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Feng J, Liu Y, Fang T, Zhu J, Wang G, Li J. Hematological and neurological expressed 1 (HN1) activates c-Myc signaling by inhibiting ubiquitin-mediated proteasomal degradation of c-Myc in hepatocellular carcinoma. Cell Biol Int 2023; 47:560-572. [PMID: 36403281 DOI: 10.1002/cbin.11957] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Revised: 10/12/2022] [Accepted: 10/30/2022] [Indexed: 11/21/2022]
Abstract
Hepatocellular carcinoma (HCC) has a poor prognosis due to the usually advanced stage at diagnosis. Sustained activation of the MYC oncogene is implicated in the development of HCC; however, the molecular mechanisms of MYC deregulation in HCC are poorly understood. Here, real-time PCR and western blotting were used to measure the expression of hematological and neurological expressed 1 (HN1) in HCC cells. Expression of HN1 and MYC in clinical specimens was analyzed using immunohistochemistry. The role of HN1 in HCC proliferation, migration, and invasion was explored in vitro and in vivo. MYC expression was measured using real-time PCR and western blotting. MYC transcriptional activity was assessed using a luciferase reporter system. Expression of MYC target genes was quantified using real-time PCR. Protein interaction between MYC and HN1 was assessed using co-immunoprecipitation and western blotting. We identified HN1 as a novel regulatory factor of the glycogen synthase kinase (GSK) 3β-MYC axis. HN1 expression is elevated in liver tumor tissues and cells, and significantly correlates with poor survival in HCC patients. Upregulation of HN1 promotes, and silencing of HN1 represses, the proliferation and metastasis of liver cancer cells in vitro and in vivo. Moreover, our results demonstrate that HN1 sustains stabilization and persistent activity of MYC via interaction with GSK3β in HCC. Importantly, the tumor-promoting effects of HN1 on HCC cells were attenuated by suppressing MYC. In conclusion, constitutive activation of MYC by HN1 promotes the progression of HCC; therefore, HN1 might be a novel therapeutic target for HCC.
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Affiliation(s)
- Jutao Feng
- Hepatobiliary Surgery Department, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Yanmin Liu
- Hepatobiliary Surgery Department, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Tianling Fang
- Hepatobiliary Surgery Department, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Jinrong Zhu
- Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China
| | - Guoying Wang
- Hepatobiliary Surgery Department, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Jun Li
- Hepatobiliary Surgery Department, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
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Capellmann S, Sonntag R, Schüler H, Meurer SK, Gan L, Kauffmann M, Horn K, Königs-Werner H, Weiskirchen R, Liedtke C, Huber M. Transformation of primary murine peritoneal mast cells by constitutive KIT activation is accompanied by loss of Cdkn2a/Arf expression. Front Immunol 2023; 14:1154416. [PMID: 37063827 PMCID: PMC10097954 DOI: 10.3389/fimmu.2023.1154416] [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: 01/30/2023] [Accepted: 03/20/2023] [Indexed: 04/18/2023] Open
Abstract
Mast cells (MCs) are immune cells of the myeloid lineage distributed in tissues throughout the body. Phenotypically, they are a heterogeneous group characterized by different protease repertoires stored in secretory granules and differential presence of receptors. To adequately address aspects of MC biology either primary MCs isolated from human or mouse tissue or different human MC lines, like HMC-1.1 and -1.2, or rodent MC lines like L138.8A or RBL-2H3 are frequently used. Nevertheless, cellular systems to study MC functions are very limited. We have generated a murine connective tissue-like MC line, termed PMC-306, derived from primary peritoneal MCs (PMCs), which spontaneously transformed. We analyzed PMC-306 cells regarding MC surface receptor expression, effector functions and respective signaling pathways, and found that the cells reacted very similar to primary wildtype (WT) PMCs. In this regard, stimulation with MAS-related G-protein-coupled receptor member B2 (MRGPRB2) ligands induced respective signaling and effector functions. Furthermore, PMC-306 cells revealed significantly accelerated cell cycle progression, which however was still dependent on interleukine 3 (IL-3) and stem cell factor (SCF). Phenotypically, PMC-306 cells adopted an immature connective tissue-like MCs appearance. The observation of cellular transformation was accompanied by the loss of Cdkn2a and Arf expression, which are both described as critical cell cycle regulators. The loss of Cdkn2a and Arf expression could be mimicked in primary bone marrow-derived mast cells (BMMCs) by sustained SCF supplementation strongly arguing for an involvement of KIT activation in the regulation of Cdkn2a/Arf expression. Hence, this new cell line might be a useful tool to study further aspects of PMC function and to address tumorigenic processes associated with MC leukemia.
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Affiliation(s)
- Sandro Capellmann
- Institute of Biochemistry and Molecular Immunology, Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
- *Correspondence: Sandro Capellmann, ; Michael Huber,
| | - Roland Sonntag
- Department of Internal Medicine III, University Hospital, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Herdit Schüler
- Institute of Human Genetics, Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Steffen K. Meurer
- Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Lin Gan
- Genomics Facility, Interdisciplinary Center for Clinical Research (IZKF), Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Marlies Kauffmann
- Institute of Biochemistry and Molecular Immunology, Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Katharina Horn
- Institute of Biochemistry and Molecular Immunology, Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Hiltrud Königs-Werner
- Electron Microscopy Facility, Institute of Pathology, Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Ralf Weiskirchen
- Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Christian Liedtke
- Department of Internal Medicine III, University Hospital, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
| | - Michael Huber
- Institute of Biochemistry and Molecular Immunology, Medical Faculty, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
- *Correspondence: Sandro Capellmann, ; Michael Huber,
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Karanth S, Zinkhan EK, Hill JT, Yost HJ, Schlegel A. FOXN3 Regulates Hepatic Glucose Utilization. Cell Rep 2016; 15:2745-55. [PMID: 27292639 DOI: 10.1016/j.celrep.2016.05.056] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Revised: 04/27/2016] [Accepted: 05/13/2016] [Indexed: 12/17/2022] Open
Abstract
A SNP (rs8004664) in the first intron of the FOXN3 gene is associated with human fasting blood glucose. We find that carriers of the risk allele have higher hepatic expression of the transcriptional repressor FOXN3. Rat Foxn3 protein and zebrafish foxn3 transcripts are downregulated during fasting, a process recapitulated in human HepG2 hepatoma cells. Transgenic overexpression of zebrafish foxn3 or human FOXN3 increases zebrafish hepatic gluconeogenic gene expression, whole-larval free glucose, and adult fasting blood glucose and also decreases expression of glycolytic genes. Hepatic FOXN3 overexpression suppresses expression of mycb, whose ortholog MYC is known to directly stimulate expression of glucose-utilization enzymes. Carriers of the rs8004664 risk allele have decreased MYC transcript abundance. Human FOXN3 binds DNA sequences in the human MYC and zebrafish mycb loci. We conclude that the rs8004664 risk allele drives excessive expression of FOXN3 during fasting and that FOXN3 regulates fasting blood glucose.
