Hepatocellular carcinoma (HCC) is one of the

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1 Hepatitis B Virus X Protein Stabilizes Amplified in Breast Cancer 1 Protein and Cooperates With It to Promote Human Hepatocellular Carcinoma Cell Invasiveness Yonghong Liu, 1 Zhangwei Tong, 1 Ting Li, 1 Qiang Chen, 1,2 Luting Zhuo, 1 Wengang Li, 3 Ray-Chang Wu, 4 and Chundong Yu 1 Chronic infection of hepatitis B virus (HBV) is closely associated with the development of human hepatocellular carcinoma (HCC). HBV X protein (HBx) plays a key role in the progression of HCC. We recently found that amplified in breast cancer 1 (AIB1) protein is overexpressed in 68% of human HCC specimens and promotes HCC progression by enhancing cell proliferation and invasiveness. Given that both HBx and AIB1 play important oncogenic roles in HCC, we aimed to determine whether they could cooperatively promote human HCC development. Herein, we show that HBx-positive HCC tissues had a higher level of AIB1 protein, compared to HBx-negative HCC tissues. A positive correlation between HBx protein level and AIB1 protein level was established in HCC specimens. Without affecting its messenger RNA level, HBx induced a significant increase of the protein level of AIB1, which correlated with a significant extension of the half-life of AIB1 protein. Mechanistically, HBx could interact with AIB1 to prevent the interaction between envelope protein 3 ubiquitin ligase F-box and WD repeat domain containing 7 (Fbw7)a and AIB1, then inhibited the Fbw7a-mediated ubiquitination and degradation of AIB1. In addition, reporter assays and chromatin immunoprecipitation assays revealed that both HBx and AIB1 were recruited to matrix metalloproteinase-9 (MMP-9) promoter to enhance MMP-9 promoter activity cooperatively. Consistently, HBx and AIB1 cooperatively enhanced MMP-9 expression in HepG2 cells, which, in turn, increased cell-invasive ability. Conclusion: Our study demonstrates that HBx can stabilize AIB1 protein and cooperate with it to promote human HCC cell invasiveness, highlighting the essential role of the cross-talk between HBx and AIB1 in HBV-related HCC progression. (HEPATOLOGY 2012;56: ) Hepatocellular carcinoma (HCC) is one of the most common human cancers worldwide, particularly in Southeast Asia. 1,2 Considering the poor prognosis and high mortality of HCC, it is imperative to understand the molecular mechanisms that trigger the progression and development of HCC. Nevertheless, the exact molecular mechanisms involved in HCC are not completely understood. Chronic infection of hepatitis B virus (HBV) is a major risk factor for HCC development. 3 The HBV Abbreviations: A, alanine; AIB1, amplified in breast cancer 1; Akt, v-akt murine thymoma viral oncogene homolog; AP-1, activator protein-1; AR, androgen receptor; ChIP, chromatin immunoprecipitation; CHX, cycloheximide; Co-IP, coimmunoprecipitation; ER, estrogen receptor; Fbw7, F-box and WD repeat domain containing 7; GSK3b, glycogen synthase kinase-3 beta; HAT, histone acetyltransferase; HBV, hepatitis B virus; HBx, HBV X protein; HCC, hepatocellular carcinoma; IgG, immunoglobulin G; MMP-9, matrix metalloproteinase-9; mrna, messenger RNA; NF-jB, nuclear factor kappa light-chain enhancer of activated B cells; PCR, polymerase chain reaction; RID, receptor interaction domain; SRC, steroid receptor coactivator; S/T, serine/threonine; TPA, 12-Otetradecanoylphorbol-13-acetate; Ub, ubiquitin; WT, wild type. From the 1 State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China; 2 The First Affiliated Hospital of Xiamen University, Xiamen, China; 3 Department of Hepatobiliary Pancreas and Vessel Surgery, Chenggong Hospital of Xiamen University, Xiamen, China; and 4 Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC. Received December 8, 2011; accepted March 23, This work was supported by grants from the Fundamental Research Funds for the Central Universities (grant no.: ; to C.Y.), the National Basic Research Program of China (973 Program; grant no.: 2009CB522200; to C.Y.), the Research Fund for the Doctoral Program of Higher Education of China (grant no.: ; to C.Y.), and the Natural Science Foundation of China (grant nos [to C.Y.] and [to W.L.]). The authors thank Dr. Jianming Xu (Baylor College of Medicine, Houston, TX) for the MMP-9-promoter/reporter plasmid and Dr. Ningshao Xia (School of Public Health at Xiamen University, Xiamen, China) for the HBx plasmid. 1015

