Hepatocellular carcinoma (HCC) is the fifthmost

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1 Hepatitis B Virus X Protein Modulates Oncogene Yes-Associated Protein by CREB to Promote Growth of Hepatoma Cells Tao Zhang, 1 Junping Zhang, 1 Xiaona You, 1 Qian Liu, 2 Yumei Du, 1 Yuen Gao, 1 Changliang Shan, 1 Guangyao Kong, 1 Youliang Wang, 3 Xiao Yang, 3 Lihong Ye, 2 and Xiaodong Zhang 1 Hepatitis B virus X protein (HBx) plays critical roles in the development of hepatocellular carcinogenesis (HCC). Yes-associated protein (YAP), a downstream effector of the Hipposignaling pathway, is an important human oncogene. In the present article, we report that YAP is involved in the hepatocarcinogenesis mediated by HBx. We demonstrated that the expression of YAP was dramatically elevated in clinical HCC samples, hepatitis B virus (HBV)-infected hepatoma HepG cell line, and liver cancer tissues of HBx-transgenic mice. Meanwhile, we found that overexpression of HBx resulted in the up-regulation of YAP in stably HBx-transfected HepG2/H7402 hepatoma cell lines, whereas HBx RNA interference reduced YAP expression in a dose-dependent manner in the above-mentioned cell lines, suggesting that HBx up-regulates YAP. Then, we investigated the mechanism underlying the up-regulation of YAP by HBx. Luciferase reporter gene assays revealed that the promoter region of YAP regulated by HBx was located at nt 2232/1115 containing cyclic adenosine monophosphate response element-binding protein (CREB) element. Chromatin immunoprecipitation (ChIP) demonstrated that HBx was able to bind to the promoter of YAP, whereas it failed to work when CREB was silenced. Moreover, we confirmed that HBx activated the YAP promoter through CREB by electrophoretic mobility shift assay and luciferase reporter gene assays. Surprisingly, we found that YAP short interfering RNA was able to remarkably block the HBx-enhanced growth of hepatoma cells in vivo and in vitro. Conclusion: YAP is a key driver gene in HBx-induced hepatocarcinogenesis in a CREB-dependent manner. YAP may serve as a novel target in HBV-associated HCC therapy. (HEPATOLOGY 2012;56: ) Hepatocellular carcinoma (HCC) is the fifthmost common cancer and the third leading cause of cancer death worldwide. 1 Hepatitis B virus (HBV) infection is one of the major causes of HCC. 2 Among the four proteins encoded by HBV, the HBV X protein (HBx) is a multifunctional regulatory protein and plays a crucial role in hepatocellular carcinogenesis. 3 Although it does not bind directly to DNA, HBx modulates transcriptional activation by interacting with nuclear transcription factors, such as activating protein 1 (AP-1), nuclear factor kappa lightchain enhancer of activated B cells (NF-jB), specificity protein 1 (Sp-1), and cyclic adenosine monophosphate (camp) response element-binding protein (CREB), and affects the cytoplasmic modulation of signal transduction pathways. 4,5 Abbreviations: Ab, antibody; AFP, alpha-fetoprotein; AP-1, activating protein 1; camp, cyclic adenosine monophosphate; ChIP, chromatin immunoprecipitation; CREB, camp response element-binding protein; DFS, disease-free survival; EdU, ethynyldeoxyuridine; EMSA, electrophoretic mobility shift assay; HBV, hepatitis B virus; HBx, HBV X protein; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; IR, immunoreactivity; mirna, microrna; mrna, messenger RNA; MTT, Methylthiazolyldiphenyl-tetrazolium bromide; NF-jB, nuclear factor kappa light-chain enhancer of activated B cells; OS, overall survival; psi-hbx, psilencer-x; qrt- PCR, quantitative real-time polymerase chain reaction; SD, standard deviation; si-creb, CREB sirna; sirna, small interfering RNA; SOX4, sex-determining region Y related high-mobility group box 4; Sp-1, specificity protein 1; TEAD, TEA domain; Tg, transgenic; WT, wild type; YAP, Yes-associated protein. From the 1 Department of Cancer Research, Key Laboratory of Molecular Microbiology and Technology of Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China; 2 Department of Biochemistry, State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China; and 3 Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China. Received January 16, 2012; accepted May 28, This study was supported, in part, by the National Basic Research Program of China (973 Program, nos. 2009CB521702, 2011CB933100, and 2011CB512113) and the Natural Scientific Foundation of China (nos and ). 2051

