HepatitisBVirusXProteinStabilizesCyclinD1and ...cancerpreventionresearch.aacrjournals.org/content/canprevres/8/5/... · HepatitisBVirusXProteinStabilizesCyclinD1and IncreasesCyclinD1NuclearAccumulationthrough

Research Article

Hepatitis BVirus XProtein Stabilizes Cyclin D1 andIncreasesCyclinD1NuclearAccumulation throughERK-Mediated Inactivation of GSK-3bXiangmei Chen1, Ling Zhang2, Sujun Zheng3, Ting Zhang1, Meng Li1, Xiaolei Zhang1,Zhenzhen Zeng1, Malcolm A. McCrae4, Jingmin Zhao5, Hui Zhuang1, and Fengmin Lu1

Abstract

The Hepatitis B virus X protein (HBx) contributes centrally tothe pathogenesis of hepatocellular carcinoma (HCC). It hasbeen suggested that the transcriptional activation of cyclin D1by HBx is implicated in the development of HCC. However,numerous studies have shown that overexpression of cyclin D1alone is not sufficient to drive oncogenic transformation. Here-in, we investigated whether HBx can stabilize cyclin D1 andinduce cyclin D1 protein nuclear accumulation, and therebyaccelerate hepatocarcinogenesis. The effects of HBx on cyclinD1 stabilization were assessed in cell-based transfection,Western blot, immunoprecipitation, immunocytofluorescencestaining, and flow-cytometric assays. The results demonstratedthat ectopic expression of HBx in HCC cells could extend thehalf-life of cyclin D1 protein from 40–60 minutes to 80–110

minutes. HBx stabilized cyclin D1 primarily in the S phase ofthe cell cycle, in a manner dependent on the inactivation ofGSK-3b, which was mediated by ERK activation. HBx alsoprompted the nuclear accumulation of cyclin D1, and cotrans-fection of the constitutively active mutant of GSK-3b along withHBx could reverse the nuclear accumulation and subsequentcell proliferation induced by HBx. Further, a positive correla-tion between HBx and nuclear cyclin D1 level was establishedin HCC specimens detected by an immunohistochemical assay.Taken together, our results indicated that HBx could stabilize andincrease cyclin D1 nuclear accumulation through ERK-mediatedinactivation of GSK-3b. This HBx-induced cyclin D1 upregulationmight play an important role in HCC development and progres-sion. Cancer Prev Res; 8(5); 455–63. �2015 AACR.

IntroductionHepatitis B virus (HBV) infection is the main risk factor for

hepatocellular carcinoma (HCC) worldwide. Epidemiologicstudies have shown that individuals who are chronic HBV carriershave a greater than 100-fold increase in the risk of developing livercancer (1). The HBV genome is a partially double-stranded,circular DNA containing four overlapping genes: S/preS, C/preC,P, and X. The X gene encodes a 17-kdHBV X protein (HBx), whichis a multifunctional transactivator of both viral and cellular genes(2). It is widely accepted that HBx plays crucial roles in thepathogenesis of HBV-induced HCC, as X gene-transgenic mousedeveloped liver cancer promptly (3, 4). However, a full under-standing of the molecular mechanism(s) responsible for HBx-induced HCC has yet to be achieved.

Perturbations in the regulation of the core cell cycle machineryare frequently observed in human cancers. As a key regulator of G1

phase reentry and progression, cyclin D1 is one of the mostfrequently altered cell cycle regulators in cancers (5). Newlysynthesized cyclin D1 associates with CDK4/6, and the cyclinD1/CDK complexes transport to and accumulate in the nucleusduring mid-G1 phase. The nuclear cyclin D1/CDK complexes canphosphorylate the retinoblastoma protein (Rb) and its relatedfamily members, thereby triggering E2F-dependent transcriptionof genes required for S-phase entry (6). During S-phase, cyclin D1level rapidly declines as a consequence of threonine-286 (Thr286)phosphorylation by glycogen synthase kinase-3b (GSK-3b; ref. 7),and this promotes the nuclear export, poly-ubiquitylation, anddegradationof cyclinD1by the 26Sproteasome (8, 9). This timelyexpression and accumulation of cyclin D1 is crucial for main-taining normal cell cycle progression, and disruption of it mayresult in uncontrolled cell proliferation.

