MYC-regulated genes involved in liver cell dysplasia identified in a transgenic model of liver cancer
Danele Hunecke
Centre for Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Department of Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, 30625 Hannover, Germany
Search for more papers by this authorFlorian Länger
Department of Pathology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Search for more papers by this authorCorresponding Author
Suk Woo Nam
Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul, Korea
Professor Juergen Borlak, Hannover Medical School, Centre for Pharmacology and Toxicology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. e-mail: [email protected]
Suk Woo Nam, Hannover Medical School, Centre for Pharmacology and Toxicology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail: [email protected]
Search for more papers by this authorCorresponding Author
Juergen Borlak
Centre for Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Department of Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, 30625 Hannover, Germany
Professor Juergen Borlak, Hannover Medical School, Centre for Pharmacology and Toxicology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. e-mail: [email protected]
Suk Woo Nam, Hannover Medical School, Centre for Pharmacology and Toxicology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail: [email protected]
Search for more papers by this authorDanele Hunecke
Centre for Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Department of Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, 30625 Hannover, Germany
Search for more papers by this authorFlorian Länger
Department of Pathology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Search for more papers by this authorCorresponding Author
Suk Woo Nam
Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul, Korea
Professor Juergen Borlak, Hannover Medical School, Centre for Pharmacology and Toxicology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. e-mail: [email protected]
Suk Woo Nam, Hannover Medical School, Centre for Pharmacology and Toxicology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail: [email protected]
Search for more papers by this authorCorresponding Author
Juergen Borlak
Centre for Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Department of Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, 30625 Hannover, Germany
Professor Juergen Borlak, Hannover Medical School, Centre for Pharmacology and Toxicology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. e-mail: [email protected]
Suk Woo Nam, Hannover Medical School, Centre for Pharmacology and Toxicology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail: [email protected]
Search for more papers by this authorNo conflicts of interest were declared.
Abstract
Foci of liver cell dysplasia (LCD) are distinct morphological entities and may evolve into hepatocellular carcinomas (HCCs). While most HCCs overexpress c-Myc, its role in LCD remains uncertain. Therefore, a c-Myc transgenic model of HCC was investigated to understand the genetic events forcing liver cells into dysplasia and subsequent malignant transformation. Specifically, whole genome scans enabled fingerprinting of genes at different stages of disease, ie LCD and HCC, while laser microdissected LCD lesions were used to validate regulation of candidate genes by quantitative real-time RT-PCR, ie Mybbp1a, Rps7, Rps19, Rpl10a, Skp1a, Tfdp1, Nhp2, and Bola2. EMSA band shift assays confirmed c-Myc DNA binding at regulatory sequences of candidate gene-specific promoters. Additionally, published ChIP-seq data helped to define the candidate genes as c-Myc bona fide targets. Treatment of the human hepatoma cell line HepG2 with hepatic growth factor (Hgf) caused c-Myc protein induction and transcriptional up-regulation of candidate genes, albeit at different levels when individual genes were compared. A significant increase of HepG2 entering the G1-phase was associated with up-regulation of the candidate genes in an Hgf concentration-dependent matter. Finally, we confirmed regulation of candidate genes in patients' samples with low- and high-grade dysplasia and HCC staged T1 to T3, while their expression was unchanged in focal nodular hyperplasia and hepatic adenoma, therefore asserting the diagnostic value and clinical significance of these candidate genes. Overall, novel c-Myc targeted genes were identified and may contribute to hepatocyte transformation by altering cell cycle control, thereby contributing to c-Myc's oncogenic activity. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Supporting Information
Filename | Description |
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path4059-sup-0001-FigureS1.tif3.2 MB | Supporting Information: Figure S1. (A) Hierarchical gene cluster analysis of differentially expressed genes in livers of c-Myc transgenic mice at the age of 2, 6, and 12 months. (B) Common regulated genes in mice aged 2 and 6 months. |
