Subcellular Mislocalization of Mutant Hepatitis B X Proteins Contributes to Modulation of STAT/SOCS Signaling in Hepatocellular Carcinoma

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Original Paper

Intervirology 2008;51:432–443 DOI: 10.1159/000209672

Subcellular Mislocalization of Mutant Hepatitis B X Proteins Contributes to Modulation ofSTAT/SOCS Signaling in Hepatocellular Carcinoma

C.-Thomas Bock a Nguyen L. Toan a, b Bernd Koeberlein a Le H. Song c

Ruth Chin d Hanswalter Zentgraf e Reinhard Kandolf a Joseph Torresi d

a Department of Molecular Pathology, Institute of Pathology, University Hospital of Tuebingen, Tuebingen , Germany; b Department of Pathophysiology, Vietnam Military Medical University, Hadong City , and c Department of Infectious Diseases, Tran Hung Dao Hospital, Hanoi , Vietnam; d Department of Medicine, Victorian Infectious Diseases Service, Royal Melbourne Hospital, The University of Melbourne, Melbourne, Vic. , Australia; e German Cancer Research Center, Applied Tumor Virology, Heidelberg , Germany

either by wt-HBx or HBx mutants. Interestingly, SOCS1 and SOCS3 expression was not activated by wt-HBx and HBx mu-tants. Conclusions: Our results suggest that atypical nucle-ar/perinuclear localization of HBx mutants might be respon-sible for an enhanced activation of STAT3, inhibition of STAT1 and silencing of SOCS1/SOCS3 expression. This observation points to an active role of HBx mutants in hepatocarcinogen-esis that involves dysregulation of STAT/SOCS signaling.

Copyright © 2009 S. Karger AG, Basel

Introduction

The hepatitis B virus X (HBx) gene encodes a viral protein with a central role in HBV infection and liver on-cogenesis [1–3] . However, the molecular mechanism by which HBx induces hepatocellular carcinoma (HCC) re-mains obscure. Previous studies have shown that HBx exhibits an oncogenic effect and can induce HCC in cer-tain lines of transgenic mice [4] . HBx is a potent transac-tivator of numerous cellular genes such as the nuclear factor (NF)- � B, activation protein 1 (AP1), AP2 and CRE

Key Words

Hepatitis B replication � Cell signaling � Chronic hepatitis � Virus variants � Hepatocarcinogenesis

Abstract

Objective: The hepatitis B virus X (HBx) protein plays an im-portant role in the pathogenesis of hepatocellular carcino-ma (HCC). One potential mechanism by which HBx can cause liver cancer may involve intracellular distribution and con-secutively modulation of the proliferative important STAT/SOCS signaling with upregulation of STAT3. Methods: 153 Vietnamese HBV-infected patients, including 48 patients with HCC, were analyzed. HBx sequences were determined by sequencing and subcloned for functional experiments. Intracellular localization of HBx mutants was determined by immunofluorescence assays. The impact of HBx mutants on JAK/STAT/SOCS signaling was investigated using Western blot and PCR analyses. Results: In 4/48 HCC patients, trun-cated HBx together with full-length mutated HBx proteins were observed. Expression of HBx mutant proteins demon-strated an atypical nuclear and perinuclear localization. Functional experiments to determine the effect of HBx mu-tants on STAT/SOCS signaling demonstrated a significantly increased upregulation of STAT3 activation (p 1 0.001) in comparison to wild-type (wt)-HBx. STAT1 was not activated

Received: September 18, 2008 Accepted: February 11, 2009 Published online: March 26, 2009

C.-Thomas Bock, PhD Department of Molecular Pathology, Institute of Pathology University Hospital of Tuebingen, DE–72076 Tuebingen (Germany) Tel. +49 7071 29 86889, Fax +49 7071 29 5334 E-Mail [email protected]

© 2009 S. Karger AG, Basel0300–5526/08/0516–0432$24.50/0

Accessible online at:www.karger.com/int

C.-T.B. and N.L.T. contributed equally to this work and share first au-thorship.

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[5–7] . Additionally, HBx has been shown to be involved in the activation of signal transduction pathways, such as Ras-Raf-MAP kinase [8, 9] , phosphoinositide 3 (PI-3) ki-nase [10] , protein kinase C, Src kinase, Janus kinase-1 (JAK-1), and signal transducer and activator of transcrip-tion (STAT) [11] . The interaction of HBx with STAT3 can lead to the activation and upregulation of various genes that contribute to cellular proliferation and carcinogen-esis [12] .

