RSUME is involved in missense VHL mutation-loss of function

GONILSKI PACIN, D; TEDESCO, L; FUERTES, M; ELGUERO, B; ARZT, E

Houston

Simposio; 13th International VHL Medical/Research Symposium; 2018

VHL Alliance

Introduction: In VHL disease HIFs-a deregulation caused by mutations in VHL results in the development of high vascularized tumors such as renal clear-cell carcinomas (RCC). Some missense mutations on VHL, described in the majority of Type 2 VHL tumors, retain a partial function on HIF downregulation. RSUME or RWDD3 (RWD-domain-containing sumoylation enhancer) is highly expressed in VHL-related tumors. A recent analysis carried out by The Human Protein Atlas shows that 20.07% of RCC tumors present high levels of RSUME mRNA, which correlates with a decrease in survival rate. Objetives: Our aim is to investigate the action of RSUME on VHL Type 2 loss of function and the mechanism by which RSUME acts on type 2 VHL-related angiogenesis.Methods: RSUME and HIF2a expression or stability in RCC-786-O and in COS-7 cells were evaluated by Western Blot (WB). HIF2a activity was evaluated by HRE-LUC. RSUME/VHL variants interaction was studied by co-immunoprecipitation. VHL sumoylation was analyzed by affinity purification of sumoylated proteins.To asses RSUME action on VHL disease, we generated RCC-786-O clones expressing either Leu188Val Type2 VHL mutant or a variant defective for sumoylation (VHLL188V/K171R), in which RSUME was silenced by shRNA. VEGF mRNA was measured in theses clones, they were used for tubulogenesis ?in vitro? assay and injected in nod-scid mice (n=4 each group, two independent experiments) for ?in vivo? angiogenesis assay.Results: VHL Type 2 representative mutants (Tyr112His, Arg167Gln and Leu188Val) failed to downregulate RSUME protein levels compared to VHL WT, generating a permissive context for high RSUME found in VHL-related tumors. RSUME potentiated missense VHL mutants loss of function, increasing HIF-2 stabilization and activity. RSUME mechanism of action involves a decrease. Type 2 VHL-HIF binding. RSUME disrupts ECV complex assembly. type 2 VHL mutants are targets of sumoylation, but HIF-2 stabilization was mediated by a VHL sumoylation independent mechanism confirmed by using type 2 VHL variants deficient for sumoylation.RCC clones in which RSUME was knocked-down resulted in a gain of function of wild type and mutant VHL. These clones showed impaired VEGF-A expression and vascularization both in vitro and in in vivo. Conclusion: These results show a mechanism for VHL type 2 loss of function and highlight RSUME as a potential biomarker of the VHL disease outcome.