USP10 Promotes Proliferation of Hepatocellular Carcinoma By

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, 2020 American Association for Cancer Research. depletion of USP10 significantly reduces YAP/TAZ abundance and

AACRJournals.org | 2204

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 8GTIIC 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 8GTIIC 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 duplexes were from Genepharma, Co. The (Tyr1079) (#8564), LATS1 (#9153), LATS2 (#5888), and b-TrCP siRNA sequences used were as follows: (#4394) antibodies were purchased from Cell Signaling Technology; anti-GAPDH (db106) and anti-HA (db2603) antibodies were pur- siRNA-USP10 #1: sense: 50-CCAUAAAGAUUGCAGAGUU- chased from Diagbio; anti-Birc5 (10508-I-AP) was purchased from TT-30, Protein Tech; anti-CYR61 (sc-13100) and anti-Cullin1 (sc-11384) antisense: 50-AACUCUGCAAUCUUUAUGGTT-30; antibodies were purchased from Santa Cruz Biotechnology. IHC siUSP10-USP10 #2: sense: 50-CCACAUAUAUUUACAGA- analyses were completed by using USP10 (ab72486) antibody CUTT-30, from Abcam, TAZ (HPA007415) antibody from Sigma, and YAP antisense: 50-AGUCUGUAAAUAUAUGUGGTT-30; (sc-15407) antibody from Santa Cruz Biotechnology. Proteasome Scramble siRNA: sense: 50-UUCUCCGAACGUGUCACGU- inhibitor MG-132 was obtained from SelleckChem. Protein syn- TT-30, thesis inhibitor cycloheximide was purchased from Sigma-Aldrich. antisense: 50-ACGUGCCACGUUCGGAGAATT-30. N-Nitrosodiethylamine (DEN) was obtained from Sigma. The transfection was performed using jetPrime and siRNA according to the manufacturer's recommendations. 8GTIIC and WWTR1 luciferase reporter activity assay The USP10 shRNAs was ligated into pLKO.1/U6 (Addgene). The The 8GTIIC luciferase reporter, which harbors eight TEAD- gene-specific shRNAs sequences used were as follows: binding sites, was utilized to indicate the transactivation YAP/TAZ, and the WWTR1 luciferase reporter was performed to depict the TAZ shRNA-USP10 #1: sense: 50-GCCTCTCTTTAGTGGCTCTTT-30, protein stability. After transfection with individual pools of DUB antisense: 50-GCCTCTCTTTAGTGGCTCTTT-30; siRNAs per well for 24 hours, the corresponding luminescence number shRNA-USP10 #2: sense: 50-GCTGTGGATAAACTACCTG- was obtained and a construct containing Renilla luciferase was used as AT-30, internal control (Promega E1960). The ratio of firefly/Renilla was antisense: 50-GCTGTGGATAAACTACCTGAT-30; normalized to the empty vector controls. shRNA-USP10 #3: sense: 50-CCTATGTGGAAACTAAGTA- TT-30, Real-time PCR antisense: 50-CCTATGTGGAAACTAAGTATT-30. Cells were harvested in TRIzol (Invitrogen) for total RNA extrac- The lentiviral vector pCDH-EF1-Puro plasmid was obtained from tion, cDNA was obtained by reverse transcription of RNA using System Biosciences and the plasmids or shRNAs were cloned into TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix pCDH plasmid by CloneEZ PCR Cloning Kit (GeneScript). All

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2205

Zhu et al.

lentiviral plasmids were transfected into HEK293FT cells for ysis between USP10 and YAP/TAZ target genes, data were performed packaging. by GEPIA (http://gepia.cancer-pku.cn/).