Therefore, it is not a surprise to find frequently overexpressedcyclin D1 in a variety of human malignancies, including HCC(10–14). The overexpression of cyclin D1 can be attributed tomany factors, including increased transcription, translation, andprotein stability. Although cyclin D1 overexpression is clearlyimplicated in the affected cancers, simple overexpression of cyclinD1 is not sufficient to drive oncogenic transformation. This stemsin part from the fact that in a number of experimental transgenicmouse models of carcinogenesis, cyclin D1 overexpression alonefailed to induce tumors (8, 15). Rather, emerging evidence sug-gests that nuclear retention of cyclin D1 resulting from alterednuclear export and proteolysis is critical for the manifestation ofits oncogenicity (8, 15, 16).

1Department of Microbiology and Infectious Disease Center, School ofBasic Medical Science, Peking University Health Science Center,Beijing, P.R. China. 2Department of Hepatobiliary Surgery, HenanCancer Hospital, Zhengzhou, P.R. China. 3Beijing YouAn hospital,Capital Medical University, Beijing, P.R. China. 4The Pirbright Institute,Pirbright,Woking, United Kingdom. 5Department of Pathology, Insti-tute of Infectious Diseases, Beijing 302 Hospital, Beijing, P.R. China.

Note: Supplementary data for this article are available at Cancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Fengmin Lu, Peking University Health Science Center,38 Xueyuan Road, Beijing 100191, P.R. China. Phone: 86-10-8280-5136; Fax: 86-10-8280-5136; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-14-0384

�2015 American Association for Cancer Research.

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Previous studies have suggested a link between HBx and cyclinD1 upregulation in HCC tumors (17–19). Park and colleagues(17) reported that HBx may upregulate cyclin D1 in an immor-talized hepatocyte cell line Chang liver cells, as well as inHepG2.2.15 cells, which is an HBV-producing cell line derivedfrom an HCC cell line HepG2. A more detailed biochemicalanalysis has shown that the upregulation of cyclin D1 by HBxis mediated by the NF-kB2 (p52)/BCL-3 complex in the nucleus,which in turn activates the transcription of the cyclin D1 gene(CCND1). In addition, Ding and colleagues (18) and Khattar andcolleagues (19) found that HBx-activated Akt or ERKs couldphosphorylate and inactivate the downstream target GSK-3b,leading to stabilization of b-catenin, which subsequently trig-gered CCND1 gene transcription and hence increased cyclin D1production. Although these findings suggested that cyclin D1overexpression in HCC might result from the increased CCND1gene transcription by HBx, these studies lacked informationregarding the role of HBx in the proteolysis and nuclear accumu-lation of cyclin D1. Given the important role of GSK-3b in cyclinD1 degradation (7), we hypothesized that the inactivation ofGSK-3b by HBx not only upregulate cyclin D1 at transcriptionallevel, but itmay also stabilize cyclinD1protein andprompt cyclinD1 to accumulate in the nucleus.

In this study, we identified a mechanism to stabilize cyclin D1and prompt its nuclear accumulation through GSK-3b phosphor-ylation and inactivation by the HBx-activated ERK pathway,which will help us further understand the roles of HBx in theHBV-related HCC.

Materials and MethodsSample collection

A total of 36 paraffin-embedded tissues of primary humanHCCs were obtained from patients who underwent surgicalresection in Henan Cancer Hospital between 2012 and 2013. Allpatients were Han Chinese and had a background of chronichepatitis B infection and cirrhosis. This studywas approved by theEthicsCommittee of PekingUniversityHealth ScienceCenter, andthe informed consent was obtained from each participant.

Recombinant plasmid preparationThe Flag-tagged pFlex-cyclin D1, pFlex-D1-T286A, and HA-

tagged pCMV GSK-3b (S9A) plasmids were all kindly providedby professor J. Alan Diehl from Medical University of SouthCarolina (20, 21). The HA-tagged full-length HBx plasmid(pCMV-HA-HBx) was constructed by inserting a PCR-amplifiedfull-length HBx fragment into the EcoRI/KpnI sites of the pCMV-HA vector, using the following primers: forward 50-TATGGC-CATGGAGGCCCGAATTCCG-30 and reverse 50-ATCCCCGCGG-CCGCGGTACCG-30.

Cell culture, synchronization, and transfectionThe human HCC cell line HepG2 and immortalized mouse

embryonic fibroblast cell line NIH3T3 were purchased from theAmerican Type Culture Collection, and human HCC cell lineSMMC7721 was from Cell Resources Center of Peking UnionMedical College (Beijing, P.R. China). HepG2 cells were authen-ticated by DNA (STR) profiling in November 2013. No authen-tication for NIH3T3 and SMMC7721 cells was done by theauthors. All cells were maintained in DMEM supplemented with10% FBS (Gibco). Cells were transfected with the indicated

plasmids by using Lipofectamine Plus (Invitrogen) or 4D-Nucleo-fector X Unit (LONZA) according to the manufacturer's instruc-tions. S-phase synchronization was achieved by culturing cells inmedium containing 2.5 mmol/L thymidine for 28 hours, afterwhich cells were released in complete media, and harvested for 4hours after release.

