Intracellular antibody targeting HBx suppresses invasion and metastasis in hepatitis B virus‐related hepatocarcinogenesis via protein phosphatase 2A‐B56γ‐mediated dephosphorylation of protein kinase B

Abstract Objectives Hepatitis B virus X (HBx) is closely associated with HBV‐related hepatocarcinogenesis via the inactivation of tumour suppressors. Protein phosphatase 2A (PP2A) regulatory subunit B56 gamma (B56γ), as a tumour suppressor, plays a critical role in regulating cellular phosphorylation signals via dephosphorylation of signalling proteins. However, the underlying mechanism that B56γ involved in regulating HBx‐associated hepatocarcinogenesis phenotypes and mediating anti‐HBx antibody‐mediated tumour suppression remains unknown. Materials and Methods We used bioinformatics analysis, paired HCC patient specimens, HBx transgenic (HBx‐Tg) mice, xenograft nude mice, HBV stable replication in the HepG2.2.15 cells, and anti‐HBx antibody intervention to systematically evaluate the biological function of protein kinase B (AKT) dephosphorylation through B56γ in HBx‐associated hepatocarcinogenesis. Results Bioinformatics analysis revealed that AKT, matrix metalloproteinase 2 (MMP2), and MMP9 were markedly upregulated, while cell migration and viral carcinogenesis pathways were activated in HBV‐infected liver tissues and HBV‐associated HCC tissues. Our results demonstrated that HBx‐expression promotes AKT phosphorylation (p‐AKTThr308/Ser473), mediating the migration and invasion phenotypes in vivo and in vitro. Importantly, in clinical samples, HBx and B56γ were downregulated in HBV‐associated HCC tumour tissues compared with peritumor tissues. Moreover, intervention with site‐directed mutagenesis (AKTT308A, AKTS473A) of p‐AKTThr308/Ser473 mimics dephosphorylation, genetics‐based B56γ overexpression, and intracellular anti‐HBx antibody inhibited cell growth, migration, and invasion in HBx‐expressing HCC cells. Conclusions Our results demonstrated that B56γ inhibited HBV/HBx‐dependent hepatocarcinogenesis by regulating the dephosphorylation of p‐AKTThr308/Ser473 in HCC cells. The intracellular anti‐HBx antibody and the activator of B56γ may provide a multipattern chemopreventive strategy against HBV‐related HCC.

Conclusions: Our results demonstrated that B56γ inhibited HBV/HBx-dependent hepatocarcinogenesis by regulating the dephosphorylation of p-AKT Thr308/Ser473 in HCC cells. The intracellular anti-HBx antibody and the activator of B56γ may provide a multipattern chemopreventive strategy against HBV-related HCC.

| INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common malignancies and ranks the third leading cause of cancer-related deaths worldwide. 1 Unfortunately, available HCC therapies are effective in a small minority of people. In addition to traditional surgery and chemotherapy, immunotherapy has been at the forefront of research into the personalized treatment of HCC. Other therapeutics, such as vaccines, oncolytic viruses, and monoclonal antibodies, are emerging for prolonging and improving patients' quality of life with advanced HCC. 2,3 It is well known that chronic hepatitis B virus (HBV) infection contributes to hepatocarcinogenesis and its outcomes, while chronic HBV infection is responsible for 50%-80% of HCC cases worldwide. 4-6 HBV X protein (HBx), a multifunctional protein encoded by the HBV X gene, is likely to be involved in several steps of the development and progression of hepatocarcinogenesis, including the characterized phenotype of intrahepatic metastases in HBVrelated HCC. 7,8 Therefore, there is a need for better understanding of the molecular mechanisms of how HBx mediates hepatocarcinogenesis, which will be critical for more effective interventional and therapeutic targets.
Protein phosphatase 2A (PP2A), a tumour suppressor with dephosphorylation function following activation, is assembled of three subunits (A, B, and C) in a holoenzyme. The regulatory B subunit can be grouped into four families with 16 isoforms. 9, 10 Xi et al. suggested that PP2A regulatory subunit B56 could increase HBV core protein Cterminal domain dephosphorylation and decrease nuclear HBV episomes, thereby inhibiting multiple stages of HBV replication. 11 Recently, we also found that B56γ could promote p53/p21 pathwaydependent cell cycle arrest, resulting in apoptosis of HBx-expressing hepatic cells through dephosphorylating p-p53 Thr55 , suggesting that B56γ could be a therapeutic target for HBV-related hepatic injury. 12 Nevertheless, the molecular mechanism by which serine/threonine (Ser/Thr) dephosphorylation of targeted substrates regulates HBVinduced HCC invasion and metastasis phenotype remains unclear.
While many kinases govern Ser/Thr phosphorylation, its dephosphorylation is regulated by only a few phosphatases. 13,14 Known as protein kinase B (a Ser/Thr protein kinase), AKT's phosphorylation is involved in cell proliferation, migration, and metabolism via regulating signal transduction pathways. 15 It has been reported that PP2A can negatively regulate tumour progression by regulating the dephosphorylation of AKT. Indeed, Umesalma et al. showed that reactivation of endogenous PP2A with a small-molecule activator of PP2A (SMAP) reduced p-AKT Ser473 in a novel RAB-like GTPase RABL6Adependent manner and blocked the growth of neuroendocrine tumour cells. 16 Furthermore, amphetamine can inhibit phosphorylation of p-AKT Thr308/Ser473 by increasing the activity of PP2A. 17 Recent studies have shown that B56γ is an integral component of the human T-cell leukaemia virus type-1 (HTLV-1) intasome, which plays an important role in HTLV-1 infection, suggesting that B56γ is a potential biomarker and target for antiviral therapy. 18 However, it remains unclear whether B56γ can regulate dephosphorylation of AKT to regulate HBx-associated hepatocarcinogenesis is not fully understood.
In this study, we first provided evidence that B56γ-dependent dephosphorylation of p-AKT Thr308/Ser473 participated in the HBxexpression cells migration and invasion phenotypes, while genetic activation of B56γ suppressed the HBV/HBx-associated hepatocarcinogenesis. Furthermore, we first adopted an anti-HBx cell-penetrating antibody to suppress hepatocarcinogenesis phenotypes via targeting blockade of intracellular HBx. Thus, our findings demonstrated that the B56γ/p-AKT Thr308/Ser473 signalling axis is a post-translational modification (PTM) mechanism that could potentially target for the multipattern chemoprevention and intervention in environmental hepatocarcinogenesis.

| Gene Expression Omnibus data analysis
Microarray data (GSE83148) and RNA sequencing (GSE94660) on mRNA expression were screened from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). Bioinformatics analysis was performed as described previously. 19 Briefly, volcano plots and heatmaps were drawn based on the obtained differential expression genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify pathways significantly affected by the hepatocarcinogenesis process of DEGs. Gene set enrichment analysis (GSEA) was used to identify signalling pathways that are differentially activated between HBV-positive infected HCC tumour tissues and their adjacent paired non-neoplastic liver tissues.

| Bioinformatics analysis of AKT and its phosphorylation modification
The amino acid sequences of AKT in different species were blasted through the Uniprot website (https://www.uniprot.org/) to screen the functional domains. The phosphorylation sites of AKT were predicted with PhosphoNET, NetPhos 3.1 Server, and the DISPHOS1.3 website.

| HBx transgenic (HBx-Tg) mice study
The HBx-Tg mice were constructed as mentioned in our previous study. 12 At 12-month-old, the C57BL/6 wide type (WT) and HBx-Tg mice were sacrificed, and the livers were collected to extract tissue lysates and make serial sections. The protein expression levels were detected by WB, and the colocalization and distribution of HBx and p-AKT Thr308/Ser473 or MMP2/MMP9 were analysed by immunofluorescence (IF) analysis. All animal experiments were approved by the Experimental Animal Ethics Committee of Xiamen University.

