VHL-P138R and VHL-L163R novel variants: mechanisms of VHL pathogenicity involving only HIF-dependent functions?

MATHÓ, CECILIA; LIU, XIANDE; VIEITES, ANA; BARONTINI, MARTA; SANSÓ, GABRIELA; JONASCH, ERIC; PENNISI, PATRICIA

Puerto Varas

Congreso; XXV Reunión Anual de la Sociedad Latinoamericana de Endocrinología Pediátrica (SLEP); 2015

Sociedad Latinoamericana de Endocrinología Pediátrica (SLEP)

Introduction: von Hippel Lindau disease is an autosomal dominantcancer syndrome caused by mutations in the VHL tumorsuppressor gene. VHL protein (pVHL) forms a complex (VBC)with elongins B-C, Cullin2 and Rbx1. The most described functionof pVHL is to recognize and target hypoxia inducible factor (HIF)degradation. We found new VHL variants in VHL families thatneed to be functionally characterized to determine their pathogenicity.Aim: Perform the in vitro functional characterization of L163Rand P138R variants of pVHL. Materials and methods: VHL variantswere generated using the VHL-wt-Venus plasmid as a templateto create 786-0 stable cell lines expressing Venus, VHL-wt-Venus, VHL-P138R-Venus and VHL-L163R-Venus. Westernblots were performed to evaluate pVHL and HIF-2α levels. VHL,EPAS1 and VEGFA expression was quantified by qPCR. VHL proteinhalf-life was determined by cycloheximide treatment. VBCcomplex formation was evaluated by immunoprecipitation (IP)using GFP-Trap beads followed by western blot.Results: Stable cell lines showed similar mRNA VHL expressionof variant and wild type VHL. However there was a markedDownloaded by:XXV Annual Meeting, SLEPPuerto Varas, Chile32 Horm Res Paediatr 2015;84(suppl 2):1?77difference in half -life of wt pVHL and P138R and L163R variantsas early as 1 h after treatment with cycloheximide indicating theseare less stable than wt pVHL. HIF-2α levels decreased when wtpVHL was reintroduced, and intermediate levels were observed inthe presence of the variants. To assess HIF-2α activity we performedqPCR of EPAS1 and VEGFA (a downstream target of HIF-2α) in 20% oxygen and observed no differences in their expressionin wt pVHL and VHL variants. VBC complex formation assessedby IP revealed complex formation was decreased for P138R andL163R variants compared to wt pVHL.Conclusions: Striking differences were observed in P138Rand L163R half-lives. We also observed decreased VBC complexformation with P138R and L163R, although HIF-2α target geneexpression was not different between wt pVHL and VHL variantsunder normoxic conditions. Taken together, our resultssuggest that P138R and L163R pathogenic mechanisms may involveHIF dependent mechanisms, but the reduced half life ofVHL mutant proteins could impact HIF independent VHL functionsas well.