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Original article
Cell cycle-related kinase mediates viral-host signalling to promote hepatitis B virus-associated hepatocarcinogenesis
  1. Zhuo Yu1,2,
  2. Yue-Qiu Gao2,
  3. Hai Feng3,
  4. Ying-Ying Lee4,
  5. May S Li3,
  6. Yuan Tian4,
  7. Minnie Y Y Go4,
  8. Dae-Yeul Yu5,
  9. Yue-Sun Cheung6,
  10. Paul B S Lai1,6,
  11. Jun Yu1,4,7,
  12. Vincent W S Wong1,4,7,
  13. Joseph J Y Sung1,7,
  14. Henry L Y Chan1,4,7,
  15. Alfred S L Cheng3,7
  1. 1Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR, P. R. China
  2. 2Department of Liver Disease, Shuguang Hospital, Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, P. R. China
  3. 3School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong SAR, P. R. China
  4. 4Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong SAR, P. R. China
  5. 5Disease Model Research Laboratory, Aging Research Center and World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea
  6. 6Department of Surgery, The Chinese University of Hong Kong, Hong Kong SAR, P. R. China
  7. 7State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR, P. R. China
  1. Correspondence to Professor Alfred Sze-Lok Cheng, School of Biomedical Sciences and State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR 999077, P. R. China; alfredcheng{at}cuhk.edu.hk and Professor Henry Lik-Yuen Chan, Institute of Digestive Disease, Department of Medicine and Therapeutics and State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR 999077, P. R. China; hlychan@cuhk.edu.hk

Abstract

Background Androgen receptor (AR) signalling contributes to male predominance in hepatocellular carcinoma (HCC), which is more pronounced in HBV-endemic areas. Cell cycle-related kinase (CCRK) is essential for AR-induced hepatocarcinogenesis but its molecular function in HBV-associated HCC remains obscure.

Objective To determine the molecular function of CCRK in HBV-associated HCC.

Design Transcriptional regulation was assessed by chromatin immunoprecipitation, promoter mutation and luciferase reporter assays. Hepatocellular proliferation and tumourigenesis were examined by colony formation, soft agar assays and using HBV X protein (HBx) transgenic mice with low-dose exposure to diethylnitrosamine. Protein expressions were examined in clinical samples and correlated with patient survival by log-rank Mantel–Cox test.

Results Overexpression of CCRK, but not its kinase-defective mutant, activated β-catenin/T cell factor signalling through phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9, led to upregulation of AR transcriptional activity and, subsequently, expression of HBx. The viral transactivator in turn induced CCRK expression through enhanced AR signalling, thus forming a positive regulatory loop. RNA interference silencing of CCRK, which suppressed the CCRK/GSK-3β/β-catenin/AR regulatory loop, significantly suppressed HBx-induced hepatocellular proliferation (p=0.001) and transformation (p<0.001) and remarkably reduced >80% diethylnitrosamine-mediated hepatocarcinogenesis in HBx transgenic mice. Finally, patients with HBV-associated HCC with concordant overexpression of CCRK, GSK-3β phosphorylation at Ser9, active dephosphorylated β-catenin and AR phosphorylation at Ser81 had poorer overall (HR=31.26, p<0.0001) and disease-free (HR=3.60, p<0.01) survival rates.

Conclusions Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates HBV-associated hepatocarcinogenesis. Further characterisation of this intricate viral-host signalling may provide new prognostic biomarkers and therapeutic targets for HCC treatment.

  • Hepatitis B
  • Hepatocellular Carcinoma
  • Gene Regulation
  • Molecular Carcinogenesis
  • Cell Signalling

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Significance of this study

What is already known about this subject?

  • Hepatocellular carcinoma (HCC) displays a pronounced male predominance in HBV-endemic regions.

  • Genetic knockout mouse models have established the causal role of androgen receptor (AR) signalling in male hepatocarcinogenesis.

  • HBV X protein (HBx) is a promiscuous transactivator that cooperates with AR signalling to increase viral load, which is believed to be a mechanism for sex disparity in chronic hepatitis B.

  • Cell cycle-related kinase (CCRK) functions as a direct AR-regulated oncogene in HCC through the upregulation of β-catenin/T cell factor (TCF) signalling.

What are the new findings?

  • CCRK mediates the HBx-AR signalling to form a viral-host oncogenic circuitry that induces hepatocellular proliferation and transformation.

  • Targeting inhibition of CCRK perturbs the CCRK/GSK-3β/β-catenin/TCF/AR regulatory loop to suppress hepatocarcinogenesis in an HBx transgenic HCC model.

  • Clinically, hyperactivation of this new regulatory loop in HBV-associated HCC tissues correlates with poor prognosis of patients.

How might it impact on clinical practice in the foreseeable future?

  • Despite the survival improvement in patients with HCC receiving a multikinase-targeted inhibitor, outcomes are still far from satisfactory. Given the importance of AR signalling in HCC and the central role of CCRK in this pathway, specific inhibition of this kinase is a promising strategy for HCC treatment.

