Xiaochen Sun

A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer.

Background & Aims The lack of a preclinical model of progressive non-alcoholic steatohepatitis (NASH) that recapitulates human disease is a barrier to therapeutic development. Methods A stable isogenic cross between C57BL/6J (B6) and 129S1/SvImJ (S129) mice were fed a high fat diet with ad libitum consumption of glucose and fructose in physiologically relevant concentrations and compared to mice fed a chow diet and also to both parent strains. Results Following initiation of the obesogenic diet, B6/129 mice developed obesity, insulin resistance, hypertriglyceridemia and increased LDL-cholesterol. They sequentially also developed steatosis (4–8 weeks), steatohepatitis (16–24 weeks), progressive fibrosis (16 weeks onwards) and spontaneous hepatocellular cancer (HCC). There was a strong concordance between the pattern of pathway activation at a transcriptomic level between humans and mice with similar histological phenotypes (FDR 0.02 for early and 0.08 for late time points). Lipogenic, inflammatory and apoptotic signaling pathways activated in human NASH were also activated in these mice. The HCC gene signature resembled the S1 and S2 human subclasses of HCC (FDR 0.01 for both). Only the B6/129 mouse but not the parent strains recapitulated all of these aspects of human NAFLD. Conclusions We here describe a diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND) that recapitulates the key physiological, metabolic, histologic, transcriptomic and cell-signaling changes seen in humans with progressive NASH. Lay summary We have developed a diet-induced mouse model of non-alcoholic steatohepatitis (NASH) and hepatic cancers in a cross between two mouse strains (129S1/SvImJ and C57Bl/6J). This model mimics all the physiological, metabolic, histological, transcriptomic gene signature and clinical endpoints of human NASH and can facilitate preclinical development of therapeutic targets for NASH.

A genomic and clinical prognostic index for hepatitis C-related early-stage cirrhosis that predicts clinical deterioration

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/mm3), 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.

β-PDGF Receptor Expressed by Hepatic Stellate Cells Regulates Fibrosis in Murine Liver Injury, but Not Carcinogenesis

Background & Aims Rapid induction of β-PDGF receptor (β-PDGFR) is a core feature of hepatic stellate cell activation, but its cellular impact in vivo is not well characterized. We explored the contribution of β-PDGFR-mediated pathway activation to hepatic stellate cell responses in liver injury, fibrogenesis, and carcinogenesis in vivo using genetic models with divergent β-PDGFR activity, and assessed its prognostic implications in human cirrhosis. Methods The impact of either loss or constitutive activation of β-PDGFR in stellate cells on fibrosis was assessed following carbon tetrachloride (CCl4) or bile duct ligation. Hepatocarcinogenesis in fibrotic liver was tracked after a single dose of diethylnitrosamine (DEN) followed by repeated injections of CCl4. Genome-wide expression profiling was performed from isolated stellate cells that expressed or lacked β-PDGFR to determine deregulated pathways and evaluate their association with prognostic gene signatures in human cirrhosis. Results Depletion of β-PDGFR in hepatic stellate cells decreased injury and fibrosis in vivo, while its auto-activation accelerated fibrosis. However, there was no difference in development of DEN-induced pre-neoplastic foci. Genomic profiling revealed ERK, AKT, and NF-kB pathways and a subset of a previously identified 186-gene prognostic signature in hepatitis C virus (HCV)-related cirrhosis as downstream of β-PDGFR in stellate cells. In the human cohort, the β-PDGFR signature was not associated with HCC development, but was significantly associated with a poorer outcome in HCV cirrhosis. Conclusions β-PDGFR is a key mediator of hepatic injury and fibrogenesis in vivo and contributes to the poor prognosis of human cirrhosis, but not by increasing HCC development.

Clinicopathological indices to predict hepatocellular carcinoma molecular classification.

Hepatocellular carcinoma (HCC) is the second most lethal cancer caused by lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti‐HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification.

The XBP1 Arm of the Unfolded Protein Response Induces Fibrogenic Activity in Hepatic Stellate Cells Through Autophagy

Autophagy and the unfolded protein response (UPR) both promote activation of hepatic stellate cells (HSC), however the link between the two stimuli remains unclear. Here we have explored the role of X-box binding protein 1 (XBP1), one of three UPR effector pathways and sought to establish the interdependence between autophagy and the UPR during HSC activation. XBP1 induction accompanied both culture-based HSC activation and ER stress induced by tunicamycin. Ectopic overexpression of XBP1 induced collagen 1-alpha expression in HSCs, which was inhibited by knockdown of ATG7, a critical autophagy mediator. Genome-wide transcriptomic profiling indicated an upregulation of collagen synthesis pathways, but not of the transforming growth factor (TGF)-b pathway, a canonical fibrogenic driver, suggesting that XBP1 activates a specific subset of fibrogenesis pathways independent of TGF-β1. XBP1 target gene signatures were significantly induced in rodent liver fibrosis models (n = 3–5) and in human samples of non-alcoholic fatty liver disease (NAFLD) (n = 72–135). Thus, XBP1-mediated UPR contributes to fibrogenic HSC activation and is functionally linked to cellular autophagy.

