Volume 70, Issue 4, Pages 623-632 (October 2016) Renal Cell Carcinoma Programmed Death-ligand 1, a New Direct Target of Hypoxia- inducible Factor-2 Alpha, is Regulated by von Hippel–Lindau Gene Mutation Status  Yosra Messai, Sophie Gad, Muhammad Zaeem Noman, Gwenael Le Teuff, Sophie Couve, Bassam Janji, Solenne Florence Kammerer, Nathalie Rioux-Leclerc, Meriem Hasmim, Sophie Ferlicot, Véronique Baud, Arnaud Mejean, David Robert Mole, Stéphane Richard, Alexander M.M. Eggermont, Laurence Albiges, Fathia Mami-Chouaib, Bernard Escudier, Salem Chouaib  European Urology  Volume 70, Issue 4, Pages 623-632 (October 2016) DOI: 10.1016/j.eururo.2015.11.029 Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 1 von Hippel–Lindau mutation status is associated with programmed death-ligand 1 expression in clear cell renal cell carcinomas. Box plots represent the distribution of programmed death-ligand 1 mRNA expression (average fold change) according to von Hippel–Lindau mutation status for the different classifications (A) loss of heterozygosity, (B) number of altered alleles (1 corresponding to 0 and 1 altered allele), and (C) loss of function (LOF) (presence of LOF=0, absence of LOF=1) (n=32). PD-L1=programmed death-ligand 1. European Urology 2016 70, 623-632DOI: (10.1016/j.eururo.2015.11.029) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 2 Differential programmed death-ligand 1 (PD-L1) expression in 786-O cells transfected with different von Hippel–Lindau mutants. (A) PD-L1, carbonic anhydrase-IX and vascular endothelial growth factor expression were evaluated by SYBR Green quantitative real-time polymerase chain reaction. (B) Western blot was performed to show hypoxia-inducible factor-2 alpha, and PD-L1 protein levels. Actin was used as a loading control. (C) 786-O cells were incubated with interferon gamma for 24h. Western blot was performed to show PD-L1 protein levels. Actin was used as a loading control. (D and E) Surface expression levels of PD-L1 and PD-L2 were determined with flow cytometry. Data represents three independent experiments with standard deviation. Statistically significant difference (indicated by asterisks) between wild-type cells and different von Hippel–Lindau mutants are shown. CA-IX=carbonic anhydrase-IX; HIF-2α=hypoxia-inducible factor-2 alpha; IFN-γ; interferon gamma; PD-L1=programmed death-ligand 1; VEGF=vascular endothelial growth factor. * p<0.05. ** p<0.005. *** p<0.0005). European Urology 2016 70, 623-632DOI: (10.1016/j.eururo.2015.11.029) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 3 Hypoxia-inducible factor-2 alpha (HIF-2α) silencing decreased programmed death-ligand (PD-L1) expression in 786-O and A498 cells. 786-O and A498 cells were transfected with two different small interfering RNAs targeting HIF-2α or scrambled control. (A) Expression levels of HIF-2α, carbonic anhydrase-IX, vascular endothelial growth factor, and PD-L1 were evaluated using SYBR Green quantitative real-time polymerase chain reaction. (B and C) Western blot was performed to show HIF-2α and PD-L1 protein levels in 786-O (B) and A498 (C) cells. Actin was used as a loading control. (D and E) Surface expression levels of PD-L1 in 786-O (D) and A498 (E) cells were determined with flow cytometry. Data represents three independent experiments with standard deviation. Statistically significant difference between cells transfected with small interfering RNA targeting scrambled control or HIF-2α are shown. CA-IX=carbonic anhydrase-IX; CT=scrambled control; HIF-2α=hypoxia-inducible factor-2 alpha; IFN-γ; interferon gamma; PD-L1=programmed death-ligand 1; VEGF=vascular endothelial growth factor. * p<0.05. ** p<0.005. *** p<0.0005). European Urology 2016 70, 623-632DOI: (10.1016/j.eururo.2015.11.029) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 4 Hypoxia-inducible factor-2 (HIF-2) regulates the expression of programmed death-ligand 1 (PD-L1) by binding directly to the hypoxia-response element-4 in the PD-L1 proximal promoter in 786-O cells. (A) Chromatin immunoprecipitation assay was performed on 786-O cells transfected with either small interfering (si)RNA against HIF-2α or scrambled control and on wild-type (WT) cells using anti-HIF-2α antibody followed by SYBR Green quantitative real-time polymerase chain reaction using vascular endothelial growth factor-A and PD-L1 hypoxia response element (HRE) sites. (B) Different HREs in human PD-L1 promoter are shown. (C) 786-O and WT cells were cotransfected with pGL4-hRluc/SV40 vector and pGL3 empty vector, pGL3 HRE-4 or pGL3 HRE-4 MUT vectors. After 48h, Firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay and the ratio of Firefly:Renilla luciferase was determined. Data represent three independent experiments with standard deviation. Statistically significant difference (indicated by asterisks) are shown. (D) WT (VHL) cells were transfected with a pcDNA3 EV or pcDNA3-HIF-2α (3μg and 5μg). HIF-2α expression was detected with western blot. Actin was used as a loading control. (E) Surface expression levels of PD-L1 were determined using flow cytometry. Data represent three independent experiments with standard deviation. Statistically significant difference between cells transfected with pcDNA3 EV pcDNA3-HIF-2α are shown. (F–H) HIF-1α and HIF-2α regulate PD-L1 expression in RCC4 cell line. RCC4 cells were transfected with siRNAs targeting HIF-1α, HIF-2α, both HIF-1α and HIF-2α or scrambled control. (F) Expression levels of vascular endothelial growth factor and PD-L1 were evaluated by SYBR Green quantitative real-time polymerase chain reaction. (G) Western blot was performed to show HIF-1α, HIF-2α, and PD-L1 protein levels in RCC4. Actin was used as a loading control. (H) Surface expression levels of PD-L1 in RCC4 were determined with flow cytometry. The graph represents percentage of positive cells. Data represent two independent experiments with standard deviation. Statistically significant difference between cells transfected with siRNA targeting scrambled control or HIF-1α, HIF-2α,and HIF-1α + HIF-2α are shown. CA-IX=carbonic anhydrase-IX; CT=scrambled control; HIF-2α=hypoxia-inducible factor-2 alpha; HRE=hypoxia response element; IFN-γ; interferon gamma; IgG=immunoglobulin-G; PD-L1=programmed death-ligand 1; VEGF=vascular endothelial growth factor; WT=wild type. * p<0.05. ** p<0.005. *** p<0.0005). European Urology 2016 70, 623-632DOI: (10.1016/j.eururo.2015.11.029) Copyright © 2015 European Association of Urology Terms and Conditions