RNA binding protein Apobec1 Complementation Factor (A1CF) regulates posttranscriptional ApoB mRNA editing but the range of RNA targets and long-term impact of altered A1CF expression on liver function are unknown. Here we studied hepatocyte-specific A1cf transgenic (A1cf +/Tg), A1cf+/Tg Apobec1–/– and A1cf –/– mice fed chow or high fat/high fructose diets using RNA-Seq, RNA-CLIP Seq and tissue microarrays from human hepatocellular cancer (HCC). A1cf +/Tg mice exhibited increased hepatic proliferation and steatosis, with increased lipogenic gene expression (Mogat1, Mogat2, Cidea, Cd36) associated with shifts in polysomal RNA distribution. Aged A1cf +/Tg mice developed spontaneous fibrosis, dysplasia and HCC, which was accelerated on a high fat/fructose diet and independent of Apobec1. RNA-Seq revealed increased expression of mRNAs involved in oxidative stress (Gstm3, Gpx3, Cbr3), inflammatory response (Il19, Cxcl14, Tnfα, Ly6c), extracellular matrix organization (Mmp2, Col1a1, Col4a1), proliferation (Kif20a, Mcm2, Mcm4, Mcm6) with a subset of mRNAs (including Sox4, Sox9, Cdh1) identified in RNA CLIP-Seq. Increased A1CF expression in human HCC correlated with advanced fibrosis and with reduced survival in a subset with nonalcoholic fatty liver disease. In conclusion, we show that hepatic A1CF overexpression selectively alters polysomal distribution and mRNA expression, promoting lipogenic, proliferative and inflammatory pathways leading to HCC.
Valerie Blanc, Jesse D. Riordan, Saeed Soleymanjahi, Joseph Nadeau, ILKe Nalbantoglu, Yan Xie, Elizabeth A. Molitor, Blair B. Madison, Elizabeth M. Brunt, Jason C. Mills, Deborah C. Rubin, Irene O.L. Ng, Yeonjung Ha, Lewis R. Roberts, Nicholas O. Davidson
Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus–mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.
Ali Dehnad, Weiguo Fan, Joy X. Jiang, Sarah R. Fish, Yuan Li, Suvarthi Das, Gergely Mozes, Kimberly A. Wong, Kristin A. Olson, Gregory W. Charville, Mohammed Ali, Natalie J. Török
BACKGROUND. Despite an increasing appreciation of the roles that myeloid cells play in tumor progression and therapy, challenges remain in interpreting the tumor-associated myeloid response balance and its translational value. We aimed to construct a simple and reliable myeloid signature for hepatocellular carcinoma (HCC). METHODS. Using in situ immunohistochemistry, we assessed the distribution of major myeloid subtypes in both peri- and intratumoral regions of HCC. A 2-feature-based, myeloid-specific prognostic signature, named the myeloid response score (MRS), was constructed using an L1-penalized Cox regression model based on data from a training subset (n=244) and in a test subset (n=244), an independent internal (n=341), and two external (n= 94; n=254) cohorts. RESULTS. The MRS and the MRS-based nomograms displayed remarkable discriminatory power, accuracy, and clinical usefulness for predicting recurrence and patient survival, superior to current staging algorithms. Moreover, an increase in MRS was associated with a shift in the myeloid response balance from antitumor to protumor activities, accompanied with enhanced CD8+ T cell exhaustion patterns. Additionally, we provide evidence that the MRS was associated with the efficacy of sorafenib treatment for recurrent HCC. CONCLUSION. We identified and validated a simple myeloid signature for HCC which showed remarkable prognostic potential and may serve as a basis for the stratification of HCC immune subtypes. FUNDING. This work was supported by the National Science and Technology Major Project of China, the National Natural Science Foundation of China, the Science and Information Technology of Guangzhou, the Fundamental Research Funds for the Central Universities, and the China Postdoctoral Science Foundation.