2 1016 LIU ET AL. HEPATOLOGY, September 2012 genome is a partial double-stranded DNA that contains four open reading frames encoding virus envelope, core protein, virus polymerase, and HBV X protein (HBx). 4 Increasing evidence suggests that HBx plays an important role in hepatocarcinogenesis. 3 Although HBx does not bind to DNA directly, as a multifunctional regulator, it modulates the transcription of a large number of cellular genes involved in cell survival and apoptosis by interacting with the components of signal pathways as well as the degradation of various proteins by interacting with components of the ubiquitin (Ub)/proteasome system. 5 For instance, it has been shown that HBx up-regulates the expression of several genes, such as c-jun and c-fos, 6 and enhances the stability of ASC-2, c-myc, and pituitary tumor transforming gene In addition, HBx has been shown to promote the activation of several transcription factors, such as activator protein-1 (AP- 1), 10,11 nuclear factor kappa light-chain enhancer of activated B cells (NF-jB), 11,12 androgen receptor (AR), 13 and activating transcription factor/cyclic adenosine monophosphate responsive element-binding transcription factor. 14 Amplified in breast cancer 1 (AIB1/steroid receptor coactivator [SRC]-3/translocation-associated membrane protein-1/activator of thyroid hormone and retinoid receptor/cbp-interacting protein/receptor-associated coactivator-3) is a member of the p160 family, which also includes SRC-1 (nuclear receptor coactivator-1) and SRC-2 (trancsiptional interacting factor 2/glucocorticoid receptor interacting protein 1). 15 It has been reported that AIB1 is amplified and overexpressed in several human cancers, such as breast, mammary, prostate, stomach, colon, lung, and pancreatic cancers. 15 AIB1 has been shown to enhance the transactivation activity of some nuclear receptors, such as AR and estrogen receptor (ER), as well as a variety of transcription factors, such as AP-1 and NF-jB. 15 Moreover, increasing evidence indicates that AIB1 is a bona fide oncogene that activates several signaling pathways, such as v-akt murine thymoma viral oncogene homolog (Akt), E2F1, NF-jB, HER2/neu, ERa, AR, and epidermal growth factor receptor, to promote cancer progression. 15,16 Recently, we reported that AIB1 protein is overexpressed in 68% of human HCC specimens and promotes HCC progression by enhancing cell proliferation and invasiveness. 17 Given that both HBx and AIB1 play important oncogenic roles in HCC, we focused on the cross-talk between AIB1 and HBx during HCC progression. In this study, we reported that the expression of AIB1 protein was positively correlated with HBx protein level in human HCC specimens; overexpression of HBx in HCC cells significantly enhanced the stability of AIB1 through inhibiting the F-box and WD repeat domain containing 7 (Fbw7)a-mediated ubiquitination pathway; HBx cooperated with AIB1 to promote HCC cell invasiveness. Patients and Methods Patients and Tissue Samples. Tumorous and adjacent nontumorous human liver specimens were collected from 32 patients who underwent surgery for HCC at the First Affiliated Hospital of Xiamen University (Xiamen, China). Informed consent was obtained from each patient, and the study protocol, which conformed to the ethical guidelines of the 1975 Declaration of Helsinki, was approved by the Institute Research Ethics Committee at Xiamen University. Plasmids and Cell Transfection. Plasmids used in this study are listed in Supporting Table 1. Cells were transfected with the indicated plasmids by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer s instructions. At 48 hours posttransfection, cells were harvested and then used for further experiments. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction. Quantitative real-time polymerase chain reaction (PCR) was performed as previously described. 17 The primers used for real-time reverse transcriptionpcrarelistedinsupportingtable2. Coimmunoprecipitation Assay and Western Blotting Analysis. For coimmunoprecipitation (Co-IP) assay, cells were lysed with lysis buffer. Cell lysates were immunoprecipitated by correspondent antibodies or control immunoglobulin G (IgG). After extensive washing, precipitates were analyzed by western blotting. Western blotting analysis was performed as previously described. 17 Antibodies used in Co-IP assay Address reprint requests to: Chundong Yu, Ph.D., State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen , China. cdyu@xmu.edu.cn; fax: 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 may be found in the online version of this article.

