Male-specific hepatitis B virus large surface protein variant W4P potentiates tumorigenicity and induces gender disparity.

BACKGROUND: The underlying mechanisms of carcinogenesis and gender disparity in hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remain unclear. Recently, we reported a novel HCC-related W4P/R mutation in the large surface protein (LHB) of HBV genotype C, which was found exclusively in male HCC patients. METHODS: LHB sequences from a carrier (wild type; WT) and W4P variant LHB sequence from an HCC patient were cloned and used to generate NIH3T3 and Huh7 cell lines. Cell proliferation and in vitro tumorigenicity were assessed by cell growth and transformation assays. Male and female nude mice were injected with the cells to determine in vivo tumorigenicity. To confirm the effect of estrogen in W4P-mediated tumorigenicity, male mice were injected with estrogen and challenged with W4P-expressing cells. The serum levels of different cytokines from the mouse model and patients were analyzed by ELISA. A critical role of interleukin (IL)-6 signaling in W4P-mediated tumorigenicity was tested by inhibition of Jak2. RESULTS: Although both WT and W4P variant LHBs enhanced cell proliferation by regulating the cell cycle and facilitated cell colony formation, the W4P variant demonstrated significantly higher activity. NIH3T3 cells expressing variant LHB, but not the WT, induced tumor in a nude mouse model. Tumor masses produced by variant LHB were significantly larger in male than female mice, and significantly reduced by estrogen. IL-6, but not tumor necrosis factor-α, was elevated in male mice harboring W4P-induced tumor, and was reduced by estrogen. IL-6 levels of HCC patients with the W4P variant were significantly higher than those of patients with WT LHB. W4P LHB induced higher production of IL-6 than WT LHB in cell lines, and the level was reduced by estrogen. The ability to reduce cell proliferation and colony formation of W4P LHB was hampered by inhibition of IL-6 signaling. CONCLUSIONS: This study suggests that the W4P mutation during the natural course of chronic hepatitis B infection may contribute to HCC development, particularly in male patients, in an IL-6-dependent manner.

Upregulation of endoplasmic reticulum stress and reactive oxygen species by naturally occurring mutations in hepatitis B virus core antigen.

Hepatitis B virus (HBV) infection is associated with a broad spectrum of clinical manifestations, including cirrhosis and hepatocellular carcinoma (HCC). Endoplasmic reticulum (ER) stress and subsequent oxidative stress have been implicated in liver carcinogenesis and disease progression with chronic inflammation. In our previous study, several mutations in the precore/core region of HBV genotype C were identified from 70 Korean chronic patients, and the mutations were associated with HCC and/or HBV e antigen serostatus. Here, we found that the naturally occurring mutations P5T/H/L of the HBV core antigen induced ER stress. The upregulation of ER stress resulted in higher reactive oxygen species production, intracellular calcium concentration, inflammatory cytokines as well as surface antigen production and apoptosis of cells. This study suggested that these mutations may contribute to the progression of liver disease in chronic patients.

Intracellular efficacy of tumor-targeting group I intron-based trans-splicing ribozyme.

BACKGROUND: Group I intron-based trans-splicing ribozyme, which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA, could be a useful tool for tumor-targeted gene therapy. In the present study, the therapeutic feasibility of this ribozyme was investigated by analyzing trans-splicing efficacy in vivo as well as in cells. METHODS: We assessed transgene activation, degree of ribozyme expression, targeted hTERT mRNA level, or the level of trans-splicing products in hTERT(+) cells or in human tumor nodules xenografted in animals after ribozyme administration. RESULTS: The activity and efficacy of the trans-splicing ribozyme in cells was dependent on the amount of endogenous hTERT mRNA and/or the accumulation of ribozyme RNA in cells. Intracellular activity of the ribozyme reached a plateau when no more targetable substrate mRNA was available or the ribozyme RNA level was fully saturated. In addition, the efficacy of ribozyme in xenografted tumor tissues was dependent on the dose of the delivered ribozyme-encoding adenoviral vector, indicating the potential of the ribozyme expression level as a determining factor for the in vivo efficacy of the trans-splicing ribozyme. On the basis of these results, we enhanced the intracellular ribozyme activity by increasing the ribozyme expression level transcriptionally and/or post-transcriptionally. CONCLUSIONS: We analyzed ribozyme efficacy and determined the most influential factors of its trans-splicing reaction in mammalian cell lines as well as in vivo. The present study could provide insights into the optimization of the trans-splicing ribozyme-based RNA replacement approach to cancer treatment.

eIF4A3 Phosphorylation by CDKs Affects NMD during the Cell Cycle.

