Description: Objective Precancerous lesion, a well-established histopathologically premalignant tissue with the highest risk for tumourigenesis, develops preferentially from activation of DNA damage checkpoint and persistent inflammation. However, little is known about the mechanisms by which precancerous lesions are initiated and their physiological significance. Design Laser capture microdissection was used to acquire matched normal liver, precancerous lesion and tumour tissues. miR-484 –/– , Ifnar1 –/– and Tgfbr2 hep mice were employed to determine the critical role of the interferon (IFN)–microRNA pathway in precancerous lesion formation and tumourigenesis. RNA immunoprecipitation (RIP), pull-down and chromatin immunoprecipitation (ChIP) assays were applied to explore the underlying mechanisms. Results miR-484 is highly expressed in over 88% liver samples clinically. DEN-induced precancerous lesions and hepatocellular carcinoma were dramatically impaired in miR-484 –/– mice. Mechanistically, ectopic expression of miR-484 initiates tumourigenesis and cell malignant transformation through synergistic activation of the transforming growth factor-β/Gli and nuclear factor-B/type I IFN pathways . Specific acetylation of H3K27 is indispensable for basal IFN-induced continuous transcription of miR-484 and cell transformation. Convincingly, formation of precancerous lesions were significantly attenuated in both Tgfbr2 hep and Ifnar1 –/– mice. Conclusions These findings demonstrate a new protumourigenic axis involving type I IFN–microRNA signalling, providing a potential therapeutic strategy to manipulate or reverse liver precancerous lesions and tumourigenesis.

Description: Background Non-alcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma (HCC). However, the mechanistic pathways that link both disorders are essentially unknown. Objective Our study was designed to investigate the role of microRNA-21 in the pathogenesis of NAFLD and its potential involvement in HCC. Methods Wildtype mice maintained on a high fat diet (HFD) received tail vein injections of microRNA-21-anti-sense oligonucleotide (ASO) or miR-21 mismatched ASO for 4 or 8 weeks. Livers were collected after that time period for lipid content and gene expression analysis. Human hepatoma HepG2 cells incubated with oleate were used to study the role of miR-21 in lipogenesis and analysed with Nile-Red staining. microRNA-21 function in carcinogenesis was determined by soft-agar colony formation, cell cycle analysis and xenograft tumour assay using HepG2 cells. Results The expression of microRNA-21 was increased in the livers of HFD-treated mice and human HepG2 cells incubated with fatty acid. MicroRNA-21 knockdown in those mice and HepG2 cells impaired lipid accumulation and growth of xenograft tumour. Further studies revealed that Hbp1 was a novel target of microRNA-21 and a transcriptional activator of p53 . It is well established that p53 is a tumour suppressor and an inhibitor of lipogenesis by inhibiting Srebp1c . As expected, microRNA-21 knockdown led to increased HBP1 and p53 and subsequently reduced lipogenesis and delayed G1/S transition, and the additional treatment of HBP1 -siRNA antagonised the effect of microRNA-21-ASO, suggesting that HBP1 mediated the inhibitory effects of microRNA-21-ASO on both hepatic lipid accumulation and hepatocarcinogenesis. Mechanistically, microRNA-21 knockdown induced p53 transcription, which subsequently reduced expression of genes controlling lipogenesis and cell cycle transition. In contrast, the opposite result was observed with overexpression of microRNA-21, which prevented p53 transcription. Conclusions Our findings reveal a novel mechanism by which microRNA-21, in part, promotes hepatic lipid accumulation and cancer progression by interacting with the Hbp1 - p53 - Srebp1c pathway and suggest the potential therapeutic value of microRNA-21-ASO for both disorders.

Description: Objective Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor ( Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. Design We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. Results Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5 neg or low cell population but was distinct from typical antral Lgr5 high stem cells. Treatment with progastrin interconverts Lgr5 neg or low CCK2R+ cells into Lgr5 high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. Conclusions CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

Description: Objective The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. Design We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (〉1 mg/dL) and platelet count (〈100 000/mm 3 ), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years). Results In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p〈0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child–Turcotte–Pugh class (HR=6.70, p〈0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p〈0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p〈0.001) and all liver-related adverse events (HR=4.98, p〈0.001). Conclusions A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.