Previous studies indicated that C-terminally truncat-ed HBx may be associated with the development of HCC in HBV-infected patients [13, 14] . Point mutations in the HBx gene at position 31 with a serine to alanine (Ser-31Ala), at position 130 with a lysine to methionine (Lys-130Met), and at position 131 with a valine to isoleucine (Val131Ile) exchange were found to be prevalent in pa-tients with HCC [15–17] . Deletions in the HBx protein have also been associated with the suppression of STAT protein transcription [11] . However, the precise function-al evaluation of HBx at the molecular level is incomplete since very little is known about the intracellular localiza-tion of both wild-type (wt)-HBx and mutant HBx pro-teins [18] .

STAT proteins represent a family of latent transcrip-tion factors that are activated upon tyrosine phosphory-lation in response to extracellular signals such as cyto-kines and growth factors [19, 20] . Ligand-dependent ac-tivation of STATs is often associated with differentiation and/or growth regulation while constitutive activation of STATs is frequently associated with growth dysregula-tion [21] . One well-characterized member of the STAT family is the STAT3 protein with a strong oncogenic po-tential which is frequently overexpressed in a varietyof solid tumors, hematological malignancies and trans-formed cell lines [22–26] .

The suppressor of cytokine signaling (SOCS) protein family is known to act as negative regulators of the JAK/STAT pathway [26] . Of these, SOCS1 and SOCS3 have been shown to inhibit phosphorylation and activation of interferon (IFN)-induced STAT proteins by inhibiting the IFN receptor-associated JAK kinases [27] . Suppres-sion of SOCS3 has been shown to be inversely correlated to STAT3 activation in HCC patients [28, 29] while HBx can upregulate STAT3 activation [11] . However, the effect of HBx proteins on SOCS1 and SOCS3 expression is still unknown.

The biological function of altered intracellular distri-bution of HBx and the consequences with regard to cell signaling and carcinogenesis is still a matter of discus-sion. Here, we investigated the intracellular localization

of HBx mutants derived from HBV-infected Vietnamese patients with HCC and analyzed their impact on STAT/SOCS signaling in functional experiments.

Materials and Methods

Patient Characteristics 153 HBV-infected Vietnamese patients from the Tran Hung

Dao Hospital, Hanoi, Vietnam, were enrolled in this study. Of these, 48 patients developed a HCC. All patients had a positive serum HBsAg and were negative for antibodies to hepatitis C vi-rus (anti-HCV) and human immunodeficiency virus (anti-HIV). None of the study participants had a history of alcohol or drug abuse and none received antiviral treatment for hepatitis B infec-tion or immunosuppressive therapy before or during the course of this study. The clinical course and severity of hepatitis infec-tions, liver biochemical tests, serological markers and serum HBV-DNA quantitation of the patients including diagnostic tests for HCC patients are shown in table 1 and have been previously described in detail [30–32] .

Polymerase Chain Reaction Viral nucleic acid was obtained from patient serum samples

using the QiaAmp blood kit (Qiagen, Hilden, Germany) accord-ing to the manufacturers’ instructions. The HBx gene regions of HBV-infected patient isolates were amplified by polymerase chain reaction (PCR). Primers and reaction conditions used for ampli-fication have been described previously in detail [31] .

Sequencing and Subcloning of the HBX Gene HBx PCR fragments were purified using PCR cycle kit (Peqlab

Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s instructions. DNA sequencing was performed as described previously [30, 31] . The sequences obtained were ana-lyzed and aligned using BioEdit (http://www.mbio.ncsu.edu/BioEdit/-bioedit.html) and matched with the National Center for Biotechnology Information GenBank. Mutant HBx cDNA frag-ments were subcloned into the pGEM-T Easy cloning vector (In-vitrogen GmbH, Karlsruhe, Germany) according to the manufac-turer’s instructions. At least 5 clones of each HBx mutant were sequenced in forward and reverse directions using pT7-forward and pM13-reverse primers.

Construction of Recombinant Plasmids HBx mutant strains were introduced into the expression vec-

tor pcDNA3.1 (Invitrogen GmbH). Construction of wt-HBx gene and HBx gene mutant expression vectors were performed with primer pairs X-F1374: 5 � -CCG CTCGAG ATGGCTGCTAGGC-TGTAC-TGCC-3 � and X-R1856: 5 � -CCG GGGCCC GGACATG-TACAAGAGATGA-3 � , HBV nt 1–483 (HBx ATG = nt 1). The primer pairs were designed with 5 � overhangs either with Xho I or Apa I restriction enzymes sites (underlined sequences) for clon-ing. SOCS1 and SOCS3 constructs were obtained by PCR from cDNA isolated from human hepatocytes and cloned into the pcDNA3.1 vector. Each construct was confirmed by DNA se-quencing.

