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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 EC deletion inhibits in vivo angiogenesis and tumorigenesisSagfl/fl mice were injected with matrigel mixed with Ad-Cre (on the left flank) or Ad-GFP control (on the right flank) in the presence or absence of VEGF. After 7 days, mice were euthanized and the matrigel plugs were harvested and photographed (A, top panel), then fixed, sectioned and stained with CD31 antibody (A, bottom panels). Sagfl/fl mice were injected with B16F10 mouse melanoma cells (5×105) mixed with Matrigel and Ad-Cre or Ad-GFP as indicated (right before injection without pre-virus infections). Twelve days later, tumors were harvested and photographed (B, left panel). Shown is mean ± SEM from 7 mice in each group, except B16F10 only control group (n=3) (B, right panel). The tumor tissues were fixed, sectioned and stained for CD31 (for blood vessels, C), Ki67 (for proliferation, D) and TUNEL assay (for apoptosis, E). Scale bar represents 100 μm.
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Figure 5: Sag EC deletion inhibits in vivo angiogenesis and tumorigenesisSagfl/fl mice were injected with matrigel mixed with Ad-Cre (on the left flank) or Ad-GFP control (on the right flank) in the presence or absence of VEGF. After 7 days, mice were euthanized and the matrigel plugs were harvested and photographed (A, top panel), then fixed, sectioned and stained with CD31 antibody (A, bottom panels). Sagfl/fl mice were injected with B16F10 mouse melanoma cells (5×105) mixed with Matrigel and Ad-Cre or Ad-GFP as indicated (right before injection without pre-virus infections). Twelve days later, tumors were harvested and photographed (B, left panel). Shown is mean ± SEM from 7 mice in each group, except B16F10 only control group (n=3) (B, right panel). The tumor tissues were fixed, sectioned and stained for CD31 (for blood vessels, C), Ki67 (for proliferation, D) and TUNEL assay (for apoptosis, E). Scale bar represents 100 μm.

Mentions: We next used two in vivo models to determine the effect of Sag endothelial deletion on angiogenesis. In an in vivo matrigel plug assay, we injected the matrigel mixed with Ad-Cre or Ad-GFP control, plus or minus VEGF, into Sagfl/fl mice (left flank for Ad-Cre and right flank for Ad-GFP). We euthanized mice seven days later and harvested the entire matrigel plug. Expectedly, VEGF induced remarkable angiogenesis when matrigel was mixed with the Ad-GFP control, as evidenced by the bloody matrigel plugs (Fig. 5A, top) and the presence of CD31-positive vessels (Fig. 5A, bottom). In contrast, VEGF-induced angiogenesis was nearly completely inhibited if Ad-Cre was present, which deleted Sag in host blood vessels during angiogenesis into matrigel plugs (Fig. 5A).


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 EC deletion inhibits in vivo angiogenesis and tumorigenesisSagfl/fl mice were injected with matrigel mixed with Ad-Cre (on the left flank) or Ad-GFP control (on the right flank) in the presence or absence of VEGF. After 7 days, mice were euthanized and the matrigel plugs were harvested and photographed (A, top panel), then fixed, sectioned and stained with CD31 antibody (A, bottom panels). Sagfl/fl mice were injected with B16F10 mouse melanoma cells (5×105) mixed with Matrigel and Ad-Cre or Ad-GFP as indicated (right before injection without pre-virus infections). Twelve days later, tumors were harvested and photographed (B, left panel). Shown is mean ± SEM from 7 mice in each group, except B16F10 only control group (n=3) (B, right panel). The tumor tissues were fixed, sectioned and stained for CD31 (for blood vessels, C), Ki67 (for proliferation, D) and TUNEL assay (for apoptosis, E). Scale bar represents 100 μm.
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Figure 5: Sag EC deletion inhibits in vivo angiogenesis and tumorigenesisSagfl/fl mice were injected with matrigel mixed with Ad-Cre (on the left flank) or Ad-GFP control (on the right flank) in the presence or absence of VEGF. After 7 days, mice were euthanized and the matrigel plugs were harvested and photographed (A, top panel), then fixed, sectioned and stained with CD31 antibody (A, bottom panels). Sagfl/fl mice were injected with B16F10 mouse melanoma cells (5×105) mixed with Matrigel and Ad-Cre or Ad-GFP as indicated (right before injection without pre-virus infections). Twelve days later, tumors were harvested and photographed (B, left panel). Shown is mean ± SEM from 7 mice in each group, except B16F10 only control group (n=3) (B, right panel). The tumor tissues were fixed, sectioned and stained for CD31 (for blood vessels, C), Ki67 (for proliferation, D) and TUNEL assay (for apoptosis, E). Scale bar represents 100 μm.
Mentions: We next used two in vivo models to determine the effect of Sag endothelial deletion on angiogenesis. In an in vivo matrigel plug assay, we injected the matrigel mixed with Ad-Cre or Ad-GFP control, plus or minus VEGF, into Sagfl/fl mice (left flank for Ad-Cre and right flank for Ad-GFP). We euthanized mice seven days later and harvested the entire matrigel plug. Expectedly, VEGF induced remarkable angiogenesis when matrigel was mixed with the Ad-GFP control, as evidenced by the bloody matrigel plugs (Fig. 5A, top) and the presence of CD31-positive vessels (Fig. 5A, bottom). In contrast, VEGF-induced angiogenesis was nearly completely inhibited if Ad-Cre was present, which deleted Sag in host blood vessels during angiogenesis into matrigel plugs (Fig. 5A).

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