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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Int J Cancer. Author manuscript; available in PMC Oct 15, 2010.
Published in final edited form as:
PMCID: PMC2745322
NIHMSID: NIHMS137457

Increased Expression of c-erbB-2 in Liver is Associated with Hepatitis B x Antigen and Shorter Survival in Patients with Liver Cancer

Abstract

Hepatitis B x antigen, or HBxAg, contributes importantly to the pathogenesis of hepatocellular carcinoma (HCC). Given that HBxAg constitutively activates β-catenin and that up-regulated ErbB2 promotes β-catenin signaling in other tumor types, experiments were designed to ask whether HBxAg was associated with up-regulated expression of ErbB2. When HBxAg positive and negative HepG2 cells were subjected to proteomics analysis, ErbB2 was shown to be up-regulated in HepG2X but not control cells. ErbB2 was also strongly up-regulated in hepatitis B infected liver, and weakly in some HCC nodules, where it correlated with HBxAg expression. Among tumor bearing patients, strong ErbB2 staining in the liver was associated with dysplasia, and a shorter survival after tumor diagnosis. This implies that elevated ErbB2 is an early marker of HCC. Treatment of HepG2X cells with ErbB2 specific siRNA not only reduced ErbB2 expression, but also reduced the expression of β-catenin, suggesting that ErbB2 contributed to the stabilization of β-catenin. ErbB2 specific siRNA also partially blocked the ability of HBxAg to promote DNA synthesis and growth of HepG2 cells. These results suggest that ErbB2/β-catenin up-regulation contributes importantly to the mechanism of HBxAg mediated hepatocellular growth.

Keywords: Hepatitis B virus, HBxAg, erbB-2, β-catenin, hepatocellular carcinoma

ErbB2 (HER2 or neu) is a member of the epidermal growth factor receptor tyrosine kinases that is involved in the transmission of differentiation and proliferation signals.1,2 High levels of ErbB2 have been documented in cancers of the breast,3 ovary,4 lung,5 bladder6 and in the gastrointestinal system.7 In some of these tumors, over-expression is associated with poor prognosis. In breast cancers, up-regulated ErbB2 appears to be an early event, since it appears in tumor and nontumor tissue.8

The finding of elevated ErbB2 in the pathogenesis of HCC differs among various studies. For example, elevated ErbB2 has been reported mostly in hyperplastic nodules, suggesting it is an early marker of hepatocarcinogenesis.9 Independent work showed elevated ErbB2 in 30-40% of HCCs analyzed.10,11 Other work showed ErbB2 in serum samples of patients with acute and chronic liver diseases, including HCC.12 Alternatively, ErbB2 was not found in HCC tissues from other groups of patients.13-15 However, ErbB2 has been detected in the embryonic liver and during liver regeneration, but not in adult hepatocytes,16 suggesting it is up-regulated in the transformed state.

There is also evidence that elevated ErbB2 is associated with hepatitis B virus (HBV) infection. One study showed a correlation between hepatitis B e antigen in the serum of chronically infected patients and the over-expression of ErbB2 in corresponding liver sections.17 Other work showed strong c-erbB-2 staining in small polygonal liver cells, dysplasic cells, and lower but elevated staining in hepatocytes and HCC cells.18 ErbB2 was also found in stored serum samples from HBV carriers but not non-carriers,19 suggesting that up-regulated ErbB2 was associated with HBV infection.

In this context, there is also a close relationship between long term HBV infection, the development and progression of chronic liver disease, and the appearance of HCC.20,21 The pathogenesis of HBV associated HCC is multi-step, and may include insertional mutagenesis and/or over-expression of preS/S polypeptides on a background of inflammatory liver disease. In addition, the HBV encoded protein, HBxAg, contributes centrally to transformation in vitro,22 and to the development of HCC in transgenic mice.23 Recently, HBxAg has been shown to up-regulate and stabilize β-catenin,24 which in some tumors is activated by elevated levels of ErbB2. Given the evidence associating elevated ErbB2 with chronic HBV infection, experiments were designed to test the hypothesis that HBxAg was associated with the up-regulated expression of ErbB2, and if so, that elevated ErbB2 was also linked to stabilization of β-catenin in HCC and liver cell lines.

