

Abstract

SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING protein required for the activity of Cullin–RING ligase (CRL). Our recent study showed that Sag total knockout caused embryonic lethality at E11.5–12.5 days with associated defects in vasculogenesis. Whether Sag is required for de novo vasculogenesis in embryos and angiogenesis in tumors is totally unknown. Here, we report that Sag endothelial deletion also causes embryonic lethality at E15.5 with poor vasculogenesis. Sag deletion in primary endothelial cells (ECs) or knockdown in MS-1 ECs inhibits migration, proliferation and tube formation, with p27 accumulation being responsible for the suppression of migration and proliferation. Furthermore, Sag deletion significantly inhibits angiogenesis in an in vivo Matrigel plug assay, and tumor angiogenesis and tumorigenesis in a B16F10 melanoma model. Finally, MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that inhibits CRL, suppresses in vitro migration, proliferation and tube formation, as well as in vivo angiogenesis and tumorigenesis. Taken together, our study, using both genetic and pharmaceutical approaches, demonstrates that Sag is essential for embryonic vasculogenesis and tumor angiogenesis, and provides the proof-of-concept evidence that targeting Sag E3 ubiquitin ligase may have clinical value for anti-angiogenesis therapy of human cancer.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Deshaies RJ, Joazeiro CA . RING domain E3 ubiquitin ligases. Annu Rev Biochem 2009; 78: 399–434.

    Article  CAS  Google Scholar 

  2. Nakayama KI, Nakayama K . Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer 2006; 6: 369–381.

    Article  CAS  Google Scholar 

  3. Zhao Y, Sun Y . Cullin-RING ligases as attractive anti-cancer targets. Curr Pharm Des 2013; 19: 3215–3225.

    Article  CAS  Google Scholar 

  4. Wu K, Fuchs SY, Chen A, Tan P, Gomez C, Ronai Z et al. The SCF(HOS/beta-TRCP)-ROC1 E3 ubiquitin ligase utilizes two distinct domains within CUL1 for substrate targeting and ubiquitin ligation. Mol Cell Biol 2000; 20: 1382–1393.

    Article  CAS  Google Scholar 

  5. Duda DM, Borg LA, Scott DC, Hunt HW, Hammel M, Schulman BA . Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell 2008; 134: 995–1006.

    Article  CAS  Google Scholar 

  6. Goldenberg SJ, Cascio TC, Shumway SD, Garbutt KC, Liu J, Xiong Y et al. Structure of the Cand1-Cul1-Roc1 complex reveals regulatory mechanisms for the assembly of the multisubunit cullin-dependent ubiquitin ligases. Cell 2004; 119: 517–528.

    Article  CAS  Google Scholar 

  7. Kamura T, Conrad MN, Yan Q, Conaway RC, Conaway JW . The Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1 modification of cullins Cdc53 and Cul2. Genes Dev 1999; 13: 2928–2933.

    Article  CAS  Google Scholar 

  8. Yamoah K, Oashi T, Sarikas A, Gazdoiu S, Osman R, Pan ZQ . Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tail. Proc Natl Acad Sci U S A 2008; 105: 12230–12235.

    Article  CAS  Google Scholar 

  9. Willems AR, Schwab M, Tyers M . A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta 2004; 1695: 133–170.

    Article  CAS  Google Scholar 

  10. Jia L, Sun Y . SCF E3 ubiquitin ligases as anticancer targets. Curr Cancer Drug Targets 2011; 11: 347–356.

    Article  CAS  Google Scholar 

  11. Duan H, Wang Y, Aviram M, Swaroop M, Loo JA, Bian J et al. SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents. Mol Cell Biol 1999; 19: 3145–3155.

    Article  CAS  Google Scholar 

  12. Sun Y, Tan M, Duan H, Swaroop M . SAG/ROC/Rbx/Hrt, a zinc RING finger gene family: molecular cloning, biochemical properties, and biological functions. Antioxid Redox Signal 2001; 3: 635–650.

    Article  CAS  Google Scholar 

  13. Swaroop M, Wang Y, Miller P, Duan H, Jatkoe T, Madore SJ et al. Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germinatton: Chip profiling implicates its role in cell cycle regulation. Oncogene 2000; 19: 2855–2866.

    Article  CAS  Google Scholar 

  14. Gu Q, Tan M, Sun Y . SAG/ROC2/Rbx2 is a novel activator protein-1 target that promotes c-Jun degradation and inhibits 12-O-tetradecanoylphorbol-13-acetate-induced neoplastic transformation. Cancer Res 2007; 67: 3616–3625.

    Article  CAS  Google Scholar 

  15. Tan M, Gu Q, He H, Pamarthy D, Semenza GL, Sun Y . SAG/ROC2/RBX2 is a HIF-1 target gene that promotes HIF-1alpha ubiquitination and degradation. Oncogene 2008; 27: 1404–1411.

