USP49/YAP1 Positive Feedback Loop Promotes Gastric Cancer Progression

Background:The importance of the Hippo-Yes-associated protein 1(YAP1)pathway in gastric carcinogenesis and metastasis has attracted considerable research attention;however,the regulatory network of YAP1 in gastric cancer(GC)is not completely understood.The ubiquitin-proteasome pathway is the most important posttranscriptional regulation of YAP1.To date,only a few deubiquitinases(DUBs),such as OTUB2,USP10,and USP47,have been reported to promote YAP1 deubiquitination and the consequent stabilization of YAP1 activity.Moreover,the regulatory effect of DUBs on YAP1 activity has not been reported in GC cells.Methods:Ubiquitin-specific peptidase 49(USP49)was identified as a new DUB contributed to the deubiquitination of YAP1 protein in GC cells by high-throughput screening,dual luciferase reporter gene,RT-q PCR and Western blotting assays.Through in vitro cell phenotype experiments such as CCK8 proliferation,Transwell invasion and migration,clone formation,and chemotherapy resistance assays,and in vivo tumor-bearing models such as cell-line-derived xenograft(CDX)and patient-derived xenograft(PDX)models,the biological function of USP49 in gastric cancer was clarified.The interaction between USP49 and YAP1 was studied by molecular docking,protein co-immunoprecipitation experiments and GST-Pull down experiments.Double luciferase reporter gene and chromatin coprecipitation were used to explore the regulatory effect of YAP1 on USP49 gene promoter.Finally,we collected tissue samples and clinical follow-up information from 482 GC patients.Further immunohistochemistry was used to identify the expression level and correlation between USP49 protein and YAP1 and its target genes connective tissue growth factor(CTGF)and cysteine-rich angiogenic inducer 61(CYR61)protein in GC and adjacent normal tissues.Kaplan-Meier plotter(KM plotter)prognostic analysis and COX regression analysis were used to study the correlation between USP49 expression and prognosis of patients with GC.Results:In this study,USP49 was identified as a novel deubiquitinase of YAP1,knockdown of USP49 inhibited the proliferation,metastasis,chemoresistance,and peritoneal metastasis of GC cells.Overexpression of USP49 showed opposing biological effects.Moreover,USP49 was transcriptionally activated by the YAP1/TEAD4 complex,which formed a positive feedback loop with YAP1 to promote the malignant progression of GC cells.The analysis results of clinical samples showed that USP49 expression was high in GC tissues and positively correlated with the expression of YAP1 and its target genes,CTGF and CYR61.KM plotter survival and COX regression analysis showed that high USP49 expression was associated with poor prognosis and was an independent prognostic factor.Moreover,patients with high USP49 and YAP1 expression had extremely shorter overall survival.Conclusion:This study demenstrated that USP49 can not only promote the cancer promoting activity of YAP1/TEAD4 complex in GC cells by direct binding and deubiquitination of YAP1,but also as a noval target of YAP1 to form USP49/YAP1 positive feedback loop thereby amplifing the cancer promoting effect.Therefore,the findings of this study reveal that the aberrant activation of the USP49/YAP1 positive feedback loop plays a critical role in the malignant progression of GC,thus providing a novel prognostic factors and therapeutic targets for GC.