Materials and methods

Construction of HBxAg positive and control liver cells

The differentiated human hepatoblastoma cell line, HepG2, was transduced with a recombinant retrovirus encoding HBxAg under control of the early cytomegalovirus promoter (pSLXCMV-HBx) or with a recombinant retrovirus encoding the bacterial chloramphenicol acetyltransferase gene (pSLXCMV-CAT). The resulting cultures, designated HepG2X and HepG2CAT, respectively, were selected for 2 weeks in G418 to eliminate uninfected cells, and then passaged without clonal selection. HBxAg expression was verified by western blotting, and CAT expression was verified by standard CAT assay.25 HBxAg positive and negative Huh7 cells were prepared the same way.

The HCC cell lines, Huh7 and HepG2, were obtained from ATCC (Manassas, VA). Cells were cultured in DMEM supplemented with 10% FCS at 37°C in 5% CO2.

Microarray analysis

Fifty micrograms of total RNA from confluent HepG2X and HepG2CAT cells was isolated using the RNeasy kit according to enclosed instructions (Qiagen, Inc., Valencia, CA). These RNAs were sent to MERGEN, Ltd (San Leandro, CA), where microarray analysis was performed using the human HO5 ExpressChip Array, which consisted of pre-spotted oligonucleotide sequences from 12,800 cDNAs. Array data was analyzed using GeneSpring™ and sent back to the applicant lab as Excel tables on CD.

Powerblot analysis

To discern patterns of differentially expressed proteins in HepG2X compared to HepG2CAT cells, cell lysates were freshly prepared and shipped to BD Transduction Labs at BD Biosciences (San Jose, CA) for PowerBlot analysis. Cell lysates were analyzed on sodium dodecyl sulfate/polyacrylamide gels and then transferred to immobilon P membranes (Millipore, Bedford, MA). The membranes were cut into strips, and each strip blotted with a mixture of monoclonal antibodies. In the entire analysis, western blotting was performed for 750 proteins. Images were captured electronically and were matched using PDQuest software (Biorad, Hercules, CA). The results were returned to the lab as images and Excel tables on CD.

Patients and clinical samples

HCC tissues from 137 patients were collected for pathological examination and then made available for these studies. Peritumor liver from tumor bearing patients with cirrhosis or chronic hepatitis was also made available. Most samples were from patients who were positive for hepatitis B surface antigen (HBsAg) in serum (Auszyme kit, Abbott Labs., North Chicago, Il). Serum samples from these patients were also tested for HBeAg and anti-HBe using standard commercially available kits (Auszyme and Ausab kits, respectively from Abbott Labs), and for HBV DNA levels by real-time RT/PCR (Smart Cycler, Cepheid, Sunnyvale, CA). Patients underwent liver transplantation or surgical wedge biopsy at the University of Witwatersrand in Johannesburg, South Africa; Chengzhen and Changhai hospitals in Shanghai, China; Thomas Jefferson University hospital in Philadelphia, PA, and Xijing hospital in Xi'an, China. Seven blocks were provided from as many individuals with HCC treated at Thomas Jefferson University who were serologically negative for HBsAg, antibodies to HBsAg (anti-HBs) and antibodies to hepatitis B core antigen (anti-HBc). An additional 7 liver blocks were provided from as many individuals from Xijing hospital who were serologically negative for HBsAg, anti-HBs, anti-HBc and antibodies against hepatitis C virus and who died of unrelated causes. Additional information about these patient samples is presented in Table 1. Retrospective follow-up was conducted on 68 patients from Xijing Hospital for a mean period of 19.0 ± 13.0 months. All tissue samples were fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA), processed through graded ethanol, and embedded in paraffin. The use of all tissue blocks for these studies was approved by the institutional review boards at each location.