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Cell Culture and Transfections The human hepatoblastoma cell line HepG2 was used in all

cell culture experiments. Cells (2.5 ! 10 5 cells per 6-well plate) grown in Dulbecco’s modified Eagle medium (Invitrogen GmbH) were transiently transfected with pHBx plasmids using the cal-cium phosphate DNA precipitation method [33] . 24, 48 and 72 h after transfection the cells were harvested for further analyses. All experiments were performed in minimum in triplicates.

Immunofluorescence Studies Immunofluorescence experiments were performed as de-

scribed earlier [34] . HBx antigen was detected with monoclonal anti-HBx antibody and anti-mouse Cy3-conjugated secondary antibodies (Sigma-Aldrich, Munich, Germany). Nuclei were stained with 4,6-diamidine-2-phenyl-indole dihydrochloride (DAPI; 1 mg/ml; Hoffmann-La Roche Ltd., Basel, Switzerland) according to the manufacturer’s instructions.

Western Blot Analysis At indicated time points after transfection, HepG2 cells were

washed with 1 ! phosphate-buffered saline, lysed and cell super-natants were subjected to Western blot analysis as described pre-viously [33] . For the detection of STAT, SOCS and HBx antigens the following monoclonal mouse antibodies were used: anti-STAT1, anti-pSTAT1, anti-STAT3, and anti-pSTAT3 (Cell Sig-naling, Calif., USA), anti-SOCS1, anti-SOCS3, and anti-HBx (Deutsches Krebsforschungszentrum, Heidelberg, Germany) an-tibodies, respectively. Monoclonal mouse antibodies for the � -tu-bulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins (Cell Signaling Technology, Inc., Danvers, Mass., USA) were used to demonstrate that equal amounts of proteins were

loaded. Membranes were analyzed using the ECL-plus detection system (Amersham Pharmacia Biotech, Munich, Germany), fol-lowed by autoradiography.

Statistical Analysis Statistical analysis was performed using the � 2 test (available

at www.stata.com); the non-parametric Mann-Whitney U test and the percentiles analysis with the unpaired comparison t test were performed using StatView, Version 4.57 (available at www.statview.com).

Ethical Approval The study was approved by the Institutional Review Board of

the Tran Hung Dao Hospital, Hanoi, Vietnam.

Results

HBx Gene Mutations Occur Frequently in HCC Patients In order to determine the frequency of HBx gene mu-

tations in Vietnamese patients with HBV infection suf-fering from HCC, we amplified the HBx coding region from serum samples by PCR. Sequence analyses of the HBx gene demonstrated random point mutations in the HBx open reading frame ( fig. 1 a). It is noteworthy that a recently reported HCC-associated Ser31Ala exchange of the HBx open reading frame [17] occurred in the major-

Table 1. Clinical and virological characteristics of HBV-infected Vietnamese patients

ASYM SYMP

(n = 12)HCC (1)(n = 48)

no HCC (2)(AHB+CHB+LC; n = 93)

p values(1) vs. (2)

Mean age, years 35 56 42.5 <0.01Male/female 8/4 40/8 62/21HBsAg 12/12 48/48 93/93Total bilirubin, mg/dl 17 39 146.5 <0.001Direct bilirubin, mg/dl ND 13 92.5 <0.001ALT, IU/l <30 118 563.5 <0.001AST, IU/l <30 82 108.3 <0.001Prothrombin, % of standard >90 85 74 NSViral load, HBV copies/ml 23,731.8 8,787.2 20,433.3 <0.001AFP ND 240 NDHBV genotypes

ABCDEFG

2141000

89

213301

18142111

303

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HBx gene5’ Ser31Ala HBx stop

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Fig. 1. Analysis of the HBx amino acid (aa) sequences derived from patients with HCC. a aa sequence analysis of the HBx gene from serum samples of HCC patients demonstrated random point mutations. The HCC-associated serine to alanine substitution (Ser31Ala) at aa position 31 is depicted (arrow). The aa sequence of the wt-HBx reference strain from HBV-C (Acc. No. X52939) is shown at the top. b Analyses of patient samples by HBx cDNA-specific PCR of HCC patients revealed four HCC samples with truncated HBx genes of 260 bp for Pat. 1–3 (lanes 2–4) and 462 bp for Pat. 4 (lane 5), respectively, in addition to full-length HBx (lanes 2–5). Representative HBx PCRs of remaining HCC patients are also shown (lanes 6–11). The percentage of full-length versus

truncated HBx calculated from semiquantitative PCR is given in the table. c HBx subclones of the four HCC patient samples dem-onstrated randomly full-length mutated (upper sequences, Pat. 1–4) and C-terminally truncated (lower sequences, Pat. 1–4) HBx genes by sequence analyses. In addition to the observed deletions, stop codon ( * ) substitutions at aa position 77 of Pat. 1 and 2, at position 78 of Pat. 3 and at position 131 of Pat. 4 were observed. The aa sequence of wt-HBx of HBV-C (acc. No. X52939) is shown at the top. The numbering of amino acids from aa 1 to aa 154is according to the methionine start codon of the HBx gene(M = 1).

