Global gene profiling of spontaneous hepatocellular carcinoma in B6C3F1 mice: similarities in the molecular landscape with human liver cancer

Abstract

Hepatocellular carcinoma (HCC) is an important cause of morbidity and mortality worldwide. Although the risk factors of human HCC are well known, the molecular pathogenesis of this disease is complex, and in general, treatment options remain poor. The use of rodent models to study human cancer has been extensively pursued, both through genetically engineered rodents and rodent models used in carcinogenicity and toxicology studies. In particular, the B6C3F1 mouse used in the National Toxicology Program (NTP) two-year bioassay has been used to evaluate the carcinogenic effects of environmental and occupational chemicals, and other compounds. The high incidence of spontaneous HCC in the B6C3F1 mouse has challenged its use as a model for chemically induced HCC in terms of relevance to the human disease. Using global gene expression profiling, we identify the dysregulation of several mediators similarly altered in human HCC, including re-expression of fetal oncogenes, upregulation of protooncogenes, downregulation of tumor suppressor genes, and abnormal expression of cell cycle mediators, growth factors, apoptosis regulators, and angiogenesis and extracellular matrix remodeling factors. Although major differences in etiology and pathogenesis remain between human and mouse HCC, there are important similarities in global gene expression and molecular pathways dysregulated in mouse and human HCC. These data provide further support for the use of this model in hazard identification of compounds with potential human carcinogenicity risk, and may help in better understanding the mechanisms of tumorigenesis resulting from chemical exposure in the NTP two-year carcinogenicity bioassay.

Figure 1

Morphologic characteristics of spontaneous HCC in the B6C3F1 mouse. Tumors were characterized by several histologic phenotypes, often within the same tumor, including (A) trabecular, composed of variably sized cords greater than three hepatocytes in thickness (arrowheads); (B) solid, composed of sheets of neoplastic cells with no apparent architecture, often exhibiting marked pleomorphism and a robust mitotic rate (arrowheads); and less frequently (C) pseudoglandular phenotypes, typified by variably sized luminal structure formation lined by few to multiple layers of neoplastic hepatocytes (arrowheads), hematoxylin and eosin, 40×.

Figure 2

Microarray analysis of spontaneous hepatocellular carcinoma (HCC) in the B6C3F1 mouse. (A) Principal component analysis of global gene expression of spontaneous HCC compared to normal liver (CTL). Principal component analysis mapping illustrates significant similarities within groups, as evidenced by tight clustering of samples, as well as significant differences in overall gene expression between groups. (B) Heat map illustrating significant differences in global gene expression between normal liver (N) and spontaneous HCC (S). Red indicates upregulation and green indicates downregulation of gene expression. Partek Genomics Suite (6.4) was used to generate heat maps to compare the normal liver (N) and spontaneous HCC (S) samples using an unsupervised learning approach. Agglomerative hierarchical clustering was used to cluster the genes (rows) and samples (columns), in which Pearson’s dissimilarity was used as the distance measure and Ward’s method was used to evaluate distance between clusters.

Figure 3

Significant concordance between spontaneous B6C3F1 mouse hepatocellular carcinoma (HCC) dataset and very advanced human HCC. Using NextBio software, there are 1,163 concordant genes shared between the mouse and human HCC datasets, with 743 similarly upregulated (p = 1.1E-95) and 420 similarly downregulated (p = 2.2E-71). Human HCC dataset: GEO Accession GSE6764.

Figure 4

A network produced by Ingenuity Pathway Analysis of upregulated and downregulated genes in spontaneous mouse hepatocellular carcinoma, illustrating the interactome of mediators implicated in the pathogenesis of human hepatocellular carcinoma (red, upregulation; green, downregulation).

Figure 5

A network produced by Ingenuity Pathway Analysis of spontaneous mouse hepatocellular carcinoma illustrating the interactome of fetal oncogenes, protooncogenes, and cell cycle mediators that are expressed in human hepatocellular carcinoma (red, upregulation of gene expression; green, downregulation). CMYC was upregulated by quantitative reverse transcriptase polymerase chain reaction but was not represented on the microarray.

Figure 6

A network produced by Ingenuity Pathway Analysis of spontaneous mouse hepatocellular carcinoma illustrating the interactome of growth factors and cell cycle mediators expressed in human hepatocellular carcinoma (red, upregulation; green, downregulation).

Figure 7

Quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) validation of important mediators in spontaneous hepatocellular carcinoma in the B6C3F1 mouse. Several mediators important in the development and progression of HCC in humans are similarly dysregulated in HCC of mice, as shown by both QRT-PCR and microarray. Other important mediators of human HCC are also shown by QRT-PCR to be dysregulated, although not differentially expressed by microarray analysis (c-Myc, Mtsu1).

Figure 8

Protein expression validation by immunohistochemistry and Western blotting for important mediators involved in hepatocellular carcinoma (HCC) development. (A)Membrane immunolabeling for β-catenin in tumor (T, white arrowheads) and adjacent normal liver parenchyma (N), hematoxylin counterstain, 20×. (B) Increased cytoplasmic and membrane immunolabeling for E-cadherin antibody in tumor tissue (T, white arrowheads) compared to adjacent normal liver (N), hematoxylin counterstain, 20×. (C) Increased Glutamine synthetase immunolabeling within tumor tissue (arrowheads) compared to centrilobular localization in normal adjacent liver (arrows), hematoxylin counterstain, 4×. (D) Increased nuclear Cyclin D immunolabeling in tumor tissue (arrows), hematoxylin counterstain, 40×. (E) Increased cytoplasmic c-Myc, hematoxylin counterstain, 20×. (F) increased nuclear Ki67, hematoxylin counterstain, 40×. (G) increased cytoplasmic Birc5 immunolabeling in tumor tissue, hematoxylin counterstain, 20×. (H) Western blot of normal liver (N) and spontaneous HCC (S1–5). There is increased protein expression of β-catenin, E-cadherin, Glutamine synthetase, and α-fetoprotein in spontaneous HCC compared to normal liver (β-actin loading control).