Table 1
Characteristics of Clinical Samples used in this Study

Immunohistochemistry

Immunohistochemistry was performed by using the DAKO EnVision+ System (Carpinteria, CA). Detection was carried out using DAKO Liquid DAB+, which is based on a horseradish peroxidase (HRP)-labeled polymer conjugated to an appropriate secondary antibody. Briefly, after slides were deparafinized and rehydrated, antigen retrieval was performed using the DAKO Antigen Retrieval Solution at 95°C for 20 min. The slides were then predigested with 0.1% trypsin in 0.1% CaCl2 (pH 7.8) at room temperature (RT) for 10 min. Endogenous peroxidase was blocked with 1% H2O2 (Fisher Scientific) at RT for 15 min. One percent bovine serum albumin was then added, and the slides blown dry for 20 min at RT. The primary antibodies for erbB2, CD31, PCNA, Ki-67 and HBx were diluted in Antibody Diluent (DAKO) and then incubated at 4°C overnight. Monoclonal c-neu (C-erbB-2) antibody (Ab3, Oncogene Science, Cambridge, MA), was used at 1:800 dilution. Mouse anti-human endothelial cell CD31 monoclonal antibody (DAKO) was used at a 1:50 dilution, mouse anti-human PCNA (PC-10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at a dilution of 1:800, mouse anti-human Ki-67 (DAKO, Carpinteria, CA) was used at a dilution of 1:500, and rabbit anti-HBx (anti- 99)26 was used at a 1:10,000 dilution. After washing three times with Tris-buffered saline (pH 7.5), the DAKO EnVision+ System was applied, followed by Liquid DAB+ Substrate-chromogen solution (DAKO). The slides were counterstained with Hematoxylin (Sigma Chemical Co., St. Louis, MO). For positive controls, sections of gastric cancer known high expressing C-erbB-2 protein were included in each staining procedure. For negative controls, normal mouse or rabbit IgG (Vector Labs, Burlingame, CA) were used in place of primary antibody to rule out false-positive responses. In addition, preabsorption of primary antibodies with corresponding antigens were performed on tissue sections to insure specificity.

Staining was evaluated as described.27 Staining intensity was scored as: weak (+1), moderate (+2), and strong (+3). The percentage of positive cells was evaluated as follows: up to 10% (+1), 11-50% (+2), 51-80% (+3), and more than 81% (+4). The final score for each included both the staining intensity plus the percentage of positive cells.

Assessment of microvessel density, Ki-67 and PCNA staining

Microvessels were stained by using mouse anti-human endothelial cell CD31 (DAKO). Microvessels were counted in three fields (×200) with the highest density. The microvessel score was the mean of the vessel counts obtained in these three fields. Vessels with a clearly defined lumen or well-defined linear vessel shape, but not single endothelial cells, were scored in microvessel counting. The immunostained tissue sections were blindly assessed by two pathologists, who provided scores for every section, and the final result was the mean of their combined observations.

Proliferation in tissue was evaluated by staining with a mouse anti-human proliferating cell nuclear antigen (PCNA) (PC-10, Santa Cruz Biotech; 1:800 dilution). The latter identified cells in the cell cycle. Cells in S phase were assessed by Ki-67 staining, as outlined above. In both cases, the proliferative index was the ratio of positively stained cells to the total cell count, expressed as a percentage. At least 1000 nuclei were counted for each sample.

Western blotting

Western blotting was performed with monoclonal anti-c-neu (C-erbB2) (Ab-3, Oncogene Science) at a 1:400 dilution, with monoclonal anti-β-catenin (Santa Cruz Biotech) at a dilution of 1:200, or with rabbit anti-phospho-AKT (ser473; Cell Signaling Technology, Inc., Beverly, MA). For secondary antibodies, HRP-conjugated goat anti-mouse Ig (1:3000 dilution) (Accurate Chemical Co., Westbury, NY) or HRP conjugated goat anti-rabbit Ig (diluted 1:4000; Accurate), were used. The results were visualized using the enhanced chemiluminescence (ECL) detection system (Amersham, Arlington Heights, Ill). Mouse anti-human β-actin monoclonal antibody (Clone AC-15, Sigma), at a dilution of 1:5,000, was used as an internal control.28

Transient transfections

Transient transfection of pcDNA3-HBx and pcDNA3 into HepG2 or Huh7 cells were conducted using SuperFect (Superfect Transfection Reagent, Qiagen) according to manufacturer's instructions.

