Multi-Omics Profiling to Assess Signaling Changes upon VHL Restoration and Identify Putative VHL Substrates in Clear Cell Renal Cell Carcinoma Cell Lines

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.

Integrative analysis of transcriptomic landscape and urinary signature reveals prognostic biomarkers for clear cell renal cell carcinoma

“…In terms of the biological activities of the dysregulated thrombus invasion-associated genes in our study, several in vitro experiments showed that TGFBI promoted adhesion, migration, and invasion in ccRCC cells (25,26). Recent study further showed that TGFBI were ubiquitinated and downregulated by VHL restoration and upregulated in human ccRCC (27). M2-related factor frequencies were regarded as robust biomarkers for predicting the renal clear cell carcinoma patient clinical phenotype and immune microenvironment.…”

Systematic identification of key extracellular proteins as the potential biomarkers in lupus nephritis

“…Research has indicated that polymorphisms in LUM are linked with the development and clinical manifestations of SLE (40). A study has confirmed that TGFBI serving as a ubiquitination substrate is reduced in clear cell renal cell carcinoma (ccRCC) (41). As a member of the collagen family, COL1A2 is significantly upregulated in metastatic ccRCC (42).…”

Multi-Omics Integrative Approach of Extracellular Vesicles: A Future Challenging Milestone

“…Many examples of multi-omics studies are available in literature, especially applied to biomarker discovery, such as Jiang et al for pancreatic cancer [ 130 ], Zheng et al for non-small cell lung cancer [ 131 ] and Buttacavoli et al for colon cancer [ 132 ]. Other studies extended from drug discovery, such as Sha et al for diabetic nephropathy [ 133 ] and Meng et al for chordoma [ 134 ], to the understanding of pathogenesis, as performed by Wang et al in clear cell renal cell carcinoma [ 135 ] and to the establishment of accessible databases of clinical and/or multi-omics data, as Answer ALS [ 136 ] or Fibromine [ 137 ]. Recently, this multi-omic approach has also been applied to the developing single-cell technology, as it could allow to capture multiple omic layers from the same cell.…”