Abstract
Abnormal expression of miRNAs has been implicated in the pathogenesis of human lung cancers, most of which are attributable to cigarette smoke. The mechanisms of action, however, remain obscure. Here, we report that there are decreased expression of miR-218 and increased expression of EZH2 and H3K27me3 during cigarette smoke extract (CSE)-induced transformation of human bronchial epithelial (HBE) cells. Depletion of EZH2 by siRNA or by the EZH2 inhibitor, 3-deazaneplanocin A, attenuated CSE-induced decreases of miR-218 levels and increases of H3K27me3, which epigenetically controls gene transcription, and BMI1, an oncogene. Furthermore, ChIP assays demonstrated that EZH2 and H3K27me3 are enriched at the miR-218-1 promoter in HBE cells exposed to CSE, indicating that EZH2 mediates epigenetic silencing of miR-218 via histone methylation. In addition, miR-218 directly targeted BMI1, through which miR-218 ablates cancer stem cells (CSCs) self-renewal in transformed HBE cells. In CSE-transformed HBE cells, the protein level of Oct-4 and mRNA levels of CD133 and CD44, indicators of the acquisition of CSC-like properties, were reduced by over-expression of miR-218, and over-expression of miR-218 decreased the malignancy of transformed HBE cells. Thus, we conclude that epigenetic silencing of miR-218 via EZH2-mediated H3K27 trimethylation is involved in the acquisition of CSC-like properties and malignant transformation of HBE cells induced by CSE and thereby contributes to the carcinogenesis of cigarette smoke.
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References
Alajez NM, Lenarduzzi M, Ito E et al (2011) MiR-218 suppresses nasopharyngeal cancer progression through downregulation of survivin and the SLIT2-ROBO1 pathway. Cancer Res 71(6):2381–2391. doi:10.1158/0008-5472.CAN-10-2754
An Y, Kiang A, Lopez JP et al (2012) Cigarette smoke promotes drug resistance and expansion of cancer stem cell-like side population. PLoS One 7(11):e47919. doi:10.1371/journal.pone.0047919
Arnold P, Scholer A, Pachkov M et al (2013) Modeling of epigenome dynamics identifies transcription factors that mediate Polycomb targeting. Genome Res 23(1):60–73. doi:10.1101/gr.142661.112
Au SL, Wong CC, Lee JM et al (2012) Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis. Hepatology 56(2):622–631. doi:10.1002/hep.25679
Barski A, Jothi R, Cuddapah S et al (2009) Chromatin poises miRNA- and protein-coding genes for expression. Genome Res 19(10):1742–1751. doi:10.1101/gr.090951.109
Breitling LP, Yang R, Korn B, Burwinkel B, Brenner H (2011) Tobacco-smoking-related differential DNA methylation: 27 K discovery and replication. Am J Hum Genet 88(4):450–457. doi:10.1016/j.ajhg.2011.03.003
Cao R, Zhang Y (2004) The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3. Curr Opin Genet Dev 14(2):155–164. doi:10.1016/j.gde.2004.02.001
Cao R, Wang L, Wang H et al (2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298(5595):1039–1043. doi:10.1126/science.1076997
Cao Q, Mani RS, Ateeq B et al (2011) Coordinated regulation of polycomb group complexes through microRNAs in cancer. Cancer Cell 20(2):187–199. doi:10.1016/j.ccr.2011.06.016
Davidson MR, Larsen JE, Yang IA et al (2010) MicroRNA-218 is deleted and downregulated in lung squamous cell carcinoma. PLoS One 5(9):e12560. doi:10.1371/journal.pone.0012560
Di Cello F, Flowers VL, Li H et al (2013) Cigarette smoke induces epithelial to mesenchymal transition and increases the metastatic ability of breast cancer cells. Mol Cancer 12:90. doi:10.1186/1476-4598-12-90
Dovey JS, Zacharek SJ, Kim CF, Lees JA (2008) Bmi1 is critical for lung tumorigenesis and bronchoalveolar stem cell expansion. Proc Natl Acad Sci USA 105(33):11857–11862. doi:10.1073/pnas.0803574105
Enfield KS, Pikor LA, Martinez VD, Lam WL (2012) Mechanistic roles of noncoding RNAs in lung cancer biology and their clinical implications. Genet Res Int 2012:737416. doi:10.1155/2012/737416
Eramo A, Lotti F, Sette G et al (2008) Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 15(3):504–514. doi:10.1038/sj.cdd.4402283
Feinberg AP, Tycko B (2004) The history of cancer epigenetics. Nat Rev Cancer 4(2):143–153. doi:10.1038/nrc1279
Glynn T, Seffrin JR, Brawley OW, Grey N, Ross H (2010) The globalization of tobacco use: 21 challenges for the 21st century. CA Cancer J Clin 60(1):50–61. doi:10.3322/caac.20052
Gupta RA, Shah N, Wang KC et al (2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464(7291):1071–1076. doi:10.1038/nature08975