  • This study also provides a panel of biomarkers whose expression levels have great prognostic value for HCC.

Introduction

Hepatocellular carcinoma (HCC) is a common manifestation of chronic HBV carriers causing about half a million deaths annually.1 ,2 A striking epidemiological characteristic of HCC is prominent male predominance, which is even more pronounced in HBV-endemic areas.2 ,3 Androgen receptor (AR) is a ligand-dependent transcription factor that has pivotal roles in a wide range of physiological and pathological conditions including HCC.46 Previous studies have shown that increased androgen activity via higher testosterone levels and AR genetic polymorphisms are associated with an increased risk of HBV-associated HCC in men.7 ,8 Prospective studies have also demonstrated a close correlation between HBV DNA level and risk of HCC among men.9 ,10 These studies point out the importance of unravelling the viral-host interactions for the development of new therapeutic strategies.

The HBV X protein (HBx) is a promiscuous transactivator important for viral replication and hepatocellular neoplastic transformation.11 ,12 HBx enhances AR transcriptional activity in an androgen-dependent manner through kinase pathways.13 ,14 The ligand-stimulated AR in turn increases the transcription of HBV RNAs through direct binding to the androgen-response element (ARE) on the viral genome,6 ,15 thus forming a positive regulatory loop. Sustained necrosis–inflammation–regeneration due to a virally induced immune response is believed to drive HBV-associated hepatocarcinogenesis.1 ,12 However, whether the cross-talk between HBx and AR promotes HCC development via a cellular signalling network is unclear.

Cell cycle-related kinase (CCRK) is the latest cyclin-dependent kinase family member (CDK20) that has been implicated in cell cycle and transcriptional regulation.16 Recent studies have shown that CCRK is overexpressed in a wide spectrum of human sporadic cancers.17 Although genome-wide location and functional analysis has identified CCRK as a key AR target gene that promotes β-catenin/T cell factor (TCF)-dependent hepatocarcinogenesis,18 its role in HBV-associated hepatocarcinogenesis remains obscure. Here, we report a viral-host oncogenic circuitry composed of HBx, AR, CCRK, glycogen synthase kinase-3β (GSK-3β), β-catenin and TCF. Perturbation of this signalling network through downregulation of CCRK prevented HBx-induced tumourigenicity in vitro and in vivo. As we also showed that the CCRK/GSK-3β/β-catenin/AR regulatory loop was perturbed in human HBV-associated HCC, our data raised the possibility that manipulation of this viral-host self-reinforcing circuitry might provide new preventive and therapeutic approaches for HCC.

Patients and methods

Patients

Thirty-four HBV surface antigen-positive patients with HCC who underwent hepatectomy at the Prince of Wales Hospital (Hong Kong) were included in this study. Histologically normal livers from patients with benign focal nodular hyperplasia served as controls. Studies using human tissue were approved by the joint CUHK-NTEC clinical research ethics committee.

Cell culture and expression vectors

The immortal human liver LO2 and hepatoma HepG2.2.15, Huh7, PLC5 and SK-Hep1 lines were maintained as previously described.18 ,19 For treatment with AR agonist R1881 (Waterstone Technology; Carmel, Indiana, USA), the cells were hormone-deprived in phenol-red free Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, New York, USA) supplemented with 5% charcoal/dextran-treated fetal bovine serum (HyClone, Waltham, Massachusetts, USA), 1% L-glutamine and 2% sodium pyruvate (Gibco). Small-interfering RNA (si RNA, 25 nM) was transfected into cells using HiPerfect (Qiagen, Hilden, Germany). Full-length HBx-expressing vector was constructed as in our previous study.20 AR-expressing vector was kindly provided by Norman Maitland (University of York). Kinase-defective CCRK T161A mutant, which produces protein with substitution of the T-loop threonine-161 with alanine, was constructed, as previously described.16 The short-hairpin RNA (shRNA) vector targeting β-catenin and the uninhibitable S9A-GSK-3β, of which the Ser9 regulatory site was mutated to alanine, were purchased from Addgene. Dominant-negative TCF was kindly provided by Alice Wong (University of Hong Kong).

Chromatin immunoprecipitation-PCR

Chromatin immunoprecipitation was performed as previously described.18 Huh7 or PLC5 cells (1×108) were transfected with HBx or empty vector for 48 h using FuGENE 6 (Roche, Indianapolis, Indiana, USA) and treated or not with 100 nM R1881 in hormone-deprived medium for the last 24 h. The cells were then crosslinked with 1% formaldehyde for 10 min at room temperature. After cell lysis, the chromatin was fragmented into 100–250 bp by a Bioruptor Sonicator (Diagenode, Denville, New Jersey, USA) and protein–DNA complexes were immunoprecipitated by 5 μg anti-AR antibody (Cell Signaling, Danvers, Massachusetts, USA) or anti-IgG antibody (Sigma-Aldrich, St Louis, Missouri, USA)–Dynal magnetic bead (Life Technologies, Oslo, Norway) mix at 4°C overnight. After washing and reversal of crosslinks, the immunoprecipitated and input DNA were purified by purification kit (Qiagen) followed by quantitative PCR. The sequences of PCR primers used in this study are listed in online supplementary table S1.