3 HEPATOLOGY, Vol. 56, No. 3, 2012 LIU ET AL Fig. 1. Expression of AIB1 protein and HBx protein in human HCCs and their surrounding nontumorous liver specimens. (A) Western blotting analysis of AIB1 protein and HBx protein in 32 human HCC specimens and their surrounding nontumorous liver samples. T represents human HCC specimens; N represents their surrounding nontumorous liver tissue specimens. Asterisk (*) indicates HCC specimens overexpressing both HBx and AIB1. (B) Quantitative analysis of relative AIB1 protein expression in HBx( ) and HBx(þ) specimens. (C) Correlation between AIB1 protein level and HBx protein level in human HCC specimens. **P < and western blotting analysis are listed in Supporting Table 3. Ubiquitination Assay. To perform ubiquitination assay, 293T cells or HepG2 cells were transfected with AIB1 expression vector (Flag-AIB1) and Ub expression vector (hemagglutinin [HA]-Ub), in combination with or without HBx expression vector (HA-HBx). Then, anti-flag antibodies were used to immunoprecipitate Flag-AIB1 protein from total cell lysates, and anti-ha antibodies were used to detect the ubiquitination of AIB1. Chromatin Immunoprecipitation Assay. To perform chromatin immunoprecipitation (ChIP) assay, HepG2 cells were processed based on the protocol described by Abcam (Cambridge, MA). The following primers were used: matrix metalloproteinase-9 (MMP-9) promoter sense strand, 5 -GTCTTGCCTGACTTGG CAGT-3; antisense strand, 5 -TGACAGGCAAGTGCT GACTC-3. Gel Zymography and Transwell Cell-Invasion Assay. MMP-9 enzymatic activity was analyzed by gel zymography as previously described. 17 Cell-invasive ability was analyzed by Transwell cell-invasion assay, which was performed as previously described. 17 Statistical Analysis. Data were gained from several independent experiments. Each experiment was replicated at least three times. All data are shown as means 6 standard deviation. Statistically significant effects (P < 0.05) were evaluated with the two-tailed Student s t test.

4 1018 LIU ET AL. HEPATOLOGY, September 2012 Fig. 2. HBx up-regulates the expression of AIB1 protein through extending its half-life. (A) Ectopic expression of HBx in 293T and HepG2 cells resulted in up-regulation of AIB1 at the protein level, but not at the mrna level. (B and C) Analysis of the stability of AIB1 protein. Western blotting (upper panel) and quantitative analysis (lower panel) of AIB1 protein in 293T and HepG2 cells in the absence or presence of HBx after CHX treatment for the indicated time. Results AIB1 Protein and HBx Protein Are Frequently Co-overexpressed in Human HCC Tissues. Recently, we reported that AIB1 is overexpressed in approximately 70% of human HCC specimens and promotes HCC progression by enhancing cell proliferation and invasiveness. 17 Because 90% of these HCC specimens were from patients who were positive for HBV (data not shown), and HBx is tightly associated with HCC, we were interested in determining the potential relationship between AIB1 and HBx. We evaluated the expression of AIB1 and HBx in a set of 32 human tumorous and adjacent nontumorous liver tissues. As determined by western blotting, levels of AIB1 protein and HBx protein were significantly up-regulated in 23 (72%) and 18 (56%) HCC tissues, compared to adjacent nontumorous liver tissues, respectively (Fig. 1A). Among them, 16 (50%) HCC tissues showed co-overexpression of both AIB1 and HBx (Fig. 1A). Quantitative analysis showed that HBx-positive tissues had higher levels of AIB1 protein (Fig. 1B), and a positive correlation between AIB1 protein level and HBx protein level was established in these HCC specimens (Fig. 1C). These results suggest that HBx might positively regulate AIB1 expression. HBx Increases AIB1 Protein Level Through Extending