| Xenograft tumour study
BALB/c nude mice (4-6 weeks old) were obtained from the SLAC Laboratory Animal Co., Ltd. (Shanghai, China). To explore the effect of HBx expression and PPP2R5C (2R5C) genetic targeting intervention on the growth of HCC cells in xenograft tumours and tumour metastasis study, an HBV genome-integrated and highly metastatic MHCC97H cell line was employed to inoculate into BALB/c nude mice. When the xenograft tumours were allowed to grow for 10 days, the mice were randomly allocated into two groups, siNC and si2R5C group (n = 3 for each group), and then injected with 10 nmol/tumour/time siNC or si2R5C at the 1st and the 8th day. The mice were sacrificed and examined for tumour metastasis in the liver until the last injection week.
Exactly 5 Â 10 6 HBx-expressing cells or control cells were suspended in cold phosphate-buffered saline (PBS) with matrigel in a 1:1 ratio.
The transfected cells were subcutaneously inoculated into the right flank of each mouse, and the tumours were allowed to grow for 10 days. Then, mice in the siNC and HBx + siNC groups were injected with 10 nmol/tumour siNC, and mice in the si2R5C and HBx + si2R5C groups were injected with 10 nmol/tumour si2R5C twice on the 11th and the 14th day (represents the 1st and the 4th day). All mice were sacrificed by cervical dislocation on the 17th day (represents the 7th day), and the xenograft tumours were removed.
Tumour width (W), tumour length (L), and body weights were measured. Tumour volume (V) was calculated with the formula: V = (W 2 Â L)/2. Xenograft tumours and livers were collected and subjected to WB, HE, and IHC assays. All animal experiments were approved by the Experimental Animal Ethics Committee of Xiamen University.

| HE and IHC analysis
The tumour tissue sections from HCC patients and BALB/c nude mice and serial sections of liver tissue from HBx-Tg mice were fixed in 4% paraformaldehyde (pH 7.4) and embedded in paraffin. HE and IHC assays were performed as described previously. 19 Briefly, the sections were rehydrated and stained with HE after dewaxing. The IHC sections were incubated with primary antibodies (anti-HBx, anti-AKT, anti-MMP2, anti-MMP9, and anti-B56γ), and images were taken by an inverted microscope (Nikon, Tokyo, Japan).

| Targeting blockade of intracellular HBx with the recombinant anti-HBx monoclonal antibody expression plasmid
The anti-HBx monoclonal antibody (mAb) expression plasmid was constructed to block intracellular HBx expression. 20 Briefly, to construct recombinant anti-HBx mAb expression plasmid, the variable region gene of the cell clone (9D11) of anti-HBx mAb was obtained from the hybridoma by reverse-PCR using a mouse Ig primer set (Merck Millipore, Darmstadt, Germany). The coding sequences for the kappa chain and heavy chain of the anti-HBx mAb were cloned into a pTT5 vector containing the constant region of mouse IgG1 and named pTT5-anti-HBx(9D11) plasmid after identification. The pTT5-anti-HBx (9D11) plasmid was transiently transfected into different HBxexpression cells (HepG2.2.15 and HepG2-Tet-ON-HBx cells) for 24 h to establish the blockade of intracellular HBx expression models, to verify the biological functions of anti-HBx mAb specific targeting to block intracellular HBx.
2.9 | Site-directed mutagenesis of p-AKT at Thr308 and Ser473 To prove the functional regulatory dephosphorylation of p-AKT at Thr308 and Ser473, we simulated dephosphorylation (alanine mutation from serine or threonine, S or T to A) and the counterpart phosphorylation (aspartate mutation from serine or threonine, S or T to D) using recombinant expression plasmids. The specific site-directed mutation method was used to convert the base in the coding sequence (CDS) by inverse PCR with KOD-Plus Mutagenesis Kit (Toyobo, Osaka, Japan). As a CDS template, pcDNA3.1-AKT was completely amplified with two primers in the opposite direction to generate base mutation from serine/threonine to alanine (S/T to A) or aspartate (S/T to D) at Thr308, or Ser473 of AKT (named as AKT T308A , AKT S473A , AKT T308D , and AKT S473D , respectively). Then, the PCR products were self-ligated by T4 polynucleotide kinase. The primers are shown in Table S1. To construct the simulation of dephosphorylated or phosphorylated AKT expression, HepG2 cells were transfected with pcDNA-3.1, pcDNA-3.1-AKT WT , -AKT T308A , -AKT S473A , -AKT T308D , or -AKT S473D (1 μg/ml) using Lipofectamine ® 2000 (Invitrogen).

