HBx induces hepatocellular carcinogenesis through ARRB1-mediated autophagy to drive the G1/S cycle

Abstract

The hepatitis B virus X protein (HBx) is involved in the process of hepatocellular carcinoma via the activation of various oncogenes. Our previous study indicated that ARBB1 (arrestin beta 1) promotes hepatocellular carcinogenesis (HCC). However, the role of ARRB1 in HBx-related HCC remains unclear. Herein, we identified that ARRB1 was upregulated by HBx in vivo and in vitro. Arrb1 deficiency suppressed HBx-induced hepatocellular carcinogenesis in several mouse models. Furthermore, knockdown of ARRB1 blocked HBx-induced macroautophagic/autophagic flux and disrupted the formation of autophagosomes. ARRB1 interacted with HBx, and the autophagic core protein MAP1LC3/LC3, a scaffolding protein, was essential for complete autophagy. Inhibition of autophagy by 3-methyladenine or interference of ATG5 or ATG7 attenuated HBx-induced cell cycle acceleration and the subsequent proliferative response via the induction of G₁/S arrest. The absence of autophagy abolished the phosphorylation of CDK2 and the activity of the CDK2-CCNE1 complex. Our results demonstrate that ARRB1 plays a critical role in HBV-related HCC via modulating autophagy and the CDKN1B-CDK2-CCNE1-E2F1 axis and indicate that ARRB1 may be a potential therapeutic target for HCC.Abbreviations: ARRB1: arrestin beta 1; ACTB: actin beta; AMPK: adenosine monophosphate (AMP)-activated protein kinase; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf A1: bafilomycin A₁; CDK2: cyclin dependent kinase 2; CDKN1B/p27Kip1: cyclin dependent kinase inhibitor 1B; CQ: chloroquine; E2F1: E2F transcription factor 1; FBS: fetal bovine serum; GPCRs: G protein-coupled receptors; GST: glutathione S-transferase; HCC: hepatocellular carcinoma; HBV: hepatitis B virus; HBx: hepatitis B virus X protein; HMGB1: high mobility group box 1; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; IHC: immunohistochemistry; JAK1: Janus kinase 1; LOX: lysyl oxidase; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MAPK: mitogen-activated protein kinase; 3-MA: 3-methyladenine; NFKB/NF-κB: nuclear factor kappa B; PIK3CA: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PHHs: primary human hepatocytes; RB1: RB transcriptional corepressor 1; SQSTM1/p62: sequestosome 1; STAT: signal transducer and activator of transcription; TACR1/NK1R: tachykinin receptor 1.

Keywords: ARRB1; autophagy; cell cycle; hepatitis B virus X protein; hepatocellular carcinoma.

Figure 1.

ARRB1 is involved in HBV-related hepatocellular carcinogenesis in patients. (A) Representative images of H&E staining and HBx and ARRB1 staining in human normal liver tissues, chronic hepatitis tissues, liver cirrhosis tissues, HCC and paracancerous tissues. Scale bar: 100 μm. (B) H-score of HBx staining in indicated tissues. (C) H-score of ARRB1 staining in indicated tissues. (D) ARRB1 mRNA levels in human normal liver tissues and HCC and paracancerous tissues. (E and F) HBx and ARRB1 protein expression in human normal liver tissues and HCC and paracancerous tissues were determined by western blotting. ACTB was used as the loading control. n = 10 in each group. (G) The correlation between HBx and ARRB1 H-score in HCC and paracancerous tissues, respectively, and the total correlation in HCC and paracancerous tissues. (H) The correlation between HBx and ARRB1 mRNA in HCC and paracancerous tissues, respectively, and the total correlation in HCC and paracancerous tissues. The Pearson correlation coefficient was used to evaluate the correlation. One-way ANOVA was used to analyze the rest of data. All values are the mean ± SD

Figure 2.

