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Hamdy H, Shen C, Xu J, Fan D, Zhang Y, Li H, Wei Y, Sun J. Hepatocyte nuclear factor 4-Alpha: a key regulator in liver carcinogenesis. Cell Oncol (Dordr) 2025:10.1007/s13402-025-01064-7. [PMID: 40392499 DOI: 10.1007/s13402-025-01064-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2025] [Accepted: 04/09/2025] [Indexed: 05/22/2025] Open
Abstract
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, associated with viral hepatitis, alcohol consumption, and non-alcoholic fatty liver disease. Hepatocyte nuclear factor 4 alpha (HNF4α), a crucial transcription factor for liver function (glucose and lipid metabolism, bile acid homeostasis, and cellular differentiation), is often dysregulated in HCC progression. This review provides a comprehensive overview of the role of HNF4α in hepatic oncogenesis, providing novel inshight into its regulatory effects on epithelial-mesenchymal transition (EMT), metabolic alterations (including the Warburg effect), cell cycle control, and tumor microenvironment. We also discuss therapeutic strategies targeting HNF4α focusing on restoring metabolic balance and inducing apoptosis. This integrated analysis advances our understanding of HNF4α's contribution to HCC and may pave the way for the development of targeted therapies (Fig. 1).
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Affiliation(s)
- Hayam Hamdy
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, The Affiliated Hospital of Yunnan University, Yunnan University, Kunming, China
- Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, New Valley University, New Valley, Egypt
| | - Chang Shen
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, The Affiliated Hospital of Yunnan University, Yunnan University, Kunming, China
| | - Jiashun Xu
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, The Affiliated Hospital of Yunnan University, Yunnan University, Kunming, China
| | - Die Fan
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, The Affiliated Hospital of Yunnan University, Yunnan University, Kunming, China
| | - Yiwen Zhang
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, The Affiliated Hospital of Yunnan University, Yunnan University, Kunming, China
| | - Hui Li
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, The Affiliated Hospital of Yunnan University, Yunnan University, Kunming, China.
| | - Yonglong Wei
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, The Affiliated Hospital of Yunnan University, Yunnan University, Kunming, China.
- Key Laboratory of Tumor Immunological Prevention and Treatment of Yunnan Province, Kunming, 650051, China.
| | - Jianwei Sun
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, The Affiliated Hospital of Yunnan University, Yunnan University, Kunming, China.
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Soubeyrand S, Lau P, McPherson R. Distinct roles of Constitutive Photomorphogenesis Protein 1 homolog (COP1) in human hepatocyte models. Front Mol Biosci 2025; 12:1548582. [PMID: 39990870 PMCID: PMC11842253 DOI: 10.3389/fmolb.2025.1548582] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Accepted: 01/21/2025] [Indexed: 02/25/2025] Open
Abstract
Introduction Constitutive Photomorphogenesis Protein 1 homolog (COP1) is a conserved E3 ligase with key roles in several biological systems. Prior work in hepatocyte-derived tumors categorized COP1 as an oncogene, but its role in untransformed hepatocytes remains largely unexplored. Here, we have investigated the role of COP1 in primary human hepatocytes and two transformed hepatocyte models, HepG2 and HuH-7 cells. Methods The role of COP1 was tested by silencing and transduction experiments in HepG2, HuH-7, and primary human hepatocytes. Transcription array data of COP1-suppressed cells were generated and analyzed using clustering analyses. Cellular impacts were examined by proliferation assays, qRT-PCR, western blotting, reporter assays, and APOB enzyme-linked immunosorbent assays. Results and Discussion COP1 suppression had no noticeable impact on HepG2 and HuH-7 proliferation and was associated with contrasting rather than congruent transcriptome changes. Transcriptomic changes were consistent with perturbed metabolism in primary hepatocytes and HepG2 cells and impaired cell cycle regulation in HuH-7 cells. In HepG2 and primary hepatocytes but not in HuH-7 cells, COP1 suppression reduced the expression of important hepatic regulators and markers. COP1 downregulation reduced hepatic nuclear factor-4 alpha (HNF4A) abundance and function, as assessed by a lower abundance of key HNF4A targets, reduced APOB secretion, and reporter assays. HNF4A function could be restored by introducing a siRNA-resistant COP1 transgene, whereas HNF4A restoration partially rescued COP1 silencing in HepG2 cells. Our results identify and detail a pivotal regulatory role of COP1 in hepatocytes, in part through HNF4A.
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Affiliation(s)
| | - Paulina Lau
- Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, Canada
| | - Ruth McPherson
- Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, Canada
- Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, Canada
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Das S, Ravi H, Devi Rajeswari V, Venkatraman G, Ramasamy M, Dhanasekaran S, Ramanathan G. Therapeutic insight into the role of nuclear protein HNF4α in liver carcinogenesis. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2024; 143:1-37. [PMID: 39843133 DOI: 10.1016/bs.apcsb.2024.05.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
Abstract
Hepatocyte nuclear factor 4-alpha (HNF4α), a well-preserved member of the nuclear receptor superfamily of transcription factors, is found in the liver. It is recognized as a central controller of gene expression specific to the liver and plays a key role in preserving the liver's homeostasis. Irregular expression of HNF4α is increasingly recognized as a crucial factor in the proliferation, cell death, invasiveness, loss of specialized functions, and metastasis of cancer cells. An increasing number of studies are pointing to abnormal HNF4α expression as a key component of cancer cell invasion, apoptosis, proliferation, dedifferentiation, and metastasis. Understanding HNF4α's intricate involvement in liver carcinogenesis provides a promising avenue for therapeutic intervention. This chapter attempts to shed light on the diverse aspects of HNF4's role in liver carcinogenesis and demonstrate how this knowledge can be harnessed for approaches to prevent and treat liver cancer. This comprehensive chapter will offer an elaborate perspective on HNF4's function in liver cancer, delineating its molecular mechanisms that aid in the emergence of liver cancer. Furthermore, it will highlight the potential to help create more effective and precisely targeted therapeutic strategies, rekindling fresh optimism in the fight against this formidable condition.
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Affiliation(s)
- Soumik Das
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India
| | - Harini Ravi
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India
| | - V Devi Rajeswari
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India
| | - Ganesh Venkatraman
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India
| | - Magesh Ramasamy
- Department of Biotechnology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
| | - Sivaraman Dhanasekaran
- School of Energy Technology, Pandit Deendayal Energy University, Knowledge Corridor, Gandhinagar, Gujarat, India
| | - Gnanasambandan Ramanathan
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India.
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Zhong Z, Du J, Zhu X, Guan L, Hu Y, Zhang P, Wang H. Highly efficient conversion of mouse fibroblasts into functional hepatic cells under chemical induction. J Mol Cell Biol 2024; 15:mjad071. [PMID: 37996395 PMCID: PMC11121195 DOI: 10.1093/jmcb/mjad071] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Revised: 06/25/2023] [Accepted: 11/22/2023] [Indexed: 11/25/2023] Open
Abstract
Previous studies have shown that hepatocyte-like cells can be generated from fibroblasts using either lineage-specific transcription factors or chemical induction methods. However, these methods have their own deficiencies that restrict the therapeutic applications of such induced hepatocytes. In this study, we present a transgene-free, highly efficient chemical-induced direct reprogramming approach to generate hepatocyte-like cells from mouse embryonic fibroblasts (MEFs). Using a small molecule cocktail (SMC) as an inducer, MEFs can be directly reprogrammed into hepatocyte-like cells, bypassing the intermediate stages of pluripotent and immature hepatoblasts. These chemical-induced hepatocyte-like cells (ciHeps) closely resemble mature primary hepatocytes in terms of morphology, biological behavior, gene expression patterns, marker expression levels, and hepatic functions. Furthermore, transplanted ciHeps can integrate into the liver, promote liver regeneration, and improve survival rates in mice with acute liver damage. ciHeps can also ameliorate liver fibrosis caused by chronic injuries and enhance liver function. Notably, ciHeps exhibit no tumorigenic potential either in vitro or in vivo. Mechanistically, SMC-induced mesenchymal-to-epithelial transition and suppression of SNAI1 contribute to the fate conversion of fibroblasts into ciHeps. These results indicate that this transgene-free, chemical-induced direct reprogramming technique has the potential to serve as a valuable means of producing alternative hepatocytes for both research and therapeutic purposes. Additionally, this method also sheds light on the direct reprogramming of other cell types under chemical induction.
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Affiliation(s)
- Zhi Zhong
- Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China
- National Center for Liver Cancer, Naval Medical University, Shanghai 201805, China
| | - Jiangchuan Du
- National Center for Liver Cancer, Naval Medical University, Shanghai 201805, China
| | - Xiangjie Zhu
- National Center for Liver Cancer, Naval Medical University, Shanghai 201805, China
- Institute of Metabolism and Integrative Biology, Fudan University, Shanghai 200438, China
| | - Lingting Guan
- National Center for Liver Cancer, Naval Medical University, Shanghai 201805, China
| | - Yanyu Hu
- National Center for Liver Cancer, Naval Medical University, Shanghai 201805, China
| | - Peilin Zhang
- National Center for Liver Cancer, Naval Medical University, Shanghai 201805, China
| | - Hongyang Wang
- Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China
- National Center for Liver Cancer, Naval Medical University, Shanghai 201805, China
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García de Herreros A. Dual role of Snail1 as transcriptional repressor and activator. Biochim Biophys Acta Rev Cancer 2024; 1879:189037. [PMID: 38043804 DOI: 10.1016/j.bbcan.2023.189037] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2023] [Revised: 11/27/2023] [Accepted: 11/27/2023] [Indexed: 12/05/2023]
Abstract
Snail1 transcriptional factor plays a key role in the control of epithelial to mesenchymal transition, a process that remodels tumor cells increasing their invasion and chemo-resistance as well as reprograms their metabolism and provides stemness properties. During this transition, Snail1 acts as a transcriptional repressor and, as growing evidences have demonstrated, also as a direct activator of mesenchymal genes. In this review, I describe the different proteins that interact with Snail1 and are responsible for these two different functions on gene expression; I focus on the transcriptional factors that associate to Snail1 in their target promoters, both activated and repressed. I also present working models for Snail1 action both as repressor and activator and raise some issues that still need to be investigated.
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Affiliation(s)
- Antonio García de Herreros
- Programa de Recerca en Càncer, Hospital del Mar Research Institute (IMIM), Unidad Asociada al CSIC, Barcelona, Spain; Departament de Medicina i Ciències de la Vida, Universitat Pompeu Fabra, Barcelona, Spain.
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Qu N, Luan T, Liu N, Kong C, Xu L, Yu H, Kang Y, Han Y. Hepatocyte nuclear factor 4 a (HNF4α): A perspective in cancer. Biomed Pharmacother 2023; 169:115923. [PMID: 38000355 DOI: 10.1016/j.biopha.2023.115923] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2023] [Revised: 11/06/2023] [Accepted: 11/20/2023] [Indexed: 11/26/2023] Open
Abstract
HNF4α, a transcription factor, plays a vital role in regulating functional genes and biological processes. Its alternative splicing leads to various transcript variants encoding different isoforms. The spotlight has shifted towards the extensive discussion on tumors interplayed withHNF4α abnormalities. Aberrant HNF4α expression has emerged as sentinel markers of epigenetic shifts, casting reverberations upon downstream target genes and intricate signaling pathways, most notably with cancer. This review provides a comprehensive overview of HNF4α's involvement in tumor progression and metastasis, elucidating its role and underlying mechanisms.
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Affiliation(s)
- Ningxin Qu
- The Breast Oncology Dept., Shengjing Hospital of China Medical University, Shenyang, China
| | - Ting Luan
- Department of Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang, China
| | - Naiquan Liu
- The Nephrological Dept., Shengjing Hospital of China Medical University, Shenyang, China
| | - Chenhui Kong
- The Breast Oncology Dept., Shengjing Hospital of China Medical University, Shenyang, China
| | - Le Xu
- The Breast Oncology Dept., Shengjing Hospital of China Medical University, Shenyang, China
| | - Hong Yu
- The Breast Oncology Dept., Shengjing Hospital of China Medical University, Shenyang, China
| | - Ye Kang
- The Pathology Dept, Shengjing Hospital of China Medical University, Shenyang, China
| | - Ye Han
- The Breast Oncology Dept., Shengjing Hospital of China Medical University, Shenyang, China.