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Affiliation(s)
- Santhosh Karanth
- University of Utah Molecular Medicine Program, University of Utah School of Medicine, Salt Lake City, UT 84112, USA; Division of Endocrinology, Metabolism and Diabetes, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
| | - Erin K Zinkhan
- Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84108, USA
| | - Jonathon T Hill
- University of Utah Molecular Medicine Program, University of Utah School of Medicine, Salt Lake City, UT 84112, USA; Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA
| | - H Joseph Yost
- University of Utah Molecular Medicine Program, University of Utah School of Medicine, Salt Lake City, UT 84112, USA; Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84108, USA; Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA
| | - Amnon Schlegel
- University of Utah Molecular Medicine Program, University of Utah School of Medicine, Salt Lake City, UT 84112, USA; Division of Endocrinology, Metabolism and Diabetes, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT 84112, USA; Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.
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Kurabe N, Murakami S, Tashiro F. SGF29 and Sry pathway in hepatocarcinogenesis. World J Biol Chem 2015; 6:139-147. [PMID: 26322172 PMCID: PMC4549758 DOI: 10.4331/wjbc.v6.i3.139] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/27/2015] [Revised: 05/31/2015] [Accepted: 07/02/2015] [Indexed: 02/05/2023] Open
Abstract
Deregulated c-Myc expression is a hallmark of many human cancers. We have recently identified a role of mammalian homolog of yeast SPT-ADA-GCN5-acetyltransferas (SAGA) complex component, SAGA-associated factor 29 (SGF29), in regulating the c-Myc overexpression. Here, we discuss the molecular nature of SFG29 in SPT3-TAF9-GCN5-acetyltransferase complex, a counterpart of yeast SAGA complex, and the mechanism through which the elevated SGF29 expression contribute to oncogenic potential of c-Myc in hepatocellularcarcinoma (HCC). We propose that the upstream regulation of SGF29 elicited by sex-determining region Y (Sry) is also augmented in HCC. We hypothesize that c-Myc elevation driven by the deregulated Sry and SGF29 pathway is implicated in the male specific acquisition of human HCCs.
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Robertson CL, Srivastava J, Rajasekaran D, Gredler R, Akiel MA, Jariwala N, Siddiq A, Emdad L, Fisher PB, Sarkar D. The role of AEG-1 in the development of liver cancer. Hepat Oncol 2015; 2:303-312. [PMID: 26798451 DOI: 10.2217/hep.15.10] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
AEG-1 is an oncogene that is overexpressed in all cancers, including hepatocellular carcinoma. AEG-1 plays a seminal role in promoting cancer development and progression by augmenting proliferation, invasion, metastasis, angiogenesis and chemoresistance, all hallmarks of aggressive cancer. AEG-1 mediates its oncogenic function predominantly by interacting with various protein complexes. AEG-1 acts as a scaffold protein, activating multiple protumorigenic signal transduction pathways, such as MEK/ERK, PI3K/Akt, NF-κB and Wnt/β-catenin while regulating gene expression at transcriptional, post-transcriptional and translational levels. Our recent studies document that AEG-1 is fundamentally required for activation of inflammation. A comprehensive and convincing body of data currently points to AEG-1 as an essential component critical to the onset and progression of cancer. The present review describes the current knowledge gleaned from patient and experimental studies as well as transgenic and knockout mouse models, on the impact of AEG-1 on hepatocarcinogenesis.
