Serum from Chronic Hepatitis B Patients Promotes Growth and Proliferation via the IGF-II/IGF-IR/MEK/ERK Signaling Pathway in Hepatocellular Carcinoma Cells

Abstract

Background/Aims: Chronic hepatitis B virus (HBV) infection (CHB) plays a central role in the etiology of hepatocellular carcinoma (HCC). Emerging evidence implicates insulin-like growth factor (IGF)-II as a major risk factor for the growth and development of HCC. However, the relationship between HBV infection and IGF-II functions remains to be elucidated. Methods: Levels of circulating IGF-II and IGF-I receptor (IGF-IR) in healthy donors (HDs) and CHB patients were tested by ELISA. Human HCC cell lines (HepG-2, SMMC-7721, MHCC97-H) were incubated with serum from HDs and CHB patients at various concentrations for 24, 48, and 72 h. MTT and plate colony formation assays, BrdU ELISA, ELISA, small-interfering RNA (siRNA) transfection, quantitative real-time PCR, and western blot were applied to assess the functional and molecular mechanisms in HCC cell lines. Results: Serum levels of IGF-II and IGF-IR were significantly higher in CHB patients than in HDs. Additionally, serum from CHB patients directly induced cell growth, proliferation, IGF-II secretion, and HDGF-related protein-2 (HRP-2) and nuclear protein 1 (NUPR1) mRNA and protein expression in HCC cells. Moreover, serum from CHB patients increased IGF-II–induced cell growth, proliferation, and HRP-2 and NUPR1 mRNA and protein expression in HCC cells. Blockade of IGF-IR clearly inhibited the above effects. Most importantly, interference with IGF-II function markedly repressed the cell proliferation and HRP-2 and NUPR1 mRNA and protein expression induced by serum from CHB patients. Furthermore, serum from CHB patients induced ERK phosphorylation via IGF-IR, with the MEK inhibitor PD98059 significantly decreasing CHB patient serum-induced IGF-II secretion, cell proliferation, and HRP-2 and NUPR1 mRNA and protein expression. Conclusion: Serum from CHB patients increases cell growth and proliferation and enhances HRP-2 and NUPR1 expression in HCC cells via the IGF-II/IGF-IR/MEK/ERK signaling pathway. These findings help to explain the molecular mechanisms underlying HBV-related HCC and may lead to the development of effective therapies.

IGF-binding proteins 3 and 4 are regulators of sprouting angiogenesis

“…For VEGF, the concentration that we used in the serum-free experiments (25 ng/mL) was similar to the concentration in medium supplemented with 20% human serum (22-45 ng/mL) [50][51][52]. However, the concentrations of IGF2 and IGFBP3 were relatively low in our experimental setup as compared to medium supplemented with 20% serum and especially that of IGFBP3 [24,[53][54][55]. A comparison of these concentrations can only be performed with caution because of the interactions between these proteins and other IGF family members [11].…”

Suppression of Hepatocyte Nuclear Factor 4 α by Long-term Infection of Hepatitis B Virus Contributes to Tumor Cell Proliferation

“…Many findings implicate HBV infection as a major risk factor in the development of HCC [33,35]. Here, we demonstrated that prolonged HBV infection in susceptible host cell line (HepG2-NTCP) or in long-term infected mice can suppress the expression of anti-tumor HNF4α, particularly at later time points.…”

“…Moreover, the effect of other cell signaling pathways, including ERK on HBV-mediated HNF4α suppression, have not been explored. Previous studies reported that serum from CHB patients enhanced cell growth and proliferation in HCC cells via the IGF-II/IGF-IR/MEK/ERK signaling pathway [33]. Moreover, HBx, through the activation of the ERK and p38 MAPK signaling pathways, promoted the metastasis of liver cancer [34].…”

Suppression of Hepatocyte Nuclear Factor 4 Alpha by Long-Term Infection of Hepatitis B Virus Contributes to Tumor Cell Proliferation

“…Moreover, the effect of other cell signaling pathways including ERK on HBVmediated HNF4α suppression have not been explored. Previous studies reported that serum from CHB patients enhanced cell growth and proliferation in HCC cells via the IGF-II/IGF-IR/MEK/ERK signaling pathway [33]. Moreover, HBx through the activation of ERK and p38 MAPK signaling pathways promoted metastasis of liver cancer [34].…”