Study Of HIF-Independent VHL Pathway In Clear Renal Cell Carcinoma (ccRCC)

Inactivation of von Hippel-Lindau(VHL)is critical to clear cell renal cell carcinoma(ccRCC)and VHL syndrome.VHL loss leads to stabilization of hypoxiainducible 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.To better understand function of pVHL,we mined putative pVHL targets and study their role in ccRCC tumor development.Therefore,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 downregulation of hypoxia signaling,glycolysis,E2 F targets and m TORC1 signaling,and 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 reexpression in 786-O.Immunoprecipitation assay showed the physical interaction between VHL and NFKB2.K72 of NFKB2 affected NFKB2 stability in a VHLdependent manner.Taken together,our study generates a comprehensive molecular catalog of VHL-restored 786-O model,and provides a list of putative VHL-dependent ubiquitination substrates including TGFBI and NFKB2 for future investigation.In the meantime,we identified KMRC2 as a highly relevant ccRCC cell line that displays hypermethylation-induced UQCRH extinction.Methylation of UQCRH showed high effect on metabolism of ccRCC,promoting Warburg effect on KMRC2.To reveal comprehensive mechanism underlying multiple organ tumor formation of VHL syndrome,we firstly generated whole-body somatic VHL knocking-out transgenic mouse.At last,we proposed potential a gene therapy design.Utilizing overaccumulation of HIFα in ccRCC promotes VHL expression,guaranteeing VHL-HIF dynamic balance systemically in vitro and in vivo.We confirmed that in vivo delivering gene cassette attenuated tumor progress in tumor-bearing mice.