Transfection of small inhibitory RNAs (siRNAs) and cell growth

HepG2X cells (1 × 105 cells) were seeded overnight, in triplicate, into 60 mm dishes. For transfection, 20 μl of a 20 μM stock containing four ErbB2 specific siRNAs or two control siRNAs (siRNA SMARTpool, Dharmacon, Lafayette, CO) were transiently transfected using oligofectamine (InVitrogen, Carlsbad, CA). Cells were incubated for 4 hrs at 37°C and then grown in complete medium. Growth was assayed over 5 days by measuring bromodeoxyuridine (BrdU) incorporation (Roche, Branchburg, NJ). Cell growth was also measured over time using the modified tetrazolium salt (MTT) assay (Cell Titer 96 Non-radioactive Cell proliferation Assay, Promega, Madison, WI). All experiments were conducted in triplicate, and the results evaluated blindly.

Inhibition of phosphatidyloinositol 3-kinase (PI3K)

To evaluate the affects of the PI3K inhibition on p-AKT levels, cell were pretreated with 50 μM of Ly294002 (Cell Signaling Technology, Inc., Danvers, MA) for 24 hrs, lysed, and then analyzed for phosphor-AKT (p-AKT) by western blotting.

Statistical analysis

Statistical analysis and graphic presentation were performed using the SPSS 10.0 for windows (SPSS Inc., Chicago, IL) program package. The χ2 test and Fisher's exact test or the unpaired two-tailed t test was used for testing relationships between categorical variables as appropriate. Spearman rank correlation coefficient analysis was used to assess the correlation between ordinal variables. Survival curves were plotted by using the Kaplan-Meier method, and statistical differences between life tables were determined by a log-rank test. P < 0.05 denoted the presence of a statistically significant difference.

Results

C-erbB-2 mRNA and protein levels in HepG2X and HepG2CAT cells

HBxAg is one of the HBV encoded proteins that contributes to hepatocarcinogenesis.29,30 To gain further insight into this process, HepG2X and HepG2CAT cells were subjected to microarray analysis. The results showed a reproducible 4-6 fold increase in ErbB-2 mRNA in HepG2X cells compared to HepG2CAT cells (data not shown). To see if this also occurs at the protein level, corresponding lysates were prepared and subjected to Powerblot analysis. When this was done, p185 ErbB-2 protein was elevated by 3.4 ± 0.27 fold in HepG2X compared to HepG2CAT cells (P < 0.005) (Fig. 1a and 1b).

Figure 1
Powerblot analysis of (a) HepG2X and (b) HepG2CAT cells. The circled spot in each blot is ErbB2. The results are representative of three independent experiments. (c) Expression of C-erbB-2 in HepG2 and Huh7 cells 48 hrs after transient transfection with ...

Expression of c-erbB-2 in cell lines transiently transfected with HBxAg

To confirm that c-erbB-2 up-regulation correlates with HBxAg expression, pcDNA3-HBx was transiently transfected into HepG2 and Huh7 cells, and the levels of ErbB-2 protein was assayed by western blotting. The results showed that HBxAg up-regulated p185 ErbB-2 by 4.2 ± 0.33 fold in HepG2 cells (P < 0.001) and by 3.2 ± 0.25 fold in Huh7 cells (P < 0.005) (Fig. 1c). In both cases, only a single immunoreactive band at the expected molecular weight was observed. Western blot analysis performed with normal IgG in place of anti-ErbB-2 did not detect p185 ErbB-2 (data not shown).

C-erbB-2 expression in liver and HCC tissues

Staining was performed on clinical samples obtained at the time of surgery from HBV infected patients. The frequency of positive samples and the intensity of ErbB-2 staining was low in normal liver, and was progressively higher in samples from patients with chronic hepatitis, cirrhosis, and highest of all in peritumor liver (Table 2). In samples where more than roughly 20% of the cells stained positive for ErbB-2, the overall staining score was also high (+2 or greater). Staining scores for ErbB2 and HBxAg were the highest in peritumor liver tissue in the majority of patient samples analyzed, suggesting that ErbB-2 was most strongly over-expressed in hepatocytes from regions of the liver where tumor nodules appeared (Table 2, Figs. 2 and and3a).3a). ErbB2 staining was exclusively cytoplasmic, and appeared to be specific, since replacement of anti-ErbB-2 with normal IgG resulted in no staining (Figs. 2c and 2g), while staining of gastric cancer for ErbB-2 yielded exclusively membranous staining, as expected (data not shown). Further, little or no ErbB-2 staining was observed in HCC or surrounding nontumor liver among 7 patients (from Philadelphia) who were serologically negative for markers of hepatitis B or C viruses (data not shown). In addition, the fact that normal human liver showed faint or no ErbB2 staining (Table 2, Fig. 2h) also suggested that the anti-ErbB-2 used herein was not binding nonspecifically to other proteins in human liver.

