Published papers

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Background: Previous research has shown a significant link between gut microbiota in children with autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD). However, much remains unknown because of the heterogeneity of disorders and the potential confounders such as dietary patterns and control group variations. Methods: Children aged 6–12 years who had been clinically diagnosed with ASD and/or ADHD, their unaffected neurotypical siblings, and non-related neurotypical volunteers were recruited cross-sectionally. The ASD diagnosis was confirmed using the Autism Diagnostic Observation Schedule-2 (ADOS-2) in all patients, including those with ADHD. Standardized DNA extraction and sequencing methods were used to compare gut microbial alpha-diversity among the groups. Dietary diversity was calculated from a standardized dietary questionnaire form. We compared the difference in gut microbiome between patients with ASD and/or ADHD with neurotypical siblings and non-related neurotypical controls. Results: Ninety-eight subjects were included in the study (18 with ASD, 19 with ADHD, 20 with both ASD and ADHD, 13 neurotypical siblings, and 28 non-related neurotypical controls). The alpha-diversity indices, such as Chao 1 and Shannon index, showed a significant difference between the groups in a Linear mixed-effect model (F(4, 93) = 4.539, p =.02), (F(4, 93) = 3.185, p =.017), respectively. In a post-hoc pairwise comparison, patients with ASD had lower alpha-diversity compared with non-related controls after Bonferroni correction. Dietary diversity shown in Shannon index did not differ among the groups (F(4, 84) = 1.494, p =.211). Conclusions: Our study indicates disorder-specific microbiome differences in patients with ASD. In future research on gut microbiota in neurodevelopmental disorders, it is necessary to consider the impact of ASD and ADHD co-occurrence, and strictly control for background information such as diet, to elucidate the gut–microbiota interaction in ASD and ADHD for exploring the potential of therapeutic interventions.

There is an unmet need for antifibrotic therapies to prevent the progression of liver cirrhosis. Previously, we conducted an exploratory trial to assess the safety and antifibrotic efficacy of PRI-724, a selective CBP/β-catenin inhibitor, in patients with liver cirrhosis. PRI-724 was well tolerated and exerted a potential antifibrotic effect. Here, we investigated whether the profiles of circulating microRNAs packaged in extracellular vesicles (EV-miRNAs) are associated with responses to liver fibrosis treatments. Eighteen patients who received PRI-724 for 12 weeks in a phase 1/2a study were classified as responders (n = 10) or non-responders (n = 8) based on changes in liver stiffness. Plasma samples were obtained before and after PRI-724 administration and the levels of EV-miRNAs were analyzed. Three miRNAs (miR-6510-5p, miR-6772-5p, and miR-4261) were identified as predictors of response or non-response to PRI-724, and the levels of three other miRNAs (miR-939-3p, miR-887-3p, and miR-7112-5p) correlated with the efficacy of treatment. Expression of miR-887-3p was detected in hepatocytes and was decreased significantly in liver tissue following PRI-724 treatment. In addition, transfection of a miR-887-3p mimic activated hepatic stellate cells. Thus, decreases in the miR-887-3p level in blood may reflect recovery from liver fibroses in patients with liver cirrhosis treated with PRI-724, although further validation studies are warranted to confirm this.

©2018 JCBN. The exact mechanisms of hepatocellular carcinoma development in nonalcoholic steatohepatitis remain unclear. In this study, we used a new class of highfat diet, which could induce hepatocellular carcinoma development without the use of general chemical carcinogens or knockout mice. We investigated the correlation between hepatocellular carcinoma and oxidative stress/anti oxidant effects after depletion of the gut microbiota by treatment with antibiotics. Mice fed with the steatohepatitisinducing high fat diet (STHD01) for 41 weeks developed hepatocellular carci noma. Antibiotictreatment in mice fed with STHD01 significantly depleted the gut microbiota and significantly ameliorated liver injury/histology. The tumor numbers of hepatocellular carcinoma were dramatically decreased by the antibioticstreatment. We analyzed the factors involved in oxidative stress and antioxidant effects. Oxidative stress was elevated in mice fed with STHD01, whereas some antioxidant factors were significantly elevated after antibiotics treatment. These results suggest that the gut microbiota is a key factor in improving oxidative stress induced by STHD01 feeding.