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Affiliation(s)
- Chadia L Robertson
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA
| | - Jyoti Srivastava
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA
| | - Devaraja Rajasekaran
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA
| | - Rachel Gredler
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA
| | - Maaged A Akiel
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA
| | - Nidhi Jariwala
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA
| | - Ayesha Siddiq
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA
| | - Luni Emdad
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA; VCU Massey Cancer Center, Virginia Commonwealth University, 401 College Street, Richmond, VA 23298, USA
| | - Paul B Fisher
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA; VCU Massey Cancer Center, Virginia Commonwealth University, 401 College Street, Richmond, VA 23298, USA; VCU Institute of Molecular Medicine, Virginia Commonwealth University, Molecular Medicine Research Building 1220 East Broad Street, 7th Floor PO Box 980033, Richmond, VA 23298-0033, USA
| | - Devanand Sarkar
- Department of Human & Molecular Genetics, Virginia Commonwealth University, Sanger Hall, Room 11-0051101 East Marshall Street, PO Box 980033, Richmond, VA 23298-0033, USA; VCU Massey Cancer Center, Virginia Commonwealth University, 401 College Street, Richmond, VA 23298, USA; VCU Institute of Molecular Medicine, Virginia Commonwealth University, Molecular Medicine Research Building 1220 East Broad Street, 7th Floor PO Box 980033, Richmond, VA 23298-0033, USA
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Srivastava J, Siddiq A, Gredler R, Shen XN, Rajasekaran D, Robertson CL, Subler MA, Windle JJ, Dumur CI, Mukhopadhyay ND, Garcia D, Lai Z, Chen Y, Balaji U, Fisher PB, Sarkar D. Astrocyte elevated gene-1 and c-Myc cooperate to promote hepatocarcinogenesis in mice. Hepatology 2015; 61:915-29. [PMID: 25065684 PMCID: PMC4309751 DOI: 10.1002/hep.27339] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/10/2014] [Accepted: 07/20/2014] [Indexed: 12/15/2022]
Abstract
UNLABELLED Astrocyte elevated gene-1 (AEG-1) and c-Myc are overexpressed in human hepatocellular carcinoma (HCC) functioning as oncogenes. AEG-1 is transcriptionally regulated by c-Myc, and AEG-1 itself induces c-Myc by activating the Wnt/β-catenin-signaling pathway. We now document the cooperation of AEG-1 and c-Myc in promoting hepatocarcinogenesis by analyzing hepatocyte-specific transgenic mice expressing either AEG-1 (albumin [Alb]/AEG-1), c-Myc (Alb/c-Myc), or both (Alb/AEG-1/c-Myc). Wild-type and Alb/AEG-1 mice did not develop spontaneous HCC. Alb/c-Myc mice developed spontaneous HCC without distant metastasis, whereas Alb/AEG-1/c-Myc mice developed highly aggressive HCC with frank metastasis to the lungs. Induction of carcinogenesis by N-nitrosodiethylamine significantly accelerated the kinetics of tumor formation in all groups. However, in Alb/AEG-1/c-Myc, the effect was markedly pronounced with lung metastasis. In vitro analysis showed that Alb/AEG-1/c-Myc hepatocytes acquired increased proliferation and transformative potential with sustained activation of prosurvival and epithelial-mesenchymal transition-signaling pathways. RNA-sequencing analysis identified a unique gene signature in livers of Alb/AEG-1/c-Myc mice that was not observed when either AEG-1 or c-Myc was overexpressed. Specifically, Alb/AEG-1/c-Myc mice overexpressed maternally imprinted noncoding RNAs (ncRNAs), such as Rian, Meg-3, and Mirg, which are implicated in hepatocarcinogenesis. Knocking down these ncRNAs significantly inhibited proliferation and invasion by Alb/AEG-1/c-Myc hepatocytes. CONCLUSION Our studies reveal a novel cooperative oncogenic effect of AEG-1 and c-Myc that might explain the mechanism of aggressive HCC. Alb/AEG-1/c-Myc mice provide a useful model to understand the molecular mechanism of cooperation between these two oncogenes and other molecules involved in hepatocarcinogenesis. This model might also be of use for evaluating novel therapeutic strategies targeting HCC.
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Affiliation(s)
- Jyoti Srivastava
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Ayesha Siddiq
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Rachel Gredler
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Xue-Ning Shen
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Devaraja Rajasekaran
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Chadia L. Robertson
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Mark A. Subler
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Jolene J. Windle
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Catherine I. Dumur
- Department of Pathology, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Nitai D. Mukhopadhyay
- Department of Biostatistics, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Dawn Garcia
- Greehey Children’s Cancer Research Institute, University of Texas Health Science Center San Antonio, San Antonio, TX 78229
| | - Zhao Lai
- Greehey Children’s Cancer Research Institute, University of Texas Health Science Center San Antonio, San Antonio, TX 78229
| | - Yidong Chen
- Computational Biology and Bioinformatics, University of Texas Health Science Center San Antonio, San Antonio, TX 78229
| | - Uthra Balaji
- Department of Pathology, Simmons Cancer Center, UT Southwestern Medical Center, Dallas, TX
| | - Paul B. Fisher
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
,VCU Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
,VCU Institute of Molecular Medicine (VIMM), Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Devanand Sarkar
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
,VCU Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
,VCU Institute of Molecular Medicine (VIMM), Virginia Commonwealth University, Richmond, VA 23298, USA
,Corresponding author: 1220 East Broad St, PO Box 980035 Richmond, VA 23298 Tel: 804-827-2339 Fax: 804-628-1176
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Akita H, Marquardt JU, Durkin ME, Kitade M, Seo D, Conner EA, Andersen JB, Factor VM, Thorgeirsson SS. MYC activates stem-like cell potential in hepatocarcinoma by a p53-dependent mechanism. Cancer Res 2014; 74:5903-13. [PMID: 25189530 DOI: 10.1158/0008-5472.can-14-0527] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Activation of c-MYC is an oncogenic hallmark of many cancers, including liver cancer, and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c-MYC activation for the biology of hepatic cancer stem cells (CSC) are undefined. Here, distinct levels of c-MYC overexpression were established by using two dose-dependent tetracycline-inducible systems in four hepatoma cell lines with different p53 mutational status. The CSCs were evaluated using side population (SP) approach as well as standard in vitro and in vivo assays. Functional repression of p53 was achieved by lentiviral shRNA transduction. The results show that c-MYC expression levels have a differential impact on liver CSC characteristics. At low levels, c-MYC activation led to increased proliferation and enhanced CSC properties including activation of reprogramming transcription factors and CSC marker expression (e.g., NANOG, OCT4, and EpCAM), expansion of SP, and acceleration of tumor growth upon subcutaneous transplantation into immunocompromised mice. However, when exceeding a threshold level, c-MYC induced a proapoptotic program and loss of CSC potential both in vitro and in vivo. Mechanistically, c-MYC-induced self-renewal capacity of liver cancer cells was exerted in a p53-dependent manner. Low c-MYC activation increased spheroid formation in p53-deficient tumor cells, whereas p53-dependent effects were blunted in the absence of c-MYC overexpression. Together, our results confirm the role of c-MYC as a master regulator during hepatocarcinogenesis and establish a new gatekeeper role for p53 in repressing c-MYC-induced CSC phenotype in liver cancer cells.
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Affiliation(s)
- Hirofumi Akita
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Jens U Marquardt
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland. Department of Medicine I, Johannes Gutenberg University, Mainz, Germany
| | - Marian E Durkin
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Mitsuteru Kitade
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Daekwan Seo
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Elizabeth A Conner
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Jesper B Andersen
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland. Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark
| | - Valentina M Factor
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Snorri S Thorgeirsson
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland.
