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.

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Background: The objective of this study was to evaluate the accuracy of fine needle aspiration cytology (FNAC) of axillary lymph nodes (LN) in breast cancer, to compare the results of FNAC and pathological examination, and to distinguish patients with 1 to 2 metastatic LNs from those with ≥3 metastatic LNs in patients with FNAC-positive patients. Patients and Methods: This study included 198 breasts of 196 patients with breast cancer who underwent FNAC and surgery for the primary and axilla without neoadjuvant chemotherapy from January 2010 to August 2016. Axillary nodal status was assessed by ultrasound (US), and whether FNAC-positive had three or more suspicious LNs on US imaging was examined. Results: The results of FNAC were positive in 75 (38%), negative in 97 (49%), suspicious in 2 (1%), indeterminate in 5 (2.5%), and insufficient in 19 patients (9.5%). FNAC sensitivity, specificity, positive predictive value, and negative predictive value were 62.6%, 100%, 100%, and 62.0%, respectively. Whereas 53% (18/34) of patients with false-negative FNAC had one metastatic LN on final pathology, 61% (47/77) patients who were FNACpositive had three or more metastatic LNs. In the FNAC-positive patients, all patients had ≥3 metastatic LNs if they had ≥3 suspicious LNs on US imaging. Conclusion: Patients with positive cytology were more likely to have ≥3 positive LNs compared to false-negative cytology patients. Patients with ≥3 abnormal LNs on US and positive FNAC might require axillary dissection.

Background and Aims: Tolvaptan, an orally active vasopressin V2-receptor antagonist, has been used for patients with di cult-to-treat ascites in Japan. In this study, we conducted a genome-wide association study (GWAS) in the Japanese population to identify genetic variants associated with tolvaptan's e cacy for patients with hepatic ascites. Methods: From 2014 through 2018, genomic DNA samples were obtained from 550 patients who were treated with tolvaptan. Of those, 80 cases (non-responder; increase of body weight [BW]) and 333 controls (responder; > 1.5 kg decrease of BW) were included in the GWAS and replication study. Results: GWAS showed 5 candidate SNPs around the miR818, KIAA1109, and SVEP1 genes. After validation and performing a replication study, an SNP (rs2991364) located in the SVEP1 gene was found to have a signi cant genome-wide association (OR = 3.55, P = 2.01 x 10 − 8 ). Univariate and multivariate analyses showed that blood urea nitrogen (BUN) and SVEP1 SNP were signi cantly associated with the response (OR = 1.03, p = 0.02 and OR = 4.24, p < 0.0001, respectively). Based on a prediction model of logistic regression analysis in a population with the rs2991364 risk allele, the failure probability (= exp (score: 22.234 + BUN*0.077 + Na*-0.179) (1 + exp (score)) was determined for the detection of nonresponders. Assuming a cutoff of failure probability at 38.6%, sensitivity was 84.4%, speci city was 70% and AUC was 0.774. Conclusion: SVEP1 rs2991364 was identi ed as the speci c SNP for the tolvaptan response. The prediction score can identify tolvaptan non-responders and help to avoid a lengthy period of futile treatment.