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.

Hepatic IFIT3 predicts interferon-α therapeutic response in patients of hepatocellular carcinoma.

Adjuvant interferon-α (IFN-α) therapy is used to control certain types of cancer in clinics. For hepatocellular carcinoma (HCC), IFN-α therapy is effective in only a subgroup of patients; therefore, identifying biomarkers to predict the response to IFN-α therapy is of high significance and clinical utility. As the induced IFN-stimulated gene expression following IFN-α treatment plays pivotal roles in IFN-α effects, we screened IFN-stimulated gene expression in HCC tissues and found that several IFN-stimulated genes were significantly decreased in HCC. Interestingly, expression of IFN-induced protein with tetratricopeptide repeats (IFIT) family members, including IFIT1, IFIT2, IFIT3, and IFIT5, was decreased in HCC tissues. We further analyzed the expression of IFIT family members in HCC and their roles in patients' responses to IFN-α therapy in two independent randomized controlled IFN-α therapy clinical trials of HCC patients. We found that higher expression of IFIT3, but not other IFITs, in HCC tissues predicts better response to IFN-α therapy, suggesting that IFIT3 may be a useful predictor of the response to IFN-α therapy in HCC patients. Mechanistically, IFIT3 enhanced the antitumor effects of IFN-α by promoting IFN-α effector responses both in vitro and in vivo. IFIT3 could bind signal transducer and activator of transcription 1 (STAT1) and STAT2 to enhance STAT1-STAT2 heterodimerization and nuclear translocation upon IFN-α treatment, thus promoting IFN-α effector signaling. CONCLUSION: Higher IFIT3 expression in HCC tissues predicts better response to IFN-α therapy in HCC patients; IFIT3 promotes IFN-α effector responses and therapeutic effects by strengthening IFN-α effector signaling in HCC. (Hepatology 2017;66:152-166).

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.

Long non-coding RNA HOTTIP is frequently up-regulated in hepatocellular carcinoma and is targeted by tumour suppressive miR-125b.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common human cancers. Recently, emerging evidence has suggested the role of long non-coding RNAs (lncRNAs) in human carcinogenesis. In this study, we aimed to investigate the expression and functional implications of lncRNAs in human HCC. METHODS: Eighty-eight well-annotated lncRNAs were profiled in primary HCC by quantitative RT-PCR. Functional relevance of lncRNAs was elucidated in HCC cell lines and nude mice models. The regulatory relationship between miRNA and lncRNA was predicted in silico and further validated by luciferase reporter assay and expression analysis. RESULTS: In our profiling study, HOTTIP was identified as the most significantly up-regulated lncRNA in human HCCs, even in early stage of HCC formation. Functionally, knock-down of HOTTIP attenuated HCC cell proliferation in vitro and markedly abrogated tumourigenicity in vivo. In addition, knock-down of HOTTIP also inhibited migratory ability of HCC cells and significantly abrogated lung metastasis in orthotopic implantation model in nude mice. HOTTIP is an antisense lncRNA mapped to the distal end of the HOXA gene cluster. Knock-down of HOTTIP significantly suppressed the expression of a number of HOXA genes. Furthermore, we identified miR-125b as a post-transcriptional regulator of HOTTIP. Ectopic expression of miR-125b reduced HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Moreover, in human HCCs, HOTTIP expression negatively correlated with that of miR-125b. CONCLUSIONS: HOTTIP is a novel oncogenic lncRNA, which negatively regulated by miR-125b. Overexpression of HOTTIP contributes to hepatocarcinogenesis by regulating the expression of its neighbouring protein-coding genes.

Hepatic RIG-I predicts survival and interferon-α therapeutic response in hepatocellular carcinoma.

