Overexpression of insulin receptor substrate-1 and hepatitis Bx genes causes premalignant alterations in the liver

Abstract

Activation of the insulin (IN)/insulin receptor substrate-1 (IRS-1)/mitogen-associated protein kinase (MAPK) and the Wnt/beta-catenin signaling cascades occurs frequently in hepatocellular carcinoma (HCC) associated with persistent viral infection. The aims of this study were to provide a chronic proliferative stimulus through IRS-1 in the context of hepatitis Bx (HBx) protein expression in transgenic mice and determine if constitutive expression of these genes is sufficient to cause hepatocyte dysplasia and cellular transformation. We generated transgenic mice in which the HBx (ATX), IRS-1, or both (ATX+/IRS-1) genes were expressed under a liver-specific promoter. We also assessed histology and oxidative damage as well as up-regulation of molecules related to these signal transduction cascades in the liver by quantitative reverse-transcriptase polymerase chain reaction. Whereas mice with a single transgene (ATX or IRS-1) did not develop tumors, ATX+/IRS-1+ double transgenic livers had increased frequency of hepatocellular dysplasia and developed HCC. All three transgenic lines had significantly increased insulin growth factor 1 (IGF-1), Wnt 1 and Wnt 3 mRNA levels, and evidence of DNA damage and oxidative stress. The ATX+/IRS+ double transgenic mice were distinguished by having the highest level of activation of Wnt 3 and Frizzled 7 and selectively increased expression of IGF-II, proliferating cell nuclear antigen, and aspartyl-(asparaginyl)-beta-hydroxylase, a gene associated with increased cell migration.

Conclusion: These results suggest that continued expression of the ATX or IRS-1 transgenes can contribute to hepatocyte transformation but are not sufficient to trigger neoplastic changes in the liver. However, dual expression that activates both the IN/IRS-1/MAPK and Wnt/beta-catenin cascades is sufficient to cause dysplasia and HCC in a previously normal liver.

Figure 1

Histopathological features of transgenic mouse livers. The histopathological features of Wt, ATX+, IRS-1+, and ATX+/IRS-1+ livers from mice 15–18 months old were examined. (A, B) Wt control livers exhibited well-organized lobular architecture (A) with minimal inflammation and relatively uniform hepatocellular morphology (B). (C, D) Livers from ATX+/IRS-1+ mice exhibited (C) focal areas of hepatocellular dysplasia juxtaposed to regions with microsteatosis. (D) Higher magnification images demonstrate hepatocellular pleomorphism, disarray, and nuclear hyperchromasia. (E–H) Dysplastic and HCC foci in ATX+/IRS-1+ livers. (E) Nodular, well-delineated dysplastic focus (right side) compressing adjacent liver tissue (arrows point to boundaries with liver tissue on left side). Note bluer hue of cells within the nodule due to increased cellularity and nuclear hyperchromasia. (F) Infiltrating HCC with nodular growth pattern. (G, H) Infiltrating HCC cells with disorganized architecture, nuclear hyperchromasia and atypical mitoses (G-arrows). Original magnifications: A-160x; B, C, F-320x; D, G, H-640x; E-100x.

Figure 2

Frequency distribution of histopathological lesions in transgenic mouse livers. The histopathological features of Wt, ATX+, IRS-1+, and ATX+/IRS-1+ livers. The slides were analyzed under code for the presence or absence of (A) lobular inflammation, (B) portal inflammation, (C) anisocoria (nuclear pleomorphism), (D) microsteatosis, (E) macrosteatosis, and (F) dysplasia. Each group consisted of 24 mice. Graphs depict the percentage of cases/specimens exhibiting the specific lesion indicated on the ordinates. Significant P-values relative to other groups are indicated above the bars.

Figure 3

Effects of ATX, IRS-1, and ATX+IRS-1 chronic over-expression on cell proliferation, β-catenin expression, DNA damage, and lipid peroxidation in the liver. (A, E, I) Wt control livers exhibited minimal or no immunoreactivity to PCNA, β-catenin, 8-OHdG, or HNE in hepatocytes. (B, F, J) ATX+ transgenic mouse livers had focally increased immunoreactivity to PCNA, β-catenin, 8-OHdG, or HNE in hepatocytes. (C, G, K) IRS-1+ transgenic mouse livers had more conspicuously increased immunoreactivity to PCNA, β-catenin, 8-OHdG, and HNE relative to ATX+ livers. (D, H, L) ATX+/IRS-1+ transgenic mouse livers had prominently increased immunoreactivity to PCNA, β-catenin, 8-OHdG, and HNE relative to all other groups. All imaged originally photographed at 400x magnification.

Figure 4

Effects of ATX, IRS-1, and ATX+IRS-1 expression in liver on mRNA levels of (A) IGF-1 receptor (R), (B) IGF-2R, (C) InsulinR (INR), (D) IGF-1, (E) IGF-2, and (F) hepatocyte growth factor (HGF). Graphs depict the mean ± S.E.M. levels of gene expression in 8–10 mice per group. Significant inter-group differences are indicated with P-values above the bars.

Figure 5

Effects of ATX, IRS-1, and ATX+IRS-1 chronic over-expression on mRNA levels of IRS-1, IRS-2 and AAH. Gene expression corresponding to (A) IRS-1, (B) IRS-2, and (C) aspartyl-asparaginyl-β-hydroxylase (AAH) by qRT-PCR. Graphs depict the mean ± S.E.M. levels of gene expression in 8–10 mice per group. Significant P-values relative to other groups are indicated above the bars.

Figure 6

Effects of ATX, IRS-1, and ATX+IRS-1 constituative expression on mRNA levels of (A) Frizzled 3 (FZD3), (B) FZD7, (C) Wnt-1, (D) Wnt-3, (E) β-catenin, and (F) p53. Graphs depict the mean ± S.E.M. levels of gene expression in 8–10 mice per group. Significant inter-group differences are indicated with P-values above the bars.