The Role And Mechanism Of USP14-TAZ Positive Feedback Loop In Driving The Progression And Metastasis Of Pancreatic Cancer

Objective:Many studies have shown that targeting Hippo signaling pathway,especially the downstream transcriptional co-activator TAZ,can effectively inhibit tumor progression.However,TAZ is not suitable for drug development targeting enzyme activity.DUB plays a very important role in the regulation of Hippo signaling pathway,and DUB is more suitable for drug research and development.Therefore,DUB library is used to screen DUB for regulating TAZ in pancreatic cancer,and providing molecular targets for the treatment of pancreatic cancer,and looking for targeted drugs.Methods:Bioinformatics analysis was used to detect the correlation between Hippo signaling pathway and its key genes and the occurrence and development of pancreatic cancer.To identify the DUB（s）that potentially regulate TAZ function,we transfected 81DUBs into human pancreatic cancer cell lines,monitored 8x GTIIC-luciferase reporter activity and monitored the protein level of TAZ by western blot.Molecular biology technology was used to overexpress or scilence USP14,and western blot and q RT-PCR were used to detect the expression of the Hippo signaling pathway.Coimmunoprecipitation（Co-IP）,immunofluorescence（IF）analysis and GST pull down were used to investigate whether there is a physical interaction between the TAZ and USP14 protein,and identify the domains involved in the USP14-TAZ interaction.To investigate the mechanism of USP14-mediated TAZ protein upregulation,PC cells were treated with the proteasome inhibitor MG-132 or the protein synthesis inhibitor cycloheximide（CHX）.To determine whether USP14-mediated stabilization of the TAZ protein is mediated by deubiquitination,in vivo and in vitro deubiquitination assays were performed to examine the ubiquitination of the TAZ protein.Western blot and q RT-PCR were used to analyse the expression level of USP14 in PC cell with TAZ overpression.To examine the potential mechanism of USP14 gene regulated by TAZ,a series of USP14 promoter with mutated TEAD binding sites was constructed and subjected to dual-luciferase reporter assays.Constructed and validated the stable USP14 overexpression and knockdown PC cell lines,and examined the role of USP14in PC cell proliferation,invasion and metastatic in vitro and in vivo.Bioinformatics analysis and fresh matched PC surgical tissues were used to examine the expression of USP14 and analyse the overall survival rates and prognosis according to USP14 m RNA expression levels.To examine the role of USP14-specific inhibitor IU1 in PC treatment,PC cells were treated with IU1 and examine the cell proliferation,invasion and metastatic in vitro and in vivo.Results:Hippo signaling pathway is dysregulated in PC,and the disorder of Hippo pathway is closely correlated with the initiation and progression of PC.The expression of TAZ was much higher in the PC specimens than in paracarcinoma and normal pancreatic tissues,and the patients with higher TAZ m RNA expression levels had decreased overall survival rates and poorer prognosis.The dual-luciferase reporter assays and western blot demonstrated that USP14 became the most promising candidate in regulating TAZ function.USP14significantly increased the TAZ protein level,but the m RNA level of TAZ was not significantly altered when USP14 expression was changed.TAZ and USP14 protein exist a physical interaction,and the domain between transactivation domain and PDZ-binding domain in TAZ and the domain between catalytic domain in USP14 contribute to their interaction with each other.Knockdown of USP14 significantly reduced the TAZ protein level,which was restored in cells treated with the proteasome inhibitor MG-132.The half-life of the TAZ protein was increased in USP14 overexpression cells,while USP14 depletion resulted in accelerated degradation of TAZ.Knockdown of USP14 resulted in increased polyubiquitination of TAZ compared with the control group,and ectopic overexpression of USP14 notably reduced the ubiquitination level of TAZ,whereas the USP14^C114A mutant lost the ability to induce this effect.USP14 significantly decreased K48-linked polyubiquitination of TAZ but had no apparent effect on its K63-linked polyubiquitination.The m RNA and protein levels of USP14 were significantly increased in TAZ-overexpression and inactivation of Hippo signalling.Konckdown of USP14 significantly decreased the proliferation and liver metastasis in vitro and in vivo,whereas ectopic overexpression of USP14 led to the opposite phenotype.USP14 was significantly upregulated in PC tissues compared to paired or unpaired adjacent tissues.Kaplan–Meier survival analysis showed that PC patients with higher USP14 m RNA expression levels had decreased overall survival rates and poorer prognosis.IU1 significantly decreased the proliferation and liver metastasis of PC cells compared to the corresponding vehicle-treated cells in vitro and in vivo.IU1 administration reduced the tumour growth rate and weight,the number of livers micrometastases and the size of nidus,which further improved the median overall survival.Conclusion:Hippo signaling pathway is dysregulated in PC;USP14 is a novel regulator of TAZ in the Hippo pathway in PC.TAZ and USP14 proteins exist a physical interaction,and USP14 increased the expression of TAZ by decreasing K48-linked polyubiquitination of TAZ.USP14 is regulated by TAZ at the transcriptional level.USP14 promotes pancreatic cancer progression in a TAZ-dependent manner.USP14 was higher expression and indicated poorer prognosis in PC.The expression abundance of USP14 and TAZ owned a positive correlation.The USP14-specific inhibitor IU1 inhibited progression and liver metastasis in PC.