Site-directed mutagenesis and luciferase assays

The wild-type (WT) and mutant CCRK-promoter luciferase reporters were constructed as in our previous study.18 The HBx-promoter luciferase reporter and its ARE (999-TATTAA-1004) deletion mutant15 were generated using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, California, USA) and verified by sequencing. The AR-responsive luciferase construct containing tandem repeats of ARE was purchased from Qiagen. The promoter luciferase assay was performed by the Dual Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA) using a GloMax microplate luminometer (Promega).

Reverse transcription-PCR (RT-PCR), immunofluorescence and western blot

RNA extraction, quantitative RT-PCR, immunofluorescence and western blot were performed as previously described.18 Protein lysates from cell lines and tissues were prepared using protease inhibitor cocktail-containing (Roche) lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate) and T-PER Tissue Protein Extraction Reagent (Pierce, Rockford, Illinois, USA), respectively. The primary antibodies used in this study were mouse anti-HBx, rabbit anti-AR, rabbit anti-CCRK (Abcam, Cambridge, Massachusetts, USA), rabbit anti-p-ARSer81, mouse anti-active (dephosphorylated) β-catenin (Upstate, Billerica, Massachusetts, USA), rabbit anti-GSK-3β, rabbit anti-p-GSK-3βSer9, rabbit anti-β-catenin (Cell Signaling), rabbit anti-CCND1 (Pierce), mouse anti-epidermal growth factor receptor (EGFR; BD Transduction laboratories, San Diego, California, USA), mouse anti-HA (Covance, Princeton, New Jersey, USA), mouse anti-Flag and mouse anti-β-actin (Sigma-Aldrich). Signals from clinical specimens were quantified by BandScan software (Glyko, Novato, California, USA) and defined as the ratio of target protein relative to β-actin. Signals of total GSK-3β and β-catenin were used to normalise the levels of p-GSK3βSer9 and active β-catenin, respectively.

Colony formation and soft agar assays

For the colony formation assay, cells seeded on a 12-well plate with 50–80% confluence were transiently transfected with vectors. After 2 days, the cells were reseeded onto a six-well plate and cultured for 3 weeks in antibiotic containing the selection medium. The resistant colonies were stained with 0.2% crystal violet and counted under the microscope. For the soft agar assay, cells seeded on a six-well plate were covered with a layer of 0.6% agar in DMEM medium supplemented with 10% fetal bovine serum. After transfection for 48 h, cells were trypsinised, gently mixed with 0.3% agar medium mixture containing selective antibiotics and reseeded in triplicate onto a six-well plate. After 4 weeks, the resistant colonies were stained with 0.2% crystal violet and counted under the microscope.

HBx transgenic (TG) mouse HCC model

Studies using male HBx homozygous TG and C57BL/6 WT mice were approved by the animal experimentation ethics committee of the Chinese University of Hong Kong. The strain of HBx TG mice was fixed to C57BL/6 by backcrossing with the C57BL/6 strain for more than 20 generations.21 Lentiviruses encoding shRNA against CCRK or a control sequence were packaged according to the manufacturer's instructions (Dharmacon, Lafayette, Colorado, USA). At 12-days-old, male HBx TG mice were given a single intraperitoneal injection of low-dose diethylnitrosamine (DEN; Sigma-Aldrich) at 5 mg/kg of body weight in comparison with other studies where 10 mg/kg of body weight was used.22 At 6- and 18-weeks-old, 5×107 transducing units of lentiviruses were administered to the mice by tail vein injection. At age 32 weeks, all mice were killed and the number, size and weight of tumours were recorded. Tissues were collected for gene expression and cell proliferation analyses.

Ki67 immunohistochemistry

Cell proliferation in tissues was assayed by immunoperoxidase staining with anti-Ki-67 antibody (Labvision, Fremont, California, USA). The proliferation index was determined by counting the number of positively stained cells per high-power field (HPF, ×400 magnification). At least 1000 tumour cells were counted for each sample.

Statistical analysis

GraphPad Prism 5 (GraphPad Software, La Jolla, California, USA) was used for statistical analysis. The independent Student t test was used to compare data between two groups. A one-way analysis of variance test with Bonferroni correction was used to compare gene expression in multiple groups of mice. The correlation between protein expressions was analysed using Pearson correlation test. The protein expression in human tissues was compared using non-parametric Wilcoxon's matched pair test or Mann–Whitney U test. The clinicopathological features of patients were compared using Fisher's exact test for categorical variables and Mann–Whitney U test for continuous data. Kaplan–Meier survival analysis was used to determine the overall and disease-free survival rates, which were calculated from the date of curative surgery to death, HCC recurrence or the last follow-up; the differences were compared by log-rank Mantel–Cox test. A two-tailed p value of <0.05 was considered statistically significant.