Quantitative real-time reverse transcription PCRQuantitative real-time reverse transcription (RT) PCR was

performed as previously described (22). The primers used forreal-time RT-PCR were listed in Supplementary Table S1.

Immunoprecipitation and Western blot assaysForWestern blot analysis, proteins were extracted from cultured

cells using a radioimmunoprecipitation assay buffer. Proteinsamples (30 mg each) were loaded onto 15% sodium dodecylsulfate-polyacrylamide gels, electrophoresed, and transferredonto nitrocellulose membranes (Bio-Rad). After blocking with5% dried milk for 2 hours, the membranes were incubated withprimary antibody. The protein–antibody complexes were visu-alized using the Odyssey Imager (LI-COR Biosciences).

Immunoprecipitation (IP) assaywas carriedout on clarified celllysates in IP buffer. The clarified supernatant was incubated withthe primary antibody overnight at 4�C, and then immunocom-plexes were bound to protein-G Sepharose 4B for 1 hour at 4�C.Proteins bound to the protein-G Sepharose 4B were eluted byadding Laemmli-SDS sample buffer and boiling for 5 minutes.After centrifugation for 2 minutes, the immunoprecipitated pro-teins present in the supernatant were analyzed using the basicWestern blot protocol described above in this section.

The antibodies used in IP andWestern blot assays were listed inSupplementary Table S2.

Flow-cytometric analysisFor cell cycle analysis, cells were harvested by trypsinization

and fixed by treatment in 70% ethanol at 4�Covernight. The fixedcells were rinsed twice with PBS, and resuspended in a propidiumiodine (PI) solution (50 mg/mL) containing 0.5 mg/mL RNase A(Sigma), and incubated at 37�C for 30 minutes in the dark. Thefluorescence of the PI-labeled cells was then measured using aFACS Calibur system (Nippon Becton Dickinson), and the per-centages of cells in G0–G1, S, and G2–M phase were determinedusing the ModFit program (Nippon Becton Dickinson).

Immunofluorescence cell stainingCells were plated on glass coverslips and after 24 hours were

permeabilized with Methanol:Acetone (1:1), washed with PBS,and incubated in primary anti-cyclinD1 antibody for 2 hours.After washing and application of the secondary FITC-conjugatedanti-mouse antibody, slides were mounted using Hoechst 33258dye and analyzed by fluorescent microscopy (Nikon EclipseE800).

Immunohistochemistry assayImmunohistochemical (IHC) assay was performed on 5-mm

thick serial sections derived from formaldehyde-fixed, paraffinwax–embeddedHCC tissueblocks. The immunostaining of cyclinD1 or HBx protein in these tissues was detected as describedpreviously (21). The antibodies used for IHC analysis were listedin Supplementary Table S2.

Chen et al.

Cancer Prev Res; 8(5) May 2015 Cancer Prevention Research456




Statistical analysisFor statistical analyses, thedifferences between twogroupswere

analyzed by the two-tailed Student t test. The correlation betweenHBx and cyclin D1 expression was analyzed by Pearson correla-tion analysis. P < 0.05 was considered statistically significant. Allanalyses were performed using the SPSS suite of computer soft-ware (version 11.0).

ResultsHBx upregulates cyclin D1 expression through inhibiting itsdegradation

To investigate the ability of HBx to upregulate cyclin D1expression, the HepG2 and SMMC7721 cells were transfectedwith either control or HA-tagged HBx expressing plasmid. Asshown in Fig. 1A and B, ectopic expression of HBx could increasecyclin D1 level both at the protein and the mRNA levels. We thentestedwhetherHBx could affect the stability of cyclinD1.We usedcycloheximide to block protein synthesis and detected the turn-over rate of cyclin D1 by Western blot assay. The results showedthat overexpression of HBx in both HepG2 and SMMC7721 cellssignificantly extended the half-life of endogenous cyclin D1protein from 40–60 minutes to 80–110 minutes (Fig. 1C), indi-cating that in addition to transcriptionally activating cyclin D1expression, HBx also appears to upregulate cyclin D1 expressionby preventing its degradation.

