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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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Sag endothelial deletion reduces migration, tube formation and proliferationPrimary ECs were isolated from lung and heart tissues of 4 week-old Sagfl/fl mice. Cells at passage 2 were infected with Ad-Cre or Ad-GFP (6×10E7 pfu) in 5 ml medium for 72 hrs. One portion of cells was subjected to PCR (A, top), a second portion for IB to detect Sag (A, bottom), and a third 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 individual mice (B, C & D). Scale bar represents 50 μm. Two independent pairs of primary cultures of ECs were subjected to IB to detect accumulation of the indicated Sag-CRL E3 substrates (E).
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Figure 2: Sag endothelial deletion reduces migration, tube formation and proliferationPrimary ECs were isolated from lung and heart tissues of 4 week-old Sagfl/fl mice. Cells at passage 2 were infected with Ad-Cre or Ad-GFP (6×10E7 pfu) in 5 ml medium for 72 hrs. One portion of cells was subjected to PCR (A, top), a second portion for IB to detect Sag (A, bottom), and a third 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 individual mice (B, C & D). Scale bar represents 50 μm. Two independent pairs of primary cultures of ECs were subjected to IB to detect accumulation of the indicated Sag-CRL E3 substrates (E).

Mentions: Given that both total KO and conditional KO of Sag showed endothelial and vascular defects, we next generated primary endothelial cells (ECs) from the lung and heart tissues of Sagfl/fl mice and assessed the potential role of Sag in endothelial migration, tube formation and proliferation in vitro. We first confirmed that the floxed Sag allele can be excised by Ad-Cre infection. Indeed, infection of ECs with Ad-Cre caused the excision of floxed Sag allele to give rise to a 257 bp PCR fragment, as compared to a ~1.6 kb PCR fragment in Ad-GFP-infected control cells which retained the floxed Sag allele (Fig 2A, top). We then confirmed that deletion of the floxed Sag allele completely eliminates the Sag protein expression (2A, bottom). Consistent with in vivo data, Sag deletion in endothelial cells significantly inhibits in vitro migration (Fig. 2B), tube formation (Fig. 2C) and proliferation (Fig. 2D), but has little, if any, effect on apoptosis (Fig. S1B). We further examined potential accumulation of known Sag substrates associated with growth suppression (p21 and p27) and apoptosis (Bim and Noxa) 24. Consistently, Sag deletion caused accumulation of p21 and p27, moderate increase in Bim, but not Noxa (Fig. 2E). Finally, we determined potential effect of Sag deletion on expression of VEGF and VEGFR-2 and found a minimal effect, if any (Fig. S2).


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 endothelial deletion reduces migration, tube formation and proliferationPrimary ECs were isolated from lung and heart tissues of 4 week-old Sagfl/fl mice. Cells at passage 2 were infected with Ad-Cre or Ad-GFP (6×10E7 pfu) in 5 ml medium for 72 hrs. One portion of cells was subjected to PCR (A, top), a second portion for IB to detect Sag (A, bottom), and a third 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 individual mice (B, C & D). Scale bar represents 50 μm. Two independent pairs of primary cultures of ECs were subjected to IB to detect accumulation of the indicated Sag-CRL E3 substrates (E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016996&req=5

Figure 2: Sag endothelial deletion reduces migration, tube formation and proliferationPrimary ECs were isolated from lung and heart tissues of 4 week-old Sagfl/fl mice. Cells at passage 2 were infected with Ad-Cre or Ad-GFP (6×10E7 pfu) in 5 ml medium for 72 hrs. One portion of cells was subjected to PCR (A, top), a second portion for IB to detect Sag (A, bottom), and a third 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 individual mice (B, C & D). Scale bar represents 50 μm. Two independent pairs of primary cultures of ECs were subjected to IB to detect accumulation of the indicated Sag-CRL E3 substrates (E).
Mentions: Given that both total KO and conditional KO of Sag showed endothelial and vascular defects, we next generated primary endothelial cells (ECs) from the lung and heart tissues of Sagfl/fl mice and assessed the potential role of Sag in endothelial migration, tube formation and proliferation in vitro. We first confirmed that the floxed Sag allele can be excised by Ad-Cre infection. Indeed, infection of ECs with Ad-Cre caused the excision of floxed Sag allele to give rise to a 257 bp PCR fragment, as compared to a ~1.6 kb PCR fragment in Ad-GFP-infected control cells which retained the floxed Sag allele (Fig 2A, top). We then confirmed that deletion of the floxed Sag allele completely eliminates the Sag protein expression (2A, bottom). Consistent with in vivo data, Sag deletion in endothelial cells significantly inhibits in vitro migration (Fig. 2B), tube formation (Fig. 2C) and proliferation (Fig. 2D), but has little, if any, effect on apoptosis (Fig. S1B). We further examined potential accumulation of known Sag substrates associated with growth suppression (p21 and p27) and apoptosis (Bim and Noxa) 24. Consistently, Sag deletion caused accumulation of p21 and p27, moderate increase in Bim, but not Noxa (Fig. 2E). Finally, we determined potential effect of Sag deletion on expression of VEGF and VEGFR-2 and found a minimal effect, if any (Fig. S2).

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