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

Tan M, Li H, Sun Y - Oncogene (2013)

Bottom Line: SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING protein required for the activity of Cullin-RING ligase (CRL).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.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.

View Article: PubMed Central - PubMed

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

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.

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p27 deletion partially rescues Sag deletion-induced defects in migration and proliferation, but not tube formationMS1 cells were infected with Lt-Sag and Lt-Cont and co-transfected with siRNA oligonucleotides targeting p27, along with scrambled control siRNA for 72 hrs. One portion was subjected to IB (A), and the other portion was used for assays of migration (B), proliferation (C) and tube formation (D). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm.
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Figure 4: p27 deletion partially rescues Sag deletion-induced defects in migration and proliferation, but not tube formationMS1 cells were infected with Lt-Sag and Lt-Cont and co-transfected with siRNA oligonucleotides targeting p27, along with scrambled control siRNA for 72 hrs. One portion was subjected to IB (A), and the other portion was used for assays of migration (B), proliferation (C) and tube formation (D). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm.

Mentions: We next investigated potential mechanisms by which Sag deletion alters endothelial properties. We focused our study on p27, since 1) p27 is a Sag substrate 16 which is accumulated upon Sag deletion in endothelial cells (Fig. 2E and 3A), and 2) p27 has been previously implicated in neoangiogenesis, invasion and vascular remodeling. 27, 28 MS-1 cells were infected with lentivirus targeting Sag, along with the scrambled control, followed by p27 knockdown via siRNA (Fig. 4A). p27 knockdown partially rescued Sag knockdown-induced suppression of endothelial migration (Fig. 4B) and proliferation (Fig. 4C), but not tube formation (Fig. 4D), suggesting that p27-induced growth suppression affects endothelial migration, but not tube formation.


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

Tan M, Li H, Sun Y - Oncogene (2013)

p27 deletion partially rescues Sag deletion-induced defects in migration and proliferation, but not tube formationMS1 cells were infected with Lt-Sag and Lt-Cont and co-transfected with siRNA oligonucleotides targeting p27, along with scrambled control siRNA for 72 hrs. One portion was subjected to IB (A), and the other portion was used for assays of migration (B), proliferation (C) and tube formation (D). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016996&req=5

Figure 4: p27 deletion partially rescues Sag deletion-induced defects in migration and proliferation, but not tube formationMS1 cells were infected with Lt-Sag and Lt-Cont and co-transfected with siRNA oligonucleotides targeting p27, along with scrambled control siRNA for 72 hrs. One portion was subjected to IB (A), and the other portion was used for assays of migration (B), proliferation (C) and tube formation (D). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm.
Mentions: We next investigated potential mechanisms by which Sag deletion alters endothelial properties. We focused our study on p27, since 1) p27 is a Sag substrate 16 which is accumulated upon Sag deletion in endothelial cells (Fig. 2E and 3A), and 2) p27 has been previously implicated in neoangiogenesis, invasion and vascular remodeling. 27, 28 MS-1 cells were infected with lentivirus targeting Sag, along with the scrambled control, followed by p27 knockdown via siRNA (Fig. 4A). p27 knockdown partially rescued Sag knockdown-induced suppression of endothelial migration (Fig. 4B) and proliferation (Fig. 4C), but not tube formation (Fig. 4D), suggesting that p27-induced growth suppression affects endothelial migration, but not tube formation.

Bottom Line: SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING protein required for the activity of Cullin-RING ligase (CRL).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.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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus