EZH2 Expression is increased in BAP1-mutant renal clear cell carcinoma and is related to poor prognosis

Abstract

Aim: BAP1 is frequently mutated in clear cell renal cell carcinoma (ccRCC) with a definitive role still unclear. Methods: In silico analysis of BAP1-mutant and wild-type gene enrichment and functional annotation in TCGA-KIRC dataset was performed. Target gene was studied based on functional clustering and was knowledge-based. Validation using in-house pathological sections were performed immunohistochemically. In vitro and in vivo studies on target gene were performed. Results: The TCGA ccRCC dataset included 534 ccRCC samples. BAP1 was frequently mutated and more frequently downregulated in ccRCC compared to normal kidney tissue or benign renal tumors. In the analysis between samples with BAP1 mutation (N = 33) and pan-negative (N = 33), we found that cancers with BAP1 mutation was significantly enriched for 14 pathways, of which 3 were DNA repair pathways, in which EZH2 played a role. CcRCC patients with lower BAP1 expression had poor prognosis and showed higher EZH2 expression, which also conferred worsened survival. Genetic and pharmaceutical inhibition of EZH2 not only inhibited BAP1-mutatn ccRCC cell viability and invasion but also abrogated genetic replenishing of BAP1 expression. Validation cohort encompassing 62 ccRCC samples confirmed the worsened phenotype for cases with higher EZH2 expression and significant positive correlation between expressions of EZH2 and BAP1. EZH2 inhibitor also inhibited tumor growth in xenograft mouse model with BAP1-mutated ccRCC cells with unremarkable toxicity. Conclusion: CcRCC with decreased BAP1 level has poor prognosis and is associated with higher EZH2 expression. Inhibition of EZH2 in BAP1-mutated entity holds promise for further investigation.

Keywords: BAP1-mutant; EZH2 Expression.

Fig 1

Loss of BAP1 is associated with renal cell carcinoma and prognosis. (A) Mutation frequency and types of BAP1 in clear cell renal cell carcinoma (ccRCC) reproduced from the cancer Genome Atlas (TCGA) database; (B) correlation between BAP methylation and mRNA expression; and B) relation between BAP1 copy number change and mRNA expression; Data from (C) HPA (left) and GDS2880 (right) and (D) ONCOMINE showing that mRNA level of BAP1 is significantly downregulated in human ccRCC. Representative immunohistochemistry (IHC) of BAP1 on benign renal and ccRCC tissues; (E) Overall survival between cases with higher or lower BAP1 expression in ccRCC and pRCC reproduced from TCGA database.

Fig 2

EZH 2 was upregulated in ccRCC with low BAP1 level. (A) Functional annotation and pathway analysis of enriched genes in BAP1 mutated ccRCC compared with pan-negative cases, reproduced from the cancer Genome Atlas (TCGA) database, showing 14 significantly changed pathways; (B) Correlation between expressions of BAP1 and EZH2 in TCGA-KIRC database; (C) Overall survival between cases with higher or lower EZH2 expression in ccRCC reproduced from TCGA database; (D) Reproduced from HPA, showing typical EZH1 positive cells in IHC.

Fig 3

Expression of EZH2 was higher in BAP1-mutant ccRCC cell line. (A) Efficacy of lentiviral delivery of BAP1 overexpression in 769-P ccRCC cells; (B) Efficacy of shRNA against BAP1 in 786-O ccRCC cells; (C) Protein levels of BAP1, EZH2, H3K27me3 in 769-P and 786-O cells with genetic manipulation of BAP1 status (**P < 0.01).

Fig 4

EZH2-KD inhibited oncogenesis of BAP1-loss in ccRCC. (A) EZH2-KD selectively effective in decreasing cell proliferation in 769-P cells harboring BAP1 mutation or 786-O cells with BAP1-KD, with similar selective effects in (B) cell cycle arrest and (C) cell apoptosis (*P<0.05, **P < 0.01).

Fig 5

Pharmaceutical inhibition of EZH2 selectively inhibited ccRCC with BAP1 mutation. EZH2 inhibitor EPZ011989 significantly and selectively inhibited (A) cell invasion, (B) migration, (C) colony formation, and (D) animal weight, and (E) tumor growth of ccRCC with BAP1 mutation or KD (*P<0.05, **P < 0.01).