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Keshavarzi M, Moradbeygi F, Mobini K, Ghaffarian Bahraman A, Mohammadi P, Ghaedi A, Mohammadi-Bardbori A. The interplay of aryl hydrocarbon receptor/WNT/CTNNB1/Notch signaling pathways regulate amyloid beta precursor mRNA/protein expression and effected the learning and memory of mice. Toxicol Res (Camb) 2021; 11:147-161. [PMID: 35237419 PMCID: PMC8882790 DOI: 10.1093/toxres/tfab120] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Revised: 11/22/2021] [Accepted: 12/01/2021] [Indexed: 12/31/2022] Open
Abstract
The amyloid beta precursor protein (APP) plays a pathophysiological role in the development of Alzheimer's disease as well as a physiological role in neuronal growth and synaptogenesis. The aryl hydrocarbon receptor (AhR)/WNT/Catenin Beta 1 (CTNNB1)/Notch signaling pathways stamp in many functions, including development and growth of neurons. However, the regulatory role of AhR-/WNT-/CTNNB1-/Notch-induced APP expression and its influence on hippocampal-dependent learning and memory deficits is not clear. Male BALB/C mice received 6-formylindolo[3,2-b]carbazole (an AhR agonist), CH223191(an AhR antagonist), DAPT (an inhibitor of Notch signaling), and XAV-939 (a WNT pathway inhibitor) at a single dose of 100 μg/kg, 1, 5 , and 5 mg/kg of body weight, respectively, via intraperitoneal injection alone or in combination. Gene expression analyses and protein assay were performed on the 7th and 29th days. To assess the hippocampal-dependent memory, all six mice also underwent contextual fear conditioning on the 28th day after treatments. Our results showed that endogenous ligand of AhR has a regulatory effect on APP gene. Also, the interaction of AhR/WNT/CTNNB1 has a positive regulatory effect, but Notch has a negative regulatory effect on the mRNA and protein expression of APP, which have a correlation with mice's learning skills and memory.
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Affiliation(s)
- Majid Keshavarzi
- Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz 7146864685, Iran,Department of Environmental Health, Faculty of Health, Sabzevar University of Medical Sciences, Sabzevar 7146864685, Iran
| | - Fatemeh Moradbeygi
- Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz 7146864685, Iran
| | - Keivan Mobini
- Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz 7146864685, Iran
| | - Ali Ghaffarian Bahraman
- Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz 7146864685, Iran,Occupational Environment Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
| | - Parisa Mohammadi
- Department of Environmental Health, Faculty of Health, Sabzevar University of Medical Sciences, Sabzevar 7146864685, Iran
| | - Afsaneh Ghaedi
- Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz 7146864685, Iran
| | - Afshin Mohammadi-Bardbori
- Correspondence address. Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz 7146864685, Iran. Tel.: +98(71)32425374; Fax: +98(71)32424326; E-mail:
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Hasanin AH, Habib EK, El Gayar N, Matboli M. Promotive action of 2-acetylaminofluorene on hepatic precancerous lesions initiated by diethylnitrosamine in rats: Molecular study. World J Hepatol 2021; 13:328-342. [PMID: 33815676 PMCID: PMC8006078 DOI: 10.4254/wjh.v13.i3.328] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Revised: 12/14/2020] [Accepted: 03/02/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Diethylnitrosamine (DEN) induces hepatic neoplastic lesions over a prolonged period. AIM To investigate the promotive action of 2-acetylaminofluorene (2-AAF) when combined with DEN in order to develop a rat model for induction of precancerous lesion and investigate the molecular mechanism underlying the activity of 2-AAF. METHODS The pre-precancerous lesions were initiated by intraperitoneal injection of DEN for three weeks consecutively, followed by one intraperitoneal injection of 2-AAF at three different doses (100, 200 and 300 mg/kg). Rats were separated into naïve, DEN, DEN + 100 mg 2-AAF, DEN + 200 mg 2-AAF, and DEN + 300 mg 2-AAF groups. Rats were sacrificed after 10 wk and 16 wk. Liver functions, level of alpha-fetoprotein, glutathione S-transferase-P and proliferating cell nuclear antigen staining of liver tissues were performed. The mRNA level of RAB11A, BAX, p53, and Cyclin E and epigenetic regulation by long-noncoding RNA (lncRNA) RP11-513I15.6, miR-1262 (microRNA), and miR-1298 were assessed in the sera and liver tissues of the rats. RESULTS 2-AAF administration significantly increased the percent area of the precancerous foci and cell proliferation along with a significant decrease in RAB11A, BAX, and p53 mRNA, and the increase in Cyclin E mRNA was associated with a marked decrease in lncRNA RP11-513I15.6 expression with a significant increase in both miR-1262 and miR-1298. CONCLUSION 2-AFF promoted hepatic precancerous lesions initiated through DEN by decreasing autophagy, apoptosis, and tumor suppression genes, along with increased cell proliferation, in a time- and dose-dependent manner. These actions were mediated under the epigenetic regulation of lncRNA RP11-513I15.6/miR-1262/miR-1298.
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Affiliation(s)
- Amany Helmy Hasanin
- Clinical Pharmacology Department, Faculty of Medicine, Ain Shams University, Cairo 11318, Egypt
| | - Eman K Habib
- Anatomy and Embryology Department, Faculty of Medicine, Ain Shams University, Cairo 11318, Egypt
| | - Nesreen El Gayar
- Clinical Pharmacology Department, Faculty of Medicine, Ain Shams University, Cairo 11318, Egypt
| | - Marwa Matboli
- Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Cairo 11381, Egypt.
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Daujat-Chavanieu M, Gerbal-Chaloin S. Regulation of CAR and PXR Expression in Health and Disease. Cells 2020; 9:E2395. [PMID: 33142929 PMCID: PMC7692647 DOI: 10.3390/cells9112395] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Revised: 10/22/2020] [Accepted: 10/28/2020] [Indexed: 12/12/2022] Open
Abstract
Pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are members of the nuclear receptor superfamily that mainly act as ligand-activated transcription factors. Their functions have long been associated with the regulation of drug metabolism and disposition, and it is now well established that they are implicated in physiological and pathological conditions. Considerable efforts have been made to understand the regulation of their activity by their cognate ligand; however, additional regulatory mechanisms, among which the regulation of their expression, modulate their pleiotropic effects. This review summarizes the current knowledge on CAR and PXR expression during development and adult life; tissue distribution; spatial, temporal, and metabolic regulations; as well as in pathological situations, including chronic diseases and cancers. The expression of CAR and PXR is modulated by complex regulatory mechanisms that involve the interplay of transcription factors and also post-transcriptional and epigenetic modifications. Moreover, many environmental stimuli affect CAR and PXR expression through mechanisms that have not been elucidated.
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Affiliation(s)
| | - Sabine Gerbal-Chaloin
- IRMB, University of Montpellier, INSERM, CHU Montpellier, 34295 Montpellier, France;
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Regulation of expression of drug-metabolizing enzymes by oncogenic signaling pathways in liver tumors: a review. Acta Pharm Sin B 2020; 10:113-122. [PMID: 31993310 PMCID: PMC6976994 DOI: 10.1016/j.apsb.2019.06.013] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2019] [Revised: 05/23/2019] [Accepted: 06/24/2019] [Indexed: 02/06/2023] Open
Abstract
Mutations in genes encoding key players in oncogenic signaling pathways trigger specific downstream gene expression profiles in the respective tumor cell populations. While regulation of genes related to cell growth, survival, and death has been extensively studied, much less is known on the regulation of drug-metabolizing enzymes (DMEs) by oncogenic signaling. Here, a comprehensive review of the available literature is presented summarizing the impact of the most relevant genetic alterations in human and rodent liver tumors on the expression of DMEs with a focus on phases I and II of xenobiotic metabolism. Comparably few data are available with respect to DME regulation by p53-dependent signaling, telomerase expression or altered chromatin remodeling. By contrast, DME regulation by constitutive activation of oncogenic signaling via the RAS/RAF/mitogen-activated protein kinase (MAPK) cascade or via the canonical WNT/β-catenin signaling pathway has been analyzed in greater depth, demonstrating mostly positive-regulatory effects of WNT/β-catenin signaling and negative-regulatory effects of MAPK signaling. Mechanistic studies have revealed molecular interactions between oncogenic signaling and nuclear xeno-sensing receptors which underlie the observed alterations in DME expression in liver tumors. Observations of altered DME expression and inducibility in liver tumors with a specific gene expression profile may impact pharmacological treatment options.
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Analysis of β-catenin gene mutations and gene expression in liver tumours of C57BL/10J mice produced by chronic administration of sodium phenobarbital. Toxicology 2019; 430:152343. [PMID: 31836555 DOI: 10.1016/j.tox.2019.152343] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Revised: 11/22/2019] [Accepted: 12/09/2019] [Indexed: 11/22/2022]
Abstract
In this study liver tumours produced in male and female mice of the low spontaneous liver tumour incidence C57BL/10J strain treated for 99 weeks with 1000 ppm in the diet with the model constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were analysed for β-catenin mutations by Western immunoblotting and DNA/RNA analysis. Some gene array analysis was also performed to identify genes involved in CAR activation and in β-catenin and Hras gene mutations. Analysis of 8 male and 2 female NaPB-induced liver tumour samples (comprising 2 adenomas, 6 carcinomas and 2 samples containing separate adenomas and carcinomas) revealed truncated β-catenin forms in just 4 male liver tumour samples, with the presence of the truncated β-catenin forms being confirmed by β-catenin exon 1-3 mutation analysis. Microarray gene expression analysis was performed with three of the NaPB-induced male mouse liver tumour samples where β-catenin mutations had not been identified by Western immunoblotting and DNA/RNA analysis and with three liver samples from both NaPB-induced non-tumour tissue and control animals. Treatment with NaPB resulted in induction of Cyp2b subfamily gene expression in both NaPB-induced mouse liver tumours and in NaPB-treated non-tumour tissue. In addition, the gene expression analysis demonstrated that the β-catenin and Hras pathways were not modified in NaPB-induced mouse liver tumours not exhibiting truncated β-catenin forms. Overall, while chronic administration of the model CAR activator NaPB results in both hepatocellular adenoma and carcinoma in the low spontaneous liver tumour incidence C57BL/10J mouse strain, only 40 % of the liver tumours evaluated in this study had β-catenin mutations. These results are in agreement with previous studies with the CAR activator oxazepam and demonstrate that mouse liver tumours induced by nongenotoxic CAR activators in the absence of initiation with a genotoxic agent are due to a number of mechanisms, including those largely independent of either the Wnt/β-catenin signalling pathway or Hras oncogene mutations.
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Laube B, Michaelsen S, Meischner V, Hartwig A, Epe B, Schwarz M. Classification or non-classification of substances with positive tumor findings in animal studies: Guidance by the German MAK commission. Regul Toxicol Pharmacol 2019; 108:104444. [PMID: 31433998 DOI: 10.1016/j.yrtph.2019.104444] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2019] [Revised: 08/10/2019] [Accepted: 08/14/2019] [Indexed: 12/19/2022]
Abstract
One of the important tasks of the German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (known as the MAK Commission) is in the evaluation of a potential for carcinogenicity of hazardous substances at the workplace. Often, this evaluation is critically based on data on carcinogenic responses seen in animal studies and, if positive tumor responses have been observed, this will mostly lead to a classification of the substance under investigation into one of the classes for carcinogens. However, there are cases where it can be demonstrated with a very high degree of confidence that the tumor findings in the experimental animals are not relevant for humans at the workplace and, therefore, the MAK Commission will not classify the respective substance into one of the classes for carcinogens. This paper will summarize the general criteria used by the MAK Commission for the categorization into "carcinogen" and "non-carcinogen" and compare this procedure with those used by other national and international organizations.
