Increased expression of ErbB-2 in liver is associated with hepatitis B x antigen and shorter survival in patients with liver cancer

Abstract

Hepatitis B x antigen, or HBxAg, contributes importantly to the pathogenesis of hepatocellular carcinoma (HCC). Given that HBxAg constitutively activates beta-catenin and that upregulated ErbB-2 promotes beta-catenin signaling in other tumor types, experiments were designed to ask whether HBxAg was associated with upregulated expression of ErbB-2. When HBxAg positive and negative HepG2 cells were subjected to proteomics analysis, ErbB-2 was shown to be upregulated in HepG2X but not control cells. ErbB-2 was also strongly upregulated in HB infected liver, and weakly in some HCC nodules, where it correlated with HBxAg expression. Among tumor bearing patients, strong ErbB-2 staining in the liver was associated with dysplasia, and a shorter survival after tumor diagnosis. This implies that elevated ErbB-2 is an early marker of HCC. Treatment of HepG2X cells with ErbB-2 specific siRNA not only reduced ErbB-2 expression, but also reduced the expression of beta-catenin, suggesting that ErbB-2 contributed to the stabilization of beta-catenin. ErbB-2 specific siRNA also partially blocked the ability of HBxAg to promote DNA synthesis and growth of HepG2 cells. These results suggest that ErbB-2/beta-catenin up-regulation contributes importantly to the mechanism of HBxAg mediated hepatocellular growth.

Figure 1

Powerblot analysis of (a) HepG2X and (b) HepG2CAT cells. The circled spot in each blot is ErbB2. The results are representative of three independent experiments. (c) Expression of C-erbB-2 in HepG2 and Huh7 cells 48 hrs after transient transfection with pcDNA3 (ctrl) or pcDNA3-HBx (HBx) as indicated.

Figure 2

Consecutive sections of a cirrhotic nodule stained with anti-HBx (a), with anti-ErbB2 (b) or with preimmune rabbit serum (c). (d) Anti-ErbB-2 staining of a liver section from a patient with chronic hepatitis. e-g. Consecutive tissue sections of an HCC nodule (lower right) and adjacent nontumor liver tissue stained with anti-HBx (e), anti-ErbB2 (f), or with preimmune serum (g). (h) Anti-ErbB-2 staining of an uninfected normal human liver. Original magnification of panel d is ×400 and for all other panels ×200.

Figure 3

Percentage of patients with strong staining scores (+2 or +3) for (a) HBxAg (gray bars) and ErbB2 (white bars) or (b) PCNA (gray bars) and Ki-67 (white bars). The corresponding histological lesions in the livers for each patient group are indicated.

Figure 4

Consecutive sections of peritumor liver stained with anti-ErbB-2 (a), anti-PCNA (b), or with normal IgG (c) (panels a-c magnification × 200). Consecutive tumor and adjacent liver sections were also stained with anti-ErbB-2 (d), anti-CD31 (e) or with an equivalent amount of normal IgG (f). Magnification of panels a-e was × 100.

Figure 5

Survival analysis of HCC patients with high or low C-erbB2 expression in peritumor liver. High ErbB2 expression had scores of +2 and +3 while low ErbB2 expression had scores of 0 and +1.

Figure 6

Treatment of HBxAg positive (HepG2X) and negative (HepG2CAT) cells with ErbB2 specific siRNA. HepG2X cells were transiently transfected with increasing amounts of ErbB2 specific siRNA as indicated, and the levels of ErbB2 protein (a) or β-catenin (b) were detected by western blotting. In panel b, both endogenous wild type (wt) and mutant (mut) β-catenin were detected. c. Growth of HepG2X and HepG2CAT (control) cells as measured by percent BrdU incorporation 72 hours after transient transfection of ErbB2 specific or control siRNA at the concentrations indicated. HepG2CAT cells transfected with irrelevant siRNA (curve 1) or with ErbB2 specific siRNA (curve 2). HepG2X cells transfected with irrelevant siRNA (curve 3) or with ErbB2 specific siRNA (curve 4). d. Growth of HepG2X cells (curves 1 and 2) and HepG2CAT cells (curves 3 and 4) transfected with control siRNA (curves 1 and 3) or ErbB-2 specific siRNA (curves 2 and 4). The experiments in panels c and d were done in triplicate and the average values plotted.

Figure 7

Western blot analysis of phospho-Akt levels in Huh7X cells (lanes 1 and 2) or HepG2X cells (lanes 3 and 4) transiently transfected with control siRNA (lanes 1 and 3) or with ErbB-2 specific siRNA (lanes 2 and 4). The data shown is representative of three independent experiments.