UCHL3 Promotes The Progression Of Anaplastic Thyroid Cancer By Stabilizing YAP

Objective Thyroid cancer is the most common endocrine-related cancer,which can be divided into three major categories based on the degree of differentiation: well differentiated thyroid cancer(WDTC),poorly-differentiated thyroid cancer(PDTC)and undifferentiated/anaplastic thyroid cancer(ATC).ATC is one of the most lethal and aggressive human malignancies with a median survival of 4 to 6 months after diagnosis.It is inherently resistant to both conventional chemotherapy and radioactive iodine.Thus,it is important to understand the underlying mechanisms during the initiation and progression of anaplastic thyroid cancer.The abnormality of the Hippo-YAP pathway is closely related to the occurrence and development of tumors.The Hippo-YAP pathway has the potential to become the target for tumor treatment.However,it is difficult to directly target YAP with small molecule compounds.It is important to find out the upstream regulators of Hippo-YAP for the development of anti-tumor drugs targeting the Hippo-YAP pathway.The deubiquitinating enzymes(DUBs)regulate the deubiquitination process of key proteins through the recognition of specific substrate.Development of DUBs selective/specific inhibitors is emerging as attractive targets for cancer treatment.In the present study,we screened a DUB si RNA library to investigate the involvement of DUBs in the Hippo pathway,and provided new targets for the treatment of ATC.Methods 1.Cell proliferation was detected using CCK8,Edu,and clone formation assays.2.The cell cycle phases determined by relative DNA content were analyzed by flow cytometer.3.Cell migration ability was measured using wound healing assay.4.Cell invasion ability was tested using transwell assay.5.Animal models were used for in vivo tumorigenic experiment.6.Western Blot analysis was performed to detect YAP protein levels and DUB si RNA library was used to screen the potential deubiquitinating enzymes that regulate the expression of YAP in ATC.7.The m RNA expression of YAP and its target genes were measured by real-time PCR.8.Luciferase activity of the YAP/TEAD luciferase reporter gene was detected using the Dual-Luciferase Reporter kit.9.Protein stability assay was used to detect YAP protein degradation and the ubiquitin-based Immuno-precipitation assays were used to detect the specific ubiquitination manner happened on YAP.10.Immuno-precipitation assay was used to detect the interaction domain between YAP and UCHL3.11.Real-time PCR and Western Blot analysis were used to detect the expression of UCHL3 induced by YAP/TEAD4.12.The effect of YAP/TEAD4 on the transcriptional regulation of UCHL3 expression was analyzed through luciferase reporter gene analysis.13.CHIP experiment was used to test the specific promoter region of UCHL3 that YAP/TEAD4 interacted.14.Immunohistochemical experiment was performed to analyze the expression of UCHL3 and YAP in clinical samples.Results 1.Depletion of YAP in ATC decreased cell proliferation,migration and invasion.2.We screened a DUB si RNA library of 98 DUBs by monitoring the levels of YAP.Among these DUBs,UCHL3 was observed to potentially deubiquitinate and stabilize YAP as its si RNA-mediated depletion markedly reduced YAP abundance.Depletion of UCHL3 significantly decreased the expression of YAP target genes and YAP transcriptional activity.3.Substrate half-life experiment and MG132 experiment confirmed that UCHL3 regulated the stability of YAP protein through the proteasome pathway.4.In vivo and in vitro deubiquitination experiments confirmed that UCHL3 can directly cleaved the ubiquitin chain on YAP protein,thereby inhibiting YAP proteolysis.5.Co-immunoprecipitation experiments showed that the carboxyl end of UCHL3 directly interacted with the WW domain of YAP.6.YAP/TEAD4 regulated the transcription level of UCHL3 by directly binding to the promoter region of the UCHL3 gene.7.Cell experiments and animal experiments confirmed that UCHL3 promoted tumor proliferation and metastasis through YAP.And overexpression of YAP can reverse the inhibitory effect caused by UCHL3 silencing.8.The immunohistochemical results of clinical samples showed that UCHL3 and YAP were highly expressed in ATC,and their expressions were positively correlated.Conclusions In summary,we found that YAP played an important role in the progression of ATC.Through the screening of a DUB si RNA library,we found that UCHL3 can interact with,deubiquitylate,and stabilize YAP in a manner dependent on its deubiquitylation activity.Further analysis indicated that YAP/TEAD4 can transcriptionally regulated the expression of UCHL3,thus forming a positive feedback loop.Inhibition of UCHL3 can inhibit the progression of ATC,and overexpression of YAP can reverse the inhibitory effect induced by UCHL3 depletion.Tissue microarray results show that the expression of YAP was positively correlated with UCHL3.Our findings provide new insights into the roles of UCHL3 in Hippo signaling pathway in ATC,and suggest that UCHL3 may prove to be a potential target for the treatment of ATC.