Abstract
The inactivation of von Hippel–Lindau (VHL) is critical for clear cell renal cell carcinoma (ccRCC) and VHL syndrome. VHL loss leads to the stabilization of hypoxia-inducible factor α (HIFα) and other substrate proteins, which, together, drive various tumor-promoting pathways. There is inadequate molecular characterization of VHL restoration in VHL-defective ccRCC cells. The identities of HIF-independent VHL substrates remain elusive. We reinstalled VHL expression in 786-O and performed transcriptome, proteome and ubiquitome profiling to assess the molecular impact. The transcriptome and proteome analysis revealed that VHL restoration caused the downregulation of hypoxia signaling, glycolysis, E2F targets, and mTORC1 signaling, and the upregulation of fatty acid metabolism. Proteome and ubiquitome co-analysis, together with the ccRCC CPTAC data, enlisted 57 proteins that were ubiquitinated and downregulated by VHL restoration and upregulated in human ccRCC. Among them, we confirmed the reduction of TGFBI (ubiquitinated at K676) and NFKB2 (ubiquitinated at K72 and K741) by VHL re-expression in 786-O. Immunoprecipitation assay showed the physical interaction between VHL and NFKB2. K72 of NFKB2 affected NFKB2 stability in a VHL-dependent manner. Taken together, our study generates a comprehensive molecular catalog of a VHL-restored 786-O model and provides a list of putative VHL-dependent ubiquitination substrates, including TGFBI and NFKB2, for future investigation.
Highlights
Kidney cancer, or renal cell carcinoma (RCC), affects nearly 300,000 individuals and causes over 130,000 deaths annually worldwide [1]
Using orthotopic injection of luciferase-labeled 786-O sublines, we confirmed that persistent extinction of von Hippel–Lindau (VHL) expression is required for Clear cell renal cell carcinoma (ccRCC) tumor maintenance (Figure 1)
We applied genome-wide transcriptomics, proteomics and large-scale ubiquitomics on 786-O-Ctrl and 786-O-VHL cells to determine the molecular impact of VHL restoration on ccRCC cells and identify potential VHL substrate proteins
Summary
Renal cell carcinoma (RCC), affects nearly 300,000 individuals and causes over 130,000 deaths annually worldwide [1]. Loss of VHL stabilizes HIF1α and HIF2α, which results in the transcriptional activation of HIF targets that promote angiogenesis (e.g., VEGFA, PDGF), metabolic reprograming toward the Warburg effect (e.g., GLUT1, hexokinase 2, LDHA), cell proliferation (e.g., TGFα, EGFR, NF-κB) and other malignancy-associated traits [5,6,7]. In the context of ccRCC, HIF2α plays a major tumor-promoting role whereas HIF1α appears to function as a tumor suppressor [8,9,10,11]. This prompts the development of HIF2α-specific inhibitor Belzutifan (MK-6482), which was recently approved to treat patients with VHL disease tumors [12,13,14,15]. It is already known that a significant fraction of ccRCC cases remain resistant to HIF2α-inhibitor treatment [12,13,14], highlighting the importance of identifying additional therapeutic vulnerabilities in ccRCC
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