Page 103 - D. Cancer biology

Hepatitis B virus X protein promotes hepatocellular carcinogenesis and
metastasis through regulating VHL binding protein1
Inho Kang, Ji Ae Kim, Sung-min Kang, Hanwoon Park and Jeong Keun Ahn*
Department of Microbiology & Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University Daejeon 305-764, KOREA

Abstract
Chronic hepatitis B virus (HBV) infection is a leading cause of various liver diseases including hepatocellular carcinoma. Among HBV proteins, hepatitis X protein (HBx) plays critical roles in the development
of hepatocellular carcinoma. However the precise mechanism of HBx involved in HCC formation is still unclear. In this study, we demonstrated that HBx binds to VHL binding protein 1 (VBP1), and
subsequently interrupts the interaction between VBP1 and Von Hippel-Lindau protein (pVHL) which enhances HIF-1α degradation. We also revealed that HBx interrupts pVHL stabilization induced by VBP1,
and consequently stabilizes HIF-1α. It has been reported that upregulated HIF-1α promotes the tumor formation and enhances the epithelial-mesenchymal transition. Taken together, this study suggest a
new mechanism of HBx which has vital roles in liver carcinogenesis and metastasis

Introducti
Chronic HBV infection is tightly associated with HCC development. Among HBV proteins, HBx is the most important factor contributing to HCC, but the underlying mechanisms are unclear. Therefore, we
on
performed yeast two-hybrid screening assay to screen the putative cellular proteins that interact with HBx. We found that HBx interacts with VBP1. VBP1 is a chaperone protein that binds to pVHL, a tumor
suppressor protein which regulates proteasomal degradation of HIF-1α.
HIF-1α plays main roles in the cancer formation and metastasis by activating a wide repertoire of genes that are related with tumor development. Increased protein level of HIF is observed in HCC with poor
prognosis.
Here, we report that HBx interacts with VBP1, destabilizes pVHL, subsequently stabilizes HIF-1α, and thereby promotes tumor formation and metastasis. These results may suggest a new mechanism of HCC
development associated with HIF-1 activation by HBV infection.
Results

(A) (B) Figure 4. Effects of HBx and VBP1 on colony formation
HCT116 cells transfected with GST-HBx and Flag-VBP1 plasmids were cultured in DMEM with 10% FBS containing 0.4% soft agar at 37°C for 7 days.
Flag-HBx - + + + +
Flag-X - + - His-VBP1 - - + - +
His-VBP1 + - - + + +
HA-VHL - - - + +
WB : Flag Flag Control VBP1 HBx VBP1+HBx
His
HA
WB : HIs
IP : α-Flag Flag
Lysate His (A) (B)
HA
WB : Flag T w is t-lu c
Lysate 2 .5 ** M o ck GFP-HBx - - - - - + + + ++ +
Flag-VBP1
WB : His 2 .0 M o ck HA-VHL - + + + +
* MG132 treatment V H L
V H L + V B P 1
F o ld 1 .5 V H L + V B P 1 + H B x WB : GFP
His Pull-down 1 .0 WB : Flag
Figure 1. Physical interaction between HBx and VBP1 0 .5 WB : HA
(A) HBx physically interacts with VBP1.
(B) HBx interacts with VBP1, but not directly with pVHL. However, HBx interacts with pVHL through VBP1. WB : HIF1α
0 .0 WB : E-cadherin
M o c k M o c k V H L H yp oxia V H L + V B P 1 V H L + V B P 1 + H B x WB : vimentin
WB : β-actin
(A) (B)
Control VBP1
His-ub + - + + +
CHX 0 2 4 8 0 2 4 8 h HA-VHL - + + + +
GST-VBP1 - - - + ++
VHL
β-actin His (C)
IP:
20 00 α-HA GST
Re… WCL HA
0 CHX
Figure 2. VBP1 enhances the stability of pVHL by repressing its ubiquitination
(A) pVHL is stabilized by VBP1. HepG2 cells were transfected with His-VBP1 plasmid. At 24h after transfection, cells were treated with 100μg of cycloheximide (CHX) for indicated periods. pVHL is stabilized in the presence of VBP1. Data were quantified by image J program.
(B) VBP1 inhibits the ubiquitination of pVHL. 293T cells were co-transfected with His-Ub, HA-VHL, and GST-VBP1 plasmids. At 36h after transfection, cells were treated with 10μM MG132, a proteasome inhibitor, for 4h and immunoprecipitation assay was performed.
Figure 5. The regulatory effect of HBx on the expression of EMT factors and cell migration
(A) Luciferase assay shows that HBx elevates the transcription of twist gene by regulating pVHL through VBP1.
(B) HBx controls the level of EMT markers by regulating pVHL stability through its binding to VBP1.
(C) HBx increases cell motility, whereas VBP1 and pVHL decrease cell mobility. Data were quantified using image J program.
Conclusio
(A) GST-HBx - + ++ (B) GFP-HBx - - - + ++
Flag-VBP1 - - + + +
HA-VHL - + + + + n
WB : GST
WB : GFP Our results show that HBx stabilizes HIF-1α by its interaction with VBP1, a chaperone
WB : VBP1
WB : Flag protein which binds to pVHL. We found that VBP1 stabilizes pVHL by inhibiting the
WB : VHL WB : HA ubiquitination of pVHL and induces the degradation of HIF-1α by stabilizing pVHL. We also
WB : HIF1α elucidated that HBx interferes the stabilizing ability of VBP1 on pVHL, and subsequently
WB : HIF1α
increases the level of HIF-1α.
WB : β-actin
WB : β-actin
Therefore, these results may suggest a new mechanism of HCC development by HBV
infection.
Figure 3. Regulatory effects of HBx and VBP1 on the stabilities of pVHL and HIF-1α
(A) HBx regulates pVHL and elevates the level of HIF-1α. HepG2 cells were transfected with GST-HBx plasmid.
(B) HBx elevates the level of HIF-1α by interfering the stabilizing activity of VBP1 on pVHL. HepG2 cells were transfected with GFP-HBx, Flag-VBP1 and HA-
VHL plasmids.
Reference
s
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