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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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CRL inhibitor MLN4924 suppresses tumor angiogenesis in vivoSagfl/fl mice were injected with B16F10 mouse melanoma cells (5×105) mixed with Matrigel. Starting on the next day MLN4924 was administrated (60 mg/kg, s.c.) once a day, 5 days a week for 12 days. Tumors were then harvested and weighed. Two pairs of tumor tissues (T) were subjected to IB with indicated antibodies (A). Tumor weight is shown as mean ± SEM from seven mice in each group (B). The tumor tissues were fixed, sectioned and stained for CD31 (for blood vessels, C), BrdU (for cell proliferation, D), and TUNEL (for apoptosis, E). **: p<0.01. Scale bar represents 100 μm. (F) B16F10 cells were treated with MLN4924 at different concentrations for 24 hrs and subjected to IB using indicated Abs.
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Figure 8: CRL inhibitor MLN4924 suppresses tumor angiogenesis in vivoSagfl/fl mice were injected with B16F10 mouse melanoma cells (5×105) mixed with Matrigel. Starting on the next day MLN4924 was administrated (60 mg/kg, s.c.) once a day, 5 days a week for 12 days. Tumors were then harvested and weighed. Two pairs of tumor tissues (T) were subjected to IB with indicated antibodies (A). Tumor weight is shown as mean ± SEM from seven mice in each group (B). The tumor tissues were fixed, sectioned and stained for CD31 (for blood vessels, C), BrdU (for cell proliferation, D), and TUNEL (for apoptosis, E). **: p<0.01. Scale bar represents 100 μm. (F) B16F10 cells were treated with MLN4924 at different concentrations for 24 hrs and subjected to IB using indicated Abs.

Mentions: Finally, we determined the effect of MLN4924 on in vivo tumor angiogenesis, again using the B16F10 model. Sagfl/fl mice were treated with MLN4924 subcutaneously at a non-toxic dose regimen (60 mg/kg, once a day, 5 days per week) 30, 31 for 12 days beginning at 24 hrs post B16F10 inoculation. Tumors were harvested at day 13, weighed and snap-frozen for Western blotting or sectioned for staining with various antibodies. We first confirmed that MLN4924 indeed reached the tumor site by showing a significant inhibition of cullin neddylation in two independent tumor tissues (Fig. 7A). Similar to Sag deletion, MLN4924 treatment caused significant reductions in tumor mass (Fig. 8B), vessel density (Fig. 8C), and proliferation (Fig. 8D), as well as extensive apoptosis (Fig. 8E). MLN4924 treatment also caused a dose dependent accumulation of p21, p27, and Bim, but not Noxa in B16F10 cells (Fig. 8F), indicating a mechanism similar to that of Sag knockdown.


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

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

CRL inhibitor MLN4924 suppresses tumor angiogenesis in vivoSagfl/fl mice were injected with B16F10 mouse melanoma cells (5×105) mixed with Matrigel. Starting on the next day MLN4924 was administrated (60 mg/kg, s.c.) once a day, 5 days a week for 12 days. Tumors were then harvested and weighed. Two pairs of tumor tissues (T) were subjected to IB with indicated antibodies (A). Tumor weight is shown as mean ± SEM from seven mice in each group (B). The tumor tissues were fixed, sectioned and stained for CD31 (for blood vessels, C), BrdU (for cell proliferation, D), and TUNEL (for apoptosis, E). **: p<0.01. Scale bar represents 100 μm. (F) B16F10 cells were treated with MLN4924 at different concentrations for 24 hrs and subjected to IB using indicated Abs.
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Figure 8: CRL inhibitor MLN4924 suppresses tumor angiogenesis in vivoSagfl/fl mice were injected with B16F10 mouse melanoma cells (5×105) mixed with Matrigel. Starting on the next day MLN4924 was administrated (60 mg/kg, s.c.) once a day, 5 days a week for 12 days. Tumors were then harvested and weighed. Two pairs of tumor tissues (T) were subjected to IB with indicated antibodies (A). Tumor weight is shown as mean ± SEM from seven mice in each group (B). The tumor tissues were fixed, sectioned and stained for CD31 (for blood vessels, C), BrdU (for cell proliferation, D), and TUNEL (for apoptosis, E). **: p<0.01. Scale bar represents 100 μm. (F) B16F10 cells were treated with MLN4924 at different concentrations for 24 hrs and subjected to IB using indicated Abs.
Mentions: Finally, we determined the effect of MLN4924 on in vivo tumor angiogenesis, again using the B16F10 model. Sagfl/fl mice were treated with MLN4924 subcutaneously at a non-toxic dose regimen (60 mg/kg, once a day, 5 days per week) 30, 31 for 12 days beginning at 24 hrs post B16F10 inoculation. Tumors were harvested at day 13, weighed and snap-frozen for Western blotting or sectioned for staining with various antibodies. We first confirmed that MLN4924 indeed reached the tumor site by showing a significant inhibition of cullin neddylation in two independent tumor tissues (Fig. 7A). Similar to Sag deletion, MLN4924 treatment caused significant reductions in tumor mass (Fig. 8B), vessel density (Fig. 8C), and proliferation (Fig. 8D), as well as extensive apoptosis (Fig. 8E). MLN4924 treatment also caused a dose dependent accumulation of p21, p27, and Bim, but not Noxa in B16F10 cells (Fig. 8F), indicating a mechanism similar to that of Sag knockdown.

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