Results

CCRK upregulates AR expression and transcriptional activity through GSK-3β/β-catenin/TCF signalling

As a first step in investigating the molecular function of CCRK in the HBx–AR positive feedback loop, we elucidated the effects of CCRK and its kinase-defective mutant16 on AR signalling in an immortal liver line LO2 and an HCC line SK-Hep1. Overexpression of CCRK phosphorylated GSK-3β at Ser9 (p-GSK3βSer9) and activated β-catenin signalling as shown by increased levels of active β-catenin and its downstream targets cyclin D1 (CCND1) and EGFR (figure 1A) as well as nuclear translocation of β-catenin (see online supplementary figure S1A), as recently reported.18 Notably, CCRK increased the expression levels of AR and phosphorylated AR at Ser81 (p-ARSer81), which regulates promoter selectivity18 (figure 1A), and also enhanced AR-responsive transcriptional activity (figure 1B). In contrast, the kinase-defective CCRK mutant, which was much less efficient in phosphorylating GSK-3β and activating β-catenin (see online supplementary figure S1A), failed to induce AR expression and transcriptional activity (figure 1A,B).

Figure 1

GSK-3β/β-catenin/T cell factor (TCF) signalling is required for cell cycle-related kinase (CCRK)-induced androgen receptor (AR) activation. (A, B) GSK-3β/β-catenin and AR signalling were examined in LO2 and SK-Hep1 cells upon ectopic expression of wild-type (WT) and kinase-defective (KD) CCRK for 48 h. (A) Whole cell lysates (50 μg) were analysed by western blot using the indicated antibodies. β-Actin, total GSK-3β and β-catenin were used as internal controls. (B) The AR-responsive luciferase (LUC) activities relative to Renilla control were measured after transfection. Data are presented as mean+SD of three experiments. (C) CCRK-induced β-catenin signalling and (D) AR transcriptional activity were suppressed by treatment of a GSK-3β activator, LY294002 (10 μM or 50 μM) for 48 h. Cotransfection of a constitutively active GSK-3β-expressing vector (GSK-3β-S9A) attenuated (E) CCRK-induced β-catenin signalling and (F) AR transcriptional activity. (G) Cotransfection of short-hairpin RNA against β-catenin (shβ-catenin) or dominant negative TCF (dnTCF) attenuated CCRK-induced AR activation as shown by western blot analysis.

To investigate the role of GSK-3β in CCRK-induced AR activation, we treated both LO2 and SK-Hep1 lines with LY294002 to activate GSK-3β. LY294002 not only abrogated CCRK-induced GSK-3β phosphorylation, but also attenuated β-catenin signalling, AR expression (figure 1C) and transcriptional activity in a dose-dependent manner (figure 1D). Concordantly, suppression of GSK-3β Ser9 phosphorylation by overexpression of the constitutively-active S9A-GSK-3β mutant also impaired CCRK-induced β-catenin and AR signalling (figure 1E,F; see online supplementary figure S1B).

We next examined the effect of β-catenin signalling on CCRK-induced AR activation. Because β-catenin binds TCF transcription factors to regulate gene expression,23 we manipulated the expression of β-catenin by using β-catenin-specific shRNA or perturbed TCF function by expressing a dominant-negative TCF.18 Inhibition of β-catenin/TCF signalling prohibited induction of AR and p-ARSer81 by CCRK (figure 1G). Taken together, these results demonstrate that CCRK upregulates AR signalling through activation of the GSK-3β/β-catenin/TCF cascade.

CCRK induces HBx expression through AR signalling

Because AR has been shown to increase HBx transcription,6 ,15 we determined whether CCRK induces HBx through AR signalling. Overexpression of CCRK in LO2 and SK-Hep1 cells strongly increased AR expression and HBx promoter activity (figure 2A,B, respectively). Deletion of the ARE in the HBx promoter15 completely abolished the HBx transcriptional activation by CCRK (figure 2B). Knockdown of AR by siRNA (figure 2C) also abrogated CCRK-induced HBx transcription (figure 2D). Conversely, knockdown of CCRK in two AR-expressing HCC lines, Huh7 and PLC5, decreased AR expression and HBx promoter activity, which could be rescued by ectopic AR expression (figure 2E,F). We further investigated the effects of CCRK on HBx expression in the HepG2.2.15 hepatoblastoma line constitutively producing HBV.19 Knockdown of CCRK markedly reduced the HBx transcript and protein levels, whereas ectopic AR expression in CCRK knockdown cells restored the HBx expression (figure 2G,H). The effect of CCRK on HBx gene expression is specific as the other HBV transcripts were unaffected (figure 2H), which is consistent with the presence of ARE solely in the HBx promoter.6 ,15 These findings demonstrate that CCRK induces HBx expression in an AR-dependent manner.