2 2052 ZHANG ET AL. HEPATOLOGY, December 2012 The Hippo-signaling pathway, initially discovered in Drosophila, is a well-conserved potent regulator of cell growth and apoptosis in mammals. 6 As a negatively regulated downstream effector of the Hippo pathway, Yes-associated protein (YAP) functions as a transcriptional modulator, 7 which regulates the activity of several transcription factors, including ErbB4, RUNX2, p73, and the TEA domain (TEAD) transcription factor family members Moreover, YAP promoted proliferation in murine livers by regulating the transcription of certain target genes, which include Ki-67, c-myc, sex-determining region Y related high-mobility group box 4 (SOX4), H19, and alpha-fetoprotein (AFP). 12 Using integrative oncogenomic and proteomic approaches, YAP was identified as a driving oncogene of the 11q22 amplicon in HCC and breast cancer. 13,14 In addition, transgenic (Tg) mice with liver-targeted YAP overexpression demonstrated a dramatic increase in liver size and eventually developed tumors. 12,15 Overexpression of YAP in the nontransformed mammary epithelial cell, MCF10A, induced epithelial-to-mesenchymal transition and enhanced anchorage-independent growth capability in soft agar. 14 Furthermore, a recent study revealed that YAP overexpression overcame cell-contact inhibition and promoted cell growth. 16 In addition, the expression and nuclear accumulation of YAP were elevated in prostate, colon, breast, esophagus, as well as in ovarian and liver cancers In clinical studies, YAP was an independent predictor associated with poor diseasefree survival (DFS) and overall survival (OS) in HCC. 19 In view of the vital roles of YAP playing in the development of HCC, it was extremely important to clarify the function of YAP in the HBV-related HCC. Thus, we presumed that HBx might regulate YAP in hepatocarcinogenesis. In the present study, we tried to gain insight into the molecular mechanisms responsible for the regulation of YAP by HBx in HBV-associated HCC. Our data indicate that HBx is able to up-regulate YAP in hepatoma cells in a CREB-dependent manner, which results in the promotion of cell proliferation. Our findings provide fascinating insights into the mechanisms of HBx-enhanced proliferation of hepatoma cells. Patients and Methods Patient Samples. Thirty-three HCC tissues and their corresponding nearby nontumorous liver tissues utilized in this study were immediately obtained from Tianjin First Center Hospital (Tianjin, China) after surgical resection. Clinicopathological information about patients was obtained from patient records and are summarized in Supporting Table 1. All patients were diagnosed with primary HCC, and none had received previous radiotherapy or chemotherapy before surgery. Written consents approving the use of their tissues for research purposes after the operation were obtained from each patient. The study protocol was approved by the institute research ethics committee at Nankai University (Tianjin, China). Statistical Analysis. Each experiment was repeated at least three times. Statistical significance was assessed by comparing mean values (6 standard deviation; SD), using a Student t test for independent groups, and was assumed for P < 0.05, P < 0.01, and P < Pearson s correlation coefficient was used to determine the correlation between YAP and HBx messenger RNA (mrna) levels in tumorous tissues. YAP expression in tumor tissues and matched adjacent nontumor tissues were compared using Wilcoxon s signedrank test. Results HBx Up-regulates YAP in Hepatoma Cells. We determined the expression of YAP protein by immunohistochemistry (IHC) in HCC tissues. YAP immunoreactivity (IR) was graded as negative (score 0) and positive (scores 1-3) according to a previous report. 19 Our data demonstrated that 70% of 40 HCC patients showed increased expression levels of YAP. Marked nuclear IR was observed in 42.5% (17 of 40) of HCC tissues. Intermediate and low nuclear staining was noted in 17.5% (7 of 40) and 10% (4 of 40), respectively. Twelve of forty (30%) HCC samples showed negative YAP nuclear staining. For cytoplasmic localization of YAP, strong and moderate IR was observed in 37.5% (15 of 40) and 27.5% (11 of 40) of HCC tissues, respectively. Weak and negative YAP Address reprint requests to: Xiaodong Zhang, M.D., Ph.D., Department of Cancer Research, Key Laboratory of Molecular Microbiology and Technology of Ministry of Education, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin , China. zhangxd@nankai.edu.cn; fax: ; or Lihong Ye, M.D., Ph.D., Department of Biochemistry, State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin , P.R. China. yelihong@nankai.edu.cn; fax: þ Copyright VC 2012 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Nothing to report. Additional Supporting Information maybe found in the online version of this article.

