RSUME stabilizes HIF through VHL tumor suppressor inhibition

DAVID GONILSKI PACIN; LUCAS TEDESCO; EDUARDO ARZT; MARIANA FUERTES

Whistler, British Columbia

Simposio; 2017 Keystone Symposia Conference "Adaptations to Hypoxia in Physiology and Disease"; 2017

Keystone

HIFs- deregulation results in development of aggressive high vascularized tumors. In particular in von Hippel-Lindau disease, inactivating mutations on VHL provide a permissive setting for pathological HIFs-alpha stabilization.RSUME (RWD-domain-containing sumoylation enhancer), or RWDD3, is expressed in hypoxic stress and increases HIF-1 stability and activity. Interestingly, RSUME is highly expressed in tissues sensitives to VHL.Our aim is to investigate the action of RSUME on VHL-dependent HIFs-alpha stabilization and VHL-related tumor progression.HIFs-alpha stability was studied in COS-7 and RCC-786-O cells (VHL -/-). RSUME reverted endogenous and overexpressed VHL action in COS-7, whereas in RCC-786-O, only expressing VHL, indicating that RSUME effect is on VHL. RSUME action involves inhibition of VHL-dependent ubiquitination. VHL/RSUME interaction was proved by pull-down and immunoprecipitation. RSUME impaired VHL/HIFalpha binding and the assembly of VHL, Elongins and Cullin-2 complex, critical for ubiquitination. Purification of sumoylated proteins showed that RSUME increases SUMO conjugation to VHL. Remarkably, RSUME effect on VHL was significantly decreased using a non-sumoylated VHL (VHLK171R), showing that RSUME-dependent sumoylation is instrumental in inhibiting VHL function.RSUME expression was found in VHL tumors (RCC, Pheochromocytoma and Hemangioblastoma). RSUME action on VHL disease was analyzed on representative VHL mutants. ODD-LUC and WB showed that RSUME retains interaction with VHL mutants, enhances their sumoylation and potentiates their loss of function.RSUME impact in VHL-related tumorigenesis was further confirmed by xenograft assay: RCC clones in which RSUME was silenced expressing VHL WT or Type2 mutation, had impaired tumor growth, HIF-2, VEGF-A and vascularization.These results describe RSUME as a new regulatory factor involved in VHL (and VHL mutation) function.