Figure 2

Cell cycle-related kinase (CCRK) induces HBx in an androgen receptor (AR)-dependent manner (A, B). The androgen-response element (ARE) in HBx promoter was essential for CCRK-induced transcriptional activation. (A) LO2 and SK-Hep1 cells were transfected with CCRK or empty vector for 48 h. Whole cell lysates (50 μg) were analysed by western blot using anti-CCRK and anti-AR. β-Actin was used as internal control. (B) The wild-type (WT) or ARE-deleted mutant of HBx-promoter construct was cotransfected in the luciferase (LUC) activity assays. Luciferase activities relative to Renilla control were measured after transfection. Data are presented as mean+SD of three experiments. (C, D) CCRK-induced HBx transcription was abrogated by knockdown of AR. The cells were cotransfected with CCRK or empty vector and short-interfering RNA against control (siCtrl) or AR (siAR), followed by (C) western blot and (D) luciferase activity assays using WT HBx-promoter construct. (E, F) Reduced HBx transcription upon CCRK knockdown could be rescued by overexpression of AR. Huh7 and PLC5 cells were cotransfected with vector expressing shRNA against control sequence (shCtrl) or CCRK (shCCRK) and AR or empty vector for 48 h, followed by (E) western blot and (F) luciferase activity assays. (G, H) HepG2.2.15 cells were cotransfected with vector expressing shCtrl or shCCRK and AR or empty vector for 48 h. The relative HBx expression was examined by (G) western blot and (H) quantitative reverse transcription-PCR (RT-PCR). The transcript levels of the HBV core (HBc), polymerase (HBp) and surface (HBs) genes were also determined by quantitative RT-PCR. β-Actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the corresponding internal controls, respectively.

HBx feedback induces CCRK expression through AR signalling

Because HBx has been shown to enhance AR-responsive gene expression,13 ,14 we investigated whether HBx feedback induces CCRK, an AR-regulated gene.18 HBx increased CCRK promoter occupancy by AR in Huh7 and PLC5 cells (figure 3A), which was further enhanced by an AR agonist R1881 (figure 3B). Deletion of the ARE in the CCRK promoter18 completely abolished the CCRK transcriptional activation by HBx (figure 3C). Further, knockdown of AR abrogated HBx-induced CCRK transcription (figure 3D) and reduced HBx-induced CCRK mRNA and protein expressions (figure 3E,F, respectively).

Figure 3

HBx feedback induces cell cycle-related kinase (CCRK) expression through androgen receptor (AR) signalling. (A) HBx increased the binding of AR to CCRK promoter in hepatocellular carcinoma cells. Huh7 and PLC5 cells were transfected with HBx or empty vector for 48 h. Data obtained by quantitative chromatin immunoprecipitation-PCR for anti-AR antibody and IgG control were plotted as percentage of input. (B) HBx-induced AR binding was enhanced by treatment with R1881. The cells transfected with HBx vector for 24 h were treated with or without R1881 (100 nM) in hormone-depleted medium for an additional 24 h before assays. (C) The androgen-response element (ARE) in CCRK promoter was essential for HBx-induced transcriptional activation. The HBx or empty vector and wild-type (WT) or ARE-deleted mutant of CCRK-promoter construct was cotransfected for 48 h, followed by the luciferase activity assays. Luciferase (LUC) activities relative to Renilla control were measured after transfection. (D) HBx-induced CCRK transcription was abrogated by knockdown of AR. The cells were cotransfected with HBx or empty vector and siCtrl or siAR, followed by luciferase activity assays using WT CCRK-promoter construct. (E, F) The cells were cotransfected with vector expressing HBx or empty vector and siCtrl or siAR for 48 h. The relative CCRK expression was examined by (E) quantitative reverse transcription-PCR and (F) western blot. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were used as the corresponding internal controls, respectively. Data are presented as mean+SD of three experiments.

We further investigated the regulatory effect of HBx on CCRK expression in HepG2.2.15 cells. SiRNA-mediated knockdown of HBx24 in this endogenously expressing line decreased CCRK promoter occupancy by AR (figure 4A) and transcriptional activity (figure 4B), which could be restored by ectopic AR expression (figure 4A,B). Deletion of the ARE rendered the CCRK promoter non-responsive to HBx induction (figure 4B). Further, knockdown of HBx markedly reduced the CCRK transcript and protein levels, whereas ectopic AR expression in HBx knockdown cells rescued the CCRK expression (figure 4C,D). Collectively, these data suggest that HBx induces CCRK expression through AR signalling.

Figure 4

Androgen receptor (AR) signalling is required for cell cycle-related kinase (CCRK) upregulation by endogenous HBx. The HepG2.2.15 cells were cotransfected with siCtrl or siHBx and AR or empty vector for 48 h, followed by chromatin immunoprecipitation (ChIP), luciferase (LUC) assays and expression analyses. (A) AR binding to CCRK promoter was measured by quantitative ChIP-PCR and the data obtained for anti-AR antibody and IgG control were plotted as percentage of input. (B) The wild-type (WT) or androgen-response element (ARE)-deleted mutant of CCRK-promoter construct was cotransfected into the HepG2.2.15 cells. Luciferase activities relative to Renilla control were measured after transfection. The relative CCRK expression was examined by (C) quantitative reverse transcription-PCR and (D) western blot using the indicated antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were used as the corresponding internal controls, respectively. Data are presented as mean+SD of three experiments.