3 HEPATOLOGY, Vol. 56, No. 6, 2012 ZHANG ET AL Fig. 1. YAP is overexpressed in HCC tissues and is significantly correlated with HBx. (A) Expression of YAP was examined by IHC staining in clinical HCC tissues and peritumor tissues. (a) Representative example of IHC observed in peritumor tissues using rabbit anti- YAP Ab. (b) Representative example of IHC observed in HCC tissues using rabbit anti-yap Ab. (B) Relative mrna levels of YAP were examined by qrt-pcr in 33 pairs of HCC tissues and corresponding nontumorous tissues (***P < 0.001; Wilcoxon s signed-rank test). Data presented are from three independent experiments. (C) Correlation between HBx mrna level and YAP mrna level was examined by qrt-pcr in 33 cases of HCC tissues (***P < 0.001, r ¼ 0.719; Pearson s correlation coefficient). Data presented are from three independent experiments. cytoplasmic IR activity accounted for 35% (14 of 40) of tumor samples. As for the paratumor tissues and healthy liver tissues, only 1 of 12 and 0 of 8 yielded a positive YAP signal, respectively (Fig. 1A). We observed that stronger nuclear YAP staining was displayed in nuclei of HCC cells and was seldom detected in nuclei of nontumor cells. Then, we examined mrna levels of YAP in 33 paired HCC and adjacent nontumorous liver tissues. Data revealed that the mrna levels of YAP were significantly elevated in HBV-related HCC samples, relative to their adjacent noncancerous hepatic tissues (P < 0.001; Wilcoxon s signed-rank test) (Fig. 1B). According to a previous report, 20 the expression of HBx mrna could be detected in HCC tissues by quantitative reverse-transcription polymerase chain reaction (qrt-pcr). Our data showed that the level of HBx mrna was detectable by RT-PCR in all clinical tissue samples from HBV-infected patients depicted in Supporting Table 1 (data not shown). Interestingly, we found that the upregulation of YAP significantly correlated with that of HBx (P < , r ¼ 0.719; Pearson s correlation coefficient) (Fig. 1C). Expression levels of YAP were elevated in HepG cells, relative to HepG2 cells. Meanwhile, YAP expression was reduced when psilencer-x (psi-hbx; 2 lg/well) was transfected into HepG cells (Fig. 2A, B). Moreover, expression of YAP was enhanced when HepG2 cells were treated with pch-9/3091 (2 lg/well) (Fig. 2C). Real-time PCR analysis showed that viral particle concentration was copies/ml in supernatants of HepG2 cells transfected with pch-9/3091 (not shown). We then investigated the expression of YAP in nontumor liver tissues from 6-month-old HBx-Tg mice and tumor tissues from 24-month-old HBx-Tg mice. 21,22 Data demonstrated that the expression of YAP was upregulated in liver tissues of 6-month-old HBx-Tg mice, relative to that of wild-type (WT) mice. Interestingly, the expression of YAP was markedly up-regulated in tumor tissues of 24-month-old HBx-Tg mice, relative to the liver tissues of 6-month-old HBx-Tg mice (Fig. 2D,E). Furthermore, western blotting analysis of nuclear YAP from HBx-Tg liver tissues revealed that there was progressive YAP nuclear localization with age or disease progression (Fig. 2E, bottom). Likewise, IHC analysis confirmed the overexpression and nuclear translocation of YAP in the murine HCC (Fig. 2F). Our data provide evidence that YAP may play important roles in the early stage of development of HCC. Next, we found that HBx was able to up-regulate the expression of YAP in stable HBx-transfected HepG2 (or H7402) cells (Fig. 3A). Strikingly, we observed that HBx knockdown by psi-hbx (1 or 2 lg/well) abolished the up-regulation of YAP in a dosedependent manner in the above-mentioned cell lines (Fig. 3B). Furthermore, our data demonstrated that knockdown of HBx mrna by psi-hbx (1 or 2 lg/well)