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ity of our HBV isolates from patients with HBV-induced HCC compared to non-HCC patients (p ! 0.01). We also identified truncated HBx genes in addition to full-length HBx genes in 4/48 HCC-related serum samples (8%) ( fig. 1 b). HBx cDNA bands corresponding to the expect-ed size of 483 bp of wt-HBx were visible ( fig. 1 b, lanes

2–11). In the samples of patients 1–3, additional HBx-spe-cific bands of approximately 260 bp were detected ( fig. 1 b, lanes 2–4), while in patient 4 a HBx-specific band of ap-proximately 462 bp ( fig. 1 b, lane 5) was obvious. Calcula-tion of the amount of truncated versus full-length HBx revealed 12.69% (Pat. 1), 36.18% (Pat. 2), 31.43% (Pat. 3), and 73.94% (Pat. 4) of truncated HBx, respectively ( fig.1 b). The serological, biochemical and virological charac-teristics of patients 1–4 are summarized in table 2 .

For functional experiments, we subcloned the HBx cDNAs from HCC patients 1–4 into eukaryotic expres-sion vectors. Each HBx mutant was sequenced showing that of the full-length HBx mutants a number of muta-tions in the HBx gene were detectable ( fig. 1 c, upper pan-el, Pat. 1–4). HBx gene species with deletions in the 3 � -ter-minal end were also detected ( fig. 1 c, lower panel, Pat. 1–4). In addition to the HBx gene deletions, stop codon substitutions at position 77 in Pat. 1 and 2 and positions 78 and 131 in Pat. 3 and 4, respectively, were obvious ( fig. 1 c).

HBx expression of the recombinant HBx constructs of HCC patients 1–4 in transfected HepG2 cells were shown by RT-PCR and Western blot analyses ( fig. 2 ). RT-PCR analyses revealed HBx RNA amplicons of 483 bp consis-tent with the expected size of wt-HBx RNA ( fig. 2 a, lanes 3 and 5). An approximate 260-bp fragment for the trun-cated HBx mutant was also detected ( fig. 2 a, lanes 2 and 4). Western blot analyses revealed HBx protein species of the expected size (17 kDa) in cells transfected with wt-HBx and randomly mutated HBx constructs ( fig. 2 b,

Table 2. Clinical characteristics of HBV-infected HCC patients representing truncated and mutated HBx

Patient 1 Patient 2 Patient 3 Patient 4

Age, years 47 77 59 39Male/female M M M MHBsAg positive positive positive positiveHBeAg negative negative positive positiveAnti-HBeAg positive positive negative negativeTotal bilirubin, mg/dl 21 84 20 19Direct bilirubin, mg/dl 9.7 55.6 11.6 11.3ALT, IU/l 44 105 143 98AST, IU/l 35 34 84 80Prothrombin

% of standard 95 60 88 100Viral load

HBV copies/ml 316.8 407.7 1,541.7 10,209.6AFP 532.3 94 95.7 484.9HBV genotype C C C C

IU = International units; AFP = �-fetoprotein; ALT = alanine aminotransferase; AST = aspartate aminotransferase.

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Fig. 2. HBx protein and HBx RNA expression in cell culture ex-periments. a RT-PCR analyses of transiently transfection experi-ments of HepG2 cells 24 h after transfection with truncated HBx (lane 2), full-length mutated HBx (lane 3), cotransfected trun-cated and full-length mutated HBx (lane 4), and wt-HBx pcDNA constructs (lane 5). Lane 6 represents the mock control of HepG2 cells transfected with pcDNA3.1 empty vector. Full-length mu-tated and wt-HBx revealed HBx RNA with an expected size of 483 bp while the truncated HBx constructs showed a band at 260 bp in agreement with sequence data ( fig. 1 ). b Representative West-ern blot analysis of HepG2 cells transiently transfected with trun-cated HBx (lane 1), full-length mutated HBx (lane 2) and cotrans-fected truncated and full-length mutated HBx (lane 3) and wt-HBx pcDNA constructs (lane 4), 48 h after transfection. Lane 5 represents the mock control transfected with pcDNA3.1 empty vector. Full-length mutated and wt-HBx revealed HBx proteins with expected size of 17-kDa of HBx antigen and truncated HBx revealed a 9-kDa polypeptide using monoclonal anti-HBx anti-bodies. The detection of the housekeeping gene � -tubulin is shown in the lower panel.