| Construction of stable B56γ-knockdown and -overexpression cell lines
The specific method was mentioned in the previous experiment. 12

| Quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed as described previously. 12 The relative expression of PPP2R5C was determined by comparing the target genes' threshold cycle (Ct) using the 2 ÀΔΔCt method. The following primers were used for detection:

| Immunofluorescence analysis
For immunofluorescence (IF) staining, serial tissue sections and cellmounted slides were permeabilized with 0.5% Triton X-100 on ice for 5 min, then blocked by 1% BSA. After incubating with the primary antibodies (anti-HBx, anti-B56γ, anti-MMP2, anti-MMP9, anti-p-AKT Thr308 , and anti-p-AKT Ser473 ) at 4 C overnight, the samples were incubated with goat anti-mouse/rabbit IgG fluorescent secondary antibody for 1 h at room temperature, and then counterstained with DAPI to visualize the cell nuclei. Finally, stained sections were visualized using a confocal microscope (Zeiss LSM 880, Wetzlar, Germany). Fluorescence curves of profile lines were generated using Zen 2010 software. It was worth noting that the sample was directly incubated with the secondary antibody containing fluorescent probes (647 nm) without first incubating the primary antibody when the cell was transfected with pTT5-anti-HBx(9D11).

| Wound healing assay
According to a previous study, cell migration was determined using the wound healing assay according to the previous study. 21 Each well was photographed using a microscope (Nikon, Tokyo, Japan) to estimate the relative cell migration. The unhealed distance was measured using Image J software.

| Transwell invasion assay
Cells were treated as described above for 24 h before being pipetted into the upper chamber of the transwell. Transwell invasion assay was performed according to the previous study. 21 We randomly chose five fields of vision to count cells under a digital camera (Canon, Tokyo, Japan). The number of invaded cells was calculated.

| Statistical analysis
Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 20.0 (SPSS, Chicago, Illinois). All data are expressed as mean ± standard deviation (SD) of at least three independent experiments. Statistical significance was determined using the Student's t-test and one-way analysis of variance (ANOVA).
Pearson's correlation analysis was performed for the correlation between variables. A P-value of <0.05 was considered statistically significant.

| Cell migration and the AKT pathway were associated with hepatocarcinogenesis in bioinformatics analysis of HBV-infected liver and HCC tissue
The GEO database (accession no.: GSE83148) of HBV-infected hepatitis samples was screened and underwent DEGs analysis to examine the gene expression landscape of HBV-related hepatocarcinogenesis.
We found 124 genes that were upregulated and 3 genes that were downregulated in the HBV-infected liver tissues (n = 122) relative to normal liver tissues (n = 6) ( Figure 1A). The levels of AKT1 (AKT isoform) and genes related to malignant cell transformation were significantly upregulated in the HBV-infected liver tissues ( Figure 1B). We used GO enrichment analysis for biological processes (BP) to identify the genes significantly affected by HBV infection to determine whether HBV infection was associated with cell migration. As a result, we found that 'response to virus', 'cell migration', 'protein kinase B signalling', and 'regulation of protein phosphorylation' terms were significantly enriched in the HBV-infected liver tissues ( Figure 1C). KEGG pathway enrichment analysis further demonstrated that 'viral carcinogenesis', 'Hepatitis B', 'cell cycle', and 'p53 signalling pathway' terms were significantly enriched in the HBV-infected liver tissues ( Figure 1D), which was consistent with the result of our previous study, which showed that HBx could regulate cell cycle arrest and apoptosis through regulating p-p53 Thr55 dephosphorylation by B56γ. 10 Moreover, in HBV-infected liver samples, the expression of AKT1, MMP2, and MMP9 were upregulated compared with that observed in normal liver samples ( Figure 1E). Upon further exploration, we identified a positive correlation between the gene expression of AKT1 and MMP2 and that of AKT1 and MMP9 ( Figure 1F).
We used another GEO database (accession no.: GSE94660) to screen and perform DEGs analysis of the relative gene expression in HBV-positive infected tumour tissues and their paired non-neoplastic liver tissues. The relative expressions of AKT1, MMP2, and MMP9 genes were higher in tumour tissues than in non-neoplastic liver tissues ( Figure 1G). Moreover, there was a positive correlation between AKT1 and MMP2 and AKT1 and MMP9 expressions in HCC samples ( Figure 1H). Furthermore, the Cancer Genome Atlas (TCGA) dataset with a higher number of HCC patients was adopted additionally. The analysis revealed that the relative expressions of AKT1, MMP2, and MMP9 genes were increased in HCC tumour tissues (n = 373) compared with the normal samples (n = 50; Figure S1A). There was a positive correlation between AKT1 and MMP2 and AKT1 and MMP9 expression in HCC samples ( Figure S1B). Moreover, the trend relationship analysis revealed that the relative expressions of AKT1 were upregulated in HCC tumour tissues compared with the normal samples ( Figure S1C). These results demonstrated that HBV infection was positively correlated with cell migration and AKT signalling, suggesting a potential regulatory mechanism of AKT signalling involved in HBxassociated hepatocarcinogenesis via regulating cell migration. bioeffect of HBx in HBV-associated apoptosis and hepatic injury. 12 We further wish to elucidate whether HBx promotes cell migration and signalling transduction of liver carcinogenesis in long-term expression HBx-Tg mice.

