Functional Study Of BAP1 In The Progression Of Clear Cell Renal Cell Carcinoma

Next-generation sequencing studies have shown that epigenetic modifiers such as PBRM1,SETD2,BAP1,and KDM5 C are frequently mutated in RCC and other tumors.These proteins are involved in regulating diverse cellular processes,including DNA repair,cytoskeletal remodeling,and transcription.BRCA1-associated protein 1(BAP1)is a member of the ubiquitin C?terminal hydrolase family of deubiquitinating enzymes and is implicated in DNA repair,cell cycle and transcription regulation.The BAP1 gene is mutated in about 10% of patients with clear cell renal cell carcinoma(ccRCC),the most common form of renal cancer.Previous studies have proposed that BAP1 functions as a tumor suppressor,but interestingly,an increasing number of studies have demonstrated that depletion of BAP1 inhibits the proliferation of various tumorigenic or nontumorigenic cell types,and germline mutation or low expression of BAP1 correlates with long-term survival of patients with mesothelioma.In the present study,through analysis of datasets in The Cancer Genome Atlas we found that low expression of WT BAP1 in ccRCC correlated with longer overall survival,suggesting that BAP1 may promote tumor progression in ccRCC.Our study showed that BAP1 was involved not only in chromatin remodeling but also in RNA processing and translation,as illustrated by enrichment of the ubiquitinated forms of these proteins in BAP1 KO cells.These results suggest that BAP1 can regulate gene expression at the epigenetic,transcriptional,and post-transcriptional levels.Our quantitative proteomic analysis revealed that BAP1 KO altered proteostasis in ccRCC cells,especially the expression of proteins associated with cell motility,cytoskeletal organization,and cell proliferation.These properties of BAP1 were confirmed in functional assays of ccRCC cell motility and invasion.The EMT markers Snail,N-cadherin,and vimentin were downregulated in BAP1 KO cells,and importantly,we demonstrated that BAP1 regulates Snail transcription by directly deubiquitinating H2 A in the Snail gene region.Consistent with the MET phenotype,the activities of Rho A,Rac,and Cdc42 GTPases were all significantly decreased by BAP1 KO,resulting in a striking loss of stress fiber and pseudopodia formation.The observed suppression of cell growth in BAP1 KO ccRCC cells could be at least partially attributed to a decrease in protein synthesis.The same phenotypes were observed in two different ccRCC cell lines,and our rescue experiments demostrted that these phenotypes were dependent of BAP1 and its catalytic activity.In addition,we also observed similar phenomena in A2780,an ovarian cancer cell line.In summary,we have demonstrated that inactivation of BAP1 in ccRCC cells altered cellular proteostasis,suppressed cell proliferation and induced a MET-like phenotype.Importantly,Snail and Rho family GTPases played fundamental roles in the biological processes regulated by BAP1.Our results also suggested that BAP1 played context-dependent roles in tumor progression.