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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chronic infection with hepatitis B virus (HBV) is strongly associated with hepatocellular carcinoma (HCC), and the viral HBx protein plays a crucial role in the pathogenesis of liver tumors. Because the protooncogene pituitary tumor–transforming gene 1 (PTTG1) is overexpressed in HCC, we investigated the regulation of this protein by HBx. We analyzed PTTG1 expression levels in liver biopsies from patients chronically infected with HBV, presenting different disease stages, and from HBx transgenic mice. PTTG1 was undetectable in biopsies from chronic hepatitis B patients or from normal mouse livers. In contrast, hyperplastic livers from transgenic mice and biopsies from patients with cirrhosis, presented PTTG1 expression which was found mainly in HBx-expressing hepatocytes. PTTG1 staining was further increased in HCC specimens. Experiments in vitro revealed that HBx induced a marked accumulation of PTTG1 protein without affecting its messenger RNA levels. HBx expression promoted the inhibition of PTTG1 ubiquitination, which in turn impaired its degradation by the proteasome. Glutathione S-transferase pull-down and co-immunoprecipitation experiments demonstrated that the interaction between PTTG1 and the Skp1–Cul1–F-box ubiquitin ligase complex (SCF) was partially disrupted, possibly through a mechanism involving protein–protein interactions of HBx with PTTG1 and/or SCF. Furthermore, confocal analysis revealed that HBx colocalized with PTTG1 and Cul1. We propose that HBx promotes an abnormal accumulation of PTTG1, which may provide new insights into the molecular mechanisms of HBV-related pathogenesis of progressive liver disease leading to HCC development. (HEPATOLOGY 2010;51:777–787.)

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide.1 Chronic infection with hepatitis B virus (HBV) is the main causal factor for HCC.1 A growing body of evidence suggests that HBV may have a direct oncogenic capacity and that expression of virally encoded proteins, in particular the HBV X protein (HBx), promotes cell growth and tumor development.2 Importantly, HBx expression is retained after viral integration into hepatocyte DNA3 and is one of the most prevalent virus antigens in the liver and tumors of HBV carriers, and may induce humoral and cellular immune responses.2 HBx alters several host functions that may lead to the carcinogenic process, including cell proliferation, viability, DNA repair, and genome stability.2 Although HBx does not bind directly to DNA, it may activate the transcription of a wide range of cellular genes by different mechanisms involving activation of signal transduction pathways or direct interaction with components of the transcriptional machinery.2 Recently, it has been proposed that HBx may also alter gene expression by promoting epigenetic changes in the DNA methylation profile4 or by enhancing the stability of transcription factors such as HIF-1α5 and c-myc.6 Thus, HBx expression results in transcriptional activation of a variety of cellular genes involved in inflammation, angiogenesis, fibrosis, oxidative stress, and tumor development and progression.2

Pituitary tumor–transforming gene 1 (PTTG1)-encoded protein, originally isolated from pituitary tumor cells,7 was later identified as a human securin, a protein implicated in inhibition of sister chromatid separation during mitosis, which has been associated with malignant transformation and tumor development.8 Furthermore, PTTG1 plays key roles in cellular growth, DNA repair, development, and metabolism.9 Mechanisms of PTTG1 action include protein–protein interactions, transcriptional activity, and paracrine/autocrine regulation.9 During mitosis and following chromosome alignment, PTTG1 is degraded by the proteasome at metaphase to anaphase transition through the anaphase-promoting complex/cyclosome, releasing inhibition of separase, which in turn mediates the proteolysis of the cohesins ring that holds sister chromatids together.8 In nonmitotic cells, the Skp1–Cul1–F-box protein ubiquitin ligase complex (SCF) is involved in the degradation of phosphorylated forms of PTTG1.10 Furthermore, the SCF complex is involved in PTTG1 turnover in cycle-arrested cells after ultraviolet radiation.11 PTTG1 overexpression has been reported in a great variety of tumors in which it correlates with invasiveness,9 and it has been identified as a key signature gene associated with tumor metastasis.12 In HCC, PTTG1 is overexpressed, and its expression levels have prognostic significance for the survival of postoperative HCC patients.13 Interestingly, it has been proposed that PTTG1 might be critically involved in the development of HCC through the promotion of angiogenesis.13 PTTG1 specifically interacts with p53, both in vitro and in vivo, and inhibits the ability of p53 to induce cell death, demonstrating its oncogenic potential.14 Additionally, PTTG1 overexpression in hepatoma cell lines negatively regulates the ability of p53 to induce apoptosis.15 Considering that HBV infection and HBx protein are associated with HCC and that a relationship between PTTG1 expression levels and HCC exists, we analyzed whether HBx may alter PTTG1 expression in chronic HBV-infected patients, HBx transgenic mice, and HBV-containing or HBx-expressing cell lines to provide new insights into our understanding of the molecular pathogenic mechanisms of advanced liver disease associated with HBV chronic infection.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patients, Transgenic Mice, and Immunohistological Assays.