| Expression of HBx promoted the phosphorylation of AKT signalling to activate the migration phenotype in the livers of HBx-Tg mice
Our results demonstrated that the protein levels of phosphorylated AKT at Thr308 (p-AKT Thr308 ) and Ser473 (p-AKT Ser473 ), which are essential for AKT pathway activation, were upregulated with increasing the expression of HBx in long-term (12-month-old) HBx-Tg mice ( Figure 2A). Immunofluorescence staining of serial sections of liver tissues showed that the levels of p-AKT Thr308 and p-AKT Ser473 were increased and highly expressed in HBx-Tg mice ( Figure 2B,C).
Matrix metalloproteinases (MMPs), including MMP2 and MMP9, have been previously reported to promote cell migration and tumour metastasis. 23 Meanwhile, the levels of MMP2 and MMP9 ( Figure 2D) and their distribution in liver cells upregulated with the increased expression of HBx ( Figure 2E,F). Concerning the phenotypic markers of metastasis in hepatocarcinogenesis, the level of EMT-related protein E-cadherin was downregulated in long-term HBx-Tg mice, while vimentin was upregulated ( Figure 2D). These results indicated that HBx-expression can promote the cell migratory phenotype during hepatocarcinogenesis and that the phosphorylation of AKT and subsequent signalling activation may be involved in this process.

| Phosphorylation of AKT Thr308/Ser473 regulated the migration and invasion of HBx-expressing cells
We performed a wound-healing assay and transwell invasion assay in vitro to determine the promoting effects of HBx on the regulation of cellular migration and invasion of HCC. We found inducible higher HBx-expression and the increased levels of p-AKT Thr308 and p-AKT-Ser473 in doxycycline (DOX)-treated HepG2-Tet-ON-HBx cells in a dose-dependent manner ( Figure 3A). It has been reported that p-AKT activation is crucial in HCC progression, 24,25 and MMP2 and MMP9 are involved in tumour invasion and metastasis. 26 As we found, the protein levels of MMP2 and MMP9 were upregulated with the We constructed the recombinant expression plasmids for the mimics phosphorylation or dephosphorylation of AKT at Thr308 and Ser473 to further verify the functional dephosphorylated regulating p-AKT Thr308/Ser473 ( Figure 4A). In vitro, the wound healing assay showed that the migration ability of AKT T308A and AKT S473A expression cells was significantly decreased compared with the AKT WT group cells, while the migration ability was significantly increased in AKT T308D and AKT S473D expression cells compared with the AKT WT group, AKT T308A , and AKT S473A group cells ( Figure 4D). A similar effect was confirmed by the results of the transwell invasion assay, which demonstrated a significant decrease in HCC-associated invasion of AKT T308A and AKT S473A expression cells compared with AKT WT group cells, while the invasion ability was significantly increased in AKT T308D and AKT S473D expression cells compared with AKT T308A and AKT S473A group cells ( Figure 4E). These results demonstrated that dephosphorylation of p-AKT Thr308/Ser473 inhibited the migration and invasion of HCC cells.
Our previous study found that HBx expression in hepatocytes upregulated the expression of B56γ (encoding by the PPP2R5C gene) via endoplasmic reticulum (ER) stress to induce cell cycle arrest and apoptosis. 12 To test whether B56γ expression was correlated with HCC progression, we analysed the TCGA dataset from 50 paired HCC and their corresponding normal samples. The analysis revealed that the relative expressions of PPP2R5C were downregulated in HCC tumour tissues compared with the normal tissues ( Figure 4F). To explore the relationship between B56γ and AKT in hepatocarcinogenesis, we used GSEA to explore the significant signalling pathways regulated by PPP2R5C and AKT genes between low and high expression groups. The EMT signalling pathway was mainly focused on in the current study. It was shown that the normalized enrichment scores (NES) for the EMT signals were À1.53 and 1.66 (P < 0.05) for PPP2R5C and AKT genes, respectively, which suggested that the expression of PPP2R5C was negatively and AKT was positively correlated with EMT ( Figure 4G). Furthermore, to investigate the potential association between B56γ and HBV-related hepatocarcinogenesis, we collected 22 pairs of HBV-related HCC tumours and adjacent peritumor tissues to analyse the levels of HBV proteins, including HBsAg, hepatitis B core antigen (HBcAg), HBx, and B56γ expression. It was shown that the expression of the B56γ subunit was significantly downregulated in tumour (T) tissues compared with the corresponding peritumor (P) tissues ( Figures 4H and S3). As shown in Figure 4I