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Shen P, Bai ZJ, Zhou L, Wang NN, Ni ZX, Sun DZ, Huang CS, Hu YY, Xiao CR, Zhou W, Zhang BL, Gao Y. A Scd1-mediated metabolic alteration participates in liver responses to low-dose bavachin. J Pharm Anal 2023; 13:806-816. [PMID: 37577386 PMCID: PMC10422113 DOI: 10.1016/j.jpha.2023.03.010] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2022] [Revised: 03/24/2023] [Accepted: 03/27/2023] [Indexed: 08/15/2023] Open
Abstract
Hepatotoxicity induced by bioactive constituents in traditional Chinese medicines or herbs, such as bavachin (BV) in Fructus Psoraleae, has a prolonged latency to overt drug-induced liver injury in the clinic. Several studies have described BV-induced liver damage and underlying toxicity mechanisms, but little attention has been paid to the deciphering of organisms or cellular responses to BV at no-observed-adverse-effect level, and the underlying molecular mechanisms and specific indicators are also lacking during the asymptomatic phase, making it much harder for early recognition of hepatotoxicity. Here, we treated mice with BV for 7 days and did not detect any abnormalities in biochemical tests, but found subtle steatosis in BV-treated hepatocytes. We then profiled the gene expression of hepatocytes and non-parenchymal cells at single-cell resolution and discovered three types of hepatocyte subsets in the BV-treated liver. Among these, the hepa3 subtype suffered from a vast alteration in lipid metabolism, which was characterized by enhanced expression of apolipoproteins, carboxylesterases, and stearoyl-CoA desaturase 1 (Scd1). In particular, increased Scd1 promoted monounsaturated fatty acids (MUFAs) synthesis and was considered to be related to BV-induced steatosis and polyunsaturated fatty acids (PUFAs) generation, which participates in the initiation of ferroptosis. Additionally, we demonstrated that multiple intrinsic transcription factors, including Srebf1 and Hnf4a, and extrinsic signals from niche cells may regulate the above-mentioned molecular events in BV-treated hepatocytes. Collectively, our study deciphered the features of hepatocytes in response to BV insult, decoded the underlying molecular mechanisms, and suggested that Scd1 could be a hub molecule for the prediction of hepatotoxicity at an early stage.
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Affiliation(s)
- Pan Shen
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Zhi-Jie Bai
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Lei Zhou
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Ning-Ning Wang
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Zhe-Xin Ni
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - De-Zhi Sun
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Cong-Shu Huang
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Yang-Yi Hu
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Cheng-Rong Xiao
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Wei Zhou
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
| | - Bo-Li Zhang
- State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China
| | - Yue Gao
- Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing, 100850, China
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Amicone L, Marchetti A, Cicchini C. The lncRNA HOTAIR: a pleiotropic regulator of epithelial cell plasticity. J Exp Clin Cancer Res 2023; 42:147. [PMID: 37308974 DOI: 10.1186/s13046-023-02725-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Accepted: 05/30/2023] [Indexed: 06/14/2023] Open
Abstract
The epithelial-to-mesenchymal transition (EMT) is a trans-differentiation process that endows epithelial cells with mesenchymal properties, including motility and invasion capacity; therefore, its aberrant reactivation in cancerous cells represents a critical step to gain a metastatic phenotype. The EMT is a dynamic program of cell plasticity; many partial EMT states can be, indeed, encountered and the full inverse mesenchymal-to-epithelial transition (MET) appears fundamental to colonize distant secondary sites. The EMT/MET dynamics is granted by a fine modulation of gene expression in response to intrinsic and extrinsic signals. In this complex scenario, long non-coding RNAs (lncRNAs) emerged as critical players. This review specifically focuses on the lncRNA HOTAIR, as a master regulator of epithelial cell plasticity and EMT in tumors. Molecular mechanisms controlling its expression in differentiated as well as trans-differentiated epithelial cells are highlighted here. Moreover, current knowledge about HOTAIR pleiotropic functions in regulation of both gene expression and protein activities are described. Furthermore, the relevance of the specific HOTAIR targeting and the current challenges of exploiting this lncRNA for therapeutic approaches to counteract the EMT are discussed.
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Affiliation(s)
- Laura Amicone
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Dipartimento di Medicina Molecolare, Sapienza University of Rome, Viale Regina Elena 324, Rome, 00161, Italy
| | - Alessandra Marchetti
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Dipartimento di Medicina Molecolare, Sapienza University of Rome, Viale Regina Elena 324, Rome, 00161, Italy
| | - Carla Cicchini
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Dipartimento di Medicina Molecolare, Sapienza University of Rome, Viale Regina Elena 324, Rome, 00161, Italy.
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Liu SQ, Deng X, Zhu CP, Cui YL, Xie WF, Zhang X. Depletion of Tgfbr2 in hepatocytes alleviates liver fibrosis and restores hepatic function in fibrotic mice. J Dig Dis 2023; 24:39-50. [PMID: 36967587 DOI: 10.1111/1751-2980.13161] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/13/2022] [Revised: 02/07/2023] [Accepted: 02/13/2023] [Indexed: 05/03/2023]
Abstract
OBJECTIVES Previous studies have demonstrated the pivotal role of transforming growth factor (TGF)-β signaling in activating hepatic stellate cells during liver fibrosis. In this study we aimed to demonstrate the effects and underlying mechanism of TGF-β signaling in hepatocytes on hepatic fibrogenesis. METHODS Hepatocyte-specific Tgfbr2-knockout (Tgfbr2HKO ) mice were generated by AAV8-TBG-Cre injection via the tail vein of Tgfbr2f/f mice. CCl4 was injected intraperitoneally twice a week for 4 weeks to establish the fibrotic mouse model. The expression of the fibrogenesis markers was evaluated by immunohistochemistry, western blot, and real-time polymerase chain reaction (PCR). RNA-seq analysis was used to detect the transcriptional profiles of primary hepatocytes isolated from Tgfbr2HKO mice and control mice. RESULTS The expression of TβR2 (Tgfbr2) was markedly upregulated in hepatocytes of the fibrotic liver. Tgfbr2 depletion in hepatocytes decreased the expressions of profibrogenic markers (Col1a1 and Acta2) in the CCl4 -treated fibrotic liver. RNA-seq analysis revealed that Tgfbr2 deletion in hepatocytes significantly reduced the inflammatory response and suppressed epithelial-mesenchymal transition of hepatocytes accompanied by upregulation of the metabolic pathways during liver fibrosis. Moreover, the expressions of hepatocyte nuclear factors (HNFs), including Hnf4α, Foxa1, Foxa2, and Foxa3, which are important for maintaining liver metabolism and homeostasis, were decreased in fibrotic livers and significantly increased after Tgfbr2 blockade. CONCLUSION Blocking the TGF-β signaling pathway in hepatocytes reduces hepatic fibrosis and improves hepatic function in fibrotic livers.
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Affiliation(s)
- Shu Qing Liu
- Department of Gastroenterology, Changzheng Hospital, Naval Medical University, Shanghai, China
| | - Xing Deng
- Department of Gastroenterology, Changzheng Hospital, Naval Medical University, Shanghai, China
| | - Chang Peng Zhu
- Department of Gastroenterology, Changzheng Hospital, Naval Medical University, Shanghai, China
| | - Ya Lu Cui
- Department of Gastroenterology, Changzheng Hospital, Naval Medical University, Shanghai, China
| | - Wei Fen Xie
- Department of Gastroenterology, Changzheng Hospital, Naval Medical University, Shanghai, China
| | - Xin Zhang
- Department of Gastroenterology, Changzheng Hospital, Naval Medical University, Shanghai, China
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Zhang X, Zhu X, Zhong Z, Du J, Fang G, Cui X, Guan L, Hu Y, Wang H, Zhang P. Small Molecule-Induced Differentiation As a Potential Therapy for Liver Cancer. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2022; 9:e2103619. [PMID: 35343115 PMCID: PMC9131429 DOI: 10.1002/advs.202103619] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/20/2021] [Revised: 03/03/2022] [Indexed: 05/07/2023]
Abstract
Despite the efficacy demonstrated by immunotherapy recently, liver cancer still remains one of the deadliest cancers, mainly due to heterogeneity of this disease. Continuous exploration of new therapeutics is therefore necessary. Chemical-induced cell differentiation can serve as a promising approach, with its ability to consistently remodel gene expression profile and alter cell fate. Inspired by advances in stem cell and reprogramming field, here it is reported that a small molecule cocktail (SMC) consisted of: SB431542 (TGFβ inhibitor), CHIR99021 (GSK3β inhibitor), BIX01294 (H3K9 methyltransferase/G9a inhibitor), and all-trans retinoic acid (ATRA), can induce differentiation of liver cancer cells including cell lines, primary cancer cells, cancer stem cells, and drug resistant cells. Treated cells lose malignant characteristics and regain hepatocyte phenotype instead. When applied in vivo, SMC induces wide range of tissue necrosis or fibrosis within the tumors, while remaining tissues begin to express hepatic nuclear factor 4α (HNF4α), the hepatic nuclear marker. SMC also leads to tumor abrogation in orthotopic xenograft models and life span extension of animals. The powerful differentiation induction of SMC is exerted through modulation of Akt/mTOR/HIF1α signaling and metabolic reprogramming, as well as suppressing Snail and enhancing HNF4α expression. Together, these results highlight that chemical-induced differentiation has the potential to effectively treat liver cancer disregard of heterogeneity.
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Affiliation(s)
- Xu Zhang
- National Center for Liver CancerShanghai201805China
| | - Xiang‐Jie Zhu
- National Center for Liver CancerShanghai201805China
- Institute of Metabolism and Integrative BiologyFudan UniversityShanghai200433China
| | - Zhi Zhong
- National Center for Liver CancerShanghai201805China
- Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical CollegeFudan UniversityShanghai200032China
| | | | - Guo‐Xu Fang
- National Center for Liver CancerShanghai201805China
| | - Xiu‐liang Cui
- National Center for Liver CancerShanghai201805China
- The International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery HospitalSecond Military Medical UniversityShanghai200438China
| | | | - Yan‐Yu Hu
- National Center for Liver CancerShanghai201805China
| | - Hong‐Yang Wang
- National Center for Liver CancerShanghai201805China
- The International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery HospitalSecond Military Medical UniversityShanghai200438China
| | - Pei‐Lin Zhang
- National Center for Liver CancerShanghai201805China
- The International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery HospitalSecond Military Medical UniversityShanghai200438China
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Garbo S, Zwergel C, Battistelli C. m6A RNA methylation and beyond - The epigenetic machinery and potential treatment options. Drug Discov Today 2021; 26:2559-2574. [PMID: 34126238 DOI: 10.1016/j.drudis.2021.06.004] [Citation(s) in RCA: 63] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2021] [Revised: 05/02/2021] [Accepted: 06/08/2021] [Indexed: 12/14/2022]
Abstract
m6A is emerging as one of the most important RNA modifications because of its involvement in pathological and physiological events. Here, we provide an overview of this epitranscriptomic modification, beginning with a description of the molecular players involved and continuing with a focus on the role of m6A in the maintenance of stemness, induction of the epithelial to mesenchymal transition (EMT), and tumor progression. Finally, we discuss the state of the art regarding the design and validation of inhibitors of m6A writers or erasers to provide a background for future investigations and for the development of specific therapeutics.
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Affiliation(s)
- Sabrina Garbo
- Istituto Pasteur Italia, Fondazione Cenci-Bolognetti, Department of Molecular Medicine, Department of Excellence 2018-2022, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy; Oncohaematology Department, IRCCS Ospedale Pediatrico Bambino Gesù, Viale di San Paolo 15, 00146 Rome, Italy
| | - Clemens Zwergel
- Department of Drug Chemistry and Technologies, Department of Excellence 2018-2022, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.
| | - Cecilia Battistelli
- Istituto Pasteur Italia, Fondazione Cenci-Bolognetti, Department of Molecular Medicine, Department of Excellence 2018-2022, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy.