Fifteen patients with HBV-related chronic liver disease (five with chronic hepatitis, five with cirrhosis, and five with HCC) were included. HBx transgenic mice were derived by microinjection the HBx gene into fertilized eggs of CD-1 mice.16 Immunohistological assays were performed by standard procedures.

Cell Culture.

Chang liver, Chang liver pX-34 (p34x), AML12 4p and AML12 4pX cells (4pX) were grown as described.17, 18

Plasmid and Transfections.

The indicated expression vectors were transfected employing Lipofectamine Transfection Reagent according to the manufacturer's instructions.

Western Blot Analysis.

Proteins were extracted and immunobloted using the indicated antibodies.

Cell Cycle Analysis.

Growth profiles of propidium iodide–labeled cells were analyzed by means of flow cytometry.

Real-Time Quantitative Reverse-Transcription Polymerase Chain Reaction Analysis.

RNA extraction and quantitative reverse-transcription polymerase chain reaction (RT-PCR) were performed as described.19

Detection of PTTG1 Ubiquitination, Coimmunoprecipitation and Pull-Down Assays.

Cleared lysates were subjected to immunoprecipitation with the indicated antibodies. The immunocomplexes were captured with protein A-sepharose. GST proteins were expressed in Escherichia coli, purified with glutathione-sepharose 4B, and incubated with cellular extracts. In both assays, bound proteins were analyzed by means of western blotting.

Immunofluorescence Analysis and Confocal Microscopy.

Cells were grown on coverslips and processed as described.19

Small Interfering RNAs and Transfections.

Cells were transfected with 100 nM ON-TARGET plus SMARTpool small interfering RNAs (siRNAs) directed against human Cul1 or a nonspecific control siRNA.

A detailed description of the protocols and reagents employed is provided in the Supporting Materials and Methods.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

PTTG1 Expression Level Increases in HBx-Immunoreactive Cells as Chronic Hepatitis B Progresses to Cirrhosis and HCC.

We first investigated the expression of PTTG1 and HBx in human liver biopsies during HBV-related hepatocarcinogenesis by staining serial liver sections with anti-PTTG1 and anti-HBx antibodies (Abs). In specimens from patients with chronic hepatitis B and weak HBx expression, PTTG1 was not detected in hepatocytes (Fig. 1A). As chronic liver disease progressed from chronic hepatitis B to cirrhosis, PTTG1 protein appeared in HBx-immunoreactive hepatocytes (Fig. 1A). PTTG1 staining increased in HCC specimens showing high HBx expression (Fig. 1A). Double immunofluorescence studies in HCC specimens revealed that the distribution of PTTG1 fit well with the pattern shown by HBx immunolabeling (Fig. 1B).

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Figure 1. PTTG1 expression levels increase as human chronic hepatitis B progresses to cirrhosis and HCC. (A) PTTG1 and HBx proteins were detected in human cirrhotic liver and HCC liver biopsies. PTTG1 protein appeared in HBx-immunoreactive hepatocytes in cirrhotic liver, and both proteins were strongly expressed in HCC specimens. Formalin-fixed, paraffin-embedded liver sections were stained with anti-PTTG1 Ab and anti-HBx. The HBx section was paired with an adjacent section stained using anti-PTTG1 Ab. Abs were then bound by goat anti-rabbit immunoglobulin G or by goat anti-mouse immunoglobulin G conjugated with peroxidase-labeled polymer. Peroxidase activity was detected using 3,3′-diaminobenzidine tetrahydrochloride. All sections were counterstained with hemotoxylin (blue). Brown coloring indicates specific Ab reactivity. We investigated five specimens of each chronic hepatitis B, cirrhosis, and HCC. Because HBx staining was not homogenous throughout the liver specimens of HCC and cirrhosis, selected fields with HBx expression are shown. Bar = 50 μm. (B) Hepatocytic PTTG1 in human HCC specimens colocalized with HBx. Human HCC sections were stained for immunofluorescence to simultaneously detect PTTG1 (red) and HBx (green). Yellow color indicates overlap of proteins. Bar = 50 μm.

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PTTG1 Expression Level Increases as HBx Transgenic Mouse Livers Progress to Hyperplasia and HCC.

