Hepatitis B virus X Protein Promotes Liver Cancer Progression through Autophagy Induction in Response to TLR4 Stimulation

Abstract

Hepatitis B virus X (HBx) protein has been reported as a key protein regulating the pathogenesis of HBV-induced hepatocellular carcinoma (HCC). Recent evidence has shown that HBx is implicated in the activation of autophagy in hepatic cells. Nevertheless, the precise molecular and cellular mechanism by which HBx induces autophagy is still controversial. Herein, we investigated the molecular and cellular mechanism by which HBx is involved in the TRAF6-BECN1-Bcl-2 signaling for the regulation of autophagy in response to TLR4 stimulation, therefore influencing the HCC progression. HBx interacts with BECN1 (Beclin 1) and inhibits the association of the BECN1-Bcl-2 complex, which is known to prevent the assembly of the pre-autophagosomal structure. Furthermore, HBx enhances the interaction between VPS34 and TRAF6-BECN1 complex, increases the ubiquitination of BECN1, and subsequently enhances autophagy induction in response to LPS stimulation. To verify the functional role of HBx in liver cancer progression, we utilized different HCC cell lines, HepG2, SK-Hep-1, and SNU-761. HBx-expressing HepG2 cells exhibited enhanced cell migration, invasion, and cell mobility in response to LPS stimulation compared to those of control HepG2 cells. These results were consistently observed in HBx-expressed SK-Hep-1 and HBx-expressed SNU-761 cells. Taken together, our findings suggest that HBx positively regulates the induction of autophagy through the inhibition of the BECN1-Bcl-2 complex and enhancement of the TRAF6-BECN1-VPS34 complex, leading to enhance liver cancer migration and invasion.

Keywords: Autophagy; Beclin-1; Hepatitis B virus; Liver neoplasms; TNF receptor-associated factor 6.

Figure 1. HBx inhibits the interaction between BECN1 and Bcl-2 protein. (A) Mock, Myc-BECN1, and Flag-Bcl-2 plasmids were transfected into HEK-293T cells, as indicated. At 38 h post-transfection, the transfected cells were extracted and the cell lysates were subjected to IP with anti-Myc Ab followed by IB using anti-Flag or anti-Myc Ab. (B) Mock, HA-HBx, and Flag-Bcl-2 plasmids were transfected into HEK-293T cells, as indicated. IP assay was performed with anti-HA Ab. (C) Mock, Flag-BECN1, and HA-HBx plasmids were transfected into HEK-293T cells, as indicated. IP assay was performed with anti-Flag Ab. (D) Truncated mutants of BECN1, as indicated, were generated as described in Materials and Methods. (E) Mock, Flag-BECN1 WT, Flag-BECN1 1-269, Flag-BECN1 1-127, and HA-HBx plasmids were transfected into HEK-293T cells, as indicated. At 38 h post-transfection, the transfected cells were extracted and the cell lysates were subjected to IP with anti-Flag Ab, followed by IB using anti-Flag or anti-HA Ab. (F) A schematic model showing the interaction between BECN1 and HBx protein. (G) Mock, Myc-BECN1, Flag-Bcl-2, and different concentrations of Flag-HBx plasmids were transfected into HEK-293T cells, as indicated. At 38 h post-transfection, the transfected cells were extracted and the cell lysates were subjected to IP with anti-Myc Ab followed by IB using anti-Flag or anti-Myc Ab. (H) A model showing how HBx inhibits the interaction between BECN1 and Bcl-2. BECN1 interacts with Bcl-2 therefore inhibiting the autophagy induction (left). In contrast, HBx interacts with BECN1 and inhibits the association of BECN1-Bcl-2 complex, leading to the autophagy induction (right).

Figure 2. HBx interacts with Vps34-BECN1 complex. (A) Mock, Myc-BECN1, and HA-Vps34 plasmids were transfected into HEK-293T cells, as indicated. At 38 h post-transfection, the transfected cells were extracted and the cell lysates were subjected to IP with anti-Myc Ab followed by IB using anti-Myc or anti-HA Ab. (B) Mock, Flag-HBx, and HA-Vps34 plasmids were transfected into HEK-293T cells, as indicated. IP assay was performed with anti-Flag Ab. (C) Truncated mutants of Vps34, were generated as described in Materials and Methods. (D) Mock, HA-Vps34, Flag-BECN1 WT, Flag-BECN1 1-269, and Flag-BECN1 1-127 plasmids were transfected into HEK-293T cells, as indicated. IP assay was performed with anti-Flag Ab, and IB assay was performed with anti-Flag or anti-HA Ab. A schematic model for the interaction between BECN1 and Vps34 protein is shown (down). (E) Mock, Flag-BECN1, HA-Vps34 WT, HA-Vps34 1-531, and HA-Vps34 1-260 plasmids were transfected into HEK-293T cells, as indicated. IP assay was performed with anti-Flag Ab, and IB assay was performed with anti-Flag or anti-HA Ab. A schematic model for the interaction between BECN1 and Vps34 protein is shown (down). (F) Mock, Flag-HBx, HA-Vps34 WT, HA-Vps34 1-531, and HA-Vps34 1-260 plasmids were transfected into HEK-293T cells, as indicated. IP assay was performed with anti-Flag Ab, and IB assay was performed with anti-Flag or anti-HA Ab. A schematic model for the interaction between HBx and Vps34 protein is shown (down).