HBx inhibits cyclinD1proteasomal degradationduring Sphaseof cell cycle

CyclinD1 has been shown to be degraded primarily via the 26Sproteasome in an ubiquitin-dependent manner (9). Consequent-ly, it is possible that HBx stabilizes cyclin D1 by inhibiting itsproteasomal degradation. To test this hypothesis, the proteasomeinhibitorMG132was used to block the degradation of proteins byubiquitin-proteasome machinery. Western blot analysis revealedthat MG132 significantly increased the cyclin D1 level in HepG2and SMMC7721 control cells (Fig. 2A, lane 2 vs. lane 1), but not inthe HBx-expressing cells (Fig. 2A, lane 4 vs. lane 3). The simplestinterpretation of this result is that HBx inhibits the proteasomal-based degradation of cyclin D1. To provide additional data insupport of this conclusion, the ubiquitination of cyclin D1 in thepresence ofHBxwasmonitored in an immunoprecipitation assay.As expected, despite the increase in cyclin D1 level in HBx-transfected cells, the ubiquitination levels of the protein dramat-ically decreased, demonstrating that HBx inhibits the ubiquitina-tion of cyclin D1 protein (Fig. 2B).

During cell cycle progression, the cyclin D1 level rises early inG1 phase and continues to accumulate until the G1–S-phaseboundary, and then it declines rapidly when DNA replicationinitiates (5). To investigate the precise cell cycle phase in whichHBx upregulated cyclin D1 level, HepG2 and SMMC7721 cellstransfected with either control or HBx expression plasmid weretreated with thymidine to block their progression into G2 phase,

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Figure 1.HBx upregulates cyclin D1 expressionthrough inhibiting its degradation.HepG2 and SMMC7721 cells weretransfectedwith pCMV-HBx or controlplasmids (Mock) using LipofectaminePlus as described in Materials andMethods. A, Western blot analysis ofcyclin D1 expression in HBx-transfected HepG2 and SMMC7721cells. B, real-time RT-PCR assay ofcyclin D1 expression in HBx-transfected HepG2 and SMMC7721cells. Data, means� SD from triplicateexperiments. C, Western blot analysisof cyclin D1 protein in the HBx-transfected HepG2 and SMMC7721cells after cycloheximide (50 mg/mL)treatment for the indicated time. Theturnover rate of cyclin D1 protein wasquantitatively analyzed by QuantityOne software (Bio-Rad) and shownonthe right.

HBx Prompts Cyclin D1 Nuclear Accumulation

www.aacrjournals.org Cancer Prev Res; 8(5) May 2015 457




thereby synchronizing the cell population in S phase (confirmedby FACS analysis). As expected, in comparison with nonsynchro-nized HepG2 cells (68.33% in G0–G1, 28.72% in S, and 2.95% inG2–M), cyclin D1 levels were dramatically reduced in synchro-nized cells (9.45% in G0–G1, 88.98% in S, and 1.57% in G2–M)transfected with control plasmid (Fig. 2Ci, lane 2 vs. lane 1). Incontrast, such reduction was much less apparent in synchronizedcell populations that express ectopic HBx (Fig. 2Ci, lane 5 vs. lane4). This result showed that HBx significantly increased the cyclinD1 level in the S phase of the cell cycle (Fig. 2Cii). It was furtherconfirmed by examining the cyclin D1 level in synchronized cellpopulations expressing ectopic HBx that had been treated withMG132. Treatment of such cells with MG132 failed to produceany significant increase in cyclinD1 levels (Fig. 2Ci, lane 6 vs. lane5), while similar treatment of synchronized S-phase cells trans-

fected with control plasmid showed a dramatic increase of cyclinD1 level (Fig. 2Ci lane 3 vs. lane 2, andFig. 2Ciii). Additionally, nodecrease of CCND1mRNA during S phase was observed in eithercontrol HepG2 cells or those expressing ectopic HBx (Supple-mentary Fig. S1). A similar phenomenon was obtained inSMMC7721 cells. These results indicated that the observeddecrease in cyclin D1 protein level seen in S-phase cells resultedprimarily from the accelerated degradation of the protein and,most likely,HBx increases cyclinD1 level during S phase primarilyvia the inhibition of proteasomal degradation of cyclin D1.

HBx stabilizes cyclin D1 through blocking GSK-3b–mediatedcyclin D1 Thr286 phosphorylation

GSK-3b–mediated cyclin D1–specific phosphorylation at Thr-286 is the initiating step for the rapid turnover of cyclinD1protein