HBx is considered one of the most important determinants of HBV-induced hepatocarcinogenesis. We further investigated the expression of PTTG1 and HBx during HBx-induced hepatocarcinogenesis in HBx transgenic mouse livers. Beginning at the age of 2 months, HBx transgenic mouse liver showed centrilobular foci of cellular alteration with cytoplasmic vacuolation surrounding the central veins where hepatocytes with increased DNA synthesis were detected.16 PTTG1 and HBx were not detected in nontransgenic normal mouse livers. In hyperplastic HBx-transgenic mouse livers, expression of PTTG1 was found mainly in the cytoplasm of hepatocytes in the centrilobular region, and distribution of PTTG1 was similar to that of HBx (Fig. 2). Strong expression of both PTTG1 and HBx was observed diffusely in HCC specimens (Fig. 2). Double immunofluorescence studies in transgenic mouse–derived HCC specimens confirmed that PTTG1 and HBx are coexpressed in cancer cells (Supporting Fig. 1).

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Figure 2. PTTG1 oncoprotein increases as HBx transgenic mouse livers progress through hyperplasia to HCC. The distribution of PTTG1 and HBx in normal nontransgenic mouse liver, hyperplasia, and HCC specimens from HBx-transgenic mice is shown. PTTG1 was present mainly in the cytoplasm of hyperplastic hepatocytes immunoreactive for HBx oncoprotein surrounding central veins in HBx-transgenic mouse liver. Strong staining of both proteins was observed diffusely in HCC specimens. Immunohistochemical analyses were performed as described above. We investigated five biopsies each of normal, hyperplastic, and HCC liver. Because all of the results were similar among the experiments, representative results are displayed.

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HBx Expression Induces PTTG1 Accumulation.

Because PTTG1 expression was increased during both HBV- and HBx-related chronic liver disease progression, we speculated that HBV and more precisely HBx might induce PTTG1 expression. We first examined whether a HBV replicon could induce PTTG1 expression. We transfected the hepatic-derived Chang liver cells with the plasmid payw1.2, which harbors 1.2 mer of the HBV genome that functions as an HBV replicon, and then evaluated PTTG1 expression by means of western blotting. The complete replicon induced the expression of PTTG1 protein (Fig. 3A). Interestingly, PTTG1 expression in cells transfected with the HBx-defective whole-genome construct (payw*7) remained unchanged, indicating a role of HBx in PTTG1 induction (Fig. 3A). To further explore the effects of HBx on PTTG1 expression, we employed two hepatocyte-derived cell lines, Chang liver p34X (p34X) and AML12 4pX (4pX), in which HBx expression was controlled by doxicycline treatment (Dox-on) or withdrawal (Dox-off), respectively. Western blot analysis revealed increased PTTG1 expression upon induction of HBx over 48 hours in both Dox-regulated systems (Fig. 3B). Similar results were obtained after 24 hours of Dox treatment (Supporting Fig. 2A). As controls, we included Chang liver and AML12 4p cells—the parental cell lines of p34X and 4pX cells, respectively—and no PTTG1 variation after Dox challenge was observed. PTTG1 levels positively correlate with cell proliferation, and its expression is controlled in a cell cycle–dependent manner.20 Several studies have also shown that HBx promotes cellular proliferation by triggering DNA synthesis and speeding up cell cycle progression.21, 22 However, evidence regarding the effects of HBx on liver cell proliferation and cell death is controversial, depending on the experimental systems and cell lines employed.23 To assess the effect of HBx expression on cell cycle progression, we analyzed the growth profiles of Chang liver p34X and AML12 4pX cells with or without Dox treatment by means of flow cytometry. In agreement with previous reports,24 our data showed that the percentages of p34X cells in G0/G1, S, and G2/M phases of the cell cycle displayed similar profiles 24 hours (Supporting Fig. 2B) and 48 hours (Fig. 3D) after induction of HBx expression. Furthermore, 4pX cells displayed a significant increase in HBx-dependent S phase entry 24 hours (Supporting Fig. 2B)17 but not 48 hours (Fig. 3D) after induction of HBx expression. Additionally, transient transfection of Chang liver cells with the HBV wild-type and HBx-defective replicons did not induce changes in the cell cycle profile (Fig. 3C). Given that HBx promoted PTTG1 accumulation without significantly affecting cell cycle (p34X and HBV complete replicon-transfected Chang liver cells), these results indicated that the HBx-promoted PTTG1 accumulation was not dependent on cell cycle modifications.

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Figure 3. Effects of HBx on PTTG1 expression. (A) Chang liver cells were transfected with the whole HBV genome payw1.2 (HBx+), an HBx-defective mutant payw7* (HBx), or control plasmid (pcDNA 3.1), and PTTG1 expression was monitored by means of western blotting. Tubulin expression was assessed to ensure equal protein loading of all samples. (B) PTTG1 protein levels were analyzed in Chang liver, Chang liver p34X, AML12 4p, and AML12 4pX cells grown with or without Dox for 48 hours by means of western blotting. (C) Flow cytometry analysis of cell cycle progression in Chang liver cells 24 hours after transient transfection with payw1.2(HBx+), payw*7(HBx), or control plasmid (pCDNA3.1). (D) Cell cycle progression in p34X and 4pX cells 48 hours after induction of HBx expression. Values represent the mean ± standard deviation of six independent experiments.

