The pVHL-associated SCF ubiquitin ligase complex: molecular genetic analysis of elongin B and C, Rbx1 and HIF-1alpha in renal cell carcinoma.
The VHL gene product (pVHL) forms a multimeric complex with the elongin B and C, Cul2 and Rbx1 proteins (VCBCR complex), which is homologous to the SCF family of ubiquitin ligase complexes. The VCBCR complex binds HIF-1alpha and HIF-2alpha, transcription factors critically involved in cellular responses to hypoxia, and targets them for ubiquitin-mediated proteolysis. Germline mutations in the VHL gene cause susceptibility to haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and other tumours. In addition somatic inactivation of the VHL gene occurs in most sporadic clear cell RCC (CC-RCC). However, the absence of somatic VHL inactivation in 30-40% of CC-RCC implies the involvement of other gatekeeper genes in CC-RCC development. We reasoned that in CC-RCC without VHL inactivation, other pVHL-interacting proteins might be defective. To assess the role of elongin B/C, Rbx1 and HIF-1alpha in RCC tumorigenesis we (a) mapped the genes to chromosomes 8q(cen) (elongin C), 16p13.3 (elongin B) and 22q11.2 (Rbx1) by FISH, monochromosomal somatic cell hybrid panel screening and in silico GenBank homology searching; (b) determined the genomic organisation of elongin C (by direct sequencing of PAC clones), Rbx1 and elongin B (by GenBank homology searching); and (c) performed mutation analysis of exons comprising the coding regions of elongins B, C and Rbx1 and the oxygen-dependent degradation domain of HIF-1alpha by SSCP screening and direct sequencing in 35 sporadic clear cell RCC samples without VHL gene inactivation and in 13 individuals with familial non-VHL clear cell RCC. No coding region sequence variations were detected for the elongin B, elongin C or Rbx1 genes. Two amino acid substitutions (Pro582Ser and Ala588Thr) were identified in the oxygen-dependent degradation/pVHL binding domain of HIF-1alpha, however neither substitution was observed exclusively in tumour samples. Association analysis in panels of CC-RCC and non-neoplastic samples using the RFLPs generated by each variant did not reveal allelic frequency differences between RCC patients and controls (P>0.32 by chi-squared analysis). Nevertheless, the significance of these variations and their potential for modulation of HIF-1alpha function merits further investigation in both other tumour types and in non-neoplastic disease. Taken together with our previous Cul2 mutation analysis these data suggest that development of sporadic and familial RCC is not commonly contributed to by genetic events altering the destruction domain of HIF-1alpha, or components of the HIF-alpha destruction complex other than VHL itself. Although (a) activation of HIF could occur through mutation of another region of HIF-a, and (b) epigenetic silencing of elongin B/C, Cul2 or Rbx1 cannot be excluded, these findings suggest that pVHL may represent the sole mutational target through which the VCBR complex is disrupted in CC-RCC. HIF response is activated in CC-RCC tumorigenesis.
Clifford
S C
SC
Section of Medical & Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, Birmingham, B15 2TT, UK.
Astuti
D
D
Hooper
L
L
Maxwell
P H
PH
Ratcliffe
P J
PJ
Maher
E R
ER
D002292:Carcinoma, Renal Cell / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002874:Chromosome Mapping; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D000075065:Elongin; D014644:Genetic Variation; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007680:Kidney Neoplasms / Q000235:genetics*; D008025:Ligases; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000235:genetics; D010453:Peptide Synthases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases; D014157:Transcription Factors / Q000235:genetics*; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D002352:Carrier Proteins; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C000616396:ELOB protein, human; C000616395:ELOC protein, human; D000075065:Elongin; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; D010453:Peptide Synthases; C497446:VHL protein, human