Molecular subclasses of hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition

A recent gene expression classification of hepatocellular carcinoma (HCC) includes a poor survival subclass termed S2 representing about one‐third of all HCC in clinical series. S2 cells express E‐cadherin and c‐myc and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between S2 and non‐S2 HCC, this study investigated whether molecular subclasses of HCC predict sensitivity to FGFR inhibition. S2 cell lines were significantly more sensitive (p < 0.001) to the FGFR inhibitors BGJ398 and AZD4547. BGJ398 decreased MAPK signaling in S2 but not in non‐S2 cell lines. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 44% or more in all five S2 cell lines (p < 0.05 for each cell line), a significantly greater decrease than seen with knockdown of FGFR1‐3 with siRNA transfection. FGFR4 knockdown decreased MAPK signaling in S2 cell lines, but little effect was seen with knockdown of FGFR1‐3. In conclusion, the S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4‐MAPK signaling plays an important role in driving proliferation of a molecular subclass of HCC. This classification system may help to identify those patients who are most likely to benefit from inhibition of this pathway.

Loss of DNA methylation in zebrafish embryos activates retrotransposons to trigger antiviral signaling

ABSTRACT Complex cytoplasmic nucleotide-sensing mechanisms can recognize foreign DNA based on a lack of methylation and initiate an immune response to clear the infection. Zebrafish embryos with global DNA hypomethylation caused by mutations in the ubiquitin-like with PHD and ring finger domains 1 (uhrf1) or DNA methyltransferase 1 (dnmt1) genes exhibit a robust interferon induction characteristic of the first line of defense against viral infection. We found that this interferon induction occurred in non-immune cells and examined whether intracellular viral sensing pathways in these cells were the trigger. RNA-seq analysis of uhrf1 and dnmt1 mutants revealed widespread induction of Class I retrotransposons and activation of cytoplasmic DNA viral sensors. Attenuating Sting, phosphorylated Tbk1 and, importantly, blocking reverse transcriptase activity suppressed the expression of interferon genes in uhrf1 mutants. Thus, activation of transposons in cells with global DNA hypomethylation mimics a viral infection by activating cytoplasmic DNA sensors. This suggests that antiviral pathways serve as surveillance of cells that have derepressed intragenomic parasites due to DNA hypomethylation. Highlighted article: Globally hypomethylated uhrf1 and dnmt1 mutants mount an interferon response, marking epigenetically damaged cells for immune clearance and limiting transposon activity.

A cell culture system for distinguishing hepatitis C viruses with and without liver cancer-related mutations in the viral core gene

BACKGROUND & AIMS Although patients infected by genotype 1b hepatitis C virus (HCV) with Q(70) and/or M(91)core gene mutations have an almost five-fold increased risk of developing hepatocellular carcinoma (HCC) and increased insulin resistance, the absence of a suitable experimental system has precluded direct experimentation on the effects of these mutations on cellular gene expression. METHODS HuH7 cells were treated long-term with human serum to induce differentiation and to produce a model system for testing high-risk and control HCV. For clinical validation, profiles of infected cells were compared to each other and to those of liver biopsies of patients with early-stage HCV-related cirrhosis followed prospectively for up to 23 years (n=216). RESULTS Long-term culture in human serum produced growth-arrested, hepatocyte-like cells whose gene profile overlapped significantly with that of primary human hepatocytes. High-risk (Q(70)/M(91)) and control (R(70)/L(91)) viruses had dramatically different effects on gene expression of these cells. The high-risk virus enhanced expression of pathways associated with cancer and type II diabetes, while the control virus enhanced pathways associated with oxidative phosphorylation. Of special clinical relevance, the transcriptome of cells replicating the high-risk virus correlated significantly with an HCC high-risk profile in patients (Bonferroni-corrected p=0.03), whereas no such association was observed for non-HCC-related clinical outcomes. CONCLUSIONS The cell-based system allowed direct head-to-head comparison of HCV variants, and provided experimental support for previous clinical data indicating an oncogenic effect of core gene mutations. This simple experimental system distinguished HCV variants and will enable future mechanistic analysis and exploration of interventional approaches.

Survival analysis tools in genomics research.

There is an increasing demand to determine the clinical implication of experimental findings in molecular biomedical research. Survival (or failure time) analysis methodologies have been adapted to the analysis of genomics data to link molecular information with clinical outcomes of interest. Genome-wide molecular profiles have served as sources for discovery of predictive/prognostic biomarkers as well as therapeutic targets in the past decade. In this review, we overview currently available software, web applications, and databases specifically developed for survival analysis in genomics research and discuss issues in assessing clinical utility of molecular features derived from genomic profiling.

Prognostic gene signature profiles of hepatitis C-related early-stage liver cirrhosis

The rate of hepatitis C virus (HCV) related liver cirrhosis and subsequent cancer development is increasing and raising the risk of related mortality and morbidity. To address this issue, we aimed to develop a prognostic index that can be used to stratify patients for risk of disease progression. This index was developed in part by using a gene signature test implemented in a clinically applicable digital transcript counting platform (NanoString nCounter system). A cohort of 145 U.S. patients with HCV-related early-stage cirrhosis was analyzed by using the assay. This dataset (GEO accession number GPL17230) provides information of expression levels of the prognostic genes in the cohort.