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HBx-Mediated PTTG1 Accumulation Is Regulated Posttranscriptionally.

It is known that HBx transcriptionally induces the expression of viral and cellular genes by activating promoter regulatory sequences.2 To determine whether HBx modulates PTTG1 transcription, its messenger RNA (mRNA) levels were measured by means of quantitative RT-PCR in p34x and 4pX cells. PTTG1 mRNA levels were unaffected by HBx expression in both p34X (Fig. 4A) and 4px (Supporting Fig. 3) cells. As expected,25 RT-PCR analysis revealed increased TNF-α mRNA levels upon induction of HBx (Fig. 4A).

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Figure 4. HBx increases PTTG1 expression levels posttranscriptionally. (A) PTTG1 (light gray bars) and TNF-α (dark gray bars) mRNA levels were measured in p34X cells 24 and 48 hours after induction of HBx expression by means of quantitative RT-PCR. Results were normalized with histone H3 and represented as fold induction over control. Results are expressed as the mean ± standard deviation of three independent experiments. (B) Hela cells were transfected with pPTTG1-CFP or pECFP-N1 plus pHBx-HA or empty vector (pcDNA 3.1). Cell lysates were analyzed by means of western blotting. (C) Hela cells were transfected with pPTTG1-CFP or pCFP-N1 plus payw1.2, payw*7, or control plasmid. Cell lysates were analyzed by means of western blotting. (D) Control or Dox-induced p34x cells (48 hours) were treated with 20 μM cycloheximide for different time intervals. Equal amounts of protein were subjected to Western blot analysis. Representative results of three independent experiments are shown.(E) Analysis of PTTG1 relative levels, assessed by scanning densitometry, was plotted (black circles, control cells; gray squares, HBx-expressing cells). Results are expressed as the percentage of values obtained without cycloheximide treatment for each experimental condition analyzed. Values represent the mean ± standard error of three independent experiments.

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Additionally, we transiently transfected Hela cells with both pPTTG1–cyan fluorescent protein (CFP), an expression vector in which PTTG1-CFP transcription is controlled by the CMV promoter, and pHBx-hemagglutinin (HA) plasmids. Western blot analysis using an anti–green fluorescent protein (GFP) Ab revealed that PTTG1-CFP was clearly accumulated in HBx-transfected cells (Fig. 4B). Interestingly, the effect of HBx was not observed when cells were cotransfected with the control plasmid pECFP-N1, coding only for the CFP protein. These results were further confirmed by cotransfecting Hela cells with wild-type or HBx-defective HBV replicons along with the pPTTG1-CFP vector (Fig. 4C). These results strongly suggested that PTTG1 accumulation induced by HBx was not mediated by transcriptional activation.

We next examined whether HBx-induced PTTG1 up-regulation could be explained through changes on protein stability by analyzing PTTG1 levels after blocking protein synthesis with cycloheximide. Western blot analysis revealed that PTTG1 protein half-life increased in p34X cells after induction of HBx expression when compared with noninduced cells (Fig. 4D,E). Taken together, these results indicated that HBx promoted PTTG1 accumulation by modulating its degradation.

HBx Inhibits the Ubiquitination of Hyperphosphorylated PTTG1 Forms.

Phosphorylation of PTTG1 leads to its ubiquitination and proteasomal degradation.10 Thus, we analyzed the levels of phosphorylated forms of PTTG1 in p34X cells treated with okadaic acid (OA), a protein phosphatase 2A (PP2A) inhibitor, and/or MG132, a proteasome inhibitor. As expected, proteasome inhibition by MG132 treatment promoted PTTG1 accumulation independently of HBx expression (Fig. 5A, lane 3 versus lane 1 and lane 7 versus lane 5). As described,10 MG132 plus OA cotreatment revealed the presence of slower migrating bands corresponding to phosphorylated forms of PTTG1 in both control or Dox-treated cells (Fig. 5A, lanes 4 and 8). OA treatment reduced PTTG1 levels in both HBx-expressing and -nonexpressing cells (Fig. 5A, lane 2 versus lane 1 and lane 6 versus lane 5). However, phosphorylated PTTG1 could be detected in the absence of MG132 after PP2A inhibition (OA treatment) only when HBx was expressed, suggesting that HBx inhibited the degradation of phosphorylated PTTG1 (Fig. 5A, lane 6 versus lane 2). In order to rule out that the differences observed between HBx-expressing and -nonexpressing cells could be due to undefined clonal properties of p34X cells or Dox-associated effects rather than the presence of HBx, parental Chang liver cells were included. As in HBx-nonexpressing p34X cells, phosphorylated PTTG1 in Chang liver cells were only detected after proteasome plus PP2A inhibition independently of Dox treatment (Supporting Fig. 4A).