Figure 2
Consecutive sections of a cirrhotic nodule stained with anti-HBx (a), with anti-ErbB2 (b) or with preimmune rabbit serum (c). (d) Anti-ErbB-2 staining of a liver section from a patient with chronic hepatitis. e-g. Consecutive tissue sections of an HCC ...
Figure 3
Percentage of patients with strong staining scores (+2 or +3) for (a) HBxAg (gray bars) and ErbB2 (white bars) or (b) PCNA (gray bars) and Ki-67 (white bars). The corresponding histological lesions in the livers for each patient group are indicated.
Table 2
Expression of C-erbB-2 and HBxAg in chronically infected liver and HCC

Relationship between ErbB-2, HBxAg and clinicopathological features

As indicated above, HBxAg staining correlated strongly with ErbB-2 staining (Table 2, Figs. 2 and and3a).3a). There was also a strong correlation between ErbB-2 and clinicopathological characteristics in patient samples. For example, peritumor liver tissue samples with high ErbB-2 scores also had elevated PCNA and Ki-67 staining, indicating that high ErbB-2 expression was associated with cell proliferation (Table 3, Figs. 3b and 4a-c). High ErbB-2 scores were also associated with elevated CD31 staining in peritumor liver compared to tumor (Table 3, Figs. 3 and 4d-f), suggesting that elevated ErbB-2 expression was associated with a higher microvessel density (indicative of angiogenesis) in the peritumor compartment. The relationship between ErbB-2 and HBx, PCNA, Ki-67, and CD31, as calculated by the Spearman correlation analysis, was 0.71, 0.88, 0.77 and 0.84, respectively. Elevated PCNA, Ki-67 and/or CD31 staining were not observed in livers from nontumor bearing patients with chronic hepatitis or cirrhosis. However, among tumor bearing patients, there was a significant correlation between high ErbB-2 scores and dysplasia in cirrhotic or peritumor liver samples (Table 3), suggesting that up-regulated expression of ErbB-2 is an early event in hepatocarcinogenesis. Analysis of ErbB-2 expression in HCC with tumor stage, histology, grade, disease free survival, overall survival rate, HBeAg status or HBV DNA levels in blood failed to show any significant relationships (data not shown). Importantly, the finding that tumor patients with high ErbB-2 scores in their livers had a significantly shorter median survival time compared to patients with low ErbB-2 scores (Table 3, Fig. 5), suggests that elevated ErbB-2 is important to the pathogenesis of HCC.

Figure 4
Consecutive sections of peritumor liver stained with anti-ErbB-2 (a), anti-PCNA (b), or with normal IgG (c) (panels a-c magnification × 200). Consecutive tumor and adjacent liver sections were also stained with anti-ErbB-2 (d), anti-CD31 (e) or ...
Figure 5
Survival analysis of HCC patients with high or low C-erbB2 expression in peritumor liver. High ErbB2 expression had scores of +2 and +3 while low ErbB2 expression had scores of 0 and +1.
Table 3
Relationship between C-erbB-2 and clinicopathological features

