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. 2016 Apr 26;7(17):24005-17.
doi: 10.18632/oncotarget.8209.

C-terminal truncated hepatitis B virus X protein promotes hepatocellular carcinogenesis through induction of cancer and stem cell-like properties

Kai-Yu Ng  1 , Stella Chai  1 , Man Tong  1 , Xin-Yuan Guan  2   3 , Chi-Ho Lin  4 , Yick-Pang Ching  1   3 , Dan Xie  5 , Alfred Sze-Lok Cheng  6 , Stephanie Ma  1   3
Affiliations

C-terminal truncated hepatitis B virus X protein promotes hepatocellular carcinogenesis through induction of cancer and stem cell-like properties

Kai-Yu Ng et al. Oncotarget. .

Abstract

Tumor relapse after chemotherapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive initial treatment. Chronic infection with hepatitis B virus (HBV) has long been linked to the development of HCC. Upon infection, random HBV genome integration can lead to truncation of hepatitis B virus X (HBx) protein at the C-terminus. The resulting C-terminal-truncated HBx (HBx-ΔC) was previously shown to confer enhanced invasiveness and diminished apoptotic response in HCC cells. Here, we found HBx-ΔC to promote the appearance of a CD133 liver CSC subset and confer cancer and stem cell-like features in HCC. HBx-ΔC was exclusively detected in HCC cell lines that were raised from patients presented with a HBV background with concomitant CD133 expression. Stable overexpression of the naturally occurring HBx-ΔC mutants, HBx-Δ14 or HBx-Δ35, in HCC cells Huh7 and immortalized normal liver cells MIHA resulted in a significant increase in the cells ability to self-renew, resist chemotherapy and targeted therapy, migrate and induce angiogenesis. MIHA cells with the mutants stably overexpressed also resulted in the induction of CD133, mediated through STAT3 activation. RNA sequencing profiling of MIHA cells with or without HBx-ΔC mutants stably overexpressed identified altered FXR activation. This, together with rescue experiments using a selective FXR inhibitor suggested that C-terminal truncated HBx can mediate cancer stemness via FXR activation. Collectively, we find C-terminal truncated HBx mutants to confer cancer and stem cell-like features in vitro and to play an important role in driving tumor relapse in HCC.

Keywords: HBx; HCC; RNA-Seq; cancer stem cells; tumor-initiating cells.

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Conflict of interest statement

Nothing to declare.