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lanes 2 and 4), while a smaller band of approximately9 kDa was detected in the cells transfected with theC-terminally truncated HBx construct ( fig. 2 b, lanes 1 and 3).

Intracellular Localization of HBx Mutants In order to determine the intracellular localization of

the HBx mutations, we performed immunofluorescence studies. In accordance with previous studies [18, 35] we found that wt-HBx protein was predominantly distrib-uted in the cytoplasm ( fig. 3 a). In contrast, the analysis of recombinant C-terminally truncated HBx protein mu-tants identified in HCC patient 1 (recombinant HBx con-struct Mut. 1) to patient 3 (Mut. 2 and Mut. 3) revealed a predominant atypical nuclear localization of the HBx protein ( fig. 3 b, panels 1–3). For Mut. 4, which only in-cluded a 21-bp deletion, the truncated protein was found mainly nuclear, but also resulted in faint cytoplasmic

staining ( fig. 3 b, panel 4). In contrast to the C-terminally truncated HBx proteins, the full-length mutated HBx proteins demonstrated a mostly perinuclear localization ( fig. 3 c, panels 1–4). As shown by the in vivo analyses above, we observed mixed HBx species in 1 patient. Co-transfection experiments with C-terminally truncated and full-length randomly mutated HBx constructs of Mut. 1 to Mut. 4 demonstrated a combination of nuclear and cytoplasmic localization for Mut. 1 to Mut. 4, which was most evident in Mut. 4. No specific fluorescence sig-nal was observed in mock-transfected cells used as a neg-ative control ( fig. 3 a, upper panel).

HBx Mutants from HCC Patients Induce STAT3 but Not STAT1 Activation We next addressed the question of whether the atypi-

cal localization of HBx mutant proteins may have an in-fluence on the cellular processes. Previous studies have

Mock

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Fig. 3. Intracellular localization of HBx mutants identified in HCC patients. Immunofluorescence experiments showing repre-sentative selected results of transiently transfected HepG2 cells with a wt-HBx, b truncated HBx (Mut. 1 to 4), c full-length mu-tated HBx (Mut. 1 to 4), and d cotransfected truncated and full-length mutated HBx pcDNA constructs using monoclonal anti-HBx antibodies 48 h after transfection. A random cytoplasmic distribution of wt-HBx ( a , lower panel) was observed, whereas the mock control of HepG2 cells shows no staining of HBx antigen ( a , upper panel). b HepG2 cells transfected with truncated HBx con-structs reveal a mainly nuclear staining of HBx antigen for Mut. 1 to 4, while Mut. 4 showed an additional to the nuclear staining

slight cytoplasmic staining for HBx antigen. c HepG2 cells tran-siently transfected with full-length mutated HBx constructs orig-inally derived from patient 1 to 4 demonstrate a mainly perinu-clear localization of HBx proteins for Mut. 1 to 4, respectively. d HepG2 cells cotransfected with truncated and full-length mu-tated HBx pcDNA constructs show mainly nuclear staining for Mut. 1, a perinuclear and nuclear staining for Mut. 2, and nuclear plus cytoplasmic distribution of HBx antigen for Mut. 3 and 4. Nuclei of corresponding cells are shown by DAPI staining. Mock control consists of HepG2 cells transfected with pcDNA3.1 emp-ty vector. Original magnification ! 200.

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Fig. 4. Expression of HBx mutants derived from HCC patients leads to activation of pSTAT3 but not pSTAT1. a pcDNA3.1 con-structs of truncated HBx, full-length mutated HBx and the mix-ture of truncated and full-length mutated HBx (Mut. 1 to 4, while Mut. 1 to 4 refer to patients 1 to 4) were transiently transfected in HepG2 cells. Activation of pSTAT3 and total STAT3 by the HBx mutants including wt-HBx and mock control are shown by West-ern blot experiments 48 h after transfection using monoclonal anti-pSTAT3 and anti-STAT3 antibodies. Experiments with trun-cated HBx (lanes 2–5; Mut. 1 to 4) demonstrate a lower, full-length mutated HBx (lanes 6–9; Mut. 1 to 4) a slightly higher and the co-transfection of truncated and full-length mutated HBx (lanes 10–13; Mut. 1 to 4) a higher pSTAT3 level in comparison to wt-HBx (lane 14). In contrast, mock-transfected cells revealed no pSTAT3 activation (lane 15). Stimulation of the cells with IFN- � served as a positive control for pSTAT3 activation (lane 1). Notably, the transfection experiments did not alter the STAT3 expression