HBx augments a CCRK/GSK-3β/β-catenin/AR regulatory loop to induce hepatocellular proliferation and tumourigenicity

Our findings suggest that CCRK mediates the HBx-AR signalling to form a viral-host circuitry. In support of this hypothesis, overexpression of HBx in LO2 liver cells increased the levels of CCRK, p-GSK3βSer9, active β-catenin, CCND1, EGFR, AR and p-ARSer81 (figure 5A). To elucidate the functional significance of this regulatory loop, we investigated the effects of CCRK inhibition on the oncogenic properties of HBx. Overexpression of HBx in LO2 cells markedly induced focus formation (p=0.001) and anchorage-independent growth (p<0.001; figure 5B,C, respectively). Notably, downregulation of CCRK abrogated HBx-stimulated GSK-3β/β-catenin/AR signalling (figure 5A) and reduced oncogenicity of the HBx-expressing cells (p<0.01; figure 5B,C).

Figure 5

The cell cycle-related kinase (CCRK)/GSK-3β/β-catenin/androgen receptor loop is perturbed during HBx-induced hepatocellular transformation and tumourigenesis. The LO2 cells were cotransfected with HBx or empty vector and shCtrl or shCCRK vector for 48 h, followed by (A) western blot, (B) colony formation and (C) soft agar assays. Whole cell lysates (50 μg) were analysed by western blot using the indicated antibodies. β-Actin, total GSK-3β and β-catenin were used as internal controls. The colonies formed were counted and are presented as mean+SD of three experiments. (D) Tumour incidence of 3-month-old wild-type (WT) mice, 3-, 10- and 15–18-month-old HBx transgenic (TG) mice was determined. Gene expressions in liver tissues of WT and HBx TG mice at the indicated age were examined by (E) western blot and (F) quantitative reverse transcription-PCR. The relative transcript levels were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each point represents one tissue and the line indicates mean expression level of each group. M, month; NL, normal liver; NT, non-tumour; T, tumour. ** p<0.01, *** p<0.001.

To investigate whether the regulatory loop is perturbed in vivo, we used an HBx TG model21 and found that 50% of the male mice developed HCC at age 15–18 months (figure 5D). While only basal levels of CCRK, p-GSK3βSer9, active β-catenin, AR and p-ARSer81 were seen in the normal livers of 3-, 10- and 15-month-old WT mice, HBx TG mice showed aberrant expressions which increased progressively with age (figure 5E; see online supplementary figure S2A). In 18-month-old TG mice, protein expressions were highly elevated, but the highest expressions were seen in the HCC tissues of the tumour-bearing mice (figure 5E; see online supplementary figure S2A). Concomitantly, the transcript levels of Ar, Ccrk, Ccnd1 and Egfr were low in WT mice but increased along with the age of TG mice and peaked when HCCs were formed (p<0.001; figure 5F; see online supplementary figure S2B). These in vivo data show that activation of the CCRK/GSK-3β/β-catenin/AR regulatory loop occurs in early hepatocarcinogenesis. Taken together, these findings suggest that HBx augments a molecular circuitry to induce hepatocellular proliferation and tumourigenicity.

Perturbation of the CCRK/GSK-3β/β-catenin/AR regulatory loop by CCRK knockdown prevents HCC development

To investigate whether perturbation of the regulatory loop affects HCC development, we tested whether lentiviral-mediated silencing of CCRK can suppress tumour growth in HBx TG mice exposed to low-dose DEN (figure 6A). The HBx TG mice exhibited significantly higher expression of the CCRK-AR loop (p<0.01; see online supplementary figure 3A) and developed significantly larger tumours (p<0.05; see online supplementary figure 3B) than WT mice treated with the same amount of DEN, thus demonstrating increased tumourigenicity by HBx in this HCC model. Downregulation of CCRK in HBx TG mice suppressed >80% tumour number (p<0.01; figure 6B), >90% tumour size (p<0.05) and >85% tumour weight (p<0.01; figure 6C) through significant reduction in hepatocellular proliferation (p<0.05; figure 6D). The reduced tumourigenicity was accompanied by inhibition of the CCRK/GSK-3β/β-catenin/AR signalling (figure 6E) and concomitant reduction in Ar, Ccrk, Ccnd1 and Egfr gene expressions (p<0.001; figure 6F). Notably, CCRK knockdown markedly suppressed HBx expression (figure 6E), which in turn significantly correlated with the reduced expression of the CCRK-AR loop (p<0.001; see online supplementary figure S3C). Taken together, these in vivo data support the proposal that activation of the CCRK/GSK-3β/β-catenin/AR regulatory loop is essential for HBx-dependent hepatocarcinogenesis.

