Limits...
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.

Show MeSH

Related in: MedlinePlus

Sag knockdown reduces migration, tube formation and proliferation in MS1 cellsMouse endothelial MS-1 cells were infected with Lt-Sag and Lt-Cont for 72 hrs. One portion was subjected to IB (A), and the other portion was used for a Boyden chamber migration assay (B), tube formation assay (C) and BrdU-based proliferation assay (D). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4016996&req=5

Figure 3: Sag knockdown reduces migration, tube formation and proliferation in MS1 cellsMouse endothelial MS-1 cells were infected with Lt-Sag and Lt-Cont for 72 hrs. One portion was subjected to IB (A), and the other portion was used for a Boyden chamber migration assay (B), tube formation assay (C) and BrdU-based proliferation assay (D). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm.

Mentions: We next extended our observation to immortalized mouse endothelial MS-1 cells and found that lentivirus-based Sag knockdown caused accumulation of p21 and p27, moderate increase in Bim, but not Noxa (Fig. 3A), and suppression of migration, tube formation and proliferation (Fig. 3B–D). Suppression of endothelial migration was also observed in human HUVEC endothelial cells upon lentivirus-mediated SAG knockdown (Fig. S3A–C).


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

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

Sag knockdown reduces migration, tube formation and proliferation in MS1 cellsMouse endothelial MS-1 cells were infected with Lt-Sag and Lt-Cont for 72 hrs. One portion was subjected to IB (A), and the other portion was used for a Boyden chamber migration assay (B), tube formation assay (C) and BrdU-based proliferation assay (D). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4016996&req=5

Figure 3: Sag knockdown reduces migration, tube formation and proliferation in MS1 cellsMouse endothelial MS-1 cells were infected with Lt-Sag and Lt-Cont for 72 hrs. One portion was subjected to IB (A), and the other portion was used for a Boyden chamber migration assay (B), tube formation assay (C) and BrdU-based proliferation assay (D). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm.
Mentions: We next extended our observation to immortalized mouse endothelial MS-1 cells and found that lentivirus-based Sag knockdown caused accumulation of p21 and p27, moderate increase in Bim, but not Noxa (Fig. 3A), and suppression of migration, tube formation and proliferation (Fig. 3B–D). Suppression of endothelial migration was also observed in human HUVEC endothelial cells upon lentivirus-mediated SAG knockdown (Fig. S3A–C).

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