Treatment of HBxAg positive (HepG2X) cells with ErbB-2 specific siRNA

The in vivo data suggests that elevated ErbB-2 expression is associated with the appearance of PCNA positive cells in the liver parenchyma (Fig. 4). To see if elevated ErbB-2 promotes DNA synthesis and growth in cell culture, HepG2X cells were transiently transfected with control or specific ErbB-2 siRNAs, and BrdU labeling was determined. There was a dose dependent inhibition of ErbB-2 protein (up to 70% inhibition) by 72 hrs post-transfection (Fig. 6a). No inhibition was observed in cells transiently transfected with control siRNAs (data not shown). Since in some tumor types, ErbB-2 binds to and activates β-catenin, the experiment was repeated and western blots were performed with anti-β-catenin. The results showed a dose dependent decrease in β-catenin levels when cells were treated with ErbB-2 specific siRNA (Fig. 6b). This was observed in both the wild type and constitutively active truncated mutant of β-catenin naturally made by HepG2 cells. Similar results were obtained using Huh7 cells stably transfected and expressing HBxAg (data not shown, but analogous to Fig. 6a and 6b). Co-immunoprecipitation failed to show a physical association between ErbB-2 and β-catenin (data not shown), but do support the hypothesis that they functionally interact. When HepG2X cells were treated with ErbB-2 specific siRNAs, there was a significant, dose dependent inhibition in BrdU incorporation (Fig. 6c). Parallel treatment of HepG2X cells with control siRNA, or treatment of HepG2CAT cells with ErbB-2 specific or control siRNAs, failed to inhibit BrdU incorporation, suggesting that the HBxAg up-regulation of ErbB-2 stabilizes β-catenin, and that the latter promotes DNA synthesis. Further, only ErbB-2 specific siRNA partially inhibited the ability of HBxAg to promote the growth of HepG2CAT cells in culture (Fig. 6d). This finding suggests that the differences in BrdU incorporation were associated with DNA replication and not due to unscheduled DNA synthesis. Further work showed that ErbB-2 activation of β-catenin was PI3K/Akt dependent, since the addition of ErbB-2 siRNA to HepG2X cells depressed the levels of phosphorylated (activated) Akt by approximately 4.3 ± 0.3 fold (P < 0.005) and 4.5 ± 0.4 fold (P < 0.005), respectively (Fig. 7). This was confirmed when PI3K activity was blocked by addition of the PI3K inhibitor, LY294002 (data not shown). Together, these results suggest that elevated ErbB-2 stabilizes β-catenin through activation of PI3K/Akt signaling.

Figure 6
Treatment of HBxAg positive (HepG2X) and negative (HepG2CAT) cells with ErbB2 specific siRNA. HepG2X cells were transiently transfected with increasing amounts of ErbB2 specific siRNA as indicated, and the levels of ErbB2 protein (a) or β-catenin ...
Figure 7
Western blot analysis of phospho-Akt levels in Huh7X cells (lanes 1 and 2) or HepG2X cells (lanes 3 and 4) transiently transfected with control siRNA (lanes 1 and 3) or with ErbB-2 specific siRNA (lanes 2 and 4). The data shown is representative of three ...

Discussion

Over-expression of ErbB-2 contributes importantly to the pathogenesis of several tumor types.3-8 However, the role of up-regulated ErbB-2 in the pathogenesis of HCC is not clear, with some studies showing up-regulated expression,9-12 while others do not.13-15 Few reports have focused upon the viral etiology of the tumors, with little work on the possible relationship between HBxAg and ErbB-2. In this context, the results herein establish a strong correlation between HBxAg and ErbB-2 expression in cell culture and in chronically infected liver tissue (Figs. 1 and and2,2, Table 2), suggesting that part of the mechanism whereby HBxAg contributes to transformation involves the up-regulated expression of ErbB-2.

The observation that strongly elevated ErbB-2 is associated with elevated PCNA and Ki-67 expression in liver (Fig. 3, Table 3), and with stimulated BrdU uptake and accelerated growth of a human liver cell line (Fig. 6), suggest that elevated ErbB-2 may promote the appearance and growth of HCC. This is further supported by the fact that patients with high ErbB-2 scores have a shorter survival time post-tumor diagnosis (Fig. 5, Table 3). Independent observations have shown that the stable over-expression of ErbB-2 also promotes the growth and metastasis of the human HCC cell line 7721.31 Hence, elevated ErbB-2 may have prognostic significance in tumor development and patient survival. It also suggests that ErbB-2 may be an important therapeutic target in the early stages of tumorigenesis, as in breast cancer.8 The finding of elevated ErbB-2 in dysplastic hepatocytes (Table 3), and in small polygonal liver cells,18 also suggests that expression in cells that may progress to HCC may be key to tumor development. Elevated ErbB-2 expression in peritumor hepatocytes suggests that it is rate limiting in the steps prior to the appearance of tumor. Once cells become transformed, and propagate through the continued accumulation of mutations, sustained HBxAg and ErbB-2 expression are no longer the driving factors in cell growth and/or survival. This hypothesis is consistent with the staining patterns of HBxAg and ErbB-2 herein. Alternatively, or in addition, once tumor develops, it may promote the sustained elevation of ErbB-2 in surrounding nontumor liver. Either way, if further work shows that ErbB-2 is an marker of HBV associated tumorigenesis, it may be amenable to chemotherapeutic intervention.