HBx induces ARRB1 expression in vitro and in vivo. (A) Representative H&E staining and HBx and ARRB1 staining of liver tissues from WT and HBx-TG mice at 4 months and 18 months. T, tumor. P, paracancerous tissue. Scale bar: 100 μm. (B and C) ARRB1 protein expression was determined by western blot analysis of liver tissues of 4- and 18-month-old mice. n = 6. Quantification from 3 independent experiments shown on the right represents the relative ARRB1 levels normalized to ACTB. (D) Relative mRNA levels of ARRB1 in LO2, HepG2, Hep3B and HepG2.2.15 cell lines. (E) Western blot analysis of ARRB1 protein levels in the indicated cells. (F) Relative mRNA levels of ARRB1 in LO2 and HepG2 cells transfected with the HA-HBx plasmid or empty vector. (G) Western blot analysis of HBx and ARRB1 proteins in cells that were transiently transfected with the HA-HBx plasmid for 48 h. (H) Western blot analysis of HBx and ARRB1 proteins in cells that were transiently transfected with the GFP-ARRB1 plasmid for 48 h. (I) LO2 cells were transiently transfected with the HA-HBx plasmid for 48 h. The cellular lysates were subjected to immunoprecipitation with anti-HBx antibody. Co-immunoprecipitated endogenous ARRB1 was detected with an anti-ARRB1 antibody as indicated. (J) HA-HBx plasmid or empty vector was co-transfected with the ARRB1 promoter construct in the luciferase activity assays. Luciferase activities relative to the Renilla control were measured after transfection. Statistical analysis was performed with Student’s t-test (two groups) or one-way ANOVA (more than two groups). All Data are mean ± SD of three experiments

Figure 3.

Arrb1 deficiency restrains spontaneous and low-dose DEN-induced hepatocellular carcinogenesis in HBx-TG mice. (A) MRI images and liver photographs of HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 4 months and 18 months. Scale bars: 5 mm. (B) Quantification of average liver weight as a percentage of body weight. Quantification of the average numbers of liver tumors in HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 18 months. Quantification of the maximal tumor size (diameter) in HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 18 months. Quantification of the average tumor size (diameter) in HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 18 months. n = 15 in each group. (C) Representative H&E staining of liver tissues from HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 4 months and 18 months. T, tumor. P, paracancerous tissue. Scale bars: 100 μm. (D) Representative MKI67 staining of liver tissues from HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 4 months and 18 months and MKI67 index score (n = 6 in each group). Scale bars: 100 μm. WT and arrb1^−/- mice were treated with a single intraperitoneal injection of DEN (15 mg/kg). HBx^+/+ Arrb1^+/+and HBx^+/+ arrb1^−/- mice were treated with a single intraperitoneal injection of DEN (5 mg/kg). All livers were harvested at 9 months. (E) Photographs of DEN-induced liver tumors at 9 months. Scale bars: 5 mm. (F) Representative H&E staining of DEN-induced liver tumors from WT and arrb1^−/- mice. Scale bars: 100 μm. (G) MKI67 staining in DEN-induced liver tumors from WT and arrb1^−/- mice at 9 months and the MKI67 index score (n = 6 in each group). Scale bars: 100 μm. (H) Photographs of low-dose DEN-induced liver tumors at 9 months. Scale bars: 5 mm. (I) Representative H&E staining of low-dose DEN-induced liver tumors from HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice. T, tumor. P, paracancerous tissue. Scale bars: 100 μm. (J) MKI67 staining in DEN-induced liver tumors from HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 9 months and the MKI67 index score (n = 6 in each group). Scale bars: 100 μm. (K) Quantification of the average liver weight as a percentage of body weight. Quantification of the average numbers of liver tumors in HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 9 months. Quantification of the maximal tumor size (diameter) in HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 9 months. Quantification of the average tumor size (diameter) in HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 9 months. n = 10 in each group. All values are the mean ± SD. P < 0.05 using Student’s t-test

Figure 4.

