RSUME is co-expressed with VHL and inhibits its tumor suppresor function

FUERTES, M.; GONILSKI, D.; TEDESCO, L.; ARZT, E.

Buenos Aires

Simposio; Fronteras en Biociencia 2; 2016

Instituto de Investigación en Biomedicina de Buenos Aires

Introduction: Inactivating mutations of pVHL provide a permissive setting for HIFs-alpha deregulation and development of high vascularized tumors. RSUME is highly expressed in tissues sensitives to VHL disease. Data from Renal Cell Carcinoma (RCC) samples (Cancer Genome Atlas Research Network) showed that 4% of the tumors expressed high RSUME levels and those patients exhibited a decrease in the survival rate. Objetives: Our aim were to investigate the action of RSUME on pVHL-dependent HIFs-alpha stabilization focused on the mechanism by which RSUME acts on pVHL, and RSUME impact on pVHL-related tumor progression.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-2alpha ubiquitination by RSUME. RSUME interacts with pVHL by, 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. Moreover RSUME effect on HIFalpha stability was impaired when non-sumoylable VHL variant was used, indicating that RSUME requires VHL SUMO modification. 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.