the Half-Life of AIB1 Protein. Because HBx protein level was positively correlated with AIB1 protein level in human HCC tissues, we speculated that overexpression of HBx might up-regulate AIB1 expression. To test this, we transfected human embryonic kidney cells (293T) and human HCC cell lines (HepG2) with control plasmids or HBx expression plasmids to determine the regulative effects of HBx on AIB1 expression. Overexpression of HBx resulted in an increase of the protein level of AIB1 without affecting its messenger RNA (mrna) level in both 293T and HepG2 cells (Fig. 2A), suggesting that HBx regulates AIB1 expression at the post-transcriptional level. To determine whether HBx affects the stability of AIB1, we transfected 293T and HepG2 cells with control plasmids or HBx expression plasmids, then used cycloheximide (CHX) to block protein synthesis. Overexpression of HBx significantly extended the halflife of AIB1 protein in both 293T and HepG2 cells (Fig. 2B,C), indicating that HBx up-regulates AIB1 protein by preventing its degradation. HBx Inhibits the Ubiquitination of AIB1 Protein. HBx has been shown to be able to interfere with the Ub/proteasome pathway to prevent protein degradation. 7,8 Therefore, it is possible that HBx stabilizes AIB1 protein by inhibiting its ubiquitination. To test this hypothesis, we performed ubiquitination assays. Ubiquitination levels of AIB1 protein were dramatically decreased in the presence of HBx in both 293T and HepG2 cells, demonstrating that HBx inhibits the ubiquitination of AIB1 (Fig. 3A,B). To determine whether HBx could interact with AIB1 to inhibit the ubiquitination of AIB1, Flagtagged AIB1 and HA-tagged HBx were coexpressed in HepG2 cells, and Co-IP assays were performed. Anti- Flag antibodies, but not control IgG, immunoprecipitated HBx from cell lysates (Fig. 3C, left panel). Reciprocally, anti-ha antibodies could also immunoprecipitate AIB1 from cell lysates (Fig. 3C, right panel).

5 HEPATOLOGY, Vol. 56, No. 3, 2012 LIU ET AL Fig. 3. HBx inhibits the ubiquitination of AIB1 protein. (A and B) Ubiquitination of AIB1 protein in 293T and HepG2 cells was inhibited after HBx transfection. (C) Co-IP analysis of the interaction between AIB1 protein and HBx protein in HepG2 cells. (D) Co-IP analysis of the interaction between AIB1 protein and HBx protein in three human HCC specimens (21T, 30T, and 32T). To further evaluate whether the interaction between AIB1 and HBx could occur in human HCC specimens, we performed Co-IP assays in three human HCC specimens (21T, 30T, and 32T), which showed a high expression of both AIB1 and HBx. Anti-AIB1 antibodies, but not control IgG, could coimmunoprecipitate HBx protein in these samples (Fig. 3D). Reciprocally, anti-hbx antibodies could coimmunoprecipitate AIB1 protein. These data suggest that the interaction between HBx and AIB1 might be involved in the HBx-mediated inhibition of AIB1 ubiquitination. HBx Inhibits Fbw7a-Mediated Ubiquitination and Degradation of AIB1. It has been reported that SCF Fbw7 E3 Ub ligases can specifically and effectively mediate the ubiquitination and degradation of its substrates. 18 AIB1 has been identified as one of the substrates of Fbw7a. 19 Therefore, we tested whether HBx could inhibit the Fbw7a-mediated ubiquitination and degradation of AIB1. Western blotting analysis revealed that Fbw7a down-regulated AIB1 in a dosedependent manner; however, the Fbw7a-mediated down-regulation of AIB1 was dramatically inhibited by HBx (Fig. 4A). To further determine whether HBx could inhibit the Fbw7a-mediated ubiquitination of AIB1, we performed ubiquitination assays. The Fbw7a-mediated increase of AIB1 ubiquitination was significantly inhibited by HBx (Fig. 4B). Because HBx can interact with AIB1 protein, it is