© 2018 The Author(s). Intrahepatic cholangiocarcinoma (IHCC) is a highly aggressive malignancy with a poor prognosis. It is thought to originate from cholangiocytes, which are the component cells of intrahepatic bile ducts. However, as patients with viral hepatitis often develop IHCC, it has been suggested that transformed hepatocytes may play a role in IHCC development. To investigate whether IHCC cells can be converted to functional hepatocytes, we established organoids derived from human IHCC and cultured them under conditions suitable for hepatocyte differentiation. IHCC organoids after hepatocyte differentiation acquired functions of mature hepatocytes such as albumin secretion, bile acid production and increased CYP3A4 activity. Studies using a mouse model of IHCC indicate that Wnt3a derived from macrophages recruited upon inflammation in the liver may promote the malignant transformation of hepatocytes to IHCC cells. The results of the present study support the recently proposed hypothesis that IHCC cells are derived from hepatocytes.

© Yamada et al. Gut microbiota plays a significant role in the development of hepatocellular carcinoma (HCC) in non-alcoholic steatohepatitis (NASH). However, understanding of the precise mechanism of this process remains incomplete. A new class steatohepatitis-inducing high-fat diet (HFD), namely STHD-01, can promote the development of HCC without the administration of chemical carcinogens. Using this diet, we comprehensively analyzed changes in the gut microbiota and its metabolic functions during the development of HCC in NASH. Mice fed the STHD-01 developed NASH within 9 weeks. NASH further progressed into HCC by 41 weeks. Treatment with antibiotics significantly attenuated liver pathology and suppressed tumor development, indicating the critical role of the gut microbiota in tumor development in this model. Accumulation of cholesterol and bile acids in the liver and feces increased after feeding the mice with STHD-01. Treatment with antibiotics did not reverse these phenotypes. In contrast, accumulation of secondary bile acids was dramatically reduced after the treatment with antibiotics, suggesting the critical role of the gut microbiota in the conversion of primary bile acids to secondary bile acids. Secondary bile acids such as deoxycholic acid activated the mTOR, pathway in hepatocytes. Activation of mTOR was observed in the liver of mice fed STHD-01, and the activation was reduced when mice were treated with antibiotics. Collectively, bile acid metabolism by the gut microbiota promotes HCC development in STHD-01- induced NASH.

© 2018 The Author(s). Background: We previously showed that knockdown of nuclear factor E2-related factor 2 (Nrf2) resulted in suppression of hepatitis C virus (HCV) infection. In this study, whether brusatol, an Nrf2 inhibitor, has dual anti-HCV and anticancer effects was explored. Methods: The anti-HCV effect of brusatol was investigated by analyzing HCV RNA and proteins in a hepatic cell line persistently-infected with HCV, HPI cells, and by analyzing HCV replication in a replicon-replicating hepatic cell line, OR6 cells. Then, dual anti-HCV and anticancer effects of brusatol and enhancement of the effects by the combination of brusatol with anticancer drugs including sorafenib, which has been reported to have the dual effects, were then investigated. Results: Brusatol suppressed the persistent HCV infection at both the RNA and protein levels in association with a reduction in Nrf2 protein in the HPI cells. Analysis of the OR6 cells treated with brusatol indicated that brusatol inhibited HCV persistence by inhibiting HCV replication. Combination of brusatol with an anticancer drug not only enhanced the anticancer effect but also, in the case of the combination with sorafenib, strongly suppressed HCV infection. Conclusions: Brusatol has dual anti-HCV and anticancer effects and can enhance the comparable effects of sorafenib. There is therefore the potential for combination therapy of brusatol and sorafenib for HCV-related hepatocellular carcinoma.