    Article  CAS  Google Scholar 

  16. He H, Gu Q, Zheng M, Normolle D, Sun Y . SAG/ROC2/RBX2 E3 ligase promotes UVB-induced skin hyperplasia, but not skin tumors, by simultaneously targeting c-Jun/AP-1 and p27. Carcinogenesis 2008; 29: 858–865.

    Article  CAS  Google Scholar 

  17. Tan M, Gallegos JR, Gu Q, Huang Y, Li J, Jin Y et al. SAG/ROC-SCFbeta-TrCP E3 ubiquitin ligase promotes pro-caspase-3 degradation as a mechanism of apoptosis protection. Neoplasia 2006; 8: 1042–1054.

    Article  CAS  Google Scholar 

  18. Tan M, Zhu Y, Kovacev J, Zhao Y, Pan ZQ, Spitz DR et al. Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-kB activation in mouse embryonic stem cells. Free Radic Biol Med 2010; 49: 976–983.

    Article  CAS  Google Scholar 

  19. Gu Q, Bowden GT, Normolle D, Sun Y . SAG/ROC2 E3 ligase regulates skin carcinogenesis by stage-dependent targeting of c-Jun/AP1 and IkappaB-alpha/NF-kappaB. J Cell Biol 2007; 178: 1009–1023.

    Article  CAS  Google Scholar 

  20. Jia L, Yang J, Hao X, Zheng M, He H, Xiong X et al. Validation of SAG/RBX2/ROC2 E3 ubiquitin ligase as an anticancer and radiosensitizing target. Clin Cancer Res 2010; 16: 814–824.

    Article  CAS  Google Scholar 

  21. Tan M, Zhao Y, Kim SJ, Liu M, Jia L, Saunders TL et al. SAG/RBX2/ROC2 E3 ubiquitin ligase is essential for vascular and neural development by targeting NF1 for degradation. Dev Cell 2011; 21: 1062–1076.

    Article  CAS  Google Scholar 

  22. Sun Y . Alteration of SAG mRNA in human cancer cell lines: requirement for the RING finger domain for apoptosis protection. Carcinogenesis 1999; 20: 1899–1903.

    Article  CAS  Google Scholar 

  23. Yang GY, Pang L, Ge HL, Tan M, Ye W, Liu XH et al. Attenuation of ischemia-induced mouse brain injury by SAG, a redox- inducible antioxidant protein. J Cereb Blood Flow Metab 2001; 21: 722–733.

    Article  CAS  Google Scholar 

  24. Sun Y, Li H . Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase. Protein Cell 2013; 4: 103–116.

    Article  Google Scholar 

  25. Duan H, Tsvetkov LM, Liu Y, Song Y, Swaroop M, Wen R et al. Promotion of S-phase entry and cell growth under serum starvation by SAG/ROC2/Rbx2/Hrt2, an E3 ubiquitin ligase component: association with inhibition of p27 accumulation. Mol Carcinog 2001; 30: 37–46.

    Article  CAS  Google Scholar 

  26. Huang Y, Duan H, Sun Y . Elevated expression of SAG/ROC2/Rbx2/Hrt2 in human colon carcinomas: SAG does not induce neoplastic transformation, but its antisense transfection inhibits tumor cell growth. Mol Carcinog 2001; 30: 62–70.

    Article  CAS  Google Scholar 

  27. Diez-Juan A, Castro C, Edo MD, Andres V . Role of the growth suppressor p27Kip1 during vascular remodeling. Curr Vasc Pharmacol 2003; 1: 99–106.

    Article  CAS  Google Scholar 

  28. Schiappacassi M, Lovat F, Canzonieri V, Belletti B, Berton S, Di Stefano D et al. p27Kip1 expression inhibits glioblastoma growth, invasion, and tumor-induced neoangiogenesis. Mol Cancer Ther 2008; 7: 1164–1175.

    Article  CAS  Google Scholar 

  29. Park AY, Shen TL, Chien S, Guan JL . Role of focal adhesion kinase ser-732 phosphorylation in centrosome function during mitosis. J Biol Chem 2009; 284: 9418–9425.

    Article  CAS  Google Scholar 

  30. Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 2009; 458: 732–736.

    Article  CAS  Google Scholar 

  31. Wei D, Li H, Yu J, Sebolt JT, Zhao L, Lawrence TS et al. Radiosensitization of human pancreatic cancer cells by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Cancer Res 2012; 72: 282–293.

