von Hippel-Lindau mutants in renal cell carcinoma are regulated by increased expression of RSUME

Abstract

Renal cell carcinoma (RCC) is the major cause of death among patients with von Hippel-Lindau (VHL) disease. Resistance to therapies targeting tumor angiogenesis opens the question about the underlying mechanisms. Previously we have described that RWDD3 or RSUME (RWD domain-containing protein SUMO Enhancer) sumoylates and binds VHL protein and negatively regulates HIF degradation, leading to xenograft RCC tumor growth in mice. In this study, we performed a bioinformatics analysis in a ccRCC dataset showing an association of RSUME levels with VHL mutations and tumor progression, and we demonstrate the molecular mechanism by which RSUME regulates the pathologic angiogenic phenotype of VHL missense mutations. We report that VHL mutants fail to downregulate RSUME protein levels accounting for the increased RSUME expression found in RCC tumors. Furthermore, we prove that targeting RSUME in RCC cell line clones carrying missense VHL mutants results in decreased early tumor angiogenesis. The mechanism we describe is that RSUME sumoylates VHL mutants and beyond its sumoylation capacity, interacts with Type 2 VHL mutants, reduces HIF-2α-VHL mutants binding, and negatively regulates the assembly of the Type 2 VHL, Elongins and Cullins (ECV) complex. Altogether these results show RSUME involvement in VHL mutants deregulation that leads to the angiogenic phenotype of RCC tumors.

Fig. 1. Increased RSUME expression is correlated with VHL missense mutations and poor survival in RCC patients.

a Representative immunohistochemistry (IHC) staining for RSUME in three ccRCC patient samples. Scale bars 100 µm and 50 µm as indicated. b Kaplan–Meier analysis in patients stratified as having high (violet line) RSUME expression or low (blue line) RSUME expression (expression cutoff 2.9 FPKM), publically available from The Human Protein Atlas. c Box plots comparison between RSUME mRNA expression in a patient stratification of the Additional file 8: Table S1 data (where VHLwt = 207 samples without mutations in VHL gene, VHLmut = 245 samples with mutations in VHL gene). *Significant differences between sample groups distribution with a P = 0.048, < 0.05 (Mann–Whitney U-test). d Box plots comparison between RSUME mRNA expression in a patient stratification of the Additional file 8: Table S1 data with mutations in VHL cohort (VHLmut) into tumor stages (where stage I–II represent samples corresponding to tumor stages I and II = 150 samples; stage III represents samples corresponding to tumor stage III = 58 samples; stage IV represents samples corresponding to tumor stage IV = 37 samples). Significant differences between sample group means with a P = 0.036 (_*) and 0.021 (#), < 0.05 (ANOVA and post hoc Tukey tests). e, f Box plots comparison between RSUME mRNA expression in a patient stratification of the Additional file 8: Table S1 data in stage IV cohort into mutation types (where VHLwt = 34 samples without mutations in VHL gene, VHLmut = 37 samples with mutations in VHL gene, VHLmiss = 18 samples with mutations missense in VHL gene only). *Significant differences between sample groups distribution with a P = 0.012 (5e), and 0.027 (5f), < 0.05 (Mann–Whitney U-test)

Fig. 2. RSUME potentiates VHL type 2 mutants loss of function.

a RCC-786-O and b–f COS-7 cells were transfected with the following vectors as indicated in each panel: 0.5 µg of Flag-VHL variants, 0.5 µg of V5-RSUME, 0.1 µg of HA-HIF-2α, 0.7 µg HRE-LUC reporter vector, 0.5 µg RSV-β-galactosidase or the corresponding empty vectors. a, b Forty-eight hours post transfection cells were lysed or c–e 48 h post transfection cells were incubated for 4 h in hypoxia (HPX) and then harvested in normoxia (NMX) at the indicated times. Cells extracts were analyzed by WB using the indicated antibodies. β-actin was used as a loading control. One representative experiment from three experiments with similar results is shown. f Twenty-four hours post transfection Firefly luciferase (LUC) was measured. Each value was normalized by RSV-β-galactosidase activity. Results are expressed as mean ± SEM of triplicates of one representative experiment of three experiments with similar results. *P < 0.05 compared with cells transfected with VHL empty vector (bar 1). **P < 0.01 compared each VHL variant with the corresponding cells transfected with RSUME empty vector. #P < 0.05 compared with cells transfected with VHL (bar 3) (one-way ANOVA followed by Scheffé’s test)

Fig. 3. RSUME acts on VHL mutants in a sumoylation independent manner.