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Kadariya Y, Tang B, Wang L, Al-Saleem T, Hayakawa K, Slifker MJ, Kruger WD. Germline Mutations in Mtap Cooperate with Myc to Accelerate Tumorigenesis in Mice. PLoS One 2013; 8:e67635. [PMID: 23840755 PMCID: PMC3694069 DOI: 10.1371/journal.pone.0067635] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2013] [Accepted: 05/20/2013] [Indexed: 11/19/2022] Open
Abstract
Objective The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (MtaplacZ) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten+/−. Methods Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of MtaplacZ/+ and Mtap+/+ mice. Results Survival in both models was significantly decreased in MtaplacZ/+ compared to Mtap+/+ mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap+/+ mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc MtaplacZ/+ and Eµ-myc Mtap+/+ animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap+/+ and MtaplacZ/+ animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer. Conclusion Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.
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Affiliation(s)
- Yuwaraj Kadariya
- Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, Unites States of America
| | - Baiqing Tang
- Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, Unites States of America
| | - Liqun Wang
- Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, Unites States of America
| | - Tahseen Al-Saleem
- Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, Unites States of America
| | - Kyoko Hayakawa
- Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, Unites States of America
| | - Michael J. Slifker
- Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, Unites States of America
| | - Warren D. Kruger
- Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, Unites States of America
- * E-mail: .
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Lee MJ, Xu DY, Li H, Yu GR, Leem SH, Chu IS, Kim IH, Kim DG. Pro-oncogenic potential of NM23-H2 in hepatocellular carcinoma. Exp Mol Med 2012; 44:214-24. [PMID: 22192927 PMCID: PMC3317485 DOI: 10.3858/emm.2012.44.3.016] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). There is abundant mRNA expression of NM23-H1, NM23-H2, or a read through transcript (NM23-LV) in the primary sites of hepatocellular carcinoma (HCC). Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study was to examine whether NM23-H2 is associated with hepatocarcinogenesis. The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Its subcellular localization was confined to mainly the cytoplasm and to a lesser extent in the nucleus. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes showed a transformed morphology, enhanced focus formation, and allowed anchorage-independent growth. Finally, NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice and showed c-Myc over-expression. In addition, NF-κB and cyclin D1 expression were also increased by NM23-H2. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Collectively, these results indicate that NM23-H2 may be pro-oncogenic in hepatocarcinogenesis.
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Affiliation(s)
- Mi-Jin Lee
- Division of GI and Hepatology, The Research Institute of Clinical Medicine, Department of Internal Medicine, Chonbuk National University Medical School and Hospital, Jeonju 561-712, Korea
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Huynh H, Ong R, Soo KC. Foretinib demonstrates anti-tumor activity and improves overall survival in preclinical models of hepatocellular carcinoma. Angiogenesis 2011; 15:59-70. [PMID: 22187171 DOI: 10.1007/s10456-011-9243-z] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2011] [Accepted: 12/08/2011] [Indexed: 02/08/2023]
Abstract
PURPOSE OF STUDY Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. The purpose of this study is to assess the anti-tumor and anti-angiogenic activities of foretinib, a vascular endothelial growth factor receptor 2 (VEGFR-2) and c-Met inhibitor using mouse models of human HCC. EXPERIMENTAL TECHNIQUES SK-HEP1 and 21-0208 HCC cells as well as patient-derived HCC models were employed to study the anti-tumor and antiangiogenic activities of foretinib. Changes of biomarkers relevant to hepatocyte growth factor (HGF) signaling pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. RESULTS Treatment of SK-HEP1 cells with foretinib resulted in growth inhibition, G2/M cell cycle arrest, reduced colony formation and blockade of HGF-induced cell migration. In both orthotopic and ectopic models of HCC, foretinib potently inhibited tumor growth in a dose-dependent manner. Inhibition of angiogenesis correlated with inactivation of VEGFR-2/c-Met signaling pathways. Foretinib also caused elevation of p27 and Bim but reduced cyclin B1 expression and p-c-Myc, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, foretinib potently inhibited primary tumor growth and significantly prolonged mouse survival. DATA INTERPRETATIONS Foretinib demonstrated significant antitumor activities in patient-derived HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC.
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Affiliation(s)
- Hung Huynh
- Laboratory of Molecular Endocrinology, Division of Molecular and Cellular Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore.
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11
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Wang Z, Lin S, Li JJ, Xu Z, Yao H, Zhu X, Xie D, Shen Z, Sze J, Li K, Lu G, Chan DTM, Poon WS, Kung HF, Lin MCM. MYC protein inhibits transcription of the microRNA cluster MC-let-7a-1~let-7d via noncanonical E-box. J Biol Chem 2011; 286:39703-14. [PMID: 21903590 DOI: 10.1074/jbc.m111.293126] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.
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Affiliation(s)
- Zifeng Wang
- Laboratory of Integrated Biosciences, School of Life Sciences, Sun Yat-sen University, Guangzhou, China
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12
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Klein A, Li N, Nicholson JM, McCormack AA, Graessmann A, Duesberg P. Transgenic oncogenes induce oncogene-independent cancers with individual karyotypes and phenotypes. ACTA ACUST UNITED AC 2010; 200:79-99. [PMID: 20620590 DOI: 10.1016/j.cancergencyto.2010.04.008] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2010] [Revised: 03/24/2010] [Accepted: 04/01/2010] [Indexed: 11/25/2022]
Abstract
Cancers are clones of autonomous cells defined by individual karyotypes, much like species. Despite such karyotypic evidence for causality, three to six synergistic mutations, termed oncogenes, are generally thought to cause cancer. To test single oncogenes, they are artificially activated with heterologous promoters and spliced into the germ line of mice to initiate cancers with collaborating spontaneous oncogenes. Because such cancers are studied as models for the treatment of natural cancers with related oncogenes, the following must be answered: 1) which oncogenes collaborate with the transgenes in cancers; 2) how do single transgenic oncogenes induce diverse cancers and hyperplasias; 3) what maintains cancers that lose initiating transgenes; 4) why are cancers aneuploid, over- and underexpressing thousands of normal genes? Here we try to answer these questions with the theory that carcinogenesis is a form of speciation. We postulate that transgenic oncogenes initiate carcinogenesis by inducing aneuploidy. Aneuploidy destabilizes the karyotype by unbalancing teams of mitosis genes. This instability thus catalyzes the evolution of new cancer species with individual karyotypes. Depending on their degree of aneuploidy, these cancers then evolve new subspecies. To test this theory, we have analyzed the karyotypes and phenotypes of mammary carcinomas of mice with transgenic SV40 tumor virus- and hepatitis B virus-derived oncogenes. We found that (1) a given transgene induced diverse carcinomas with individual karyotypes and phenotypes; (2) these karyotypes coevolved with newly acquired phenotypes such as drug resistance; (3) 8 of 12 carcinomas were transgene negative. Having found one-to-one correlations between individual karyotypes and phenotypes and consistent coevolutions of karyotypes and phenotypes, we conclude that carcinogenesis is a form of speciation and that individual karyotypes maintain cancers as they maintain species. Because activated oncogenes destabilize karyotypes and are dispensable in cancers, we conclude that they function indirectly, like carcinogens. Such oncogenes would thus not be valid models for the treatment of cancers.