© The Author(s) 2017. Background: The increased use of generic drugs is a good indicator of the need to reduce the increasing costs of prescription drugs. Since there are more expensive drugs compared with other therapeutic areas, “oncology” is an important one for generic drugs. The primary objective of this article was to quantify the extent to which generic drugs in Japan occupy each level of the Anatomical Therapeutic Chemical (ATC) classification system. Methods: The dataset used in this study was created from publicly available information obtained from the IMS Japan Pharmaceutical Market database. Data on the total amount of sales and number of prescriptions for anti-cancer drugs between 2010 and 2016 in Japan were selected. The data were categorized according to the third level of the ATC classification system. Results: All categories of the ATC classification system had increased market shares in Japan between 2010 and 2016. The barriers to market entry were relatively low in L01F (platinum anti-neoplastics), L01C (plant-based neoplastics), L02B (cytostatic hormone antagonists), and L01D (anti-neoplastic antibiotics) but were high in L02A (cytostatic hormones), L01H (protein kinase inhibitors), and L01B (anti-metabolites). Conclusions: Generic cancer drugs could bring savings to Japanese health care systems. Therefore, their development should be directed toward niche markets, such as L02A, L01H, and L01B, and not competitive markets.

© The Author(s) 2018. Background: Despite their benefits, the rapid development of new cancer treatments has been a significant driver of increasing health care expenditures in the face of limited health care budgets. In this study, we analyzed the prescribing trends for anticancer drugs from 2010 through 2016 in Japan and sought to identify unique trends that could provide a basis for future medical economic research aiming to develop more efficacious and cost-effective cancer therapies. Methods: We used publicly available marketing data for anticancer drugs in Japan for 2010-2016. The drugs selected for this research were categorized according to the Anatomical Therapeutic Chemical Classification System. We investigated the overall anticancer drug market size, the number of anticancer drugs, the top 30 selling anticancer categories, sales and prescription volumes, and changes in sales and prescription volumes between 2010 and 2016 in the country. Results: The anticancer agent market expanded each year from 2010 to 2016, with sales exceeding 1 trillion yen in 2015. The proportion of molecular targeted drugs (antineoplastic mAbs and protein kinase inhibitors) among the top 30 selling anticancer categories has continued to increase, and both the sales and prescription volumes of these drugs exceeded those of drugs in other categories, suggesting that these treatments play a dominant role in cancer pharmacotherapy. Conclusion: The availability and increasing use of innovative but more expensive targeted therapies were major drivers of increases in pharmaceutical expenditures for cancer treatment in Japan. Therefore, the effective use of genetic testing can mitigate these rising costs.

© 2019 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd Biliary tract cancers and pancreatic cancers are aggressive malignancies that are difficult to diagnose early and have a poor prognosis. Patients with inoperable biliary tract and pancreatic cancers generally receive chemotherapy regimens including gemcitabine. However, the effects of these drugs are limited, and the 5-year survival rates of patients are very low. The newly developed three-dimensional culture system known as “organoid culture” allows long-term expansion of stem cells into cyst-like structures (organoids) with properties resembling those of the original tissues. We and other groups have successfully established long-term in vitro cultures of organoids derived from biliary tract and pancreatic cancers. Organoids derived from biliary tract and pancreatic cancers closely recapitulate the properties of the original tumors including genetic alterations, gene expression profiles, and histopathological structures. These patient-derived cancer organoids can be applied for drug sensitivity testing, drug screening, epigenetic therapy, and differentiation-inducing therapy to identify therapeutic agents optimal for each patient. We intend to further establish organoids derived from various cancer cases and construct an organoid bank of biliary tract and pancreatic cancers. These powerful in vitro preclinical models of refractory cancers may bridge the gap between basic research and clinical trials and allow personalized therapy for patients.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Eradication of Helicobacter pylori (H. pylori) is an effective strategy for preventing various gastrointestinal diseases such as gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. However, the eradication success rate is decreasing because of a recent increase in drug-resistant strains of H. pylori. Here, we evaluated the success rate of eradication therapy with vonoprazan (VPZ), a new potassium-competitive acid blocker, against drug-resistant H. pylori. In total, 793 patients who received H. pylori eradication therapy were investigated retrospectively. All underwent esomeprazole (EPZ)-based triple therapy (n = 386) or VPZ-based triple therapy (n = 407) for first-, second- and third-line H. pylori eradication for 7 days. The overall success rates of first- and third-line H. pylori eradication were significantly higher for VPZ-based triple therapy (88.4% and 93.0%, respectively, per protocol (PP)) than for EPZ-based triple therapy (69.5% and 56.5%, respectively, PP). Moreover, the success rates of first- and third-line eradication of clarithromycin (CLR)- and sitafloxacin (STFX)-resistant H. pylori were significantly higher for VPZ-based triple therapy (72.0% and 91.7%, PP) than for EPZ-based triple therapy (38.5% and 20.0%, PP). In addition, patient age did not affect the eradication rate of VPZ-based first-line therapy, whereas the success rate of EPZ-based therapy was lower in patients under 65 years of age. Our results clearly demonstrated that VPZ-based therapy achieved a higher eradication rate even against CLR- and STFX-resistant H. pylori, and that patient age did not affect the eradication rate of VPZ-based therapy. These findings suggest that dual therapy using VPZ and amoxicillin may be sufficient for standard H. pylori eradication, and may thus also be beneficial for avoiding antibiotic misuse.

