USP10 Promotes Proliferation Of Hepatocellular Carcinoma By Deubiquitinating And Stabilizing YAP/TAZ

ObjectivesLack of clinically actionable targets and effective drugs lead to high mortality of Hepatocellular carcinoma(HCC).Aberrant activation of Yes-associated protein(YAP)and its paralogue,transcriptional coactivator with PDZ-binding motif(TAZ)play pivotal roles in promoting the initiation and progression of HCC.Therefore,suppressing both YAP and TAZ will provoke an effective antitumor response for HCC treatment.However,as transcriptional coactivators,both YAP and TAZ remain technically challenging to target.Seeking for alternative therapeutic strategies,emerging pathogenic insights unveiled the landscape of molecular aberrations of YAP/TAZ regulation.Deubiquitinating enzymes(DUBs)are intrinsically appealing for drug targeting with well-defined catalytic clefts,capable of cleaving ubiquitins on its substrates,and altering their abundance level and transcriptional activities.In our preliminary study,we identified Uubiquitin-specific peptidase 10(USP10)as a novel regulator which physically deubiquitinated and functionally stabilized YAP/TAZ.Therefore,our study aims to clarify the mechanism between USP10 and YAP/TAZ,and determine the pathological roles of USP10 in HCC tumorigenesis to provide a rationale for potential therapeutic interventions in the treatment of HCC patients harboring a high level of YAP/TAZ.Methods(1)Identification of USP10 as a new regulator YAP/TAZ signaling: To search for DUBs critical for YAP/TAZ signaling,we performed a gain-of-function screen by monitoring YAP/TAZ-dependent transcriptional luciferase reporter(8× GTIIC)and TAZ abundance luciferase signal(WWTR1-Luciferase).We screened 98 DUBs for which we employed a pool of four non-overlapping siRNA oligos for transfection experiments in HepG2 cells.Western Blotting was performed to detect the effect of USP10 on YAP/TAZ protein abundance.qRT-PCR assay was used to investigate the mRNA levels of YAP/TAZ by USP10 overexpression or silencing.(2)Molecular mechanism of USP10 regulation on YAP/TAZ: Western Blotting was performed to test the deubiquitinase activity of USP10 on YAP/TAZ regulation.Proteasome inhibitor MG132 were utilized to detect whether USP10 regulated the turnover of YAP/TAZ via the ubiquitin-proteasome pathway.The interaction between USP10 and YAP/TAZ were detected via immunoprecipitation and Western Blotting.In vivo and in vitro deubiquitinating assay combined immunoprecipitation and Western Blotting were performed to investigate the role of USP10 on YAP/TAZ ubiquitination.(3)USP10 promoted HCC proliferation through stabilizing YAP/TAZ: In vitro cell proliferation assay were performed to determine the effect of silencing USP10 on multiple HCC cell lines.Clony formation assay was performed to determine the effect of silencing USP10 on multiple HCC cell lines.Nude mouse xenograft model of HepG2 cell and patient-derived xenografted(PDX)were introduced to evaluate the role of USP10 in the malignant progression of HCC in vivo.(4)DEN-induced tumorigenesis of liver and clinical significance analysis of USP10-YAP/TAZ activation in liver cancer tissues: Immunohistochemistry was performed to detect the expression and correlation of USP10-YAP/TAZ in clinical HCC patients.DEN-treated tumorigenesis of liver was introduced to detect the expression of USP10 and YAP/TAZ in tumoral tissues and normal liver tissues.Clinical TCGA database analysis(Ualcan,Kaplan meier,and GEPIA)were performed to detect USP10-YAP/TAZ axis activation in the malignant progression of HCC and correlation of the prognosis of HCC patients.Results(1)Identification of USP10 as a new regulator YAP/TAZ signaling: DUBs siRNA screening assay showed that,among 98 DUBs,USP10 siRNA exert the strongest inhibitory effect on YAP/TAZ transcriptional activity and abundance.Overexpression of USP10 significantly increased YAP/TAZ protein levels while silencing USP10 downregulated YAP/TAZ abundance.qRT-PCR assay showed that USP10 had no impact on YAP/TAZ mRNA levels.(2)Molecular mechanism of USP10 regulation on YAP/TAZ: USP10 regulated YAP/TAZ protein levels through its deubiquitinase activity.USP10 depletion induced YAP/TAZ to be subjected to proteasomal degradation.Immunoprecipitation assay suggested USP10 directly interacted with YAP/TAZ.In vitro and in vivo deubiquitinating assay showed that USP10 removed the ubiquitin chains on YAP/TAZ through interaction.(3)USP10 promoted HCC proliferation through stabilizing YAP/TAZ in vitro and in vivo: In vitro cell proliferation assay showed that USP10 depletion significantly impaired HCC cell proliferation and colony formation.In vivo HepG2 xenografted and PDX models showed that USP10 depletion significantly reduces YAP/TAZ abundance and consequently suppresses tumor growth while the introduction of YAP-5SA attenuated the growth inhibition mediated by USP10 shRNA.(4)DEN-induced tumorigenesis of liver and clinical significance analysis of USP10-YAP/TAZ activation in liver cancer tissues: Immunohistochemistry of USP10 and YAP/TAZ showed that both USP10 and YAP/TAZ were highly evelated in clinical HCC patients and statistically significant correlations were found between USP10 and YAP/TAZ levels.DEN-treated hepatocellular tumorigenesis suggested that the expressions of both USP10 and YAP/TAZ were substantially elevated in the tumor tissues compared with normal liver tissues.In order to confirm the clinical significance of USP10 by using The Cancer Genome Atlas(TCGA)database,most human HCC samples exhibited markedly higher expression levels of USP10 than non-tumorous tissues.Moreover,the USP10 expression level was correlated with the risk of HCC in patients.In addition,an apparent positive correlation was detected between the expression level of USP10 and those of YAP/TAZ target genes(CTGF,AMOTL2,and CYR61)in HCC patients.ConclusionIn this study,we identify that USP10 directly interacts with and stabilizes YAP/TAZ by reverting their proteolytic ubiquitination and promotes HCC proliferation.Depletion of USP10 arrests the proliferation of HCC both in vitro and in vivo.Introduction of active YAP attenuates the growth inhibition mediated by USP10 depletion.The high abundance of USP10 in both HCC tissues and DEN-induced hepatocellular tumorigenesis are significantly positive correlated with YAP/TAZ abundance and transcription activity.Aberrant activation of USP10 in HCC confirmed the key role of USP10 as a profound tumor-promoting factor,and therefore provided a novel therapeutic target for the HCC intervention.