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LncRNA HOTAIR recruits SNAIL to inhibit the transcription of HNF4α and promote the viability, migration, invasion and EMT of colorectal cancer. Transl Oncol 2021; 14:101036. [PMID: 33588137 PMCID: PMC7901038 DOI: 10.1016/j.tranon.2021.101036] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2020] [Revised: 01/07/2021] [Accepted: 02/01/2021] [Indexed: 02/06/2023] Open
Abstract
HOTAIR recruited SNAIL and reduced the expression of HNF4α to promote EMT of colorectal cancer. Provided potential novel long non-coding RNA-directed early diagnosis and therapy for colorectal cancer. Provided further insight into the regulatory mechanism of HOTAIR in colorectal cancer. Colorectal cancer causes severe burdensome on the health by its high fatality and poor prognosis. Hox transcript antisense intergenic RNA (HOTAIR) was believed closely related with the genesis and development of colorectal cancer, but the regulatory mechanism is still to be investigated. The expression of HOTAIR was analyzed in colorectal cancer using both qRT-PCR and ISH assay. The cell viability, migration, invasion and apoptosis rate were evaluated using MTT, BrdU,Transwell and flow cytometryexperiments. The interaction between HOTAIR and SNAIL was detected using RIP and RNA pull-down. The binding of SNAIL to HNF4α promoter was assessed by ChIP. The cell lines that knock down HOTAIR, SNAIL or overexpress HNF4α were constructed using retroviral vector system. The tumorigenic and metastatic capacity of colorectal cancer cells after knocking down HOTAIR were evaluated based on xenograft assay and liver metastases model. HOTAIR was highly expressed in both tissue and cell lines of colorectal cancer, indicated a regulatory function in colorectal cancer. Knock-down of HOTAIR suppressed cell viability, migration, invasion and epithelial-mesenchymal transition (EMT) of colorectal cancer cells in vitro, and inhibited the growth and metastasis of colorectal tumor in nude mice. We further found that HOTAIR suppressed HNF4α via recruiting SNAIL, and the overexpression of HNF4α inhibited cell viability, migration, invasion and EMT of colorectal cancer cells. We demonstrated that HOTAIR regulates the level of HNF4α via recruiting SNAIL, knocking down HOTAIR repressed the cell viability and metestasis of colorectal cancer cell line in vitro, and suppressed the tomorgenesis and migration/invasion of colorectal cancer in vivo.
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13
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Lv DD, Zhou LY, Tang H. Hepatocyte nuclear factor 4α and cancer-related cell signaling pathways: a promising insight into cancer treatment. Exp Mol Med 2021; 53:8-18. [PMID: 33462379 PMCID: PMC8080681 DOI: 10.1038/s12276-020-00551-1] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2020] [Revised: 10/23/2020] [Accepted: 11/19/2020] [Indexed: 02/06/2023] Open
Abstract
Hepatocyte nuclear factor 4α (HNF4α), a member of the nuclear receptor superfamily, is described as a protein that binds to the promoters of specific genes. It controls the expression of functional genes and is also involved in the regulation of numerous cellular processes. A large number of studies have demonstrated that HNF4α is involved in many human malignancies. Abnormal expression of HNF4α is emerging as a critical factor in cancer cell proliferation, apoptosis, invasion, dedifferentiation, and metastasis. In this review, we present emerging insights into the roles of HNF4α in the occurrence, progression, and treatment of cancer; reveal various mechanisms of HNF4α in cancer (e.g., the Wnt/β-catenin, nuclear factor-κB, signal transducer and activator of transcription 3, and transforming growth factor β signaling pathways); and highlight potential clinical uses of HNF4α as a biomarker and therapeutic target for cancer.
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Affiliation(s)
- Duo-Duo Lv
- Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
| | - Ling-Yun Zhou
- Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
| | - Hong Tang
- Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China.
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14
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Battistelli C, Garbo S, Riccioni V, Montaldo C, Santangelo L, Vandelli A, Strippoli R, Tartaglia GG, Tripodi M, Cicchini C. Design and Functional Validation of a Mutant Variant of the LncRNA HOTAIR to Counteract Snail Function in Epithelial-to-Mesenchymal Transition. Cancer Res 2021; 81:103-113. [PMID: 33158813 PMCID: PMC7611326 DOI: 10.1158/0008-5472.can-20-1764] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2020] [Revised: 10/05/2020] [Accepted: 11/03/2020] [Indexed: 12/18/2022]
Abstract
HOTAIR is a lncRNA overexpressed in several epithelial cancers and strongly correlated with invasion. This lncRNA was proven a pivotal element of the epithelial-to-mesenchymal transition (EMT), a transdifferentiation process triggering metastasis. Snail, master inducer of EMT, requires HOTAIR to recruit EZH2 on specific epithelial target genes (i.e., HNF4α, E-cadherin, and HNF1α) and cause their repression. Here, we designed a HOTAIR deletion mutant form, named HOTAIR-sbid, including the putative Snail-binding domain but depleted of the EZH2-binding domain. HOTAIR-sbid acted as a dominant negative of the endogenous HOTAIR. In both murine and human tumor cells, HOTAIR-sbid impaired the ability of HOTAIR to bind Snail and, in turn, trigger H3K27me3/EZH2-mediated repression of Snail epithelial target genes. Notably, HOTAIR-sbid expression was proven to reduce cellular motility, invasiveness, anchorage-independent growth, and responsiveness to TGFβ-induced EMT. These data provide evidence on a lncRNA-based strategy to effectively impair the function of a master EMT-transcriptional factor. SIGNIFICANCE: This study defines an innovative RNA-based strategy to interfere with a pivotal function of the tumor-related lncRNA HOTAIR, comprising a dominant negative mutant that was computationally designed and that impairs epithelial-to-mesenchymal transition.
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Affiliation(s)
- Cecilia Battistelli
- Department of Molecular Medicine, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.
| | - Sabrina Garbo
- Department of Molecular Medicine, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - Veronica Riccioni
- Department of Molecular Medicine, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - Claudia Montaldo
- National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
| | - Laura Santangelo
- National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
| | - Andrea Vandelli
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology and Universitat Pompeu Fabra (UPF), Barcelona, Spain
- Department of Biochemistry and Molecular Biology, Systems Biology of Infection Lab, Universitat Autònoma de Barcelona, Spain
| | - Raffaele Strippoli
- Department of Molecular Medicine, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
- National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
| | - Gian Gaetano Tartaglia
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology and Universitat Pompeu Fabra (UPF), Barcelona, Spain
- Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genoa, Italy
- Department of Biology 'Charles Darwin', Sapienza University of Rome, Rome, Italy
- Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
| | - Marco Tripodi
- Department of Molecular Medicine, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.
- National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
| | - Carla Cicchini
- Department of Molecular Medicine, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
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15
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Ríos-López DG, Aranda-López Y, Sosa-Garrocho M, Macías-Silva M. La plasticidad del hepatocito y su relevancia en la fisiología y la patología hepática. TIP REVISTA ESPECIALIZADA EN CIENCIAS QUÍMICO-BIOLÓGICAS 2020. [DOI: 10.22201/fesz.23958723e.2020.0.225] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
El hígado es uno de los principales órganos encargados de mantener la homeostasis en vertebrados, además de poseer una gran capacidad regenerativa. El hígado está constituido por diversos tipos celulares que de forma coordinada contribuyen para que el órgano funcione eficientemente. Los hepatocitos representan el tipo celular principal de este órgano y llevan a cabo la mayoría de sus actividades; además, constituyen una población heterogénea de células epiteliales con funciones especializadas en el metabolismo. El fenotipo de los hepatocitos está controlado por diferentes vías de señalización, como la vía del TGFβ/Smads, la ruta Hippo/YAP-TAZ y la vía Wnt/β-catenina, entre otras. Los hepatocitos son células que se encuentran normalmente en un estado quiescente, aunque cuentan con una plasticidad intrínseca que se manifiesta en respuesta a diversos daños en el hígado; así, estas células reactivan su capacidad proliferativa o cambian su fenotipo a través de procesos celulares como la transdiferenciación o la transformación, para contribuir a mantener la homeostasis del órgano en condiciones saludables o desarrollar diversas patologías.
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16
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Holmgren G, Ulfenborg B, Asplund A, Toet K, Andersson CX, Hammarstedt A, Hanemaaijer R, Küppers-Munther B, Synnergren J. Characterization of Human Induced Pluripotent Stem Cell-Derived Hepatocytes with Mature Features and Potential for Modeling Metabolic Diseases. Int J Mol Sci 2020; 21:ijms21020469. [PMID: 31940797 PMCID: PMC7014160 DOI: 10.3390/ijms21020469] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2019] [Revised: 01/06/2020] [Accepted: 01/07/2020] [Indexed: 01/17/2023] Open
Abstract
There is a strong anticipated future for human induced pluripotent stem cell-derived hepatocytes (hiPS-HEP), but so far, their use has been limited due to insufficient functionality. We investigated the potential of hiPS-HEP as an in vitro model for metabolic diseases by combining transcriptomics with multiple functional assays. The transcriptomics analysis revealed that 86% of the genes were expressed at similar levels in hiPS-HEP as in human primary hepatocytes (hphep). Adult characteristics of the hiPS-HEP were confirmed by the presence of important hepatocyte features, e.g., Albumin secretion and expression of major drug metabolizing genes. Normal energy metabolism is crucial for modeling metabolic diseases, and both transcriptomics data and functional assays showed that hiPS-HEP were similar to hphep regarding uptake of glucose, low-density lipoproteins (LDL), and fatty acids. Importantly, the inflammatory state of the hiPS-HEP was low under standard conditions, but in response to lipid accumulation and ER stress the inflammation marker tumor necrosis factor α (TNFα) was upregulated. Furthermore, hiPS-HEP could be co-cultured with primary hepatic stellate cells both in 2D and in 3D spheroids, paving the way for using these co-cultures for modeling non-alcoholic steatohepatitis (NASH). Taken together, hiPS-HEP have the potential to serve as an in vitro model for metabolic diseases. Furthermore, differently expressed genes identified in this study can serve as targets for future improvements of the hiPS-HEP.
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Affiliation(s)
- Gustav Holmgren
- Systems biology research center, School of Bioscience, University of Skövde, 54128 Skövde, Sweden; (G.H.); (J.S.)
| | - Benjamin Ulfenborg
- Systems biology research center, School of Bioscience, University of Skövde, 54128 Skövde, Sweden; (G.H.); (J.S.)
- Correspondence: (B.U.); (B.K.-M.)
| | - Annika Asplund
- R&D, Hepatocyte Product Development, Takara Bio Europe AB, 41346 Gothenburg, Sweden; (A.A.)
| | - Karin Toet
- Department of Metabolic Health Research, TNO, 2333 Leiden, The Netherlands; (K.T.); (R.H.)
| | - Christian X Andersson
- R&D, Hepatocyte Product Development, Takara Bio Europe AB, 41346 Gothenburg, Sweden; (A.A.)
| | - Ann Hammarstedt
- The Lundberg Laboratory for Diabetes Research, Departments of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, 41345 Gothenburg, Sweden;
| | - Roeland Hanemaaijer
- Department of Metabolic Health Research, TNO, 2333 Leiden, The Netherlands; (K.T.); (R.H.)
| | - Barbara Küppers-Munther
- R&D, Hepatocyte Product Development, Takara Bio Europe AB, 41346 Gothenburg, Sweden; (A.A.)
- Correspondence: (B.U.); (B.K.-M.)
| | - Jane Synnergren
- Systems biology research center, School of Bioscience, University of Skövde, 54128 Skövde, Sweden; (G.H.); (J.S.)
