Proteome Characteristics of Non-Alcoholic Steatohepatitis Liver Tissue and Associated Hepatocellular Carcinomas

Abstract

To uncover mechanisms of nonalcoholic steatohepatitis (NASH) associated hepatocarcinogenesis, we compared the proteomes of human NASH-associated liver biopsies, resected hepatocellular carcinomas (HCCs) and HCCs of HCV⁺ patients with normal liver tissue of patients with gastrointestinal tumor metastasis, in formalin-fixed paraffin-embedded samples obtained after surgery in our hospital during the period from 2006 to 2011. In addition, proteome analysis of liver tumors in male STAM NASH-model mice was performed. Similar changes in the proteome spectrum such as overexpression of enzymes involved in lipid, cholesterol and bile acid biosynthesis and examples associated with suppression of fatty acid oxidation and catabolism, alcohol metabolism, mitochondrial function as well as low expression levels of cytokeratins 8 and 18 were observed in both human NASH biopsies and NASH HCCs, but not HCV⁺ HCCs. Alterations in downstream protein expression pointed to significant activation of transforming growth factor β, SMAD family member 3, β-catenin, Nrf2, SREBP-LXRα and nuclear receptor-interacting protein 1 (NRIP1), and inhibition of PPARs and p53 in human NASH biopsies and/or HCCs, suggesting their involvement in accumulation of lipids, development of fibrosis, oxidative stress, cell proliferation and suppression of apoptosis in NASH hepatocarcinogenesis. In STAM mice, PPARs inhibition was not obvious, while expression of cytokeratins 8 and 18 was elevated, indicative of essential differences between human and mouse NASH pathogenesis.

Keywords: NRIP1; PPARs; biopsy; cytokeratins 8/18; hepatocellular carcinoma; nonalcoholic steatohepatitis (NASH); oxidative stress; β-catenin.

Figure 1

(A) Summary of comparative analysis of upstream regulators which activation (z-score ≥ 2.0) or inhibition (z-score ≤ 2.0) predicted by IPA; (B) functional; and (C) canonical pathways analyses of NASH-associated biopsies, HCCs and HCV⁺ HCCs.

Figure 2

Histopathological and immunohistochemical analyses of human NASH-associated and HCV⁺ HCCs and STAM mice PPFs and HCCs: (A) immunohistochemical assessment of β-catenin and wnt1; (B) Immunohistochemistry for PHB1 and PHB2; and (C) immunohistochemistry for CK8/18 in the PPFs and HCCs STAM mice. Note the overexpression of β-catenin and wnt1 in the cell membrane, cytoplasm and/or nuclei of tumor cells in both human and STAM mouse HCCs.

Figure 2

Histopathological and immunohistochemical analyses of human NASH-associated and HCV⁺ HCCs and STAM mice PPFs and HCCs: (A) immunohistochemical assessment of β-catenin and wnt1; (B) Immunohistochemistry for PHB1 and PHB2; and (C) immunohistochemistry for CK8/18 in the PPFs and HCCs STAM mice. Note the overexpression of β-catenin and wnt1 in the cell membrane, cytoplasm and/or nuclei of tumor cells in both human and STAM mouse HCCs.

Figure 3

Events potentially contributing to NASH and Hepatitis C Virus-associated hepatocarcinogenicity. Black up and down arrows indicate increase/decrease in expression or activity for proteins or up-stream regulators.