4 2054 ZHANG ET AL. HEPATOLOGY, December 2012 Fig. 2. YAP is up-regulated in HepG cells and HBx-Tg mice. (A and B) Relative mrna and protein levels of YAP were detected by qrt- PCR and western blotting in HepG2, HepG2.2.15, and HepG cells transfected with psilencer-control (psi-con; 2 lg/well) or psi-hbx (2 lg/ well), respectively. (C) mrna and protein expression levels of YAP were detected in HepG2 transiently transfected with control vector (2 lg/well) or pch-9/3091 (2 lg/well) by qrt-pcr and western blotting. (D) mrna levels of YAP were examined by qrt-pcr in nontumor liver tissues from 6- month-old HBx-Tg mice and tumor tissues from 24-month-old murine HCC versus WT mice liver tissues, respectively. (E) Total and nuclear YAP expression levels were examined by western blotting in 6-month-old HBx-Tg mice and 24-month-old murine HCC versus WT mice, respectively. (F) YAP expression was examined by IHC analysis in WT mice, 6-month-old HBx-Tg mice, and 24-month-old HCC, respectively. Data are shown as mean þ SD of three independent experiments. Statistically significant differences are indicated: *P < 0.05; **P < 0.01; Student t test. resulted in the down-regulation of AFP, a downstream target gene of YAP (Fig. 3C). 12,23 Taken together, we conclude that HBx is able to up-regulate YAP in hepatoma cells. HBx Is Able to Activate YAP Promoter. We further identified the YAP promoter core region. Various lengths of the YAP 5 -flanking region, including 1420/þ115 (pgl3-1536), 1000/þ115 (pgl3-1116), 604/þ115 (pgl3-720), 354/þ115 (pgl3-470), 232/þ115 (pgl3 348), 62/þ115 (pgl3-178), and 41/þ115 (pgl3-157), were cloned and transiently transfected into HepG2 (or H7402) cells to determine promoter activity, respectively. The luciferase reporter gene assays indicated that pgl3-348 exhibited the maximum luciferase activity among pgl3-1536, pgl3-1116, pgl3-720, pgl3-470, pgl3-348, pgl3-178, and pgl3-157 (Fig. 4A), indicating that the region of 232/þ115 is the promoter core region of YAP. Cotransfection of HBx expression plasmid (pcmv-x) (or psi-hbx) with various constructs (0.2 lg/well) or pgl3-basic control (0.2 lg/ well) was performed in HepG2/H7402/293T (or HepG2-X/H7402-X) cell lines, respectively. Data demonstrated that the relative luciferase activities of pgl increased, on average, by 5- or 10-fold in the 0.2- or 0.3-lg/well HBx-transfected HepG2/H7402/ 293T cells, respectively (Fig. 4B and Supporting Fig. 1). Meanwhile, we found that the relative luciferase

5 HEPATOLOGY, Vol. 56, No. 6, 2012 ZHANG ET AL Fig. 3. HBx increases endogenous mrna and protein levels of YAP in hepatoma cells. (A) Expression of YAP was measured by qrt- PCR and western blotting in HepG2/H7402, HepG2-P/H7402-P, and HepG2-X/H7402-X cell lines, respectively. (B) Expression of YAP was examined by qrt-pcr and western blotting in HepG2-X and H7402-X cell lines transiently transfected with psilencer-control (psi- Con; 2 lg/well) or psi-hbx (1 or 2 lg/well), respectively. (C) Expression of AFP was detected by qrt-pcr and western blotting in HepG2-X and H7402-X cell lines transfected with psi-con (2 lg/well) or psi-hbx (1 or 2 lg/well), respectively. Data are shown as mean þ SD of three independent experiments. Statistically significant differences are indicated: *P < 0.05; **P < 0.01; Student t test. activities of pgl decreased, on average, by 1- or 3-fold in 0.2- or 0.3-lg/well psi-hbx-transfected HepG2-X/H7402-X cells, respectively (Fig. 4B, right). Moreover, the promoter activities of pgl3-470 and pgl3-348 were enhanced by HBx as well (Fig. 4C,D and Supporting Fig. 1). However, the pgl3-basic control construct showed no activation by HBx (Supporting Fig. 2). Thus, we conclude that the regulation site of HBx is located at 232/þ115 of YAP, which is consistent with the promoter core region of YAP. HBx Activates YAP Promoter in a CREB-Dependent Manner. We then performed a search for possible transcription-factor binding sites in the promoter region 232/þ115 using MatInspector ( genomatix.de/online_help/help_matinspector/matinspector_ help). The promoter region 232/þ115 contains various different promoter elements, such as Ets and CREB. It has been reported that HBx is able to interact with CREB to regulate transcription. 