path4059-sup-0002-TableS1.pdf11.6 KB | Supporting Information: Table S1. Patient characteristics and histological grading of liver lesions. The procedures followed were in accordance with the ethical standards of the committee on human experimentation at the Catholic University of Korea and with the Helsinki Declaration of 1975, as revised in 1983. Immediately after hepatectomy, freshly removed livers were serially sliced from the top edge to the bottom edge at 7- to 8-mm intervals and examined by a pathologist for the presence of nodular lesions. The haematoxylin and eosin-stained sections were examined independently by two pathologists and classified as HCC with different histological grading according to the Edmondson and Steiner method or dysplastic nodules of low or high grade according to the guidelines of the International Working Party. |
path4059-sup-0003-TableS2.xls50.5 KB | Supporting Information: Table S2. Gene expression of differentially expressed genes in either 2- or 6-month-old animals. |
path4059-sup-0004-TableS3.xls40.5 KB | Supporting Information: Table S3. Expression of cancer marker genes. |
path4059-sup-0005-TableS4.doc64 KB | Supporting Information: Table S4. Primer sequence and conditions for qRT-PCR assays. |
path4059-sup-0006-TableS5.doc31.5 KB | Supporting Information: Table S5. Oligonucleotide sequence for EMSA assays at gene-specific promoters of candidate genes. Nuclear extracts were isolated and EMSA assay were performed as follows: Liver tissue was homogenized in (10 mM Tris, pH 7.4, 2 mM MgCl2, 140 mM NaCl, 1 mM DTT, 4 mM Pefabloc, 1% v/v aprotinin, 40 mM β-glycerophosphate, 1 mM sodium orthovanadate, and 0.5% TX100) followed by homogenization and centrifugation (24,000 rpm, 2 °C, 1 hour). The pellet was transferred into a 50% sucrose hypotonic buffer vortexed and centrifuged (14,000 ×g, 4 °C, 10 minutes). Nuclei were re-suspended in 100 μl/g tissue in Dignam C buffer (20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 4 mM Pefabloc, 1% v/v aprotinin, 40 mM β-glycerophosphate, 1 mM sodium orthovanadate) and shaken at 4 °C for 45 minutes. Nuclear debris was removed and extracts were aliquoted and stored at -70 °C. 15 μg of nuclear extract and 105 cpm 32P-labeled oligonucleotides (MWG Biotech, Muenchen, Germany) were incubated in buffer that consisted of 25 mM Hepes, pH 7.6, 5 mM MgCl2, 34 mM KCl, 2 mM DTT, 2 mM Pefabloc, 2% v/v aprotinin, 40 ng of poly (dI-dC)/μl, and 100 ng of bovine serum albumin/μl. Oligonucleotides and nuclear proteins were incubated for 20 minutes on ice. Free DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel and shifted with a c-Myc antisera (kind gift of Dr. Bister, University of Innsbruck, Austria) or Max Antibody (purchased from Santa Cruz). The EMSA was visualized with the phosphor imaging system (Molecular Imager FX pro plus; Bio-Rad). |
path4059-sup-0007-micrdatas1.xls56 KB | Supporting Information: Microarray Data |
path4059-sup-0008-micrdatas2.xls64.5 KB | Supporting Information: Microarray Data |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1Bosch FX, Ribes J, Diaz M, et al. Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004; 127: S5–S16.
- 2Park YN, Roncalli M. Large liver cell dysplasia: a controversial entity. J Hepatol 2006; 45: 734–743.
- 3Lin CP, Liu CR, Lee CN, et al. Targeting c-Myc as a novel approach for hepatocellular carcinoma. World J Hepatol 2010; 2: 16–20.
- 4Dalemans W, Perraud F, Le Meur M, et al. Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse). Biologicals 1990; 18: 191–198.
- 5Desiderio MA, Pogliaghi G, Dansi P. Hepatocyte growth factor-induced expression of ornithine decarboxylase, c-met, and c-myc is differently affected by protein kinase inhibitors in human hepatoma cells HepG2. Exp Cell Res 1998; 242: 401–409.
- 6Borlak J, Meier T, Halter R, et al. Epidermal growth factor-induced hepatocellular carcinoma: gene expression profiles in precursor lesions, early stage and solitary tumours. Oncogene 2005; 24: 1809–1819.
- 7Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402–408.
- 8Niehof M, Borlak J. RSK4 and PAK5 are novel candidate genes in diabetic rat kidney and brain. Mol Pharmacol 2005; 67: 604–611.
- 9Reymann S, Borlak J. Transcription profiling of lung adenocarcinomas of c-myc-transgenic mice: identification of the c-myc regulatory gene network. BMC Syst Biol 2008; 2: 46.
- 10Coleman SL, Buckland PR, Hoogendoorn B, et al. Experimental analysis of the annotation of promoters in the public database. Hum Mol Genet 2002; 11: 1817–1821.
- 11Kel AE, Gossling E, Reuter I, et al. MATCH: a tool for searching transcription factor binding sites in DNA sequences. Nucleic Acids Res 2003; 31: 3576–3579.
- 12Quandt K, Frech K, Karas H, et al. MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 1995; 23: 4878–4884.
- 13Blais A, Dynlacht BD. E2F-associated chromatin modifiers and cell cycle control. Curr Opin Cell Biol 2007; 19: 658–662.
- 14Yasui K, Okamoto H, Arii S, et al. Association of over-expressed TFDP1 with progression of hepatocellular carcinomas. J Hum Genet 2003; 48: 609–613.
