| Background Uveal melanoma(UM)is the most common ocular malignancy in adults,accounting for approximately 85% of all ocular tumors [1] Uveal melanoma is characterized by a high prevalence of liver metastasis and a poor prognosis[2].Once metastasized,there is no effective therapy,with average survival of 2 to 8 months [3].Molecular genetic analyses have shown that GNAQ or GNA11 mutations are the major driver mutations in UM [4].GNAQ/11 mutations lead to the constitutive activation of the encoded Gaq/11 proteins,which activate the downstream YAP signaling pathway and finally drive the occurrence and development of UM [5].The survival of GNAQ/11 mutant UM cells depends heavily on the transcriptional activity of YAP.As a transcriptional coactivator,YAP mainly regulates gene transcription by binding to the transcription factor TEAD proteins [6].The bromodomain and extraterminal domain(BET)family,including BRD2,BRD3,BRD4 and BRDT,are epigenetic readers and regulate gene expression.BET proteins have two highly conserved bromodomains(BRDs)that recognize and bind acetylated lysines of histones or other proteins,recruiting transcription factors to regulate gene transcription.BET inhibitors inhibit the function of BET proteins by competing for the bromodomains of BET proteins and thus preventing their binding to acetylated histones [7].Recently,it has been shown that BET inhibitors inhibit the growth of GNAQ/11 mutant UM cells [8].However,the underlying mechanisms remain unclear.Objectives Based on the findings that GNAQ/11 mutant cells are highly sensitive to BET inhibitors and that UM cells carrying GNAQ/11 mutations are dependent on the transcriptional activity of YAP for survival,this study aims to explore whether BET inhibitors inhibit GNAQ mutant UM cells through the Hippo-YAP pathway,and investigate the antitumor effects of BET inhibitors on GNAQ mutant UM cells in zebrafish embryo xenograft models and nude mice subcutaneous xenograft models.Our study is expected to provide scientific evidence for BET inhibitors as targeted therapeutic agents for UM patients with GNAQ mutations.Methods MTT method was used to study the sensitivity of UM cells with different mutant backgrounds to BET inhibitors.2.CRISPR/Cas9 and repair template were used to establish isogenic cell models of GNAQ mutations(knock-in).3.Western Blot was used to measure the protein levels of LATS1/2,p-YAP and YAP in cells treated with BET inhibitors.4.Rescue experiment was used to verify the potential link between YAP and hypersensitivity of GNAQ mutant UM cells to BET inhibitors.5.Dual-Luciferase Reporter Assay System,qPCR,CRISPR/Cas9 technology,Co-IP,ChIP-qPCR and Western Blot were used to explore the underlying molecular mechanism by which GNAQ mutant UM cells are hypersensitive to BET inhibitors.6.The in vivo antitumor activity of BET inhibitors was determined by zebrafish embryo xenograft models and nude mice subcutaneous xenograft models.Results1.GNAQ mutant UM cells were highly sensitive to BET inhibitors.2.BET inhibitors or knockdown of BRD4 inhibited YAP promoter luciferase activity and reduced the mRNA level of YAP.3.BET inhibitors blocked the interaction between BRD4 and YAP,and reduced YAP deposit on the promoter regions of its target genes CTGF and CYR61.4.Knockdown of YAP reduced BRD4 promoter luciferase activity and protein level.5.Zebrafish embryo xenograft models showed that BET inhibitor inhibited the migration of GNAQ mutant UM cells.Nude mice subcutaneous xenograft models showed that BET inhibitor suppressed the growth of GNAQ mutant UM.Conclusion1.GNAQ mutations render cells highly sensitive to BET inhibitors.2.BET inhibitors decrease YAP expression via targeting BRD4 instead of regulating Hippo pathway.3.BET inhibitors block the interaction of BRD4 and YAP,and inhibit the transcription-promoting activity of YAP on downstream target genes..4.BET inhibitors reduce BRD4 expression by inhibiting YAP.5.BET inhibitors display anti-migrative and anti-growth effects on GNAQ mutant UM xenografts. |
PDF Full Text Request