Page 105 - D. Cancer biology

Hepatitis B virus X protein enhances the metastasis of
hepatocellular carcinoma by interacting with SOCS1

Inho Kang, Jung Sun Min, Heemin Lee, Ju Hyeon Lee and Jeong Keun Ahn*
Department of Microbiology & Molecular Biology, College of Bioscience and Biotechnology,
Chungnam National University, Daejeon 305-764, KOREA

Abstract Introduction
High metastasis rate of hepatocellular carcinoma (HCC) is the reason of Since high metastasis of HCC causes of poor prognosis of liver cancer and
poor prognosis of liver cancer and hepatitis B virus (HBV) is the most HBV is most common factor of HCC, we attempted to reveal the correlation
important pathogen for HCC development. In this study, we attempted to between HBV and metastasis. We found that HBx, interacts with Suppressor
reveal the correlation between HBV and metastasis of HCC. We found that of cytokine signaling 1 (SOCS1). SOCS1 modulates NF-κB through
HBV X protein (HBx), one of the viral proteins of HBV, interacts with ubiquitination and degradation of NF-κB’s subunit p65. NF-κB
suppressor of cytokine signaling 1 (SOCS1). SOCS1 negatively regulates modulates the transcription of EMT factors. EMT is a cellular process
NF-κB by degradation of p65, one of the subunits of NF-κB. NF-κB regulates inducing the metastasis of cancer cell. Therefore, it is possible that HBx may
various epithelial-mesenchymal transition (EMT) transcription factors such modulate p65 via SOCS1 to induce the metastasis of HCC. To verify this
as Snail, Slug, and Twist. EMT is a cellular process which controls possibility, we confirmed the physical interactions and reciprocal stability
invasiveness and motility of cancer cells. Here, we report that HBx interacts effect among these proteins. We also tested the effect of interaction
with SOCS1, subsequently prevents the ubiquitination of p65, and activates between HBx with SOCS1 on the expression of EMT factors and cell
EMT transcription factors, suggesting new metastasis mechanism of HBV- migration. Here, we report that HBx induces EMT process and increases cell
associated HCC. motility by regulating SOCS1.

Result
NF-kB activity (293T)
(A) HBx siRNA - + (B) Flag-HBx (A) 14 (B) 14 NF-kB activity (Chang)
WB:α- - 12 12
HBx WB: α-p65 10
WB:α- 10
WB:α- WB: α-Flag RLU 8 8
p65
IĸBα 6 RLU
6
WB:α- WB: α-β- 4 4
IKK actin 2
WB:α-p- . 0 2
WB:α-p- 0
IKK
IĸBα His-P65 - + + + His-P65 - + + +
Myc-SOCS1 -
-
+ +
-
+
-
Flag-HBx -
Myc-SOCS1 - - + +
Flag-HBx - - - +
WB:α-β-
actin (
Fig. 1. The effect of HBx on NF-κB signaling. C ) Myc-SOCS1 - -
(A) The correlation between HBx and NF-κB activity in human liver HepG2.2.15 cells producing Flag-HBx - - - - - - - - + + + + + + + +
HBV. HepG2.2.15 cells were transfected with siRNA against HBx. (B) HBx increases p65 level
in a dose-dependent manner.
(A) (B) Fig. 3. HBx inhibits SOCS1 induced degradation of NF-ĸB.
(A-B) SOCS1 mediated repression of NF-ĸB activity was recovered by HBx. NF-ĸB activities
Flag-HBx - Flag-HBx - + were determined by dual luciferase reporter assay in 293T cells (A) and Chang cells (B). (C)
+ The degradation of p65 by SOCS1 is reduced in liver cells expressing HBx.
WB:α- Myc-SOCS1 + +
IP: α-Flag SOCS1
WB:α-Flag WB:α-
IP: α-Flag Myc
WB:α- WB:α-Flag
Ly SOCS1
sat WB:α-Flag Ly WB:α-Myc
e sat
e
Reference (D)
(C)
His-p65 - + + + His-p65 + + + + +
Myc-SOCS1 + + + + Myc-SOCS1 - - + + +
Flag-HBx - - + ++ Flag-HBx - - - + ++
His pull down WB: HA-Ub - + + + +
α-
Ly WB: IP: WB:
Myc
sa α- α-His α-HA
te Myc
WB:
ly α-HA
sa
te ly WB:
sa α-p65
te
Fig. 2. HBx prevents the ubiquitin-mediated degradation of p65 through interaction with SOCS1.
(A) HBx interacts with SOCS1. At 36 h after transfection, cells were harvested and
immunoprecipitated with anti-Flag antibody. (B) HBx interacts with endogenous SOCS1. The
extracts of 293T cells transfected with Flag-HBx were immunoprecipitated with anti-Flag antibody.
(C) HBx interrupts the interaction between SOCS1 and p65. The cell lysates were incubated with
Ni-NTA-agarose and analyzed by Western blotting with anti-Myc antibody. (D) HBx inhibits p65
ubiquitination which is induced by SOCS1. Fig. 4. The effects of HBx and SOCS1 on the expression of EMT factors and cell migration.
(A-C) HBx and SOCS1 interferes with each other to regulate the promoter activities of EMT
factor genes. (D) HBx regulates the migration of HCC cells. HepG2 cells were transfected with
Flag-HBx and Myc-SOCS1 plasmids, and chemotactic cell migration assay was performed. (E)
Effect of HBx and SOCS1 on the expression of EMT markers.
Conclusion Reference
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