RSUME is co-expressed with VHL and inhibits its function

TEDESCO, L.; GEREZ, J.; BARONTINI, M.; ARZT, E.; GEREZ, J.; FUERTES, M.; ARZT, E.; FUERTES, M.; TEDESCO, L.; BARONTINI, M.

Boston

Simposio; 12th International VHL Medical Symposium; 2016

VHL Alliance

Introduction: Inactivating mutations of pVHL provide a permissive setting for HIFs-alpha deregulation and development of high vascularized tumors, such as hemangioblastomas, renal clear-cell carcinomas (RCC) and pheochromocytomas. RSUME (RWD-domain-containing sumoylation enhancer) is highly expressed in tissues sensitives to VHL disease. Data from RCC samples (Cancer Genome Atlas Research Network) showed that 4% of the tumors expressed high levels of RSUME and those patients exhibited a decrease in the survival rate. In hypoxia, RSUME increases HIF-1 stability and activity. Objetives: Our aim were to investigate: a) the action of RSUME on pVHL-dependent HIFs-alpha stabilization, b) the mechanism by which RSUME acts on pVHL, and c) RSUME impact on pVHL-related tumor progression.Methods: RSUME expression in human tumor samples and RCC-786-O cells was evaluated by RT-PCR, Western Blot (WB) and Immunofluorescence. HIFalpha stability was evaluated by WB and HIF stability reporter (ODD-LUC). VHL/RSUME interaction was evaluated by pull-down of recombinant proteins or by co-immunoprecipitation of COS-7 and RCC cell extracts. VHL sumoylation studies were performed by affinity purification of sumoylated proteins from transfected cells expressing His-SUMO-2 and by in vitro sumoylation assay.To asses RSUME action on VHL disease, RCC cells were transfected with VHL WT or mutants (Tyr112His, Arg167Gln or Leu188Val) and overexpressed or silenced RSUME. RCC-786-O stable clones expressing VHL (wt or mutated) and shRSUME or Scramble were injected in nude mice (n=4 each group, two independent experiments). HIF-2alpha, VEGF-A and vascularization were studied by immunostaining of tumor slides. Results: Human tumor samples analysis revealed that RSUME is expressed in pheochromocytoma and Hemangioblastoma, as well as RCC-786-O cells. In this cell line, RSUME stabilized endogenous HIF-2alpha only when pVHL was expressed, indicating that RSUME effect is on pVHL. The mechanism of action involves the inhibition of VHL-dependent HIF-2 ubiquitination by RSUME. RSUME interacts with pVHL by its β domain, impairing VHL/HIFalpha binding and the assembly the complex between pVHL, Elongins and Cullin-2 (ECV complex), critical for HIFalpha ubiquitination. Both in vitro and in vivo sumoylation assays showed that RSUME increases SUMO conjugation to pVHL. Furthermore RSUME increased HIFalpha stability similar to SUMO, supporting RSUME effect on pVHL. In line with its role on pVHL, RSUME exerts its action on another pVHL target. Thus, RSUME inhibited ERalpha VHL-dependent degradation.RSUME action on VHL disease was analyzed on representative VHL mutants (Tyr112His, Arg167Gln or Leu188Val). ODD-LUC showed that RSUME produces loss of pVHL function of VHL mutants and by RSUME knockdown all pVHL mutants became functional as pVHL. In vivo sumoylation assays revealed that VHL mutants are still targets of sumoylation. RSUME/pVHL interaction in VHL-related tumor progression was further confirmed by xenograft assay. RCC clones in which RSUME was knocked down expressing either pVHL WT or Type 2 mutation, had an impaired tumor growth, as well as HIF-2alpha, VEGF-A and vascularization. These results describe that RSUME is necessary for VHL mutation functional impact. Conclusion: This work shows a novel mechanism that contributes to VHL tumor progression and presents a new factor that could be target for pathologies with pVHL/HIF altered function.