Figure 6

Suppression of the cell cycle-related kinase (CCRK)/GSK-3β/β-catenin/androgen receptor loop prevents hepatocellular carcinoma development in mice. (A) Timeline of the intervention study by CCRK knockdown. The HBx transgenic mice were injected with diethylnitrosamine (DEN) at 14-days-old, followed by tail vein injection of lentivirus expressing short-hairpin RNA against control sequence (shCtrl) or CCRK (shCCRK; n=9 per group) at weeks 6 and 18. All mice were killed at 32 weeks. (B) The numbers of tumours per mouse in shCtrl-treated and shCCRK-treated groups were determined. Representative images of livers in both groups are shown. (C) The average tumour volume and weight were determined. Data are presented as mean+SD. (D) The average number of proliferative cells per high-power field (HPF) was detected by Ki67 immunostaining. Data are presented as mean+SD. Representative immunohistochemical images are shown at ×200 magnification. Gene expressions in liver tissues of shCtrl- and shCCRK-treated mice were examined by (E) western blot and (F) quantitative reverse transcription-PCR. Whole cell lysates (50 μg) were analysed by western blot using the indicated antibodies. β-Actin, total GSK-3β and β-catenin were used as internal controls. The relative transcript levels were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each point represents one tissue and the line indicates the mean expression level of each group. *p<0.05, **p<0.01, ***p<0.001.

The CCRK/GSK-3β/β-catenin/AR regulatory loop is activated in human HBV-associated HCC tissues

To investigate the clinical relevance of our findings, the protein levels of CCRK, p-GSK3βSer9, active β-catenin, AR and p-ARSer81 in 34 paired HBV-associated HCC liver tissues were examined and compared with those of five normal liver tissues. We found that the proteins were highly upregulated in all HBx-expressing HCC tissues, starting from the precancerous liver tissues adjacent to the HCCs (figure 7A). Intriguingly, when the results were stratified by gender, we found that protein expressions in both precancerous liver (p<0.01) and HCC tissues (p<0.001) were even higher in male patients than in those of the female counterparts (figure 7B).

Figure 7

The cell cycle-related kinase (CCRK)/GSK-3β/β-catenin/androgen receptor loop is perturbed during human hepatocarcinogenesis. (A) Whole cell lysates (50 μg) of human tissues were analysed by western blot using the indicated antibodies. β-Actin, total GSK-3β and β-catenin were used as internal controls. Representative blots from normal liver (NL), adjacent non-tumour (NT) and tumour (T) tissues of both sexes are shown. (B) Relative protein expression levels of 25 male and nine female paired hepatocellular carcinoma and NT tissues as well as five NL tissues are represented as box plots; lines within boxes and whiskers denote the median and 95% CI, respectively. *p<0.05; **p<0.01; ***p<0.001.

Hyperactivation of the regulatory loop correlates with poor prognosis of patients

Finally, we investigated whether the activity of the CCRK/GSK-3β/β-catenin/AR loop correlates with HCC prognosis. The activity was determined by the averaged fold-change of CCRK, p-GSK3βSer9, active β-catenin and p-ARSer81 expressions in tumour versus non-tumour tissues (online supplementary table S2). Strikingly, Kaplan–Meier analysis showed that patients with HBV-associated HCC with high activity of the regulatory loop were significantly associated with shorter overall (HR=31.26, p<0.0001; figure 8A) and disease-free survival rates (HR=6.30, p<0.01; figure 8B). Moreover, hyperactivation was significantly associated with poor tumour differentiation (p=0.002) and reduced serum albumin level (p=0.031; online supplementary table S3).

Figure 8

Hyperactivation of the cell cycle-related kinase (CCRK)/GSK-3β/β-catenin/androgen receptor (AR) loop correlates with short survival of patients with HBV-associated hepatocellular carcinoma (HCC). Kaplan–Meier analyses showed that high activity of the CCRK/GSK-3β/β-catenin/AR loop was significantly associated with poorer (A) overall and (B) disease-free survivals in patients with HCC. Patients with the averaged tumour versus non-tumour fold-changes of CCRK, p-GSK3βSer9, active β-catenin and p-ARSer81 expressions ranked in the upper (n=11) and middle/lower tertiles (n=23) were categorised as having high and low activity of the regulatory loop, respectively. (C) Schematic representation of the molecular circuitry in HBV-associated hepatocarcinogenesis. TCF, T cell factor.

Discussion

Although an intriguing interaction between HBx and the androgen pathway provides a plausible explanation for the male predominance of HBV-associated HCC,3 its regulation and mode of action remain poorly understood. Our data disclose a new molecular circuitry that involves not only HBx and AR, but also CCRK, GSK-3β, β-catenin and TCF in the regulation of hepatocellular proliferation and transformation (figure 8C). The first component of this circuit is the reciprocal regulation of HBx and CCRK expression through transcriptional control by AR. The second component of the circuit links both HBx and CCRK expression to AR activation via concomitant regulation of the GSK-3β/β-catenin/TCF signalling. We demonstrate for the first time that CCRK mediates the HBx-AR signalling to form a viral-host oncogenic circuitry in liver tumourigenicity. Targeting inhibition of CCRK perturbs the CCRK/GSK-3β/β-catenin/TCF/AR regulatory loop to suppress hepatocarcinogenesis in an HBx TG HCC model. Clinically, hyperactivation of this new regulatory loop in HBV-associated HCC tissues correlates with poor prognosis of patients. Thus, this study highlights the key role of CCRK as a hitherto unidentified regulator and enhancer of AR oncogenic activity in HBV-associated hepatocarcinogenesis.