Figure 3. HBx enhances the ubiquitination of BECN1 and autophagy induction. (A) A schematic model showing the association among HBx, BECN1, and Vps34 protein. (B) Mock, HA-Vps34, Myc-BECN1, and different concentrations of Flag-HBx plasmids were transfected into HEK-293T cells, as indicated. At 38 h post-transfection, the transfected cells were extracted and the cell lysates were subjected to IP with anti-HA Ab followed by IB using anti-HA, anti-Flag, or anti-Myc Ab. (C) Mock, Myc-BECN1, Flag-TRAF6, HA-Ub, HA-Vps34, and different concentrations of Flag-HBx plasmids were transfected into HEK-293T cells, as indicated. At 38 h post-transfection, the transfected cells were extracted and the cell lysates were subjected to IP with anti-Myc Ab followed by IB using anti-HA or anti-Myc Ab. (D, E) Ctrl HepG2 and HBx-HepG2 cells were treated with or without LPS (5 or 10 µg/mL) in the presence or absence of CQ (10 μM) for 6 hours, as indicated. The cells were lysed and subjected to SDS-PAGE followed by immunoblotting with LC3-I/-II, HBx, or GAPDH antibodies (D). Band intensity was quantified using Image J software (E) (^*p<0.05, ^**p<0.01, ±SEM, n=3). (F) A schematic model showing how HBx facilitate the association of BECN1-Vps34 and the ubiquitination of BECN1, therefore enhancing autophagy induction.

Figure 4. HBx-HepG2 cells exhibited increased cell migration and invasion in response to TLR4 stimulation. (A) Ctrl HepG2 and HBx-HepG2 cells were extracted, and the cell lysates were subjected to WB with anti-HBx or anti-GAPDH Ab. (B, C) Ctrl HepG2 and HBx-HepG2 cells were suspended in culture medium including vehicle, LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 μg/mL), and CQ (10 μM) plus LPS (10 μg/mL), and the invasive assay was performed as described in the Materials and Methods. Fixed cells were stained with crystal violet (B). The number of migrated cells was counted, and presented as the mean±SEM (C) (^*p<0.05 and ^**p<0.01). (D, E) Ctrl HepG2 and HBx-HepG2 cells were seeded into 12-well cell culture plates, scraped with a sterile yellow Gilson-pipette tip, and treated with a vehicle (DMSO, <0.2% in culture medium), LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 μg/mL), and CQ (10 μM) plus LPS (10 μg/mL) for different time periods, as indicated. A representative experiment is shown (D). The residual gap between the migrating cells from the opposing wound edge was expressed as a percentage of the initial scraped area (E) (±SEM, n=3; ^*p<0.05, ^**p<0.01, and ^***p<0.001).

Figure 5. HBx-HepG2 cells exhibited increased cell mobility and colony formation in response to TLR4 stimulation. (A, B) Ctrl HepG2 and HBx-HepG2 cells were seeded into 6-well culture plate, treated with vehicle (DMSO), and 3-MA (5 mM) in the presence or absence of LPS (10 μg/mL), and time-lapse imaging analysis was performed for different times by using phase-contrast microscope (A), as described in Materials and Methods. Data analysis on the speed of cell mobility was performed following protocols provided by the Bio-protocol (B) (www.bio-protocol.org/e3586) (^*p<0.05). (C, D) Ctrl HepG2 and HBx-HepG2 cells were harvested with trypsin-EDTA and re-suspended in a singular form. The 1×10³ cells (per well) were plated in a 6-well plate and treated with a vehicle (DMSO), 3-MA (5 mM), and CQ (10 μM) in the presence or absence of LPS (10 μg/mL). After incubation for 18 days (C), colonies were stained with 0.5% crystal violet (Sigma) for 30 minutes at room temperature and counted (D) (±SEM, n=3; ^*p<0.05, ^**p<0.01, ^***p<0.001, and ^****p<0.0001). (E, F) Ctrl HepG2 and HBx-HepG2 cells (1.0×10⁴ cells per well) mixed with 0.3% Difco Noble Agar in complete medium were plated on the top of 0.5% agar layer in a 6-well plate with complete medium. Growth medium (1.5 mL) with a vehicle (DMSO), LPS (10 μg/mL), 3-MA (5 mM), or CQ (10 μM) was added on top of the layer and the cells were incubated at 37°C for 4 weeks. For visualization, the foci were stained with 0.0005% crystal violet (E). The number of colonies were counted (F) (±SEM, n=3; ^*p<0.05 and ^**p<0.01).