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Affiliation(s)
- Andreas Klein
- Charité-Universitätsmedizin Berlin, Campus Charité Mitte, Institut für Biochemie, Monbijoustrasse 2, Berlin, Germany
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13
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Wei W, Chua MS, Grepper S, So S. Small molecule antagonists of Tcf4/beta-catenin complex inhibit the growth of HCC cells in vitro and in vivo. Int J Cancer 2010; 126:2426-36. [PMID: 19662654 DOI: 10.1002/ijc.24810] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide. It is intrinsically resistant toward standard chemotherapy, making it imperative to develop novel selective chemotherapeutic agents. The Wnt/beta-catenin pathway plays critical roles in development and oncogenesis, and is dysregulated in HCC. Our study aims to evaluate the activity of 3 small molecule antagonists of the Tcf4/beta-catenin complex (PKF118-310, PKF115-584 and CGP049090) on HCC cell lines in vitro and in vivo. All 3 chemicals displayed dose-dependent cytotoxicity in vitro against all 3 HCC cell lines (HepG2, Hep40 and Huh7), but were at least 10 times less cytotoxic to normal hepatocytes (from 3 donors) by using ATP assay. In HepG2 and Huh7 cells, treatment with the antagonists decreased Tcf4/beta-catenin binding capability and transcriptional activity, associated with downregulation of the endogenous Tcf4/ beta-catenin target genes c-Myc, cyclin D1 and survivin. In HepG2 and Huh7 cells, treatment with the antagonists induced apoptosis and cell cycle arrest at the G1/S phase. All antagonists suppressed in vivo tumor growth in a HepG2 xenograft model, associated with apoptosis and reduced c-Myc, cyclin D1 and survivin expressions. Our results suggest that these 3 antagonists of the Tcf4/beta-catenin complex are potential chemotherapeutic agents which may offer a pathway specific option for the clinical management of HCC.
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Affiliation(s)
- Wei Wei
- Asian Liver Center, Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305-5655, USA
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14
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Zimonjic DB, Ullmannova-Benson V, Factor VM, Thorgeirsson SS, Popescu NC. Recurrent and nonrandom DNA copy number and chromosome alterations in Myc transgenic mouse model for hepatocellular carcinogenesis: implications for human disease. ACTA ACUST UNITED AC 2009; 191:17-26. [PMID: 19389504 DOI: 10.1016/j.cancergencyto.2008.12.014] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2008] [Accepted: 12/30/2008] [Indexed: 12/11/2022]
Abstract
Mouse models for hepatocellular carcinoma (HCC) provide an experimental ground for dissecting the genetic and biological complexities of human liver cancer and contribute to our ability to gain insights into the relevance of candidate cancer genes. We examined, using spectral karyotyping (SKY) and array-based CGH (aCGH), seven cell lines derived from HCC spontaneously developed in transgenic Myc mice (Myc), and four cell lines established from tumors induced in nude mice by inoculation with the original Myc cells (nuMyc). All the cell lines exhibited gain of material from chromosomes 5, 6, 8, 10, 11, 15, and 19 and DNA copy-number loss from chromosomes 2, 4, 7, 9, 12, 14, and X. In addition, several recurrent chromosome reorganizations were found, including del(3), t(3;8), del(4), t(4;11), t(6;5), del(7), del(8), del(9), t(10;14), del(11), and del(16). Chromosome breakpoints underlying rearrangements clustered in the regions previously identified as important for the early stages of Myc-induced hepatocarcinogenesis. The results strongly suggest the importance of recurrent breakage and loss of chromosomes 4, 9, and 14 and gain of chromosomes 15 and 19 in mouse liver neoplasia. Genomic changes observed in Myc HCC cell lines are also recurrent in HCC developed in other transgenic mouse models, in mouse spontaneous HCC and derivative cell lines, and in preneoplastic liver lesions induced with chemical carcinogens. Overall, the present results document selective, nonrandom genomic changes involving chromosomal regions homologous to those implicated in human HCC.
