APOBEC3B promotes hepatocarcinogenesis and metastasis through novel deaminase-independent activity.

Cytidine deaminase APOBEC3B (A3B) is known to play important roles in creating de novo genomic C-to-T mutations in cancers and contribute to induction of genomic instability. Our study evaluated the roles of A3B in the progression and metastasis of human hepatocellular carcinoma (HCC). Using whole-transcriptome and whole-exome sequencing, and quantitative PCR, we found that A3B was overexpressed in human HCCs and A3B expression was significantly correlated with the proportion of genomic C-to-A and G-to-T mutations. Upon clinicopathological correlation, higher A3B expression was associated with more aggressive tumor behavior. Wild-type A3B (wt-A3B) overexpression in HCC cells promoted cell proliferation, and cell migratory and invasive abilities in vitro, and tumorigenicity and metastasis in vivo. On the other hand, knockdown of A3B suppressed cell proliferation, migratory, and invasive abilities of HCC cells with high endogenous A3B level. However, to our surprise, overexpression of A3B deaminase-dead double mutant (E68A/E255Q) led to similar results as wt-A3B in HCC. Furthermore, overexpression of wt-A3B and mutant A3B both enhanced cell cycle progression in HCC cells. Altogether, our data demonstrated a novel deaminase-independent role of A3B in contributing to HCC tumorigenesis and metastasis.

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. METHODS: Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. RESULTS: Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. CONCLUSIONS: A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. LAY SUMMARY: HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC.

Virus-Clip: a fast and memory-efficient viral integration site detection tool at single-base resolution with annotation capability.

Viral integration into the human genome upon infection is an important risk factor for various human malignancies. We developed viral integration site detection tool called Virus-Clip, which makes use of information extracted from soft-clipped sequencing reads to identify exact positions of human and virus breakpoints of integration events. With initial read alignment to virus reference genome and streamlined procedures, Virus-Clip delivers a simple, fast and memory-efficient solution to viral integration site detection. Moreover, it can also automatically annotate the integration events with the corresponding affected human genes. Virus-Clip has been verified using whole-transcriptome sequencing data and its detection was validated to have satisfactory sensitivity and specificity. Marked advancement in performance was detected, compared to existing tools. It is applicable to versatile types of data including whole-genome sequencing, whole-transcriptome sequencing, and targeted sequencing. Virus-Clip is available at http://web.hku.hk/~dwhho/Virus-Clip.zip.

C-terminal truncated hepatitis B virus x protein is associated with metastasis and enhances invasiveness by C-Jun/matrix metalloproteinase protein 10 activation in hepatocellular carcinoma.

UNLABELLED: Random integration of hepatitis B virus (HBV) DNA into the host genome is frequent in human hepatocellular carcinoma (HCC) and this leads to truncation of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). In this study, we investigated the frequency of this natural C-terminal truncation of HBx in human HCCs and its functional significance. In 50 HBV-positive patients with HCC, full-length HBx was detected in all nontumorous livers. However, full-length HBx was found in only 27 (54%) of the HCC tumors, whereas natural carboxylic acid (COOH)-truncated HBx was found in the remaining 23 (46%) tumors. Upon clinicopathological analysis, the presence of natural COOH-truncated HBx significantly correlated with the presence of venous invasion, a hallmark of metastasis (P = 0.005). Inducible stable expression of the COOH-truncated HBx protein (with 24 amino acids truncated at the C-terminal end) enhanced the cell-invasive ability of HepG2 cells, as compared to full-length HBx, using the Matrigel cell-invasion assay. It also resulted in increased C-Jun transcriptional activity and enhanced transcription of matrix metalloproteinase 10 (MMP10), whereas activation of the MMP10 promoter by COOH-truncated HBx was abolished when the activator protein 1-binding sites on the MMP10 promoter were mutated. Furthermore, silencing of MMP10 by short interfering RNA in HBxΔC1-expressing HepG2 cells resulted in significant reduction of cell invasiveness. CONCLUSIONS: Our data suggest that COOH truncation of HBx, particularly with 24 amino acids truncated at the C-terminal end, plays a role in enhancing cell invasiveness and metastasis in HCC by activating MMP10 through C-Jun.