Description: Objective The investigation regarding the clinical significance of quantitative hepatitis B core antibody (anti-HBc) during chronic hepatitis B (CHB) treatment is limited. The aim of this study was to determine the performance of anti-HBc as a predictor for hepatitis B e antigen (HBeAg) seroconversion in HBeAg-positive CHB patients treated with peginterferon (Peg-IFN) or nucleos(t)ide analogues (NUCs), respectively. Design This was a retrospective cohort study consisting of 231 and 560 patients enrolled in two phase IV, multicentre, randomised, controlled trials treated with Peg-IFN or NUC-based therapy for up to 2 years, respectively. Quantitative anti-HBc evaluation was conducted for all the available samples in the two trials by using a newly developed double-sandwich anti-HBc immunoassay. Results At the end of trials, 99 (42.9%) and 137 (24.5%) patients achieved HBeAg seroconversion in the Peg-IFN and NUC cohorts, respectively. We defined 4.4 log 10 IU/mL, with a maximum sum of sensitivity and specificity, as the optimal cut-off value of baseline anti-HBc level to predict HBeAg seroconversion for both Peg-IFN and NUC. Patients with baseline anti-HBc ≥4.4 log 10 IU/mL and baseline HBV DNA 〈9 log 10 copies/mL had 65.8% (50/76) and 37.1% (52/140) rates of HBeAg seroconversion in the Peg-IFN and NUC cohorts, respectively. In pooled analysis, other than treatment strategy, the baseline anti-HBc level was the best independent predictor for HBeAg seroconversion (OR 2.178; 95% CI 1.577 to 3.009; p〈0.001). Conclusions Baseline anti-HBc titre is a useful predictor of Peg-IFN and NUC therapy efficacy in HBeAg-positive CHB patients, which could be used for optimising the antiviral therapy of CHB.

Description: Dear Editor, We read with interest the paper by Angeli et al 1 proposing the value of the acute-on-chronic liver failure (ACLF) stratification in the prediction of short-term mortality in patients with acute decompensation of cirrhosis. Other studies 2 3 indicated that the dynamic changes in serological markers are associated with patients who have cirrhosis. We hypothesised that the pathological processes of HBV-related ACLF (HBV-ACLF) would result in specific changes in the levels of signalling proteins in the blood. Thus, we aimed to search detectable biomarkers to predict the outcome of HBV-ACLF. We collected serum samples from 420 patients with HBV-ACLF, 121 patients with chronic hepatitis B (CHB) and 25 normal controls to identify novel serological biomarkers of HBV-ACLF (see online supplementary table S1). As an initial screening group, 15 subjects were included in the cytokine antibody array analyses (n=5 per group). The remaining 551...

Description: Background Ovarian serous carcinoma (OSC) is the most common ovarian epithelial malignancy. Despite new medical and surgical advances, the overall 5-year survival for OSC remains poor. There is an important need to determine diagnostic and prognostic markers for this disease. Eph receptors are the largest known family of receptor tyrosines characterised in humans. These receptors are involved in the development and progression of various diseases including cancer. EphB6 contains kinase domains that are altered in several conserved amino acids and is catalytically inactive. The aim of the present study was to correlate the immunohistological expression of EphB6 in a cohort of patients with epithelial ovarian tumours with clinicopathological parameters and survival. Methods In this study we examined the expression of EphB6 protein in 55 cases of OSC, 24 cases of benign ovarian serous tumours, 37 cases of serous borderline tumours and 20 cases with normal fallopian tubes by immunohistochemical staining with a polyclonal anti-EphB6 antibody. The relationship between EphB6 expression and pathological parameters was analysed. Kaplan–Meier survival function was used to analyse the prognosis. Results High expression of EphB6 was observed in 100% (20/20) of normal fallopian tube samples, 100% (24/24) of benign epithelial ovarian tumours, 78% (29/37) of ovarian serous borderline tumours and 18% (10/55) of OSCs (p〈0.001). The expression of EphB6 was significantly associated with grade (p〈0.001) and TNM stage (p=0.017). EphB6 expression was reversely related to Ki-67 (p=0.021). The survival analysis showed that patients with negative or weak expression of EphB6 protein had a poorer outcome than those with positive expression (p=0.046). Conclusions Our results show that EphB6 is a new biomarker for distinguishing high- and low-grade OSC, and may be a potential prognostic marker in OSCs.