Immunoblotting analysis and immunoprecipitation Statistical analysis Immunoblotting analysis and immunoprecipitation were per- All statistical analyses were performed using SigmaPlot 10.0. The formed as described previously (25) with details in the Supplementary values were presented as means SD and two-tailed unpaired Student Information. t tests were used for statistical analysis. Results were considered significant when P < 0.05 (, P < 0.05, , P < 0.01, and , P < 0.001). Sulforhodamine B, cell proliferation, and colony formation assays Cell proliferation was assessed by sulforhodamine B (SRB) assay Results and colony formation assay. HepG2 cells infected with control Identification of USP10 as a new regulator of YAP/TAZ signaling or shUSP10 lentivirus were seeded 1,000/well in 96-well plates and To search for DUBs critical to YAP/TAZ signaling, we first per- optical density value was measured every day for 6 days. Then cell formed a gain-of-function screen by monitoring YAP/TAZ- n proliferation of day was calculated as A510day n/A510day 1 100% by dependent transcriptional luciferase reporter (8 GTIIC; Fig. 1A). SRB assay. For colony formation assay, lentivirus infected cells were We screened 98 DUBs, for which we employed a pool of four seeded 1,000/well in 6-well plates. Two weeks later, cells were stained nonoverlapping siRNA oligos for transfection experiments in HepG2 with SRB and numbers of colonies were counted. cells. Among those tested, USP10 siRNA exerted the strongest inhibitory effect on YAP/TAZ-TEADs luciferase activity (inhibition ratio ¼ Antitumor activity in vivo 52.8%; Fig. 1B). To further confirm the regulatory effect of USP10 on Female nude mice and C57BL/6 (4–5 weeks old) were obtained from YAP/TAZ signaling, we performed another screen using WWTR1– National Rodent Laboratory Animal Resource and were maintained in Luciferase fusion construct wherein the abundance of TAZ (encoded a pathogen-free animal facility and all animal experiments were by WWTR1 gene) could be indicated by luciferase signal (Supple- approved by the Animal Care and Use Committee of Zhejiang mentary Fig. S1A and S1B). As expected, USP10 siRNA substantially University (Hangzhou, China), with the ethical approval number reduced TAZ protein level (inhibition ratio ¼ 57.4%; Supplementary IACUC-S18006, IACUC-S19-047, IACUC-S19-061, and IACUC- Fig. S1C). Both luciferase reporter systems suggested that USP10 was S19-062. Mice were randomized into indicated groups (5 animals/ required to optimally induce YAP/TAZ-mediated transcriptional condition) and were inoculated with corresponding lentivirus-infected responses (Supplementary Table S1). This finding was further vali- cells subcutaneously. Tumor volume (V) was calculated as V ¼ (length dated by the experiments transfecting two independent shRNAs of width2)/2. USP10 in above-mentioned reporter systems: deletion of endogenous USP10 robustly reduced the transcriptional activities as well as the Patient-derived xenograft models protein abundance of YAP/TAZ (Fig. 1C and D; Supplementary Tumors from patients with hepatocellular carcinoma cancer (P0) Fig. S1D–S1F). Consistently, the introduction of exogenous USP10 were fragmented and then subcutaneously transplanted into immu- in HepG2 and Bel-7402 cells greatly upregulated the protein levels of nodeficient mice (P1) for engraftment. After grown, the tumors YAP/TAZ (Fig. 1E; Supplementary Fig. S1G). These results collec- were cut into pieces of 3 3 3mm3 and subcutaneously tively indicate that USP10 positively regulates the protein stability and transplanted per mouse. When xenografted tumors reached a mean transcriptional function of YAP/TAZ. group size of about 80 mm3, mice were randomized into control or treatment groups with 10 mice per cohort, relative tumor volume USP10 stabilizes YAP/TAZ through the deubiquitinase activity (RTV) and therapeutic treatment effects (T/C) were calculated as To test whether the deubiquitinase activity of USP10 is required for described previously. its function in YAP/TAZ regulation, we overexpressed WT or cata- lytically inactive mutant (CA) of USP10 in HepG2 and Bel-7402 cell Ethical approval lines and found that WT USP10, but not CA mutant, increased YAP/ All procedures in this study were in accordance with recognized TAZ protein level (Fig. 1F; Supplementary Fig. S2A). Next, cells stably ethical guidelines of the responsible committee on human experimen- expressing control shRNA or USP10 shRNA were treated with cyclo- tation (institutional and national) and with the Helsinki Declaration. heximide, a general inhibitor of protein synthesis. The following half- Written informed consent was obtained from the patient included in life analyses showed that TAZ protein was much more stable in the study. The patient-derived xenografted study was approved by the USP10-deficient cells (Fig. 1G; Supplementary Fig. S2B). Notably, Ethics Committee of the Second Affiliated Hospital, School of Med- USP10 stabilized YAP/TAZ with no appreciable change at the tran- icine Zhejiang University (Hangzhou, China; Ethics Approval License: scriptional level (Supplementary Fig. S2C and S2D). Taken together, 2019-325). these results suggested USP10 regulates YAP/TAZ stability through deubiquitinase activity. Clinical significance analysis Because YAP/TAZ was previously reported to be subjected to To compare USP10 expression between primary tumors and non- proteasomal degradation activated by upstream kinase (15), we sought tumor tissues, USP10 expression values were extracted from Ualcan to further confirm that USP10 regulates the turnover of YAP/TAZ via (http://ualcan.path.uab.edu/). For USP10 expression analyzed by the the ubiquitin-proteasome pathway. Upon treating USP10-deficient risks of patients with hepatocellular carcinoma, USP10 expression cells with proteasome inhibitor MG132, we restored the protein levels values were extracted from SurvExpress (http://bioinformatica.mty. of YAP/TAZ, which were previously decreased by USP10 depletion itesm.mx:8080/Biomatec/SurvivaX.jsp). Kaplan–Meier curves were (Fig. 1H, lane 4; Supplementary Fig. S2E). We next examined the calculated with autoselective best cutoff and censored at threshold downstream target of YAP/TAZ and found that the deletion of USP10 was not checked (http://kmplot.com/analysis/). For correlation anal- markedly decreased the expression levels of Birc5 and CYR61, two

2206 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

USP10 Stabilizes YAP/TAZ to Regulate HCC Cell Growth

Figure 1. USP10 is a novel YAP/TAZ regulator that protects YAP/TAZ from ubiquitin-proteasome degradation through its deubiquitinase activity. A, The schematic diagram of 8GTIIC luciferase reporter system was used to screen YAP/TAZ-regulating DUB(s). 8GTIIC contained eight TEAD-binding sites, which could reﬂect the YAP/TAZ transcriptional activity. B, HepG2 cells were transfected to express the 8GTIIC luciferase reporter together with siRNA pools targeting 98 DUB(s). USP10 RNAi, which robustly inhibited 8GTIIC luciferase, is indicated (empty circle). C, Luciferase reporter assays showed the effects of USP10 knockdown. HepG2 cells infected with lentivirus encoding indicated shRNAs were transfected to express by shRNA targeting two sequences. Data are shown as mean SD derived from three independent biological replicates. , P < 0.01. D, Immunoblotting analysis of YAP and TAZ protein expression in HepG2 cells (left) and Bel-7402 cells (right) depleted of USP10 by siRNA. E, HepG2 and Bel-7402 cells were transfected with USP10. Immunoblotting analysis was performed with the indicated antibodies. F, Cells were transfected with control vector, USP10 WT and USP10 CA.YAP and TAZ were upregulated by USP10 WT (lane 2) but not on USP10 CA (lane 3). G, HepG2 cells stably expressing control shRNA or USP10 shRNA were treated with or without cycloheximide (40 mg/mL) and harvested at the indicated times. Protein levels of USP10 and TAZ were analyzed by immunoblotting (left). H and I, HEK293FT cells transfected with indicated siRNA were left untreated or treated with MG132 (10 mmol/L) for 8 hours, followed by cell lysates being immunoblotted as indicated. J, HEK293FT cells were transfected with indicated constructs. Twenty-four hours later, cell lysates were extracted and subjected to immunoblotting analysis performed with the indicated antibodies.