Chong Wu, Jie Lin, Yulan Weng, Dan-Ni Zeng, Jing Xu, Shufeng Luo, Li Xu, Mingyu Liu, Qiaomin Hua, Chao-Qun Liu, Jin-Qing Li, Jing Liao, Cheng Sun, Jian Zhou, Min-Shan Chen, Chao Liu, Zhenhong Guo, Shi-Mei Zhuang, Jin-Hua Huang, Limin Zheng
Nonalcoholic fatty liver disease (NAFLD) is becoming a major health issue as obesity increases around the world. We studied the effect of a circadian locomotor output cycles kaput (CLOCK) mutant (ClkΔ19/Δ19) protein on hepatic lipid metabolism in C57Bl6 Clkwt/wt and apolipoprotein E–deficient (Apoe−/−) mice. Both ClkΔ19/Δ19 and ClkΔ19/Δ19Apoe−/− mice developed a full spectrum of liver diseases (steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma) recognized in human NAFLD when challenged with a Western diet, lipopolysaccharide, or CoCl2. We identified induction of cluster of differentiation 36 (CD36) and hypoxia-inducible factor 1α (HIF1α) proteins as contributing factors for NAFLD. Mechanistic studies showed that wild-type CLOCK protein interacted with the E-box enhancer elements in the promoters of the proline hydroxylase domain (PHD) proteins to increase expression. In ClkΔ19/Δ19 mice, PHD levels were low, and HIF1α protein levels were increased. When its levels were high, HIF1α interacted with the Cd36 promoter to augment expression and enhance fatty acid uptake. Thus, these studies establish a novel regulatory link among circadian rhythms, hypoxia response, fatty acid uptake, and NAFLD. The mouse models described here may be useful for further mechanistic studies in the progression of liver diseases and in the discovery of drugs for the treatment of these disorders.
Xiaoyue Pan, Joyce Queiroz, M. Mahmood Hussain
The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide. Although gene-environment interactions have been implicated in the etiology of several disorders, the impact of paternal and/or maternal metabolic syndrome on the clinical phenotypes of offspring and the underlying genetic and epigenetic contributors of NAFLD have not been fully explored. To this end, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique nondietary model manifesting 3 hallmarks that confer high risk for the development of NAFLD: hyperglycemia, insulin resistance, and dyslipidemia. We report that parental metabolic syndrome epigenetically reprograms members of the TGF-β family, including neuronal regeneration–related protein (NREP) and growth differentiation factor 15 (GDF15). NREP and GDF15 modulate the expression of several genes involved in the regulation of hepatic lipid metabolism. In particular, NREP downregulation increases the protein abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and ATP-citrate lyase (ACLY) in a TGF-β receptor/PI3K/protein kinase B–dependent manner, to regulate hepatic acetyl-CoA and cholesterol synthesis. Reduced hepatic expression of NREP in patients with NAFLD and substantial correlations between low serum NREP levels and the presence of steatosis and nonalcoholic steatohepatitis highlight the clinical translational relevance of our findings in the context of recent preclinical trials implicating ACLY in NAFLD progression.
Dario F. De Jesus, Kazuki Orime, Dorota Kaminska, Tomohiko Kimura, Giorgio Basile, Chih-Hao Wang, Larissa Haertle, Renzo Riemens, Natalie K. Brown, Jiang Hu, Ville Männistö, Amélia M. Silva, Ercument Dirice, Yu-Hua Tseng, Thomas Haaf, Jussi Pihlajamäki, Rohit N. Kulkarni
BACKGROUND HBV-related acute-on-chronic liver failure (HBV-ACLF) is hallmarked by high short-term mortality rates, calling for accurate prognostic biomarkers for initial risk stratification.METHODS Three tandem mass tag–labeled (TMT-labeled) quantitative proteomic studies were performed on 10 patients with HBV-related acute hepatic decompensation and on 20 patients with HBV-ACLF. Candidate biomarkers were preliminarily verified in a cross-sectional cohort (n = 144) and further confirmed in 2 prospective cohorts (n = 207 and n = 148).RESULTS Plasminogen, a potential prognostic biomarker for HBV-ACLF, was identified by TMT quantitative proteomics and preliminarily verified in the cross-sectional cohort. Further validation with a prospective cohort (n = 207) showed that plasminogen levels at admission were significantly lower (P < 0.001) in HBV-ACLF nonsurvivors than in survivors. The cumulative survival duration of patients with high plasminogen levels was significantly longer (P < 0.001) than that of patients with low plasminogen levels. During hospitalization, plasminogen levels significantly decreased (P = 0.008) in the deterioration group but significantly increased (P < 0.001) in the improvement