Cache Domain Containing 1 Is a Novel Marker of Non-Alcoholic Steatohepatitis-Associated Hepatocarcinogenesis

Abstract

In the present study, potential molecular biomarkers of NASH hepatocarcinogenesis were investigated using the STAM mice NASH model, characterized by impaired insulin secretion and development of insulin resistance. In this model, 2-days-old C57BL/6N mice were subjected to a single subcutaneous (s.c.) injection of 200 μg streptozotocin (STZ) to induce diabetes mellitus (DM). Four weeks later, mice were administered high-fat diet (HFD) HFD-60 for 14 weeks (STAM group), or fed control diet (STZ group). Eighteen-week-old mice were euthanized to allow macroscopic, microscopic, histopathological, immunohistochemical and proteome analyses. The administration of HFD to STZ-treated mice induced significant fat accumulation and fibrosis development in the liver, which progressed to NASH, and rise of hepatocellular adenomas (HCAs) and carcinomas (HCCs). In 18-week-old animals, a significant increase in the incidence and multiplicity of HCAs and HCCs was found. On the basis of results of proteome analysis of STAM mice HCCs, a novel highly elevated protein in HCCs, cache domain-containing 1 (CACHD1), was chosen as a potential NASH-HCC biomarker candidate. Immunohistochemical assessment demonstrated that STAM mice liver basophilic, eosinophilic and mixed-type altered foci, HCAs and HCCs were strongly positive for CACHD1. The number and area of CACHD1-positive foci, and cell proliferation index in the area of foci in mice of the STAM group were significantly increased compared to that of STZ group. In vitro siRNA knockdown of CACHD1 in human Huh7 and HepG2 liver cancer cell lines resulted in significant inhibition of cell survival and proliferation. Analysis of the proteome of knockdown cells indicated that apoptosis and autophagy processes could be activated. From these results, CACHD1 is an early NASH-associated biomarker of liver preneoplastic and neoplastic lesions, and a potential target protein in DM/NASH-associated hepatocarcinogenesis.

Keywords: CACHD1; HCC; NASH; STAM mice; hepatocarcinogenesis.

Figure 1

(A) Steatosis, lobular inflammation, hepatocyte ballooning and total non-alcoholic fatty liver disease (NAFLD) activity scores in 18-week-old Stellic Animal Model (STAM) and streptozotocin (STZ) control mice. (B) Serum blood adiponectin, leptin and α-SMA levels in 18-week-old mice, (C) Blood glucose levels, (D) Numbers and areas of CACHD1⁺ foci developed in 10 and 18-weeks old mice. (E) Representative pictures of H&E, PAS, AZAN staining, and CACHD1 immunohistochemistry in the livers of STAM mice. CACHD1⁺ preneoplastic lesions included basophilic, eosinophilic, mixed-cell type, and those which were undetectable histopathologically. Note the elevation of PAS-positive (glycogenation) areas in all lesions, Azan-positive (fibrosis) areas in AF (mainly eosinophilic and mixed-cell type foci) and liver tumors, and strongly positive for CACHD1 nuclei and cytoplasm of the ballooned cells in CACHD1⁺ alone and mixed-cell type foci. *, p < 0.05, **, p < 0.01. Scale Bar: 100 μm (B,D); 50 μm, 100 μm and 200 μm in foci, HCA and HCC images, respectively, in (E).

Figure 2

Alterations of cell proliferation (PCNA) (A) in the area of CACHD1⁺ foci, HCAs, HCCs, and livers of 18-week-old STAM and STZ control mice. (B) Representative pictures of double IHC for PCNA/CACHD1, p62/CACHD1, Atg12/CACHD1, and single IHC for P-PERK and P-mTOR in STAM and STZ control mice. Note the elevation of PCNA and p62 in CACHD1⁺ foci, HCAs and HCCs, strong Atg12 and P-PERK positivity in the surrounding liver of STAM mice and lower expression in CACHD1⁺ foci and tumors, and P-mTOR-positive staining of STAM mice HCC. Scale Bar: 50 μm.

Figure 3

Effects of CACHD1 siRNA knockdown in Huh7 and HepG2 human hepatoma cell lines. Western blot (A) and RT-PCR analysis (B) of CACHD1 expression, (C) cell viability detected by WST8 assay, (D) cyclin D1 and p21^Waf1/Cip1 mRNA expression in CACHD1kn-2 cell lines, and (E) results of upstream regulator and pathway analysis by IPA in CACHD1kn Huh7/HepG2 cells. Note the significant suppression of cell viability, decrease of cyclin D1 and elevation of p21^Waf1/cip1 mRNA expression in CACHD1kn cell lines. Uncropped Western Blot of subfigure (A) is available in Figure S1.