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Figure 5. HBx reduces the ubiquitination of hyperphosphorylated PTTG1 forms. (A) Chang liver p34x cells were treated with OA (1 μM), MG132 (10 μM), or both for 2 hours after 48 hours of Dox induction. Lysates were subsequently analyzed by means of western blotting for the detection of PTTG1. (B) Confocal immunofluorescence analysis of the distribution of PTTG1 (green) and HBx (red) in OA-treated (OA+) or nontreated (OA−) p34x cells. 4′,6-Diamidino-2-phenylindole staining is shown in blue. Bar = 5 μm. (C) Twenty-four hours after transfection of Chang liver cells with pCMS-EGFP-HBx (CMS-X) or pCMS-EGFP (CMS-0) plasmids, cells were treated with OA, and PTTG1 accumulation was compared by means of immunofluorescence. Percentages of transfected cells (GFP positive) displaying PTTG1 accumulation are shown. Values are expressed as the mean ± standard deviation of four independent experiments in which at least 50 GFP-positive cells were analyzed. (D) Control or Dox-induced p34X cells were treated or not for 4 hours with MG132 (10 μM). Cell lysates were immunoprecipitated (IP) with anti-PTTG1 pAb and immunoblotted with anti-PTTG1 (bottom) and anti-ubiquitin (top) Abs. (E) Cells were treated as in (D), and lysates were immunoprecipitated with anti-occludin pAb and immunoblotted with anti-occludin (bottom) and anti-ubiquitin (top). Representative results of at least two independent experiments are shown.

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Additionally, we compared PTTG1 distribution between HBx-expressing versus HBx-nonexpressing cells after OA treatment by immunofluorescence experiments. Of note, not all p34X cells expressed HBx in response to Dox treatment. In the absence of OA, PTTG1 was diffusely localized in both the nucleus and cytoplasm of HBx-positive and -negative p34X cells (Fig. 5B, top). As mentioned, OA treatment reduced PTTG1 levels (Fig. 5B, bottom). However, we observed a PTTG1 accumulation in HBx-positive cells that colocalized with the viral protein. To quantify the effect of HBx on PTTG1 accumulation after OA treatment, Chang liver cells were transfected with the bicistronic plasmids pCMS-EGFP-HBx (HBx-expressing vector; CMS-X) or pCMS-EGFP (control vector; CMS-O) and processed for immunofluorescence after PP2A inhibition. As shown in Fig. 5C, there was a marked increase of PTTG1-positive cells when transfected with HBx-expressing vector compared with control vector.

It has been shown that HBx is an inhibitor of both proteasome complex26 and ubiquitin ligases.6 Therefore, HBx could promote PTTG1 accumulation through proteasome and/or ubiquitin ligase inhibition. Because ubiquitination targets proteins to proteasomal degradation, we analyzed the ubiquitination of PTTG1 in the presence of HBx. For this purpose, unstimulated or Dox-treated p34X cells were incubated with the proteasome inhibitor MG132 and used for immunoprecipitacion using an anti-PTTG1 Ab. Membranes were blotted with anti-ubiquitin monoclonal Ab to detect ubiquitinated forms of PTTG1. As expected, MG132-mediated proteasome inhibition promoted the accumulation of polyubiquinated PTTG1 forms in cells that did not express HBx (Fig. 5D, lane 7 versus lane 5). In contrast, incubation of HBx-expressing cells with MG132 did not significantly increase the levels of ubiquitinated forms of PTTG1 (Fig. 5D, lane 8 versus lane 6). Similar results were obtained in Hela cells cotransfected with expression vectors coding for HA-tagged ubiquitin (HA-ubiquitin), PTTG1 (pcDNA-PTTG1), and either an HBx-coding vector (pSVX) or the control plasmid (pSVHygro) (Supporting Fig. 4B). The tight junction–associated protein occludin is ubiquitinated, and its degradation is sensitive to proteasome inhibition.27 To analyze whether HBx affected general ubiquitination events, we determined the influence of HBx on occludin ubiquitination. As shown in Fig. 5E, the accumulation of polyubiquitinated occludin was not affected by HBx expression. Together, these results strongly suggested that HBx specifically reduced PTTG1 ubiquitination.

HBx Disrupts the Interaction Between PTTG1 and the SCF Protein Complex.