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Affiliation(s)
- Britta Laube
- Scientific Secretariat of the Senate Commission on the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission), Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Department of Food Chemistry and Toxicology, Karlsruhe, Germany
| | - Sandra Michaelsen
- Scientific Secretariat of the Senate Commission on the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission), Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Department of Food Chemistry and Toxicology, Karlsruhe, Germany
| | - Veronika Meischner
- Scientific Secretariat of the Senate Commission on the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission), Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Department of Food Chemistry and Toxicology, Karlsruhe, Germany
| | - Andrea Hartwig
- Food Chemistry and Toxicology, Institute of Applied Bioscience, Karlsruhe Institute of Technology (KIT), Adenauerring 20a, 76131, Karlsruhe, Germany
| | - Bernd Epe
- Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099, Mainz, Germany
| | - Michael Schwarz
- Dept. of Experimental and Clinical Pharmacology and Toxicology, Dept. Toxicology, Eberhard Karls University, Wilhelmstr. 56, 72074, Tübingen, Germany.
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Connor F, Rayner TF, Aitken SJ, Feig C, Lukk M, Santoyo-Lopez J, Odom DT. Mutational landscape of a chemically-induced mouse model of liver cancer. J Hepatol 2018; 69:840-850. [PMID: 29958939 PMCID: PMC6142872 DOI: 10.1016/j.jhep.2018.06.009] [Citation(s) in RCA: 99] [Impact Index Per Article: 14.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/04/2018] [Revised: 06/04/2018] [Accepted: 06/05/2018] [Indexed: 01/09/2023]
Abstract
BACKGROUND & AIMS Carcinogen-induced mouse models of liver cancer are used extensively to study the pathogenesis of the disease and are critical for validating candidate therapeutics. These models can recapitulate molecular and histological features of human disease. However, it is not known if the genomic alterations driving these mouse tumour genomes are comparable to those found in human tumours. Herein, we provide a detailed genomic characterisation of tumours from a commonly used mouse model of hepatocellular carcinoma (HCC). METHODS We analysed whole exome sequences of liver tumours arising in mice exposed to diethylnitrosamine (DEN). Mutational signatures were compared between liver tumours from DEN-treated and untreated mice, and human HCCs. RESULTS DEN-initiated tumours had a high, uniform number of somatic single nucleotide variants (SNVs), with few insertions, deletions or copy number alterations, consistent with the known genotoxic action of DEN. Exposure of hepatocytes to DEN left a reproducible mutational imprint in resulting tumour exomes which we could computationally reconstruct using six known COSMIC mutational signatures. The tumours carried a high diversity of low-incidence, non-synonymous point mutations in many oncogenes and tumour suppressors, reflecting the stochastic introduction of SNVs into the hepatocyte genome by the carcinogen. We identified four recurrently mutated genes that were putative oncogenic drivers of HCC in this model. Every neoplasm carried activating hotspot mutations either in codon 61 of Hras, in codon 584 of Braf or in codon 254 of Egfr. Truncating mutations of Apc occurred in 21% of neoplasms, which were exclusively carcinomas supporting a role for deregulation of Wnt/β-catenin signalling in cancer progression. CONCLUSIONS Our study provides detailed insight into the mutational landscape of tumours arising in a commonly used carcinogen model of HCC, facilitating the future use of this model to better understand the human disease. LAY SUMMARY Mouse models are widely used to study the biology of cancer and to test potential therapies. Herein, we have described the mutational landscape of tumours arising in a carcinogen-induced mouse model of liver cancer. Since cancer is a disease caused by genomic alterations, information about the patterns and types of mutations in the tumours in this mouse model should facilitate its use to study human liver cancer.
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Affiliation(s)
- Frances Connor
- Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK
| | - Tim F Rayner
- Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK
| | - Sarah J Aitken
- Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK; Department of Histopathology, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Hills Road, Cambridge CB2 0QQ, UK
| | - Christine Feig
- Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK
| | - Margus Lukk
- Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK
| | - Javier Santoyo-Lopez
- Edinburgh Genomics (Clinical), The Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK
| | - Duncan T Odom
- Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK.
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Chiyonobu N, Shimada S, Akiyama Y, Mogushi K, Itoh M, Akahoshi K, Matsumura S, Ogawa K, Ono H, Mitsunori Y, Ban D, Kudo A, Arii S, Suganami T, Yamaoka S, Ogawa Y, Tanabe M, Tanaka S. Fatty Acid Binding Protein 4 (FABP4) Overexpression in Intratumoral Hepatic Stellate Cells within Hepatocellular Carcinoma with Metabolic Risk Factors. THE AMERICAN JOURNAL OF PATHOLOGY 2018; 188:1213-1224. [PMID: 29454748 DOI: 10.1016/j.ajpath.2018.01.012] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/04/2017] [Revised: 12/25/2017] [Accepted: 01/16/2018] [Indexed: 01/21/2023]
Abstract
Metabolic syndrome is a newly identified risk factor for hepatocellular carcinoma (HCC); however, tumor-specific biomarkers still remain unclear. We performed cross-species analysis to compare gene signatures of HCC from human patients and melanocortin 4 receptor-knockout mice, which develop HCC with obesity, insulin resistance, and dyslipidemia. Unsupervised hierarchical clustering and principle component analysis of 746 differentially expressed orthologous genes classified HCC of 152 human patients and melanocortin 4 receptor-knockout mice into two distinct subgroups, one of which included mouse HCC and was causatively associated with metabolic risk factors. Nine genes commonly overexpressed in human and mouse metabolic disease-associated HCC were identified; fatty acid binding protein 4 (FABP4) was remarkably enriched in intratumoral activated hepatic stellate cells (HSCs). Subclones constitutively expressing FABP4 were established from a human HSC cell line in which expression levels of inflammatory chemokines, including IL-1A and IL-6, were up-regulated through NF-κB nuclear translocation, resulting in recruitment of macrophages. An immunohistochemical validation study of 106 additional human HCC samples indicated that FABP4-positive HSCs were distributed in tumors of 38 cases, and the FABP4-high group consisted of patients with nonviral and nonalcoholic HCC (P = 0.027) and with multiple metabolic risk factors (P < 0.001) compared with the FABP4-low group. Thus, FABP4 overexpression in HSCs may contribute to hepatocarcinogenesis in patients with metabolic risk factors by modulation of inflammatory pathways.
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Affiliation(s)
- Norimichi Chiyonobu
- Department of Molecular Oncology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Shu Shimada
- Department of Molecular Oncology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Yoshimitsu Akiyama
- Department of Molecular Oncology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Kaoru Mogushi
- Department of Molecular Oncology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Michiko Itoh
- Department of Organ Network and Metabolism, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Keiichi Akahoshi
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Satoshi Matsumura
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Kosuke Ogawa
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Hiroaki Ono
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Yusuke Mitsunori
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Daisuke Ban
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Atsushi Kudo
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Shigeki Arii
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Takayoshi Suganami
- Department of Molecular Medicine and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
| | - Shoji Yamaoka
- Department of Molecular Virology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Yoshihiro Ogawa
- Department of Medical and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Molecular and Cellular Metabolism, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Tokyo, Japan
| | - Minoru Tanabe
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
| | - Shinji Tanaka
- Department of Molecular Oncology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
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Felter SP, Foreman JE, Boobis A, Corton JC, Doi AM, Flowers L, Goodman J, Haber LT, Jacobs A, Klaunig JE, Lynch AM, Moggs J, Pandiri A. Human relevance of rodent liver tumors: Key insights from a Toxicology Forum workshop on nongenotoxic modes of action. Regul Toxicol Pharmacol 2018; 92:1-7. [PMID: 29113941 PMCID: PMC11350555 DOI: 10.1016/j.yrtph.2017.11.003] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2017] [Revised: 10/31/2017] [Accepted: 11/02/2017] [Indexed: 12/21/2022]
Abstract
The Toxicology Forum sponsored a workshop in October 2016, on the human relevance of rodent liver tumors occurring via nongenotoxic modes of action (MOAs). The workshop focused on two nuclear receptor-mediated MOAs (Constitutive Androstane Receptor (CAR) and Peroxisome Proliferator Activated Receptor-alpha (PPARα), and on cytotoxicity. The goal of the meeting was to review the state of the science to (1) identify areas of consensus and differences, data gaps and research needs; (2) identify reasons for inconsistencies in current regulatory positions; and (3) consider what data are needed to demonstrate a specific MOA, and when additional research is needed to rule out alternative possibilities. Implications for quantitative risk assessment approaches were discussed, as were implications of not considering MOA and dose in hazard characterization and labeling schemes. Most, but not all, participants considered the CAR and PPARα MOAs as not relevant to humans based on quantitative and qualitative differences. In contrast, cytotoxicity is clearly relevant to humans, but a threshold applies. Questions remain for all three MOAs concerning what data are necessary to determine the MOA and to what extent it is necessary to exclude other MOAs.
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Affiliation(s)
- Susan P Felter
- Procter and Gamble, Central Product Safety, Mason, OH, United States.
| | | | - Alan Boobis
- Department of Medicine, Imperial College London, London, UK
| | - J Christopher Corton
- National Health and Environmental Effects Research Lab, US EPA, Durham, NC, United States
| | - Adriana M Doi
- BASF Corporation, Research Triangle Park, NC, United States
| | - Lynn Flowers
- Office of Science Policy, US EPA, Washington DC, United States
| | - Jay Goodman
- Michigan State University, Dept. Pharmacology and Toxicology, East Lansing, MI, United States
| | - Lynne T Haber
- Risk Science Center, Dept. of Environmental Health, University of Cincinnati, Cincinnati, OH, United States
| | - Abigail Jacobs
- Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States
| | | | | | - Jonathan Moggs
- Novartis Institutes for BioMedical Research, Preclinical Safety, Translational Medicine, Basel, Switzerland
| | - Arun Pandiri
- National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
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Garcia K, Regan D. Bigger Is Better: Refinement of an Animal Model of Hepatocellular Carcinoma and Transfemoral Arterial Embolization. J Vasc Interv Radiol 2017. [PMID: 28645501 DOI: 10.1016/j.jvir.2017.04.020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Affiliation(s)
- Kelly Garcia
- Biologic Resources Laboratory, University of Illinois at Chicago, (MC533) 1840 West Taylor Street, Chicago, IL 60612.
| | - Dan Regan
- Flint Animal Cancer Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado
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Vitamin D 3 supplementation attenuates the early stage of mouse hepatocarcinogenesis promoted by hexachlorobenzene fungicide. Food Chem Toxicol 2017. [PMID: 28634113 DOI: 10.1016/j.fct.2017.06.030] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Hexachlorobezene (HCB), a fungicide widely distributed in the environment, promotes the development of hepatocellular preneoplastic lesions (PNL) and tumors in rodents. In contrast, vitamin D3 (VD3) supplementation presents a potential role for the prevention/treatment of chronic liver diseases. Thus, we investigated whether VD3 supplementation attenuates the early stage of HCB-promoted hepatocarcinogenesis. Female Balb/C mice were injected a single dose of diethylnitrosamine (DEN, 50 mg/kg) at postnatal day 15. From day 40 onwards, mice were fed with a standard diet containing 0.02% HCB alone or supplemented with VD3 (10,000 or 20,000 IU/Kg diet) for 20 weeks. Untreated mice were fed just standard diet. After this period, mice were euthanized and liver and serum samples were collected. Compared to the untreated group, DEN/HCB treatment decreased total hepatic glutathione levels and glutathione peroxidase (GSH-Px) activity while increased lipid peroxidation, p65 protein expression, cell proliferation/apoptosis and the PNL development. In contrast, dietary VD3 supplementation enhanced vitamin D receptor (VDR) protein expression, total glutathione levels and GSH-Px activity while diminished lipid hydroperoxide levels. Also, VD3 supplementation decreased p65 protein expression, hepatocyte proliferation, the size and the liver area occupied by PNL. Therefore, our findings indicate that VD3 supplementation attenuates the early stage of HCB-promoted hepatocarcinogenesis.