possible that HBx inhibits the Fbw7a-mediated ubiquitination and degradation of AIB1 through disruption of the interaction between AIB1 and SCF Fbw7a. To test this hypothesis, we performed Co-IP assays to examine the interaction between Fbw7a, AIB1, and HBx. Western blotting analysis revealed that Fbw7a interacted with AIB1 in the absence of HBx (Fig. 4C, lane 5); however, the interaction between Fbw7a and AIB1 was dramatically reduced in the presence of HBx (Fig. 4C, lane 6 versus lane 5), demonstrating that HBx inhibits the interaction between Fbw7a and AIB1. It is reported that phosphorylations of S505 and S509 in AIB1 are important for Fbw7a to regulate AIB1 turnover, and the down-regulation effect of Fbw7a on AIB1 is attenuated or completely abolished when either S505 or S509 is mutated to alanine (A). 19 We found that HBx significantly up-regulated the protein level of wild-type (WT) AIB1, as expected (Fig. 4D, lanes 1 and 2), whereas this up-regulation was severely abolished when either S505 (S505A) (Fig. 4D, lanes 3 and 4) or S509 (S509A) (Fig. 4D, lanes 5 and 6) was mutated to A, suggesting that the up-regulation effect of HBx on WT AIB1 is mainly the result of its inhibition of Fbw7a function. HBx Inhibits the Interaction Between Fbw7a and the Serine/Threonine Domain of AIB1. It is known that full-length AIB1 contains five functional domains: basic-helix-loop-helix domain; serine/threonine (S/T) domain; receptor interaction domain (RID); CBP/ P300 interaction domain; and histone acetyltransferase

6 1020 LIU ET AL. HEPATOLOGY, September 2012 Fig. 4. HBx inhibits Fbw7a-mediated ubiquitination and degradation of AIB1. (A) HBx inhibited Fbw7a-mediated down-regulation of AIB1 protein. (B) HBx inhibited Fbw7a-mediated ubiquitination of AIB1. (C) Co-IP analysis of the interaction between AIB1 and Fbw7a in the absence or presence of HBx. (D) Effects of HBx on protein levels of WT AIB1, AIB1-S505A, and AIB1-S509A, respectively. (HAT) domain (Fig. 5A). To identify which domains of AIB1 interact with Fbw7a or HBx, each of these five domains of AIB1 was coexpressed with Fbw7a or HBx in 293T cells, and Co-IP assays were performed. Western blotting analysis revealed that AIB1 interacted with HBx through its S/T and HAT domains (Fig. 5B) and interacted with Fbw7a through its S/T and RID domains (Fig. 5C). Because the S/T domain of AIB1 not only interacted with Fbw7a, but also interacted with HBx, it is possible that HBx inhibits the interaction between AIB1 and Fbw7a through the S/T domain. To test this hypothesis, Fbw7a was coexpressed with full-length AIB1, S/T domain, or RID of AIB1 alone or in combination with HBx in 293T cells; then, Co-IP assays were performed. In the presence of HBx, the amount of Fbw7a protein coimmunoprecipitated with full-length AIB1, as well as the S/T domain of AIB1, was significantly reduced (Fig. 5D, lane 9 versus lane 8, and lane 11 versus lane 10), whereas the amount of Fbw7a protein coimmunoprecipitated with RID of AIB1 was comparable in the absence or presence of HBx, as expected (Fig. 5D, lane 13 versus lane 12), because RID could not interact with HBx. Taken together, these data indicate that HBx inhibits the Fbw7a-mediated ubiquitination and degradation of AIB1 by competitively inhibiting the interaction between Fbw7a and the S/T domain of AIB1. HBx Cooperates With AIB1 to Promote HCC Cell Invasiveness Through Enhancing MMP-9 Expression. It has been reported that AIB1 plays an important role in the transactivation process mediated by transcription factors, such as NF-jB and AP-1, which can also be activated by HBx. 10,12 Thus, we hypothesized that HBx and AIB1 can cooperatively promote the transactivation activities of these transcription factors. To test this hypothesis, we cotransfected