It has been reported that phosphorylated forms of PTTG1 are degraded by the proteasome after ubiquitination by SCF ubiquitin ligase complex.28 In agreement with our previous results using other cell lines,11 coimmunoprecipitation assays using lysates of unstimulated p34X cells treated with OA plus MG132 revealed that the SCF core component Cul1 coimmunoprecipitated with PTTG1 (Fig. 6A, top, lane 4). Interestingly, treatment of p34X cells with Dox to induce HBx expression partially disrupted the interaction between PTTG1 and Cul1 (Fig. 6A, lane 5 versus lane 4). GST-based pull-down assays revealed that the fusion protein GST-PTTG1, but not GST, interacted with endogenous Cul1 from a cellular lysate of noninduced p34X (Fig. 6B, top, lane 5). As above, this interaction was also reduced in the presence of HBx (Fig. 6B, top, lane 6 versus lane 5). These data suggested that HBx could reduce PTTG1 ubiquitination, at least partially, by interfering the interaction between PTTG1 and SCF. In addition, these results indicated that the interaction of HBx with PTTG1 and/or SCF complex might be operating in the disruption of PTTG1/SCF association. To further explore this issue, additional pull-down assays were performed. As shown in Fig. 6D, GST-HBx interacted with endogenous PTTG1 and GST-PTTG1 associated with HBx protein (Fig. 6B bottom, lane 6). Furthermore, an interaction between GST-HBx and Cul1 could also be demonstrated (Fig. 6D). The specificity of these GST-HBx interactions was confirmed by observing no interaction of HBx with occludin and other cell cycle–regulating proteins as cyclin B1 or STAG2/SA2 (Fig. 6D). The association between HBx and Cul1 was further confirmed by confocal double-label immunofluorescence in Chang liver p34X cells in which HBx significantly colocalized with Cul1 in dot-like structures (Fig. 6E).

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Figure 6. HBx inhibits the interaction between PTTG1 and SCF complex. (A) Lysates from p34X cells, grown with or without Dox for 48 hours, were immunoprecipitated (IP) using anti-PTTG1 or rabbit immunoglobulin G as a control. Western blot analysis of cell lysates and immunoprecipitates was performed with anti-Cul1 (top) or anti-PTTG1 (bottom) Abs. (B) Pull-down assay with GST or GST-PTTG1 and Dox-treated (+) or untreated (−) p34X extracts. Cell lysates and bound proteins were subjected to western blotting using anti-Cul1 (top) or anti-HA (bottom) Abs. (C) Coomassie brilliant blue staining of 1/10 of the GST proteins used is shown. Molecular weight markers (kDa) are indicated on the left. (D) Pull-down assay with GST or GST-HBx and noninduced p34X cell extracts. Cell lysates and bound proteins were subjected to western blot analysis using anti-Cul1, anti-PTTG1, anti-occludin, anti-SA2, and anti–cyclin B1 Abs. (E) Confocal immunofluorescence analysis of the distribution of Cul1 (green; monoclonal Ab anti-Cul-1) and HBx (red; byotinilated Ab anti-HA epitope) in OA-treated (OA+) or untreated (OA−) p34x cells. 4′,6-Diamidino-2-phenylindole staining is shown in blue. Bar = 7.5 μm. All results are representative of at least two independent experiments.

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HBx Does Not Affect PTTG1 Stabilization in Cul1 Knockdown Cells.

The SCF ubiquitin ligase complex is involved in the degradation of phosphorylated forms of PTTG1.10 To analyze the specific role of Cul1 on HBx-mediated PTTG1 accumulation, an siRNA-based knockdown approach was employed. First, we determined the levels of PTTG1 in Chang liver cells transiently transfected with control or Cul1-specific siRNAs, and then treated or not with OA and/or MG132. Western blot analysis revealed that Cul1 knockdown promoted PTTG1 accumulation in both control and OA-treated cells. Additionally, OA treatment of Cul1 knockdown cells resulted in the formation of phosphorylated PTTG1 forms (Supporting Fig. 5). We then analyzed the effect of HBx expression on PTTG1 accumulation in Cul1-silenced cells. In both Chang liver and p34x cells, PTTG1 expression levels were increased after Cul1 silencing (Fig. 7). As above, Dox-induced HBx increased PTTG1 levels in p34X control siRNA-treated cells (Fig. 7, lane 7 versus lane 5). Interestingly, PTTG1 accumulation after Cul1 silencing was not further enhanced by HBx (Fig. 7, lane 8 versus lane 6), suggesting that the stabilization of PTTG1 by HBx was Cul1-dependent, not being likely that other ubiquitin ligase was involved. Given that HBx expression mimicked the effects of Cul-1 knockdown on PTTG1, it can be hypothesized that HBx interferes Cul1-asscociated functions. Overall, these data strongly suggest that HBx promotes the disruption of the PTTG1/SCF association and prevents its ubiquitination and subsequent degradation by the proteasome (Fig. 8).