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Santos NP, Colaço AA, Oliveira PA. Animal models as a tool in hepatocellular carcinoma research: A Review. Tumour Biol 2017; 39:1010428317695923. [PMID: 28347231 DOI: 10.1177/1010428317695923] [Citation(s) in RCA: 76] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
Cancer is the first cause of death in developed countries and the second in developing countries. Concerning the most frequent worldwide-diagnosed cancer, primary liver cancer represents approximately 4% of all new cancer cases diagnosed globally. However, among primary liver cancer, hepatocellular carcinoma is by far the most common histological subtype. Notwithstanding the health promotion and disease prevention campaigns, more than half a million new hepatocellular carcinoma cases are reported yearly, being estimated to growth continuously until 2020. Taking this scenario under consideration and the fact that some aspects concerning hepatocellular carcinoma evolution and metastasize process are still unknown, animal models assume a crucial role to understand this disease. The animal models have also provided the opportunity to screen new therapeutic strategies. The present review was supported on research and review papers aiming the complexity and often neglected chemically induced animal models in hepatocarcinogenesis research. Despite the ongoing debate, chemically induced animal models, namely, mice and rat, can provide unique valuable information on the biotransformation mechanisms against xenobiotics and apprehend the deleterious effects on DNA and cell proteins leading to carcinogenic development. In addition, taking under consideration that no model achieves all hepatocellular carcinoma research purposes, criteria to define the " ideal" animal model, depending on the researchers' approach, are also discussed in this review.
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Affiliation(s)
- Nuno Paula Santos
- 1 Department of Veterinary Sciences, Veterinary and Animal Science Research Center (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal.,2 Center for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal
| | - Aura Antunes Colaço
- 1 Department of Veterinary Sciences, Veterinary and Animal Science Research Center (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal
| | - Paula Alexandra Oliveira
- 1 Department of Veterinary Sciences, Veterinary and Animal Science Research Center (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal.,2 Center for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal
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13
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A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis. Nat Commun 2016; 7:13781. [PMID: 28000790 PMCID: PMC5187498 DOI: 10.1038/ncomms13781] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2016] [Accepted: 10/28/2016] [Indexed: 02/06/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, but its molecular heterogeneity hampers the design of targeted therapies. Currently, the only therapeutic option for advanced HCC is Sorafenib, an inhibitor whose targets include RAF. Unexpectedly, RAF1 expression is reduced in human HCC samples. Modelling RAF1 downregulation by RNAi increases the proliferation of human HCC lines in xenografts and in culture; furthermore, RAF1 ablation promotes chemical hepatocarcinogenesis and the proliferation of cultured (pre)malignant mouse hepatocytes. The phenotypes depend on increased YAP1 expression and STAT3 activation, observed in cultured RAF1-deficient cells, in HCC xenografts, and in autochthonous liver tumours. Thus RAF1, although essential for the development of skin and lung tumours, is a negative regulator of hepatocarcinogenesis. This unexpected finding highlights the contribution of the cellular/tissue environment in determining the function of a protein, and underscores the importance of understanding the molecular context of a disease to inform therapy design.
The kinase RAF1 usually exerts pro-tumorigenic functions promoting proliferation in RAS-driven cancers. Here, the authors using a mouse model of HCC and clinical data describe an unexpected oncosuppressor role of RAF1 in hepatocarcinoma development linked to a gp130-dependent Stat3 activation and YAP1 regulation.
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Henderson JM, Zhang HE, Polak N, Gorrell MD. Hepatocellular carcinoma: Mouse models and the potential roles of proteases. Cancer Lett 2016; 387:106-113. [PMID: 27045475 DOI: 10.1016/j.canlet.2016.03.047] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2016] [Revised: 03/24/2016] [Accepted: 03/24/2016] [Indexed: 02/07/2023]
Abstract
Primary liver cancer is the second most common cause of mortality from cancer. The most common models of hepatocellular carcinoma, which use a chemical and/or metabolic insult, xenograft, or genetic manipulation, are discussed in this review. In the tumour microenvironment lymphocytes, fibroblasts, endothelial cells and antigen presenting cells are important determinants of cell fate. These cells make a range of proteases that modify the biological activity of other proteins, particularly extracellular matrix proteins that alter cell migration of tumour cells, fibroblasts and leucocytes, and chemokines that alter leucocyte migration. The DPP4 family of post-proline peptidase enzymes modifies cell movement and the activities of many bioactive molecules including growth factors and chemokines.
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Affiliation(s)
- James M Henderson
- Centenary Institute and Sydney Medical School, University of Sydney, Sydney, New South Wales 2006 Australia
| | - Hui Emma Zhang
- Centenary Institute and Sydney Medical School, University of Sydney, Sydney, New South Wales 2006 Australia
| | - Natasa Polak
- Centenary Institute and Sydney Medical School, University of Sydney, Sydney, New South Wales 2006 Australia
| | - Mark D Gorrell
- Centenary Institute and Sydney Medical School, University of Sydney, Sydney, New South Wales 2006 Australia.
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15
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Marx-Stoelting P, Braeuning A, Buhrke T, Lampen A, Niemann L, Oelgeschlaeger M, Rieke S, Schmidt F, Heise T, Pfeil R, Solecki R. Application of omics data in regulatory toxicology: report of an international BfR expert workshop. Arch Toxicol 2015; 89:2177-84. [PMID: 26486796 DOI: 10.1007/s00204-015-1602-x] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Accepted: 09/15/2015] [Indexed: 02/07/2023]
Abstract
Advances in omics techniques and molecular toxicology are necessary to provide new perspectives for regulatory toxicology. By the application of modern molecular techniques, more mechanistic information should be gained to support standard toxicity studies and to contribute to a reduction and refinement of animal experiments required for certain regulatory purposes. The relevance and applicability of data obtained by omics methods to regulatory purposes such as grouping of chemicals, mode of action analysis or classification and labelling needs further improvement, defined validation and cautious expert judgment. Based on the results of an international expert workshop organized 2014 by the Federal Institute for Risk Assessment in Berlin, this paper is aimed to provide a critical overview of the regulatory relevance and reliability of omics methods, basic requirements on data quality and validation, as well as regulatory criteria to decide which effects observed by omics methods should be considered adverse or non-adverse. As a way forward, it was concluded that the inclusion of omics data can facilitate a more flexible approach for regulatory risk assessment and may help to reduce or refine animal testing.
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Affiliation(s)
- P Marx-Stoelting
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany.
| | - A Braeuning
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - T Buhrke
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - A Lampen
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - L Niemann
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - M Oelgeschlaeger
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - S Rieke
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - F Schmidt
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - T Heise
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - R Pfeil
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
| | - R Solecki
- Federal Institute for Risk Assessment, Max-Dohrn-Str 8-10, 10589, Berlin, Germany
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16
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Merhi A, De Mees C, Abdo R, Victoria Alberola J, Marini AM. Wnt/β-Catenin Signaling Regulates the Expression of the Ammonium Permease Gene RHBG in Human Cancer Cells. PLoS One 2015; 10:e0128683. [PMID: 26029888 PMCID: PMC4452261 DOI: 10.1371/journal.pone.0128683] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2015] [Accepted: 04/29/2015] [Indexed: 11/18/2022] Open
Abstract
Ammonium is a metabolic waste product mainly detoxified by the liver. Hepatic dysfunction can lead to cytotoxic accumulation of circulating ammonium and to subsequent encephalopathy. Transmembrane ammonium transport is a widely spread process ensured by the highly conserved proteins of the Mep-Amt-Rh superfamily, including the mammalian Rhesus (Rh) factors. The regulatory mechanisms involved in the control of RH genes expression remain poorly studied. Here we addressed the expression regulation of one of these factors, RHBG. We identify HepG2 hepatocellular carcinoma cells and SW480 colon adenocarcinoma cells as expressing RHBG and show that its expression relies on β-catenin signaling. siRNA-mediated β-catenin knockdown resulted in significant reduction of RHBG mRNA in both cell lines. Pharmaceutical inhibition of the TCF4/β-catenin interaction or knockdown of the transcription factor TCF4 also downregulated RHBG expression. We identify a minimal RHBG regulatory sequence displaying a promoter activity and show that β-catenin and TCF4 bind to this fragment in vivo. We finally characterize the role of potential TCF4 binding sites in RHBG regulation. Taken together, our results indicate RHBG expression as a direct target of β-catenin regulation, a pathway frequently deregulated in many cancers and associated with tumorigenesis.
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Affiliation(s)
- Ahmad Merhi
- Biology of Membrane Transport Laboratory, IBMM, Université Libre de Bruxelles, Gosselies, Belgium
| | - Christelle De Mees
- Biology of Membrane Transport Laboratory, IBMM, Université Libre de Bruxelles, Gosselies, Belgium
| | - Rami Abdo
- Biology of Membrane Transport Laboratory, IBMM, Université Libre de Bruxelles, Gosselies, Belgium
| | | | - Anna Maria Marini
- Biology of Membrane Transport Laboratory, IBMM, Université Libre de Bruxelles, Gosselies, Belgium
- * E-mail:
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17
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Mercer KE, Hennings L, Ronis MJJ. Alcohol consumption, Wnt/β-catenin signaling, and hepatocarcinogenesis. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2015; 815:185-95. [PMID: 25427908 DOI: 10.1007/978-3-319-09614-8_11] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Alcohol is a well-established risk factor for hepatocellular carcinoma, and the mechanisms by which alcohol liver cancer is complex. It has been suggested that ethanol (EtOH) metabolism may enhance tumor progression by increasing hepatocyte proliferation. To test this hypothesis, ethanol (EtOH) feeding of male mice began 7 weeks post-injection of the chemical carcinogen diethylnitrosamine (DEN), and continued for 16 weeks, with a final EtOH concentration of 28% of total calories. As expected, EtOH increased the total number of cancerous foci and liver tumors identified in situ fixed livers from the EtOH+DEN group compared to corresponding pair-fed (PF)+DEN and chow+DEN control groups. In the EtOH+DEN group, tumor multiplicity corresponded to a 3- to 4-fold increase in proliferation and immunohistochemical staining of β-catenin in non-tumorigenic hepatocytes when compared to the PF+DEN and chow+DEN groups, p<0.05. Analysis of EtOH-treated livers from a previously published rat model of chronic liver disease revealed increases in hepatocyte proliferation accompanied by a hepatic depletion of retinol and retinoic acid stores (p<0.05), nuclear accumulation of β-catenin (p<0.05), increased cytosolic expression p-GSK3β (p<0.05), significant upregulation of soluble Wnts, Wnt2, and Wnt7a, and increased expression of several β-catenin targets involved in tumor promotion and progression, cyclin D1, c-myc, WISP1, and MMP7 (p<0.05). These data suggest that chronic EtOH consumption activates the Wnt/β-catenin signaling pathway, which increases hepatocyte proliferation thus promoting tumorigenesis following an initiating insult in the liver.