a, b COS-7 cells were transfected with the following vectors: 0.6 μg of each Flag-pVHL variant, 0.6 μg of 6xHis-SUMO-2, 0.1 μg of V5-Ubc-9 expression vectors, 20 μM siRNA against RSUME or Scramble as a control. Forty-eight hours post transfection cells were harvested, lysed and an aliquot of the lysates was analyzed by WB (Input). The remaining extracts were subjected to Ni²⁺ affinity chromatography to purify 6xHis-SUMO-2 (Ni-NTA). Purified fraction and Inputs were analyzed by WB using Flag antibody. One representative experiment from two independent experiments with similar results is shown. c–e COS-7 cells were transfected with the following vectors: 0.7 μg of HRE-LUC reporter vector, 0.2 μg of RSV-β-galactosidase control vector and/or 0.5 μg of V5-RSUME and/or 0.5 μg of Flag-pVHL or Flag-VHL_K171R, 0.5 μg of indicated Flag-VHL variants and/or respective empty vector as control. c Twenty-four hours post transfection Firefly luciferase (LUC) was measured. Each value was normalized by RSV-β-galactosidase activity. Results are expressed as mean ± SEM of triplicates of one representative experiment of three experiments with similar results. *P < 0.05 and **P < 0.01 compared with cells transfected with the corresponding RSUME empty vector, ^#P < 0.05 compared to VHL empty vector without RSUME (one-way ANOVA followed by Scheffé’s test). d, e Forty-eight hours post transfection cells extracts were Immunoprecipitated with anti-FLAG antibodies. Immunoprecipitated fractions and extract aliquots (Input) were analyzed by WB using the indicated antibodies. One representative experiment from three independent experiments with similar results is shown. f–h RCC 786-O cells were transfected with 1.0 μg of Flag-VHL indicated variant and/or 1.5 μg of V5-RSUME expression vectors. Forty-eight hours post transfection cells were lysed and extracts were analyzed by WB using the indicated antibodies. β-actin was used as a loading control. One representative experiment from two independent experiments with similar results is shown

Fig. 4. RSUME potentiates VHL mutants loss of function by decreasing VHL–HIF interaction and ECV complex assembly.

a–d COS-7 cells were transfected with 0.5 µg of Flag-VHL variants, 0.5 µg of V5-RSUME, 0.5 µg of HA-HIF-2α, 0.1 μg of Gam-1, Gam-1_MUT control vector. a Forty-eight hours post transfection cells were incubated in 2% serum, 5 μM MG-132 for 6 h and lysed. A first immunoprecipitation (IP) using anti-FLAG antibody was performed. Immunoprecipitated fractions were eluted with soluble FLAG peptide and a second IP using anti-V5 antibodies was performed. Immunoprecipitated fractions from the second IP “Tandem IP” and extract aliquots (Input) were analyzed by WB using the indicated antibodies. b–d Cells extracts were immunoprecipitated with anti-FLAG antibodies. Immunoprecipitated fractions and extract aliquots (Input) were analyzed by WB using the indicated antibodies. One representative experiment from three independent experiments with similar results is shown. e Cells were transfected with 0.5 μg of the indicated Flag-VHL-GFP variants, 0.5 μg of Flag-Elongin C, 0.5 μg of HA-Elongin B, 0.5 μg of Cullin-2 and/or 0.5 μg of V5-RSUME expression vectors. Forty-eight hours post transfection cells were lysed and lysates immunoprecipitated with GFP antibody. Immunoprecipitated fractions and extract aliquots (Input) were analyzed by WB using the indicated antibodies. One representative experiment from two independent experiments with similar results is shown

Fig. 5. RSUME impacts on VHL mutants function and increases early angiogenesis.

a RCC 786-O clones were lysed and VEGF mRNA levels were analyzed by quantitative real-time RT-PCR in triplicates. Values are given as mean ± SEM after normalization to RPL19. *P < 0.05 and **P < 0.01 compared with the corresponding shScramble clone (one-way ANOVA followed by Scheffè’s test). b, c The conditioned medium from RCC 786-O clones was tested for tube formation assay in vitro. In all, 15,000 Eahy.926 cells/well were incubated with 100 µl of conditioned media. Photographs were taken 18 h post incubation using an inverted microscope (magnification: 5 × ). Scale bar 400 µm. One representative picture for each clone of two independent experiments is shown. d, e Quantification of number of capillary/tube-like structures in b and c. Values are mean from two experiment ± SEM. Three independents wells were analyzed per experiment. P-value was determined by Kruskal–Wallis followed by a Dunn’s test. *P < 0.05 and ** = P < 0.01 compared with the corresponding shScramble clone. f, g RCC 786-O clones (10⁶ cells, each clone) were intradermal injected in the right flank of 6–8-weeks male NOD/SCID mice. Vehicle was injected in the left flank (contralateral flank). After 7 days, the skin was removed and photographs were taken under magnification glass. Pictures representative of each condition with similar results to the others of each group are shown. Scale bar 5 mm. h, i Quantification of vessel density calculated (number of vessels_{cells side} – number of vessels_{vehicle side})/Area. Results obtained from two independent experiments with four independent pictures for each condition are expressed as mean ± SEM. *P < 0.05 and **P < 0.01 compared with the corresponding Scramble clone (Kruskal–Wallis followed by a Dunn’s test)

Fig. 6. Proposed model for RSUME increase in VHL tumors and its action on VHL mutants function.

VHL missense Type 2 mutants fail in negatively regulate RSUME levels, leading to a permissive context for increased RSUME in VHL-related tumors, like RCC. By protein–protein interaction with VHL mutants, RSUME reduces HIF-2α-VHL binding and negatively regulates the assembly of the ECV complex. By this mean, RSUME regulates VHL Type 2 mutants loss of function and worsens HIF transcriptional activity deregulation, thus leading to increased angiogenesis and tumor progression