He X, Dong Y, Wu CW et al (2012) MicroRNA-218 inhibits cell cycle progression and promotes apoptosis in colon cancer by downregulating BMI1 polycomb ring finger oncogene. Mol Med 18:1491–1498. doi:10.2119/molmed.2012.00304
Hochedlinger K, Yamada Y, Beard C, Jaenisch R (2005) Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121(3):465–477. doi:10.1016/j.cell.2005.02.018
Holm K, Grabau D, Lovgren K et al (2012) Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes. Mol Oncol 6(5):494–506. doi:10.1016/j.molonc.2012.06.002
Hsu TC, Cherry LM, Bucana C, Shirley LR, Gairola CG (1991) Mitosis-arresting effects of cigarette smoke condensate on human lymphoid cell lines. Mutat Res 259(1):67–78
Iorio MV, Croce CM (2012) MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 4(3):143–159. doi:10.1002/emmm.201100209
Izzotti A, Calin GA, Arrigo P, Steele VE, Croce CM, De Flora S (2009a) Downregulation of microRNA expression in the lungs of rats exposed to cigarette smoke. FASEB J 23(3):806–812. doi:10.1096/fj.08-121384
Izzotti A, Calin GA, Steele VE, Croce CM, De Flora S (2009b) Relationships of microRNA expression in mouse lung with age and exposure to cigarette smoke and light. FASEB J 23(9):3243–3250. doi:10.1096/fj.09-135251
Izzotti A, Balansky R, D’Agostini F et al (2013) Relationships between pulmonary micro-RNA and proteome profiles, systemic cytogenetic damage and lung tumors in cigarette smoke-exposed mice treated with chemopreventive agents. Carcinogenesis 34(10):2322–2329. doi:10.1093/carcin/bgt178
Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60(5):277–300. doi:10.3322/caac.20073
Kang MK, Kim RH, Kim SJ et al (2007) Elevated Bmi-1 expression is associated with dysplastic cell transformation during oral carcinogenesis and is required for cancer cell replication and survival. Br J Cancer 96(1):126–133. doi:10.1038/sj.bjc.6603529
Kawaguchi A, Ochi H, Sudou N, Ogino H (2012) Comparative expression analysis of the H3K27 demethylases, JMJD3 and UTX, with the H3K27 methylase, EZH2, in Xenopus. Int J Dev Biol 56(4):295–300. doi:10.1387/ijdb.113360ak
Kikuchi J, Kinoshita I, Shimizu Y et al (2010) Distinctive expression of the polycomb group proteins Bmi1 polycomb ring finger oncogene and enhancer of zeste homolog 2 in nonsmall cell lung cancers and their clinical and clinicopathologic significance. Cancer 116(12):3015–3024. doi:10.1002/cncr.25128
Ku M, Koche RP, Rheinbay E et al (2008) Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet 4(10):e1000242. doi:10.1371/journal.pgen.1000242
Leung EL, Fiscus RR, Tung JW et al (2010) Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS One 5(11):e14062. doi:10.1371/journal.pone.0014062
Li CH, To KF, Tong JH et al (2013) Enhancer of zeste homolog 2 silences microRNA-218 in human pancreatic ductal adenocarcinoma cells by inducing formation of heterochromatin. Gastroenterology 144(5):1086–1097. doi:10.1053/j.gastro.2013.01.058
Malumbres M (2013) miRNAs and cancer: an epigenetics view. Mol Aspects Med 34(4):863–874. doi:10.1016/j.mam.2012.06.005
Mani SA, Guo W, Liao MJ et al (2008) The epithelial–mesenchymal transition generates cells with properties of stem cells. Cell 133(4):704–715. doi:10.1016/j.cell.2008.03.027
Markou A, Liang Y, Lianidou E (2011) Prognostic, therapeutic and diagnostic potential of microRNAs in non-small cell lung cancer. Clin Chem Lab Med CCLM/FESCC 49(10):1591–1603. doi:10.1515/CCLM.2011.661
Miranda TB, Cortez CC, Yoo CB et al (2009) DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation. Mol Cancer Ther 8(6):1579–1588. doi:10.1158/1535-7163.MCT-09-0013
Mitchell PS, Parkin RK, Kroh EM et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105(30):10513–10518. doi:10.1073/pnas.0804549105
Murata K, Yoshitomi H, Tanida S et al (2010) Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis. Arthritis Res Therapy 12(3):R86. doi:10.1186/ar3013
Narayan S, Jaiswal AS, Kang D, Srivastava P, Das GM, Gairola CG (2004) Cigarette smoke condensate-induced transformation of normal human breast epithelial cells in vitro. Oncogene 23(35):5880–5889. doi:10.1038/sj.onc.1207792
Pillsbury HC, Bright CC (1972) Comparison of aliquot and complete sample procedure for the determination of nicotine in cigarette smoke. J Assoc Off Anal Chem 55(3):636–638