- 15Olson MF, Ashworth A, Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science 1995; 269: 1270–1272.
- 16Arai M, Kondoh N, Imazeki N, et al. The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cells. Liver Int 2009; 29: 55–62.
- 17Kang HS, Jung HM, Jun DY, et al. Expression of the human homologue of the small nucleolar RNA-binding protein NHP2 gene during monocytic differentiation of U937 cells. Biochim Biophys Acta 2002; 1575: 31–39.
- 18Robinson K, Asawachaicharn N, Galloway DA, et al. c-Myc accelerates S-phase and requires WRN to avoid replication stress. PLoS One 2009; 4: e5951.
- 19Guerzoni C, Bardini M, Mariani SA, et al. Inducible activation of CEBPB, a gene negatively regulated by BCR/ABL, inhibits proliferation and promotes differentiation of BCR/ABL-expressing cells. Blood 2006; 107: 4080–4089.
- 20Wang XW, Zhan Q, Coursen JD, et al. GADD45 induction of a G2/M cell cycle checkpoint. Proc Natl Acad Sci U S A 1999; 96: 3706–3711.
- 21Jin S, Tong T, Fan W, et al. GADD45-induced cell cycle G2–M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene 2002; 21: 8696–8704.
- 22Amundson SA, Zhan Q, Penn LZ, et al. Myc suppresses induction of the growth arrest genes gadd34, gadd45, and gadd153 by DNA-damaging agents. Oncogene 1998; 17: 2149–2154.
- 23Bush A, Mateyak M, Dugan K, et al. c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets. Genes Dev 1998; 12: 3797–3802.
- 24Marhin WW, Chen S, Facchini LM, et al. Myc represses the growth arrest gene gadd45. Oncogene 1997; 14: 2825–2834.
- 25Bangoura Gassimou, Liu Zhi-Su, Qian Qun, et al. Prognostic significance of HIF-2α/EPAS1 expression in hepatocellular carcinoma. World J Gastroenterol 2007; 13: 3176–3182.
- 26Prathapam T, Tegen S, Oskarsson T, et al. Activated Src abrogates the Myc requirement for the G0/G1 transition but not for the G1/S transition. Proc Natl Acad Sci U S A 2006; 103: 2695–2700.
- 27Kidder BL, Yang J, Palmer S. Stat3 and c-Myc genome-wide promoter occupancy in embryonic stem cells. PLoS One 2008; 3: e3932.
- 28Li Z, Van Calcar S, Qu C, et al. A global transcriptional regulatory role for c-Myc in Burkitt's lymphoma cells. Proc Natl Acad Sci U S A 2003; 100: 8164–8169.
- 29Fernandez PC, Frank SR, Wang L, et al. Genomic targets of the human c-Myc protein. Genes Dev 2003; 17: 1115–1129.
- 30Eisenman RN. Deconstructing myc. Genes Dev 2001; 15: 2023–2030.
- 31Patel JH, Loboda AP, Showe MK, et al. Analysis of genomic targets reveals complex functions of MYC. Nature Rev Cancer 2004; 4: 562–568.
- 32Dang CV, Resar LM, Emison E, et al. Function of the c-Myc oncogenic transcription factor. Exp Cell Res 1999; 253: 63–77.
- 33Dang CV, O'Donnell KA, Zeller KI, et al. The c-Myc target gene network. Semin Cancer Biol 2006; 16: 253–264.
- 34Wu CH, Sahoo D, Arvanitis C, et al. Combined analysis of murine and human microarrays and ChIP analysis reveals genes associated with the ability of MYC to maintain tumourigenesis. PLoS Genet 2008; 4: e1000090.
- 35Smith CM, Steitz JA. Classification of gas5 as a multi-small-nucleolar-RNA (snoRNA) host gene and a member of the 5′-terminal oligopyrimidine gene family reveals common features of snoRNA host genes. Mol Cell Biol 1998; 18: 6897–6909.
- 36Rozental R, Srinivas M, Gokhan S, et al. Temporal expression of neuronal connexins during hippocampal ontogeny. Brain Res Brain Res Rev 2000; 32: 57–71.
- 37Gokhan S, Song Q, Mehler MF. Generation and regulation of developing immortalized neural cell lines. Methods 1998; 16: 345–358.
- 38Lee TC, Li L, Philipson L, et al. Myc represses transcription of the growth arrest gene gas1. Proc Natl Acad Sci U S A 1997; 94: 12886–12891.
- 39Mamidipudi V, Zhang J, Lee KC, et al. RACK1 regulates G1/S progression by suppressing Src kinase activity. Mol Cell Biol 2004; 24: 6788–6798.