| Inhibition of B56γ promoted the migration and invasion of HBV-related HCC cells in vitro and in vivo
We used MHCC97H cells, HBV genome-integrated, and highly metastatic HCC cells, 27

| B56γ targeted the dephosphorylation of p-AKT to negatively regulate migration and invasion of HBx-expressing HCC cells
HBx-associated cell migration and invasion depend on the phosphorylation of AKT at Thr308 and Ser473 in HBx-expressing HCC cells. We constructed a B56γ-knockdown expression model using si2R5C F I G U R E 5 Legend on next page.
( Figure 7A). Interestingly, knockdown of B56γ promoted HBx-induced cell migration and invasion ( Figures 7B,C and S6A,B). To further demonstrate the impact of loss-of-function of B56γ, stable B56γknockdown HepG2-sh2R5C (sh268, sh417, and sh1416) cells were constructed and verified ( Figure 7D). Compared with HepG2-shGFP control cells, the growth rate of HepG2-sh268 and -sh1416 cells were significantly increased, and the plate colony-forming ability was increased ( Figure S6C). HepG2-sh268 cells with the fastest growth were used as the representative HepG2-sh2R5C cells for follow-up experiments ( Figure 7D). The level of B56γ was decreased, and the expressions of p-AKT Thr308 , p-AKT Ser473 , MMP2, and MMP9 were increased in B56γ-knockdown HepG2-sh2R5C cells compared with the control group ( Figure 7E). Moreover, inhibition of B56γ further enhanced p-AKT Thr308 and p-AKT Ser473 , and MMP2 and MMP9 were induced by HBx-expression ( Figure 7E). In addition, knockdown of To explore the targeted effect of B56γ on the regulation of functional p-AKT Thr308/Ser473 dephosphorylation in hepatocarcinogenesis, we further constructed stable B56γ-overexpression HepG2-2R5C cells for a targeted genetic intervention study ( Figure 7H). Compared with the HepG2-pBabe control cells, the growth rate of HepG2-2R5C cells was significantly decreased (Figure 7H), while the plate colonyforming ability was inhibited ( Figure S6E). Moreover, B56γ was further increased in HBx-expressing HepG2-2R5C cells and significantly inhibited the increase in p-AKT Thr308/Ser473 , MMP2, and MMP9 induced by HBx-expression ( Figure 7I). In addition, consistent with the inhibition of cell growth and plate colony formation, the migration and invasion ability of HepG2-2R5C cells were significantly reduced, while that of HBx-expressing HepG2-2R5C cells was significantly inhibited ( Figures 7J,K and S6F). These results indicated that B56γ might especially target the dephosphorylation of p-AKT Thr308 and p-AKT Ser473 to negatively regulate the activation of p-AKT signalling, while and gain-of-function of B56γ may represent a potential approach to inhibit the migratory and invasive phenotypes of HBxexpressing HCC cells. Furthermore, the targeting blockade of HBx expression as the source intervention for HBV infection-derived carcinogenesis should be further explored.