Pulling LC, Vuillemenot BR, Hutt JA, Devereux TR, Belinsky SA (2004) Aberrant promoter hypermethylation of the death-associated protein kinase gene is early and frequent in murine lung tumors induced by cigarette smoke and tobacco carcinogens. Cancer Res 64(11):3844–3848. doi:10.1158/0008-5472.CAN-03-2119
Reddel RR, Ke Y, Gerwin BI et al (1988) Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res 48(7):1904–1909
Russ R, Slack FJ (2012) Cigarette-smoke-induced dysregulation of MicroRNA expression and its role in lung carcinogenesis. Pulm Med 2012:791234. doi:10.1155/2012/791234
Schembri F, Sridhar S, Perdomo C et al (2009) MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium. Proc Natl Acad Sci USA 106(7):2319–2324. doi:10.1073/pnas.0806383106
Siddique HR, Saleem M (2012) Role of BMI1, a stem cell factor, in cancer recurrence and chemoresistance: preclinical and clinical evidences. Stem Cells 30(3):372–378. doi:10.1002/stem.1035
Sing A, Pannell D, Karaiskakis A et al (2009) A vertebrate Polycomb response element governs segmentation of the posterior hindbrain. Cell 138(5):885–897. doi:10.1016/j.cell.2009.08.020
Tan J, Yang X, Zhuang L et al (2007) Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells. Genes Dev 21(9):1050–1063. doi:10.1101/gad.1524107
Thun MJ, DeLancey JO, Center MM, Jemal A, Ward EM (2010) The global burden of cancer: priorities for prevention. Carcinogenesis 31(1):100–110. doi:10.1093/carcin/bgp263
Tie J, Pan Y, Zhao L et al (2010) MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor. PLoS Genet 6(3):e1000879. doi:10.1371/journal.pgen.1000879
Tokar EJ, Diwan BA, Waalkes MP (2010) Arsenic exposure transforms human epithelial stem/progenitor cells into a cancer stem-like phenotype. Environ Health Perspect 118(1):108–115. doi:10.1289/ehp.0901059
Tsai MC, Manor O, Wan Y et al (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329(5992):689–693. doi:10.1126/science.1192002
Tu Y, Gao X, Li G et al (2013) MicroRNA-218 inhibits glioma invasion, migration, proliferation, and cancer stem-like cell self-renewal by targeting the polycomb group gene Bmi1. Cancer Res 73(19):6046–6055. doi:10.1158/0008-5472.CAN-13-0358
Uesugi A, Kozaki K, Tsuruta T et al (2011) The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer. Cancer Res 71(17):5765–5778. doi:10.1158/0008-5472.CAN-11-0368
Venkataraman S, Birks DK, Balakrishnan I et al (2013) MicroRNA 218 acts as a tumor suppressor by targeting multiple cancer phenotype-associated genes in medulloblastoma. J Biol Chem 288(3):1918–1928. doi:10.1074/jbc.M112.396762
Vire E, Brenner C, Deplus R et al (2006) The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439(7078):871–874. doi:10.1038/nature04431
Woo CJ, Kharchenko PV, Daheron L, Park PJ, Kingston RE (2010) A region of the human HOXD cluster that confers polycomb-group responsiveness. Cell 140(1):99–110. doi:10.1016/j.cell.2009.12.022
Yoo KH, Hennighausen L (2012) EZH2 methyltransferase and H3K27 methylation in breast cancer. Int J Biol Sci 8(1):59–65
Youlden DR, Cramb SM, Baade PD (2008) The international epidemiology of lung cancer: geographical distribution and secular trends. J Thorac Oncol 3(8):819–831. doi:10.1097/JTO.0b013e31818020eb
Zhao J, Sun BK, Erwin JA, Song JJ, Lee JT (2008) Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322(5902):750–756. doi:10.1126/science.1163045
Zhao Y, Xu Y, Li Y et al (2013) NF-kappaB-mediated inflammation leading to EMT via miR-200c is involved in cell transformation induced by cigarette smoke extract. Toxicol Sci 135(2):265–276. doi:10.1093/toxsci/kft150
Acknowledgments
The authors wish to thank Donald L. Hill (University of Alabama at Birmingham, USA) for editing. This work was supported by the Natural Science Foundations of China (81473011), the Key Program of Educational Commission of Jiangsu Province of China (11KJA330002), the open project of Key Laboratory of Modern Toxicology (NJMU), Ministry of Education (NJMUMT201403), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (2010).
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Bairu Wang, Yi Liu, and Fei Luo have contributed equally to this work.
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Wang, B., Liu, Y., Luo, F. et al. Epigenetic silencing of microRNA-218 via EZH2-mediated H3K27 trimethylation is involved in malignant transformation of HBE cells induced by cigarette smoke extract. Arch Toxicol 90, 449–461 (2016). https://doi.org/10.1007/s00204-014-1435-z
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DOI: https://doi.org/10.1007/s00204-014-1435-z