© 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group. Helicobacter pylori-derived CagA, a type IV secretion system effector, plays a role as an oncogenic driver in gastric epithelial cells. However, upon delivery into gastric epithelial cells, CagA is usually degraded by macroautophagy/autophagy. Hence, the induction of autophagy in H. pylori-infected epithelial cells is an important host-protective ability against gastric carcinogenesis. However, the mechanisms by which autophagosome-lysosome fusion is regulated, are unknown. Here, we report that enhancement of LAMP1 (lysosomal associated membrane protein 1) expression is necessary for autolysosome formation. LAMP1 expression is induced by nuclear translocated LRP1 (LDL receptor related protein 1) intracellular domain (LRP1-ICD) binding to the proximal LAMP1 promoter region. Nuclear translocation of LRP1-ICD is enhanced by H. pylori infection. In contrast, CAPZA1 (capping actin protein of muscle Z-line alpha subunit 1) inhibits LAMP1 expression via binding to LRP1-ICD in the nuclei. The binding of CAPZA1 to LRP1-ICD prevents LRP1-ICD binding to the LAMP1 proximal promoter. Thus, in CAPZA1-overexpressing gastric epithelial cells infected with H. pylori, autolysosome formation is inhibited and CagA escapes autophagic degradation. These findings identify CAPZA1 as a novel negative regulator of autolysosome formation and suggest that deregulation of CAPZA1 expression leads to increased risk of gastric carcinogenesis. Abbreviations: CagA: cytotoxin-associated gene A; CAPZA1: capping actin protein of muscle Z-line alpha subunit 1; ChIP: chromatin immunoprecipitation; GTF2I: general transcription factor IIi; HDAC: histone deacetylase; LAMP1: lysosomal associated membrane protein 1; LRP1: LDL receptor related protein 1; LRP1-ICD: CagA intracellular domain; qPCR: quantitative polymerase chain reaction; VacA: vacuolating cytotoxin.

© 2019 The Japan Society of Hepatology Aims: Early tumor recurrence (ETR) after hepatic resection is a crucial predictor of poor prognosis in patients with hepatocellular carcinoma (HCC). The aim of this study was to identify clinically significant serum microRNAs (miRNAs) involved in the ETR of HCC. Methods: We compared expression profiles of circulating miRNAs from serum samples between five HCC patients with ETR (recurrence within 12 months after hepatectomy) and five HCC patients without recurrence using microarray analysis of miRNA. The identified miRNA associated with ETR was further verified in 121 HCC patients, 73 liver disease patients, and 15 health controls by real-time quantitative reverse transcription–polymerase chain reaction (PCR). Results: Of the approximately 2000 miRNAs analyzed, we identified 15 miRNAs for which expression levels correlated significantly with ETR. Of these miRNAs, we further investigated expression of miRNA-1246 (miR-1246). Quantitative PCR confirmed that miR-1246 was upregulated in HCC with ETR, compared to the level in HCC without ETR (P < 0.001). Serum miR-1246 showed a receiver operating characteristic curve area of 0.762, with 77.4% specificity and 54.1% sensitivity in discriminating HCC patients with ETR from HCC patients without ETR. Altered expression of miR-1246 was associated with aggressive tumor characteristics, including tumor–node–metastasis classification (P = 0.0413), tumor differentiation (P = 0.0419), and portal vein invasion (P = 0.0394). Moreover, multivariate Cox regression analysis identified serum miR-1246 level as an independent risk factor for overall survival (hazard ratio, 2.784; 95% confidence interval, 1.528–5.071; P = 0.0008). Conclusion: Circulating miR-1246 in serum has strong potential as a novel ETR and prognostic biomarker for HCC.