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Affiliation(s)
- Drazen B Zimonjic
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, 37 Convent Drive MSC 4262, Building 37, Room 4128B, Bethesda, MD 20892, USA
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15
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Hwang-Verslues WW, Sladek FM. Nuclear receptor hepatocyte nuclear factor 4alpha1 competes with oncoprotein c-Myc for control of the p21/WAF1 promoter. Mol Endocrinol 2007; 22:78-90. [PMID: 17885207 PMCID: PMC2194635 DOI: 10.1210/me.2007-0298] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The dichotomy between cellular differentiation and proliferation is a fundamental aspect of both normal development and tumor progression; however, the molecular basis of this opposition is not well understood. To address this issue, we investigated the mechanism by which the nuclear receptor hepatocyte nuclear factor 4alpha1 (HNF4alpha1) regulates the expression of the human cyclin-dependent kinase inhibitor gene p21/WAF1 (CDKN1A). We found that HNF4alpha1, a transcription factor that plays a central role in differentiation in the liver, pancreas, and intestine, activates the expression of p21 primarily by interacting with promoter-bound Sp1 at both the proximal promoter region and at newly identified sites in a distal region (-2.4 kb). Although HNF4alpha1 also binds two additional regions containing putative HNF4alpha binding sites, HNF4alpha1 mutants deficient in DNA binding activate the p21 promoter to the same extent as wild-type HNF4alpha1, indicating that direct DNA binding by HNF4alpha1 is not necessary for p21 activation. We also observed an in vitro and in vivo interaction between HNF4alpha1 and c-Myc as well as a competition between these two transcription factors for interaction with promoter-bound Sp1 and regulation of p21. Finally, we show that c-Myc competes with HNF4alpha1 for control of apolipoprotein C3 (APOC3), a gene associated with the differentiated hepatic phenotype. These results suggest a general model by which a differentiation factor (HNF4alpha1) and a proliferation factor (c-Myc) may compete for control of genes involved in cell proliferation and differentiation.
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Affiliation(s)
- Wendy W Hwang-Verslues
- Environmental Toxicology Graduate Program, University of California, Riverside, California 92521, USA
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16
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Rekha RD, Amali AA, Her GM, Yeh YH, Gong HY, Hu SY, Lin GH, Wu JL. Thioacetamide accelerates steatohepatitis, cirrhosis and HCC by expressing HCV core protein in transgenic zebrafish Danio rerio. Toxicology 2007; 243:11-22. [PMID: 17997003 DOI: 10.1016/j.tox.2007.09.007] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2007] [Revised: 09/03/2007] [Accepted: 09/03/2007] [Indexed: 12/19/2022]
Abstract
Hepatocellular carcinoma (HCC) is one of the common cancers worldwide, caused by Hepatitis C virus (HCV) and hepatotoxins. Here we report the development of HCC in wild type as well as HCV core protein (HCP)-transgenic zebrafish upon treatment with a hepatotoxin, thioacetamide (TAA). Two-fold accelerated HCC development could be achieved in the TAA-treated transgenic fish, that is, the progression of the disease in TAA-treated wild type zebrafish developed HCC in 12 weeks whereas that of HCP-transgenic zebrafish shortened the HCC progression to 6 weeks. Histopathological observation showed the specific pathological features of HCC. The HCC progression was confirmed through RT-PCR that revealed an up and down regulation of different marker genes at various stages of HCC progression such as, steatohepatitis, fibrosis and HCC. Moreover, HCV core protein expressed in the HCP-transgenic zebrafish and TAA synergistically accelerate the HCC development. It must be mentioned that, this is the first report revealing HCV core protein along with TAA to induce HCC in zebrafish, particularly, in a short period of time comparing to mice model. As zebrafish has already been considered as a good human disease model and in this context, this HCC-zebrafish model may serve as a powerful preclinical platform to study the molecular events in hepatocarcinogenesis, therapeutic strategies and for evaluating chemoprevention strategies in HCC.
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Affiliation(s)
- Ravikumar Deepa Rekha
- Laboratory of Marine Molecular Biology and Biotechnology, Institute of Cellular and Organismic Biology, Academia Sinica, NanKang, Taipei 11529, Taiwan
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17
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Wootton SK, Metzger MJ, Hudkins KL, Alpers CE, York D, DeMartini JC, Miller AD. Lung cancer induced in mice by the envelope protein of jaagsiekte sheep retrovirus (JSRV) closely resembles lung cancer in sheep infected with JSRV. Retrovirology 2006; 3:94. [PMID: 17177996 PMCID: PMC1764900 DOI: 10.1186/1742-4690-3-94] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2006] [Accepted: 12/19/2006] [Indexed: 11/23/2022] Open
Abstract
Background Jaagsiekte sheep retrovirus (JSRV) causes a lethal lung cancer in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse lung, by using a replication-defective adeno-associated virus type 6 (AAV6) vector, induces tumors resembling those seen in sheep. However, the mouse and sheep tumors have not been carefully compared to determine if Env expression alone in mice can account for the disease features observed in sheep, or whether additional aspects of virus replication in sheep are important, such as oncogene activation following retrovirus integration into the host cell genome. Results We have generated mouse monoclonal antibodies (Mab) against JSRV Env and have used these to study mouse and sheep lung tumor histology. These Mab detect Env expression in tumors in sheep infected with JSRV from around the world with high sensitivity and specificity. Mouse and sheep tumors consisted mainly of well-differentiated adenomatous foci with little histological evidence of anaplasia, but at long times after vector exposure some mouse tumors did have a more malignant appearance typical of adenocarcinoma. In addition to epithelial cell tumors, lungs of three of 29 sheep examined contained fibroblastic cell masses that expressed Env and appeared to be separate neoplasms. The Mab also stained nasal adenocarcinoma tissue from one United States sheep, which we show was due to expression of Env from ovine enzootic nasal tumor virus (ENTV), a virus closely related to JSRV. Systemic administration of the AAV6 vector encoding JSRV Env to mice produced numerous hepatocellular tumors, and some hemangiomas and hemangiosarcomas, showing that the Env protein can induce tumors in multiple cell types. Conclusion Lung cancers induced by JSRV infection in sheep and by JSRV Env expression in mice have similar histologic features and are primarily characterized by adenomatous proliferation of peripheral lung epithelial cells. Thus it is unnecessary to invoke a role for insertional mutagenesis, gene activation, viral replication, or expression of other viral gene products in sheep lung tumorigenesis, although these processes may play a role in other clinically less important sequelae of JSRV infection such as metastasis observed with variable frequency in sheep.