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Affiliation(s)
- K E Mercer
- Arkansas Children's Nutrition Center, University of Arkansas for Medical Sciences, Little Rock, AR, USA,
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18
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Schneider AJ, Branam AM, Peterson RE. Intersection of AHR and Wnt signaling in development, health, and disease. Int J Mol Sci 2014; 15:17852-85. [PMID: 25286307 PMCID: PMC4227194 DOI: 10.3390/ijms151017852] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2014] [Revised: 09/04/2014] [Accepted: 09/18/2014] [Indexed: 12/16/2022] Open
Abstract
The AHR (aryl hydrocarbon receptor) and Wnt (wingless-related MMTV integration site) signaling pathways have been conserved throughout evolution. Appropriately regulated signaling through each pathway is necessary for normal development and health, while dysregulation can lead to developmental defects and disease. Though both pathways have been vigorously studied, there is relatively little research exploring the possibility of crosstalk between these pathways. In this review, we provide a brief background on (1) the roles of both AHR and Wnt signaling in development and disease, and (2) the molecular mechanisms that characterize activation of each pathway. We also discuss the need for careful and complete experimental evaluation of each pathway and describe existing research that explores the intersection of AHR and Wnt signaling. Lastly, to illustrate in detail the intersection of AHR and Wnt signaling, we summarize our recent findings which show that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced disruption of Wnt signaling impairs fetal prostate development.
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Affiliation(s)
- Andrew J Schneider
- School of Pharmacy and Molecular and Environmental Toxicology Center University of Wisconsin, Madison, WI 53705, USA.
| | - Amanda M Branam
- School of Pharmacy and Molecular and Environmental Toxicology Center University of Wisconsin, Madison, WI 53705, USA.
| | - Richard E Peterson
- School of Pharmacy and Molecular and Environmental Toxicology Center University of Wisconsin, Madison, WI 53705, USA.
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Xu B, Wang F, Song C, Sun Z, Cheng K, Tan Y, Wang H, Zou H. Large-scale proteome quantification of hepatocellular carcinoma tissues by a three-dimensional liquid chromatography strategy integrated with sample preparation. J Proteome Res 2014; 13:3645-54. [PMID: 24972180 DOI: 10.1021/pr500200s] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatocellular carcinoma is one of the most fatal cancers worldwide. In this study, a reversed-phase-strong cation exchange-reversed-phase three-dimensional liquid chromatography strategy was established and coupled with mass spectrometry to investigate the differential proteome expression of HCC and normal liver tissues. In total, 2759 proteins were reliably quantified, of which, 648 proteins were dysregulated more than 3-fold in HCC liver tissues. Some important proteins that relate to HCC pathology were significantly dysregulated, such as NAT2 and AKR1B10. Furthermore, 2307 phosphorylation sites from 1264 phosphoproteins were obtained in our previous phosphoproteome quantification, and the nonphosphorylated counterparts of 445 phosphoproteins with 983 phosphorylation sites were reliably quantified in this work. It was observed that 337 (34%) phosphorylation sites exhibit significantly different expression trends from that of their corresponding nonphosphoproteins. Some novel phosphorylation sites with important biological functions in the progression of HCC were reliably quantified, such as the significant downregulation of pT185 for ERK2 and pY204 for ERK1.
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Affiliation(s)
- Bo Xu
- Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China
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20
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Zhang J, Pan Z, Moloney S, Sheppard A. RNA-Seq analysis implicates detoxification pathways in ovine mycotoxin resistance. PLoS One 2014; 9:e99975. [PMID: 24936865 PMCID: PMC4061066 DOI: 10.1371/journal.pone.0099975] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2014] [Accepted: 05/21/2014] [Indexed: 11/19/2022] Open
Abstract
Mycotoxin induced hepatoxocity has been linked to oxidative stress, resulting from either an increase in levels of reactive oxygen species (ROS) above normal levels and/or the suppression of antioxidant protective pathways. However, few detailed molecular studies of mycotoxicoses in animals have been carried out. This study use current RNA-seq based approaches to investigate the effects of mycotoxin exposure in a ruminant model. Having first assembled a de novo reference transcriptome, we use RNA-Seq technology to define in vivo hepatic gene expression changes resulting from mycotoxin exposure in relationship to pathological effect. As expected, characteristic oxidative stress related gene expression is markedly different in animals exhibiting poorer outcomes. However, expression of multiple genes critical for detoxification, particularly members of the cytochrome P450 gene family, was significantly higher in animals exhibiting mycotoxin tolerance ('resistance'). Further, we present novel evidence for the amplification of Wnt signalling pathway activity in 'resistant' animals, resulting from the marked suppression of multiple key Wnt inhibitor genes. Notably, 'resistance' may be determined primarily by the ability of an individual to detoxify secondary metabolites generated by the metabolism of mycotoxins and the potentiation of Wnt signalling may be pivotal to achieving a favourable outcome upon challenge.
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Affiliation(s)
- Jinbi Zhang
- Liggins Institute, University of Auckland, Auckland, New Zealand
| | - Zengxiang Pan
- Liggins Institute, University of Auckland, Auckland, New Zealand
| | | | - Allan Sheppard
- Liggins Institute, University of Auckland, Auckland, New Zealand
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21
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Genetic ablation of β-catenin inhibits the proliferative phenotype of mouse liver adenomas. Br J Cancer 2014; 111:132-8. [PMID: 24874479 PMCID: PMC4090738 DOI: 10.1038/bjc.2014.275] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2014] [Revised: 04/10/2014] [Accepted: 04/25/2014] [Indexed: 01/22/2023] Open
Abstract
BACKGROUND Aberrant activation of Wnt/β-catenin has been implicated in various cancer-related processes, for example, proliferation or tumour cell survival. However, the exact mechanism by which β-catenin provides liver tumour cells with a selective advantage is still unclear. This study was aimed to analyse growth behaviour and survival of β-catenin-driven mouse liver tumours after β-catenin ablation. METHODS Transgenic mice with a controllable hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) were generated and liver tumours were induced by means of a N-nitrosodiethylamine/phenobarbital tumour initiation/promotion protocol, which leads to the outgrowth of hepatocellular tumours with activated β-catenin. Cre recombinase was activated and the effects of the knockout in the tumours were studied. RESULTS Activation of Cre recombinase led to the knockout of Ctnnb1 in a fraction of tumour cells, thus resulting in the formation of two different tumour cell subpopulations, with or without β-catenin. Comparative analysis of the two subpopulations revealed that cell proliferation was significantly decreased in Ctnnb1-deleted hepatoma cells, compared with the corresponding non-deleted cell population, whereas no increased rate of apoptosis after knockout of Ctnnb1 was observed. CONCLUSIONS β-catenin-dependent signalling is an important regulator of hepatoma cell growth in mice, but not a crucial factor in the regulation of tumour survival.
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22
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Mercer KE, Hennings L, Sharma N, Lai K, Cleves MA, Wynne RA, Badger TM, Ronis MJJ. Alcohol consumption promotes diethylnitrosamine-induced hepatocarcinogenesis in male mice through activation of the Wnt/β-catenin signaling pathway. Cancer Prev Res (Phila) 2014; 7:675-85. [PMID: 24778325 DOI: 10.1158/1940-6207.capr-13-0444-t] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Although alcohol effects within the liver have been extensively studied, the complex mechanisms by which alcohol causes liver cancer are not well understood. It has been suggested that ethanol (EtOH) metabolism promotes tumor growth by increasing hepatocyte proliferation. In this study, we developed a mouse model of tumor promotion by chronic EtOH consumption in which EtOH feeding began 46 days after injection of the chemical carcinogen diethylnitrosamine (DEN) and continued for 16 weeks. With a final EtOH concentration of 28% of total calories, we observed a significant increase in the total number of preneoplastic foci and liver tumors per mouse in the EtOH+DEN group compared with corresponding pair-fed (PF)+DEN and chow+DEN control groups. We also observed a 4-fold increase in hepatocyte proliferation (P < 0.05) and increased cytoplasmic staining of active-β-catenin in nontumor liver sections from EtOH+DEN mice compared with PF+DEN controls. In a rat model of alcohol-induced liver disease, we found increased hepatocyte proliferation (P < 0.05); depletion of retinol and retinoic acid stores (P < 0.05); increased expression of cytosolic and nuclear expression of β-catenin (P < 0.05) and phosphorylated-glycogen synthase kinase 3β (p-GSK3β), P < 0.05; significant upregulation in Wnt7a mRNA expression; and increased expression of several β-catenin targets, including, glutamine synthetase (GS), cyclin D1, Wnt1 inducible signaling pathways protein (WISP1), and matrix metalloproteinase-7(MMP7), P < 0.05. These data suggest that chronic EtOH consumption activates the Wnt/β-catenin signaling pathways to increase hepatocyte proliferation, thus promoting tumorigenesis following an initiating insult to the liver.
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Affiliation(s)
- Kelly E Mercer
- Authors' Affiliations: Departments of Pediatrics and Arkansas Children's Nutrition Center, Little Rock, Arkansas
| | - Leah Hennings
- Pathology at the University of Arkansas for Medical Sciences; and
| | - Neha Sharma
- Arkansas Children's Nutrition Center, Little Rock, Arkansas
| | - Keith Lai
- Pathology at the University of Arkansas for Medical Sciences; and
| | - Mario A Cleves
- Arkansas Children's Nutrition Center, Little Rock, Arkansas
| | | | - Thomas M Badger
- Authors' Affiliations: Departments of Pediatrics and Arkansas Children's Nutrition Center, Little Rock, Arkansas
| | - Martin J J Ronis
- Authors' Affiliations: Departments of Pediatrics and Arkansas Children's Nutrition Center, Little Rock, Arkansas
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23
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Santos NP, Oliveira PA, Arantes-Rodrigues R, Faustino-Rocha AI, Colaço A, Lopes C, Gil da Costa RM. Cytokeratin 7/19 expression in N-diethylnitrosamine-induced mouse hepatocellular lesions: implications for histogenesis. Int J Exp Pathol 2014; 95:191-8. [PMID: 24730441 DOI: 10.1111/iep.12082] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2013] [Accepted: 03/06/2014] [Indexed: 01/20/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is a common malignancy with poor clinical outcome, whose histogenesis is the subject of intense debate. Specifically, expression of cytokeratins (CKs) 7 and 19, associated with aggressive biological behaviour, is proposed to reflect a possible progenitor cell origin or tumour dedifferentiation towards a primitive phenotype. This work addresses that problem by studying CKs 7 and 19 expression in N-diethylnitrosamine (DEN)-induced mouse HCCs. ICR mice were divided into six DEN-exposed and six matched control groups. Samples were taken from each group at consecutive time points. Hyperplastic foci (13 lesions) occurred at 29-40 weeks (groups 8, 10 and 12) with diffuse dysplastic areas (19 lesions) and with one hepatocellular adenoma (HCA) (at 29 weeks). HCCs (4 lesions) were observed 40 weeks after the first DEN administration (group 12). CKs 7 and 19 showed identical expression patterns and located to large, mature hepatocytes, isolated or in small clusters. Hyperplastic foci and the single HCA were consistently negative for both markers, while dysplastic areas and HCCs were positive. These results support the hypothesis that CKs 7 and 19 expression in hepatocellular malignancies results from a dedifferentiation process rather than from a possible progenitor cell origin.