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Figure 2.HBx inhibits cyclin D1 proteasomal degradation mainly in the S phase of the cell cycle. A, Western blot analysis of cyclin D1 protein in HBx-transfected HepG2and SMMC7721 cells after MG132 (10 mmol/L) treatment for 4 hours. Lower panel, quantitative analysis of cyclin D1 protein. B, Immunoprecipitation analysis of cyclin D1ubiquitination in pCMV-HBx–transfected HepG2 and SMMC7721 cells. Anti-cyclin D1 antibodies were used to immunoprecipitate the protein from cell lysates,and anti-ubiquitin antibodies were used to detect the ubiquitination of cyclin D1. Ten percent of lysates were subjected to direct Western blot analysis to confirmexpression levels of cyclin D1, HBx, and tubulin (input). C, HepG2 and SMMC7721 cells transfected with pCMV-HBx or control plasmids were cultured in mediumcontaining 2.5mmol/L thymidine for 28 hours. Then, cellswere released in completemedia for 4 hours (synchronized in Sphase) andharvested after being treatedwithor without MG132 (10 mmol/L) for 4 hours. Western blot analysis was used to detect the expression of cyclin D1 (i). ii, quantitative analysis of cyclin D1 protein innonsynchronized cells and synchronized in S-phase cells. Iii, quantitative analysis of cyclin D1 protein in S-phase cells with or without MG132 treatment.

Chen et al.





(7). Thus, we proposed that HBx would disrupt the GSK-3b–promoted cyclin D1 Thr286 phosphorylation and thereby extendits half-life. To test this hypothesis, HepG2 and SMMC7721 cellswere cotransfected with pCMV-HA-HBx and pFlex-cyclin D1 orpFlex-D1-T286A plasmids, then exogenous cyclin D1 expressionwas detected by Western blot analysis using anti-Flag antibody.The cyclin D1 T286A mutant is known to produce a highly stableform of the protein that is refractory to phosphorylation by GSK-3b. Western blot analysis showed that HBx can upregulate wild-type cyclin D1 expression in a dose-dependent manner (Fig. 3A).However, no change in cyclin D1 T286A mutant was seen at thedifferent dosages of pCMV-HA-HBx used (Fig. 3B), indicating thatthe HBx-mediated increase in cyclin D1 levels is linked with theThr286 phosphorylation of the protein. Strongly supporting thisnotion, further Western blot analysis demonstrated that the Thr-286 phosphorylation of cyclin D1 was significantly inhibited byHBx when MG132 was used to block the proteasomal degrada-tion of protein (Fig. 3C, lane 4 vs. lane 3). Because the exogenouscyclin D1 expression was driven by CMVpromoter in pFlex vectorand HBx did not affect promoter activity of the pFlex-cyclin D1,the detected differences of exogenous cyclin D1 expression levelsin the presence ofHBx are not associatedwith the change of cyclinD1 transcription. Taken together, these results suggested that HBxstabilized cyclin D1 through blocking the Thr286 phosphoryla-tion of the protein.

We next investigated whether the decreased Thr286 phosphor-ylation of cyclin D1 in HBx-expressing cells resulted from theinhibition ofGSK-3b kinase activity. To this end,we compared theGSK-3b kinase activity in HepG2 and SMMC7721 cells with orwithout HBx overexpression. We found that the phosphorylationof the GSK-3b Ser9 residue, which is known to inactivate GSK-3bkinase, was much stronger in both HepG2 and SMMC7721 cellstransfected with HBx than that detected in control cells (Fig. 4Aand B, lane 2 vs. lane 1). Furthermore, cotransfected with HBx,cyclin D1 and different doses of constitutively active S9A mutantof GSK-3b kinase (GSK-3b-S9A) into HepG2 and SMMC7721cells resulted in a dose-dependent suppression of exogenouscyclin D1 expression, indicating that constitutively active GSK-3b can reverse HBx-induced cyclin D1 stability (Fig. 4C). Thesedata suggested that HBx stabilized cyclin D1 and increased cyclinD1 expression through inactivation of GSK-3b.

Active ERKs are required for the HBx-mediated inactivation ofGSK-3b

It has been reported that HBx can activate either Akt or ERKs,and both Akt and ERKs can phosphorylate the Ser9 residue andinactiveGSK-3b (18, 19). To delineatewhich kinase is responsiblefor the phosphorylation of GSK-3b and cyclin D1, specific inhi-bitors of the Akt and ERK pathways were used to block thesekinases. As shown in Fig. 4, increasing expression of activephosphorylated ERKs, coupled with inactive phosphorylation ofGSK-3b, was found in the presence of HBx in both HepG2 andSMMC7721 cells. Pretreatment of cells with the ERK inhibitorU0126 significantly reduced HBx-mediated increase of endoge-nous cyclin D1, accompanied with the decrease of the phospho-GSK-3b (Ser9) (Fig. 4A, lane 3 vs. lane 2), whereas the PI3K/Aktinhibitor LY294002 did not (Supplementary Fig. S2). Consistent-ly, Western blot assays showed that the ERK inhibitor U0126 alsoinhibitedupregulated ectopic expression of cyclinD1byHBx (Fig.4B, lane 3 vs. lane 2). Taken together, these results suggested thatthe HBx-activated ERK pathway played a major role in theinactivation of GSK-3b and upregulation of cyclin D1 expression.