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2207

Zhu et al.

target genes of YAP/TAZ (Fig. 1I, lanes 2 and 3; Supplementary lane 3; Supplementary Fig. S2I), whereas USP10 overexpression Fig. S2F). In contrast, disruption of YAP/TAZ degradation by MG132 abrogated TAZ degradation (Fig. 1J, lane 4). Furthermore, we also abated the reduction of Birc5 and CYR61 level (Fig. 1I, lanes 4 and 5). detected whether USP10 can protect YAP from ubiquitination. As Taken together, these findings confirmed that USP10 modulates YAP/ shown in Supplementary Fig. S2J, YAP ubiquitination was increased TAZ stability and its downstream activity in a proteasome-dependent by MG132 treatment and USP10 significantly decreased ubiquitin manner. chains on YAP. To determine whether USP10 directly regulates YAP/TAZ, we In aggregate, these results validated that USP10 directly stabilizes further investigated how USP10 may affect LATS1/2, the upstream YAP/TAZ protein in hepatocellular carcinoma cells in a deubiquiti- kinases of YAP/TAZ. As a result, neither USP10 overexpression nor nase activity-dependent manner. USP10 knockdown could cause significant changes in LATS1, LATS2, or p-LATS1 (T1079; Supplementary Fig. S2G and S2H), thus excluding USP10 interacts with and deubiquitinates YAP/TAZ the possibility that these canonical upstream regulators were engaged We then asked whether USP10 modulates YAP/TAZ by directly in the modulation of YAP/TAZ by USP10. interacting with the pair. We cotransfected exogenous TAZ and USP10 In addition, we further evaluated the ability of USP10 antagonizing into HEK293FT cells, and the Coimmunoprecipitation (co-IP) assay E3 ligase-mediated TAZ degradation, by transfecting b-TrCP and showed that TAZ indeed physically interacted with USP10 (Fig. 2A). b Cullin 1, the subunits of E3 ligase SCF -TrCP, into HEK293FT cells, and Importantly, we transfected TAZ alone, and the results showed that found TAZ protein levels were downregulated as expected (Fig. 1J, TAZ could also interact with endogenous USP10 (Fig. 2A, lane 2). In

Figure 2. USP10 interacts with YAP and TAZ. A and B, Interaction between exogenous USP10 and TAZ. HEK293FT cells were cotransfected with indicated constructs. Cellular extracts were immunoprecipitated with FLAG Sepharose and immunoprecipitations were performed with antibodies against the indicated proteins. C, HEK293FT cells were transfected with FLAG-tagged TAZ. Extracts were immunoprecipitated with FLAG Sepharose and examined by immunoblotting. Endogenous USP10 was detected to interact with TAZ. D and E, Interaction between exogenous USP10 and YAP. HEK293FT cells were cotransfected with indicated constructs. Cellular extracts were immunoprecipitated with FLAG Sepharose and immunoprecipitations were performed with antibodies against the indicated proteins. F, HEK293FT cells were transfected with FLAG-tagged YAP. Extracts were immunoprecipitated with FLAG Sepharose and examined by immunoblotting. Endogenous USP10 was detected to interact with YAP. G, Endogenous YAP/TAZ was immunoprecipitated with USP10 antibody and examined by immunoblotting.

2208 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

USP10 Stabilizes YAP/TAZ to Regulate HCC Cell Growth

reciprocal co-IP with anti-Flag antibodies, exogenous USP10 was also tion assay in vitro using bacterial-expressed USP10 (Fig. 3D). We found in contact with TAZ–GFP fusion protein wherein GFP tagging purified ubiquitinated YAP/TAZ from cells expressing YAP/TAZ-Flag was introduced to better illustrate the TAZ signal (Fig. 2B). Consis- and HA-Ub and incubated recombinant USP10 and ubiquitinated tently, endogenous USP10 co-IPed with TAZ as well (Fig. 2C). And YAP/TAZ in a cell-free system. As shown in Fig. 3E and F, purified similar results attested YAP–USP10 interaction (Fig. 2D–F). Further- USP10 effectively deubiquitinated YAP/TAZ in vitro (Supplementary more, endogenous interaction between USP10 and YAP/TAZ was Fig. S3D and S3E). further confirmed (Fig. 2G), which was in line with the data obtained Taken together, these results indicated that USP10 directly interacts from the co-IP experiments at endogenous levels (Fig. 2C and F). with and deubiquitinates YAP/TAZ, further confirming that USP10 Next, we sought to examine whether USP10 could remove the regulates YAP/TAZ as a DUB. polyubiquitination of YAP/TAZ. As shown in Fig. 3A and B, overexpression of USP10, but not of USP10-CA, significantly reduced the USP10 depletion impairs the growth of hepatocellular ubiquitination level of YAP/TAZ (Fig. 3A, lane 4; Fig. 3B, lane 3; carcinoma cells and xenograft tumors through inhibiting Supplementary Fig. S3A and S3B). Consistently, USP10 knockdown YAP/TAZ pathway markedly increased the level of ubiquitinated TAZ (Fig. 3C, lanes 4 Many studies have well demonstrated that YAP/TAZ promotes and 5; Supplementary Fig. S3C). We then performed a deubiquitina- hepatocellular carcinoma cell proliferation (26–28). In this context, we

Figure 3. USP10 removes the ubiquitin chains on YAP/TAZ. A and B, HEK293FT cells transfected with indicated constructs were treated with MG132 (10 mmol/L) for 8 hours before harvest. Cell lysates were immunoprecipitated with FLAG Sepharose to detect the ubiquitin chains on YAP/TAZ. C, HEK293FT cells stably expressed control or USP10 shRNA and were transfected with indicated constructs, followed by MG132 treatment. Cell lysates were immunoprecipitated and subjected to immunoblotting analysis of ubiquitin. D, The in vitro deubiquitination assay mode is presented. E and F, Deubiquitination of YAP/TAZ in vitro by recombinant USP10. HEK293FT cells transfected with YAP/TAZ and ubiquitin constructs were treated with MG132 (10 mmol/L) for 8 hours before harvest. Cell lysates were incubated with FLAG Sepharose, then ubiquitinated YAP/TAZ was incubated with or without puriﬁed recombinant USP10 and blotted with anti-HA antibodies to indicate the ubiquitin on TAZ (E) or YAP (F).