(middle panel, lanes 2–15). The housekeeping gene � -tubulin was used to standardize the system and indicates that equal amounts of total protein were loaded in each lane (lower panel, lanes 2–15). b Densitometric evaluation of pSTAT3 activation of 3–5 different Western blot experiments as shown in a is given. The analysis was performed using the percentiles analysis with the unpaired com-parison t test (table on the right). c Western blot analyses of pSTAT1 activation (upper panel) and total STAT1 expression (middle panel) using monoclonal anti-pSTAT1 and anti-STAT1 antibodies demonstrated that pSTAT1 was not activated by wt-HBx (lane 15) and HBx mutants (lanes 2–13, Mut. 1 to 4). Stimu-lation of the cells with IFN- � served as a positive control for pSTAT1 activation (lane 1). The housekeeping gene � -tubulin was used to standardize the system and indicates that equal amounts of total protein were loaded in each lane (lower panel, lanes 2–15).

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shown that persistent activation of STAT3 is detectable in various human cancers and in HCC [23, 24, 36] . We therefore analyzed the effect of both wt-HBx and HBx mutants (Mut. 1 to Mut. 4) on the STAT/SOCS signaling. Activation of STAT3 (pSTAT3) was analyzed by Western blot 24, 48 and 72 h after transfection of HepG2 cells with HBx clones. Truncated Mut. 1 to Mut. 4 showed an over-all reduction of pSTAT3 activation compared to wt-HBx 48 h after transfection ( fig. 4 a, lanes 2–4). Densitometric evaluation demonstrated a 3.4% reduction of pSTAT3 for

truncated Mut. 1, 17% for truncated Mut. 2, 44.5% for truncated Mut. 3 and 41.4% for truncated Mut. 4 com-pared to wt-HBx ( fig. 4 b). Taken together, the level of STAT3 activation in the truncated HBx protein group (Mut. 1 to 4) was significantly lower than wt-HBx (p = 0.04; fig. 4 b). In contrast, the full-length mutated HBx proteins (Mut. 1 to Mut. 4) resulted in an overall induc-tion of pSTAT3 activation compared to wt-HBx ( fig 4 a, lanes 6–9). However, densitometric evaluation revealed that there is no significant difference in pSTAT3 level of

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Fig. 5. Time-dependent effect of HBx mutants on pSTAT3 and pSTAT1 activation. Recombinant pcDNA3.1 constructs of trun-cated HBx, full-length mutated HBx, and a mixture of truncated and full-length mutated HBx were transiently transfected in HepG2 cells. Western blot analysis for the detection of a pSTAT3 and b pSTAT1 activation at 24 h (column I), 48 h (column II) and 72 h (column III) after transfection. Experiments at 24 h after transfection demonstrated an approximately 30% induction of pSTAT3 for wt-HBx and HBx mutants in comparison to HepG2

cells ( a , column I). 48 h after transfection pSTAT3 levels were ac-tivated by approximately 46% for wt-HBx and mutated as well as truncated HBx and up to 81% for the combination of truncated and full-length mutated HBx in comparison to HepG2 cells ( a , column II). The pSTAT3 levels were reduced at 72 h after transfec-tion for wt-HBx and mutated HBx ( a , column III). b No pSTAT1 activation could be detected 24 h (column I), 48 h (column I) and 72 h after transfection (column III).

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full-length mutated HBx proteins compared to wt-HBx (p = 0.06; fig. 4 b). The strongest upregulation of pSTAT3 was observed in cells cotransfected with both truncated and full-length HBx mutants ( fig. 4 a, lanes 10–13). When compared to wt-HBx, pSTAT3 was increased by 75% for Mut. 1, 58.6% for Mut. 2, 65.5% for Mut. 3, and 43% for Mut. 4, respectively ( fig. 4 a). Overall the level of STAT3 activation was significantly higher in comparison to wt-HBx (p ! 0.001; fig. 4 b). In contrast to the changes in pSTAT3, the total STAT3 expression was not altered by wt-HBx nor any of the HBx mutants ( fig. 4 a, panel 2, lanes 2–14).