- 40Goto Y, Matsuzaki Y, Kurihara S, et al. A new melanoma antigen fatty acid-binding protein 7, involved in proliferation and invasion, is a potential target for immunotherapy and molecular target therapy. Cancer Res 2006; 66: 4443–4449.
- 41Kim KR, Choi HN, Lee HJ, et al. A peroxisome proliferator-activated receptor gamma antagonist induces vimentin cleavage and inhibits invasion in high-grade hepatocellular carcinoma. Oncol Rep 2007; 18: 825–832.
- 42Sebastian T, Malik R, Thomas S, et al. C/EBPbeta cooperates with RB:E 2F to implement Ras(V12)-induced cellular senescence. EMBO J 2005; 24: 3301–3312.
- 43Johansen LM, Iwama A, Lodie TA, et al. c-Myc is a critical target for c/EBPalpha in granulopoiesis. Mol Cell Biol 2001; 21: 3789–3806.
- 44Iakova P, Awad SS, Timchenko NA. Aging reduces proliferative capacities of liver by switching pathways of C/EBPalpha growth arrest. Cell 2003; 113: 495–506.
- 45Barondes SH, Cooper DN, Gitt MA, et al. Galectins. Structure and function of a large family of animal lectins. J Biol Chem 1994; 269: 20807–20810.
- 46Wang L, Bhattacharyya N, Chelsea DM, et al. A novel nuclear protein, MGC5306, interacts with DNA polymerase beta and has a potential role in cellular phenotype. Cancer Res 2004; 64: 7673–7677.
- 47O'Connell BC, Cheung AF, Simkevich CP, et al. A large scale genetic analysis of c-Myc-regulated gene expression patterns. J Biol Chem 2003; 278: 12563–12573.
- 48Yamauchi T, Keough RA, Gonda TJ, et al. Ribosomal stress induces processing of Mybbp1a and its translocation from the nucleolus to the nucleoplasm. Genes Cells 2008; 13: 27–39.
- 49Kim MJ, Kim HS, Lee JK, et al. Regulation of septation and cytokinesis during resumption of cell division requires uvi31+, a UV-inducible gene of fission yeast. Mol Cells 2002; 14: 425–430.
- 50Kim SH, Kim M, Lee JK, et al. Identification and expression of uvi31+, a UV-inducible gene from Schizosaccharomyces pombe. Environ Mol Mutagen 1997; 30: 72–81.
- 51Lee JK, Park EJ, Chung HK, et al. Isolation of UV-inducible transcripts from Schizosaccharomyces pombe. Biochem Biophys Res Commun 1994; 202: 1113–1119.
- 52Kuramitsu M, Hamaguchi I, Takuo M, et al. Deficient RPS19 protein production induces cell cycle arrest in erythroid progenitor cells. Br J Haematol 2008; 140: 348–359.
- 53Guo QM, Malek RL, Kim S, et al. Identification of c-myc responsive genes using rat cDNA microarray. Cancer Res 2000; 60: 5922–5928.
- 54Badhai J, Frojmark AS, J Davey E, et al. Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond–Blackfan anemia. Biochim Biophys Acta 2009; 1792: 1036–1042.
- 55Chen D, Zhang Z, Li M, et al. Ribosomal protein S7 as a novel modulator of p53–MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Oncogene 2007; 26: 5029–5037.
- 56Boon K, Caron HN, van Asperen R, et al. N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis. EMBO J 2001; 20: 1383–1393.
- 57Fishman-Jacob T, Reznichenko L, Youdim MB, et al. A sporadic Parkinson disease model via silencing of the ubiquitin-proteasome/E3 ligase component SKP1A. J Biol Chem 2009; 284: 32835–32845.
- 58Bertwistle D, Sugimoto M, Sherr CJ. Physical and functional interactions of the Arf tumour suppressor protein with nucleophosmin/B23. Mol Cell Biol 2004; 24: 985–996.
- 59Chan JA, Olvera M, Lai R, et al. Immunohistochemical expression of the transcription factor DP-1 and its heterodimeric partner E2F-1 in non-Hodgkin lymphoma. Appl Immunohistochem Mol Morphol 2002; 10: 322–326.
- 60Chen CR, Kang Y, Siegel PM, et al. E2F4/5 and p107 as Smad cofactors linking the TGFbeta receptor to c-myc repression. Cell 2002; 110: 19–32.
- 61Ogawa H, Ishiguro K, Gaubatz S, et al. A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells. Science 2002; 296: 1132–1136.
- 62Campanero MR, Armstrong MI, Flemington EK. CpG methylation as a mechanism for the regulation of E2F activity. Proc Natl Acad Sci U S A 2000; 97: 6481–6486.