| Intracellular expression of anti-HBx mAb inhibited HBV-related hepatocarcinogenesis phenotypes via blocking HBx expression in HCC cells
A previous study has reported that intracellularly expressed anti-HBx antibody, a mAb generated by the fusion of a cell-penetrating peptide (CPP) on the C-terminus of the heavy chain of a potent mAb specific targeting to HBx, can promote the degradation of intracellular HBx. 20 In the current study, this anti-HBx mAb plasmid (pTT5-anti-HBx) was

| DISCUSSION
Hepatitis viruses are associated with diseases that cause liver injury, especially hepatocarcinogenesis. 28,29 HBV infection leads to chronic Many studies have shown that B56γ is an antitumour protein, whereas HBx is a pro-tumour protein. [31][32][33] This study focused on the role of B56γ and HBx expression in the migration and invasion phenotypes during hepatocarcinogenesis and screened for the regulation of AKT phosphorylation/dephosphorylation associated with B56γ and HBx expression. Therefore, we hypothesized that the dephosphorylation mechanism of B56γ on specific sites of p-AKT Thr308/Ser473 is involved in the HBx-related hepatocarcinogenesis signalling pathways in HCC cells. A study in HBV-positive patient liver samples also demonstrated that AKT levels were elevated in HBV-related HCC. 34 It has been reported that regulating PTM, especially the balance of phosphorylation and dephosphorylation, is involved in cellular activities.
Abnormal alterations in phosphorylation and the disruption of dephosphorylated homeostasis result in diseases such as cancer. 35 In this study, we found that HBx expression promoted the migration and its related invasion in HCC cells via a mechanism related to AKT expression and its modification of functional p-AKT Thr308 and p-AKT-Ser473 status. Similar to our results, Han et al. recently reported that AMPK fully activated AKT by enhancing its phosphorylation at Thr308 and Ser473 and, as a result, promoted cellular migration in breast cancer MDA-MB-231 cells. 36 In addition, Liu H et al. showed that HBx promoted hepatoma cell invasion and metastasis by stabilizing Snail protein to enhance the expression of p-AKT Ser473 by activating the PI3K/AKT/GSK-3β signal pathway in Huh7 cells. 37 In our study, to investigate the role of regulatory dephosphorylation of p-AKT Thr308/Ser473 in hepatocarcinogenesis, the HBx-Tg mice and a variety of HBV/HBx expression in vivo and in vitro experimental models have been established. Our results demonstrated that the levels of phosphorylated AKT (p-AKT) at Thr308 (p-AKT Thr308 ) and Ser473 (p-AKT Ser473 ), which are essential for AKT pathway activation, were upregulated with the increased expression of HBx in vivo and in vitro.
We further constructed the HCC cell models to mimic the dephosphorylation of AKT T308A and AKT S473A . These results showed that dephosphorylation of p-AKT Thr308/Ser473 inhibited the migration and invasion of HBx-expressing HCC cells.
Our previous study found that B56γ, a protein phosphatase to target dephosphorylation, regulated cell cycle arrest and apoptosis in HBx-expression hepatocytes. 12 Functionally, PP2A has been studied for its role in tumour suppression via tumour metastasis signalling. 31 Chen et al. reported that suppressing B56γ expression by Simian virus cell viability and cell cycle progression in Huh7 cells. 40 In this study, we provided the first evidence that B56γ specifically targeted p-AKT as a substrate to regulate the dephosphorylation of p-AKT Thr308 and p-AKT Ser473 and inhibit the MMP2/9-associated metastasis phenotype of HBx-expressing HCC cells in vitro and in vivo. So far, no specific agonist targeting the B56γ mediates PP2A-B56γ/AC holoenzyme activation, although there is a specific activator for the PP2A-B56α and PP2A-B56ε holoenzyme. Therefore, selective activation of the PP2A holoenzyme, depending on the specific targeting activator for different PP2A-B subunits, may be a potential strategy for the intervention and control of liver injuries and HBV/HBx-related hepatocarcinogenesis.
Consistent with our results, Xi et al. suggested that PP2A regulatory subunit B56 could increase HBV core protein C-terminal domain dephosphorylation and decrease nuclear HBV episomes, thereby inhibiting multiple stages of HBV replication. 11 Xiao et al. demonstrated that PP2A protein phosphatase agonist D-erythro-Sphingosine could attenuate microcystins-LR and C-terminally truncated HBx-induced the migration and invasion of HepG2 cells via regulating the dephosphorylation of PP2A/p-p38 MAPK Thr180/Tyr182 axis. 41 These studies found that although targeted activation of PP2A was found to rescue the malignant phenotype of HBx-expressing cells, the expression level of PP2A protein itself was not detected, which needs further investigation. Surprisingly, we found that the HBx increased p-AKT Thr308/ Ser473 to drive HCC and then, at the same time, upregulated B56γ in hepatocarcinogenesis. The upregulation of B56γ might be a dephosphorylation defence mechanism caused by HBX-induced cellular stress, a new question that remains to be solved.
Moreover, in the current study, we used the HBx-Tg mouse model to explore the pathogenesis of HBV infection-related HCC.
Different HBV mouse models (e.g., human-mouse liver chimeric mice) developed to study infection and pathogenesis still have certain limitations. 42,43 Therefore, combining in vitro and in vivo models of HBxexpressing cells and HBx-Tg mice in this study, we further rigorously evaluated the applicability of these mouse models to the exploration of the mechanisms by which HBx-associated hepatocarcinogenesis phenotypes, which will help provide important insights for the devel- reprogramming in HCC cells. 47 In the current study, we further used this recombinant plasmid (pTT5-anti-HBx) to verify that the production of intracellular anti-HBx mAb could block HBx expression and its induced B56γ-mediated dephosphorylation activity, which, in turn, could inhibit the HBV-associated migration and invasion of HBx-expressing HCC cells. Our results showed a feasible strategy for inhibiting the bioeffects of HBx-expression using a specific intracellular anti-HBx antibody.