group. Additionally, plasminogen levels gradually increased in survivors but gradually decreased in nonsurvivors. The P5 score, a prognostic panel incorporating plasminogen levels, hepatic encephalopathy occurrence, age, international normalized ratio (INR), and total bilirubin, was significantly superior to the Child-Pugh, Model for End-stage Liver Disease (MELD), Chronic Liver Failure Consortium ACLF (CLIF-C ACLF), Chinese Group on the Study of Severe Hepatitis B (COSSH), and HINT (a prognostic score based on hepatic encephalopathy occurrence, INR, neutrophil count, and thyroid-stimulating hormone) scores (all P < 0.05). The performances of the plasminogen level and P5 score were validated in a second multicenter, prospective cohort (n = 148).CONCLUSIONS Plasminogen is a promising prognostic biomarker for HBV-ACLF, and sequential plasminogen measurements could profile the clinical course of HBV-ACLF. P5 is a high-performance prognostic score for HBV-ACLF.FUNDING The National Key Research and Development Program (2017YFC1200204); the National Natural Science Foundation of China (81400589, 81600497); the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81121002); the Chinese High-Tech Research and Development Programs (2012AA020204); the National S&T Major Project (2012ZX10002004); and the Zhejiang Provincial Medicine and Health Science and Technology Project (2016147735).
Daxian Wu, Sainan Zhang, Zhongyang Xie, Ermei Chen, Qunfang Rao, Xiaoli Liu, Kaizhou Huang, Jing Yang, Lanlan Xiao, Feiyang Ji, Zhengyi Jiang, Yalei Zhao, Xiaoxi Ouyang, Danhua Zhu, Xiahong Dai, Zhouhua Hou, Bingjie Liu, Binbin Deng, Ning Zhou, Hainv Gao, Zeyu Sun, Lanjuan Li
In patients with HBV and HCV coinfection, HBV reactivation leading to severe hepatitis has been reported with the use of direct-acting antivirals (DAAs) to treat HCV infection. Here we study the molecular mechanisms behind this viral interaction. In coinfected cell culture and humanized mice, HBV replication was suppressed by HCV coinfection. In vitro, HBV suppression was attenuated when interferon signaling was blocked. In vivo, HBV viremia, after initial suppression by HCV super-infection, rebounded following HCV clearance by DAA treatment that was accompanied by a reduced hepatic interferon response. Using blood samples of coinfected patients, interferon-stimulated gene products including C-X-C motif chemokine 10 (CXCL10) and C-C motif chemokine ligand 5 (CCL5), and alanine aminotransferase (ALT) were identified to have predictive value for HBV reactivation after HCV clearance. Taken together, our data suggest that HBV reactivation is a result of diminished hepatic interferon response following HCV clearance and identifies serologic markers that can predict HBV reactivation in DAA-treated HBV-HCV coinfected persons.
Xiaoming Cheng, Takuro Uchida, Yuchen Xia, Regina Umarova, Chun-Jen Liu, Pei-Jer Chen, Anuj Gaggar, Vithika Suri, Marcus Maximilian Mücke, Johannes Vermehren, Stefan Zeuzem, Yuji Teraoka, Mitsutaka Osawa, Hiroshi Aikata, Keiji Tsuji, Nami Mori, Shuhei Hige, Yoshiyasu Karino, Michio Imamura, Kazuaki Chayama, T. Jake Liang
De novo lipogenesis is tightly regulated by insulin and nutritional signals to maintain metabolic homeostasis; excessive lipogenesis induces lipotoxicity, leading to nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Genetic lipogenic programs have been extensively investigated, but epigenetic regulation of lipogenesis is poorly understood. Here, we identified Slug as an important epigenetic regulator of lipogenesis. Hepatic Slug levels were markedly upregulated in mice by either feeding or insulin treatment. In primary hepatocytes, insulin stimulation increased Slug expression, stability, and interactions with epigenetic enzyme lysine-specific demethylase-1 (Lsd1). Slug bound to the fatty acid synthase (Fasn) promoter where Slug-associated Lsd1 catalyzed H3K9 demethylation, thereby stimulating Fasn expression and lipogenesis. Ablation of Slug blunted insulin-stimulated lipogenesis; conversely, overexpression of Slug, but not a Lsd1 binding-defective Slug mutant, stimulated Fasn expression and lipogenesis. Lsd1 inhibitor treatment also blocked Slug-stimulated lipogenesis. Remarkably, hepatocyte-specific deletion of Slug inhibited the hepatic lipogenic program and protected against obesity-associated NAFLD, insulin resistance, and glucose intolerance in mice. Conversely, liver-restricted overexpression of Slug, but not the Lsd1 binding-defective Slug mutant, had the opposite effects. These results unveil an insulin/Slug/Lsd1/H3K9 demethylation lipogenic pathway that promotes NAFLD and type 2 diabetes.
