The pVHL-associated SCF ubiquitin ligase complex: Molecular genetic analysis of elongin B and C, Rbx1 and HIF-1 alpha in renal cell carcinoma

The VHL gene product (pVHL) forms a multimeric complex with the elongin B a nd C, Cul2 and Rbx1 proteins (VCBCR complex), which is homologous to the SC F family of ubiquitin ligase complexes. The VCBCR complex binds HIF-1 alpha and HIF-2 alpha, transcription factors critically involved in cellular res ponses to hypoxia, and targets them for ubiquitin-mediated proteolysis. Ger mline mutations in the VHL gene cause susceptibility to haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and other tumours. In additi on 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 develop ment. We reasoned that in CC-RCC without VHL inactivation, other pVHL-inter acting proteins might be defective. To assess the role of elongin B/C, Rbx1 and HIF-1 alpha in RCC tumorigenesis we (a) mapped the genes to chromosome s 8q(cen) (elongin C), 16p13.3 (elongin B) and 22q11.2 (Rbx1) by FISH, mono chromosomal somatic cell hybrid panel screening and in silico GenBank homol ogy searching; (b) determined the genomic organisation of elongin C (by dir ect sequencing of PAC clones), Rbx1 and elongin B (by GenBank homology sear ching); and (c) performed mutation analysis of exons comprising the coding regions of elongins B, C and Rbx1 and the oxygen-dependent degradation doma in of HIF-1 alpha by SSCP screening and direct sequencing in 35 sporadic cl ear cell RCC samples without VHL gene inactivation and in 13 individuals wi th familial non-VHL clear cell RCC. No coding region sequence variations we re detected for the elongin 13, elongin C or Rbx1 genes. Two amino acid sub stitutions (Pro582Ser and Ala588Thr) were identified in the oxygen-dependen t degradation/pVHL binding domain of HIF-1 alpha, however neither substitut ion was observed exclusively in tumour samples. Association analysis in pan els of CC-RCC and non-neoplastic samples using the RFLPs generated by each variant did not reveal allelic frequency differences between RCC patients a nd controls (P >0.32 by chi-squared analysis). Nevertheless, the significan ce of these variations and their potential for modulation of HIF-1 alpha fu nction merits further investigation in both other tumour types and in non-n eoplastic disease. Taken together with our previous Cul2 mutation analysis these data suggest that development of sporadic and familial RCC is not com monly contributed to by genetic events altering the destruction domain of H IF-1 alpha, or components of the HIF-alpha destruction complex other than V HL itself. Although (a) activation of HIF could occur through mutation of a nother region of HIF-a, and (b) epigenetic silencing of elongin B/C, Cul2 o r Rbx1 cannot be excluded, these findings suggest that pVHL may represent t he sole mutational target through which the VCBR complex is disrupted in CC -RCC. HIF response is activated in CC-RCC tumorigenesis.