Published OnlineFirst March 26, 2020; DOI: 10.1158/0008-5472.CAN-19-2388 CANCER RESEARCH | MOLECULAR CELL BIOLOGY USP10 Promotes Proliferation of Hepatocellular Carcinoma by Deubiquitinating and Stabilizing YAP/TAZ Hong Zhu1, Fangjie Yan1, Tao Yuan1, Meijia Qian1, Tianyi Zhou1, Xiaoyang Dai2,JiCao1, Meidan Ying1, Xiaowu Dong1, Qiaojun He1, and Bo Yang1 ABSTRACT ◥ Yes-associated protein (YAP) and its paralog, transcriptional tocellular carcinoma in vitro and in vivo. Expression levels coactivator with PDZ-binding motif (TAZ), play pivotal roles of USP10 positively correlated with the abundance of YAP/TAZ in promoting the progression of hepatocellular carcinoma. How- in hepatocellular carcinoma patient samples as well as in N- ever, the regulatory mechanism underpinning aberrant activation nitrosodiethylamine (DEN)-induced liver cancer mice models. of YAP/TAZ in hepatocellular carcinoma remains unclear. In Collectively, this study establishes the causal link between USP10 this study, we globally profiled the contribution of deubiquiti- andhyperactivatedYAP/TAZinhepatocellular carcinoma cells nating enzymes (DUB) to both transcriptional activity and and provides a rationale for potential therapeutic interventions in protein abundance of YAP/TAZ in hepatocellular carcinoma the treatment of patients with hepatocellular carcinoma harbor- models and identified ubiquitin-specificpeptidase10(USP10) ing a high level of YAP/TAZ. as a potent YAP/TAZ-activating DUB. Mechanistically, USP10 directly interacted with and stabilized YAP/TAZ by reverting Significance: These findings identify USP10 as a DUB of YAP/ their proteolytic ubiquitination. Depletion of USP10 enhanced TAZ and its role in hepatocellular carcinoma progression, which polyubiquitination of YAP/TAZ, promoted their proteasomal may serve as a potential therapeutic target for hepatocellular degradation, and ultimately arrested the proliferation of hepa- carcinoma treatment. Introduction TEAD-dependent transcription of the cell proliferation gene CTGF, induce cancer stem-like properties, and promote tumor cell prolifer- Hepatocellular carcinoma is the third leading cause of cancer ation (11). Suppressing the aberrant expression of YAP/TAZ is deaths (1). The global burden of hepatocellular carcinoma is increasing therefore essential to alleviate tumor progression. However, both notably due to advanced stages of diagnosis and limited treatment transcriptional coactivators YAP and TAZ are technically challenging options (2). The multitarget tyrosine kinase inhibitor sorafenib was the to be directly targeted (12), which gives rise to the necessity to examine first systemic therapy approved for the treatment of unresectable their posttranslational modifications (PTM) barcodes (13), and hepatocellular carcinoma (3, 4). However, most patients with hepa- explore the core enzymatic regulators of PTMs as potential targets (14). tocellular carcinoma who received systemic therapy eventually devel- The classic Hippo pathway reveals the critical roles of PTMs, partic- oped into therapy resistance with unfavorable prognosis and poor ularly phosphorylation and ubiquitination, in biological processes. survival (5, 6). Seeking for alternative therapeutic strategies, emerging Phosphorylation of YAP/TAZ, induced by large tumor suppressor pathogenic insights unveiled the landscape of molecular aberrations in kinase 1/2 (LATS1/2), is a key canonical PTM that inhibits hepatocellular carcinoma models; however, the majority of the targets the transcriptional activity of YAP/TAZ, but the state of phosphor- engaged in these mechanisms are not clinically actionable (7). Quest ylation remains hard to evoke due to the difficulty associated with for novel hepatocellular carcinoma interventions therefore remains. LATS1/2 activation (15–17). Here we focus on deubiquitinating Yes-associated protein (YAP) and transcriptional coactivator with enzymes (DUB), of which, the catalytic inhibition has been demon- PDZ-binding motif (TAZ), are a pair of critical downstream effectors strated to offer a novel strategy addressing the undruggability of their of the Hippo pathway that play important oncogenic roles in human substrates (18). cancers, particularly in hepatocellular carcinoma (8–10). Overex- Ubiquitin-specific peptidase 10 (USP10) is a highly conserved pressed YAP and TAZ coordinately enhance the activation of deubiquitinating enzyme (19), extensively involved in the initiation and progression of a broad spectrum of cancer types (20–22). How- ever, the roles of USP10 in tumorigenesis have varied, dictated by the 1Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of substrate(s) it interacts with. According to Yuan study, in renal cell Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. 