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17
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YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation. Cell Death Dis 2019; 10:768. [PMID: 31601778 PMCID: PMC6787001 DOI: 10.1038/s41419-019-2000-8] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2019] [Revised: 05/29/2019] [Accepted: 06/24/2019] [Indexed: 12/17/2022]
Abstract
Yes-associated protein (YAP) is a transcriptional co-factor involved in many cell processes, including development, proliferation, stemness, differentiation, and tumorigenesis. It has been described as a sensor of mechanical and biochemical stimuli that enables cells to integrate environmental signals. Although in the liver the correlation between extracellular matrix elasticity (greatly increased in the most of chronic hepatic diseases), differentiation/functional state of parenchymal cells and subcellular localization/activation of YAP has been previously reported, its role as regulator of the hepatocyte differentiation remains to be clarified. The aim of this study was to evaluate the role of YAP in the regulation of epithelial/hepatocyte differentiation and to clarify how a transducer of general stimuli can integrate tissue-specific molecular mechanisms determining specific cell outcomes. By means of YAP silencing and overexpression we demonstrated that YAP has a functional role in the repression of epithelial/hepatocyte differentiation by inversely modulating the expression of Snail (master regulator of the epithelial-to-mesenchymal transition and liver stemness) and HNF4α (master regulator of hepatocyte differentiation) at transcriptional level, through the direct occupancy of their promoters. Furthermore, we found that Snail, in turn, is able to positively control YAP expression influencing protein level and subcellular localization and that HNF4α stably represses YAP transcription in differentiated hepatocytes both in cell culture and in adult liver. Overall, our data indicate YAP as a new member of the HNF4/Snail epistatic molecular circuitry previously demonstrated to control liver cell state. In this model, the dynamic balance between three main transcriptional regulators, that are able to control reciprocally their expression/activity, is responsible for the induction/maintenance of different liver cell differentiation states and its modulation could be the aim of therapeutic protocols for several chronic liver diseases.
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18
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Bisceglia F, Battistelli C, Noce V, Montaldo C, Zammataro A, Strippoli R, Tripodi M, Amicone L, Marchetti A. TGFβ Impairs HNF1α Functional Activity in Epithelial-to-Mesenchymal Transition Interfering With the Recruitment of CBP/p300 Acetyltransferases. Front Pharmacol 2019; 10:942. [PMID: 31543815 PMCID: PMC6728925 DOI: 10.3389/fphar.2019.00942] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Accepted: 07/24/2019] [Indexed: 12/24/2022] Open
Abstract
The cytokine transforming growth factor β (TGFβ) plays a crucial role in the induction of both epithelial-to-mesenchymal transition (EMT) program and fibro-cirrhotic process in the liver, where it contributes also to organ inflammation following several chronic injuries. All these pathological situations greatly increase the risk of hepatocellular carcinoma (HCC) and contribute to tumor progression. In particular, late-stage HCCs are characterized by constitutive activation of TGFβ pathway and by an EMT molecular signature leading to the acquisition of invasive and metastatic properties. In these pathological conditions, the cytokine has been shown to induce the transcriptional downregulation of HNF1α, a master regulator of the epithelial/hepatocyte differentiation and of the EMT reverse process, the mesenchymal-to-epithelial transition (MET). Therefore, the restoration of HNF1α expression/activity has been proposed as targeted therapeutic strategy for liver fibro-cirrhosis and late-stage HCCs. In this study, TGFβ is found to trigger an early functional inactivation of HNF1α during EMT process that anticipates the effects of the transcriptional downregulation of its own gene. Mechanistically, the cytokine, while not affecting the HNF1α DNA-binding capacity, impaired its ability to recruit CBP/p300 acetyltransferases on target gene promoters and, consequently, its transactivating function. The loss of HNF1α capacity to bind to CBP/p300 and HNF1α functional inactivation have been found to correlate with a change of its posttranslational modification profile. Collectively, the results obtained in this work unveil a new level of HNF1α functional inactivation by TGFβ and contribute to shed light on the early events triggering EMT in hepatocytes. Moreover, these data suggest that the use of HNF1α as anti-EMT tool in a TGFβ-containing microenvironment may require the design of new therapeutic strategies overcoming the TGFβ-induced HNF1α inactivation.
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Affiliation(s)
- Francesca Bisceglia
- Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy
| | - Cecilia Battistelli
- Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy
| | - Valeria Noce
- Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy
| | - Claudia Montaldo
- National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
| | - Agatino Zammataro
- Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy
| | - Raffaele Strippoli
- Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy
- National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
| | - Marco Tripodi
- Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy
- National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
| | - Laura Amicone
- Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy
| | - Alessandra Marchetti
- Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy
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19
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New Insights into the Role of Epithelial⁻Mesenchymal Transition during Aging. Int J Mol Sci 2019; 20:ijms20040891. [PMID: 30791369 PMCID: PMC6412502 DOI: 10.3390/ijms20040891] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2019] [Revised: 02/08/2019] [Accepted: 02/15/2019] [Indexed: 12/29/2022] Open
Abstract
Epithelial–mesenchymal transition (EMT) is a cellular process by which differentiated epithelial cells undergo a phenotypic conversion to a mesenchymal nature. The EMT has been increasingly recognized as an essential process for tissue fibrogenesis during disease and normal aging. Higher levels of EMT proteins in aged tissues support the involvement of EMT as a possible cause and/or consequence of the aging process. Here, we will highlight the existing understanding of EMT supporting the phenotypical alterations that occur during normal aging or pathogenesis, covering the impact of EMT deregulation in tissue homeostasis and stem cell function.
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20
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Ruoß M, Damm G, Vosough M, Ehret L, Grom-Baumgarten C, Petkov M, Naddalin S, Ladurner R, Seehofer D, Nussler A, Sajadian S. Epigenetic Modifications of the Liver Tumor Cell Line HepG2 Increase Their Drug Metabolic Capacity. Int J Mol Sci 2019; 20:347. [PMID: 30654452 PMCID: PMC6358789 DOI: 10.3390/ijms20020347] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Revised: 01/12/2019] [Accepted: 01/14/2019] [Indexed: 01/31/2023] Open
Abstract
Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards 'normal' liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.
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Affiliation(s)
- Marc Ruoß
- Siegfried Weller Institute, BG Trauma Clinic, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
| | - Georg Damm
- Department of Hepatobiliary Surgery and Visceral Transplantation, University of Leipzig, 04103 Leipzig, Germany.
| | - Massoud Vosough
- Royan Institute for Stem Cell Biology and Technology, Department of Stem Cells and Developmental Biology, Tehran 16635-148, Iran.
| | - Lisa Ehret
- Siegfried Weller Institute, BG Trauma Clinic, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
| | - Carl Grom-Baumgarten
- Siegfried Weller Institute, BG Trauma Clinic, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
| | - Martin Petkov
- Siegfried Weller Institute, BG Trauma Clinic, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
| | - Silvio Naddalin
- Department of General, Visceral and Transplant Surgery, University Hospital Tübingen, 72076 Tübingen, Germany.
| | - Ruth Ladurner
- Department of General, Visceral and Transplant Surgery, University Hospital Tübingen, 72076 Tübingen, Germany.
| | - Daniel Seehofer
- Department of Hepatobiliary Surgery and Visceral Transplantation, University of Leipzig, 04103 Leipzig, Germany.
| | - Andreas Nussler
- Siegfried Weller Institute, BG Trauma Clinic, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
| | - Sahar Sajadian
- Siegfried Weller Institute, BG Trauma Clinic, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
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21
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The lncRNA HOTAIR transcription is controlled by HNF4α-induced chromatin topology modulation. Cell Death Differ 2018; 26:890-901. [PMID: 30154449 PMCID: PMC6461983 DOI: 10.1038/s41418-018-0170-z] [Citation(s) in RCA: 65] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2018] [Revised: 07/02/2018] [Accepted: 07/03/2018] [Indexed: 01/16/2023] Open
Abstract
The expression of the long noncoding RNA HOTAIR (HOX Transcript Antisense Intergenic RNA) is largely deregulated in epithelial cancers and positively correlates with poor prognosis and progression of hepatocellular carcinoma and gastrointestinal cancers. Furthermore, functional studies revealed a pivotal role for HOTAIR in the epithelial-to-mesenchymal transition, as this RNA is causal for the repressive activity of the master factor SNAIL on epithelial genes. Despite the proven oncogenic role of HOTAIR, its transcriptional regulation is still poorly understood. Here hepatocyte nuclear factor 4-α (HNF4α), as inducer of epithelial differentiation, was demonstrated to directly repress HOTAIR transcription in the mesenchymal-to epithelial transition. Mechanistically, HNF4α was found to cause the release of a chromatin loop on HOTAIR regulatory elements thus exerting an enhancer-blocking activity.
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22
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Yoshida K, Matsuzaki K, Murata M, Yamaguchi T, Suwa K, Okazaki K. Clinico-Pathological Importance of TGF-β/Phospho-Smad Signaling during Human Hepatic Fibrocarcinogenesis. Cancers (Basel) 2018; 10:cancers10060183. [PMID: 29874844 PMCID: PMC6025395 DOI: 10.3390/cancers10060183] [Citation(s) in RCA: 60] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Revised: 05/19/2018] [Accepted: 06/01/2018] [Indexed: 12/20/2022] Open
Abstract
Chronic viral hepatitis is a global public health problem, with approximately 570 million persons chronically infected. Hepatitis B and C viruses increase the risk of morbidity and mortality from liver cirrhosis, hepatocellular carcinoma (HCC), and extrahepatic complications that develop. Hepatitis virus infection induces transforming growth factor (TGF)-β, which influences microenvironments within the infected liver. TGF-β promotes liver fibrosis by up-regulating extracellular matrix production by hepatic stellate cells. TGF-β is also up-regulated in patients with HCC, in whom it contributes importantly to bringing about a favorable microenvironment for tumor growth. Thus, TGF-β is thought to be a major factor regulating liver fibrosis and carcinogenesis. Since TGF-β carries out regulatory signaling by influencing the phosphorylation of Smads, we have generated several kinds of phospho-specific antibodies to Smad2/3. Using these, we have identified three types of phospohorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C), linker phosphorylated Smad2/3 (pSmad2L and pSmad3L), and dually phosphorylated Smad3 (pSmad2L/C and pSmad3L/C). TGF-β-mediated pSmad2/3C signaling terminates cell proliferation; on the other hand, cytokine-induced pSmad3L signaling accelerates cell proliferation and promotes fibrogenesis. This review addresses TGF-β/Smad signal transduction in chronic liver injuries and carcinogenic processes. We also discuss the reversibility of Smad signaling after antiviral therapy.
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Affiliation(s)
- Katsunori Yoshida
- Department of Gastroenterology and Hepatology, Kansai Medical University 2-5-1, Shin-Machi, Hirakata, Osaka 573-1010, Japan.
| | - Koichi Matsuzaki
- Department of Gastroenterology and Hepatology, Kansai Medical University 2-5-1, Shin-Machi, Hirakata, Osaka 573-1010, Japan.
| | - Miki Murata
- Department of Gastroenterology and Hepatology, Kansai Medical University 2-5-1, Shin-Machi, Hirakata, Osaka 573-1010, Japan.
| | - Takashi Yamaguchi
- Department of Gastroenterology and Hepatology, Kansai Medical University 2-5-1, Shin-Machi, Hirakata, Osaka 573-1010, Japan.
| | - Kanehiko Suwa
- Department of Gastroenterology and Hepatology, Kansai Medical University 2-5-1, Shin-Machi, Hirakata, Osaka 573-1010, Japan.
| | - Kazuichi Okazaki
- Department of Gastroenterology and Hepatology, Kansai Medical University 2-5-1, Shin-Machi, Hirakata, Osaka 573-1010, Japan.
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23
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Amicone L, Marchetti A. Microenvironment and tumor cells: two targets for new molecular therapies of hepatocellular carcinoma. Transl Gastroenterol Hepatol 2018; 3:24. [PMID: 29971255 DOI: 10.21037/tgh.2018.04.05] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2018] [Accepted: 04/11/2018] [Indexed: 12/17/2022] Open
Abstract
Hepatocellular carcinoma (HCC), is one of the most frequent human cancer and is characterized by a high mortality rate. The aggressiveness appears strictly related to the liver pathological background on which cancer develops. Inflammation and the consequent fibro/cirrhosis, derived from chronic injuries of several origins (viral, toxic and metabolic) and observable in almost all oncological patients, represents the most powerful risk factor for HCC and, at the same time, an important obstacle to the efficacy of systemic therapy. Multiple microenvironmental cues, indeed, play a pivotal role in the pathogenesis, evolution and recurrence of HCC as well as in the resistance to standard therapies observed in most of patients. The identification of altered pathways in cancer cells and of microenvironmental changes, strictly connected in pathogenic feedback loop, may permit to plan new therapeutic approaches targeting tumor cells and their permissive microenvironment, simultaneously.
