Elevated levels of circulating ITIH4 are associated with hepatocellular carcinoma with nonalcoholic fatty liver disease: from pig model to human study

Abstract

Background: Noninvasive biomarkers are urgently needed for optimal management of nonalcoholic fatty liver disease (NAFLD) for the prevention of disease progression into nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). In order to identify the biomarkers, we generated the swine hepatocellular carcinoma (HCC) model associated with NAFLD and performed serum proteomics on the model.

Methods: Microminipigs were fed a high-fat diet to induce NAFLD and a normal diet as the control. To induce HCC, diethylnitrosamine was intraperitoneally administered. Biopsied liver samples were histopathologically analyzed every 12 weeks. Serum proteins were separated by blue native two-dimensional gel electrophoresis and proteins of interest were subsequently identified by MALDI-TOF MS/MS. Human serum samples were analyzed to validate the candidate protein using antibody-mediated characterization.

Results: In the NAFLD pigs, hepatic histology of nonalcoholic steatohepatitis (NASH) was observed at 36 weeks, and HCC developed at 60 weeks. Among serum proteins identified with MALDI-TOF MS/MS, serum inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), an acute response protein which is secreted primarily by liver, was identified as the most characteristic protein corresponding with NAFLD progression and HCC development in the NAFLD pigs. With immunoassay, serum ITIH4 levels in the NAFLD pigs were chronologically increased in comparison with those in control animal. Furthermore, immunohistochemistry showed ITIH4 expression in hepatocytes also increased in both the cancer lesions and parenchyma as NAFLD progressed. Human study is also consistent with this observation because serum ITIH4 levels were significantly higher in HCC-NAFLD patients than in the simple steatosis, NASH, and virus-related HCC patients. Of note, HCC-NAFLD patients who had higher serum ITIH4 levels exhibited poorer prognosis after hepatectomy.

Conclusions: We established an HCC pig model associated with NAFLD. Serum proteomics on the swine HCC with NAFLD model implicated ITIH4 as a non-invasive biomarker reflecting NAFLD progression as well as subsequent HCC development. Most importantly, the results in the swine study have been validated in human cohort studies. Dissecting speciation of serum ITIH4 promises to have clinical utility in monitoring the disease.

Keywords: Blue native/SDS gel electrophoresis; Hepatocellular carcinoma; Inter-alpha-trypsin inhibitor heavy chain 4; MALDI-TOF MS/MS; Nonalcoholic fatty liver disease; Pig model.

Fig. 1

Changes in histological findings and NAFLD activity scores over time in the NAFLD group and control. Hematoxylin and eosin stained sections of samples from the NAFLD group (× 200) revealed that hepatocyte ballooning and lobular inflammation with lymphocyte infiltration appeared in the perivenular region (zone 3) at 12 weeks and became diffuse at 36 weeks. Microvesicular steatosis also appeared at 36 weeks. Masson trichrome staining (× 20) revealed slight fibrosis in zone 3 at 12 weeks; pericellular fibrosis, at 36 weeks; and complete bridging fibrosis, at 60 weeks. In the control animal, lobular inflammation was observed at 24 and 36 weeks. Fibrosis appeared in zone 3 at 24 weeks, and cirrhosis developed at 36 weeks. NAFLD activity scores in the NAFLD group are expressed as mean values. The NAFLD group; HFD feeding with DEN injection. The control; normal diet feeding without DEN injection

Fig. 2

Histological features of liver tumors in the NAFLD group. (a) Macroscopic view of the liver in the NAFLD group. All animals in the NAFLD group developed multiple liver tumors at 60 weeks. Encapsulated tumor nodules with various sizes were observed in the liver. (b) Histological features of tumor lesions with hematoxylin and eosin staining. Tumor lesions compressed the surrounding liver parenchyma and exhibited fatty changes (a: × 20). Cellular atypia with large nuclei, small cells, and trabecular structures were observed in the tumor lesions (b: × 200). (c) Immunohistochemistry for well-differentiated HCC markers in tumor lesions. Glutamine synthetase showed diffuse cytoplasmic staining (a: × 200). Glypican-3 showed faint staining (b: × 200). Heat-shock-protein 70 showed strongly diffuse nucleo-cytoplasmic staining, not only in the tumor lesions but also in the non-tumor lesions (c: × 200). Arginase-1 showed focal nucleo-cytoplasmic staining (d: × 200)

Fig. 3

Protein identifications from the BN/SDS 2D-PAGE gels by MALDI-TOF MS/MS and validation for serum ITIH4, ceruloplasmin, and haptoglobin. a ITIH4, ceruloplasmin (Cp), and haptoglobin (Hpt) were identified from protein spots on a vertical line in the NAFLD group from the BN/SDS 2D-PAGE gels by MALDI-TOF MS/MS. b Chronological changes of serum expression of ITIH4, ceruloplasmin, and haptoglobin in the NAFLD group and control animal by western blotting. Cropped blots are showed in the figure. Full-length blots are presented in Additional file 4: Figure S3. c Chronological changes of serum ITIH4 levels by enzyme-linked immunosorbent assay. ITIH4 levels in the NAFLD group are expressed as the mean values. The mean values for 0, 12, 24, 36, and 60 weeks in the NAFLD group and the control were 0.48, 0.45, 0.73, 0.77, and 1.70 ng/ml, and 0.60, 0.44, 0.49, 0.34, and 0.01 ng/ml, respectively. Inter-α-trypsin inhibitor heavy chain 4: ITIH4, ceruloplasmin: Cp, haptoglobin: Hpt

Fig. 4

Relationship between serum ITIH4 levels and NAFLD activity scores (NAS). a Comparison of serum ITIH4 levels according to NAS [14] and the presence of HCC. * P <  0.05 compared with NAS = 0. b Comparison of serum ITIH4 levels according to fibrosis stage. There was no significant difference between fibrosis stages

Fig. 5

Hepatic expression of ITIH4 in the NAFLD group and control. Changes in ITIH4 immunoreactivity in the liver of both groups over time (× 200)

Fig. 6

Validation for serum expression of ITIH4 in patients. a Serum ITIH4 intensity in the simple steatosis (SS), NASH, HCC with NAFLD, and virus-related HCC groups from western blotting quantified by densitometry. SS group: n = 40, NASH group: n = 40, HCC with NAFLD group: n = 55, virus-related HCC group: n = 35. Not significant: N.S. b The probability of overall survival in NAFLD with HCC patients according to serum ITIH4 intensity. The 55 patients in the HCC with NAFLD group were divided into low ITIH4 and high ITIH4 groups. The low ITIH4 and high ITIH4 groups were defined as serum ITIH4 intensity < 14,000 and serum ITIH4 intensity ≥14,000, respectively. Overall survival (OS) was defined as the period from the day of hepatectomy for HCC until the day of death caused by HCC or liver failure. For patients who survived, the date of the last follow-up was set as March 31, 2017. Cumulative 5-year overall survival rates of patients in the low ITIH4 and high ITIH4 groups were 95.0 and 72.5%, respectively (P = 0.0353: Log-rank, P = 0.0222: Wilcoxon)

Fig. 7

Serum expression of 35 kDa fragment of ITIH4 in patients by western blotting. a Expression of full-length 120 kDa and 35 kDa fragment of ITIH4 in representative blot of the SS, NASH, HCC with NAFLD group. b Serum 35 kDa fragment intensity in the simple steatosis (SS), NASH, and HCC with NAFLD groups from western blotting quantified by densitometry. SS group: n = 40, NASH group: n = 40, HCC with NAFLD group: n = 55