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Affiliation(s)
- Nuno P Santos
- Veterinary Sciences Department, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal; Veterinary Science Department, Veterinary and Animal Science Research Centre (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal
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24
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Unterberger EB, Eichner J, Wrzodek C, Lempiäinen H, Luisier R, Terranova R, Metzger U, Plummer S, Knorpp T, Braeuning A, Moggs J, Templin MF, Honndorf V, Piotto M, Zell A, Schwarz M. Ha-ras and β-catenin oncoproteins orchestrate metabolic programs in mouse liver tumors. Int J Cancer 2014; 135:1574-85. [PMID: 24535843 DOI: 10.1002/ijc.28798] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Accepted: 02/06/2014] [Indexed: 01/08/2023]
Abstract
The process of hepatocarcinogenesis in the diethylnitrosamine (DEN) initiation/phenobarbital (PB) promotion mouse model involves the selective clonal outgrowth of cells harboring oncogene mutations in Ctnnb1, while spontaneous or DEN-only-induced tumors are often Ha-ras- or B-raf-mutated. The molecular mechanisms and pathways underlying these different tumor sub-types are not well characterized. Their identification may help identify markers for xenobiotic promoted versus spontaneously occurring liver tumors. Here, we have characterized mouse liver tumors harboring either Ctnnb1 or Ha-ras mutations via integrated molecular profiling at the transcriptional, translational and post-translational levels. In addition, metabolites of the intermediary metabolism were quantified by high resolution (1)H magic angle nuclear magnetic resonance. We have identified tumor genotype-specific differences in mRNA and miRNA expression, protein levels, post-translational modifications, and metabolite levels that facilitate the molecular and biochemical stratification of tumor phenotypes. Bioinformatic integration of these data at the pathway level led to novel insights into tumor genotype-specific aberrant cell signaling and in particular to a better understanding of alterations in pathways of the cell intermediary metabolism, which are driven by the constitutive activation of the β-Catenin and Ha-ras oncoproteins in tumors of the two genotypes.
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Affiliation(s)
- Elif B Unterberger
- Institute of Experimental and Clinical Pharmacology and Toxicology Department of Toxicology, Eberhard Karls University of Tübingen, Tübingen, 72074, Germany
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25
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Hoenerhoff MJ, Pandiri AR, Snyder SA, Hong HHL, Ton TV, Peddada S, Shockley K, Witt K, Chan P, Rider C, Kooistra L, Nyska A, Sills RC. Hepatocellular carcinomas in B6C3F1 mice treated with Ginkgo biloba extract for two years differ from spontaneous liver tumors in cancer gene mutations and genomic pathways. Toxicol Pathol 2013; 41:826-41. [PMID: 23262642 PMCID: PMC4799723 DOI: 10.1177/0192623312467520] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Ginkgo biloba leaf extract (GBE) has been used for centuries in traditional Chinese medicine and today is used as an herbal supplement touted for improving neural function and for its antioxidant and anticancer effects. Herbal supplements have the potential for consumption over extended periods of time, with a general lack of sufficient data on long-term carcinogenicity risk. Exposure of B6C3F1 mice to GBE in the 2-year National Toxicology Program carcinogenicity bioassay resulted in a dose-dependent increase in hepatocellular tumors, including hepatocellular carcinoma (HCC). We show that the mechanism of hepatocarcinogenesis in GBE exposed animals is complex, involving alterations in H-ras and Ctnnb1 mutation spectra, WNT pathway dysregulation, and significantly altered gene expression associated with oncogenesis, HCC development, and chronic xenobiotic and oxidative stress compared to spontaneous HCC. This study provides a molecular context for the genetic changes associated with hepatocarcinogenesis in GBE exposed mice and illustrates the marked differences between these tumors and those arising spontaneously in the B6C3F1 mouse. The molecular changes observed in HCC from GBE-treated animals may be of relevance to those seen in human HCC and other types of cancer, and provide important data on potential mechanisms of GBE hepatocarcinogenesis.
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Affiliation(s)
- Mark J Hoenerhoff
- Cellular and Molecular Pathology Branch, National Institute of Environmental Health Sciences and National Toxicology Program, Research Triangle Park, NC 27519, USA.
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Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CEP, Gómez-Lechón MJ, Groothuis GMM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EHK, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 2013; 87:1315-530. [PMID: 23974980 PMCID: PMC3753504 DOI: 10.1007/s00204-013-1078-5] [Citation(s) in RCA: 965] [Impact Index Per Article: 80.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2013] [Accepted: 05/06/2013] [Indexed: 12/15/2022]
Abstract
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
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Affiliation(s)
- Patricio Godoy
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | | | - Ute Albrecht
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Melvin E. Andersen
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Nariman Ansari
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Sudin Bhattacharya
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Johannes Georg Bode
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Jennifer Bolleyn
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Christoph Borner
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
| | - Jan Böttger
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Albert Braeuning
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Wilhelmstr. 56, 72074 Tübingen, Germany
| | - Robert A. Budinsky
- Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI USA
| | - Britta Burkhardt
- BG Trauma Center, Siegfried Weller Institut, Eberhard Karls University Tübingen, 72076 Tübingen, Germany
| | - Neil R. Cameron
- Department of Chemistry, Durham University, Durham, DH1 3LE UK
| | - Giovanni Camussi
- Department of Medical Sciences, University of Torino, 10126 Turin, Italy
| | - Chong-Su Cho
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - Yun-Jaie Choi
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - J. Craig Rowlands
- Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI USA
| | - Uta Dahmen
- Experimental Transplantation Surgery, Department of General Visceral, and Vascular Surgery, Friedrich-Schiller-University Jena, 07745 Jena, Germany
| | - Georg Damm
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
| | - Olaf Dirsch
- Institute of Pathology, Friedrich-Schiller-University Jena, 07745 Jena, Germany
| | - María Teresa Donato
- Unidad de Hepatología Experimental, IIS Hospital La Fe Avda Campanar 21, 46009 Valencia, Spain
- CIBERehd, Fondo de Investigaciones Sanitarias, Barcelona, Spain
- Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain
| | - Jian Dong
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Steven Dooley
- Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Dirk Drasdo
- Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, 04107 Leipzig, Germany
- INRIA (French National Institute for Research in Computer Science and Control), Domaine de Voluceau-Rocquencourt, B.P. 105, 78153 Le Chesnay Cedex, France
- UPMC University of Paris 06, CNRS UMR 7598, Laboratoire Jacques-Louis Lions, 4, pl. Jussieu, 75252 Paris cedex 05, France
| | - Rowena Eakins
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Karine Sá Ferreira
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
- GRK 1104 From Cells to Organs, Molecular Mechanisms of Organogenesis, Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Valentina Fonsato
- Department of Medical Sciences, University of Torino, 10126 Turin, Italy
| | - Joanna Fraczek
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Rolf Gebhardt
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Andrew Gibson
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Matthias Glanemann
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
| | - Chris E. P. Goldring
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - María José Gómez-Lechón
- Unidad de Hepatología Experimental, IIS Hospital La Fe Avda Campanar 21, 46009 Valencia, Spain
- CIBERehd, Fondo de Investigaciones Sanitarias, Barcelona, Spain
| | - Geny M. M. Groothuis
- Department of Pharmacy, Pharmacokinetics Toxicology and Targeting, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands
| | - Lena Gustavsson
- Department of Laboratory Medicine (Malmö), Center for Molecular Pathology, Lund University, Jan Waldenströms gata 59, 205 02 Malmö, Sweden
| | - Christelle Guyot
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - David Hallifax
- Centre for Applied Pharmacokinetic Research (CAPKR), School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT UK
| | - Seddik Hammad
- Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt
| | - Adam Hayward
- Biological and Biomedical Sciences, Durham University, Durham, DH13LE UK
| | - Dieter Häussinger
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Claus Hellerbrand
- Department of Medicine I, University Hospital Regensburg, 93053 Regensburg, Germany
| | | | - Stefan Hoehme
- Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, 04107 Leipzig, Germany
| | - Hermann-Georg Holzhütter
- Institut für Biochemie Abteilung Mathematische Systembiochemie, Universitätsmedizin Berlin (Charité), Charitéplatz 1, 10117 Berlin, Germany
| | - J. Brian Houston
- Centre for Applied Pharmacokinetic Research (CAPKR), School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT UK
| | | | - Kiyomi Ito
- Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo, 202-8585 Japan
| | - Hartmut Jaeschke
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 USA
| | - Verena Keitel
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | | | - B. Kevin Park
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Claus Kordes
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Gerd A. Kullak-Ublick
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Edward L. LeCluyse
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Peng Lu
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | | | - Anna Lutz
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | - Daniel J. Maltman
- Reinnervate Limited, NETPark Incubator, Thomas Wright Way, Sedgefield, TS21 3FD UK
| | - Madlen Matz-Soja
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Patrick McMullen
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Irmgard Merfort
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | | | - Christoph Meyer
- Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Jessica Mwinyi
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Dean J. Naisbitt
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Andreas K. Nussler
- BG Trauma Center, Siegfried Weller Institut, Eberhard Karls University Tübingen, 72076 Tübingen, Germany
| | - Peter Olinga
- Division of Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands
| | - Francesco Pampaloni
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Jingbo Pi
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Linda Pluta
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Stefan A. Przyborski
- Reinnervate Limited, NETPark Incubator, Thomas Wright Way, Sedgefield, TS21 3FD UK
- Biological and Biomedical Sciences, Durham University, Durham, DH13LE UK
| | - Anup Ramachandran
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 USA
| | - Vera Rogiers
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Cliff Rowe
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Celine Schelcher
- Department of Surgery, Liver Regeneration, Core Facility, Human in Vitro Models of the Liver, Ludwig Maximilians University of Munich, Munich, Germany
| | - Kathrin Schmich
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | - Michael Schwarz
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Wilhelmstr. 56, 72074 Tübingen, Germany
| | - Bijay Singh
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - Ernst H. K. Stelzer
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Bruno Stieger
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Regina Stöber
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | - Yuichi Sugiyama
- Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Yokohama Biopharmaceutical R&D Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045 Japan
| | - Ciro Tetta
- Fresenius Medical Care, Bad Homburg, Germany
| | - Wolfgang E. Thasler
- Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich, Germany
| | - Tamara Vanhaecke
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Mathieu Vinken
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Thomas S. Weiss
- Department of Pediatrics and Juvenile Medicine, University of Regensburg Hospital, Regensburg, Germany
| | - Agata Widera
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | - Courtney G. Woods
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | | | | | - Jan G. Hengstler
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
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Wang J, Park JS, Wei Y, Rajurkar M, Cotton JL, Fan Q, Lewis BC, Ji H, Mao J. TRIB2 acts downstream of Wnt/TCF in liver cancer cells to regulate YAP and C/EBPα function. Mol Cell 2013; 51:211-25. [PMID: 23769673 DOI: 10.1016/j.molcel.2013.05.013] [Citation(s) in RCA: 123] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2012] [Revised: 02/25/2013] [Accepted: 05/08/2013] [Indexed: 01/12/2023]
Abstract
Dysregulation of Wnt signaling is closely associated with human liver tumorigenesis. However, liver cancer-specific Wnt transcriptional programs and downstream effectors remain poorly understood. Here, we identify tribbles homolog 2 (TRIB2) as a direct target of Wnt/TCF in liver cancer and demonstrate that transcription of Wnt target genes, including TRIB2, is coordinated by the TCF and FoxA transcription factors in liver cancer cells. We show that Wnt-TRIB2 activation is critical for cancer cell survival and transformation. Mechanistically, TRIB2 promotes protein stabilization of the YAP transcription coactivator through interaction with the βTrCP ubiquitin ligase. Furthermore, we find that TRIB2 relieves the liver tumor suppressor protein C/EBPα-mediated inhibition of YAP/TEAD transcriptional activation in liver cancer cells. Altogether, our study uncovers a regulatory mechanism underlying liver cancer-specific Wnt transcriptional output, and suggests that TRIB2 functions as a signaling nexus to integrate the Wnt/β-catenin, Hippo/YAP, and C/EBPα pathways in cancer cells.