HBx enhances autophagy in hepatocellular carcinogenesis. (A) Representative images and quantification of HBx, BECN1 and SQSTM1 staining in human normal liver tissues, HCC and paracancerous tissues. Scale bar, 100 μm. (B) BECN1, LC3B and SQSTM1 protein expression in human normal liver tissues, HCC and paracancerous tissues were determined by western blotting assay. Quantification of proteins was shown in the right graph. n = 6 in each group. (C) Double immunofluorescence staining for HBx and BECN1 were performed in HCC. Nuclei was stained with DAPI in blue. Scale bar: 100 μm. (D) Representative images and quantification of BECN1 and SQSTM1 staining in liver tissues from WT and HBx-TG mice at 4 months and 18 months. scale bar: 100 μm. (E) HBx, BECN1, LC3B and SQSTM1 protein expression in the WT and HBx-TG mice liver tissues at 4 months and 18 months were determined by western blotting assay. Quantification of proteins was shown in the right graph. n = 6 in each group. (F) Autophagosomes and autolysosomes (yellow arrows indicated) were detected in the WT and HBx-TG mice liver tissues at 18 months by electron microscopy. Scale bar: 500 nm. (G) Autophagosomes and autolysosomes (yellow arrows indicated) were detected in the HA-HBx plasmid or empty vector transfected LO2 and HepG2 cells. Scale bar: 500 nm. (H) LC3 immunofluorescent staining in HA-HBx plasmid or empty vector transfected LO2 and HepG2 cells. Scale bar: 10 μm. LC3B puncta were quantified as described in Materials and Methods. (I) Western blotting analysis of HBx, BECN1, LC3B and SQSTM1 protein expression in LO2 and HepG2 cells transfected with the HA-HBx plasmid or empty vector. (J) LO2 and HepG2 cells were transfected with the HA-HBx plasmid or empty vector with or without chloroquine (CQ, 10 μM 1 h). LC3B protein was determined by Western blotting. Quantification of LC3B was shown in the graph below. All quantitative data are the mean ± SD and were obtained from 3 independent experiments. Statistical analysis was performed with Student’s t-test (two groups) or one-way ANOVA (more than two groups)

Figure 5.

Deletion of ARRB1 inhibits HBx-induced autophagosome formation. (A) LO2 and HepG2 cells with stable HBx expression were infected with stubRFP-sensGFP-LC3 lentivirus, followed by siARRB1 or siNC for 48 h. Representative confocal microscopy images of LC3 fluorescence intensity in cells. Nuclei are highlighted with DAPI. Scale bar: 20 μm. (B) Quantification of LC3 puncta in each field. (C) ARRB1, BECN1, LC3B and SQSTM1 protein expression in cells was determined by western blotting. (D) Relative expression levels of the indicated proteins in different groups. (E) Representative images of BECN1 and SQSTM1 staining of liver tissues from HBx^+/+ Arrb1^+/+and HBx^+/+ arrb1^−/- mice at 4 months and 18 months. Scale bar: 100 μm. (F) The index of indicated positive cells was determined by counting 1000 cells/sample. n = 6 in each group. (G) Autophagosomes and autolysosomes were detected in liver tissues from HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 18 months by electron microscopy. Scale bar: 500 nm. Quantification of autophagosomes was shown in the right graph. n = 6 in each group. (H) Western blot analysis and quantification of BECN1, LC3B and SQSTM1 protein expression in liver tissues from HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 18 months. n = 6 in each group. All data are the mean ± SD. Statistical analysis was performed with Student’s t test (two groups) or one-way ANOVA (more than two groups)

Figure 6.

ARRB1 recruits HBx and LC3B to form a complex, resulting in the induction of autophagy. (A) Double immunofluorescent staining of ARRB1 and LC3B in stable HBx-expressing HepG2 cells. DAPI was used to stain nuclei. Scale bar: 10 μm. (B) Analysis of the interactions among LC3B, ARRB1, and HBx in HBx-expressing HepG2 cells by immunoprecipitation. At 48 h after transfection with the HA-HBx plasmid, cell lysates were immunoprecipitated with anti-ARRB1 anti-HA and anti-LC3B antibodies, and proteins were detected by immunoblot assay. IP, immunoprecipitation. (C) SiARRB1 was transfected into stable HBx-expressing cells for 48 h. Analysis of the interaction between HBx and LC3B by immunoprecipitation with anti-HA antibody. (D) The HA-ARRB1 construct was transfected into stable HBx-expressing cells for 48 h. Analysis of the interaction between HBx and LC3B by immunoprecipitation with anti-HA antibody. (E) Recombinant GST or GST-ARRB1 immobilized on glutathione resin was incubated with purified recombinant LC3B. Analysis of a direct interaction between ARRB1 and LC3B in vitro by using a GST affinity isolation assay. (F) Recombinant GST or GST-ARRB1 immobilized on glutathione resin was incubated with purified recombinant HBx. Analysis of a direct interaction between ARRB1 and HBx in vitro by using a GST affinity isolation assay. (G) Recombinant GST or GST-HBx immobilized on glutathione resin was incubated with purified recombinant LC3B. Analysis of a direct interaction between HBx and LC3B in vitro by using a GST affinity isolation assay

Figure 7.

