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Published on: July 20, 2019
pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development
Natalie H Stickle1, Jacky Chung, Jeffery M Klco
1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada. michael.ohh@utoronto.ca
Abstract:
Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is the cause of the familial VHL disease and most sporadic renal clear-cell carcinomas (RCC). pVHL has been shown to play a role in the destruction of hypoxia-inducible factor alpha (HIF-alpha) subunits via ubiquitin-mediated proteolysis and in the regulation of fibronectin matrix assembly. Although most disease-causing pVHL mutations hinder the regulation of the HIF pathway, every disease-causing pVHL mutant tested to date has failed to promote the assembly of the fibronectin matrix, underscoring its potential importance in VHL disease. Here, we report that a ubiquitin-like molecule called NEDD8 covalently modifies pVHL. A nonneddylateable pVHL mutant, while retaining its ability to ubiquitylate HIF, failed to bind to and promote the assembly of the fibronectin matrix. Expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. These results suggest that NEDD8 modification of pVHL plays an important role in fibronectin matrix assembly and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent VHL-associated tumorigenesis.
Insights
NEDD8 modification of the VHL protein is crucial for fibronectin matrix assembly. Without this modification, the hypoxia-inducible factor (HIF) pathway alone cannot prevent VHL-associated tumor formation.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- The von Hippel-Lindau (VHL) tumor suppressor gene inactivation causes VHL disease and renal clear-cell carcinoma (RCC).
- pVHL regulates hypoxia-inducible factor alpha (HIF-alpha) degradation and fibronectin matrix assembly.
- Most VHL mutations impair HIF regulation, but all fail to promote fibronectin assembly, suggesting its importance.
Purpose of the Study:
- To investigate the role of NEDD8 modification in pVHL function.
- To determine if NEDD8 modification is essential for fibronectin matrix assembly and tumor suppression.
Main Methods:
- Utilized a non-neddylateable pVHL mutant.
- Assessed HIF ubiquitylation and fibronectin matrix assembly.
- Evaluated pVHL function in RCC cells using 3D growth and SCID mouse tumor formation assays.
Main Results:
- A non-neddylateable pVHL mutant retained HIF ubiquitylation but lost fibronectin matrix assembly binding.
- NEDD8-defective pVHL restored HIF regulation but failed to induce differentiated morphology in 3D cultures.
- Expression of neddylation-defective pVHL did not suppress tumor formation in SCID mice.
Conclusions:
- NEDD8 modification of pVHL is critical for fibronectin matrix assembly.
- Intact HIF pathway regulation is insufficient to prevent VHL-associated tumorigenesis without proper fibronectin matrix assembly.
- NEDD8-mediated regulation of pVHL is a key factor in VHL disease pathogenesis.
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