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2209

Zhu et al.

asked whether USP10 could influence hepatocellular carcinoma pro- the SRB assay and colony formation assay (Fig. 4A). Moreover, in gression by mediating YAP/TAZ. First, we found that stably silencing those USP10-deficient cells, the protein level of TAZ was evidently USP10 in HepG2 significantly suppressed cell growth, as measured by downregulated (Fig. 4A), consistent with the aforementioned findings.

Figure 4. USP10 regulates hepatocellular carcinoma cell proliferation in vitro. A–C, The proliferation of hepatocellular carcinoma cells with/without USP10 shRNA (#1 and #2) were determined using SRB assay. The cell proliferations were measured by SRB assay (mean SD of three independent experiments). Immunoblotting analysis of YAP and TAZ protein expression in hepatocellular carcinoma cells transfected with shUSP10. D, The proliferation of USP10 stable knockdown HepG2 cells with indicated plasmids was determined using SRB assay (mean SD of three independent experiments). Indicated groups of HepG2 cells were subjected to colony formation assay (mean SD of three independent experiments). Immunoblotting analysis of YAP and TAZ protein expression in hepatocellular carcinoma cells transfected with indicated plasmids. , P < 0.01; , P < 0.001; ###, P < 0.001.

2210 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

USP10 Stabilizes YAP/TAZ to Regulate HCC Cell Growth

Similar results were obtained in another two hepatocellular carcinoma USP10 almost completely inhibited the growth of HepG2-xenografted cell lines (SNU-387 and Li-7; Fig. 4B and C). To further corroborate tumors (P ¼ 0.0073; Fig. 5A–C). Together, these data confirmed that that YAP/TAZ participates in the regulation of USP10 on tumor inhibition of USP10 would significantly arrest the proliferation and growth, we introduced YAP WT or YAP-5SA mutant into USP10- growth of hepatocellular carcinoma cells and xenografted tumors. deficient cells. YAP-5SA lacks five serine phosphorylation residues and Having shown that USP10 inhibition displayed potent antiproli- is predominantly located in the nucleus. As reflected in Fig. 4D, only feration of hepatocellular carcinoma in vitro and in vivo, we further the nuclear-residing YAP-5SA, but not YAP WT, escaped the degra- examined USP10 function in hepatocellular carcinoma patient- dation in cytoplasm enhanced by USP10 depletion and reversed the derived xenografted (PDX) model, a more predictive and relevant reduction of cell proliferation and colony formation. preclinical model to evaluate cancer therapy. Intratumoral injection Next, to validate the influence of USP10 on cell growth in vivo,we of lentiviral-vector delivered USP10 shRNA significantly delayed the subcutaneously injected USP10-deficient HepG2 cells into nude mice, PDX tumor growth, with T/C value of 0.55 (mean RTV: shUSP10 vs. and measured tumor sizes every 2 days. Strikingly, the depletion of Control: 3.72 vs. 6.80; P ¼ 0.0205; Fig. 5D–F). To further clarify

Figure 5. USP10 regulates hepatocellular carcinoma cell proliferation in vitro and vivo. A and B, HepG2 cells with/without USP10 shRNA (#1 and #2) were injected into the immunodeﬁcient nude mice as described in Materials and Methods. Tumor volume was measured every 3 days (mean SEM; n ¼ 5/group). C, Tumorweightof each mouse. , P < 0.01. D, The in vivo anticancer activities of shUSP10 on patient-derived xenografted hepatocellular carcinoma mice. E, shUSP10 arrested the growth of PDX tumors. RTV is expressed as mean SEM. F, Tumorweightofeachmouse., P < 0.05 vs. Ctrl. G, Intratumor injection of shUSP10 virus distinctly reduced expression levels of YAP and TAZ of PDX tumors. H, YAP-5SA attenuated the tumor growth inhibition mediated by USP10 shRNA. RTV is expressed as mean SEM. I, Immunoblotting analysis carried out with Con, shUSP10, and shUSP10þYAP-5SA cells in H. n.s., not signiﬁcant; , P < 0.05; , P < 0.01; #, P < 0.05; ##, P < 0.01.

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2211

Zhu et al.