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Affiliation(s)
- Laura Amicone
- Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy
| | - Alessandra Marchetti
- Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy
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24
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Guo R, Tang W, Yuan Q, Hui L, Wang X, Xie X. Chemical Cocktails Enable Hepatic Reprogramming of Mouse Fibroblasts with a Single Transcription Factor. Stem Cell Reports 2017; 9:499-512. [PMID: 28757167 PMCID: PMC5550014 DOI: 10.1016/j.stemcr.2017.06.013] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2017] [Revised: 06/25/2017] [Accepted: 06/26/2017] [Indexed: 12/31/2022] Open
Abstract
Liver or hepatocytes transplantation is limited by the availability of donor organs. Functional hepatocytes independent of the donor sources may have wide applications in regenerative medicine and the drug industry. Recent studies have demonstrated that chemical cocktails may induce reprogramming of fibroblasts into a range of functional somatic cells. Here, we show that mouse fibroblasts can be transdifferentiated into the hepatocyte-like cells (iHeps) using only one transcription factor (TF) (Foxa1, Foxa2, or Foxa3) plus a chemical cocktail. These iHeps show typical epithelial morphology, express multiple hepatocyte-specific genes, and acquire hepatocyte functions. Genetic lineage tracing confirms the fibroblast origin of these iHeps. More interestingly, these iHeps are expandable in vitro and can reconstitute the damaged hepatic tissues of the fumarylacetoacetate hydrolase-deficient (Fah−/−) mice. Our study provides a strategy to generate functional hepatocyte-like cells by using a single TF plus a chemical cocktail and is one step closer to generate the full-chemical iHeps.
Fibroblasts are converted to iHeps using only one TF plus a chemical cocktail Genetic lineage tracing confirms the fibroblast origin of these iHeps The iHeps are expendable and functional both in vitro and in vivo
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Affiliation(s)
- Ren Guo
- CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China; University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
| | - Wei Tang
- Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Qianting Yuan
- CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China
| | - Lijian Hui
- Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy for Sciences, 320 Yueyang Road, 200031 Shanghai, China
| | - Xin Wang
- Key Laboratory of National Education, Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010021, China; Department of Laboratory Medicine and Pathology, Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | - Xin Xie
- CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China; Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
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25
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Munker S, Wu YL, Ding HG, Liebe R, Weng HL. Can a fibrotic liver afford epithelial-mesenchymal transition? World J Gastroenterol 2017; 23:4661-4668. [PMID: 28765687 PMCID: PMC5514631 DOI: 10.3748/wjg.v23.i26.4661] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/25/2017] [Revised: 04/04/2017] [Accepted: 05/19/2017] [Indexed: 02/06/2023] Open
Abstract
The question whether epithelial-mesenchymal transition (EMT) occurs during liver fibrogenesis is a controversial issue. In vitro studies confirm that hepatocytes or cholangiocytes undergo EMT upon transforming growth factor β (TGF-β) stimulation, whereas in vivo experiments based on genetic fate mapping of specific cell populations suggest that EMT does not occur in fibrotic animal models. In this review we present current data supporting or opposing EMT in chronic liver disease and discuss conditions for the occurrence of EMT in patients. Based on the available data and our clinical observations we hypothesize that EMT-like alterations in liver cirrhosis are a side effect of high levels of TGF-β and other pro-fibrotic mediators rather than a biological process converting functional parenchyma, i.e., hepatocytes, into myofibroblasts at a time when essential liver functions are deteriorating.
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26
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De Santis Puzzonia M, Cozzolino AM, Grassi G, Bisceglia F, Strippoli R, Guarguaglini G, Citarella F, Sacchetti B, Tripodi M, Marchetti A, Amicone L. TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure. PLoS One 2016; 11:e0167158. [PMID: 27893804 PMCID: PMC5125678 DOI: 10.1371/journal.pone.0167158] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2016] [Accepted: 11/09/2016] [Indexed: 12/21/2022] Open
Abstract
In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.
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Affiliation(s)
- Marco De Santis Puzzonia
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy
| | - Angela Maria Cozzolino
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy.,L. Spallanzani National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Germana Grassi
- L. Spallanzani National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Francesca Bisceglia
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy
| | - Raffaele Strippoli
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy
| | - Giulia Guarguaglini
- Institute of Molecular Biology and Pathology, CNR National Research Council, c/o Department of Biology and Biotechnology, Sapienza University of Rome, Rome, Italy
| | - Franca Citarella
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy
| | | | - Marco Tripodi
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy.,L. Spallanzani National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Alessandra Marchetti
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy
| | - Laura Amicone
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy
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27
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Vasconcellos R, Alvarenga ÉC, Parreira RC, Lima SS, Resende RR. Exploring the cell signalling in hepatocyte differentiation. Cell Signal 2016; 28:1773-88. [DOI: 10.1016/j.cellsig.2016.08.011] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2016] [Revised: 08/18/2016] [Accepted: 08/18/2016] [Indexed: 02/08/2023]
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28
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Battistelli C, Cicchini C, Santangelo L, Tramontano A, Grassi L, Gonzalez FJ, de Nonno V, Grassi G, Amicone L, Tripodi M. The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition. Oncogene 2016; 36:942-955. [PMID: 27452518 PMCID: PMC5318668 DOI: 10.1038/onc.2016.260] [Citation(s) in RCA: 138] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2016] [Revised: 05/30/2016] [Accepted: 06/13/2016] [Indexed: 12/18/2022]
Abstract
The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.
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Affiliation(s)
- C Battistelli
- Department of Cellular Biotechnologies and Haematology, Sezione di Genetica Molecolare, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - C Cicchini
- Department of Cellular Biotechnologies and Haematology, Sezione di Genetica Molecolare, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - L Santangelo
- Department of Cellular Biotechnologies and Haematology, Sezione di Genetica Molecolare, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - A Tramontano
- Department of Physics, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - L Grassi
- Department of Physics, Sapienza University of Rome, Rome, Italy
| | - F J Gonzalez
- Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
| | - V de Nonno
- Department of Cellular Biotechnologies and Haematology, Sezione di Genetica Molecolare, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - G Grassi
- National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
| | - L Amicone
- Department of Cellular Biotechnologies and Haematology, Sezione di Genetica Molecolare, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
| | - M Tripodi
- Department of Cellular Biotechnologies and Haematology, Sezione di Genetica Molecolare, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.,National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy
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29
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Li D, Zhao X, Xiao Y, Mei H, Pu J, Xiang X, Jiao W, Song H, Qu H, Huang K, Zheng L, Tong Q. Intelectin 1 suppresses tumor progression and is associated with improved survival in gastric cancer. Oncotarget 2016; 6:16168-82. [PMID: 25965823 PMCID: PMC4599263 DOI: 10.18632/oncotarget.3753] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2014] [Accepted: 04/06/2015] [Indexed: 12/30/2022] Open
Abstract
Recent evidence shows the emerging roles of intelectin 1 (ITLN1), a secretory lectin, in human cancers. Our previous studies have implicated the potential roles of ITLN1 in the aggressiveness of gastric cancer. Herein, we investigated the functions, downstream targets, and clinical significance of ITLN1 in the progression of gastric cancer. We demonstrated that ITLN1 increased the levels of hepatocyte nuclear factor 4 alpha (HNF4α), resulting in suppression of nuclear translocation and transcriptional activity of β-catenin in gastric cancer cells. Mechanistically, ITLN1 attenuated the activity of nuclear factor-kappa B, a transcription factor repressing the HNF4α expression, in gastric cancer cells through inactivating the phosphoinositide 3-kinase/AKT/Ikappa B kinase signaling. Gain- and loss-of-function studies demonstrated that ITLN1 suppressed the growth, invasion, and metastasis of gastric cancer cells in vitro and in vivo. In addition, restoration of HNF4α expression prevented the gastric cancer cells from ITLN1-mediated changes in these biological features. In clinical gastric cancer tissues, HNF4α expression was positively correlated with that of ITLN1. Patients with high ITLN1 or HNF4α expression had greater survival probability. Taken together, these data indicate that ITLN1 suppresses the progression of gastric cancer through up-regulation of HNF4α, and is associated with improved survival in patients with gastric cancer.
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Affiliation(s)
- Dan Li
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Xiang Zhao
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Yong Xiao
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Hong Mei
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Jiarui Pu
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Xuan Xiang
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Wanju Jiao
- Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Huajie Song
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Hongxia Qu
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Kai Huang
- Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China.,Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Liduan Zheng
- Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China.,Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Qiangsong Tong
- Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China.,Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
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30
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David CJ, Huang YH, Chen M, Su J, Zou Y, Bardeesy N, Iacobuzio-Donahue CA, Massagué J. TGF-β Tumor Suppression through a Lethal EMT. Cell 2016; 164:1015-30. [PMID: 26898331 DOI: 10.1016/j.cell.2016.01.009] [Citation(s) in RCA: 480] [Impact Index Per Article: 53.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2015] [Revised: 12/06/2015] [Accepted: 01/07/2016] [Indexed: 01/06/2023]
Abstract
TGF-β signaling can be pro-tumorigenic or tumor suppressive. We investigated this duality in pancreatic ductal adenocarcinoma (PDA), which, with other gastrointestinal cancers, exhibits frequent inactivation of the TGF-β mediator Smad4. We show that TGF-β induces an epithelial-mesenchymal transition (EMT), generally considered a pro-tumorigenic event. However, in TGF-β-sensitive PDA cells, EMT becomes lethal by converting TGF-β-induced Sox4 from an enforcer of tumorigenesis into a promoter of apoptosis. This is the result of an EMT-linked remodeling of the cellular transcription factor landscape, including the repression of the gastrointestinal lineage-master regulator Klf5. Klf5 cooperates with Sox4 in oncogenesis and prevents Sox4-induced apoptosis. Smad4 is required for EMT but dispensable for Sox4 induction by TGF-β. TGF-β-induced Sox4 is thus geared to bolster progenitor identity, whereas simultaneous Smad4-dependent EMT strips Sox4 of an essential partner in oncogenesis. Our work demonstrates that TGF-β tumor suppression functions through an EMT-mediated disruption of a lineage-specific transcriptional network.
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Affiliation(s)
- Charles J David
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Yun-Han Huang
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Mo Chen
- The Rockefeller University, New York, NY 10065, USA
| | - Jie Su
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Yilong Zou
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Nabeel Bardeesy
- Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA
| | | | - Joan Massagué
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
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31
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Yoshida K, Murata M, Yamaguchi T, Matsuzaki K, Okazaki K. Reversible Human TGF-β Signal Shifting between Tumor Suppression and Fibro-Carcinogenesis: Implications of Smad Phospho-Isoforms for Hepatic Epithelial-Mesenchymal Transitions. J Clin Med 2016; 5:jcm5010007. [PMID: 26771649 PMCID: PMC4730132 DOI: 10.3390/jcm5010007] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2015] [Revised: 12/16/2015] [Accepted: 01/04/2016] [Indexed: 12/23/2022] Open
Abstract
Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are observed during both physiological liver wound healing and the pathological fibrotic/carcinogenic (fibro-carcinogenetic) process. TGF-β and pro-inflammatory cytokine are considered to be the major factors accelerating liver fibrosis and promoting liver carcinogenesis. Smads, consisting of intermediate linker regions connecting Mad homology domains, act as the intracellular mediators of the TGF-β signal transduction pathway. As the TGF-β receptors, c-Jun N-terminal kinase and cyclin-dependent kinase, differentially phosphorylate Smad2/3, we have generated numerous antibodies against linker (L) and C-terminal (C) phosphorylation sites in Smad2/3 and identified four types of phosphorylated forms: cytostatic COOH-terminally-phosphorylated Smad3 (pSmad3C), mitogenic pSmad3L (Ser-213) signaling, fibrogenic pSmad2L (Ser-245/250/255)/C signaling and migratory pSmad2/3L (Thr-220/179)/C signaling. After acute liver injury, TGF-β upregulates pSmad3C signaling and terminates pSmad3L (Ser-213)-mediated hepatocyte proliferation. TGF-β and pro-inflammatory cytokines cooperatively enhance collagen synthesis by upregulating pSmad2L (Thr-220)/C and pSmad3L (Thr-179)/C pathways in activated hepatic stellate cells. During chronic liver injuries, hepatocytes persistently affected by TGF-β and pro-inflammatory cytokines eventually become pre-neoplastic hepatocytes. Both myofibroblasts and pre-neoplastic hepatocyte exhibit the same carcinogenic (mitogenic) pSmad3L (Ser-213) and fibrogenic pSmad2L (Ser-245/250/255)/C signaling, with acquisition of fibro-carcinogenic properties and increasing risk of hepatocellular carcinoma (HCC). Firstly, we review phospho-Smad-isoform signalings in epithelial and mesenchymal cells in physiological and pathological conditions and then consider Smad linker phosphorylation as a potential target for pathological EMT during human fibro-carcinogenesis, because human Smad phospho-isoform signals can reverse from fibro-carcinogenesis to tumor-suppression in a process of MET after therapy.
