Concomitant downregulation of the imprinted genes DLK1 and MEG3 at 14q32.2 by epigenetic mechanisms in urothelial carcinoma

Clinical Epigenetics

Wolfgang A Schulz¹  Günter Niegisch¹  Teodora Ribarska¹  Judith Knievel¹  Annemarie Greife¹ 

[1] Department of Urology, Medical Faculty, Heinrich-Heine University, Moorenstr. 5, Düsseldorf 40225, Germany

关键词: Histone modification;    DNA methylation;    Imprinted genes;    Urothelial cancer;    MEG3;    DLK1;   

Others  :  1092775
DOI  :  10.1186/1868-7083-6-29

 received in 2014-06-18, accepted in 2014-11-07,  发布年份 2014

【 摘 要 】

Background

The two oppositely imprinted and expressed genes, DLK1 and MEG3, are located in the same gene cluster at 14q32. Previous studies in bladder cancer have suggested that tumor suppressor genes are located in this region, but these have not been identified.

Results

We observed that both DLK1 and MEG3 are frequently silenced in urothelial cancer tissues and cell lines. The concomitant downregulation of the two genes is difficult to explain by known mechanisms for inactivating imprinted genes, namely deletion of active alleles or epitype switching. Indeed, quantitative PCR revealed more frequent copy number gains than losses in the gene cluster that were, moreover, consistent within each sample, excluding gene losses as the cause of downregulation. Instead, we observed distinctive epigenetic alterations at the three regions controlling DLK1 and MEG3 expression, namely the DLK1 promoter; the intergenic (IG) and MEG3 differentially methylated regions (DMRs). Bisulfite sequencing and pyrosequencing revealed novel patterns of DNA methylation in tumor cells, which were distinct from that of either paternal allele. Furthermore, chromatin immunoprecipitation demonstrated loss of active and gain of repressive histone modifications at all regulatory sequences.

Conclusions

Our data support the idea that the main cause of the prevalent downregulation of DLK1 and MEG3 in urothelial carcinoma is epigenetic silencing across the 14q32 imprinted gene cluster, resulting in the unusual concomitant inactivation of oppositely expressed and imprinted genes.