whether USP10 inhibition was capable of interfering with YAP/TAZ finding consistent with our IHC outcomes analyzing hepatocellular pathway in the xenograft model, we next monitored the intratumor carcinoma clinical samples (Fig. 6B–F). protein levels of YAP and TAZ, and found significant reduction of Collectively, these data not only validate our hypothesis that USP10 YAP and TAZ in shUSP10-treated PDX tumors (Fig. 5G; Supple- promotes hepatocellular tumorigenesis through stabilizing YAP/TAZ, mentary Fig. S4A–S4C). These data collectively suggested that inhi- but also represent USP10 as a potential therapeutic target for hepa- bition of USP10 arrested tumor growth in vivo through the inhibition tocellular carcinoma treatment. on YAP and TAZ abundance (activation). To confirm the roles of YAP/TAZ in USP10-regulated tumor growth in vivo, we evaluated the influence of nuclear-residing YAP- Discussion 5SA mutant on USP10 shRNA-mediated tumor inhibition of HepG2- The lack of intervention targets remains an elusive, yet pivotal xenografted tumors. Consistent with the previous data (Fig. 5A–C), challenge in drug discovery for hepatocellular carcinoma treatment. USP10 shRNA completely block the tumor growth, while the intro- Given the critical and pervasive functions of YAP/TAZ signaling in duction of YAP-5SA attenuated the growth inhibition mediated by hepatocellular carcinoma cells, the exploration of the upstream reg- USP10 shRNA (P ¼ 0.0073; Fig. 5H and I). These findings corroborate ulators emerges as a very attractive way to develop potential thera- the notion that the regulatory effects on YAP are critical for USP10 to peutic strategies. In this study, we employed an in vitro functional promote the growth of hepatocellular carcinoma tumors in vivo. screening on YAP/TAZ transcriptional activity and identified USP10 In conclusion, the high profile of USP10 tends to facilitate hepa- as a novel regulator that physically deubiquitinates and functionally tocellular carcinoma progression by stabilizing YAP and TAZ. stabilizes YAP/TAZ, hence promoting hepatocellular carcinoma cell proliferation both in vitro and in vivo (Fig. 7G). USP10 expression is positively correlated with YAP/TAZ level YAP and TAZ are equivalent downstream effectors of the Hippo and risk for hepatocellular carcinoma in patients pathway for their structural and functional similarities (8). Increasing To further investigate whether USP10 was involved in the patho- evidence has revealed the pair act coordinately and complementarily to genetic alterations underlying the formation of hepatocellular carci- promote cancer progression, highlighting the importance of cotarget- noma, we established a model of DEN-induced hepatocellular tumor- ing YAP/TAZ in anticancer therapy (11). Both YAP and TAZ are igenesis in C57BL/6 mice by injecting a 75 mg/kg single dose of DEN negatively regulated by the upstream kinases LATS1/2 and MST1/2, into the mice at their age of 2 weeks. The formation of liver cancers was and are subjected to ubiquitination and proteasomal degradation observed after 8 months (Supplementary Fig. S5A), followed by under physiologic or pathophysiologic conditions. Although protein hematoxylin-eosin staining to validate the tumor formation, with kinases are generally regarded as paradigm targets for small-molecular tumorous and nontumorous regions indicated respectively (Supple- therapeutics, it is frustratingly difficult to activate these kinases to show mentary Fig. S5B). We found that the expressions of both USP10 and tumor-suppressive responses (33). Hence, new amenable regulators of YAP/TAZ were more substantially elevated in the tumorous region YAP/TAZ need to be explored beyond the scope of core Hippo kinases. than in the nontumorous region from the same liver (Fig. 6A; DUBs are intrinsically appealing for drug targeting with well- Supplementary Fig. S5C). defined catalytic clefts (18), capable of cleaving ubiquitins on its Furthermore, we evaluated USP10 and YAP/TAZ expression in substrates, and altering their abundance level and transcriptional microarray tissues from 109 liver patients with cancers using IHC activities. We were therefore interested to explore how DUBs regulate staining. Most tumor specimens expressed high volume of USP10 and YAP/TAZ in hepatocellular carcinoma models. Among the two YAP/TAZ (Fig. 6B–D). Moreover, statistically significant correlations coactivators, TAZ is much more unstable with a half-life of less than were found between USP10 and YAP/TAZ levels (Fig. 6E, USP10 and 2 hours, compared with that of YAP longer than 9 hours (34), out- YAP, r ¼ 0.3259, P < 0.001; Fig. 6F, USP10 and TAZ, r ¼ 0.5596, P < performing Luciferase protein. In this regard, we constructed 0.001; n ¼ 109). Collectively, these data not only validate our hypoth- WWTR1–Luciferase fusion whereby luciferase signal could precisely esis that USP10 promotes hepatocellular tumorigenesis through sta- indicate the protein level of TAZ. WWTR1–Luciferase, together with bilizing YAP/TAZ, but also represents USP10 as a potential thera- 8GTIIC luciferase reporter system was employed to screen DUB(s) peutic target for hepatocellular carcinoma treatment. with the ability to regulate YAP/TAZ, and the cross-comparison Next, we sought to confirm the clinical significance of USP10 by optimally diminishes “false positive hit.” Among the 98 DUBs tested, using The Cancer Genome Atlas (TCGA) database and a Kaplan– USP10 stood out for its superior capability to suppress both YAP and Meier plotter analysis (http://kmplot.com/analysis/; refs. 29, 30). As a TAZ, according to the data from the transcriptional activity and result, most human hepatocellular carcinoma samples exhibited protein abundance analyses. Follow-up studies further validated that markedly higher expression levels of USP10 than nontumorous tissues USP10 indeed interacts with YAP/TAZ in hepatocellular carcinoma (P < 0.001, data from TCGA dataset; http://ualcan.path.uab.edu/ cells,in a catalytic activity-dependent manner. analysis.html; Fig. 7A; ref. 31). Notably, the USP10 expression level USP10 exert diverse functions in tumorigenesis and cancer was correlated with the risk of hepatocellular carcinoma in patients (32; progression (21–23), and the tumor-promoting or tumor- P < 0.001; Fig. 7B), which prompted us to investigate whether an suppressive function of USP10 was largely dictated by its sub- elevated USP10 expression could predict the poor prognosis in patients strate(s) in the specific cellular context of cancer models. Previous with hepatocellular carcinoma. As shown by the Kaplan–Meier plotter study revealed that USP10 deubiquitinates both WT and mutant analysis, the expression of USP10 was inversely correlated with the p53, thus promoting or exacerbating the proliferation in cells with progression-free survival (PFS) of patients with hepatocellular carci- WT p53 and mutant p53, respectively (23). The oncogenic function noma, highlighting the pivotal role of USP10 in the development and of USP10 was supported by a recent study conducted by Weisberg prognosis of hepatocellular carcinoma (Fig. 7C). In addition, an and colleagues, which identified USP10 as the critical DUB required apparent positive correlation was detected between the expression to stabilize mutant feline McDonough sarcoma–like tyrosine kinase level of USP10 and those of YAP/TAZ target genes (CTGF, AMOTL2, 3 (FLT3) to aggravate the progression of acute myeloid leukemia and CYR61) in patients with hepatocellular carcinoma (Fig. 7D–F), a harboring the mutations in FLT3 (21).