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Affiliation(s)
- Katsunori Yoshida
- Department of Gastroenterology and Hepatology, Kansai Medical University, 2-5-1, Shin-machi, Hirakata, Osaka 573-1010, Japan.
| | - Miki Murata
- Department of Gastroenterology and Hepatology, Kansai Medical University, 2-5-1, Shin-machi, Hirakata, Osaka 573-1010, Japan.
| | - Takashi Yamaguchi
- Department of Gastroenterology and Hepatology, Kansai Medical University, 2-5-1, Shin-machi, Hirakata, Osaka 573-1010, Japan.
| | - Koichi Matsuzaki
- Department of Gastroenterology and Hepatology, Kansai Medical University, 2-5-1, Shin-machi, Hirakata, Osaka 573-1010, Japan.
| | - Kazuichi Okazaki
- Department of Gastroenterology and Hepatology, Kansai Medical University, 2-5-1, Shin-machi, Hirakata, Osaka 573-1010, Japan.
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32
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Role of Tribbles Pseudokinase 1 (TRIB1) in human hepatocyte metabolism. Biochim Biophys Acta Mol Basis Dis 2015; 1862:223-32. [PMID: 26657055 DOI: 10.1016/j.bbadis.2015.12.003] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Revised: 11/18/2015] [Accepted: 12/01/2015] [Indexed: 01/23/2023]
Abstract
Genome-wide association studies for plasma triglycerides and hepatic steatosis identified a risk locus on chromosome 8q24 close to the TRIB1 gene, encoding Tribbles Pseudokinase 1 (TRIB1). In previous studies conducted in murine models, hepatic over-expression of Trib1 was shown to increase fatty acid oxidation and decrease triglyceride synthesis whereas Trib1 knockdown mice exhibited hypertriglyceridemia. Here we have examined the impact of TRIB1 suppression in human and mouse hepatocytes. Examination of a panel of lipid regulator transcripts revealed species-specific effects, prompting us to focus on human models for the remainder of the study. Acute knockdown of TRIB1 in human primary hepatocytes resulted in decreased expression of MTTP and APOB, required for very low density lipoprotein (VLDL) assembly although particle secretion was not significantly affected. A parallel analysis performed in HepG2 revealed reduced MTTP, but not APOB, protein as a result of TRIB1 suppression. Global gene expression changes of human primary hepatocytes upon TRIB1 suppression were analyzed by clustering algorithms and found to be consistent with dysregulation of several pathways fundamental to liver function, including altered CEBPA and B transcript levels and impaired glucose handling. Indeed, TRIB1 expression in HepG2 cells was found to be inversely proportional to glucose concentration. Lastly TRIB1 downregulation in primary hepatocytes was associated with suppression of the HNF4A axis. In HepG2 cells, TRIB1 suppression resulted in reduced HNF4A protein levels while HNF4A suppression increased TRIB1 expression. Taken together these studies reveal an important role for TRIB1 in human hepatocyte biology.
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33
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New Tools for Molecular Therapy of Hepatocellular Carcinoma. Diseases 2015; 3:325-340. [PMID: 28943628 PMCID: PMC5548255 DOI: 10.3390/diseases3040325] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Revised: 10/15/2015] [Accepted: 10/22/2015] [Indexed: 12/12/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is the most common type of liver cancer, arising from neoplastic transformation of hepatocytes or liver precursor/stem cells. HCC is often associated with pre-existing chronic liver pathologies of different origin (mainly subsequent to HBV and HCV infections), such as fibrosis or cirrhosis. Current therapies are essentially still ineffective, due both to the tumor heterogeneity and the frequent late diagnosis, making necessary the creation of new therapeutic strategies to inhibit tumor onset and progression and improve the survival of patients. A promising strategy for treatment of HCC is the targeted molecular therapy based on the restoration of tumor suppressor proteins lost during neoplastic transformation. In particular, the delivery of master genes of epithelial/hepatocyte differentiation, able to trigger an extensive reprogramming of gene expression, could allow the induction of an efficient antitumor response through the simultaneous adjustment of multiple genetic/epigenetic alterations contributing to tumor development. Here, we report recent literature data supporting the use of members of the liver enriched transcription factor (LETF) family, in particular HNF4α, as tools for gene therapy of HCC.
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34
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Kodama S, Yamazaki Y, Negishi M. Pregnane X Receptor Represses HNF4α Gene to Induce Insulin-Like Growth Factor-Binding Protein IGFBP1 that Alters Morphology of and Migrates HepG2 Cells. Mol Pharmacol 2015; 88:746-57. [PMID: 26232425 PMCID: PMC4576682 DOI: 10.1124/mol.115.099341] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2015] [Accepted: 07/23/2015] [Indexed: 12/27/2022] Open
Abstract
Upon treatment with the pregnane X receptor (PXR) activator rifampicin (RIF), human hepatocellular carcinoma HepG2-derived ShP51 cells that stably express PXR showed epithelial-mesenchymal transition (EMT)-like morphological changes and migration. Our recent DNA microarrays have identified hepatocyte nuclear factor (HNF) 4α and insulin-like growth factor-binding protein (IGFBP) 1 mRNAs to be downregulated and upregulated, respectively, in RIF-treated ShP51 cells, and these regulations were confirmed by the subsequent real-time polymerase chain reaction and Western blot analyses. Using this cell system, we demonstrated here that the PXR-HNF4α-IGFBP1 pathway is an essential signal for PXR-induced morphological changes and migration. First, we characterized the molecular mechanism underlying the PXR-mediated repression of the HNF4α gene. Chromatin conformation capture and chromatin immunoprecipitation (ChIP) assays revealed that PXR activation by RIF disrupted enhancer-promoter communication and prompted deacetylation of histone H3 in the HNF4α P1 promoter. Cell-based reporter and ChIP assays showed that PXR targeted the distal enhancer of the HNF4α P1 promoter and stimulated dissociation of HNF3β from the distal enhancer. Subsequently, small interfering RNA knockdown of HNF4α connected PXR-mediated gene regulation with the PXR-induced cellular responses, showing that the knockdown resulted in the upregulation of IGFBP1 and EMT-like morphological changes without RIF treatment. Moreover, recombinant IGFBP1 augmented migration, whereas an anti-IGFBP1 antibody attenuated both PXR-induced morphological changes and migration in ShP51 cells. PXR indirectly activated the IGFBP1 gene by repressing the HNF4α gene, thus enabling upregulation of IGFBP1 to change the morphology of ShP51 cells and cause migration. These results provide new insights into PXR-mediated cellular responses toward xenobiotics including therapeutics.
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Affiliation(s)
- Susumu Kodama
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
| | - Yuichi Yamazaki
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
| | - Masahiko Negishi
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
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Grassi G, Di Caprio G, Santangelo L, Fimia GM, Cozzolino AM, Komatsu M, Ippolito G, Tripodi M, Alonzi T. Autophagy regulates hepatocyte identity and epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions promoting Snail degradation. Cell Death Dis 2015; 6:e1880. [PMID: 26355343 PMCID: PMC4650445 DOI: 10.1038/cddis.2015.249] [Citation(s) in RCA: 97] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2015] [Revised: 07/24/2015] [Accepted: 07/28/2015] [Indexed: 01/16/2023]
Abstract
Epithelial-to-mesenchymal transition (EMT) and the reverse process mesenchymal-to-epithelial transition (MET) are events involved in development, wound healing and stem cell behaviour and contribute pathologically to cancer progression. The identification of the molecular mechanisms underlying these phenotypic conversions in hepatocytes are fundamental to design specific therapeutic strategies aimed at optimising liver repair. The role of autophagy in EMT/MET processes of hepatocytes was investigated in liver-specific autophagy-deficient mice (Alb-Cre;ATG7fl/fl) and using the nontumorigenic immortalised hepatocytes cell line MMH. Autophagy deficiency in vivo reduces epithelial markers' expression and increases the levels of mesenchymal markers. These alterations are associated with an increased protein level of the EMT master regulator Snail, without transcriptional induction. Interestingly, we found that autophagy degrades Snail in a p62/SQSTM1 (Sequestosome-1)-dependent manner. Moreover, accordingly to a pro-epithelial function, we observed that autophagy stimulation strongly affects EMT progression, whereas it is necessary for MET. Finally, we found that the EMT induced by TGFβ affects the autophagy flux, indicating that these processes regulate each other. Overall, we found that autophagy regulates the phenotype plasticity of hepatocytes promoting their epithelial identity through the inhibition of the mesenchymal programme.
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Affiliation(s)
- G Grassi
- National Institute for Infectious Diseases L. Spallanzani IRCCS, Rome, Italy
| | - G Di Caprio
- National Institute for Infectious Diseases L. Spallanzani IRCCS, Rome, Italy.,Department of Cellular Biotechnologies and Hematology, Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - L Santangelo
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - G M Fimia
- National Institute for Infectious Diseases L. Spallanzani IRCCS, Rome, Italy.,Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce, Italy
| | - A M Cozzolino
- National Institute for Infectious Diseases L. Spallanzani IRCCS, Rome, Italy.,Department of Cellular Biotechnologies and Hematology, Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - M Komatsu
- Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata, 951-8510, Japan
| | - G Ippolito
- National Institute for Infectious Diseases L. Spallanzani IRCCS, Rome, Italy
| | - M Tripodi
- National Institute for Infectious Diseases L. Spallanzani IRCCS, Rome, Italy.,Department of Cellular Biotechnologies and Hematology, Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - T Alonzi
- National Institute for Infectious Diseases L. Spallanzani IRCCS, Rome, Italy
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Liu K, Guo MG, Lou XL, Li XY, Xu Y, Ji WD, Huang XD, Yang JH, Duan JC. Hepatocyte nuclear factor 4α induces a tendency of differentiation and activation of rat hepatic stellate cells. World J Gastroenterol 2015; 21:5856-5866. [PMID: 26019449 PMCID: PMC4438019 DOI: 10.3748/wjg.v21.i19.5856] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/18/2014] [Revised: 01/15/2015] [Accepted: 02/11/2015] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effect of hepatocyte nuclear factor 4α (HNF4α) on the differentiation and transformation of hepatic stellate cells (HSCs).
METHODS: By constructing the recombinant adenovirus vector expressing HNF4α and HNF4α shRNA vector, and manipulating HNF4α expression in HSC-T6 cells, we explored the influence of HNF4α and its induction capacity in the differentiation of rat HSCs into hepatocytes.
RESULTS: With increased expression of HNF4α mediated by AdHNF4α, the relative expression of Nanog was downregulated in HSC-T6 cells (98.33 ± 12.33 vs 41.33 ± 5.67, P < 0.001). Consequently, the expression of G-P-6 and PEPCK was upregulated (G-P-6: 14.34 ± 3.33 vs 42.53 ± 5.87, P < 0.01; PEPCK: 10.10 ± 4.67 vs 56.56 ± 5.25, P < 0.001), the expression of AFP and ALB was positive, and the expression of Nanog, Type I collagen, α-SMA, and TIMP-1 was significantly decreased. HNF4α also downregulated vimentin expression and enhanced E-cadherin expression. The ultrastructure of HNF4α-induced cells had more mitochondria and ribosomes compared with the parental cells. After silencing HNF4α expression, EPCK, E-cadherin, AFP, and ALB were downregulated and α-SMA and vimentin were upregulated.
CONCLUSION: HNF4α can induce a tendency of differentiation of HSCs into hepatocyte-like cells. These findings may provide an effective way for the treatment of liver diseases.
