Cancer Res Treat.  2018 Jul;50(3):646-657. 10.4143/crt.2016.550.

Inter-alpha Inhibitor H4 as a Potential Biomarker Predicting the Treatment Outcomes in Patients with Hepatocellular Carcinoma

Affiliations
  • 1Department of Radiation Oncology, Yonsei University Health System, Seoul, Korea. jsseong@yuhs.ac
  • 2Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul, Korea.
  • 3Korea and the Brain Korea 21 Plus Project for Medical Science, Yonsei University, Seoul, Korea.

Abstract

PURPOSE
Early prediction of treatment outcomes represents an essential step towards increased treatment efficacy and survival in patients with hepatocellular carcinoma (HCC). In this study, we performed two-dimensional electrophoresis (2-DE) followed by protein profiling to identify biomarkers predictive of therapeutic outcomes in patients with HCC who received liver-directed therapy (LDTx) involving local radiotherapy (RT), and studied the underlying mechanisms of the identified proteins.
MATERIALS AND METHODS
2-DE analysis was conducted by pooling sera from patients with a good or poor prognosis; serum proteomic profiles of the two groups were compared and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Identified proteins were confirmed via enzyme-linked immunosorbent assay. An invasion assay was performed after overexpression and knockdown of target protein in Huh7 cells.
RESULTS
Levels of inter-alpha inhibitor H4 (ITIH4), fibrinogen gamma chain, keratin 9/1 complex, carbonic anhydrase I, and carbonmonoxyhemoglobin S were changed by more than 4-fold in response to LDTx. In particular, pre-LDTx ITIH4 expression was more than 5-fold higher in patients with a good prognosis, compared to patients with a poor prognosis. The migration ability of Huh7 cells was significantly suppressed and enhanced by ITIH4 overexpression and knockdown, respectively. The tumors of patients with HCC and a good prognosis expressed high levels of ITIH4, compared to those of patients with a poor prognosis.
CONCLUSION
Taken together, ITIH4 may be a potential therapeutic target that could inhibit cancer metastasis, as well as a prognostic marker for patients with HCC who are receiving LDTx.

Keyword

Prognosis; Hepatocellular carcinoma; Liver-directed therapy; ITIH4; Biomarkers

MeSH Terms

Biomarkers
Carbonic Anhydrase I
Carboxyhemoglobin
Carcinoma, Hepatocellular*
Electrophoresis
Enzyme-Linked Immunosorbent Assay
Fibrinogen
Humans
Mass Spectrometry
Neoplasm Metastasis
Prognosis
Radiotherapy
Treatment Outcome
Biomarkers
Carbonic Anhydrase I
Carboxyhemoglobin
Fibrinogen

Figure

  • Fig. 1. Flow chart of patient selection. HCC, hepatocellular carcinoma; LDTx, liver-directed therapy.

  • Fig. 2. Differentially expressed proteins identified in pre- and post-LDTx samples according to treatment outcomes in patients with hepatocellular carcinoma. Protein spots were identified using two-dimensional electrophoresis. Green and red circles indicate paired and non-paired spots, respectively. LDTx, liver-directed therapy; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

  • Fig. 3. Inter-alpha inhibitor 4 (ITIH4) inhibited the migration of Huh7 cells. (A) ITIH4 mRNA expression in Huh7 cells. ITIH4 was downregulated in Huh7 cells infected with ITIH4 shRNA lentiviral vectors. For ITIH4 overexpression, Huh7 cells were transfected with the ITIH4 pCNS-D2 vector. (B) Cell viability of ITIH4 knockdown or overexpressing Huh7 cells. (C) Migration ability increased in response to ITIH4 downregulation in Huh7 cells (×200). (D) Migration ability decreased in ITIH4 overexpressing Huh7 cells (×200). Data are representative of the means and standard deviations of three independent experiments. n.s., not significant. **p < 0.01, ***p < 0.001 compared to control.

  • Fig. 4. Immunohistochemical analysis of inter-alpha inhibitor H4 (ITIH4) in surgical specimens of human hepatocellular carcinoma. (A) ITIH4 expression was significantly higher in cancer tissues than in non-tumor liver tissues (×200). (B) Higher levels of ITIH4 were detected in the tumors of patients with a good prognosis (representative: patient 1), compared with tumors of patients with a poor prognosis (representative: patient 2) (×200).


Reference

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