Exon junction complexes (EJCs) loaded onto spliced mRNAs during splicing serve as molecular markers for various post-transcriptional gene-regulatory processes, including nonsense-mediated mRNA decay (NMD). Although the composition and structure of EJCs are well characterized, the mechanism regulating EJC deposition remains unknown. Here we find that threonine 163 (T163) within the RNA-binding motif of eIF4A3 (a core EJC component) is phosphorylated by cyclin-dependent protein kinases 1 and 2 in a cell cycle-dependent manner. T163 phosphorylation hinders binding of eIF4A3 to spliced mRNAs and other EJC components. Instead, it promotes association of eIF4A3 with CWC22, which guides eIF4A3 to an active spliceosome. These molecular events ensure the fidelity of specific deposition of the EJC ∼20-24 nt upstream of an exon-exon junction. Accordingly, NMD is affected by T163 phosphorylation. Collectively, our data provide evidence that T163 phosphorylation affects EJC formation and, consequently, NMD efficiency in a cell cycle-dependent manner.

Targeted retardation of hepatocarcinoma cells by specific replacement of alpha-fetoprotein RNA.

Although hepatocellular carcinoma (HCC) is one of the world-wide common malignancies, development of more specific and controlled therapeutic methods should be warranted. In this study, we describe a novel approach to HCC therapy that is based on trans-splicing ribozyme-mediated replacement of HCC-associated specific RNAs. We have developed a specific ribozyme that can target and replace human alpha-fetoprotein (AFP) RNA, which is highly expressed in HCC, with new transcript exerting therapeutic activity selectively in AFP-expressing liver cancer cells. The RNA replacement was employed via a high-fidelity trans-splicing reaction with the targeted residue in the AFP-expressing cells. Noticeably, the ribozyme could selectively deliver activity of suicide gene, herpes simplex virus thymidine kinase gene, into the liver cancer cells expressing the AFP RNA and thereby specifically and effectively retarded the survival of these cells with ganciclovir treatment. Simultaneously with the specific induction of therapeutic gene activity, the ribozyme reduced expression level of the targeted AFP RNA in the cells. These results suggest that the AFP RNA-targeting trans-splicing ribozyme could be a useful genetic agent for HCC-targeted efficient gene therapy.

Targeted anticancer effect through microRNA-181a regulated tumor-specific hTERT replacement.

We previously generated a group I intron-based ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to stimulate transgene activity in cancer cells expressing the target RNA via an accurate and specific trans-splicing reaction. One of the major concerns of the hTERT RNA targeting anti-cancer approach is the potential side effects to hTERT(+) hematopoietic stem cell-derived blood cells. Thus, here we modified the ribozyme by inserting target sites against microRNA-181a, which is a blood cell-specific microRNA, downstream of its 3' exon. The specificity of transgene induction and anticancer activity in hTERT(+) cancer cells improved significantly with the modified ribozyme, resulting in selective targeting of hTERT(+) cancer cells, but not hematopoietic cells even if they are hTERT-positive. Importantly, the trans-splicing reaction of the microRNA-regulated ribozyme worked equally well in a nude mouse model of hepatocarcinoma-derived intrasplenic carcinomatosis, inducing highly specific expression of a therapeutic transgene and efficiently regressing hTERT-positive liver tumors with minimal liver toxicity when systemically delivered with an adenoviral vector encoding the ribozyme. These results suggest that a combined approach of microRNA regulation with targeted RNA replacement is more useful for effective anti-cancer treatment.

Induction of endoplasmic reticulum-derived oxidative stress by an occult infection related S surface antigen variant.

AIM: To investigate the mechanism of endoplasmic reticulum (ER) stress induction by an occult infection related hepatitis B virus S surface antigen (HBsAg) variant. METHODS: We used an HBsAg variant with lower secretion capacity, which was a KD variant from a Korean subject who was occultly infected with the genotype C. We compared the expression profiles of ER stress-related proteins between HuH-7 cells transfected with HBsAg plasmids of a wild-type and a KD variant using Western blot. RESULTS: Confocal microscopy indicated that the KD variant had higher levels of co-localization with ER than the wild-type HBsAg. The KD variant up-regulated ER stress-related proteins and induced reactive oxygen species (ROS) compared to the wild-type via an increase in calcium. The KD variant also down-regulated anti-oxidant proteins (HO-1, catalase and SOD) compared to the wild-type, which indicates positive amplification loops of the ER-ROS axis. The KD variant also induced apoptotic cell death via the up-regulation of caspase proteins (caspase 6, 9 and 12). Furthermore, the KD variant induced a higher level of nitric oxide than wild-type HBsAg via the up-regulation of the iNOS protein. CONCLUSION: Our data indicate that occult infection related HBsAg variants can lead to ER-derived oxidative stress and liver cell death in HuH-7 cells.

Occult infection related hepatitis B surface antigen variants showing lowered secretion capacity.