HBx and AIB1 with p65 or c-jun together with NF-jB reporter or AP- 1 reporter into HepG2 cells, respectively. Compared to control transfection, the NF-jB reporter activities induced by HBx and AIB1 alone were 3- and 2-fold, respectively, whereas it was induced more than 5-fold by the coexpression of HBx and AIB1 (Fig. 6A). Similarly, the AP-1 reporter activities induced by HBx and AIB1 were less than 15- and 2-fold, respectively, whereas the coexpression of HBx and AIB1 dramatically increased AP-1 reporter activity more than 30-fold (Fig. 6B). These results suggest that HBx cooperates with AIB1 to promote the activities of NF-jB and AP-1. Because both NF-jB and AP-1 are major regulators that activate the promoter of the mmp-9 gene, and both HBx and AIB1 can activate MMP-9 expression in HCC cells, 17,20 we examined whether HBx could cooperate with AIB1 to promote MMP-9 transcription by transfecting HBx and AIB1 together with the MMP-9

7 HEPATOLOGY, Vol. 56, No. 3, 2012 LIU ET AL Fig. 5. HBx inhibits the interaction between Fbw7a and the S/T domain of AIB1. (A) Schematic representation of the five functional domains of AIB1. (B) AIB1 interacted with HBx through its S/T and HAT domains. (C) AIB1 interacted with Fbw7a through its S/T and RID domains. (D) HBx inhibited the interaction between Fbw7a and the S/T domain of AIB1. promoter/reporter into HepG2 cells. After transfection, cells were treated with MMP-9 inducer 12-O-tetradecanoylphorbol-13-acetate (TPA), and then luciferase activities were determined. HBx or AIB1 alone can induce MMP-9 promoter activity to 5- or 3-fold, whereas the coexpression of HBx and AIB1 dramatically increased MMP-9 promoter activity to more than 10- fold (Fig. 6C). These results suggest that HBx can cooperate with AIB1 to increase MMP-9 promoter activity. To determine whether the cooperative effect of HBx and AIB1 on MMP-9 promoter activity is simply the result of elevated AIB1 protein levels, MMP-9 promoter/reporter assays were performed after HBx was transfected along with WT AIB1, AIB1-S505A, and AIB1-S509A, respectively. Similar to WT AIB1, HBx could cooperate with AIB1-S505A and AIB1-S509A to promote MMP-9 promoter activity (Fig. 6D). These results exclude the possibility that the cooperative effect of HBx and AIB1 on MMP-9 promoter activity is solely dependent on the elevation of AIB1 protein levels, because the protein levels of AIB1-S505A and AIB1- S509A were almost not affected by HBx (Fig. 4D). We previously showed that AIB1 could be recruited to the MMP-9 promoter. 17 Therefore, it is possible that HBx and AIB1 can co-occupy the MMP-9 promoter if these two proteins are stably associated. To test this hypothesis, ChIP assays were performed. Results showed that HBx was recruited to the MMP-9 promoter, and that recruitment was enhanced after the overexpression of AIB1 (Fig. 6E, lane 6 versus lane 5); similarly, AIB1 was recruited to the MMP-9 promoter, and recruitment was enhanced by HBx (Fig. 6F, lane 6 versus lane 5). These results indicate that both HBx and AIB1 are recruited to the MMP-9 promoter to cooperatively enhance MMP-9 promoter activity. To further confirm the cooperative role of HBx and AIB1 in MMP-9 expression, HepG2 cells, which highly express AIB1 (AIB1 WT ), but do not contain the HBx gene, were stably transfected with AIB1-knockdown (AIB1 KD ) plasmids to establish AIB1 KD /HBx cell lines, HBx expression plasmids to establish AIB1 WT /HBx þ cell lines, both AIB1-knockdown plasmids and HBx expression plasmids to establish AIB1 KD /HBx þ cell lines, and control plasmids to establish AIB1 WT /HBx cell lines, respectively. The expression of AIB1 protein was dramatically reduced in AIB1 KD /HBx cells, compared to AIB1 WT /HBx cells; ectopic expression of HBx significantly up-regulated AIB1 protein levels in both AIB1 KD /HBx þ and AIB1 WT /HBx þ cells, compared to AIB1 KD /HBx and

