Telaprevir is a potent inhibitor of hepatitis C virus (HCV) NS3-4A protease. However, the emergence of drug-resistant strains during therapy is a serious problem, and the susceptibility of resistant strains to interferon (IFN), as well as the details of the emergence of mutant strains in vivo, is not known. We previously established an infectious model of HCV using human hepatocyte chimeric mice. Using this system we investigated the biological properties and mode of emergence of mutants by ultra-deep sequencing technology. Chimeric mice were injected with serum samples obtained from a patient who had developed viral breakthrough during telaprevir monotherapy with strong selection for resistance mutations (A156F [92.6%]). Mice infected with the resistant strain (A156F [99.9%]) developed only low-level viremia and the virus was successfully eliminated with interferon therapy. As observed in patients, telaprevir monotherapy in viremic mice resulted in breakthrough, with selection for mutations that confer resistance to telaprevir (e.g., a high frequency of V36A [52.2%]). Mice were injected intrahepatically with HCV genotype 1b clone KT-9 with or without an introduced resistance mutation, A156S, in the NS3 region, and treated with telaprevir. Mice infected with the A156S strain developed lower-level viremia compared to the wildtype strain but showed strong resistance to telaprevir treatment. Although mice injected with wildtype HCV showed a rapid decline in viremia at the beginning of therapy, a high frequency (11%) of telaprevir-resistant NS3 V36A variants emerged 2 weeks after the start of treatment. Conclusion: Using deep sequencing technology and a genetically engineered HCV infection system, we showed that the rapid emergence of telaprevir-resistant HCV was induced by mutation from the wildtype strain of HCV in vivo. (HEPATOLOGY 2011;54:781-788)

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A need exists for seeking effective treatments for castration-resistant prostate cancer (CRPC) in response to its emergence following androgen deprivation therapy as a major clinical problem. In the present study, we investigated the chemopreventive and chemotherapeutic potential of luteolin, a flavonoid with antioxidative properties, on prostate cancer, including CRPC. Luteolin inhibited the progression of rat prostate carcinogenesis by induction of apoptosis in a transgenic rat for adenocarcinoma of prostate (TRAP) model. Luteolin decreased cell proliferation in a dose-dependent manner and induced apoptosis with the activation of caspases 3 and 7 in both rat (PCai1, established from a TRAP prostate tumor) and human (22Rv1) CRPC cells. Dietary luteolin also suppressed tumor growth via an increase in apoptosis and inhibition of angiogenesis in PCai1 and 22Rv1 xenografts implanted in castrated nude mice. We also focused on androgen receptor splice variant 7 (AR-V7), which contributes to cell proliferation and therapeutic resistance in CRPC. Luteolin dramatically suppressed AR-V7 protein expression in 22Rv1 cells in vitro and ex vivo. Microarray analysis identified MiR-8080, which contains a possible target sequence for AR-V7 3′-UTR, as a gene upregulated by luteolin. MiR-8080 transfection decreased the AR-V7 expression level and the induction of apoptosis in 22Rv1 cells. Furthermore, miR-8080 knockdown canceled luteolin decreasing AR-V7 and the cell growth of 22Rv1. MiR-8080 induced by luteolin intake enhanced the therapeutic effect of enzalutamide on 22Rv1 xenografts under castration conditions. These results indicate luteolin inhibits CRPC by AR-V7 suppression through miR-8080, highlighting luteolin and miR-8080 as promising therapeutic agents for this disease.

The spatially organized gene expression program within the liver specifies hepatocyte functions according to their relative distances to the bloodstream (i.e., zonation), contributing to liver homeostasis. Despite the knowledge that solid cancers remotely disrupt liver homeostasis, it remains unexplored whether solid cancers affect liver zonation. Here, using spatial transcriptomics, we thoroughly investigate the abundance and zonation of hepatic genes in cancer-bearing mice. We find that breast cancers affect liver zonation in various distinct manners depending on biological pathways. Aspartate metabolism and triglyceride catabolic processes retain relatively intact zonation patterns, but the zonation of xenobiotic catabolic process genes exhibits a strong disruption. The acute phase response is induced in zonated manners. Furthermore, we demonstrate that breast cancers activate innate immune cells in particular neutrophils in distinct zonated manners, rather than in a uniform fashion within the liver. Collectively, breast cancers disorganize hepatic transcriptomes in zonated manners, thereby disrupting zonated functions of the liver.

Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers’ protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.