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Affiliation(s)
- Sarah K Wootton
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
| | - Michael J Metzger
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
| | - Kelly L Hudkins
- Department of Pathology, University of Washington, Seattle, Washington 98195, USA
| | - Charles E Alpers
- Department of Pathology, University of Washington, Seattle, Washington 98195, USA
| | - Denis York
- Molecular Diagnostic Services, Westville 3630, South Africa
| | - James C DeMartini
- Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523, USA
| | - A Dusty Miller
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
- Department of Pathology, University of Washington, Seattle, Washington 98195, USA
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18
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Wang S, Huang Q, Lian Y, Lan F. Experimental study on siRNA expressing vector-based RNA interference targeting c-Myc in human hepatocellular carcinoma cell line BEL-7402. ACTA ACUST UNITED AC 2006. [DOI: 10.1007/s10330-006-0519-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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19
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Jang JW, Boxer RB, Chodosh LA. Isoform-specific ras activation and oncogene dependence during MYC- and Wnt-induced mammary tumorigenesis. Mol Cell Biol 2006; 26:8109-21. [PMID: 16908535 PMCID: PMC1636749 DOI: 10.1128/mcb.00404-06] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
We have previously shown that c-MYC-induced mammary tumorigenesis in mice proceeds via a preferred secondary pathway involving spontaneous activating mutations in Kras2 (C. M. D'Cruz, E. J. Gunther, R. B. Boxer, J. L. Hartman, L. Sintasath, S. E. Moody, J. D. Cox, S. I. Ha, G. K. Belka, A. Golant, R. D. Cardiff, and L. A. Chodosh, Nat. Med. 7:235-239, 2001). In contrast, we now demonstrate that Wnt1-induced mammary tumorigenesis proceeds via a pathway that preferentially activates Hras1. In addition, we find that expression of oncogenic forms of Kras2 and Hras1 from their endogenous promoters has markedly different consequences for the progression of tumors to oncogene independence. Spontaneous activating Kras2 mutations occurring in either MYC- or Wnt1-induced tumors were strongly associated with oncogene-independent tumor growth following MYC or Wnt1 downregulation. In contrast, Hras1-mutant Wnt1-induced tumors consistently remained oncogene dependent. Additionally, Kras2-mutant tumors exhibited substantially higher levels of ras-GTP, phospho-Erk1/2, and phospho-Mek1/2 compared to Hras1-mutant tumors, suggesting the involvement of the ras/mitogen-activated protein kinase (MAPK) pathway in the acquisition of oncogene independence. Consistent with this, by use of carcinogen-induced ras mutations as well as knock-in mice harboring a latent activated Kras2 allele, we demonstrate that Kras2 activation strongly synergizes with both c-MYC and Wnt1 in mammary tumorigenesis and promotes the progression of tumors to oncogene independence. Together, our findings support a model for tumorigenesis in which c-MYC and Wnt1 select for the outgrowth of cells harboring mutations in specific ras isoforms and that these secondary mutations, in turn, determine the extent of ras/MAPK pathway activation and the potential for oncogene-independent growth.
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Affiliation(s)
- Joanne W Jang
- Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA
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20
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Pelengaris S, Khan M. The c-MYC oncoprotein as a treatment target in cancer and other disorders of cell growth. Expert Opin Ther Targets 2005; 7:623-42. [PMID: 14498825 DOI: 10.1517/14728222.7.5.623] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The c-MYC proto-oncogene is essential for cellular proliferation but, paradoxically, may also promote cell death. Deregulated expression of c-MYC is present in most, if not all, human cancers, and is associated with a poor prognosis. However, given that human tumours at diagnosis generally carry multiple genetic lesions that have accumulated during (although they are not necessarily essential for) tumour progression, it has proved difficult to attribute a specific role to any given single factor or indeed to explore the therapeutic potential of selectively mitigating their biological functions. Regulatable transgenic mouse models of oncogenesis have shed light on these issues, influenced our thinking about cancer and provided encouragement for the future development of cancer therapies based on targeting individual oncogenes such as c-MYC. Although still in its infancy, encouraging results have been reported using antisense oligodeoxynucleotide-based methods, as well as other approaches to interfere with MYC expression both in vitro and in vivo.
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Affiliation(s)
- Stella Pelengaris
- Molecular Medicine, Biomedical Research Institute, University of Warwick, Coventry, CV4 7AL, UK.
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21
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Chen CF, Yeh SH, Chen DS, Chen PJ, Jou YS. Molecular genetic evidence supporting a novel human hepatocellular carcinoma tumor suppressor locus at 13q12.11. Genes Chromosomes Cancer 2005; 44:320-8. [PMID: 16075462 DOI: 10.1002/gcc.20247] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
A novel 1-cM (1.8 Mb) homozygous deletion (HD) on 13q12.11 was identified in a human hepatocellular carcinoma (HCC) cell line, SK-Hep-1, after high-density genetic marker scan and Southern blotting analysis. A loss of heterozygosity (LOH) analysis indicated that LOH frequency of the HD region in 48 pairs of HCC tissues was 52%. Interestingly, the occurrence of LOH in the 13q12.11 HD region is significantly associated with early-onset HCC, inferred from Fisher's exact test (P = 0.0047) and Mann-Whitney test (P = 0.023). Since the novel 1-cM (1.8 Mb) HD region is gene-rich with more than 37 predicted transcripts, we used a candidate gene approach by examining down-regulation of known tumor suppressor genes (TSGs), including LATS2, TG737, CRYL1, and GJB2, in HCC tissues. We detected only 14% down-regulation of the LAST2 gene that flanks the outside of the HD, in HCC tissues, by quantitative RT-PCR assays. However, we observed significant down-regulation of the TG737, CRYL1, and GJB2 genes located within the HD in 59, 64, and 71% of HCC tissues, respectively. Together, our results indicated that the identified 13q12.11 HD region contained at least three significant down-regulated TSGs, and preferential LOH in early-onset HCC patients is a putative tumor suppressor locus in HCC.