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Affiliation(s)
- Jiayi Wang
- Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA
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28
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Ganzenberg K, Singh Y, Braeuning A. The time point of β-catenin knockout in hepatocytes determines their response to xenobiotic activation of the constitutive androstane receptor. Toxicology 2013; 308:113-21. [DOI: 10.1016/j.tox.2013.03.019] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2013] [Revised: 03/27/2013] [Accepted: 03/30/2013] [Indexed: 12/13/2022]
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Zeller E, Hammer K, Kirschnick M, Braeuning A. Mechanisms of RAS/β-catenin interactions. Arch Toxicol 2013; 87:611-32. [PMID: 23483189 DOI: 10.1007/s00204-013-1035-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2013] [Accepted: 02/28/2013] [Indexed: 12/20/2022]
Abstract
Signaling through the WNT/β-catenin and the RAS (rat sarcoma)/MAPK (mitogen-activated protein kinase) pathways plays a key role in the regulation of various physiological cellular processes including proliferation, differentiation, and cell death. Aberrant mutational activation of these signaling pathways is closely linked to the development of cancer in many organs, in humans as well as in laboratory animals. Over the past years, more and more evidence for a close linkage of the two oncogenic signaling cascades has accumulated. Using different experimental approaches, model systems, and experimental conditions, a variety of molecular mechanisms have been identified by which signal transduction through WNT/β-catenin and RAS interact, either in a synergistic or an antagonistic manner. Mechanisms of interaction comprise an upstream crosstalk at the level of pathway-activating ligands and their receptors, interrelations of cytosolic kinases involved in either pathways, as well as interaction in the nucleus related to the joint regulation of target gene transcription. Here, we present a comprehensive review of the current knowledge on the interaction of RAS/MAPK- and WNT/β-catenin-driven signal transduction in mammalian cells.
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Affiliation(s)
- Eva Zeller
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Germany
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Singh Y, Braeuning A, Schmid A, Pichler BJ, Schwarz M. Selective poisoning of Ctnnb1-mutated hepatoma cells in mouse liver tumors by a single application of acetaminophen. Arch Toxicol 2013; 87:1595-607. [DOI: 10.1007/s00204-013-1030-8] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2012] [Accepted: 02/25/2013] [Indexed: 01/26/2023]
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Abstract
Hepatocellular carcinoma (HCC), the most common form of primary liver cancer is the third leading cause of cancer-related cell death in human and the fifth in women worldwide. The incidence of HCC is increasing despite progress in identifying risk factors, understanding disease etiology and developing anti-viral strategies. Therapeutic options are limited and survival after diagnosis is poor. Therefore, better preventive, diagnostic and therapeutic tools are urgently needed, in particular given the increased contribution from systemic metabolic disease to HCC incidence worldwide. In the last three decades, technological advances have facilitated the generation of genetically engineered mouse models (GEMMs) to mimic the alterations frequently observed in human cancers or to conduct intervention studies and assess the relevance of candidate gene networks in tumor establishment, progression and maintenance. Because these studies allow molecular and cellular manipulations impossible to perform in patients, GEMMs have improved our understanding of this complex disease and represent a source of great potential for mechanism-based therapy development. In this review, we provide an overview of the current state of HCC modeling in the mouse, highlighting successes, current challenges and future opportunities.
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Cellular and molecular mechanisms of hepatocellular carcinoma: an update. Arch Toxicol 2012; 87:227-47. [PMID: 23007558 DOI: 10.1007/s00204-012-0931-2] [Citation(s) in RCA: 175] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2012] [Accepted: 08/27/2012] [Indexed: 12/11/2022]
Abstract
Hepatocellular carcinoma (HCC) is the most common primary malignant tumor that accounts for ~80 % of all liver cancer cases worldwide. It is a multifactorial disease caused by a variety of risk factors and often develops in the background of underlying cirrhosis. A number of cellular phenomena, such as tumor microenvironment, inflammation, oxidative stress, and hypoxia act in concert with various molecular events to facilitate tumor initiation, progression, and metastasis. The emergence of microRNAs and molecular-targeted therapies adds a new dimension in our efforts to combat this deadly disease. Intense research in this multitude of areas has led to significant progress in our understanding of cellular processes and molecular mechanisms that occur during multistage events that lead to hepatocarcinogenesis. In this review, we discuss the current knowledge of HCC, focusing mainly on advances that have occurred during the past 5 years and on the development of novel therapeutics for liver cancer.
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Schmid A, Rignall B, Pichler BJ, Schwarz M. Quantitative analysis of the growth kinetics of chemically induced mouse liver tumors by magnetic resonance imaging. Toxicol Sci 2012; 126:52-9. [PMID: 22273797 DOI: 10.1093/toxsci/kfs018] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Noninvasive methods for the early detection of tumor onset and progression in rodent liver would be of high value for pharmaceutical and chemical industry and would, at the same time, foster one of the 3 Rs (replacement, reduction, refinement) by reducing the number of animals in the bioassay. We have induced liver tumors in mice by single injection of diethylnitrosamine (DEN) either in 2-week- (experiment 1) or 6-week-old (experiment 2) male C3H mice. In the latter, mice were also chronically treated with 0.05% phenobarbital in diet according to an initiation/promotion protocol. Starting at 16 weeks after DEN injection (18 weeks after DEN in experiment 2), mice were routinely scanned by noninvasive magnetic resonance imaging (MRI) using a T2-weighted 3D sequence in regular intervals. Liver tumors became detectable in both experiments when they exceeded a diameter of ∼1 mm. Exponential increases in total tumor volume per liver were observed in both experiments. The onset of tumor development was similar with respect to DEN treatment. Although mice in experiment 1 had developed a mean total tumor volume of ∼100 mm³ approximately 24 weeks after DEN, it took ∼4 weeks longer to reach this tumor mass in experiment 2. Determination of time-dependent growth of individual tumors demonstrated strong tumor heterogeneity. In vivo MRI data were further correlated with tumor histology. The phenotype of tumors differed strongly between the two experiments, but our results demonstrate that tumors can be reliably detected by MRI when they exceed a certain size independent of their phenotype.
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Affiliation(s)
- Andreas Schmid
- Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-Foundation, Department of Preclinical Imaging and Radiopharmacy, University of Tübingen, Röntgenweg 13, 72076 Tübingen, Germany
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Braeuning A, Köhle C, Buchmann A, Schwarz M. Coordinate regulation of cytochrome P450 1a1 expression in mouse liver by the aryl hydrocarbon receptor and the beta-catenin pathway. Toxicol Sci 2011; 122:16-25. [PMID: 21498875 DOI: 10.1093/toxsci/kfr080] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The expression of cytochrome P450 (CYP) 1a1 and other drug-metabolizing enzymes is controlled by the aryl hydrocarbon receptor (AhR), which is activated by dioxin-type inducers leading to transcriptional induction of target genes. Here, we show that a second level of transcriptional control exists in hepatocytes, which is tightly linked to the Wnt/β-catenin/T-cell factor (TCF) signaling pathway. In transgenic mice, hepatic expression of CYP1A (and other CYP isoforms) is stimulated by the expression of mutationally activated β-catenin(S33Y) in the absence of AhR-activating compounds but repressed after knockout of β-catenin. These effects were further analyzed in vitro, and the stimulatory role of β-catenin was ascribed to a TCF-binding site within the CYP1A1 promoter. Moreover, β-catenin signaling acted cooperatively with AhR agonists via AhR-binding sites on the DNA during the induction of Cyp1a1 in vivo and in vitro. Activation of β-catenin enhanced the transactivation potential of ligand-activated AhR at its DNA-binding sites without altering the total amount of DNA-bound AhR. Coimmunoprecipitation demonstrated a physical interaction between AhR and β-catenin. Furthermore, the present results suggest that transcriptional induction of the AhR by β-catenin does not play a major role in β-catenin-dependent regulation of Cyp1a1 expression and that inhibition of β-catenin signaling by ligand-activated AhR, as recently observed in the intestine does not occur in mouse liver. In conclusion, signaling through β-catenin activates basal CYP1A1 expression and augments CYP1A1 induction by AhR ligands through enhancement of the transactivation potential of the AhR.
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Affiliation(s)
- Albert Braeuning
- Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Tübingen, Germany
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Stein TJ, Jochem A, Holmes KE, Sandgren EP. Effect of mutant β-catenin on liver growth homeostasis and hepatocarcinogenesis in transgenic mice. Liver Int 2011; 31:303-12. [PMID: 21281430 PMCID: PMC3093768 DOI: 10.1111/j.1478-3231.2010.02430.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND Mutations in the Wnt signalling pathway molecule β-catenin are associated with liver cancer. AIMS Our aim was to confirm the effects of stabilized β-catenin on liver growth, identify whether those effects were reversible and cell autonomous or non-cell autonomous and to model β-catenin-induced liver cancer in mice. METHODS Using a liver-specific inducible promoter, we generated transgenic mice in which the expression of mutant β-catenin can be induced or repressed within hepatocytes in mice of different ages. RESULTS Similar to other models, the hepatic expression of mutant β-catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two-fold liver enlargement and development of disease with a latency of 1-5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant β-catenin could not increase the growth of transgene-expressing foci in either growth-permissive or -restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year. CONCLUSIONS Our findings indicate that hepatocyte gene expression is directly affected by mutant β-catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte-specific expression of mutant β-catenin is secondary to liver functional alteration or non-cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene-expressing cells progressed to carcinoma, confirming the association of mutant β-catenin with liver cancer.
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Affiliation(s)
- Timothy J. Stein
- Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | - Adam Jochem
- Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | - Katie E. Holmes
- Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | - Eric P. Sandgren
- Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA
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Lahousse SA, Hoenerhoff M, Collins J, Ton TVT, Masinde T, Olson D, Rebolloso Y, Koujitani T, Tomer KB, Hong HHL, Bucher J, Sills RC. Gene expression and mutation assessment provide clues of genetic and epigenetic mechanisms in liver tumors of oxazepam-exposed mice. Vet Pathol 2010; 48:875-84. [PMID: 21147764 DOI: 10.1177/0300985810390019] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Liver tumors from a previous National Toxicology Program study were examined using global gene expression and mutation analysis to define the mechanisms of carcinogenesis in mice exposed to oxazepam. Five hepatocellular adenomas and 5 hepatocellular carcinomas from male B6C3F1 mice exposed to 5000 ppm oxazepam and 6 histologically normal liver samples from control animals were examined. One of the major findings in the study was upregulation of the Wnt/β-catenin signaling pathway. Genes that activate β-catenin, such as Sox4, were upregulated, whereas genes that inhibit Wnt signaling, such as APC and Crebbp, were downregulated. In addition, liver tumors from oxazepam-exposed mice displayed β-catenin mutations and increased protein expression of glutamine synthetase, a downstream target in the Wnt signaling pathway. Another important finding in this study was the altered expression of oxidative stress-related genes, specifically increased expression of cytochrome p450 genes, including Cyp1a2 and Cyp2b10, and decreased expression of genes that protect against oxidative stress, such as Sod2 and Cat. Increased oxidative stress was confirmed by measuring isoprostane expression using mass spectrometry. Furthermore, global gene expression identified altered expression of genes that are associated with epigenetic mechanisms of cancer. There was decreased expression of genes that are hypermethylated in human liver cancer, including tumor suppressors APC and Pten. Oxazepam-induced tumors also exhibited decreased expression of genes involved in DNA methylation (Crebbp, Dnmt3b) and histone modification (Sirt1). These data suggest that formation of hepatocellular adenomas and carcinomas in oxazepam-exposed mice involves alteration of the Wnt signaling pathway, oxidative stress, and potential epigenetic alterations.