Results from both in vitro and in vivo studies show that the HBx oncoprotein augments a CCRK/GSK-3β/β-catenin/AR regulatory loop. In addition to the multilayered genetic and epigenetic regulation of Wnt signalling in HCC,25 ,26 this viral-host circuitry further stimulates the β-catenin signals through a phosphorylation and transcriptional relay, resulting in overexpression of pro-proliferative targets such as CCND1 and EGFR. Importantly, the components that constitute the molecular circuitry also have many other mitogenic targets that undoubtedly contribute to sustained proliferation and survival of hepatocytes. Activation of this regulatory loop precedes tumour formation in HBx TG mice. Expression analysis of human precancerous liver and HCC tissues also supports its possible involvement in the early carcinogenic process. Taken together, our findings suggest that HBx participates in a signalling network composed of protein kinases and transcriptional regulators that drives the initiation and maintenance of the hepatocyte-transformed phenotype.

The critical role of AR signalling in male hepatocarcinogenesis has been established using liver-specific knockout mouse models.5 ,6 Our findings consolidate our previous observation that a self-reinforcing circuitry, not simply a unidirectional pathway, is essential for AR-induced hepatocellular transformation and tumourigenesis.27 Of note, we found that the expression levels of the CCRK/GSK-3β/β-catenin/AR loop in both tumour and adjacent liver tissues of male patients are significantly higher than those of the female patients. These new findings extend our previous observation that AR, CCRK and β-catenin are concordantly overexpressed in human HCC tissues.18 The proposal of a gender-difference expression pattern is in line with a recent observation of an AR-regulated oncomir, microRNA-216a, which is preferentially increased in the precancerous liver tissues of male patients with HCC.28 The analogous male-predominant elevation patterns indicate that cooperation among transcriptional regulators and non-transcriptional elements (microRNAs) orchestrates gene expression programme in male hepatocarcinogenesis.

By means of kinase-defective CCRK and uninhibitable GSK-3β mutants, this study emphasises that GSK-3β phosphorylation by CCRK is a prerequisite for β-catenin and AR activation in the molecular circuitry. A previous study has shown that HBx inhibits GSK-3β activity to enhance AR dimerisation, thus increasing AR transcriptional activity.13 ,14 Decreased activity of GSK-3β has also been shown to contribute to HCC development upon exposure to environmental carcinogen,29 further emphasising the important role of the CCRK/GSK-3β kinase cascade in hepatocarcinogenesis.

Despite the improved survival of patients with HCC receiving the multikinase inhibitor sorafenib,30 the treatment outcomes are still far from satisfactory. Moreover, patients with HBV-associated HCC may receive less clinical benefit from sorafenib treatment than patients with HCV-associated HCC.31 These findings that lentiviral-mediated knockdown of CCRK prevents HBx-dependent hepatocellular carcinogenesis via suppression of the cancer-driving β-catenin and AR pathways,32 ,33 pinpoint CCRK as a promising candidate for kinase-targeted treatment of HCC.

The relatively small number of HBV-associated HCC specimens used in this study may limit the interpretation of the clinicopathological data. Specifically, this might have precluded a significant relationship between the activity of the regulatory loop and tumour recurrence (p=0.060). Nevertheless, the striking correlations with worse overall and disease-free survivals of patients have emphasised the clinical significance of this circuitry. While a larger cohort will be required to validate its prognostic power, detailed characterisation of this intricate viral-host signalling network may provide new preventive and therapeutic targets for treatment of HBV-associated HCC. Although our data highlight the critical role of AR signalling in the positive feedback regulation between HBx and CCRK, the possibility that HBx directly activates CCRK cannot be excluded. Moreover, as AR is probably not the sole mechanism, we are now investigating the role of other transcription factors in CCRK upregulation. Further studies are also warranted to determine whether this tumour-initiating circuitry hardwired in men contributes to liver cancers arising from other causes.

References

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Footnotes

  • Correction notice This article has been corrected since it was published Online First. Study supervision in the Contributors section has been updated to read: study supervision: HLYC, ASLC.

  • Contributors Study concept and design: JJYS, HLYC, ASLC; acquisition of data: ZY, HF, Y-YL, MSL, YT, MYYG; analysis and interpretation of data: ZY, HLYC, ASLC; drafting of the manuscript: ZY, ASLC; critical revision of the manuscript for important intellectual content: Y-QG, PBSL, JY, VWSW, HLYC; statistical analysis: ZY, VWSW, ASLC; obtained funding: Y-QG, HLYC, ASLC; administrative, technical, or material support: D-YY, Y-SC, PBSL; study supervision: HLYC, ASLC.

  • Funding Research fund for the control of infectious diseases (12110532); general research fund (CUHK462710); National Natural Science Foundation of China (373492); focused investments scheme—scheme B (1907301) and research fellowship scheme of CUHK.

  • Competing interests None.

  • Ethics approval Joint CUHK-NTEC clinical research ethics committee.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement The authors would like to share the reagents used in this article—for example, plasmids, with the academic community. Send requests by email.

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