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Figure 7. HBx does not enhance PTTG1 stabilization after Cul1 knockdown. Chang liver and p34x cells were transfected with control or Cul1 siRNA and treated with Dox for 48 hours. PTTG1, Cul1, and tubulin protein levels were analyzed by means of western blotting. Results are representative of two independent experiments.

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Figure 8. Possible mechanism for HBx-mediated PTTG1 stabilization. (A) PTTG1 undergoes proteasomal degradation via ubiquitination by the SCF ubiquitin ligase complex (A. PTTG1 degradation). Cul1 interacts with PTTG1, and in the presence of HBx this interaction is disrupted. As a result, there is an impairment of PTTG1 ubiquitination that leads to an increase of its half-life (B. HBx-induced PTTG1 accumulation). The proliferative actions of PTTG1 and HBx could act synergistically in cell transformation.

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

HBV-associated carcinogenesis is a multifactorial process. Liver inflammation results in hepatocellular death and regeneration processes that lead to the accumulation of critical mutations in the host genome. In addition, the regulatory protein HBx has been involved in hepatocarcinogenesis by altering cellular processes. In the present study, we have demonstrated that PTTG1 expression levels increase in HBx-immunoreactive cells as chronic hepatitis B progresses to cirrhosis and HCC. Furthermore, PTTG1 expression increases as HBx transgenic mouse livers progress through hyperplasia to HCC. In addition, PTTG1 accumulates in human and mouse HBx-expressing cell lines and in HBV replicon-containing cells, but not in cells harboring an HBx-defective genome construct. Together, these data strongly suggest that PTTG1 accumulation is, at least partially, an HBx-mediated effect.

Several viruses, including HBV, have the ability to stimulate the cell cycle progression in order to facilitate their own replication. In doing so, viruses generally disrupt the normal cell cycle checkpoints and in turn extend proliferative signals to host cells to establish a carcinogenic environment.29 HBx has been demonstrated to suppress serum dependence for cell cycle activation.30 Furthermore, HBx has been shown to promote transit through G1 in G0-arrested cells and to alter G1-to-S and G2-to-M progression.17, 22 However, in Chang liver p34X cells, the cell cycle profile was unaffected after HBx induction.24 In addition, it is known that HBx transcriptionally induces the expression of viral and cellular genes.2 However, our data strongly suggest that HBx-promoted PTTG1 protein accumulation is not strictly dependent on cell cycle modifications or transcriptional up-regulation.

Through interactions with host factors, HBx alters different cellular processes implicated in the development of HCC. Protein degradation by the proteasome complex is a strictly regulated key event of cellular homeostasis. Oncogenic viruses alter the proteasomal activity of target cells, affecting viral entry, replication, and release and enhancing cell survival.31 Targeting of proteins to the proteasome through interactions with ubiquitin ligases is essential for normal protein turnover. In this context, HBx is able to down-regulate both proteasome26 and ubiquitin ligase functions.6 Our data show that HBx induces a marked accumulation of PTTG1 protein by reducing its ubiquitination and subsequent degradation.

It has been demonstrated that the SCF ubiquitin ligase complex is involved in the degradation of phosphorylated forms of PTTG1 in nonmitotic cells. In addition, HBx affects SCF ubiquitin ligase functions through mechanisms involving protein–protein interactions.6 Confocal microscopy analysis and biochemical data strongly suggest that HBx may interact with both the SCF component Cul1 and PTTG1. Interestingly, the association between PTTG1 and Cul1 is disrupted in the presence of HBx. However, HBx expression does not enhance PTTG1 accumulation after Cul1 silencing. Together, these data suggest that HBx may alter the formation of the SCF/PTTG1 complex, leading to an impairment of PTTG1 ubiquitination. Thus, in the presence of HBx, PTTG1 is not targeted to proteasome-mediated degradation resulting in an abnormal protein accumulation (Fig. 8). It is tempting to speculate that by affecting the normal turnover of PTTG1, HBx could alter some of the PTTG1-related functions and promote cellular transformation.

The SCF ubiquitin ligases are mammalian cullin RING ubiquitin ligases in which F-box proteins provide the substrate targeting specificity of the complex. Skp2 is the F-box protein that targets key regulatory proteins, such as c-myc, for degradation.32 Interestingly, it has been shown that HBx is able to block ubiquitination of c-myc through a direct interaction with Skp2 and destabilization of the SCF/Skp2 complex. An association between HBx-mediated PTTG1 stabilization and HBx/Skp2 interaction may also exist, but this issue requires further study.