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Epigenetic control of EMT/MET dynamics: HNF4α impacts DNMT3s through miRs-29. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2015; 1849:919-29. [PMID: 26003733 DOI: 10.1016/j.bbagrm.2015.05.005] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/17/2014] [Revised: 04/17/2015] [Accepted: 05/13/2015] [Indexed: 01/11/2023]
Abstract
BACKGROUND AND AIMS Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B. METHODS Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity. RESULTS HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR-29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype. CONCLUSIONS HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels.
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Pilli VS, Gupta K, Kotha BP, Aradhyam GK. Snail-mediated Cripto-1 repression regulates the cell cycle and epithelial-mesenchymal transition-related gene expression. FEBS Lett 2015; 589:1249-56. [PMID: 25889638 DOI: 10.1016/j.febslet.2015.04.005] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2014] [Revised: 03/23/2015] [Accepted: 04/05/2015] [Indexed: 10/23/2022]
Abstract
Transcription factor Snail mediates epithelial to mesenchymal transitions (EMT) by coordinate repression of epithelial markers, facilitating mass cell movement during germ layer formation. Aberrant reprogramming in its signaling pathways causes metastatic cancer. Snail's involvement in "fate-changing" decisions is however not understood. Cripto-1 shares a common temporal expression pattern with Snail during development. While Cripto-1 is required for mammary morphogenesis and hematopoietic stem cell renewal, its unregulated expression causes metastatic cancers. Therefore, we suspected that Snail regulates the expression of Cripto-1 controlling decisions such as motility, transformation and differentiation. We demonstrate that Snail represses Cripto-1 gene by direct transcriptional interaction and propose a novel mechanism by which it co-ordinately regulates cell fate decisions during development and could be causal of cancers.
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Affiliation(s)
- Vijaya S Pilli
- Department of Biotechnology, Bhupat & Jyoti Mehta School of Bioscience, Indian Institute of Technology Madras, Chennai 600036, India
| | - K Gupta
- Department of Biotechnology, Bhupat & Jyoti Mehta School of Bioscience, Indian Institute of Technology Madras, Chennai 600036, India
| | - Bhanu P Kotha
- Department of Biotechnology, Bhupat & Jyoti Mehta School of Bioscience, Indian Institute of Technology Madras, Chennai 600036, India
| | - Gopala Krishna Aradhyam
- Department of Biotechnology, Bhupat & Jyoti Mehta School of Bioscience, Indian Institute of Technology Madras, Chennai 600036, India.
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Shen H, Zhong F, Zhang Y, Yu H, Liu Y, Qin L, He F, Tang Z, Yang P. Transcriptome and proteome of human hepatocellular carcinoma reveal shared metastatic pathways with significant genes. Proteomics 2015; 15:1793-800. [PMID: 25652264 DOI: 10.1002/pmic.201400275] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2014] [Revised: 12/23/2014] [Accepted: 01/30/2015] [Indexed: 11/12/2022]
Abstract
Previously isolated pathways screened from individual genes were investigated at either the transcriptional or translational level; however, the consistency between the pathways screened at the gene expression levels was obscure in metastatic human hepatocellular carcinoma (HCC). To elucidate this question, we performed a transcriptomic (16,353 genes) and proteomic (7861 proteins) analysis simultaneously on six metastatic HCC cell lines against two nonmetastatic HCC cell lines, with all HBV traceable and close genetic-backgrounds for a comparative study. The quantitative and integrated results showed that significant genes were screened differentially with 351 transcripts from the transcriptome and 304 proteins from the proteome, with limited overlapping genes (7%). However, we discovered that these discrete 351 transcripts and 304 proteins screened share extrusive significant-pathways/networks with a 77% overlap, including active TGF-β, RAS, NFκB, and Wnt, and inactive HNF4A, which are responsible for HCC metastasis. We conclude that the discrete, but significant genes predicted by either ome play intrinsically important roles in the linkage of responsible pathways shared by both omes in HCC metastasis.
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Affiliation(s)
- Huali Shen
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China.,Department of Chemistry, Fudan University, Shanghai, P. R. China
| | - Fan Zhong
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China
| | - Yang Zhang
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China
| | - Hongxiu Yu
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China
| | - Yinkun Liu
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China.,Zhongshan Hospital, Fudan University, Shanghai, P. R. China
| | - Lunxiu Qin
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China.,Zhongshan Hospital, Fudan University, Shanghai, P. R. China
| | - Fuchu He
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China.,State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing, P. R. China
| | - Zhaoyou Tang
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China.,Zhongshan Hospital, Fudan University, Shanghai, P. R. China
| | - Pengyuan Yang
- Department of Systems Biology for Medicine and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China.,Department of Chemistry, Fudan University, Shanghai, P. R. China.,Children Hospital, Fudan University, Shanghai, P. R. China
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40
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Teng NY, Liu YS, Wu HH, Liu YA, Ho JH, Lee OKS. Promotion of mesenchymal-to-epithelial transition by Rac1 inhibition with small molecules accelerates hepatic differentiation of mesenchymal stromal cells. Tissue Eng Part A 2015; 21:1444-54. [PMID: 25625545 DOI: 10.1089/ten.tea.2014.0320] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
In vitro differentiation of stem cells into specific cell lineages provides a stable cell supply for cell therapy and tissue engineering. Therefore, understanding the mechanisms underlying such differentiation processes is critical for generating committed lineage-specific cell progenies effectively. We previously developed a two-step protocol to differentiate mesenchymal stromal cells (MSCs) into hepatocyte-like cells. Since hepatic differentiation involves mesenchymal-epithelial transition (MET), we hypothesize that promoting MET could further accelerate the differentiation process. Ras-related C3 botulinum toxin substrate 1 (Rac1) is involved in actin polymerization and its role in MET was investigated in the study. Our results showed that inhibition of Rac1 activation by Rac1-specific inhibitor, NSC23766, led to cells favoring epithelial morphology and being more packed during hepatic differentiation. In addition, Rac1 inhibition accelerated the upregulation of hepatic marker genes accompanied by more mature hepatic functions. Taken together, promotion of MET by inhibiting Rac1 accelerates the hepatic differentiation of MSCs. Our findings open a new prospect of directing the commitment of MSCs by manipulating cell morphology and cytoskeleton arrangement through small molecules. The results provide further insight into scaffold design for rapid production of MSC-differentiated hepatocytes.
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Affiliation(s)
- Nan-Yuan Teng
- 1 Institute of Clinical Medicine, National Yang-Ming University , Taipei, Taiwan
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Cicchini C, Amicone L, Alonzi T, Marchetti A, Mancone C, Tripodi M. Molecular mechanisms controlling the phenotype and the EMT/MET dynamics of hepatocyte. Liver Int 2015; 35:302-10. [PMID: 24766136 PMCID: PMC4344819 DOI: 10.1111/liv.12577] [Citation(s) in RCA: 75] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2013] [Accepted: 04/19/2014] [Indexed: 02/06/2023]
Abstract
The complex spatial and paracrine relationships between the various liver histotypes are essential for proper functioning of the hepatic parenchymal cells. Only within a correct tissue organization, in fact, they stably maintain their identity and differentiated phenotype. The loss of histotype identity, which invariably occurs in the primary hepatocytes in culture, or in vivo in particular pathological conditions (fibrosis and tumours), is mainly because of the phenomenon of epithelial-to-mesenchymal transition (EMT). The EMT process, that occurs in the many epithelial cells, appears to be driven by a number of general, non-tissue-specific, master transcriptional regulators. The reverse process, the mesenchymal-to-epithelial transition (MET), as yet much less characterized at a molecular level, restores specific epithelial identities, and thus must include tissue-specific master elements. In this review, we will summarize the so far unveiled events of EMT/MET occurring in liver cells. In particular, we will focus on hepatocyte and describe the pivotal role in the control of EMT/MET dynamics exerted by a tissue-specific molecular mini-circuitry. Recent evidence, indeed, highlighted as two transcriptional factors, the master gene of EMT Snail, and the master gene of hepatocyte differentiation HNF4α, exhorting a direct reciprocal repression, act as pivotal elements in determining opposite cellular outcomes. The different balances between these two master regulators, further integrated by specific microRNAs, in fact, were found responsible for the EMT/METs dynamics as well as for the preservation of both hepatocyte and stem/precursor cells identity and differentiation. Overall, these findings impact the maintenance of stem cells and differentiated cells both in in vivo EMT/MET physio-pathological processes as well as in culture.
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Affiliation(s)
- Carla Cicchini
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of RomeRome, Italy
| | - Laura Amicone
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of RomeRome, Italy
| | - Tonino Alonzi
- National Institute for Infectious Diseases L. Spallanzani, IRCCSRome, Italy
| | - Alessandra Marchetti
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of RomeRome, Italy
| | - Carmine Mancone
- National Institute for Infectious Diseases L. Spallanzani, IRCCSRome, Italy
| | - Marco Tripodi
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of RomeRome, Italy,National Institute for Infectious Diseases L. Spallanzani, IRCCSRome, Italy
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The aPKCι blocking agent ATM negatively regulates EMT and invasion of hepatocellular carcinoma. Cell Death Dis 2014; 5:e1129. [PMID: 24651432 PMCID: PMC3973203 DOI: 10.1038/cddis.2014.91] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2013] [Revised: 01/08/2014] [Accepted: 02/10/2014] [Indexed: 12/23/2022]
Abstract
Epithelial-to-mesenchymal transition (EMT) has an important role in invasion and metastasis of hepatocellular carcinoma (HCC). To explore the regulatory mechanism of atypical protein kinase C ι (aPKCι) signaling pathways to HCC development, and find an agent for targeted therapy for HCC, immortalized murine hepatocytes were employed to establish an EMT cell model of HCC, MMH-RT cells. Our study showed that EMT took place in MMH-R cells under the effect of transforming growth factor-β1 (TGF-β1) overexpressing aPKCι. Furthermore, we showed that the aPKCι blocking agent aurothiomalate (ATM) inhibited EMT and decreased invasion of hepatocytes. Moreover, ATM selectively inhibited proliferation of mesenchymal cells and HepG2 cells and induced apoptosis. However, ATM increased proliferation of epithelial cells and had little effect on apoptosis and invasion of epithelial cells. In conclusion, our result suggested that aPKCι could be an important bio-marker of tumor EMT, and used as an indicator of invasion and malignancy. ATM might be a promising agent for targeted treatment of HCC.
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Yang M, Li SN, Anjum KM, Gui LX, Zhu SS, Liu J, Chen JK, Liu QF, Ye GD, Wang WJ, Wu JF, Cai WY, Sun GB, Liu YJ, Liu RF, Zhang ZM, Li BA. A double-negative feedback loop between Wnt-β-catenin signaling and HNF4α regulates epithelial-mesenchymal transition in hepatocellular carcinoma. J Cell Sci 2013; 126:5692-703. [PMID: 24101726 DOI: 10.1242/jcs.135053] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Wnt-β-catenin signaling participates in the epithelial-mesenchymal transition (EMT) in a variety of cancers; however, its involvement in hepatocellular carcinoma (HCC) and downstream molecular events is largely undefined. HNF4α is the most prominent and specific factor maintaining the differentiation of hepatic lineage cells and a potential EMT regulator in HCC cells. However, the molecular mechanisms by which HNF4α maintains the differentiated liver epithelium and inhibits EMT have not been completely defined. In this study, we systematically explored the relationship between Wnt-β-catenin signaling and HNF4α in the EMT process of HCC cells. Our results indicated that HNF4α expression was negatively regulated during Wnt-β-catenin signaling-induced EMT through Snail and Slug in HCC cells. In contrast, HNF4α was found to directly associate with TCF4 to compete with β-catenin but facilitate transcription co-repressor activities, thus inhibiting expression of EMT-related Wnt-β-catenin targets. Moreover, HNF4α may control the switch between the transcriptional and adhesion functions of β-catenin. Overexpression of HNF4α was found to completely compromise the Wnt-β-catenin-signaling-induced EMT phenotype. Finally, we determined the regulation pattern between Wnt-β-catenin signaling and HNF4α in rat tumor models. Our studies have identified a double-negative feedback mechanism controlling Wnt-β-catenin signaling and HNF4α expression in vitro and in vivo, which sheds new light on the regulation of EMT in HCC. The modulation of these molecular processes may be a method of inhibiting HCC invasion by blocking Wnt-β-catenin signaling or restoring HNF4α expression to prevent EMT.