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Affiliation(s)
- S A Lahousse
- Cellular and Molecular Pathology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
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Braeuning A, Schwarz M. Zonation of heme synthesis enzymes in mouse liver and their regulation by β-catenin and Ha-ras. Biol Chem 2010; 391:1305-13. [DOI: 10.1515/bc.2010.115] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Abstract
Cytochrome P450 (CYP) hemoproteins play an important role in hepatic biotransformation. Recently, β-catenin and Ha-ras signaling have been identified as players controlling transcription of various CYP genes in mouse liver. The aim of the present study was to analyze the role of β-catenin and Ha-ras in the regulation of heme synthesis. Heme synthesis-related gene expression was analyzed in normal liver, in transgenic mice expressing activated β-catenin or Ha-ras, and in hepatomas. Regulation of the aminolevulinate dehydratase promoter was studied in vitro. Elevated expression of mRNAs and proteins involved in heme biosynthesis was linked to β-catenin activation in perivenous hepatocytes, in transgenic hepatocytes, and in hepatocellular tumors. Stimulation of the aminolevulinate dehydratase promoter by β-catenin was independent of the β-catenin/T-cell-specific transcription factor dimer. By contrast, activation of Ha-ras repressed heme synthesis-related gene expression. The present data suggest that β-catenin enhances the expression of both CYPs and heme synthesis-related genes, thus coordinating the availability of CYP apoprotein and its prosthetic group heme. The reciprocal regulation of heme synthesis by β-catenin and Ha-ras-dependent signaling supports our previous hypothesis that antagonistic action of these pathways plays a major role in the control of zonal gene expression in healthy mouse liver and aberrant expression patterns in hepatocellular tumors.
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Dal Bello B, Rosa L, Campanini N, Tinelli C, Torello Viera F, D'Ambrosio G, Rossi S, Silini EM. Glutamine synthetase immunostaining correlates with pathologic features of hepatocellular carcinoma and better survival after radiofrequency thermal ablation. Clin Cancer Res 2010; 16:2157-66. [PMID: 20233882 DOI: 10.1158/1078-0432.ccr-09-1978] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
PURPOSE Activation of the wnt pathway identifies a subgroup of hepatocellular carcinomas (HCC) with specific epidemiologic and genetic profiles. Wnt activation is predicted by mutation and/or nuclear translocation of beta-catenin and by glutamine synthetase (GS) immunoreactivity. We investigated whether GS staining associates with specific pathologic features of HCC and with survival after radiofrequency thermal ablation. EXPERIMENTAL DESIGN Monoistitutional retrospective-prospective study in a tertiary hospital setting. Two hundred and seven cirrhotics (mean age, 70 years; 63% males, 82.1% hepatitis C virus positive) with early HCC were consecutively treated with radiofrequency thermal ablation (RFTA). Mean tumor size was 2.7 cm; 20.3% of patients had multiple nodules; and median follow-up was 36 months with 54.6% overall mortality. Tumor samples were mainly obtained by biopsy (92,5%) and examined by H&E and immunostaining for beta-catenin and GS. Main outcome measures were overall and tumor-specific mortality by Kaplan-Meier analysis and Cox proportional hazard models corrected for competing risks. RESULTS Ninety-one patients (43.9%) had GS-positive HCCs by immunostaining. These tumors had larger size (P = 0.012) and characteristic histology (low grade, pseudoacini, hydropic changes, bile staining, lack of steatosis, and fibrosis). Other clinical or treatment variables were similar between groups. Variables correlating with tumor-specific and overall mortality by univariate analysis were tumor recurrence, advanced disease, posttreatment alpha-fetoprotein levels, and GS staining. Yearly, overall mortality rate was lower in GS-positive patients (12.4 versus 20% yearly; P = 0.006). By multivariate analysis, GS immunostaining correlated with reduced specific (hazard ratio, 0.58; 95% confidence interval, 0.34-0.97) and overall mortality (hazard ratio, 0.62; 95% confidence interval, 0.40-0.96). CONCLUSIONS Standard histology and GS status identify a HCC subset with distinct clinical and pathologic features.
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Affiliation(s)
- Barbara Dal Bello
- Departments of Pathology, VI Internal Medicine, and Biostatistics, Istituto Di Ricovero e Cura a Carattere Scientifico-Fondazione Policlinico San Matteo and University of Pavia, Pavia, Italy.
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Giera S, Braeuning A, Köhle C, Bursch W, Metzger U, Buchmann A, Schwarz M. Wnt/beta-catenin signaling activates and determines hepatic zonal expression of glutathione S-transferases in mouse liver. Toxicol Sci 2010; 115:22-33. [PMID: 20118494 DOI: 10.1093/toxsci/kfq033] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Glutathione S-transferases (GSTs) play an essential role in the elimination of xenobiotic-derived electrophilic metabolites and also catalyze certain steps in the conversion of endogenous molecules. Their expression is controlled by different transcription factors, such as the antioxidant-activated Nrf2 or the constitutive androstane receptor. Here, we show that the Wnt/beta-catenin pathway is also involved in the transcriptional regulation of GSTs: GSTm2, GSTm3, and GSTm6 are overexpressed in mouse hepatomas with activating Ctnnb1 (encoding beta-catenin) mutations and in transgenic hepatocytes expressing activated beta-catenin. Inversely, GSTm expression is reduced in mice with hepatocyte-specific knock out of Ctnnb1. Activation of beta-catenin-dependent signaling stimulates GSTm expression in vitro. Activation of beta-catenin in mouse hepatoma cells activates GSTm3 promoter-driven reporter activity, independently of beta-catenin/T-cell factor sites, via a retinoid X receptor-binding site. By contrast, GSTm expression is inhibited upon Ras activation in mouse liver tumors and transgenic hepatocytes. Recent studies by different groups have shown that beta-catenin-dependent signaling is involved in the transcriptional control of "perivenous" expression of various cytochrome P450s in mouse liver, whereas Ras signaling was hypothesized to antagonize the perivenous hepatocyte phenotype. In synopsis with our present results, it now appears that the Wnt/beta-catenin pathway functions as a master regulator of the expression of both phase I and phase II drug-metabolizing enzymes in perivenous hepatocytes from mouse liver.
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Affiliation(s)
- Stefanie Giera
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Tübingen, Germany
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Miyoshi H, Deguchi A, Nakau M, Kojima Y, Mori A, Oshima M, Aoki M, Taketo MM. Hepatocellular carcinoma development induced by conditional beta-catenin activation in Lkb1+/- mice. Cancer Sci 2009; 100:2046-53. [PMID: 19671058 PMCID: PMC11159713 DOI: 10.1111/j.1349-7006.2009.01284.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2009] [Revised: 07/04/2009] [Accepted: 07/06/2009] [Indexed: 12/26/2022] Open
Abstract
The development of hepatocellular carcinomas (HCC) appears to be a multistep process that takes several decades in humans. However, the identities of specific gene alterations and their contribution to HCC pathogenesis remain poorly understood. We previously reported that Lkb1(+/-) mice spontaneously develop multiple hepatic nodular foci (NdFc) followed by HCC, and that the conditional activation of beta-catenin in Catnb(lox(ex3)) mouse livers alone does not cause tumor formation. We show here that the conditional activation of beta-catenin accelerates HCC development in Catnb(+/lox(ex3))Lkb1(+/-) compound mutant mice, affecting displastic hepatocytes in NdFc that suffered LOH at the Lkb1 locus. We further show that beta-catnin activation provides HCC with a growth advantage as well as transplantability. These results suggest that the loss of Lkb1 contributes to the formation of dysplastic NdFc, and that Wnt signaling activation is involved in ensuing progression toward HCC. A combination of these sequential changes can be a practical model for a subset of human HCC.
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Affiliation(s)
- Hiroyuki Miyoshi
- Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan
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Heindryckx F, Colle I, Van Vlierberghe H. Experimental mouse models for hepatocellular carcinoma research. Int J Exp Pathol 2009; 90:367-86. [PMID: 19659896 DOI: 10.1111/j.1365-2613.2009.00656.x] [Citation(s) in RCA: 289] [Impact Index Per Article: 18.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Every year almost 500,000 new patients are diagnosed with hepatocellular carcinoma (HCC), a primary malignancy of the liver that is associated with a poor prognosis. Numerous experimental models have been developed to define the pathogenesis of HCC and to test novel drug candidates. This review analyses several mouse models useful for HCC research and points out their advantages and weaknesses. Chemically induced HCC mice models mimic the injury-fibrosis-malignancy cycle by administration of a genotoxic compound alone or, if necessary, followed by a promoting agent. Xenograft models develop HCC by implanting hepatoma cell lines in mice, either ectopically or orthotopically; these models are suitable for drug screening, although extrapolation should be considered with caution as multiple cell lines must always be used. The hollow fibre assay offers a solution for limiting the number of test animals in xenograft research because of the ability for implanting multiple cell lines in one mouse. There is also a broad range of genetically modified mice engineered to investigate the pathophysiology of HCC. Transgenic mice expressing viral genes, oncogenes and/or growth factors allow the identification of pathways involved in hepatocarcinogenesis.
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Affiliation(s)
- Femke Heindryckx
- Department of Gastroenterology and Hepatology, Ghent University Hospital, 9000 Ghent, Belgium.
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42
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Braeuning A, Buchmann A. The glycogen synthase kinase inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) is a partial agonist of the aryl hydrocarbon receptor. Drug Metab Dispos 2009; 37:1576-80. [PMID: 19448134 DOI: 10.1124/dmd.109.027821] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2025] Open
Abstract
Kinase inhibitors are frequently used tools in signal transduction research. 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), a potent inhibitor of glycogen synthase kinase 3beta (GSK3beta), is frequently used to activate beta-catenin signaling by mimicking the action of Wnt molecules. beta-Catenin is a crucial player in the regulation of hepatic drug metabolism. Thus, it is of particular importance to know whether the tools used to study the effects of beta-catenin signaling may affect the respective drug-metabolizing target enzymes in an unwanted manner. In this study, we show that SB216763 is able to induce cytochrome P450 1a1 (Cyp1a1) expression in a dose-dependent manner in mouse hepatoma cells. Moreover, SB216763 is able to inhibit Cyp1a1 induction by the prototype aryl hydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachloro-p-dibenzodioxin. Cyp1a1 induction by SB216763 is independent of GSK3beta and the beta-catenin pathway. Instead, SB216763 induces Cyp1a1 by activation of AhR-mediated transcription. The present results suggest that SB216763 acts as a partial agonist of the AhR.
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Affiliation(s)
- Albert Braeuning
- University of Tübingen, Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Toxicology, Wilhelmstrasse 56, 72074 Tübingen, Germany.