24 Thus, we presumed that HBx might activate the YAP promoter by CREB. Interestingly, we found that the YAP promoter activity decreased in a dose-dependent manner when HepG2-X or H7402-X cells were treated with CREB short interfering RNA (sirna) (si-creb; 50 or 100 nm) (Fig. 5A). However, HBx failed to activate the YAP promoter mutant (pgl3-348 MUT), which disrupted the CREB-binding site (Fig. 5B). Moreover, expression levels of YAP were down-regulated by si-creb (50 or 100 nm) (Fig. 5C,D). To further determine the interaction of HBx with the promoter of YAP, we performed chromatin immunoprecipitation (ChIP) assays. Data indicated that the YAP promoter fragment could be detected in anti-hbx antibody (Ab)-immunoprecipited candidates, whereas the YAP promoter fragment was undetectable when cells were treated with si-creb (Fig. 5E). It suggests that HBx is able to bind to the promoter region of YAP by CREB. To test DNA-protein interactions in the promoter region of YAP, we performed electrophoretic mobility shift assay (EMSA) using the probe of DNA sequence from 231 to 200 bearing the CREBbinding site. Data revealed that HBx could bind to the CREB consensus sequence of the YAP promoter, and that the interaction could be blocked when the

6 2056 ZHANG ET AL. HEPATOLOGY, December 2012 lines. We found that HBx reduced mirna-375 expression (Supporting Fig. 3A,B), indicating that HBx up-regulates YAP expression through down-regulating mirna-375, rather than activating the YAP promoter. YAP Is Required for HBx-Induced Proliferation of Hepatoma Cells. Based on the hypothesis that YAP is involved in HBx-enhanced cell proliferation, we performed Methylthiazolyldiphenyl-tetrazolium bromide (MTT), ethynyldeoxyuridine (EdU), and colony-formation assays in HepG2-X or H7402-X cells by sirnamediated knockdown of YAP expression. Data clearly indicated that the proliferation ability of HepG2-X/ H7402-X cells was reduced by YAP sirna-1 (100 nm) or YAP sirna-2 (100 nm) (Fig. 6A-C). In addition, we found that YAP sirna was available to inhibit the growth of HepG2/H7402 cells in the absence of HBx (Supporting Fig. 4A,B), suggesting that YAP sirna is a nonspecific growth inhibitor in hepatoma cells. Surprisingly, we observed that tumorigenicity was remarkably suppressed in nude mice injected with HepG2-X cells pretreated with YAP sirna-1 (100 nm) in vivo (Fig. 7A-C). Meanwhile, the silence efficiency of YAP was confirmed by western blotting analysis in tumor tissues from mice (Fig. 7D). Taken together, we conclude that YAP is a key driver gene in the promotion of growth of hepatoma cells mediated by HBx. Fig. 4. HBx is able to activate the YAP promoter. (A) HepG2 and H7402 cells were transiently transfected with pgl3-basic (0.2 lg/ well) or reporter constructs containing various lengths of the 5 0 -flanking region of the YAP gene, as indicated (pgl3-1536, pgl3-1116, pgl3-720, pgl3-470, pgl3-348, pgl3-178, and pgl3-157, 0.2 lg/ well, respectively). Results were obtained as relative luciferase activity against the activity of pgl3-basic. Data are shown as mean þ SD of three independent experiments. (B-D) HepG2/H7402 or HepG2-X/ H7402-X cell lines were cotransfected with pgl (0.2 lg/well) or pgl3-470 (0.2 lg/well) or pgl3-348 (0.2 lg/well) and HBx expression plasmid (pcmv-hbx; 0.2 or 0.3 lg/well) or psi-hbx (0.2 or 0.3 lg/well), respectively. Promoter activities of YAP were measured by luciferase reporter gene assays. Data are shown as mean þ SD of three independent experiments. Statistically significant differences are indicated: *P < 0.05; **P < 0.01; Student t test. cold competitor or anti-hbx Ab or anti-creb Ab was added into the nuclear extracts (Fig. 5F), suggesting that HBx is able to activate the YAP promoter through CREB. In addition, it has been shown that microrna (mirna)-375, which is down-regulated in HCC, is able to target YAP. 25 To explore another mechanism by which HBx up-regulates YAP, we examined the effect of HBx on mirna-375 levels in HBx-Tg mice and HBx-transfected HepG2/H7402 hepatoma cell Discussion HBx plays an important role in hepatocellular carcinogenesis. 