Inhibition of autophagy triggers G₁/S arrest in HBx-induced proliferation. (A) The HA-HBx construct or empty vector was transfected into LO2 or HepG2 cells with or without 3-MA (5 mM) for 24 h. Cell proliferation was examined by EdU incorporation assay after treatment. Scale bar: 100 μm. (B) Quantification of positive cells in the EdU assay. The percentage of positive cells was determined by counting 1000 cells/sample. (C) Western blot analysis of HBx and PCNA protein expression in LO2 or HepG2 cells with the above treatments. (D) Quantification of the relative PCNA protein expression in LO2 or HepG2 cells. (E) Synchronized HBx-expressing LO2 and HepG2 cells were stimulated with 10% FBS with or without 3-MA (5 mM) for 24 h, followed by PI staining and flow cytometry. Cell cycle distribution among different groups. (F) mRNA levels of CCNE1 and CDK2 in different groups were detected by real-time PCR. (G) Western blot analysis of HBx, LC3B, SQSTM1, CCND1, CCNE1 and CDK2 protein expression in LO2 or HepG2 cells with the above treatments. All values are the mean ± SD. Statistical analysis was performed with one-way ANOVA

Figure 8.

Deletion of ARRB1 promotes G₁/S arrest by downregulating autophagy in HBx-induced proliferation. (A) ARRB1 was downregulated by siRNA in LO2 and HepG2 cells infected with HA-HBx lentivirus. Cell proliferation was examined by EdU incorporation assay. Scale bar: 100 μm. (B) ARRB1 was downregulated by shRNA lentivirus in stable HBx-expressing LO2 and HepG2 cells. Cell growth curve after treatment by CCK-8 assay. (C) LO2 and HepG2 cells with the above treatment were stained with PI and examined by flow cytometry and cell cycle distribution among different groups. (D) Western blot analysis of HBx, CCNE1 and CDK2 protein expression in LO2 or HepG2 cells with the above treatments. (E) Representative images and quantification of CCNE1, CDK2 and MKI67 staining in liver tissues from 18-month-old HBx^+/+ Arrb1^+/+and HBx^+/+ arrb1^−/-mice. Black triangles indicated CDK2 strong positive cells. Scale bar: 100 μm. n = 6 in each group. (F) Western blot analysis of HBx, CCNE1 and CDK2 protein expression in HBx^+/+ Arrb1^+/+ and HBx^+/+ arrb1^−/- mice at 4 months and 18 months. Densitometry of the proteins was shown in the graph below. n = 6 in each group. (G and H) Stable ARRB1-expressing Hep3B and HepG2.2.15 cells were deprived of serum for 48 h and then stimulated with 10% FBS for 24 h or deprived of serum for 24 h, followed by PI staining and flow cytometry. Cell cycle distribution among the different groups. (I and J) Western blot analysis of CCNE1, CDK2, and PCNA protein expression in stable ARRB1-expressing Hep3B and HepG2.2.15 cells with the above treatment. All values are the mean ± SD. Statistical analysis was performed with Student’s t-test (two groups) or one-way ANOVA (more than two groups)

Figure 9.

HBx promotes ARRB1-mediated autophagy to drive G₁/S in a manner that is dependent on the CDKN1B-CDK2-CCNE1-E2F1 axis. (A) Western blot analysis of HBx, CDKN1B, CCNE1, p-CDK2, CDK2, p-RB1, E2F1, and PCNA protein expression in stable HBx-expressing LO2 and HepG2 cells treated with 3-MA (5 mM, 24 h). (B) Western blot analysis of ARRB1, CDKN1B, CCNE1, p-CDK2, CDK2, p-RB1, E2F1, and PCNA protein expression in stable ARRB1-expressing Hep3B and HepG2.2.15 cells treated with 3-MA (5 mM, 24 h). (C and D) Analysis of the interactions between CCNE1 and CDK2 in stable HBx-expressing LO2 and HepG2 cells with the above 3-MA treatment by immunoprecipitation. (E and F) Analysis of the interactions between CCNE1 and CDK2 in stable ARRB1-expressing HepG2 and HepG2.2.15 cells with the above 3-MA treatment by immunoprecipitation

Figure 10.

Proposed mechanism by which ARRB1-mediated autophagy drives the G₁/S cycle in HBx-induced HCC. HBx enhances the expression level of ARRB1 in HCC. HBx facilitates autophagy and promotes LC3B esterification to form autophagosomes via interaction with ARRB1. HBx-induced autophagy expedites CDKN1B degradation. Inhibition of ARRB1-mediated autophagy suppresses CDK2-CCNE1 complex activity and then blocks the G₁/S cycle in HBx-induced cell proliferation