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.
Highlights
Chronic hepatitis B virus (HBV) infection is a major risk factor in the development of hepatocellular carcinoma (HCC) in Southeast Asia
HBxΔC14 and HBxΔC35 were chosen for studies because these C-terminal truncated hepatitis B virus X (HBx) variants have previously been shown to abrogate the growth suppressive effects induced by full-length HBx, and as a result, can effectively promote cell transformation and enhance the proliferative activity of neoplastic cells [8, 11, 25]. They have been identified as natural deletion mutants in HCC tissues [8, 11, 25]. We found these two HBx-∆C mutants to promote the appearance of a CD133 liver cancer stem cell subset and confer cancer and stem celllike properties in HCC cell line models
As compared to parental HepG2 cells, we found HepG2.2.15 cells to preferentially express stemness associated genes including NANOG, SMO, ABCB1, ABCC2, aldehyde dehydrogenase 1A1 (ALDH1A1) as well as the functional liver cancer stem cells (CSCs) marker CD133, as detected by qRT-PCR (Figure 1A)
Summary
Chronic hepatitis B virus (HBV) infection is a major risk factor in the development of hepatocellular carcinoma (HCC) in Southeast Asia. A recent study by Quetier et al found that the HBx protein with C-terminal deletions was more susceptible to DENinduced hepatocarcingoenesis than the full-length HBx protein in a mice model, through increased expression of IL-6, TNF-α and IL-1β transcripts as well as activation of STAT3, ERK and JNK proteins [14]. These results demonstrate that, in addition to the full-length HBx, HBxΔC plays an important, and likely a more critical role, in HCC development
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