3 It has been reported that HBx regulates a wide variety of cellular genes and contributes to the multiple functions in modulating transcription, signal transduction, cell-cycle progress, protein-degradation pathway, apoptosis, and genetic stability by interaction with various transcription factors or components of signal transduction pathways. 4,5,26 YAP is an important oncogene and is able to promote proliferation in murine livers by regulating the transcription of certain target genes, such as Ki-67, c-myc, SOX4, H19, and AFP. 12 Thus, we supposed that YAP may be involved in HBx-induced hepatocarcinogenesis. Zhao et al. reproted that 54% of 115 American HCC patients showed overexpression of YAP. 16 Xu et al. reproted that 62% of 177 Hong Kong HCC patients showed overexpression of YAP. 19 Intriguingly, our data demonstrated that 70% of 40 HCC patients showed increased expression levels of YAP by IHC, which might be relevant to high HBV infection in mainland China. Moreover, we showed that YAP was mainly located in the nucleus of hepatoma cells and

7 HEPATOLOGY, Vol. 56, No. 6, 2012 ZHANG ET AL Fig. 5. HBx activates the YAP promoter in a CREB-dependent manner. (A) Luciferase activities were measured in HepG2-X and H7402-X cell lines cotransfected with pgl3-348 (0.2 lg/well) and control sirna (NC; 100 nm) or si-creb (50 or 100 nm), respectively. (B) Luciferase activities were measured in HepG2-X and H7402-X cell lines transfected with pgl3-348 (WT; 0.2 lg/ well) or YAP mutant promoter pgl3-348-mut (MUT; 0.2 lg/well). (C and D) Expression of YAP and CREB were detected by qrt-pcr and western blotting in HepG2-X and H7402-X cell lines transfected with control sirna (NC; 100 nm) or si-creb (50 or 100 nm), respectively. (E) ChIP assays were performed to confirm the interaction of HBx with the promoter region of YAP. (F) Interaction between HBx and CREB element in the promoter region of YAP was examined by EMSA assay. Data are shown as mean þ SD of three independent experiments. Statistically significant differences are indicated: *P < 0.05; **P < 0.01; Student t test. that the majority of nontumor cells demonstrated very weak staining, which is consistent with above-cited reports. 16,19 Interestingly, we found that the mrna levels of YAP were significantly correlated with those of HBx in 33 paired HCC tissues. The follow-up information of patients is partially summarized in Supporting Table 1. However, it is unavailable to evaluate the correlation of survival of patients with YAP expression. Our results showed that YAP was up-regulated in hepatoma cells mediated by HBV. We further observed that YAP was up-regulated markedly in liver tissues of HBx-Tg mice and that nuclear translocation of YAP occurred in HCC, indicating that YAP expression is elevated with age or disease progression in the model of HBx-Tg mice. Thus, our finding supports the notion that YAP overexpression and prominent nuclear localization are closely associated with viral-mediated hepatocarcinogenesis. We observed that YAP was upregulated in 6-month-old HBx-Tg mice, suggesting that YAP is up-regulated in the early stage of development of HCC. In addition, expression and nuclear accumulation of YAP were elevated in other multiple types of cancer, including prostate, colon, breast, ovarian, and esophagus It has been reported that YAP serves as an independent predictor for HCC-specific, DFS and OS. 19 Taken together, YAP is an important oncoprotein in human cancers. We further demonstrated that HBx up-regulated YAP in hepatoma cells. Meanwhile, we also revealed that HBx up-regulated the expression of AFP, a downstream target gene of YAP. 12,23 Thus, we conclude that HBx is able to upregulate YAP in hepatoma cells, which may contribute to the development of HBV-related HCC. Next, we identified that the fragment of 232/ þ115 contained the core region of the YAP promoter. Moreover, we observed that HBx was indeed able to activate the region of 232/þ115. Intriguingly, we predicted the CREB element in the YAP promoter region 232/þ115. As a camp-responsive transcription factor, CREB is a ubiquitous transcription factor that activates the transcriptional activity of various promoters through its binding site. The activated CREB protein binds to a camp response element region and is then bound to by CREB-binding protein, which coactivates it, allowing it to switch certain genes on or off. 24 Moreover, CREB has been implicated in hepatocarcinogenesis. 27,28 It has been reported that HBx acts as a transcriptional coactivator by directly interacting