An unexpected feature of up-regulated ErbB-2 expression in the liver is that its' distribution is predominantly cytoplasmic instead of membranous (Fig. 2). Cytoplasmic ErbB-2 has been observed in several other tumor types,32-34 with distribution in both tumor and surrounding nontumor cells. In breast cancer, for example, cytoplasmic ErbB-2 staining had no prognostic significance,35 while in patients with ovarian cancer, cytoplasmic staining was associated with poor survival.36 In chronically infected liver, elevated ErbB-2 is associated with evidence of increased cell growth, increased microvessel density, liver cell dysplasia, and decreased survival (Figs. 4 and and5,5, Table 3), suggesting that it may be an early prognostic marker in HCC development. In studies on breast cancer, elevated ErbB-2 is associated with increased microvessel density by stimulating synthesis of vascular endothelial growth factor,37 which is also elevated in the serum of HCC patients.38 Further, the fact that ErbB-2 is normally ubiquitinated and degraded by the proteasome,39 and that HBxAg inhibits the proteasome,40 may partially explain the accumulation of ErbB-2 in the chronically infected liver and its' close association with HBxAg (Figs. 2 and and4,4, Tables 2 and and3).3). Given that HBxAg is a trans-activating protein,29,30 activation of one or more cytoplasmic signaling pathways may result in transcriptional activation of ErbB-2 expression. Preliminary work herein showed that the accumulation of wild type β-catenin in the presence of elevated ErbB-2 correlated with the activation of PI3K/Akt signaling (Fig. 7), which has previously been documented to be activated by HBxAg and ErbB-2.2,24,41 PI3K/Akt activity may also be stimulated by src, the latter of which is activated by HBxAg, early in tumor development.42,43 Further, the peptidyl prolyl isomerase, Pin1, is up-regulated in HCC, and is known to stabilize both HBxAg44 and ErbB-2,45 suggesting a variety of possible mechanisms underlying the close HBxAg/ErbB-2 relationship observed herein. Future work may reveal other signaling pathways that up-regulates ErbB-2 expression. A possible candidate is EGFR (ErbB-1), which is up-regulated by HBxAg,46 and also stabilizes β-catenin.47 This will help to identify novel targets for therapeutic intervention.

Although the function of cytoplasmic ErbB-2 has not been established, the ability of ErbB-2 specific siRNA to suppress β-catenin (Fig. 6) suggests a functional relationship between them. Independent observations suggest that cytoplasmic ErbB-2 may be a truncated form of the receptor with altered or no function,34 but the present study showed only wild type sized ErbB-2 in HepG2X and Huh7X cells that had an impact upon cell growth and survival (Figs. 1 and and6).6). Hence, in the presence of HBxAg, elevated ErbB-2 may be activated by mechanisms other than its normal binding to the other ErbB receptors (ErbB-1, -3 and/or 4), which may explain the cytoplasmic distribution of ErbB-2 in this study. Additional work is underway to test this hypothesis.

Acknowledgments

This work was supported by NIH grants CA104025 and CA111427 to MF.

Abbreviations

BrdU
bromodeoxyuridine
CAT
chloramphenicol
ECL
enhanced chemilluminescence
HBV
hepatitis B virus
HBxAg
hepatitis B x antigen
HCC
hepatocellular carcinoma
HRP
horseradish peroxidase
MTT
modified tetrazolium salt assay
PCNA
proliferating cell nuclear antigen
PI3K
phosphatidylinositol 3-kinase
RT
room temperature
siRNA
small, inhibitory RNA

Footnotes

There are no conflicts of interest to report.

References

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