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. 2009 Aug 15;18(16):2975-88.
doi: 10.1093/hmg/ddp236. Epub 2009 May 19.

Diet-induced Hepatocellular Carcinoma in Genetically Predisposed Mice

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Free PMC article

Diet-induced Hepatocellular Carcinoma in Genetically Predisposed Mice

Annie E Hill-Baskin et al. Hum Mol Genet. .
Free PMC article

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with approximately 70% of cases resulting from hepatitis B and C viral infections, aflatoxin exposure, chronic alcohol use or genetic liver diseases. The remaining approximately 30% of cases are associated with obesity, type 2 diabetes and related metabolic diseases, although a direct link between these pathologies and HCCs has not been established. We tested the long-term effects of high-fat and low-fat diets on males of two inbred strains of mice and discovered that C57BL/6J but not A/J males were susceptible to non-alcoholic steatohepatitis (NASH) and HCC on a high-fat but not low-fat diet. This strain-diet interaction represents an important model for genetically controlled, diet-induced HCC. Susceptible mice showed morphological characteristics of NASH (steatosis, hepatitis, fibrosis and cirrhosis), dysplasia and HCC. mRNA profiles of HCCs versus tumor-free liver showed involvement of two signaling networks, one centered on Myc and the other on NFkappaB, similar to signaling described for the two major classes of HCC in humans. miRNA profiles revealed dramatically increased expression of a cluster of miRNAs on the X chromosome without amplification of the chromosomal segment. A switch from high-fat to low-fat diet reversed these outcomes, with switched C57BL/6J males being lean rather than obese and without evidence for NASH or HCCs at the end of the study. A similar diet modification may have important implications for prevention of HCCs in humans.

Figures

Figure 1.
Figure 1.
Patterns of weight gain. Males were weighed at 2 week intervals. Vertical dashed lines at 135 days show the points when diets were switched from H to L or from L to H chow for groups of A/J and C57BL/6J males. Vertical bars show the SEM at the end of the study. See the legend of Table 1 for details.
Figure 2.
Figure 2.
Examples of a nodule (A) and an HCC (B).
Figure 3.
Figure 3.
Frequency of histological features and pathological conditions. (A) Reference features. (B) Frequency of features. Sequence of diets—conception, 35 days of age and 135 days of age; H, high fat and L, low fat. Features in each sample were scored on the scale that is shown. The percentage of males in each group is provided with a Y-axis scale that ranges from 0 (bottom) to 100% (top). Information about diet composition can be found in the Materials and Methods.
Figure 4.
Figure 4.
Development of NASH. (AH) Histopathological features of NASH in livers of C57BL/6J mice fed high-fat (H) chow. (I-J) Normal morphology of livers from A/J mice fed H chow. (A) Mixed intralobular inflammatory infiltrate (arrow) in fatty liver (H&E, ×400). (B) Pronounced inflammatory infiltrate composed of mononuclear cells and blue wave-like bands of fibrotic tissue visualized by Masson's trichrome stain in the portal region; an arrow points to a bile duct (Masson's trichrome, ×400). (C) Perivenular and pericellular fibrosis (blue) in zone 3 (Masson's trichrome, ×400). (D) Bands of fibrotic tissue (blue) connect several portal triads (bridging fibrosis) (Masson's trichrome, ×200). (E) Mallory hyaline visible in the cytoplasm of degenerating hepatocyte (arrow) (H&E, ×600). (F) Ballooning degeneration of hepatocytes (arrow head) and giant mitochondria visible as round red cytoplasmic inclusions (arrow) (H&E, ×400). (G) Intra-nuclear inclusion (arrow) (Masson trichrome, ×400). (H) Ductular reaction in portal triad, arrows point to hypertrophic, proliferating bile ducts (H&E, ×400). (I) Normal morphology of liver (H&E, ×40) from an A/J mouse. (J) Unaltered portal triad (arrow) and central veins (arrow heads) from the same mouse (H&E, ×200).
Figure 5.
Figure 5.
Pre-neoplastic and neoplastic lesions in livers from C57BL/6J males fed high-fat (H) chow. (A) Low-grade dysplastic changes found in the liver with features of NASH; arrows point to the border between dysplastic and non-dysplastic liver parenchyma (H&E, ×400). (B) A nodular lesion compressing surrounding the parenchyma (bulging growth) in a liver with histological features of NASH; arrows point to the border between a nodule and the remaining parenchyma (H&E, ×40). (C) The same nodule as shown in (B); arrows point to the fibrotic tissue separating the lesion from surrounding liver tissue (Masson's trichrome, ×40). (D) Plate thickening and small cell dysplasia in a nodular lesion found in a liver with NASH (H&E, ×200). (E) Significant cellular atypia observed in the nodular lesion found in a liver with NASH (H&E, ×400). (F) Hepatocellular carcinoma (HCC), which developed in a liver with NASH, compressing surrounding liver parenchyma (H&E, ×40). (G) Pseudo-glandular structures and necrosis (asterisk) in HCC shown in F (H&E, ×200). (H) Mitotic activity in the same HCC as shown in (F) and (G); arrows point to mitotic figures (H&E, ×400).
Figure 6.
Figure 6.
Gene expression pattern of HCCs resemble an undifferentiated cancer. Each column represents one sample (chip) and each row represents one gene. The range of colors (red to blue) corresponds to the range of expression values (high to low). Genes contributing to the core enrichment are shown in red.
Figure 7.
Figure 7.
Genes on human 4q25 are over-represented among those that are up-regulated in HCCs. Each column represents one sample and each row represents one gene. The range of colors (red to blue) corresponds to the range of expression values (high to low). Genes contributing to the core enrichment are shown in red.
Figure 8.
Figure 8.
mRNA profiles. (A) Hierarchical clustering of mRNA expression profiles of human HCCs and mouse models. The analyses were augmented with newly identified orthologous genes that were identified since the original report (20); see Lee et al. (20) for details about the data and definition of gene symbols and carcinogen treatments. Colored cells (black for humans and gray for mice) were used to show the subclass membership. Subclass membership for diet-induced HCCs is shown at the bottom. H, human; M, mouse. (B) Myc network. The Myc network was the top-ranked network for the tumors (15,19,22,25). (C) NFκB network. NFκB is the top-ranked network for HCC19 but not the four others.
Figure 9.
Figure 9.
Hierarchical analysis of mRNA expression patterns in developing liver, regeneration liver and diet-induced HCCs.
Figure 10.
Figure 10.
X-linked miRNA cluster. (A) Hierarchical cluster analysis of mRNA expression in the four HCC tumors. miRNAs (black) are located in the X-linked cluster and those in red are located elsewhere in the genome. (B) Sequence-based map of the X-linked miRNA cluster. (A)–(E) shows the location of PCR primers that were used to test for genomic amplification (Supplementary Material, Table S1). Slitrk2 and Gm1140 are the closest flanking genes on the proximal and distal sides, respectively. Scale bar represents 5 kb.

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