AIM: To elucidate the molecular mechanisms underlying hepatitis B virus (HBV) occult infection of genotype C. METHODS: A total of 10 types of hepatitis B surface antigen (HBsAg) variants from a Korean occult cohort were used. After a complete HBV genome plasmid mutated such that it does not express HBsAg and plasmid encoding, each HBsAg variant was transiently co-transfected into HuH-7 cells. The secretion capacity and intracellular expression of the HBV virions and HBsAgs in their respective variants were analyzed using real-time quantitative polymerase chain reaction assays and commercial HBsAg enzyme-linked immunosorbent assays, respectively. RESULTS: All variants exhibited lower levels of HBsAg secretion into the medium compared with the wild type. In particular, in eight of the ten variants, very low levels of HBsAg secretion that were similar to the negative control were detected. In contrast, most variants (9/10) exhibited normal virion secretion capacities comparable with, or even higher than, the wild type. This provided new insight into the intrinsic nature of occult HBV infection, which leads to HBsAg sero-negativeness but has horizontal infectivity. Furthermore, most variants generated higher reactive oxidative species production than the wild type. This finding provides potential links between occult HBV infection and liver disease progression. CONCLUSION: The presently obtained data indicate that deficiency in the secretion capacity of HBsAg variants may have a pivotal function in the occult infections of HBV genotype C.

Naturally occurring mutations in the nonstructural region 5B of hepatitis C virus (HCV) from treatment-naïve Korean patients chronically infected with HCV genotype 1b.

The nonstructural 5B (NS5B) protein of the hepatitis C virus (HCV) with RNA-dependent RNA polymerase (RdRp) activity plays a pivotal role in viral replication. Therefore, monitoring of its naturally occurring mutations is very important for the development of antiviral therapies and vaccines. In the present study, mutations in the partial NS5B gene (492 bp) from 166 quasispecies of 15 genotype-1b (GT) treatment-naïve Korean chronic patients were determined and mutation patterns and frequencies mainly focusing on the T cell epitope regions were evaluated. The mutation frequency within the CD8+ T cell epitopes was significantly higher than those outside the CD8+ T cell epitopes. Of note, the mutation frequency within predicted CD4+ T cell epitopes, a particular mutational hotspot in Korean patients was significantly higher than it was in patients from other areas, suggesting distinctive CD4+ T cell-mediated immune pressure against HCV infection in the Korean population. The mutation frequency in the NS5B region was positively correlated with patients with carrier-stage rather than progressive liver disease (chronic hepatitis, liver cirrhosis and hepatocellular carcinoma). Furthermore, the mutation frequency in four codons (Q309, A333, V338 and Q355) known to be related to the sustained virological response (SVR) and end-of treatment response (ETR) was also significantly higher in Korean patients than in patients from other areas. In conclusion, a high degree of mutation frequency in the HCV GT-1b NS5B region, particularly in the predicted CD4+ T cell epitopes, was found in Korean patients, suggesting the presence of distinctive CD4+ T cell pressure in the Korean population. This provides a likely explanation of why relatively high levels of SVR after a combined therapy of pegylated interferon (PEG-IFN) and ribavirin (RBV) in Korean chronic patients with GT-1b infections are observed.

Selective regression of cancer cells expressing a splicing variant of AIMP2 through targeted RNA replacement by trans-splicing ribozyme.

AIMP2/p38 is a scaffolding protein critical for the assembly of the macromolecular tRNA synthetase complex. Moreover, AIMP2 harbors anti-proliferative activity and promotes cell death as a proapototic factor. A splicing variant of AIMP2 lacking exon 2 (AIMP2-DX2) is specifically generated by an alternative splicing process and is highly expressed in human lung cancer cells and the tissues of cancer patients. AIMP2-DX2 induces tumorigenesis by compromising the tumor suppressor function of normal AIMP2. Here, we describe a novel approach to cancer therapy that is based on trans-splicing ribozyme-mediated replacement of specific RNAs. We developed a specific ribozyme that can target and replace the splicing variant AIMP2-DX2 RNA with a new transcript selectively exerting therapeutic activity in AIMP2-DX2-expressing lung cancer cells. The RNA replacement was employed via a high-fidelity trans-splicing reaction with the targeted residue of the AIMP2-DX2 transcript, but not with normal AIMP2 RNA, in the cells. Noticeably, the ribozyme could selectively deliver the activity of a suicide gene into the AIMP2-DX2 RNA expressing lung cancer cells and thereby specifically and effectively retard the growth of the cancer cells with prodrug treatment. Therefore, the AIMP2-DX2 RNA-targeting trans-splicing ribozyme could be a useful genetic agent for efficient therapy targeting lung cancer.

Cancer-specific induction of adenoviral E1A expression by group I intron-based trans-splicing ribozyme.

In this study, we describe a novel approach to achieve replicative selectivity of conditionally replicative adenovirus that is based upon trans-splicing ribozyme-mediated replacement of cancer-specific RNAs. We developed a specific ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to induce adenoviral E1A gene expression selectively in cancer cells that express the RNA. Western blot analysis showed that the ribozyme highly selectively triggered E1A expression in hTERT-expressing cancer cells. RT-PCR and sequencing analysis indicated that the ribozyme-mediated E1A induction was caused via a high fidelity trans-splicing reaction with the targeted residue in the hTERTexpressing cells. Moreover, reporter activity under the control of an E1A-dependent E3 promoter was highly transactivated in hTERT-expressing cancer cells. Therefore, adenovirus containing the hTERT RNA-targeting transsplicing ribozyme would be a promising anticancer agent through selective replication in cancer cells and thus specific destruction of the infected cells.