Analyses of time-dependent induction of pSTAT3 by HBx mutants demonstrated that 24 h after transfection with HBx mutants the level of pSTAT3 was approximate-ly 30% higher compared to HepG2 cells stimulated with IFN- � . However, there was no significant detectable dif-

ference in the pSTAT3 levels of HBx mutants compared to wt-HBx (p 1 0.05). At 48 h the level of pSTAT3 was higher in cells transfected with the combination of trun-cated and full-length mutated HBx (81%) compared to wt-HBx (46%) (p ! 0.01; fig. 5 a). 72 h after transfection of HBV mutants the level of pSTAT3 dropped to approxi-mately 20% compared to HepG2 cells stimulated with IFN- � . Notably, there was no significant difference in pSTAT3 levels observed using either mutated HBx or wt-HBx constructs (p 1 0.05; fig. 5 a).

Recent studies have also indicated that STAT1 plays an important role in the development of cancer [25, 37] . We therefore analyzed whether wt-HBx and HBx mutant proteins have a role in modulating STAT1 activation. These experiments demonstrated that neither wt-HBx nor HBx mutant proteins affected pSTAT1 activation ( fig. 4 c, 5 b).

SOCS1

SOCS1

GAPDH

Lane:

pcDNASOCS1

1 2a 3 4

Mut.1

Mut.2

Mut.3

Mut.4

5 6 7 8 9 10 11 12 13 14

wt Mock

15

Mut.1

Mut.2

Mut.3

Mut.4

Mut.1

Mut.2

Mut.3

Mut.4

Truncated HBx Mutated HBxTruncated +

mutated HBx

SOCS1

GAPDH

24 hb

Tr

Lane: 1 2 3 4 5 6

pcDNASOCS1 M Tr + M wt Mock

48 h

Tr

7 8 9 10 11 12

72 h

13 14 15 16 17 18

pcDNASOCS1 M Tr + M wt Mock Tr

pcDNASOCS1 M Tr + M wt Mock

SOCS3

SOCS3

GAPDH

Lane:

pcDNASOCS3

1 2c 3 4

Mut.1

Mut.2

Mut.3

Mut.4

5 6 7 8 9 10 11 12 13 14

wt Mock

15

Mut.1

Mut.2

Mut.3

Mut.4

Truncated +mutated HBx

Mut.1

Mut.2

Mut.3

Mut.4

Mutated HBxTruncated HBx

SOCS3

GAPDH

24 hd

Tr

Lane: 1 2 3 4 5 6

pcDNASOCS3 M Tr + M wt Mock

48 h

Tr

7 8 9 10 11 12

72 h

13 14 15 16 17 18

pcDNASOCS3 M Tr + M wt Mock Tr

pcDNASOCS3 M Tr + M wt Mock

Fig. 6. No SOCS1 and SOCS3 activation by HBx proteins. Recombinant pcDNA3.1 constructs of truncated HBx, full-length mutated HBx, and a mixture of truncated and full-length mutated HBx were tran-siently transfected in HepG2 cells. Analy-sis of SOCS1 ( a , b ) and SOCS3 ( c , d ) ex-pression by HBx mutants (as shown for STAT activation in figure 4) including wt-HBx and mock control are shown by West-ern blot experiments 48 h after transfec-tion ( a , c ) and by time course ( b , d ; 24, 48 and 72 h) using monoclonal SOCS1 and SOCS3 antibodies. The housekeeping gene GAPDH was used to standardize the sys-tem and indicates that equal amounts of total protein were loaded in each lane (lower panels in a – d ). As a positive control, recombinant SOCS1 and 3 plasmids were used.

HBx Mutants Dysregulate STAT/SOCS Signaling

Intervirology 2008;51:432–443 441

STAT1 and STAT3 signaling are negatively regulated by SOCS1 and SOCS3 proteins, respectively [26] . Cur-rently, there is no information available of the interaction of HBx protein with the activation of SOCS1 and SOCS3 proteins. We therefore determined whether or not wt-HBx and mutant HBx proteins (Mut. 1 to Mut. 4) could alter the expression of SOCS1 and SOCS3 proteins. Nei-ther wt-HBx nor HBx mutants (Mut. 1 to Mut. 4) resulted in the upregulation of SOCS1 and SOCS3 expression ( fig. 6 ).

Discussion

Previous studies have shown that specific HBx gene mutations and truncated HBx proteins may be associated with the development of HCC in patients with chronic hepatitis B infection [13–15, 38] . In accordance with these studies, we found more frequently HBV variants with mutated HBx proteins in patients with chronic hepatitis B infection and HCC in comparison to patients without HCC. It is also noteworthy that the previously described Ser31Ala mutation in the HBx protein [15] , which has been shown to be associated with HCC development, was frequently observed in our patients with HCC. In addi-tion to full-length HBx proteins bearing random muta-tions, we also found in the same HCC patients C-termi-nally truncated HBx proteins.