| CONCLUSIONS
In conclusion, HBV/HBx induced the phosphorylation of specific AKT functional sites (Thr308 and Ser473) involved in cell migration and invasion in HBV-related hepatocarcinogenesis. PP2A-B56γ inhibited the p-AKT Thr308/Ser473 -dependent hepatocarcinogenesis via a specific dephosphorylation regulatory mechanism, while the genetic intervention of PPP2R5C function mediated the targeted modulation of p-AKT-MMP2/9-EMT-mediated HCC cell phenotypes. Intracellular anti-HBx mAb-mediated specific blockade of HBx expression and its induced activation of B56γ would help target the B56γ-p-AKT Thr308/Ser473 -MMP2/9 signalling axis to mediate the intervention in HBx-associated hepatocarcinogenesis ( Figure 9).
Altogether, our findings indicated that B56γ might be a potential therapeutic target and that anti-HBx mAb may be the multipattern chemoprevention and therapeutical strategy for HBV-related hepatocarcinogenesis.
F I G U R E 9 Schematic diagram of PP2A-B56γ mediated the dephosphorylation of p-AKT Thr308/Ser473 in HBx-expressing HCC cells to regulate the migration and invasion phenotypes of HBV/HBx-related hepatocarcinogenesis. In the current study, HBx-expression induced the phosphorylation of specific AKT sites (p-AKT Thr308/Ser473 ) involved in mediating the migration and invasion phenotypes of HCC cells. The inducible upregulation of B56γ mediated the dephosphorylation of p-AKT Thr308/Ser473 in HBx-expressing HCC cells. Specific blockade of HBxexpression via pTT5-anti-HBx plasmid-mediated targeting intracellular anti-HBx mAb production and genetic activation of B56γ would help target the p-AKT Thr308/Ser473 -MMP2/9 signalling axis to mediate the multipattern chemoprevention and intervention in HBV/HBx-related hepatocarcinogenesis.