2Center for carcinoma models, harboring wild-type (WT) p53, USP10 deubiqui- Drug Safety Evaluation and Research of Zhejiang University, Hangzhou, China. tinates and stabilizes its substrate p53, a tumor suppressor, and exerts Note: Supplementary data for this article are available at Cancer Research an inhibitory effect on cancer cell growth. Conversely, in those RCC Online (http://cancerres.aacrjournals.org/). cells containing oncogenic mutant of p53, USP10 promotes tumor cell H. Zhu and F. Yan contributed equally to this article. proliferation by elevating the mutant abundance (23). Therefore, determining the pathologic roles of USP10 in tumorigenesis requires Corresponding Authors: Bo Yang, Zhejiang University, #866 Yuhangtang Road, Hangzhou 310058, China. Phone/Fax: 86-571-88208400; E-mail: mechanistic investigations on its substrate. [email protected]; and Qiaojun He, [email protected] Here we report that USP10 can potently activate both YAP and TAZ by directly removing their polyubiquitin chains, a catalytic activity that Cancer Res 2020;80:2204–16 stabilizes the protein levels of YAP and TAZ, and ultimately reinforces doi: 10.1158/0008-5472.CAN-19-2388 their oncogenic functions in hepatocellular carcinoma. Conversely, Ó2020 American Association for Cancer Research. depletion of USP10 significantly reduces YAP/TAZ abundance and AACRJournals.org | 2204 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 26, 2020; DOI: 10.1158/0008-5472.CAN-19-2388 USP10 Stabilizes YAP/TAZ to Regulate HCC Cell Growth consequently suppresses tumor growth in hepatocellular carcinoma Kit (Transgen Biotech). SYBR Premix Ex TaqTMa (TaKaRa) was used xenograft models. Furthermore, the expression of USP10 positively to carry out real-time qPCR analyses with QuantStudio 6 Flex and correlates with the YAP/TAZ level in liver tumor tissues, and high QuantStudio Real-time PCR software. Expression levels of the target USP10 level predicts poor prognosis in patients with hepatocellular genes were given relative to Actin. The sequences of primers were used carcinoma. We therefore believe that USP10 would be a feasible as follows: therapeutic target for patients with hepatocellular carcinoma with prolonged activation of YAP/TAZ. ACTIN, forward primer: 50-GGTCATCACTATTGGCAACG-30, ACTIN, reverse primer: 50-ACGGATGTCAACGTCACACT-30; WWTR1, forward primer: 50-ATCCCCAACAGACCCGTTTC-30, Materials and Methods WWTR1, reverse primer: 50-ACAGCCAGGTTAGAAAGGGC-30; Cell culture YAP, forward primer: 50-TAGCCCTGCGTAGCCAGTTA-30, Human liver tumor–derived cell lines HepG2, Bel-7402, and human YAP, reverse primer: 50-TCATGCTTAGTCCACTGTCTGT-30; embryonic kidney HEK293 cells were originally obtained from the Cell USP10, forward primer: 50-ATTGAGTTTGGTGTCGATGAA- Bank of China Science Academy in 2016. Human liver tumor–derived GT-30, cell lines SNU-387 and Li-7 were obtained from China Science USP10, reverse primer: 50-GGAGCCATAGCTTGCTTCTT- Academy in 2019. All cell lines were maintained in DMEM or TAG-30. RPMI1640 medium (Gibco) supplemented with 10% FBS (Gibco BRL) at 37 C, in 5 CCO2 humid atmosphere. Cell Lines were Immunohistochemistry routinely monitored for Mycoplasma (4A Biotech Co.). Authentica- Tissue microarrays were purchased from Aleano. All steps were tion of the cell lines was confirmed by short tandem repeating profiling performed as described previously (24) and details were provided in every 6 months. The cells used for experiments were passaged within the Supplementary Information. 10 times after thawing. In vitro ubiquitination assay Plasmids and reagents HEK293FT cells were transfected with both HA-UB and YAP/TAZ- The human USP10, YAP, and TAZ were amplified from the FLAG. After 24 hours, ubiquitinated TAZ/YAP was purified from the HepG2 cDNA library and were subsequently subcloned into the cell extracts with anti-FLAG Sepharose in FLAG-lysis buffer. For the pCDNA3.0 plasmid. The 8ÂGTIIC plasmid was purchased from in vitro deubiquitination assay, ubiquitinated TAZ/YAP protein was Addgene. The pCDNA3.0-WWTR1-Luc plasmid was cloned as incubated with recombinant USP10 in a deubiquitination buffer for following: the luciferase sequence was amplified from 8ÂGTIIC 2 hours at 37C. The buffer contains 50 mmol/L Tris-HCl, 5 mmol/L and subsequently subcloned into the pCDNA3.0-TAZ plasmid MgCl2, 2 mmol/L DTT, and 2 mmol/L ATP-Na2 with proteasome (fusion plasmid). YAP-5SA (phosphorylation mutation sites: inhibitors. S61A, S109A, S127A, S164A, and S381A) plasmid was cloned into pCDH vector. Primary antibodies used for immunoblotting were as Gene transfection and RNA interference follows: USP10 (#8501), YAP/TAZ (#8418), YAP (#4912), p-LATS1 The siRNA sequences