2212 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

USP10 Stabilizes YAP/TAZ to Regulate HCC Cell Growth

Figure 6. USP10 is upregulated in tumor samples and correlates with the protein levels of YAP and TAZ. A, Samples were obtained from DEN-induced mice. Immunoblotting analysis of paired samples of nontumorous region (N) and tumor region (T) from the same mouse. B–D, IHC staining of YAP, TAZ, and USP10 (n ¼ 109/group). E and F, Correlations of expression between USP10 and YAP/TAZ were performed (n ¼ 109/group).

In this study, we found that USP10 promoted cell proliferation and associated with increased YAP/TAZ activity (36), thus the regulatory tumor growth in hepatocellular carcinoma models. However, Lu study machinery related to these two proteins by USP10 would impose suggested that USP10 could suppress the hepatocellular carcinoma cell robust promoting effects in hepatocellular carcinoma cells. Therefore, proliferation, through the deubiquitination of PTEN and AMPK (35). targeting USP10 as opposed to inhibition of its enzyme activity will be The seemingly contradicting conclusions of these two independent potentially beneﬁcial for overcoming “undruggable” of YAP/TAZ in studies may arise from the different cellular context of the hepato- hepatocellular carcinoma. cellular carcinoma cells. In addition, because PDX models, hepato- Recent studies characterized several small-molecule inhibitors cellular carcinoma patients’ specimens, and comprehensive TCGA Spautin-1, HBX19818, and P22077 with promising anti-cancer activity database analyses were introduced in our study, we were encouraged to against a variety of cancer types by targeting USP10 (21, 37). Inter- conclude that USP10 indeed plays essential tumor-promoting roles in estingly, although HBX19818 and P22077 also target USP7, yet this patients with hepatocellular carcinoma. More importantly, mounting part of function seems to be dispensable for their anticancer evidence showed that approximately 60% of human liver cancer was effects (21). This is in line with our ﬁndings that USP7 depletion

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2213

Zhu et al.

Figure 7. High USP10 expression is associated with poor prognosis in patients with hepatocellular carcinoma. A, USP10 expression levels in hepatocellular carcinoma (HCC) and nontumorous tissue based on TCGA RNA-sequencing data. B, The gene expression level of USP10 positively correlates with the risk for hepatocellular carcinoma in patients (TCGA database). C, The gene expression level of USP10 negatively correlates with the PFS of patients with hepatocellular carcinoma (Kaplan–Meier curve was calculated with autoselective best cutoff and censored at threshold was not checked). D-F, Spearman correlation test showing a signiﬁcant positive correlation between the USP10 expression and the YAP target genes (CTGF, AMOTL2,andCYR61) levels in hepatocellular carcinoma tissues. G, Scheme for the regulatory mechanism of USP10 on YAP/TAZ. , P < 0.001.

2214 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

USP10 Stabilizes YAP/TAZ to Regulate HCC Cell Growth

imposed minimal on both the transcriptional activity and protein level exerted little inhibitory effect in our screening models (8GTIIC: of YAP/TAZ (Supplementary Table S1). As USP10 may represent an 1.656 of OTUB2 siRNA, 0.472 of USP10 siRNA; WWTR1–Luc: 0.885 attractive intervention target for patients with hepatocellular carcino- of OTUB2 siRNA, 0.426 of USP10 siRNA), and that recombinant ma, further exploration of more specific and potent USP10 inhibitors is OTUB2 failed to cleave the ubiquitins on YAP/TAZ in vitro (39). On merited to improve the clinical treatment of patients with hepatocel- the contrary, we found that recombinant USP10 robustly removed the lular carcinoma. polyubiquitin chains from YAP/TAZ. Taken together, these data In addition to USP10, we also noticed a few other DUBs with demonstrated that OTUB2 and USP10 affect YAP/TAZ differently potential effect on YAP/TAZ signaling. USP9X (ubiquitin-specific not only due to varied cancer themes, but also in regulatory mode. peptidase 9X) and OTUB2 (Otubain-2) were recently found to revert In conclusion, our study identifies USP10 as a novel deubiquitinat- the proteolytic ubiquitination of YAP in breast cancer models (38, 39). ing mediator that stabilizes YAP/TAZ in hepatocellular carcinoma and However, siUSP9X or siOTUB2 imposed minimal effect on YAP/TAZ enhances downstream oncogenic response. The high abundance of in our screening systems. The seemingly contradicting results may USP10 in both hepatocellular carcinoma tissues and DEN-induced arise from the variation of cellular context in different cancer types hepatocellular tumorigenesis confirmed the key role of USP10 as a studied by these two independent investigations, as well as from the profound tumor-promoting factor, and therefore a novel therapeutic varied targeting substrate(s) involved. Li and colleagues identified that target for the hepatocellular carcinoma intervention. USP9X deubiquitinates and stabilizes YAP1 in breast cancer models, thus promoting breast cancer growth and chemoresistance toward Disclosure of Potential Conflicts of Interest paclitaxel (38). Nevertheless, the study primarily focused on YAP1, No potential conflicts of interest were disclosed. sparing TAZ untested in their models, thus the influence of USP9X on TAZ remains unknown. Given that TAZ could compensate for the Authors’ Contributions transactivation function of prohibited YAP (11), it is unsurprising that Conception and design: H. Zhu, F. Yan, X. Dong, Q. He, B. Yang USP9X silence imposed little inhibitory effect on 8xGTIIC–Luciferase Development of methodology: H. Zhu, F. Yan, T. Yuan Acquisition of data (provided animals, acquired and managed patients, provided activity. In addition, USP9X may also interact with and stabilizes a facilities, etc.): H. Zhu, F. Yan, M. Qian, T. Zhou, X. Dai variety of Hippo pathway components including LATS, WW45, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, KIBRA, and Angiomotins (40), which are important negative regu- computational analysis): H. Zhu, F. Yan, T. Yuan, M. Qian, M. Ying, B. Yang lators of YAP/TAZ. By activating these regulators, USP9X was Writing, review, and/or revision of the manuscript: H. Zhu, F. Yan, J. Cao, Q. He, reported to suppress cell proliferation in pancreatic cancer models (40). B. Yang Collectively, these studies showed that USP9X may exert dual func- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Zhu, T. Yuan, J. Cao, M. Ying, Q. He, B. Yang tions in varied cancer types when deubiquitinating different substrates, Study supervision: H. Zhu, B. Yang which may have compromised the inhibitory effects on the YAP Other (assisted some experiments): T. Zhou pathway in our hepatocellular carcinoma models too. It is therefore Other (material support): X. Dai critical to determine the specificity of how DUB(s) regulates in its pathway. Here in this study, we found that the upstream kinase Acknowledgments LATS1/2 was minimally affected by USP10, whether silenced or This work was supported by the State Key Program of the Natural Science overexpressed. This finding not only further confirms the direct Foundation of China (81830107), the Natural Science Foundation for Distin- modulation of USP10 on YAP/TAZ in a LATS1/2-independent man- guished Young Scholar of China (81625024), the Natural Science Foundation of fi China (81773753), and the Zhejiang Provincial Natural Science Foundation ner, but also identi es USP10 as a potentially promising intervention (LR19H310002). target for hepatocellular carcinoma. Zhang and colleagues reported that OTUB2, a member of ovarian The costs of publication of this article were defrayed in part by the payment of page tumor proteases (OTU) family, could also deubiquitinate YAP/TAZ, charges. This article must therefore be hereby marked advertisement in accordance but it requires the SUMOylation of OTUB2 caused by oncogenic RAS with 18 U.S.C. Section 1734 solely to indicate this fact. activation(39). However, to our knowledge, RAS proteins, especially K-, N-, and H-RAS, are rarely mutated in most hepatocellular carci- Received August 13, 2019; revised January 28, 2020; accepted March 17, 2020; noma models (41). This may explain the findings that OTUB2 siRNA published first March 26, 2020.