In hepatocellular carcinoma (HCC), biomarkers for prediction of prognosis and response to immunotherapy such as interferon-α (IFN-α) would be very useful in the clinic. We found that expression of retinoic acid-inducible gene-I (RIG-I), an IFN-stimulated gene, was significantly downregulated in human HCC tissues. Patients with low RIG-I expression had shorter survival and poorer response to IFN-α therapy, suggesting that RIG-I is a useful prognosis and IFN-α response predictor for HCC patients. Mechanistically, RIG-I enhances IFN-α response by amplifying IFN-α effector signaling via strengthening STAT1 activation. Furthermore, we found that RIG-I deficiency promotes HCC carcinogenesis and that hepatic RIG-I expression is lower in men than in women. RIG-I may therefore be a tumor suppressor in HCC and contribute to HCC gender disparity.

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.

Genetic and epigenetic inactivation of T-cadherin in human hepatocellular carcinoma cells.

T-cadherin is an atypical cadherin and growing evidence has indicated that T-cadherin exerts tumor-suppressive effects on cancers of epithelial cell type and also causes positive effects on tumor angiogenesis. Human hepatocellular carcinoma (HCC) is a hypervascular tumor and T-cadherin has been shown to be overexpressed in intratumoral endothelial cells of HCCs. However, the expression status and functions of T-cadherin in hepatocytes or HCC cells remain unclear. Here, we demonstrated that T-cadherin was underexpressed in HCC cells (26.5%, 13/49 cases), but was frequently (77.6%, 38/49) overexpressed in intratumoral endothelial cells immunohistochemically. Semiquantitative RT-PCR analysis also showed that the T-cadherin gene was underexpressed in 7 of 11 HCC cell lines. Loss of heterozygosity analysis revealed that 32-38% of the 42 human HCC samples had allelic losses at this locus. Upon pharmacological treatment with demethylating agent 5-aza-2'-deoxycytidine or histone deacetylase inhibitor trichostatin A, T-cadherin promoter hypermethylation and/or histone deacetylation was frequently observed in HCC samples and cell lines. Functionally, enforced expression of T-cadherin induced G(2)/M cell cycle arrest, reduced cell proliferation in low serum medium, suppressed anchorage-independent growth in soft agar and increased sensitivity to TNFalpha-mediated apoptosis in HCC cells. Intriguingly, we found that T-cadherin significantly suppressed the activity of c-Jun, a crucial oncoprotein constitutively activated in HCC cells. To conclude, T-cadherin was differentially expressed in human HCCs. The underexpression of T-cadherin in HCC cells suggests it may be another critical event in addition to T-cadherin-mediated angiogenesis during HCC development.

Allelic alterations in nontumorous liver tissues and corresponding hepatocellular carcinomas from chinese patients.

Allelic imbalance may play an important in tumor progression in hepatocarcinogenesis, but the genetic background of the corresponding nontumorous liver in hepatocellular carcinoma (HCC) is not well defined. We studied the incidence of loss of heterozygosity (LOH) and microsatellite instability (MSI) by microsatellite analysis in both nontumorous livers and the corresponding tumors, by comparing them with the normal DNA from Chinese patients with resected primary HCCs. We also evaluated the pathologic significance of the alterations. We used 18 highly polymorphic microsatellite markers on chromosomes 1, 3, 4, 7, 8, 9, 13, 16, 17, and 18. Our results showed that 70.6% (24 of 34) of the HCCs exhibited LOH at 1 or more loci, and that the overall fractional allelic loss (FAL) was 0.169. MSI was observed in only 1 tumor. In contrast, the nontumorous livers of the HCCs showed a very low incidence of LOH, with only a single LOH detected in 1 of 34 (2.9%) of the nontumorous livers, with an overall FAL index of 0.005. Tumors with LOH at 1 or more loci had significantly more frequent venous invasion (P = 0.019). Allelic loss at locus D9S199 (9p23) was seen more frequently in larger tumors (P = 0.031), and, less significantly, allelic loss at locus D16S516 (16q24.1) was seen more frequently in larger tumors (P = 0.059). LOH was common in predominantly hepatitis B virus-associated HCCs from Chinese patients. However, LOH or MSI in the corresponding cirrhotic or noncirrhotic livers was uncommon.