PP2A is an important serine/threonine phosphatase family involved in essential cellular processes such as cell division, gene regulation, protein synthesis, and cytoskeleton organization. PP2A enzymes typically exist as heterotrimers comprising a common catalytic subunit (PP2Ac) and different structural and regulatory subunits.33 It has been shown that hepatotropic viruses, including hepatitis C virus and HBV, alter PP2Ac activity.34 HBx protein is the most likely candidate responsible for HBV-mediated PP2Ac modulation.34 Our results show that HBx promotes PTTG1 accumulation, inhibiting the degradation of phosphorylated forms of PTTG1 after chemical inhibition of PP2A. Further experiments are necessary to analyze whether HBx could affect PTTG1 expression levels by up-regulating PP2A activity.

Several lines of evidence suggest that an important transforming mechanism underlying PTTG1 overexpression is the induction of chromosomal instability.9 Thus, it has been demonstrated that PTTG1 accumulation inhibits mitosis progression and chromosome segregation, but does not directly affect cytokinesis, resulting in aneuploidy.35 It has been shown that HBx can transform cultured cells21 and induce liver cancer in transgenic mice.36 Genetic instability is frequently accompanied with the acquisition of transformation ability and malignant progression of tumors. Moreover, recent reports have shown that HBx expression induces chromosomal aberrations such as chromosome rearrangements and micronuclei formation.37 Furthermore, HBx promotes multipolar spindle formation and chromosomal missegregation during mitosis, and increases multinucleated cells.18 Interestingly, it has been determined that HBx binds to BubR1, a component of the mitotic checkpoint complex, and attenuates the association between BubR1 and CDC20, an activator of the anaphase-promoting complex/cyclosome, resulting in chromosomal instability.38 Our results demonstrate that HBx induces the accumulation of PTTG1 in interphase cells. Further experiments are necessary to study the effects of HBx on PTTG1 functions during mitotic events.

In conclusion, we propose that HBx promotes alterations of PTTG1 expression levels, which may improve our understanding of the molecular mechanisms of HBV-related pathogenesis of progressive liver disease leading to cirrhosis and HCC development.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Drs. O. M. Andrisani, H. Cho, E. Lara-Pezzi, M. Levrero, S. Murakami, K. I. Nakayama, B. L. Slagle, and J. R. Wands for providing critical reagents and R. López-Rodríguez for statistical analysis.

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  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_23468_sm_Suppfig1.tif598KSupplementary figure S1. HCC sections of HBx transgenic mouse livers were stained for immunofluorescence to simultaneously detect PTTG1 (red) and HBx (green). Yellow color indicates overlap of proteins. Bar, 50 μm.
HEP_23468_sm_Suppfig2.tif183KSupplementary figure S2. (A) Chang liver, p34X, AML12 4p and 4pX cells were cultured with or without Dox for 24 h, and HBx and PTTG1 protein levels were analyzed by Western blot. Tubulin expression was assessed to ensure equal protein loading of all samples. (B) Flow cytometry analysis of cell cycle progression of p34X and 4pX cells 24 h after induction of HBx expression. Values represent the mean ± SD of three independent experiments performed at least in duplicate. (*p≤0,05 vs HBx non-expressing cells, Mann-Whitney U test).
HEP_23468_sm_Suppfig3.tif25KSupplementary figure S3. PTTG1 mRNA levels in 4pX cells in response to HBx expression were measured by quantitative RT-PCR. The results were normalized with histone H3 and represented as fold induction over control. Results are presented as the mean ± SD of three independent experiments.
HEP_23468_sm_Suppfig4.tif278KSupplementary figure S4. (A) Chang liver cells were treated with okadaic acid (OA) (1 μM), MG132 (10 μM) or both for 2 h after 48 h of doxycycline treatment. Lysates were subsequently analyzed by Western blot for the detection of PTTG1. Tubulin expression was assessed to ensure equal protein loading. (B) Hela cells were co-transfected with pCGN-HA-ubiquitin, pcDNA-PTTG1 and either pSV-HBx or pSV-hygro plasmids and treated for 4 h with MG132 (10 μM). Cell lysates were immunoprecipitated with anti-PTTG1 mAb and immunoblotted with anti-PTTG1 (bottom) and anti-HA (top) Abs. Representative results of at least two independent experiments are shown.
HEP_23468_sm_Suppfig5.tif135KSupplementary figure S5. Chang liver cells were treated for 2 h with okadaic acid (OA; 1μM) and/or MG132 (10μM) after 48 h of transfection with control or Cul1 siRNA. Lysates were subsequently analyzed by Western blot for the detection of PTTG1 and Cul1. Tubulin expression was assessed to ensure equal protein loading.
HEP_23468_sm_Supptext.doc79KSUPPLEMENTARY DATA

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