References 1. Forner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet 2018;391:1301–14. 7. Schulze K, Imbeaud S, Letouze E, Alexandrov LB, Calderaro J, Rebouissou S, et al. 2. Llovet JM, Montal R, Sia D, Finn RS. Molecular therapies and precision medicine Exome sequencing of hepatocellular carcinomas identifies new mutational for hepatocellular carcinoma. Nat Rev Clin Oncol 2018;15:599–616. signatures and potential therapeutic targets. Nat Genet 2015;47:505–11. 3. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc J-F, et al. Sorafenib in 8. Moya IM, Halder G. Hippo-YAP/TAZ signalling in organ regeneration and advanced hepatocellular carcinoma. N Engl J Med 2008;359:378–90. regenerative medicine. Nat Rev Mol Cell Biol 2019;20:211–26. 4. FinnRS,ZhuAX,FarahW,AlmasriJ,ZaiemF,ProkopLJ,etal.Therapiesfor 9. Yimlamai D, Christodoulou C, Galli GG, Yanger K, Pepe-Mooney B, Gurung B, advanced stage hepatocellular carcinoma with macrovascular invasion or et al. Hippo pathway activity influences liver cell fate. Cell 2014;157:1324–38. metastatic disease: a systematic review and meta-analysis. Hepatology 2018; 10. Juan WC, Hong W. Targeting the hippo signaling pathway for tissue regener- 67:422–35. ation and cancer therapy. Genes 2016;7:55. 5. Llovet JM, Pena~ CEA, Lathia CD, Shan M, Meinhardt G, Bruix J. Plasma 11. Hayashi H, Higashi T, Yokoyama N, Kaida T, Sakamoto K, Fukushima Y, et al. biomarkers as predictors of outcome in patients with advanced hepatocellular An imbalance in TAZ and YAP expression in hepatocellular carcinoma confers carcinoma. Clin Cancer Res 2012;18:2290. cancer stem cell-like behaviors contributing to disease progression. Cancer Res 6. He AR, Goldenberg AS. Treating hepatocellular carcinoma progression 2015;75:4985–97. following first-line sorafenib: therapeutic options and clinical observations. 12. Bradner JE, Hnisz D, Young RA. Transcriptional addiction in cancer. Cell 2017; Therap Adv Gastroenterol 2013;6:447–58. 168:629–43.

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2215

Zhu et al.