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Affiliation(s)
- Chian-Feng Chen
- Graduate Institute of Life Sciences, National Defense Medical Center, National Defense University Taipei, Taiwan
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22
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Kramer MG, Hernandez-Alcoceba R, Qian C, Prieto J. Evaluation of hepatocellular carcinoma models for preclinical studies. ACTA ACUST UNITED AC 2005. [DOI: 10.1016/j.ddmod.2005.05.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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Shachaf CM, Kopelman AM, Arvanitis C, Karlsson A, Beer S, Mandl S, Bachmann MH, Borowsky AD, Ruebner B, Cardiff RD, Yang Q, Bishop JM, Contag CH, Felsher DW. MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature 2004; 431:1112-7. [PMID: 15475948 DOI: 10.1038/nature03043] [Citation(s) in RCA: 677] [Impact Index Per Article: 32.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2004] [Accepted: 09/21/2004] [Indexed: 01/18/2023]
Abstract
Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker alpha-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.
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MESH Headings
- Animals
- Apoptosis
- Bile Ducts/cytology
- Bile Ducts/metabolism
- Biomarkers, Tumor/analysis
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Carcinoma, Hepatocellular/therapy
- Cell Differentiation
- Cell Transformation, Neoplastic
- Gene Expression Regulation, Neoplastic
- Genes, myc/genetics
- Luminescent Measurements
- Mice
- Mice, SCID
- Mice, Transgenic
- Proto-Oncogene Proteins c-myc/genetics
- Proto-Oncogene Proteins c-myc/metabolism
- Stem Cells/cytology
- Stem Cells/metabolism
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Affiliation(s)
- Catherine M Shachaf
- Division of Medical Oncology, Department of Medicine, Stanford University, California 94305, USA
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Calvisi DF, Factor VM, Ladu S, Conner EA, Thorgeirsson SS. Disruption of beta-catenin pathway or genomic instability define two distinct categories of liver cancer in transgenic mice. Gastroenterology 2004; 126:1374-86. [PMID: 15131798 DOI: 10.1053/j.gastro.2004.02.014] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
BACKGROUND & AIMS Human liver cancer can be divided into 2 categories that are characterized by activation of beta-catenin and genomic instability. Here we investigate whether similar categories exist among 5 transgenic models of liver cancer, including c-myc, transforming growth factor-alpha, E2F-1, c-myc/transforming growth factor-alpha, and c-myc/E2F-1 mice. METHODS The random amplified polymorphic DNA method was used to assess the overall genomic instability, and chromosomal loci affected by genomic alterations were determined by microsatellite analysis. beta-Catenin mutations and deletions were analyzed by polymerase chain reaction and sequencing screening. Cellular localization of beta-catenin and expression of alpha-fetoprotein, a prognostic marker of hepatocellular carcinoma, were investigated by immunohistochemistry. RESULTS Liver tumors from the transgenic mice could be divided into 2 broad categories characterized by extensive genomic instability (exemplified by the c-myc/transforming growth factor-alpha mouse) and activation of beta-catenin (exemplified by the c-myc/E2F-1 mouse). The c-myc/transforming growth factor-alpha tumors displayed extensive genomic instability with recurrent loss of heterozygosity at chromosomes 1, 2, 4, 6, 7, 9, 12, 14, and X and a low rate of beta-catenin activation. The genomic instability was evident from the early dysplastic stage and occurred concomitantly with increased expression of alpha-fetoprotein. The c-myc/E2F-1 tumors were characterized by a high frequency of beta-catenin activation in the presence of a relatively stable genome and low alpha-fetoprotein levels. CONCLUSIONS We have identified 2 prototype experimental models, i.e., c-myc/transforming growth factor-alpha and c-myc/E2F-1 mice, for the 2 categories of human hepatocellular carcinoma characterized by genomic instability and beta-catenin activation, respectively. These mouse models will assist in the elucidation of the molecular basis of human hepatocellular carcinoma.
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Affiliation(s)
- Diego F Calvisi
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute/NIH, Building 37, 37 Convent Drive, Bethesda, MD 20892, USA
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Schreiner B, Greten FR, Baur DM, Fingerle AA, Zechner U, Böhm C, Schmid M, Hameister H, Schmid RM. Murine pancreatic tumor cell line TD2 bears the characteristic pattern of genetic changes with two independently amplified gene loci. Oncogene 2003; 22:6802-9. [PMID: 14555993 DOI: 10.1038/sj.onc.1206836] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
TGFalpha/p53(+/-) transgenic mice represent a genetically engineered mouse model for pancreatic adenocarcinoma. The tumors develop a characteristic pattern of secondary genetic changes. From one of these tumors, the permanent cell line TD2 was established. Here, we describe in detail the genetic changes by molecular-cytogenetic techniques. The original tumor-specific CGH profile has been retained unchanged. The most characteristic aberration pattern bears chromosome 11. Egfr, localized on proximal chromosome 11, is amplified two to three times and leads to an easily identifiable, stable marker chromosome with a large amplification unit, which is present in each metaphase. The wild-type p53 gene on distal chromosome 11 is lost. The p16Ink4a locus on chromosome 4 is hypermethylated. For c-Myc a 15-fold amplification, present in a 1.65 Mb amplification unit, is detected on chromosome 15. Transition between presence in the form of several double minutes, DMs, or a single homogeneously staining region, HSR, was observed for c-Myc. Molecular-cytogenetic analysis of both amplification units show that Egfr amplification and c-Myc amplification represent two alternative modes by which genes get amplified in tumor cells. The expression level of the respective genes was proven by Northern blot analysis. The cell line TD2 represents a valuable in vitro model for pancreatic adenocarcinoma.
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Affiliation(s)
- Bettina Schreiner
- Department of Human Genetics, University of Ulm, D-89069 Ulm, Germany
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Abstract
Deregulated expression of c-MYC occurs in a broad range of human cancers and is often associated with poor prognosis, indicating a key role for this oncogene in tumour progression. However, as established human tumours often bear multiple genetic lesions, it is difficult to determine whether c-MYC is instrumental in the initiation/progression of the tumour, or indeed whether inactivating c-MYC would lead to tumour regression. Regulatable transgenic mouse models of oncogenesis have shed light on these issues and provide hope for effective cancer therapies.
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Affiliation(s)
- Stella Pelengaris
- Molecular Medicine Research Centre, University of Warwick, Coventry CV4 7AL, UK.
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