© 2019 The Author(s). Liver and hepatocyte transplantation are the only effective therapies for late-stage liver diseases, in which the liver loses its regenerative capacity. However, there is a shortage of donors. As a potential alternative approach, functional hepatocytes were recently generated from various cell sources. Analysis of drug metabolism in the human liver is important for drug development. Consequently, cells that metabolize drugs similar to human primary hepatocytes are required. This review discusses the current challenges and future perspectives concerning hepatocytes and hepatic progenitor cells that have been reprogrammed from various cell types, focusing on their functions in transplantation models and their ability to metabolize drugs.

© Katsuda et al. Hepatocytes are regarded as the only effective cell source for cell transplantation to treat liver diseases; however, their availability is limited due to a donor shortage. Thus, a novel cell source must be developed. We recently reported that mature rodent hepatocytes can be reprogrammed into progenitor-like cells with a repopulative capacity using small molecule inhibitors. Here, we demonstrate that hepatic progenitor cells can be obtained from human infant hepatocytes using the same strategy. These cells, named human chemically induced liver progenitors (hCLiPs), had a significant repopulative capacity in injured mouse livers following transplantation. hCLiPs redifferentiated into mature hepatocytes in vitro upon treatment with hepatic maturation-inducing factors. These redifferentiated cells exhibited cytochrome P450 (CYP) enzymatic activities in response to CYP-inducing molecules and these activities were comparable with those in primary human hepatocytes. These findings will facilitate liver cell transplantation therapy and drug discovery studies.

© 2019 The Author(s) Biological Sciences; Genetics; Genomics; Cancer

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Cancer cells are strongly dependent on the glycolytic pathway for generation of energy even under aerobic condition through a phenomenon known as the Warburg effect. Rapid proliferation of cancer cells is often accompanied by high glucose consumption and abnormal angiogenesis, which may lead to glucose depletion. In the present study, we investigated how cholangiocarcinoma cells adapt to glucose depletion using a 3D organoid culture system. We cultured organoids derived from cholangiocarcinoma under glucose-free condition and investigated cell proliferation, expression of stem cell markers and resistance to gemcitabine. Cholangiocarcinoma organoids cultured under glucose-free condition showed reduced proliferation but were able to survive. We also observed an increase in the expression of stem cell markers including LGR5 and enhancement of stem cell phenotypic characteristics such as resistance to gemcitabine through AKT phosphorylation and reactive oxygen species. These findings indicate that cholangiocarcinoma cells are able to adapt to glucose depletion through enhancement of their stem cell phenotype in response to changes in microenvironmental conditions.

© 2018, The Author(s). To understand the molecular features underlying stem cell aging, we established intestinal epithelial organoids derived from both young and aged mice and investigated alterations in their senescence and epigenetic status. Senescence-related changes including accumulation of senescence-associated β-galactosidase and up-regulation of Cdkn1a (p21) by DNA demethylation were observed in intestinal epithelial organoids derived from aged mice. We also demonstrated that the important stem cell marker Lgr5 was epigenetically silenced by trimethylation of histone H3 lysine 27, inducing suppression of Wnt signaling and a decrease of cell proliferation in organoids from aged mice. We further treated intestinal epithelial organoids from aged mice with nicotinamide mononucleotide (NMN), a key NAD + intermediate. As a result, the organoids showed a higher NAD + level, increased cell proliferative ability, activation of Lgr5 and suppression of senescence-associated genes, indicating that treatment with NMN could ameliorate senescence-related changes in intestinal epithelia. These findings suggest that organoids derived from aged animals could be a powerful research tool for investigating the molecular mechanisms underlying stem cell aging and for development of some form of anti-aging intervention, thus contributing to prolongation of healthy life expectancy.