THE ROLES OF HBX IN NOTCH SIGNALING PATHWAY IN HEPATOCELLULAR CARCINOMA

Abstract

Hepatocellular carcinoma (HCC) is one of the top five cancers leading cause of deaths in the world. Hepatitis B virus infection is the major cause of HCC in Thailand. The essential protein for HBV infection and replication in hepatocytes is HBx. Previous reports demonstrated that HBx is the multi-functional protein because it induces expression of various proto-oncogenes and inhibits expression of tumor suppressor genes. In addition, this protein regulates various transcription factors and signal transduction pathways leading to malignant transformation in infected cells. One of the signaling pathways involved in HCC development is a well conserved Notch signaling pathway. Previous studies showed that HBx induces the activation of Notch signaling in HBx-transfected cell lines but how HBx regulates Notch signaling remained unknown. In this study, we investigated how HBx regulates the activation of Notch signaling pathway in HBV-genome transfected HCC cell line, (HepG2.2.15). We detected the expression of Notch receptors, ligands and one of its target gene in HepG2.2.15, compared with the control parental cell line HepG2 and immortalized hepatocyte THLE-2. We found that both mRNA and proteins of Notch1 and Delta-like4 (Dll4) increased in HepG2.2.15. In addition, cleaved Notch1, a cleaved form of Notch1, was detected only in HepG2.2.15, suggesting that Notch signaling is activated in this cell line. Silencing of HBx in HepG2.2.15 reduced the expression of all Notch receptors and ligands. Moreover, we studied the mRNA expression of Notch receptor and ligands in tumor lesion of HBV-associated HCC tissue biopsy (N = 8), compared with non-tumor lesions, and found that 50% and 62.5% of specimens have increased Notch1 and Dll4 in tumor lesions, respectively. Next, we inhibited the activation of Notch signaling by using gamma-secretase inhibitor (GSI) or silencing Dll4 in HepG2.2.15. The result showed that both treatments suppressed cell proliferation and induced apoptosis. No difference in viral replication was found in both treatments. Interestingly, only the knock-down of Dll4 induced cell cycle arrest at the G1 phase. To investigate the upstream pathways responsible for the activation of Notch signaling via Dll4/Notch1 axis, HepG2.2.15 was treated with specific inhibitors, i.e. BAY 11-7082 (NF-kB inhibitor), LY-294002 (PI3K/Akt inhibitor), SB-203580 (p38 MAP kinase inhibitor) and U0126 (MEK1/2 inhibitor) and cleaved Notch1, Notch1 and Dll4 were detected by Western Blot. The results revealed that at high dose of both LY-294002 and U0126 suppressed the appearance of cleaved Notch1, Notch1 and Dll4. At low concentration, U0126 was the only inhibitor to suppress the appearance of cleaved Notch1, Notch1 and Dll4. Furthermore, we investigated the effect of these inhibitors on cell survival of HepG2.2.15. U0126 treatment, even at low dose, decreased the viability of HepG2.2.15 compared to parental HepG2. Taken together, these data strongly suggested that HBx/Dll4/Notch1 axis operates in HBV-infected HCC and regulates cell survival and cell proliferation.