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Affiliation(s)
- Meng Yang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, 361102, China
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Yao D, Peng S, Dai C. The role of hepatocyte nuclear factor 4alpha in metastatic tumor formation of hepatocellular carcinoma and its close relationship with the mesenchymal-epithelial transition markers. BMC Cancer 2013; 13:432. [PMID: 24059685 PMCID: PMC3852538 DOI: 10.1186/1471-2407-13-432] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2013] [Accepted: 09/17/2013] [Indexed: 12/27/2022] Open
Abstract
Background Mesenchymal–epithelial transition (MET) is now suggested to participate in the process of metastatic tumor formation. However, in hepatocellular carcinoma (HCC) the process is still not well revealed. Methods Paraffin-embedded tissue samples were obtained from 13 patients with HCC in Shengjing Hospital of China Medical University. The expression of E-cadherin, Fibronectin, N-cadherin, Vimentin, Hepatocyte nuclear factor 4alpha (HNF4alpha), Snail and Slug was assessed in primary tumors and their corresponding metastases by immunohistochemical staining. Next, the expression of HNF4alpha and E-cadherin in four HCC cell lines was examined. Furthermore, SK-Hep-1 cells were transfected with human HNF4alpha expression vector, and the change of E-cadherin expression was assessed. Results 45.2% (14/31) of the lesions in the metastases showed increased E-cadherin expression compared with the primaries, suggesting the possible occurrence of MET in metastatic tumor formation of HCC, as re-expression of E-cadherin is proposed to be the important hallmark of MET. The occurrence of MET was also confirmed by the reduced expression of Fibronectin (54.8%, 17/31), N-cadherin (38.7%, 12/31) and Vimentin (61.3%, 19/31) in the metastases. 45.2% (14/31) of the lesions in the metastases also showed increased HNF4alpha expression, and 67.7% (21/31) and 48.4% (15/31) of metastases showed decreased Snail and Slug expression respectively. Statistical results showed that the expression of HNF4alpha was positively related with that of E-cadherin, and negatively correlated with that of Snail, Slug and Fibronectin, suggesting that the expression change of the MET markers in the metastatic lesions might be associated with HNF4alpha. Among the four HCC cell lines, both HNF4alpha and E-cadherin expressed high in Hep3B and Huh-7 cells, but low in SK-Hep-1 and Bel-7402 cells. Furthermore, the expression of E-cadherin increased accordingly when SK-Hep-1 cells were transfected with human HNF4alpha expression vector, further confirming the role of HNF4alpha in the regulation of E-cadherin expression. Conclusions Our clinical observations and experimental data indicate that HNF4alpha might play a crucial role in the metastatic tumor formation of HCC, and the mechanism may be related with the process of phenotype transition.
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Affiliation(s)
- Dianbo Yao
- Department of Hepatobiliary and Splenic Surgery, Shengjing Hospital, China Medical University, Shenyang 110004, Liaoning Province, China.
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Cozzolino AM, Alonzi T, Santangelo L, Mancone C, Conti B, Steindler C, Musone M, Cicchini C, Tripodi M, Marchetti A. TGFβ overrides HNF4α tumor suppressing activity through GSK3β inactivation: implication for hepatocellular carcinoma gene therapy. J Hepatol 2013; 58:65-72. [PMID: 22960426 DOI: 10.1016/j.jhep.2012.08.023] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/04/2012] [Revised: 08/20/2012] [Accepted: 08/23/2012] [Indexed: 02/06/2023]
Abstract
BACKGROUND & AIMS The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor β (TGFβ) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity. METHODS Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3β activity was modulated by chemical inhibition and constitutive active mutant expression. RESULTS We demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation: GSK3β chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor suppressor activity. CONCLUSIONS Our data identify in the dominance of TGFβ a limit for the HNF4α-mediated gene therapy of HCC.
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Affiliation(s)
- Angela Maria Cozzolino
- Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, University La Sapienza, Rome, Italy
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Hong MH, Chou YC, Wu YC, Tsai KN, Hu CP, Jeng KS, Chen ML, Chang C. Transforming growth factor-β1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4α expression. PLoS One 2012; 7:e30360. [PMID: 22276183 PMCID: PMC3262823 DOI: 10.1371/journal.pone.0030360] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2011] [Accepted: 12/14/2011] [Indexed: 12/13/2022] Open
Abstract
Several studies have demonstrated that cytokine-mediated noncytopathic suppression of hepatitis B virus (HBV) replication may provide an alternative therapeutic strategy for the treatment of chronic hepatitis B infection. In our previous study, we showed that transforming growth factor-beta1 (TGF-β1) could effectively suppress HBV replication at physiological concentrations. Here, we provide more evidence that TGF-β1 specifically diminishes HBV core promoter activity, which subsequently results in a reduction in the level of viral pregenomic RNA (pgRNA), core protein (HBc), nucleocapsid, and consequently suppresses HBV replication. The hepatocyte nuclear factor 4alpha (HNF-4α) binding element(s) within the HBV core promoter region was characterized to be responsive for the inhibitory effect of TGF-β1 on HBV regulation. Furthermore, we found that TGF-β1 treatment significantly repressed HNF-4α expression at both mRNA and protein levels. We demonstrated that RNAi-mediated depletion of HNF-4α was sufficient to reduce HBc synthesis as TGF-β1 did. Prevention of HNF-4α degradation by treating with proteasome inhibitor MG132 also prevented the inhibitory effect of TGF-β1. Finally, we confirmed that HBV replication could be rescued by ectopic expression of HNF-4α in TGF-β1-treated cells. Our data clarify the mechanism by which TGF-β1 suppresses HBV replication, primarily through modulating the expression of HNF-4α gene.
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Affiliation(s)
- Ming-Hsiang Hong
- Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
- Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan
| | - Yu-Chi Chou
- Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
| | - Yi-Chieh Wu
- Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan
| | - Kuen-Nan Tsai
- Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan
| | - Cheng-po Hu
- Department of Life Science, Tunghai University, Taichung, Taiwan
| | - King-Song Jeng
- Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
| | - Mong-Liang Chen
- Center for Molecular Medicine, China Medical University and Hospital, Taichung, Taiwan
| | - Chungming Chang
- Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
- Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan
- * E-mail:
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An epistatic mini-circuitry between the transcription factors Snail and HNF4α controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs. Cell Death Differ 2011; 19:937-46. [PMID: 22139130 DOI: 10.1038/cdd.2011.175] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal transition program, whereas maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular mini-circuitry between master elements of these biological processes might provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4α, directly represses the expression of the epithelial microRNAs (miRs)-200c and -34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4α, previously identified as a transcriptional repressor of Snail, induces the miRs-34a and -200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4α and miRs-200a, b, c and -34a as epistatic elements controlling hepatic stem cell maintenance/differentiation.
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Pelletier L, Rebouissou S, Vignjevic D, Bioulac-Sage P, Zucman-Rossi J. HNF1α inhibition triggers epithelial-mesenchymal transition in human liver cancer cell lines. BMC Cancer 2011; 11:427. [PMID: 21975049 PMCID: PMC3203860 DOI: 10.1186/1471-2407-11-427] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2011] [Accepted: 10/05/2011] [Indexed: 12/31/2022] Open
Abstract
Background Hepatocyte Nuclear Factor 1α (HNF1α) is an atypical homeodomain-containing transcription factor that transactivates liver-specific genes including albumin, α-1-antitrypsin and α- and β-fibrinogen. Biallelic inactivating mutations of HNF1A have been frequently identified in hepatocellular adenomas (HCA), rare benign liver tumors usually developed in women under oral contraceptives, and in rare cases of hepatocellular carcinomas developed in non-cirrhotic liver. HNF1α-mutated HCA (H-HCA) are characterized by a marked steatosis and show activation of glycolysis, lipogenesis, translational machinery and mTOR pathway. We studied the consequences of HNF1α silencing in hepatic cell lines, HepG2 and Hep3B and we reproduced most of the deregulations identified in H-HCA. Methods We transfected hepatoma cell lines HepG2 and Hep3B with siRNA targeting HNF1α and obtained a strong inhibition of HNF1α expression. We then looked at the phenotypic changes by microscopy and studied changes in gene expression using qRT-PCR and Western Blot. Results Hepatocytes transfected with HNF1α siRNA underwent severe phenotypic changes with loss of cell-cell contacts and development of migration structures. In HNF1α-inhibited cells, hepatocyte and epithelial markers were diminished and mesenchymal markers were over-expressed. This epithelial-mesenchymal transition (EMT) was related to the up regulation of several EMT transcription factors, in particular SNAIL and SLUG. We also found an overexpression of TGFβ1, an EMT initiator, in both cells transfected with HNF1α siRNA and H-HCA. Moreover, TGFβ1 expression is strongly correlated to HNF1α expression in cell models, suggesting regulation of TGFβ1 expression by HNF1α. Conclusion Our results suggest that HNF1α is not only important for hepatocyte differentiation, but has also a role in the maintenance of epithelial phenotype in hepatocytes.
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Affiliation(s)
- Laura Pelletier
- Inserm U674 Génomique fonctionnelle des tumeurs solides, Paris, France
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Santangelo L, Marchetti A, Cicchini C, Conigliaro A, Conti B, Mancone C, Bonzo JA, Gonzalez FJ, Alonzi T, Amicone L, Tripodi M. The stable repression of mesenchymal program is required for hepatocyte identity: a novel role for hepatocyte nuclear factor 4α. Hepatology 2011; 53:2063-74. [PMID: 21384409 PMCID: PMC6624426 DOI: 10.1002/hep.24280] [Citation(s) in RCA: 97] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
UNLABELLED The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial-to-mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal-to-epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real-time quantitative polymerase chain reaction. HNF4α recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4α depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4α. HNF4α, in cooperation with its target HNF1α, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4α-mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. CONCLUSION The pivotal role of HNF4α in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4α activator and repressor functions are necessary for the identity of hepatocytes.
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Affiliation(s)
- Laura Santangelo
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Alessandra Marchetti
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Carla Cicchini
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Alice Conigliaro
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Beatrice Conti
- National Institute for Infectious Diseases L. Spallanzani, Institute of Research and Cure of Scientific Character, Rome, Italy
| | - Carmine Mancone
- National Institute for Infectious Diseases L. Spallanzani, Institute of Research and Cure of Scientific Character, Rome, Italy
| | - Jessica A. Bonzo
- Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD
| | - Frank J Gonzalez
- Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD
| | - Tonino Alonzi
- National Institute for Infectious Diseases L. Spallanzani, Institute of Research and Cure of Scientific Character, Rome, Italy
| | - Laura Amicone
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Marco Tripodi
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
- National Institute for Infectious Diseases L. Spallanzani, Institute of Research and Cure of Scientific Character, Rome, Italy
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Hepatocyte-derived Snail1 propagates liver fibrosis progression. Mol Cell Biol 2011; 31:2392-403. [PMID: 21482667 DOI: 10.1128/mcb.01218-10] [Citation(s) in RCA: 106] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Chronic exposure of the liver to hepatotoxic agents initiates an aberrant wound healing response marked by proinflammatory, as well as fibrotic, changes, leading to compromised organ structure and function. In a variety of pathological states, correlative links have been established between tissue fibrosis and the expression of transcription factors associated with the induction of epithelial-mesenchymal cell transition (EMT) programs similar to those engaged during development. However, the role played by endogenously derived, EMT-associated transcription factors in fibrotic states in vivo remains undefined. Using a mouse model of acute liver fibrosis, we demonstrate that hepatocytes upregulate the expression of the zinc finger transcriptional repressor, Snail1, during tissue remodeling. Hepatocyte-specific ablation of Snail1 demonstrates that this transcription factor plays a key role in liver fibrosis progression in vivo by triggering the proximal genetic programs that control multiple aspects of fibrogenesis, ranging from growth factor expression and extracellular matrix biosynthesis to the ensuing chronic inflammatory responses that characterize this class of pathological disorders.
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