Figure 6. HBx-expressed SK-Hep-1 and HBx-expressed SNU-761 cells exhibited increased cell invasion in response to TLR4 stimulation. (A) SK-Hep-1 cells were transfected with mock (Ctrl) or Flag-HBx plasmid, extracted, and the cell lysates were subjected to WB with anti-Flag or anti-GAPDH Ab. (B) SNU-761 cells were transfected with mock (Ctrl) or Flag-HBx plasmid, extracted, and the cell lysates were subjected to WB with anti-Flag or anti-GAPDH Ab. (C, D) Ctrl SK-Hep-1 and HBx-SK-Hep-1 cells were suspended in culture medium including vehicle, LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 μg/mL), and CQ (10 μM) plus LPS (10 μg/mL), and the invasive assay was performed as described in the Materials and Methods. Fixed cells were stained with crystal violet (C). The number of migrated cells was counted, and presented as the mean±SEM (D) (^*p<0.05 and ^**p<0.01). (E, F) Ctrl SNU-761 and HBx-SNU-761 cells were suspended in culture medium with a vehicle, LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 μg/mL), and CQ (10 μM) plus LPS (10 μg/mL), and the invasive assay was performed as described in the materials and methods. Fixed cells were stained with 4,6-diamidino-2-phenylindole (E). The number of migrated cells was counted, and presented as the mean±SEM (F) (^*p<0.05 and ^**p<0.01).

Figure 7. HBx-expressed SK-Hep-1 and HBx-expressed SNU-761 cells exhibited increased cell migration in response to TLR4 stimulation. (A, B) Ctrl SK-Hep-1 and HBx-SK-Hep-1 cells were seeded into 12-well cell culture plates, scraped with a sterile yellow Gilson-pipette tip, and treated with vehicle (DMSO, <0.2% in culture medium), LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 μg/mL), and CQ (10 μM) plus LPS (10 μg/mL) for different time periods, as indicated. A representative experiment is shown (A). The residual gap between the migrating cells from the opposing wound edge was expressed as a percentage of the initial scraped area (B) (±SEM, n=3; ^*p<0.05 and ^**p<0.01). (C, D) Ctrl SNU-761 and HBx-SNU-761 cells were seeded into 12-well cell culture plates, scraped with a sterile yellow Gilson-pipette tip, and treated with vehicle (DMSO, <0.2% in culture medium), LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 μg/mL), and CQ (10 μM) plus LPS (10 μg/mL) for different time periods, as indicated. A representative experiment is shown (C). The residual gap between the migrating cells from the opposing wound edge was expressed as a percentage of the initial scraped area (D) (±SEM, n=3; ^*p<0.05, ^**p<0.01, and ^***p<0.001).

Figure 8. HBx-expressed SK-Hep-1 and HBx-expressed SNU-761 cells exhibited increased cell mobility and colony formation in response to TLR4 stimulation. (A, B) Ctrl SK-Hep-1 and HBx-SK-Hep-1 cells were seeded into 6-well culture plate, treated with vehicle (DMSO), and 3-MA (5 mM) in the presence or absence of LPS (10 μg/mL), and time-lapse imaging analysis was performed for different times by using phase-contrast microscope (A), as described in Materials and Methods. Data analysis for measuring the speed of cell mobility was performed as following protocols provided by Bio-protocol (B) (www.bio-protocol.org/e3586) (^*p<0.05). (C, D) Ctrl SNU-761 and HBx-SNU-761 cells were harvested with trypsin-EDTA and re-suspended in a singular form. The 1×10³ cells (per well) were plated in a 6-well plate and treated with the vehicle (DMSO), 3-MA (5 mM), and CQ (10 μM) in the presence or absence of LPS (10 μg/mL). After incubation for 12 days (C), colonies were stained with 0.5% crystal violet (Sigma) for 30 minutes at room temperature and counted (D) (±SEM, n=3; ^*p<0.05 and ^**p<0.01).

Figure 9. A model of how HBx is positively implicated in autophagy. BECN1 is either positively or negatively implicated in the autophagy through the formation of BECN1-Vps34 (left; induction of autophagy by BECN1-Vps34 complex) or BECN1-Bcl-2 complex (right; inhibition of autophagy induction by BECN1-Bcl-2 complex), respectively. HBx interacts with Vps34 and BECN1, and that facilitates the formation of BECN1-Vps34 complex for the induction of autophagy. Simultaneously, HBx interacts with BECN1 and that interrupts the formation of BECN1-Bcl-2 complex, resulting in the induction of autophagy. The HBx-mediated positive regulation of the formation of BECN1-Vps34 complex and negative regulation of the formation of BECN1-Bcl-2 complex eventually lead to cancer progression through the autophagy induction.