8 1022 LIU ET AL. HEPATOLOGY, September 2012 Fig. 6. AIB1 cooperates with HBx to enhance the activities of NF-jB reporter, AP-1 reporter, and MMP-9 promoter/reporter. (A) NF-jB reporter activity. (B) AP-1 reporter activity. (C) MMP-9 promoter/reporter activity. (D) WT AIB1, AIB1-S505A, and AIB1-S509A cooperated with HBx to enhance MMP-9 promoter/reporter activity. (E and F) AIB1 and HBx were recruited to the MMP-9 promoter, as measured by ChIP assays. AIB1 WT /HBx cells, respectively (Fig. 7A). The protein levels of HBx in AIB1 KD /HBx þ and AIB1 WT / HBx þ cells were comparable to that in human HBxpositive HCC tissues (Supporting Fig. 1). Compared to AIB1 KD /HBx cells, AIB1 KD /HBx þ cells exhibited a 7-fold increase, AIB1 WT /HBx exhibited a 5-fold increase, and AIB1 WT /HBx þ cells exhibited a 15-fold increase in TPA-induced MMP-9 mrna expression (Fig. 7B), indicating that HBx cooperates with AIB1 to increase MMP-9 expression. Consistent with mrna results, TPA-induced MMP-9 enzymatic activity in the conditioned medium of AIB1 WT /HBx þ cells was the highest (Fig. 7C). To determine whether increased MMP-9 expression would lead to an increase of cell-invasive ability, we measured the invasive ability of AIB1 KD /HBx, AIB1 KD /HBx þ, AIB1 WT /HBx, and AIB1 WT /HBx þ cells by using Transwell cell-invasion assays. Compared to AIB1 KD /HBx cells, AIB1 WT /HBx, AIB1 KD / HBx þ, and AIB1 WT /HBx þ cells exhibited higher invasive ability; among them, AIB1 WT /HBx þ cells exhibited the highest invasive ability (Fig. 7D). Collectively, these data demonstrate that HBx stabilizes AIB1 protein by inhibiting the Fbw7a-mediated ubiquitination and degradation of AIB1, and HBx cooperates with AIB1 to promote HCC cell invasiveness, at least in part, through up-regulating MMP-9 expression (Fig. 8). Discussion As an oncogene, AIB1 is frequently overexpressed in human cancers and plays a crucial role in promoting the progression of several human cancers, including HCC. 17 Meanwhile, HBx, a multifunctional regulatory protein, has been considered as a causative factor in the progression of HBV-related HCC. 3 In this study, we found that AIB1 protein level was dramatically increased in HBx-positive HCC tissues, compared to that in HBx-negative HCC tissues. In addition, the AIB1-positive rate in HBx-positive HCC tissues (88.9%) was apparently higher than that in HBx-negative HCC tissues (50.0%) (data not shown). These results indicate that HBx might regulate AIB1 expression. This speculation was supported by the fact that

9 HEPATOLOGY, Vol. 56, No. 3, 2012 LIU ET AL Fig. 7. AIB1 cooperates with HBx to enhance the invasiveness of HepG2 cells through up-regulation of MMP-9 expression. (A) Western blotting analysis of AIB1 protein and HBx protein in AIB KD / HBx, AIB KD /HBx þ, AIB1 WT /HBx, and AIB1 WT /HBx þ cells. (B and C) TPA-induced MMP-9 mrna expression and enzymatic activity were increased in AIB1 WT /HBx þ cells, compared to AIB KD /HBx, AIB KD / HBx þ, and AIB1 WT /HBx cells. (D) TPA-induced invasive ability was increased in AIB1 WT /HBx þ cells, compared to AIB KD /HBx, AIB KD / HBx þ, and AIB1 WT /HBx cells. *P < 0.05; **P < 0.01; ***P < overexpression of HBx led to the up-regulation of AIB1 protein in cells. Furthermore, overexpression of HBx resulted in an increased AIB1 protein stability and a reduced AIB1 protein ubiquitination. SCF Fbw7 E3 Ub ligase has been shown to play an important role in the ubiquitination and degradation of AIB1 through directly ubiquitinating AIB1 at K723 and K786 Fig. 8. Schematic model for the mechanism by which HBx inhibits AIB1 degradation and cooperates with it to promote HCC