The Hepatitis B Virus Pre-Core Protein p22 Activates Wnt Signaling - PubMed (original) (raw)
. 2020 May 31;12(6):1435.
doi: 10.3390/cancers12061435.
Dustin James Flanagan 1 2, Gregor Ebert 3 4, Nadia Warner 5, Hoanh Tran 3 4, Theodora Fifis 6, Georgios Kastrappis 6, Christopher Christophi 6, Marc Pellegrini 3 4, Joseph Torresi 7, Toby James Phesse 1 8, Elizabeth Vincan 1 5 9
Affiliations
- PMID: 32486480
- PMCID: PMC7352296
- DOI: 10.3390/cancers12061435
The Hepatitis B Virus Pre-Core Protein p22 Activates Wnt Signaling
Bang Manh Tran et al. Cancers (Basel). 2020.
Abstract
An emerging theme for Wnt-addicted cancers is that the pathway is regulated at multiple steps via various mechanisms. Infection with hepatitis B virus (HBV) is a major risk factor for liver cancer, as is deregulated Wnt signaling, however, the interaction between these two causes is poorly understood. To investigate this interaction, we screened the effect of the various HBV proteins for their effect on Wnt/β-catenin signaling and identified the pre-core protein p22 as a novel and potent activator of TCF/β-catenin transcription. The effect of p22 on TCF/β-catenin transcription was dose dependent and inhibited by dominant-negative TCF4. HBV p22 activated synthetic and native Wnt target gene promoter reporters, and TCF/β-catenin target gene expression in vivo. Importantly, HBV p22 activated Wnt signaling on its own and in addition to Wnt or β-catenin induced Wnt signaling. Furthermore, HBV p22 elevated TCF/β-catenin transcription above constitutive activation in colon cancer cells due to mutations in downstream genes of the Wnt pathway, namely APC and CTNNB1. Collectively, our data identifies a previously unappreciated role for the HBV pre-core protein p22 in elevating Wnt signaling. Understanding the molecular mechanisms of p22 activity will provide insight into how Wnt signaling is fine-tuned in cancer.
Keywords: HBV; TCF/LEF; Wnt signaling; cancer; hepatitis B virus; liver cancer; β-catenin.
Conflict of interest statement
The authors declare no conflicts of interest.
Figures
Figure 1
Wnt signaling activation is induced by hepatitis B virus (HBV) precore protein p22. (a) Effect of various HBV proteins on TCF/β-catenin transcription activity in Huh7 cells, was determined by reporter activity (sTOPflash reporter) and is shown as fold change relative to empty vector (EV) (mean ± SEM, * p < 0.05, *** p < 0.0001 Student _t_-test, n ≥ 3 independent experiments for each data point) (b) Expression of protein from the indicated plasmids transfected in Huh7 cells was confirmed by immunoblot. Lysates prepared from Huh7 cells transfected with EV and the parental, un-transfected cells served as negative controls. Lysate from HBV core p21 transfected Huh7 cells was used as a positive control. The membrane was stained with anti-HBc antibody first, then re-probed with anti α-tubulin antibody. (c) Huh7 cells were transfected with p22 plasmid and p22 protein expression (red) and localization detected with anti-HBV core antibody and confocal microscopy (nuclei are blue). Scale bars = 20 µM.
Figure 2
HBV p22 stimulates Wnt signaling in Huh7 cells. (a) The effect of HBV p22 alone or in addition to stimulation by Wnt 3a or wildtype β-catenin (β-cat-WT) on TCF/β-catenin transcription in Huh7 cells, was determined by reporter activity (sTOPflash reporter) and is shown as fold change relative to empty vector (EV) (mean ± SEM, *** p < 0.0001 Student _t_-test, n = 8 independent experiments). (b) Huh7 cells were transfected with the indicated amounts of p22 expression plasmid. The figure shows the dose-dependent effect of HBV p22 on TCF/β-catenin transcription activity (sTOPflash reporter) (mean ± SEM, * p < 0.05, ** p < 0.001 Student _t_-test, n = 4 independent experiments). (c) Huh7 cells were transfected with the indicated amounts of p22 and 100 ng of wild-type β-catenin expression plasmids. Co-expression of 5–50 ng p22 increased TCF/β-catenin transcription activity (sTOPflash reporter) mediated by wild-type β-catenin; reporter activity decreased when 100 or 200 ng p22 was co-transfected with wild-type β-catenin (mean ± SEM, ** p < 0.001, *** p < 0.0001 Student _t_-test, n = 3 independent experiments). (d) Immunoblot analysis for the transcriptionally active form of β-catenin (β-cat-ACT) on lysates prepared from Huh7 cells co-transfected with 100 ng wild-type β-catenin, 100 ng of p22 or equivalent EV expression plasmids. The membrane was stripped and re-probed with anti-actin antibody. The bar graph shows quantitative analysis for the levels of detected active β-catenin using Image Lab software and normalized for β-actin levels (mean ± SEM, ** p < 0.001 Student _t_-test, n = 3 samples).
Figure 3
HBV p22 activates TCF/β-target gene native promoters. (a) Effect of HBV p22 on FZD7-native promoter reporter activity, with and without stimulation with Wnt3a or 100 ng wild-type β-catenin (β-cat-WT), in Huh7 cells was determined by luciferase activity (pFz7-prom reporter) and is shown as fold change relative to empty vector (EV) (mean ± SEM, ** p < 0.001, *** p < 0.0001 Student _t_-test, n = 6 independent experiments). (b) Schematic diagram of hydrodynamic tail-vein injection in mice (adapted from [31]). (c) Expression of TCF/β-target genes Fzd7 and glutamine synthase (Glul) was increased in mouse livers 20 days post HDI injection of p22. Gene expression was determined by qRT-PCR and is shown relative to empty vector (EV) (mean ± SEM, * p < 0.05 Student _t_-test, n ≥ 4 mice).
Figure 4
HBV p22 increases TCF/β-catenin signaling in the context of oncogenic activation of the Wnt pathway. (a) Effect of 100 ng p22 expression plasmid on TCF/β-catenin transcription activity (sTOPflash reporter) in HEK293T cells with no known mutation or aberrant modulation of Wnt signaling; SW480 cells with truncated, mutant APC, rendering Wnt signaling constitutively active and HCT116 cells with mutation at the N-terminus of β-catenin, making Wnt signaling constitutively active (mean ± SEM, * p < 0.05, *** p < 0.0001 Student _t_-test, n = 3, 5 and 3 experiments, respectively). Reporter activity is expressed relative to empty vector (EV). (b) HBV p22 upregulates TCF/β-catenin transcription (sTOPflash reporter) in the context of truncated APC and this upregulation is blocked by dnTCF4. SW480 cells were co-transfected with 100 ng of p22 and dnTCF4 expression plasmids and the reporter activity is expressed relative to EV (mean ± SEM, *** p < 0.0001 Student _t_-test, n = 5 experiments). (c) HBV p22 upregulates TCF/β-catenin transcription (sTOPflash reporter) in the context of mutant, oncogenic β-catenin in Huh7 cells. The effect of co-transfection of 100 ng p22 expression plasmid with 100 ng of wild-type or mutant β-catenin on TCF/β-catenin is shown relative to EV (mean ± SEM, *** p < 0.0001 _t_-test, n = 4 experiments).
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