    Article  CAS  Google Scholar 

  32. Tan M, Davis SW, Saunders TL, Zhu Y, Sun Y . RBX1/ROC1 disruption results in early embryonic lethality due to proliferation failure, partially rescued by simultaneous loss of p27. Proc Natl Acad Sci U S A 2009; 106: 6203–6208.

    Article  CAS  Google Scholar 

  33. Baldassarre G, Belletti B, Nicoloso MS, Schiappacassi M, Vecchione A, Spessotto P et al. p27(Kip1)-stathmin interaction influences sarcoma cell migration and invasion. Cancer Cell 2005; 7: 51–63.

    Article  CAS  Google Scholar 

  34. Wander SA, Zhao D, Slingerland JM . p27: a barometer of signaling deregulation and potential predictor of response to targeted therapies. Clin Cancer Res 2011; 17: 12–18.

    Article  CAS  Google Scholar 

  35. Wang XQ, Lui EL, Cai Q, Ching WY, Liu KS, Poon RT et al. p27Kip1 promotes migration of metastatic hepatocellular carcinoma cells. Tumour Biol 2008; 29: 217–223.

    Article  CAS  Google Scholar 

  36. Folkman J . Tumor angiogenesis. Adv Cancer Res 1985; 43: 175–203.

    Article  CAS  Google Scholar 

  37. Grunstein J, Roberts WG, Mathieu-Costello O, Hanahan D, Johnson RS . Tumor-derived expression of vascular endothelial growth factor is a critical factor in tumor expansion and vascular function. Cancer Res 1999; 59: 1592–1598.

    CAS  PubMed  Google Scholar 

  38. Carmeliet P . Angiogenesis in health and disease. Nat Med 2003; 9: 653–660.

    Article  CAS  Google Scholar 

  39. Folkman J . Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 2007; 6: 273–286.

    Article  CAS  Google Scholar 

  40. Deshaies RJ, Emberley ED, Saha A . Control of cullin-RING ubiquitin ligase activity by Nedd8. In: Groettrup M ed. Conjugation and Deconjugation of Ubiquitin Family Modifiers. Subcellular Biochemistry, New York, 2010. 41–56.

    Chapter  Google Scholar 

  41. Xirodimas DP . Novel substrates and functions for the ubiquitin-like molecule NEDD8. Biochem Soc Trans 2008; 36: 802–806.

    Article  CAS  Google Scholar 

  42. Rabut G, Peter M . Function and regulation of protein neddylation. 'Protein modifications: beyond the usual suspects' review series. EMBO Rep 2008; 9: 969–976.

    Article  CAS  Google Scholar 

  43. Soucy TA, Dick LR, Smith PG, Milhollen MA, Brownell JE . The NEDD8 conjugation pathway and its relevance in cancer biology and therapy. Genes Cancer 2010; 1: 708–716.

    Article  CAS  Google Scholar 

  44. Soucy TA, Smith PG, Rolfe M . Targeting NEDD8-activated cullin-RING ligases for the treatment of cancer. Clin Cancer Res 2009; 15: 3912–3916.

    Article  CAS  Google Scholar 

  45. Nawrocki ST, Griffin P, Kelly KR, Carew JS . MLN4924: a novel first-in-class inhibitor of NEDD8-activating enzyme for cancer therapy. Expert Opin Investig Drugs 2012; 21: 1563–1573.

    Article  CAS  Google Scholar 

  46. Shen TL, Park AY, Alcaraz A, Peng X, Jang I, Koni P et al. Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis. J Cell Biol 2005; 169: 941–952.

    Article  CAS  Google Scholar 

  47. Zhu Y, Romero MI, Ghosh P, Ye Z, Charnay P, Rushing EJ et al. Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Genes Dev 2001; 15: 859–876.

    Article  CAS  Google Scholar 

  48. Zhao X, Peng X, Sun S, Park AY, Guan JL . Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development. J Cell Biol 2010; 189: 955–965.

    Article  CAS  Google Scholar 

  49. Bockbrader KM, Tan M, Sun Y . A small molecule Smac-mimic compound induces apoptosis and sensitizes TRAIL- and etoposide-induced apoptosis in breast cancer cells. Oncogene 2005; 24: 7381–7388.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Millennium Pharmaceuticals, Inc. for providing MLN4924. This work is supported by the NCI grants (CA118762, CA156744, CA170995, and CA171277) to YS.

Author information

Authors and Affiliations

  1. Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA

    M Tan, H Li & Y Sun

Authors
  1. M Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Y Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Y Sun.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tan, M., Li, H. & Sun, Y. Endothelial deletion of Sag/Rbx2/Roc2 E3 ubiquitin ligase causes embryonic lethality and blocks tumor angiogenesis. Oncogene 33, 5211–5220 (2014). https://doi.org/10.1038/onc.2013.473

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.473

Keywords

This article is cited by

Search

Advanced search

Quick links