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. 2020 Jul;39(29):5228-5239.
doi: 10.1038/s41388-020-1359-4. Epub 2020 Jun 17.

Mdm2-mediated neddylation of pVHL blocks the induction of antiangiogenic factors

Eric R Wolf  1 , Alexander R Mabry  1 , Blossom Damania  2 , Lindsey D Mayo  3   4
Affiliations

Mdm2-mediated neddylation of pVHL blocks the induction of antiangiogenic factors

Eric R Wolf et al. Oncogene. 2020 Jul.

Abstract

Mutations in the tumor suppressor TP53 are rare in renal cell carcinomas. p53 is a key factor for inducing antiangiogenic genes and RCC are highly vascularized, which suggests that p53 is inactive in these tumors. One regulator of p53 is the Mdm2 oncogene, which is correlated with high-grade, metastatic tumors. However, the sole activity of Mdm2 is not just to regulate p53, but it can also function independent of p53 to regulate the early stages of metastasis. Here, we report that the oncoprotein Mdm2 can bind directly to the tumor suppressor VHL, and conjugate nedd8 to VHL within a region that is important for the p53-VHL interaction. Nedd8 conjugated VHL is unable to bind to p53 thereby preventing the induction of antiangiogenic factors. These results highlight a previously unknown oncogenic function of Mdm2 during the progression of cancer to promote angiogenesis through the regulation of VHL. Thus, the Mdm2-VHL interaction represents a pathway that impacts tumor angiogenesis.

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Conflict of interest statement

Competing Interests

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.. p53 target genes for apoptosis and angiogenesis are altered by VHL status
(A-B) Log counts per million for BAX, CDKN1A, and BBC3 (A) or THBS1, COL18A1, and SERPINE1 (B). Only samples with wild-type p53 were included in the analysis. WT refers to wild type VHL status, and α mutant refers to any point mutation within the α domain of VHL. Error bars represent SEM. P values were calculated using an unpaired t test. (C) Luciferase assay in 786-O and RCC4 cells with or without VHL for thrombospondin-1 plotted as relative luciferase units. Means are calculated from three biological replicates. Error bars represent SEM. Statistics were calculated using an unpaired t test (***p<0.001). (D) Dot blot assay to measure protein level of thrombospondin-1 in media secreted from 786-O or RCC4 cells with or without VHL. Means are calculated from three biological replicates. Error bars represent SEM. Statistics were calculated using an unpaired t test (***p<0.001).
Figure 2.
Figure 2.. Mdm2 binds to VHL and inhibits VHL-p53 complex formation under hypoxia
(A-B) Transient transfection in H1299 cells followed by incubation in either normoxia or hypoxia. p53 (A) or VHL (B) was immunoprecipitated from cell lysates and subjected to analysis by Western blot. (C) Schematic of VHL protein showing the primary site of neddylation, as well as the region that has been shown to be indispensable for binding to p53. (D-E) Transfection of 786-O cells with VHL or VHL and Mdm2 under normoxia (D) or hypoxia (E). Cells were fixed and stained with antibody against p53 and VHL and subjected to immunofluorescence confocal microscopy. Scale bar represents 25 μm.
Figure 3.
Figure 3.. Neddylation of VHL by Mdm2 interferes with VHL-p53 complex formation
(A) Transient transfection in H1299 cells under normoxia or hypoxia. Cell lysates were immunoprecipitated for HA and subjected to analysis by Western blot. (B) Immunoprecipitation of VHL from 786-O cells treated with the neddylation inhibitor MLN4924 and Western blotted for p53 and VHL. (C) Immunoprecipitation of p53 or IgG control from MCF7 cells treated with MLN4924 for various indicated times and Western blotted for VHL and p53. (D) GST pulldown of GST, GST-alpha VHL, GST-beta VHL, or GST-VHL with His-Mdm2 and Western blotted for GST and Mdm2. (E) Cell free neddylation assay using recombinant proteins. Reactions were separated by SDS-PAGE and analyzed by Western blot. (F) Immunoprecipitation of endogenous nedd8 from 786-O cells transfected with Mdm2 and subjected to hypoxia and Western blotted for VHL and Mdm2. (G) Ni-NTA pulldown for His-nedd8 in H1299 cells in a transient transfection and subjected to treatment with MLN4924. Samples were separated with SDS-PAGE and analyzed by Western blot for VHL and Mdm2. (H) Schematic of the p53-pVHL-Thrombospondin pathway disrupted by c-Src-Mdm2.
Figure 4.
Figure 4.. VHL forms a complex with p53 under hypoxia or with treatment of MLN4924
(A-B) Transfection of 786-O cells with WT VHL (A) or K159E VHL (B) and Mdm2 under normoxia. Cells were treated with MLN4924 or a control, fixed, and stained with antibodies against p53 and VHL and subjected to immunofluorescence confocal microscopy. Scale bar represents 25 μm. (C-D) Transfection of 786-O cells with WT VHL (C) or K159E (D) VHL and Mdm2 under hypoxia. Cells were treated with MLN4924 or a control, fixed, and stained with antibodies against p53 and VHL and subjected to immunofluorescence confocal microscopy. Scale bar represents 25 μm. (E-F) Immunoflourescence microscopy of Caki-1 cells treated with MLN4924 or a control under normoxia (E) or hypoxia (F). Cells were fixed and stained with antibodies against VHL and p53. Scale bar represents 50 μm.
Figure 5.
Figure 5.. VHL increases the activation of p53 under hypoxia leading to increased transcription and secretion of TSP-1
(A) Luciferase assay for thrombospondin-1 in 786-O cells treated with MLN4924 and subjected to hypoxia. Means are plotted as relative luciferase units and calculated from three biological replicates. Error bars represent SEM. Statistics were calculated using an unpaired t test. (B) Enzyme-linked immunosorbent assay for TSP-1 using a 10% dilution of conditioned media from Caki-1 cells treated with MLN4924 under hypoxia. Means from three biological replicates are plotted. Error bars represent SEM. Statistics were calculated using an unpaired t test. (C-D) Enzyme-linked immunosorbent assay for TSP-1 using a 10% dilution of conditioned media from 786-O (C) or RCC4 (D) cells with knockdown p53 under hypoxia. Means from three biological replicates are plotted. Error bars represent SEM. Statistics were calculated using an unpaired t test (*p<0.05, **p<0.01, ***p<0.001).
Figure 6.
Figure 6.. p53 and TSP-1 inhibit Human Umbilical Vein Endothelial Cell network formation
(A) Network formation assay using conditioned media from transiently transfected H1299 cells. Network branch points were quantified and plotted as the mean of three biological replicates. Error bars represent SEM. Statistics were calculated with an unpaired t test. (B-C) Network formation assay using conditioned media from 786-O (B) or RCC4 (C) cells with VHL, VHL with knockdown p53, or VHL and an antibody against TSP-1 under normoxia or hypoxia. Network branch points were quantified and plotted as the mean of three biological replicates. Error bars represent SEM. Statistics were calculated with an unpaired t test (*p<0.05, **p<0.01, ***p<0.001).
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