Regulation of ERK and AKT pathways by hepatitis B virus X protein via the Notch1 pathway in hepatocellular carcinoma

Abstract

Hepatitis B virus (HBV) is the dominant risk factor for hepatocellular carcinoma (HCC). HBV X protein (HBx) plays crucial roles in HCC carcinogenesis. HBx interferes with several signaling pathways including the Notch1 pathway in HCC. In this study, we found that Notch1 was highly expressed in HCC, especially in large HCCs. Notch1 and HBx co-localized in HCC and their levels were positively correlated with each other. Notch1 expression was more elevated in HepG2.2.15 cells than that in HepG2 cells. HBx activated the Notch1 pathway in HepG2.2.15 cells. Suppression of HBx and the Notch1 pathway attenuated the growth of HepG2.2.15 cells. Notch1, ERK, and AKT pathways were inhibited after γ-secretase inhibitor treatment. Dual-specificity phosphatase 1 (DUSP1) and phosphatase and tensin homolog (PTEN) were upregulated after γ-secretase inhibitor treatment and Hes1 inhibition. Luciferase reporter assays showed that Hes1 suppressed the promoters of DUSP1 and PTEN genes, which was reversed by γ-secretase inhibitor treatment. Western blotting demonstrated that DUSP1 dephosphorylated pERK and PTEN dephosphorylated pAKT. Collectively, we found a link among HBx, the Notch1 pathway, DUSP1/PTEN, and ERK/AKT pathways, which influenced HCC cell survival and could be a therapeutic target for HCC treatment.

Figure 1

Notch1 is highly expressed in HCC tissues. (A) Immunohistochemical staining of Notch1 in HCC (a), adjacent non-tumor (b), cirrhosis (c) and normal liver tissues (d). (e) Negative control. Scale bar, 100 µm. Magnification, ×200 and ×400. (B) The immunohistochemistry score of Notch1 in each immunostained liver section. (C) Western blotting of Notch1 in HCC, adjacent non-tumor, cirrhosis and normal liver tissues. ^**P<0.01, ^***P<0.001; NS, not significant.

Figure 2

Notch1 and HBx co-expressed in HCC tissues and HepG2.2.15 cells. (A) Co-localization of Notch1 and HBx detected by immunohistochemical staining. Scale bar, 100 µm. Magnification, ×200 and ×400. (B) Correlation of expression levels of Notch1 and HBx in 108 HBV-positive HCC tissues. The Spearman's rho = 0.584, P<0.001. (C) Co-expression of Notch1 with HBx in HepG2.2.15 cells detected by double-fluorescence immunostaining.

Figure 3

HBx activates the Notch1 pathway and DAPT attenuates cell growth via inhibition of ERK and AKT pathways. HBx gene (A) and protein (B) were expressed in HepG2.2.15 cells. (B) The Notch1 pathway was activated in HepG2.2.15. (C) HBx was knocked down by SiRNA and its mRNA level was tested by qRT-PCR. The changes were presented as fold change in comparison to the control. (D) The Notch1 pathway was inhibited after SiHBx. (E) Cell proliferation of HepG2.2.15 cells was inhibited after SiHBx. (F) Cell proliferation of HepG2.2.15 cells was inhibited after DAPT treatment. (G) Alteration of expression of the Notch1 pathway after DAPT treatment. (H) Expressions of pERK and pAKT were decreased after DAPT treatment. (I and J) Expression of DUSP1 and PTEN was increased after DAPT treatment. Data were collected in at least three independent experiments. ^**P<0.01, ^***P<0.001; NS, not significant.

Figure 4

Hes1 represses the expressions of DUSP1 and PTEN. (A) Hes1 mRNA level was effectively reduced after SiHes1. The changes were presented as fold change in comparison to the control. (B) Cell growth of HepG2.2.15 was inhibited after SiHes1. (C) DUSP1 mRNA level was increased after SiHes1. The changes were presented as fold change in comparison to the control. (D) PTEN mRNA level was increased after SiHes1. The changes were presented as fold change in comparison to the control. (E) Protein levels of DUSP1 and PTEN were increased after SiHes1. (F and G) Luciferase activity of HepG2.2.15 cells transfected with a DUSP1-luc reporter or PTEN-luc reporter together with a plasmid expressing HES1 or its corresponding empty control. Cells were treated with DAPT or its vehicle. Data were collected in at least three independent experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 5

Sequences of the upstream regions from the ATG (bold) of human DUSP1 and PTEN promoters. The 5′-UTRs of DUSP1 and PTEN are in blue letters. The primers used to amplify the PCR products of the human DUSP1 and PTEN promoters are in red letters and correspond to regions A and B.

Figure 6

Quantitative ChIP analysis of Hes1 binding to the promoter sequences of DUSP1 and PTEN. (A) Schematic diagram of the DUSP1 promoter region cantaining potential binding sites of Hes1. Immunoprecipitated DNA was amplified by PCR using primers specific for regions A and B. The arrow indicates the transcription initiation site (TIS). ATG, the translation start codon. (B) Schematic diagram of the PTEN promoter region cantaining potential binding sites of Hes1. (C) HepG2.2.15 cells were subjected to ChIP assay with mixed Hes1 antibodies or IgG antibody to identify Hes1 binding sites on the DUSP1 promoter. Two different DNA regions from the DUSP1 promoter were analyzed by qPCR. Cells were treated with DAPT or vehicle. (D) ChIP assay was performed using Hes1 antibodies or IgG to identify Hes1 binding sites on the PTEN promoter in HepG2.2.15 cells. ^**P<0.01, ^***P<0.001.

Figure 7

DUSP1 regulates the ERK pathway and PTEN regulates the AKT pathway. (A and B) DUSP1 mRNA and protein levels were significantly reduced after SiDUSP1. (B) Expression of pERK was increased after SiDUSP1. (C and D) PTEN mRNA and protein levels were significantly reduced after SiPTEN. (D) Expression of pAKT was increased after SiPTEN. The changes of DUSP1 and PTEN mRNA levels were presented as fold change in comparison to the control. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 8

Schematic illustrations of the role of HBx and the Notch1 pathway in HCC proliferation explored in this study. Detailed explanation can be seen in Discussion.