HBx-induced cyclin D1 nuclear accumulation prompts HCCcell proliferation

The cyclin D1 T286Amutant, which localizes specifically to thenucleus of the cell, exhibits a cellular transformation capacity,suggesting that disruption of cyclin D1 nuclear export is anoncogenic event (23). To investigate whether the oncogenicpotential of HBx is linked with the nuclear localization of cyclinD1, we detected the subcellular distribution of cyclin D1 in HBx-transfected cells by immunocytofluorescent staining. As shownin Fig. 5A, cyclinD1 accumulates predominantly in the nucleus ofHepG2 cells and mouse fibroblast NIH3T3 cells transfected withHBx during S phase, whereas in control cells cyclin D1 showed anexclusively cytoplasmic pattern of localization. Cotransfection ofthe constitutively active GSK-3b-S9A together with HBx was ableto reverse the nuclear accumulation of cyclin D1 induced by HBx.Consistently, flow-cytometric analysis confirmed the more rapidprogression in HBx-expressing HepG2 cells, whereby the percent-age of cells in S-phase entry was greater than control cells (51.13%vs. 30.53%) and the percentage of cells in G0–G1 phase decreasedfrom 67.85% to 40.45% (Fig. 5B). A similar phenomenonwas observed in SMMC7721 cells. It is also noteworthy that,consistent with the reversal of HBx-induced cyclin D1 nuclearlocalization, cotransfection of GSK-3b-S9Awas able to reverse the

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Figure 3.HBx stabilizes cyclin D1 through blocking its phosphorylation at Thr286.HepG2 and SMMC7721 cells were transfected with pFlex-cyclin D1 (A) orpFlex-D1-T286A (B) together with increasing doses of pCMV-HBx. Ectopiccyclin D1 protein was detected by Western blot analysis using M2 antibody.C, Western blot analysis of cyclin D1 phosphorylation at Thr286 by usinganti-cyclin D1 (phosphor T286) antibody in HBx-transfected HepG2 andSMMC7721 cells after treatment with MG132 (10 mmol/L).






proliferative activity induced by HBx alone. These results dem-onstrated that the nuclear localization of cyclin D1 induced byHBx was required for the cell proliferation and might be anoncogenic driver in HCC development.

Thenuclear cyclinD1 expression is significantly correlatedwithHBx in HBV-related HCC

To further examine whether this relationship identified in vitrocan also be observed in human tumor tissues, we compared thelevels of HBx and nuclear expressions of cyclin D1 in 36 pairs ofHBV-related HCC tissues by IHC. The results showed that thepositive rates of nuclear cyclin D1 and HBx in HCC tissues were33.3% (12 of 36) and 38.9% (14 of 36), respectively. A significantcorrelation was found between the accumulation of cyclin D1 innucleus and the expression of HBx in these HBV-related HCCsamples (P ¼ 0.0002; Fig. 6A and B), suggesting that the nuclearlocalization of cyclin D1 induced by HBx may be a generalphenomenon in HBV-related HCC.

DiscussionAlthough the multifunctional characteristics of viral protein

HBx have been extensively studied, the pathogenic mechanism ofHBx in human liver cancer remains largely unknown (2, 3). In thisstudy,weprovide thefirst evidence that by inactivatingGSK-3b viainhibiting the ERK pathways, HBx is able to inhibit cyclin D1nucleus export and subsequent proteasomal degradation. Impor-

tantly, our data suggest that the nuclear accumulation of cyclinD1induced by HBx is an oncogenic driver in HCC cells. Theseobservations highlighted the essential role of the cross-talkbetweenHBx and cyclinD1 inHBV-related hepatocarcinogenesis.