cell invasiveness. In the absence of HBx, AIB1 undergoes proteasomal degradation through SCF Fbw7a -mediated ubiquitination. In the presence of HBx, HBx inhibits the interaction between Fbw7a and AIB1 to prevent the Fbw7- mediated ubiquitination and degradation of AIB1. Furthermore, HBx cooperates with AIB1 to promote HCC cell invasiveness by up-regulating MMP-9 expression. (located in the RID domain) in an S505/S509 (located in the S/T domain) phosphorylation-dependent manner, 19 suggesting that the Fbw7 phosphodegron of AIB1 is located in the S/T domain, and that the S/T domain is the primary binding site of Fbw7a. Consistent with these results, we found that Fbw7a can bind to the S/T domain of AIB1. Furthermore, we showed that HBx could also bind to the S/T domain of AIB1, and that overexpression of HBx abolished the binding of Fbw7a to the S/T domain of AIB1, suggesting that HBx may have higher binding affinity to the S/T domain of AIB1, compared to Fbw7a, thus preventing the formation of the SCF Fbw7a /AIB1 complex to inhibit the Fbw7a-mediated ubiquitination and degradation of AIB1. Glycogen synthase kinase-3 beta (GSK3b) is an Fbw7a priming kinase, 21 which phosphorylates AIB1 at S505 to promote the Fbw7a-mediated ubiquitination and degradation of AIB1. 19 It has been shown that HBx is able to inactivate GSK3b through Akt activation. 22 Thus, it is also possible that HBx inhibits the Fbw7a-mediated ubiquitination and degradation of AIB1 through the inactivation of GSK3b. However, our data showed that overexpression of HBx in 293T and HepG2 cells cannot further increase the levels of phosphorylated Akt (active form) as well as the levels of phosphorylated GSK3b (inactive form) (Supporting Fig. 2), indicating that the HBx-induced up-regulation of AIB1 protein is not the result of the inactivation of GSK3b in this study.

10 1024 LIU ET AL. HEPATOLOGY, September 2012 Because several oncogenic transcription factors, such as NF-jB, AP-1, and AR, can be activated by both AIB1 and HBx, we speculated that AIB1 and HBx may have synergistic effects on the activation of these transcription factors. In agreement with this notion, we found that the coexpression of AIB1 and HBx synergistically induced MMP-9 expression through enhancement of the transactivation activity of NF-jB and AP-1 and, subsequently, promoted HCC invasion. In addition, we found that AIB1 and HBx could synergistically activate AR (Supporting Fig. 3), a nuclear receptor that plays an important role in promoting HCC progression, 13,23,24 suggesting that AIB1 and HBx may cooperatively activate oncogenic transcription factors other than NF-jB and AP-1 to promote HCC progression. Further study is needed to verify this implication. Cooperative effects of HBx and AIB1 on HCC progression may result in an earlier onset and diagnosis of the disease in patients with AIB1/HBx double-positive HCC. Indeed, we found that the rate of AIB1/HBx double-positive HCC in patients between the ages 30 and 45 (10 cases) was 80.0%, between 45 and 60 (11 cases) was 54.5%, and between 60 and 75 (9 cases) was 22.2%, suggesting that the rate of AIB1/HBx doublepositive HCC in patients is inversely correlated with age. Furthermore, the average age of patients with AIB1/HBx double-positive HCC at the time of diagnosis ( years old) was dramatically lower than that of patients with AIB1-negative or HBx-negative HCC ( years old) (Supporting Fig. 4). As in most cancers, multiple oncogenic pathways have been implicated in HCC progression. Discovering the cross-talk between different oncogenic pathways in HCC not only helps to understand the molecular mechanisms of HCC progression, but also provides new clues for therapeutic intervention. 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