8 2058 ZHANG ET AL. HEPATOLOGY, December 2012 mechanism underlying the increase of YAP expression mediated by HBx in HCC, indicating that HBx upregulates YAP expression through down-regulating mirna-375, rather than activating the YAP promoter. The mechanism of the down-regulation of mirna- 375 mediated by HBx should be further investigated. In terms of function, MTT, EdU analysis, and colony-formation assays revealed that cell proliferation capability was decreased in vitro when YAP expression was knocked down in hepatoma cells. Furthermore, in accord with our in vitro data, we found that tumor formation of hepatoma cells was remarkably suppressed by YAP sirna in animals. Expression levels of YAP were extremely decreased in tumor tissues from the test group, whereas expression levels of HBx were invariant in tissues, suggesting that the HBx-enhanced growth of hepatoma cells is strongly blocked by YAP RNA Fig. 6. YAP mediates HBx-induced cell proliferation in hepatoma cells in vitro. (A) Effect of YAP sirna (100 nm) on HBx-induced cell proliferation was determined by MTT assay in HepG2-X/H7402-X cells. (B) Effect of YAP sirna (100 nm) on HBx-induced cell proliferation was determined by EdU incorporation assay in HepG2-X/H7402-X cells. (C) Effect of YAP sirna (100 nm) on HBx-induced cell proliferation was determined by colony-formation in HepG2-X/H7402-X cells. Data are shown as mean þ SD of three independent experiments. Statistically significant differences are indicated: *P < 0.05; **P < 0.01; Student t test. with CREB. 5,24 Then, we were interested in whether CREB was involved in HBx-enhanced YAP expression. Our data indicated that the promoter activity of YAP could be reduced by CREB sirna, and that HBx failed to activate the YAP promoter when the CREBbinding site in the promoter was mutated. Meanwhile, expression levels of YAP were down-regulated by CREB sirna. Accordingly, we validated that HBx was able to indirectly bind to the promoter of YAP by CREB using ChIP and EMSA assays. Therefore, we conclude that HBx is able to activate the YAP promoter through CREB. In addition, it has been reported that YAP can be regulated by mirna-375 as a target gene. 25 Our data thus define another possible Fig. 7. YAP mediates HBx-induced cell proliferation in hepatoma cells in vivo. (A) Growth curve of tumors transplanted with HepG2-X cells pretreated with YAP sirna-1 (100 nm) or control sirna (NC; 100 nm) in nude mice. (B) Diagram of average weight of tumors. (C) Photographs of dissected tumors from nude mice (D) Protein expression levels of YAP and HBx were examined by western blotting in tumor tissues from nude mice, respectively. Data are shown as mean þ SD of three independent experiments. Statistically significant differences are indicated: *P < 0.05; **P < 0.01; Student t test.

9 HEPATOLOGY, Vol. 56, No. 6, 2012 ZHANG ET AL interference. Accordingly, it suggests that YAP is a key driver gene in HBx-induced hepatocarcinogenesis. In summary, we demonstrate that HBx promotes the growth of hepatoma cells through up-regulating oncogene YAP in the development of HCC involving CREB. The HBx-YAP connection may be a key central driver in the development of HCC. Our findings provide new insights into viral-mediated hepatocarcinogenesis. YAP may serve as a crucial target in HBVassociated HCC therapy. References 1. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132: Engelke M, Mills K, Seitz S, Simon P, Gripon P, Schn olzer M, Urban S. Characterization of a hepatitis B and hepatitis delta virus receptor binding site. HEPATOLOGY 2006;43: Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci 2006;97: Ng SA, Lee C. 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