In normal cells, the proteolysis of cyclin D1 protein is subjectto a precise regulation by several mechanisms involving GSK3b-mediated phosphorylation of Thr286 (7), nuclear exportby CRM1 (8, 24), and subsequent ubiquitylation bySCFFbx4/aB-crystallin ligase targeting the cyclin D1 to the 26S protea-some (9). Deregulations of cyclin D1 proteolysis can lead toaberrant accumulation of cyclin D1 independent of alterationsin CCND1 gene expression and translation. Indeed, mutationsthat directly disrupt on the phosphorylation of Thr286 (25, 26) orinactivate SCFFbx4-aB-crystallin ligase have been identified in esoph-ageal cancer and endometrial cancer (9, 27). In addition to thesemutations, an alternative splice variant of cyclin D1, cyclin D1b,was identified in human cancer–derived cell lines. The character-ization of cyclin D1b revealed that cyclin D1b lacked Thr286 andremained in nucleus through the cell cycle, where its constitutiveexpression facilitates cellular transformation (21). Additionally,the upstream oncogenic events, such as those targeting the Ras orWnt pathway, can inhibit GSK-3b activity and thereby decreasecyclin D1 turnover (7, 28). Because no mutations at Thr286 ofcyclin D1 in HCC tissues have been reported, it is reasonable topredict that frequent alterations in regulation of GSK-3b activitymay play a major role in the nuclear accumulation of cyclin D1.Consistent with this notion, we observed that in HCC cell lines,

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0 0.5 1.0 1.5 2.0 μg2.0 1.5 1.0 0.5 0 μg

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Figure 4.HBx-inactivating GSK-3b isdependent on the ERK parthway.A, Western blot analysis ofphosphorylated GSK-3b,phosphorylated ERK, andendogenous cyclin D1 in pCMV-HBx–transfected HepG2 and SMMC7721cells treated with or without theERK inhibitor U0126 (50 mmol/L).B, Western blot analysis ofphosphorylated GSK-3b,phosphorylated ERK, and exogenouscyclin D1 in pCMV-HBx–transfectedcells treated with or without U0126(50mmol/L). C, HepG2 andSMMC7721cells were cotransfected with pFlex-cyclinD1 and pCMV-HBx togetherwith increasing doses of pCMV-GSK-3b (S9A) expression plasmid.Ectopic cyclin D1 expression wasdetected by Western blot analysisusing M2 antibody.

Chen et al.





HBx can stabilize cyclin D1 and increase cyclin D1 nuclear accu-mulation through inactivationofGSK-3bbySer9phosphorylation.Importantly, more than 30% of primary HBV-related HCC speci-mens examined in this study showed an abnormal cyclin D1accumulation in the nucleus, and a positive correlation betweenHBx and nuclear cyclin D1 protein level was established in theseHCC specimens. These data provide the first evidence of HBxinvolvement in the GSK-3b–mediated cyclin D1 stabilization.

GSK-3b is a serine/threonine kinase that can phosphorylateseveral proteins that are involved in controlling the structuralcharacteristics of the cells. Aberrant activity of GSK-3b has beenimplicated in the pathologies of many diseases and disorders(29). GSK-3b can be inactivated via phosphorylation at Ser9 bydiverse kinases, including Akt and ERKs. It is known that theinsulin signaling causes inactivation of GSK-3b (Ser9) by activat-ed Akt following receptor tyrosine kinase (RTK) and G protein–coupled receptor stimulation of PI3K, while growth factors causeinactivation of GSK-3b (Ser9) by activated Raf/MEK/ERK signal-ing (30, 31). Recent work has revealed that both Akt and ERKs canbe activated by HBx (18, 19); we therefore predicted that theinactivation of GSK-3bmight correlate with the activation of Aktor ERKs triggered byHBx. Our analysis using specific inhibitors of

the Akt and ERK pathways revealed that the HBx-activated ERKpathway plays an indispensable role in the inactivation ofGSK-3band nuclear accumulation of cyclin D1.

Accumulating data suggest that oncogenic functions of cyclinD1 depend upon its nuclear accumulation during S phase(16, 23). To date, there seem to be at least twomajormechanismsof cyclin D1–mediated tumorigenesis attributed to its associationwith CDKs. The first mechanism is based on the capacity of cyclinD1 to activate CDKs and consequently promote S-phase entry.This hypothesis is supported by the observations that the consti-tutively active nuclear cyclin D1–T286A/CDK4 complex mayperturb critical temporal and spatial timing of Rb-dependentgrowth control, resulting in S-phase entry and continuous pro-liferation (8, 32). Recently, experimental evidence linking thenuclear cyclin D1/CDK4 complex with induction of genomicinstability (33) provided a novel mechanism wherein cyclinD1 stabilization initiates tumor formation. Inmouse models andhuman cancer-derived cells, the cyclinD1–T286A/CDK4 complexwas found to be able to trigger DNA rereplication during S phase,resulting fromCdt1 stabilization, which in turn induced genomicinstability characterized by aneuploidy (33). Although the precisemechanism of this regulation remains to be uncovered, this

B

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2,000

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G0–G1: 67.85%G2–M: 1.62%S: 30.53%

