HCV tumor promoting effect is dependent on host genetic background

Abstract

Background: The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo.

Methodology/principal findings: We crossed HCV-Tg mice that do not develop HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and investigated the potential contributing factors for the generated phenotype by gene expression and protein analyses. The Mdr2-KO/HCV-Tg females from the N2 generation of this breeding (having 75% of the FVB/N genome and 25% of the C57BL/6 genome) produced significantly larger tumors in comparison with Mdr2-KO mice. In parallel, the Mdr2-KO/HCV-Tg females had an enhanced inflammatory gene expression signature. However, in the N7 generation (having 99.2% of the FVB/N genome and 0.8% of the C57BL/6 genome) there was no difference in tumor development between Mdr2-KO/HCV-Tg and Mdr2-KO animals of both sexes. The HCV transgene was similarly expressed in the livers of Mdr2-KO/HCV-Tg females of both generations, as revealed by detection of the HCV transcript and the core protein.

Conclusion: These findings suggest that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in a host genetic background-dependent manner.

Figure 1. HCV transgene expression and incidence of large liver tumors in female mice from the N2 and N7 generations.

(A) Similar expression levels of the HCV transcript in 14-month-old Mdr2-KO/HCV mice from the N2 and N7 generations. Average levels of the HCV transcript were determined by semi-quantitative RT-PCR relative to expression level of the housekeeping gene Hprt in 5 females from the N2 generation and in 15 females from the N7 generation. (B) Similar expression of the HCV core protein in 14-month-old Mdr2-KO/HCV mice from the N2 and N7 generations. Representative immunohistochemical staining against HCV core protein in females from the N2 and N7 generations (magnification: ×200). (C) Higher incidence of large liver tumors (diameter over 1 cm) per mouse in 14-month-old Mdr2-KO/HCV compared to Mdr2-KO female mice from the N2, but not from the N7 generations. Incidence of all tumors is shown in Table 2. Red bars – Mdr2-KO/HCV group, blue bar – Mdr2-KO group.

Figure 2. Levels of liver enzymes in the serum of 14-month-old Mdr2-KO female mice.

Liver enzymes: Alkaline Phosphatase (ALP) Alanine aminotransferase (ALT), and Aspartate aminotransferase (AST) were measured in serum of 14 month old female mice. Graph represents average levels ±SD. (A) Comparison of enzymes levels (U/l) between Mdr2-KO and Mdr2^+/− with and without HCV-Tg from the N2 generation. (B) Comparison of enzymes levels (U/l) of Mdr2-KO/HCV and Mdr2-KO females from N2 and N7 generation. Statistically significant difference was found in ALP levels between Mdr2-KO/HCV females and Mdr2-KO females of the N2 generation only (* p = 0.04).

Figure 3. Mitotic and apoptotic events in the livers of mice from the N2 and N7 generations.

Events were counted per 50 HPF ±SD in H&E stained liver sections of tumors (0.3–1 cm diameter) and non-tumorous tissue samples from 14-month-old mice. (A) Reduced mitotic events (p = 0.016) in non-tumorous liver tissues of Mdr2-KO/HCV females compared to Mdr2-KO female mice from the N2, but not from the N7 generations. (B) Reduced apoptotic events (p = 0.002) in non-tumorous liver tissues of Mdr2-KO/HCV females compared to Mdr2-KO female mice from the N2, but not from the N7 generations. (C) Similar intensities of mitotic and apoptotic events in the livers of male mice from the N2 generation. There was no statistically significant difference between groups.

Figure 4. Hierarchical clustering of expression profiles of liver tissue samples subjected to whole genome expression profiling.

Average linkage hierarchical clustering of liver tissue samples from females of the N2 generation was performed on 5,000 genes with highest STD values using the software from Michael Eisen's Laboratory (Lawrence Berkeley National Laboratory and University of California, Berkeley; http://rana.lbl.gov/EisenSoftware.htm). The Mdr2 genotype and the tumor phenotype were major clustering forces, whereas the contribution of the HCV transgene in clustering was only marginal.

Figure 5. Higher expression of galectin-1 in the livers of Mdr2-KO/HCV-Tg N2 females.

(A–C) Immunohistochemical staining of non-tumorous liver tissues of 14-month-old Mdr2-KO and Mdr2-KO/HCV-Tg females from N2 generation with an antibody specific to galectin-1. (A) Left panel: Mdr2-KO/HCV-Tg; right panel: Mdr2-KO females. Magnification: upper panel ×40, lower panel ×100. (B) Cytoplasmic staining of hepatocytes in Mdr2-KO/HCV livers. Magnification ×400. (C) Staining of luminal side of cholangiocytes' membranes (indicated by arrows) in Mdr2-KO/HCV livers. Magnification ×400. (D) Scores of the immunostaining level for all liver samples in each group are shown in blue circles; average scores of each group are shown in red squares; p = 0.007 between the groups.

Figure 6. Nuclear expression of diubiquitin in tumors and its cytoplasmic expression in non-tumorous hepatocytes.

Immunohistochemical staining of 14-months-old Mdr2-KO/HCV-Tg N2 females with an antibody specific to diubiquitin. (A) Nuclear staining of hepatocytes in tumorous tissue (arrows). (B) Cytoplasmic staining of hepatocytes in non-tumorous liver tissue (arrows). Magnification: ×400.

Figure 7. Immunohistochemical detection of the HCV core, β-catenin and cyclin D1 proteins in the liver of Mdr2-KO/HCV-Tg mice.

(A) A typical example of the absence of correlation between the expression of the HCV core and either nuclear level of cyclin D1, or cytoplasmic/nuclear level of β-catenin proteins in hepatocytes. Magnification: ×100. Inserts contain the framed regions magnified ×400. (B) A rare example of the correlation between the expression of the HCV core and nuclear level of β-catenin proteins in hepatocytes (first two panels; magnification: ×200). Third panel: a typical membrane-associated pattern of β-catenin expression in hepatocytes of Mdr2-KO/HCV-Tg mice. Magnification: ×400.