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. 2014 Oct 30;33(44):5211-20.
doi: 10.1038/onc.2013.473. Epub 2013 Nov 11.

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

M Tan  1 , H Li  1 , Y Sun  1
Affiliations

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

M Tan et al. Oncogene. .

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) VSports手机版. 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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Conflicts of Interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Sag endothelial deletion disrupts vascular development in mouse embryos
(A) Appearance of mouse embryos at E15.5 (left) and E13.5 (right). (B) Appearance of the brain regions of mouse embryos at E13.5 after CD31 whole-mount staining. Scale bar represents 3 mm. (C) Sagittal sections of control and mutant embryos were stained with antibodies against CD31, Ki67, and DAPI at the spinal cord areas. Scale bar represents 100 μm. (D and E) Quantification of CD31 positive cells (D) and Ki67 positive cells (E). The data were normalized as the ratio of CD31+/DAPI+ cells and represented as fold change with CD31+/DAPI+ value setting at 1 in Tie2-Cre;Sagfl/+ embryos (mean ± SEM, n=3) (D). The number of cells expressing Ki67 was normalized to the total number of DAPI positive cells as a percentage (mean ± SEM, n=3) (E).
Figure 2
Figure 2. Sag endothelial deletion reduces migration, tube formation and proliferation
Primary 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).
Figure 3
Figure 3. Sag knockdown reduces migration, tube formation and proliferation in MS1 cells
Mouse 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.
Figure 4
Figure 4. p27 deletion partially rescues Sag deletion-induced defects in migration and proliferation, but not tube formation
MS1 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.
Figure 5
Figure 5. Sag EC deletion inhibits in vivo angiogenesis and tumorigenesis
Sagfl/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.
Figure 6
Figure 6. CRL inhibitor MLN4924 suppresses migration, tube formation and proliferation in vitro
Mouse endothelial MS-1 cells were treated with MLN4924 for 24 hrs. One portion was used for assays of migration (A), tube formation (B) and BrdU incorporation (C). Shown is mean ± SEM from three independent experiments. Scale bar represents 50 μm. The other portion was used for IB with indicated Abs (D).
Figure 7
Figure 7. p27 knockdown partially rescues MLN4924-induced migration and proliferation, but not tube formation
MS1 cells were first transfected with siRNA targeting p27 and si-Cont for 72 hrs, followed by MLN4924 treatment for 24 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.
Figure 8
Figure 8. CRL inhibitor MLN4924 suppresses tumor angiogenesis in vivo
Sagfl/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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