Description: Objective Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD. Design We determined BVES promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured BVES mRNA levels in a tissue microarray consisting of normal colons and CAC samples. Bves –/– and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels. Results BVES mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, BVES promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, Bves was underexpressed in experimental inflammatory carcinogenesis, and Bves –/– mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of Bves –/– tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction. Conclusion Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. BVES promoter methylation status may serve as a CAC biomarker.

Description: Objective Stem cell transplantation provides a promising alternative for the treatment of fulminant hepatic failure (FHF). However, it lacks fundamental understanding of stem cells’ activities. Our objective was to clarify stem cell-recipient interactions for overcoming barriers to clinical application. Design We used an in-house large-animal (pig) model of FHF rescue by human bone marrow mesenchymal stem cells (hBMSCs) and profiled the cells’ activities. The control and transplantation groups of pigs (n=15 per group) both received a D-galactosamine (D-Gal) injection (1.5 g/kg). The transplantation group received hBMSCs via intraportal vein infusion (3 x 10 6 cells/kg) immediately after D-Gal administration. The stem cell-recipient interactions were quantitatively evaluated by biochemical function, cytokine array, metabolite profiling, transcriptome sequencing and immunohistochemistry. Results All pigs in the control group died within an average of 3.22 days, whereas 13/15 pigs in the transplantation group lived 〉14 days. The cytokine array and metabolite profiling analyses revealed that hBMSC transplantation suppressed D-Gal-induced life-threatening cytokine storms and stabilised FHF within 7 days, while human-derived hepatocytes constituted only ~4.5% of the pig hepatocytes. The functional synergy analysis of the observed profile changes indicated that the implanted hBMSCs altered the pigs’ cytokine responses to damage through paracrine effects. Delta-like ligand 4 was validated to assist liver restoration in both pig and rat FHF models. Conclusions Our results delineated an integrated model of the multifaceted interactions between stem cells and recipients, which may open a new avenue to the discovery of single molecule-based therapeutics that simulate stem cell actions.

Description: Background Usher syndrome is a genetically heterogeneous disorder featured by combined visual impairment and hearing loss. Despite a dozen of genes involved in Usher syndrome having been identified, the genetic basis remains unknown in 20–30% of patients. In this study, we aimed to identify the novel disease-causing gene of a distinct subtype of Usher syndrome. Methods Ophthalmic examinations and hearing tests were performed on patients with Usher syndrome in two consanguineous families. Target capture sequencing was initially performed to screen causative mutations in known retinal disease-causing loci. Whole exome sequencing (WES) and whole genome sequencing (WGS) were applied for identifying novel disease-causing genes. RT-PCR and Sanger sequencing were performed to evaluate the splicing-altering effect of identified CEP78 variants. Results Patients from the two independent families show a mild Usher syndrome phenotype featured by juvenile or adult-onset cone–rod dystrophy and sensorineural hearing loss. WES and WGS identified two homozygous rare variants that affect mRNA splicing of a ciliary gene CEP78 . RT-PCR confirmed that the two variants indeed lead to abnormal splicing, resulting in premature stop of protein translation due to frameshift. Conclusions Our results provide evidence that CEP78 is a novel disease-causing gene for Usher syndrome, demonstrating an additional link between ciliopathy and Usher protein network in photoreceptor cells and inner ear hair cells.