Yan Liu, Haiyan Lin, Lin Jiang, Qingsen Shang, Lei Yin, Jiandie D. Lin, Wen-Shu Wu, Liangyou Rui
Hepatocellular carcinoma (HCC) is clearly age-related and represents one of the deadliest cancer types worldwide. Due to globally increasing risk factors including metabolic disorders, the incidence rates of HCC are still rising. However, the molecular hallmarks of HCC remain poorly understood. Neuropeptide Y (NPY) and NPY-receptors represent a highly conserved, stress-activated system which is involved in diverse cancer-related hallmarks including aging and metabolic alterations, but its impact on liver cancer had been unclear. Here, we observed increased NPY5-receptor (Y5R) expression in HCC which correlated with tumor growth and survival. Furthermore, we found that its ligand NPY was secreted by peri-tumorous hepatocytes. Hepatocyte-derived NPY promoted HCC progression by Y5R-activation. Transforming growth factor beta 1 (TGFβ1) was identified as a regulator of NPY in hepatocytes and induced Y5R in invasive cancer cells. Moreover, NPY-conversion by dipeptidylpeptidase 4 (DPP4) augmented Y5R-activation and function in liver cancer. The TGFβ-NPY-Y5R-axis and DPP4 represent attractive therapeutic targets for controlling liver cancer progression.
Peter Dietrich, Laura Wormser, Valerie Fritz, Tatjana Seitz, Monica De Maria, Alexandra Schambony, Andreas E. Kremer, Claudia Günther, Timo Itzel, Wolfgang E. Thasler, Andreas Teufel, Jonel Trebicka, Arndt Hartmann, Markus F. Neurath, Stephan von Hörsten, Anja Bosserhoff, Claus Hellerbrand
Severe alcoholic hepatitis (SAH) is a deadly liver disease without an effective medical therapy. Although SAH mortality is known to correlate with hepatic accumulation of immature liver cells, why this occurs, and how it causes death is unclear. Here, we demonstrated that expression of epithelial splicing regulatory protein-2 (ESRP2), an RNA splicing factor that maintains the non-proliferative, mature phenotype of adult hepatocytes, was suppressed in both human SAH and various mouse models of SAH in parallel with the severity of alcohol consumption and liver damage. Inflammatory cytokines released by excessive alcohol ingestion reprogrammed adult hepatocytes into proliferative, fetal-like cells by suppressing ESRP2. Sustained loss of ESRP2 permitted re-emergence of a fetal RNA splicing program that attenuates the Hippo signaling pathway and thus, allows fetal transcriptional regulators to accumulate in adult liver. We further showed that depleting ESRP2 in mice exacerbated alcohol-induced steatohepatitis, enabling surviving hepatocytes to shed adult hepatocyte functions and become more regenerative but threatens overall survival by populating the liver with functionally-immature hepatocytes. Our findings revealed a novel mechanism that explains why liver failure develops in patients with the clinical syndrome of SAH, suggesting that recovery from SAH might be improved by limiting adult-to-fetal reprogramming in hepatocytes.
Jeongeun Hyun, Zhaoli Sun, Ali Reza Ahmadi, Sushant Bangru, Ullas V. Chembazhi, Kuo Du, Tianyi Chen, Hidekazu Tsukamoto, Ivan Rusyn, Auinash Kalsotra, Anna Mae Diehl