13. Benayoun BA, Veitia RA. A post-translational modification code for tran- 28. Lu L, Li Y, Kim SM, Bossuyt W, Liu P, Qiu Q, et al. Hippo signaling is a potent scription factors: sorting through a sea of signals. Trends Cell Biol 2009;19: in vivo growth and tumor suppressor pathway in the mammalian liver. Proc Natl 189–97. Acad Sci U S A 2010;107:1437–42. 14. Yan C, Higgins PJ. Drugging the undruggable: transcription therapy for cancer. 29. Rich JT, Neely JG, Paniello RC, Voelker CCJ, Nussenbaum B, Wang EW. A Biochim Biophys Acta 2013;1835:76–85. practical guide to understanding Kaplan-Meier curves. Otolaryngol Head Neck 15. Pan D. The hippo signaling pathway in development and cancer. Dev Cell 2010; Surg 2010;143:331–6. 19:491–505. 30. Menyhart O, Nagy A, Gyorffy B. Determining consistent prognostic biomarkers 16. He M, Zhou Z, Shah AA, Hong Y, Chen Q, Wan Y. New insights into of overall survival and vascular invasion in hepatocellular carcinoma. R Soc Open posttranslational modifications of Hippo pathway in carcinogenesis and ther- Sci 2018;5:181006. apeutics. Cell division 2016;11:4. 31. Chandrashekar DS, Bashel B, Balasubramanya SAH, Creighton CJ, Ponce- 17. Yan F, Qian M, He Q, Zhu H, Yang B. The posttranslational modifications of Rodriguez I, Chakravarthi BVSK, et al.UALCAN:aportalforfacilitating Hippo-YAP pathway in cancer. Biochim Biophys Acta Gen Subj 2020;1864: tumor subgroup gene expression and survival analyses. Neoplasia 2017;19: 129397. 649–58. 18. Harrigan JA, Jacq X, Martin NM, Jackson SP. Deubiquitylating enzymes 32. Aguirre-Gamboa R, Gomez-Rueda H, Martínez-Ledesma E, Martínez-Torteya and drug discovery: emerging opportunities. Nat Rev Drug Discov 2018;17: A, Chacolla-Huaringa R, Rodriguez-Barrientos A, et al. SurvExpress: an online 57–78. biomarker validation tool and database for cancer gene expression data using 19. Luo P, Qin C, Zhu L, Fang C, Zhang Y, Zhang H, et al. Ubiquitin-specific survival analysis. PLoS One 2013;8:e74250. peptidase 10 (USP10) inhibits hepatic steatosis, insulin resistance, and inflam- 33. Moroishi T, Hansen CG, Guan K-L. The emerging roles of YAP and TAZ in mation through Sirt6. Hepatology 2018;68:1786–803. cancer. Nat Rev Cancer 2015;15:73–9. 20. Ko A, Han SY, Choi CH, Cho H, Lee MS, Kim SY, et al. Oncogene-induced 34. Liu C-Y, Zha Z-Y, Zhou X, Zhang H, Huang W, Zhao D, et al. The hippo tumor senescence mediated by c-Myc requires USP10 dependent deubiquitination and pathway promotes TAZ degradation by phosphorylating a phosphodegron and stabilization of p14ARF. Cell Death Differ 2018;25:1050–62. recruiting the SCFb-TrCP E3 ligase. J Biol Chem 2010;285:37159–69. 21.WeisbergEL,SchauerNJ,YangJ,LambertoI,DohertyL,BhattS,etal. 35. Lu C, Ning Z, Wang A, Chen D, Liu X, Xia T, et al. USP10 suppresses tumor Inhibition of USP10 induces degradation of oncogenic FLT3. Nat Chem Biol progression by inhibiting mTOR activation in hepatocellular carcinoma. 2017;13:1207–15. Cancer Lett 2018;436:139–48. 22. Deng M, Yang X, Qin B, Liu T, Zhang H, Guo W, et al. Deubiquitination and 36. Zhang S, Zhou D. Role of the transcriptional coactivators YAP/TAZ in liver activation of AMPK by USP10. Mol Cell 2016;61:614–24. cancer. Curr Opin Cell Biol 2019;61:64–71. 23. Yuan J, Luo K, Zhang L, Cheville JC, Lou Z. USP10 regulates p53 localization and 37. Liu J, Xia H, Kim M, Xu L, Li Y, Zhang L, et al. Beclin1 controls the levels of p53 by stability by deubiquitinating p53. Cell 2010;140:384–96. regulating the deubiquitination activity of USP10 and USP13. Cell 2011;147: 24. Zhu H, Wang DD, Yuan T, Yan FJ, Zeng CM, Dai XY, et al. Multikinase inhibitor 223–34. CT-707 targets liver cancer by interrupting the hypoxia-activated IGF-1R-YAP 38. Li L, Liu T, Li Y, Wu C, Luo K, Yin Y, et al. The deubiquitinase USP9X promotes axis. Cancer Res 2018;78:3995–4006. tumor cell survival and confers chemoresistance through YAP1 stabilization. 25. Cao J, Wang Y, Dong R, Lin G, Zhang N, Wang J, et al. Hypoxia-induced WSB1 Oncogene 2018;37:2422–31. promotes the metastatic potential of osteosarcoma cells. Cancer Res 2015;75: 39. Zhang Z, Du J, Wang S, Shao L, Jin K, Li F, et al. OTUB2 promotes cancer 4839–51. metastasis via hippo-independent activation of YAP and TAZ. Mol Cell 2019;73: 26. Zhou D, Conrad C, Xia F, Park J-S, Payer B, Yin Y, et al. Mst1 and Mst2 7–21. maintain hepatocyte quiescence and suppress hepatocellular carcinoma 40. Toloczko A, Guo F, Yuen HF, Wen Q, Wood SA, Ong YS, et al. Deubiquitinating development through inactivation of the Yap1 oncogene. Cancer Cell enzyme USP9X suppresses tumor growth via LATS Kinase and core components 2009;16:425–38. of the hippo pathway. Cancer Res 2017;77:4921–33. 27. Song H, Mak KK, Topol L, Yun K, Hu J, Garrett L, et al. Mammalian Mst1 and 41. Taketomi A, Shirabe K, Muto J, Yoshiya S, Motomura T, Mano Y, et al. A rare Mst2 kinases play essential roles in organ size control and tumor suppression. point mutation in the Ras oncogene in hepatocellular carcinoma. Surg Today Proc Natl Acad Sci U S A 2010;107:1431. 2013;43:289–92.

2216 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

USP10 Promotes Proliferation of Hepatocellular Carcinoma by Deubiquitinating and Stabilizing YAP/TAZ

Hong Zhu, Fangjie Yan, Tao Yuan, et al.

Cancer Res 2020;80:2204-2216. Published OnlineFirst March 26, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-2388

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/03/26/0008-5472.CAN-19-2388.DC1

Cited articles This article cites 41 articles, 8 of which you can access for free at: http://cancerres.aacrjournals.org/content/80/11/2204.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/80/11/2204. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.