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Rignall B, Ittrich C, Krause E, Appel KE, Buchmann A, Schwarz M. Comparative Transcriptome and Proteome Analysis of Ha-ras and B-raf Mutated Mouse Liver Tumors. J Proteome Res 2009; 8:3987-94. [DOI: 10.1021/pr9002933] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Affiliation(s)
- Benjamin Rignall
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany, German Cancer Research Center, Central Unit of Biostatistics, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and Federal Institute for Risk Assessment, Center for Experimental Toxicology, Thielallee 88-92, 14195 Berlin, Germany
| | - Carina Ittrich
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany, German Cancer Research Center, Central Unit of Biostatistics, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and Federal Institute for Risk Assessment, Center for Experimental Toxicology, Thielallee 88-92, 14195 Berlin, Germany
| | - Eberhard Krause
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany, German Cancer Research Center, Central Unit of Biostatistics, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and Federal Institute for Risk Assessment, Center for Experimental Toxicology, Thielallee 88-92, 14195 Berlin, Germany
| | - Klaus E. Appel
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany, German Cancer Research Center, Central Unit of Biostatistics, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and Federal Institute for Risk Assessment, Center for Experimental Toxicology, Thielallee 88-92, 14195 Berlin, Germany
| | - Albrecht Buchmann
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany, German Cancer Research Center, Central Unit of Biostatistics, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and Federal Institute for Risk Assessment, Center for Experimental Toxicology, Thielallee 88-92, 14195 Berlin, Germany
| | - Michael Schwarz
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany, German Cancer Research Center, Central Unit of Biostatistics, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and Federal Institute for Risk Assessment, Center for Experimental Toxicology, Thielallee 88-92, 14195 Berlin, Germany
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Yip-Schneider MT, Klein PJ, Wentz SC, Zeni A, Menze A, Schmidt CM. Resistance to mitogen-activated protein kinase kinase (MEK) inhibitors correlates with up-regulation of the MEK/extracellular signal-regulated kinase pathway in hepatocellular carcinoma cells. J Pharmacol Exp Ther 2009; 329:1063-70. [PMID: 19258520 DOI: 10.1124/jpet.108.147306] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/09/2025] Open
Abstract
The extracellular signal-regulated (ERK), mitogen-activated protein kinase (p42/p44 MAPK) pathway is up-regulated in hepatocellular carcinoma (HCC). Molecular targeting of this critical mitogenic pathway may have therapeutic potential for the treatment of HCC; however, chemoresistance to long-term therapy may develop. In the present study, we employed small-molecule MAPK kinase (MEK) inhibitors, including U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] and PD184161 (Neoplasia 8:1-8, 2006), in HepG2 and Hep3B human HCC cell lines to identify potential mechanism(s) of resistance. U0126 dose-dependently suppressed ERK phosphorylation at both 1- and 24-h time points in HepG2 cells, previously shown to be sensitive to growth inhibition by U0126. In contrast, ERK phosphorylation was only decreased at the 1-h time point but not at 24 h in the more resistant Hep3B cells. It is interesting that the lack of prolonged phospho-ERK suppression was associated with MEK hyperphosphorylation in Hep3B cells. Several MEK/ERK pathway intermediates were up-regulated in Hep3B cells; furthermore, transfection of Raf-1 small interfering RNA to suppress MEK/ERK pathway activation sensitized Hep3B cells to U0126. MEK inhibitor resistance was independent of p53 or hepatitis Bx protein status. Finally, we showed that combining two chemically distinct MEK inhibitors enhanced growth inhibition and apoptosis compared with the single agents. Taken together, these results suggest that up-regulated expression or activity of the MEK/ERK pathway contributes to MEK inhibitor resistance in HCC cells. Our findings also provide preclinical evidence suggesting that the status of the MEK/ERK pathway in patients may predict response to MEK/ERK-targeted therapeutics.
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Braeuning A, Sanna R, Huelsken J, Schwarz M. Inducibility of drug-metabolizing enzymes by xenobiotics in mice with liver-specific knockout of Ctnnb1. Drug Metab Dispos 2009; 37:1138-45. [PMID: 19237511 DOI: 10.1124/dmd.108.026179] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Basal as well as xenobiotic-induced expression of the main enzymes from phase I and phase II of drug metabolism is confined to the perivenous areas of the mammalian liver lobule. Whereas signal transduction pathways that govern xenobiotic-induced expression of these enzymes via ligand-activated transcription factors such as constitutive androstane receptor (CAR) or the aryl hydrocarbon receptor (AhR) have been intensively studied, the mechanisms regulating zone-specific basal expression of genes related to drug metabolism and preferential response of perivenous hepatocytes to xenobiotic inducers are still largely unknown. Recent publications by our and other groups point to an important role for the Wnt/beta-catenin pathway in the maintenance of the perivenous hepatocyte gene expression profile including the main hepatic detoxification enzymes, and beta-catenin signaling was recently implicated in the expression of several cytochrome P450 isoenzymes. To analyze, whether the beta-catenin pathway would also affect inducible expression of drug-metabolizing enzymes, mice with liver-specific knockout of the Ctnnb1 gene (encoding beta-catenin) were treated with different model inducers of xenobiotic metabolism. Knockout of beta-catenin led to alterations in basal expression of most drug metabolism-related genes analyzed and resulted in strongly diminished responses to agonists of CAR-, AhR-, and nuclear factor erythroid-related factor 2-dependent transcription. Taken together, the data presented in this study indicate that beta-catenin not only regulates basal expression of drug-metabolizing enzymes but also determines the magnitude and hepatic localization of response to xenobiotic inducers in vivo.
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Affiliation(s)
- Albert Braeuning
- Institute of Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany
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Comparative analysis of proteome and transcriptome in human hepatocellular carcinoma using 2D-DIGE and SAGE. Protein J 2009; 27:409-19. [PMID: 19048362 DOI: 10.1007/s10930-007-9123-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Proteome analysis of human hepatocellular carcinoma was conducted using two-dimensional difference gel electrophoresis, and the protein expression profiles were compared to the mRNA expression profiles made from serial analysis of gene expression (SAGE) in identical samples from a single patient. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the protein expression profiles. A total of 188 proteins were identified, and the expression profiles of 164 proteins which had the corresponding SAGE data were compared to the mRNA expression profiles. Among them, 40 proteins showed significant differences in the mRNA expression levels between non HCC and HCC. We compared expression changes of proteins with those of mRNAs. We found that the expression tendency of 24 proteins were similar to that of mRNA, whereas 16 proteins showed different or opposite tendency to the mRNA expression.
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Macheiner D, Gauglhofer C, Rodgarkia-Dara C, Grusch M, Brachner A, Bichler C, Kandioler D, Sutterlüty H, Mikulits W, Schulte-Hermann R, Grasl-Kraupp B. NORE1B is a putative tumor suppressor in hepatocarcinogenesis and may act via RASSF1A. Cancer Res 2009; 69:235-42. [PMID: 19118008 DOI: 10.1158/0008-5472.can-08-2144] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Recently, we found epigenetic silencing of the Ras effector genes NORE1B and/or RASSF1A in 97% of the hepatocellular carcinoma (HCC) investigated. This is strong evidence that the two genes are of major significance in hepatocarcinogenesis. Although RASSF1A serves as a tumor suppressor gene, the functions of NORE1B are largely unknown. Here, we studied the role of NORE1B for growth and transformation of cells. To understand the molecular mechanisms of action of the gene, we used the wild-type form and deletion mutants without the NH(2) terminus and CENTRAL domain, the Ras association (RA) domain, or the COOH-terminal SARAH-domain. Intact RA and SARAH-domains were found to be necessary for NORE1B (a) to increase the G(0)-G(1) fraction in hepatoma cells, (b) to suppress c-Myc/Ha-Ras-induced cell transformation, and (c) to interact closely with RASSF1A, as determined with fluorescence resonance energy transfer. In further studies, cell cycle delay by NORE1B was equally effective in hepatocyte cell lines with wild-type or mutant Ras suggesting that NORE1B does not interact with either Ras. In conclusion, NORE1B suppresses replication and transformation of cells as effectively as RASSF1A and thus is a putative tumor suppressor gene. NORE1B interacts physically with RASSF1A and functional loss of one of the interacting partners may lead to uncontrolled growth and transformation of hepatocytes. This may explain the frequent epigenetic silencing of NORE1B and/or RASSF1A in HCC.
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Affiliation(s)
- Doris Macheiner
- Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria
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Cairo S, Armengol C, De Reyniès A, Wei Y, Thomas E, Renard CA, Goga A, Balakrishnan A, Semeraro M, Gresh L, Pontoglio M, Strick-Marchand H, Levillayer F, Nouet Y, Rickman D, Gauthier F, Branchereau S, Brugières L, Laithier V, Bouvier R, Boman F, Basso G, Michiels JF, Hofman P, Arbez-Gindre F, Jouan H, Rousselet-Chapeau MC, Berrebi D, Marcellin L, Plenat F, Zachar D, Joubert M, Selves J, Pasquier D, Bioulac-Sage P, Grotzer M, Childs M, Fabre M, Buendia MA. Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer. Cancer Cell 2008; 14:471-84. [PMID: 19061838 DOI: 10.1016/j.ccr.2008.11.002] [Citation(s) in RCA: 366] [Impact Index Per Article: 21.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/13/2007] [Revised: 08/13/2008] [Accepted: 11/03/2008] [Indexed: 12/25/2022]
Abstract
Hepatoblastoma, the most common pediatric liver cancer, is tightly linked to excessive Wnt/beta-catenin signaling. Here, we used microarray analysis to identify two tumor subclasses resembling distinct phases of liver development and a discriminating 16-gene signature. beta-catenin activated different transcriptional programs in the two tumor types, with distinctive expression of hepatic stem/progenitor markers in immature tumors. This highly proliferating subclass was typified by gains of chromosomes 8q and 2p and upregulated Myc signaling. Myc-induced hepatoblastoma-like tumors in mice strikingly resembled the human immature subtype, and Myc downregulation in hepatoblastoma cells impaired tumorigenesis in vivo. Remarkably, the 16-gene signature discriminated invasive and metastatic hepatoblastomas and predicted prognosis with high accuracy.
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Affiliation(s)
- Stefano Cairo
- Oncogenesis and Molecular Virology Unit, Institut Pasteur, Paris Cedex 15, France
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Sagmeister S, Drucker C, Losert A, Grusch M, Daryabeigi A, Parzefall W, Rohr-Udilova N, Bichler C, Smedsrød B, Kandioler D, Grünberger T, Wrba F, Schulte-Hermann R, Grasl-Kraupp B. HB-EGF is a paracrine growth stimulator for early tumor prestages in inflammation-associated hepatocarcinogenesis. J Hepatol 2008; 49:955-64. [PMID: 18929421 DOI: 10.1016/j.jhep.2008.06.031] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2008] [Accepted: 06/27/2008] [Indexed: 12/11/2022]
Abstract
BACKGROUND/AIMS We studied the impact of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on inflammation-driven hepatocarcinogenesis. METHODS HB-EGF expression was determined by qRT-PCR and immunodetection in hepatocellular adenoma and carcinoma and in mesenchymal (MC) and parenchymal liver cells obtained from different models of inflammation. The functions of HB-EGF in early hepatocarcinogenesis were assessed in co-cultures of unaltered and initiated/premalignant hepatocytes. RESULTS In human and rat (pre)malignant liver lesions, HB-EGF levels were comparable to that of the surrounding tissue. In inflamed livers HB-EGF was expressed predominantly in MC and was further increased by pro-inflammatory lipopolysaccharide (LPS) or linoleic acid hydroperoxide (LOOH). In culture, DNA-replication occurred rather in initiated/premalignant than unaltered hepatocytes and was further elevated by LOOH- or LPS-stimulated MC-supernatants. The supernatant effects were abrogated by pre-incubation with HB-EGF-neutralizing antisera. HB-EGF itself induced DNA-replication and mitosis preferentially in the initiated/premalignant cells. When transducing hepatocytes with a dominant-negative ErbB1-construct, HB-EGF-induced DNA-replications were blocked completely in unaltered hepatocytes but incompletely in initiated/premalignant cells, which suggests elevated ErbB-mediated signal transduction in first stages of hepatocarcinogenesis. CONCLUSIONS Pro-inflammatory stimuli induce the release of HB-EGF from MC, which stimulates DNA-replication in initiated/premalignant hepatocytes. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.
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Affiliation(s)
- Sandra Sagmeister
- Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria
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50
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Abstract
To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFβ-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.
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