The HBx protein has been proposed to act as a tran-scriptional transactivator that can activate a wide variety of viral and cellular promoters [39] . The functional activ-ity and subcellular distribution of HBx seemed to be con-trolled by its phosphorylation status, e.g., mediated by ERK1/2 [18, 40] . It has been reported that cytoplasmic HBx is involved in the activation of iNOS, a factor that plays an important role in inflammatory disorders and regulation of the Ras-Map kinase signaling [35, 41, 42] . Nuclear localization of the HBx protein has been shown to modulate the activity of cellular factors like AP-1, NF- � B, tumor suppressor P53, DDB proteins (damaged DNA binding protein), and p21(WAF1/Cip1) [40, 43] . These findings indicate that different subcellular localization and the phosphorylation status of HBx will play a major role in dysregulation of cell cycle progression and hepa-tocarcinogenesis.

We have shown that truncated HBx proteins localized to the cell nucleus in contrast to the predominant cyto-plasmic distribution of wt-HBx. Moreover, full-length mutated HBx proteins isolated from our HCC patients showed perinuclear localization, possibly within the en-

doplasmic reticulum (ER). The accumulation of viral proteins in the ER has been reported to induce ER stress and possibly enhance the oncogenic potential [44] . Re-cently, the wt-HBx protein has been shown to act as a tu-mor suppressor abrogating the transforming ability of oncogenes [45, 46] . However, Tu et al. [14] demonstrated that C-terminally truncated HBx protein enhances onco-genic transformation by inducing interaction between ras and myc . From this we can speculate that nuclear lo-calization of mutated HBx proteins may contribute to the process of the HCC development.

wt-HBx is able to interact with JAK1-tyrosine kinase leading to the activation of JAK/STAT signaling [11, 12] . It has been demonstrated that the ability of HBx to trans-activate Src kinase providing a possible mechanism by which HBx may mediates the activation of the JAK/STAT signaling, especially STAT3 [47–49] . Our results demon-strated that wt-HBx and full-length mutated HBx medi-ated activation of STAT3 while the truncated HBx mu-tants resulted in a reduction in STAT3 activation. By con-trast, coexistence of truncated and full-length mutated HBx proteins strongly induced the activation of STAT3 while this was not a consequence of increased expression of STAT3 protein. From these findings we would postu-late that the atypical nuclear/perinuclear localization of the mutant HBx proteins and the ensuing effects on the ER induces oxidative stress and upregulation of pSTAT3. A comparable mechanism of ER stress-induced STAT3 activation has recently been described for the hepatitis C virus protein NS5A [50] .

STAT1 has been proposed to act as a tumor suppressor [47] as evidenced by the development of tumors in STAT1-deficient mice and the association of mutations in STAT1 within various human tumors [37, 47, 51] . However, in contrast to these reports, we found that STAT1 was not altered in cells expressing wt-HBx and mutated HBx pro-tein.

In various human cancers, SOCS1 and SOCS3 expres-sion is inhibited by hypermethylation [52, 53] pointing to an important role of silencing of SOCS1 and SOCS3 ex-pression/activation during carcinogenesis. Our results have shown that neither wt-HBx nor mutant HBx pro-teins resulted in the activation of SOCS1 and SOCS3 ex-pression, as would have been expected following the in-duction of pSTAT3 by HBx. As inhibition of SOCS1 and SOCS3 expression supports the constitutive activation of STAT3, HBx-mediated suppression of these SOCS pro-teins may play a more important role in the development of HBV-associated HCC.

Bock /Toan /Koeberlein /Song /Chin /Zentgraf /Kandolf /Torresi

Intervirology 2008;51:432–443 442

In summary, we have shown that HBx mutants occur frequently in Vietnamese HCC patients revealing trun-cated HBx proteins together with full-length mutated HBx. These finding indicate that mutations in the HBx gene may be associated with hepatocarcinogenesis. The HBx mutants modulate cellular signaling and especially the STAT/SOCS pathway. This might be a consequence of the atypical nuclear localization of the HBx mutants. Consequently, HBx mutants supporting the upregulation of pSTAT3 while down-regulating SOCS3 may in part contribute to the multistep process of hepatocarcinogen-esis.

Acknowledgments

We are grateful to Dr. L.V. Don, Ms. D.T. Lan and Ms. D.T. Binh for their help in collecting samples, and H. Kaiser for excel-lent technical assistance. This work was supported by grants of the Deutsche Krebshilfe, ‘Dr. Mildred-Scheel-Stiftung für Krebs-forschung’, grant No. 10-2142-Bo1. Nguyen Linh Toan was sup-ported by a scholarship from Project 322 of the Vietnam Ministry of Education and Training.

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