G0–G1: 40.45%G2–M: 8.42%S: 51.13%

G0–G1: 69.78%G2–M: 4.22%S: 25.98%

G0–G1: 63.96%G2–M: 13.56%S: 22.48%

G0–G1: 47.98%G2–M: 20.05%S: 31.97%

G0–G1: 59.32%G2–M: 15.26%S: 25.42%

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bers

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pCMV-HBxpCMV-HBx+GSK-3β(S9A)pCMV-Mock

Cyclin D1 Hoest Merge Cyclin D1 Hoest Merge

Figure 5.The nuclear cyclin D1 promptedHCC cell proliferation. A,immunofluorescence assay wasperformed tomeasure the expressionand cellular distribution of cyclin D1 inHepG2 and NIH-3T3 cells transfectedwith pCMV-HBx alone or pCMV-HBxplus pCMV-GSK-3b-S9A. B, flow-cytometric analysis of HepG2 andSMMC7721 cells transfected withplasmids as in A.






observation suggests that the cyclin D1/CDK4 kinase is likely toparticipate more directly in the neoplastic process. Consistentwith these findings, our study demonstrated that the nuclearlocalization of cyclin D1 induced by HBx was required for theS-phase entry and might be an oncogenic driver in HCC cells.Further study should investigate whether the HBx-induced nucle-ar cyclin D1/CDK complex could trigger genomic instability andcontribute to neoplastic growth.

In conclusion, our results, together with those in previousreports (18), provided a plausiblemechanism for howHBxmightfacilitate cell proliferation andmalignant transition (Fig. 6C). Onthe one hand, HBx activates ERKs, resulting in GSK-3b inacti-vation and consequently b-catenin stabilization, which in turnactives CCND1 transcription; on the other hand, the HBx-mediated GSK-3b inactivation abolishes its capability to phos-phorylate cyclin D1 at Thr286 and blocks the phosphorylation-dependent nuclear export of the protein. As a consequence,

cyclin D1 protein accumulates in the nucleus, where it caninduce genomic instability and neoplastic growth. The nuclearretention of cyclin D1 may contribute to the malignant trans-formation of hepatocytes in HBV-related hepatocarcinogenesisand may be a potential target in the design of HCC therapy.However, further studies are needed to verify these results inHBx-expressing HCC cell lines by knocking down the expres-sion of HBx protein.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: X. Chen, T. Zhang, F. LuDevelopment of methodology: T. Zhang, M. LiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Zhang, S. Zheng

B

A

C

Cyclin D1+ (n = 12) Cyclin D1– (n = 24) P value

Correlation analysis of HBx and cyclin D1 expression in HCC

Cyclin D1

HBx+ (n = 14)

HBx– (n = 22)

HBx ERK

Cyc D1 Cyc D1

Nuclearexport

26S Proteasome

S-phase entryDNA rereplication

Hepatocyte with HBx expression Cell transformation

NuclearretentionCyc D1

CDK4

GSK-3b

P

PP

UbUb

Ub

Ser9

10

2

40.0002

20

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10x

40x

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Positive expression Negative expression

Figure 6.Expression of cyclin D1 and HBx proteindetermined by IHC staining in human HCCsamples. A, representative IHC images ofHBx and cyclin D1 in primary human HBV-HCC tissues. B, correlation analysis of HBxand nuclear cyclin D1 expression in primaryhuman HBV-HCC tissues. C, schematicdiagram of the possible role of HBx in theregulation of cyclin D1 during HBV infection–related hepatocarcinogenesis.


Chen et al.




Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Chen, T. Zhang, M. Li, X. Zhang, Z. ZengWriting, review, and/or revision of the manuscript: X. Chen, M.A. McCrae,H. Zhuang, F. LuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Zhang, J. ZhaoStudy supervision: H. Zhuang, F. Lu

AcknowledgmentsThe authors thank professor J. Alan Diehl from the Medical University of

South Carolina for providing the pFlex-cyclin D1, pFlex-D1-T286A, and pCMV-GSK-3b (S9A) plasmids.

Grant SupportThis work was supported by grants from the Natural Science Foundation

of China (nos. 30771099 and 81372603; to F. Lu and X. Chen), the NationalS&T Major Project for Infectious Diseases (nos. 2013ZX10002-002 and2012ZX10004-904; to X. Chen), and 111 project (B07001 to F. Lu).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 27, 2014; revised January 25, 2015; accepted February 9,2015; published OnlineFirst February 24, 2015.

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2015;8:455-463. Published OnlineFirst February 24, 2015.Cancer Prev Res Xiangmei Chen, Ling Zhang, Sujun Zheng, et al.

βInactivation of GSK-3Cyclin D1 Nuclear Accumulation through ERK-Mediated Hepatitis B Virus X Protein Stabilizes Cyclin D1 and Increases

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