Figures

Figure 1
Figure 1
(A) Relative expression of stemness associated genes in HepG2 and HepG2.2.15 HCC cells. (B) Flow cytometry dot plot analysis for CD133 expression in HepG2 and HepG2.2.15 HCC cells. (C) Flow cytometry analysis for ALDH activity using the ALDEFLUOR kit in HepG2 and HepG2.2.15 cells. DEAB stands for negative control when cells were treated with an ALDH inhibitor, diethylaminobenzaldehyde. (D) Representative image and quantification of hepatospheres generated from HepG2 and HepG2.2.15 HCC cells. Scale bar = 100 μm. ***p < 0.001. (E) Detection of full-length and C-terminal truncated HBx by RT-PCR using two sets of primers. 44F and XR1 flanks 197 nucleotides. 44F and 465R flanks 465 nucleotides. (F) RT-PCR analysis of full-length and C-terminal truncated HBx in a panel of immortalized normal and HCC cell lines. 18S was amplified as an internal control. CD133 expression was determined by flow cytometry analysis.
Figure 2
Figure 2
(A) Validation of HBx-Δ14 and HBx-Δ35 (expressing 14- and 35-amino acid C-terminal truncation) overexpression into HBV negative, CD133 absent MIHA cells and HBV negative, CD133 present Huh7 cells at genomic levels by RT-PCR and proteomic levels by Western blot. Empty vector (EV) transfected as control. 18S and beta-actin as internal controls for RT-PCR and Western blot, respectively. (B) Representative image and quantification of hepatospheres (primary and secondary passages) in MIHA or Huh7 cells with HBx-Δ14 and HBx-Δ35 stably overexpressed. Scale bar = 100 μm. ***p < 0.001, **p < 0.01, *p < 0.05. (C) Percentage of Annexin V positive cells in MIHA or Huh7 cells with HBx-Δ14 and HBx-Δ35 stably overexpressed, following 5-fluorouracil (5-FU) or sorafenib treatment. (D) Representative image and quantification of number of cells that migrated in MIHA or Huh7 cells with HBx-Δ14 and HBx-Δ35 stably overexpressed. Scale bar = 100 μm. ***p < 0.001, **p < 0.01. (E) Representative image and quantification of capillary tubes formed by HUVECs following treatment with supernatant collected from MIHA or Huh7 cells with HBx-Δ14 and HBx-Δ35 stably overexpressed. Scale bar = 100 μm. *p < 0.05. (F) Flow cytometry dot plot analysis for CD133 expression in MIHA cells with HBx-Δ14 and HBx-Δ35 stably overexpressed. (G) Western blot analysis of MIHA with EV, HBx-Δ14 or HBx-Δ35 stably overexpressed for phosphorylated and total STAT3 expression. (H) Relative expression of SOX2 in MIHA cells with or without HBx-Δ14 and HBx-Δ35 stably overexpressed.
Figure 3
Figure 3
(A) Venn diagram of differentially regulated genes based on gene fold change ≥ 1.5 and FDR ≤ 0.05 between EV vs. HBx-Δ14 and EV vs. HBx-Δ35 in MIHA cells. Genes that were significantly and commonly deregulated are shown in the overlapping area. (B) Unique gene signatures of MIHA cells with or without expression of HBx-Δ14 and HBx-Δ35, as shown by hierarchical cluster analysis (fold change ≥ 1.5 and FDR ≤ 0.05). Each cell in the matrix represents a particular expression level, where red and green cells indicate high and low gene expression, respectively. (C) Functional analysis of C-terminal truncated HBx mutants regulated genes by IPA. The p value was calculated using Fisher exact test to show the likelihood of association between our dataset of deregulated genes and a biological or toxicological function.
Figure 4
Figure 4
(A) Pathway analysis of C-terminal truncated HBx mutants regulated genes by IPA. The p value was calculated using Fisher exact test to show the likelihood of association between our dataset of deregulated genes and canonical pathways. (B) FXR/RXR interaction and functional network identified by IPA. Genes in red and green indicate up- and down-regulated genes found in C-terminal truncated HBx mutants, respectively. Genes in white are not included in our dataset, but added by IPA to complete the network connections. Molecules predicted to be activated and inhibited by the IPA molecule activity predictor are labeled in orange and blue, respectively. (C) Heatmap showing fold change of individual genes involved in FXR/RXR pathway and drug metabolism. A total of 22 and 5 genes were found commonly up- and down-regulated, respectively. Each cell in the matrix represents a particular expression level of genes, where red and green cells indicate high and low gene expression, respectively. (D) Drug metabolism interaction and functional network identified by IPA. Genes in red and green indicate up- and down-regulated genes found in C-terminal truncated HBx mutants, respectively. Genes in pink are related to drug metabolism. Genes in white are not included in our dataset, but added by IPA to complete the network connections. Molecules predicted to be activated and inhibited by the IPA molecule activity predictor are labeled in orange and blue, respectively. (E) qRT-PCR validation of identified differentially expressed genes relating to FXR/RXR pathway and drug metabolism in MIHA cells with or without HBx-Δ14 and HBx-Δ35 stably overexpressed.
Figure 5
Figure 5
(A) Representative image and quantification of hepatospheres in MIHA cells with HBx-Δ14 and HBx-Δ35 stably overexpressed, in the absence or presence of FXR inhibitor, Z-guggulsterone (Z-gugg; 25 μM). Scale bar = 100 μm. *compared to EV with ***p < 0.001, **p < 0.01, *p < 0.05. #compared to DMSO with ##p < 0.01, #p < 0.05. (B) Representative image and quantification of number of cells that migrated in MIHA cells with HBx-Δ14 and HBx-Δ35 stably overexpressed in the absence or presence of FXR inhibitor, Z-guggulsterone (Z-gugg; 25 μM). Scale bar = 100 μm. *compared to EV with ***p < 0.001, *p < 0.05. #compared to DMSO with ###p < 0.001, #p < 0.05. (C) Cartoon summary of the role of C-terminal truncated HBx variants, in particular HBx-Δ14 and HBx-Δ35, in promoting cancer and stem cell-like features in vitro in HCC.
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References

    1. Tanaka M, Katayama F, Kato H, Tanaka H, Wang J, Qiao YL, Inoue M. Hepatitis B and C virus infection and hepatocellular carcinoma in China: a review of epidemiology and control measures. J Epidemiol. 2011;21:401–416. - PMC - PubMed
    1. Matsubara K, Tokino T. Integration of HBV DNA and its implications for hepatocarcinogenesis. Mol Biol Med. 1990;7:243–260. - PubMed
    1. Feitelson MA, Duan LX. Hepatitis B virus X antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma. Am J Pathol. 1997;150:1141–1157. - PMC - PubMed
    1. Takada S, Koike K. Trans-activation function of a 3' truncated X gene-cell fusion product from integrated HBV DNA in chronic hepatitis tissues. Proc Natl Acad Sci USA. 1990;87:5628–5632. - PMC - PubMed
    1. Diamantis ID, McGandy CE, Chen TJ, Liaw YF, Gudat F, Bianchi L. Hepatitis B X-gene expression in hepatocellular carcinoma. J Hepatol. 1992;15:400–403. - PubMed

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