【 授权许可】

   
2014 Greife et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Sun M, Xia R, Jin F, Xu T, Liu Z, De W, Liu X: Downregulated long noncoding RNA MEG3 is associated with poor prognosis and promotes cell proliferation in gastric cancer. Tumour Biol 2014, 35:1065-1073.
• [2]Hoffmann MJ, Florl AR, Seifert HH, Schulz WA: Multiple mechanisms downregulate CDKN1C in human bladder cancer. Int J Cancer 2005, 114:406-413.
• [3]Kavanagh E, Joseph B: The hallmarks of CDKN1C (p57, KIP2) in cancer. Biochim Biophys Acta 1816, 2011:50-56.
• [4]Bena F, Gimelli S, Migliavacca E, Brun-Druc N, Buiting K, Antonarakis SE, Sharp AJ: A recurrent 14q32.2 microdeletion mediated by expanded TGG repeats. Hum Mol Genet 2010, 19:1967-1973.
• [5]Kawakami T, Chano T, Minami K, Okabe H, Okada Y, Okamoto K: Imprinted DLK1 is a putative tumor suppressor gene and inactivated by epimutation at the region upstream of GTL2 in human renal cell carcinoma. Hum Mol Genet 2006, 15:821-830.
• [6]Sanlaville D, Aubry MC, Dumez Y, Nolen MC, Amiel J, Pinson MP, Lyonnet S, Munnich A, Vekemans M, Morichon-Delvallez N: Maternal uniparental heterodisomy of chromosome 14: chromosomal mechanism and clinical follow up. J Med Genet 2000, 37:525-528.
• [7]Temple IK, Shrubb V, Lever M, Bullman H, Mackay DJ: Isolated imprinting mutation of the DLK1/GTL2 locus associated with a clinical presentation of maternal uniparental disomy of chromosome 14. J Med Genet 2007, 44:637-640.
• [8]da Rocha ST, Edwards CA, Ito M, Ogata T, Ferguson-Smith AC: Genomic imprinting at the mammalian Dlk1-Dio3 domain. Trends Genet 2008, 24:306-316.
• [9]Hagan JP, O’Neill BL, Stewart CL, Kozlov SV, Croce CM: At least ten genes define the imprinted Dlk1-Dio3 cluster on mouse chromosome 12qF1. PLoS One 2009, 4:e4352.
• [10]Royo H, Cavaille J: Non-coding RNAs in imprinted gene clusters. Biol Cell 2008, 100:149-166.
• [11]Takada S, Paulsen M, Tevendale M, Tsai CE, Kelsey G, Cattanach BM, Ferguson-Smith AC: Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19. Hum Mol Genet 2002, 11:77-86.
• [12]Schmidt JV, Matteson PG, Jones BK, Guan XJ, Tilghman SM: The Dlk1 and Gtl2 genes are linked and reciprocally imprinted. Genes Dev 2000, 14:1997-2002.
• [13]Kagami M, O’Sullivan MJ, Green AJ, Watabe Y, Arisaka O, Masawa N, Matsuoka K, Fukami M, Matsubara K, Kato F, Ferguson-Smith AC, Ogata T: The IG-DMR and the MEG3-DMR at human chromosome 14q32.2: hierarchical interaction and distinct functional properties as imprinting control centers. PLoS Genet 2010, 6:e1000992.
• [14]Khoury H, Suarez-Saiz F, Wu S, Minden MD: An upstream insulator regulates DLK1 imprinting in AML. Blood 2010, 115:2260-2263.
• [15]Lopez-Terrada D, Gunaratne PH, Adesina AM, Pulliam J, Hoang DM, Nguyen Y, Mistretta TA, Margolin J, Finegold MJ: Histologic subtypes of hepatoblastoma are characterized by differential canonical Wnt and Notch pathway activation in DLK + precursors. Hum Pathol 2009, 40:783-794.
• [16]Anwar SL, Krech T, Hasemeier B, Schipper E, Schweitzer N, Vogel A, Kreipe H, Lehmann U: Loss of imprinting and allelic switching at the DLK1-MEG3 locus in human hepatocellular carcinoma. PLoS One 2012, 7:e49462.
• [17]Gordon FE, Nutt CL, Cheunsuchon P, Nakayama Y, Provencher KA, Rice KA, Zhou Y, Zhang X, Klibanski A: Increased expression of angiogenic genes in the brains of mouse meg3-null embryos. Endocrinology 2010, 151:2443-2452.
• [18]Lu KH, Li W, Liu XH, Sun M, Zhang ML, Wu WQ, Xie WP, Hou YY: Long non-coding RNA MEG3 inhibits NSCLC cells proliferation and induces apoptosis by affecting p53 expression. BMC Cancer 2013, 13:461. BioMed Central Full Text
• [19]Qin R, Chen Z, Ding Y, Hao J, Hu J, Guo F: Long non-coding RNA MEG3 inhibits the proliferation of cervical carcinoma cells through the induction of cell cycle arrest and apoptosis. Neoplasma 2013, 60:486-492.
• [20]Bray SJ, Takada S, Harrison E, Shen SC, Ferguson-Smith AC: The atypical mammalian ligand Delta-like homologue 1 (Dlk1) can regulate Notch signalling in Drosophila. BMC Dev Biol 2008, 8:11. BioMed Central Full Text
• [21]D’Souza B, Miyamoto A, Weinmaster G: The many facets of Notch ligands. Oncogene 2008, 27:5148-5167.
• [22]Zhang X, Rice K, Wang Y, Chen W, Zhong Y, Klibanski A: Maternally expressed gene 3 (MEG3) noncoding ribonucleic acid: isoform structure, expression, and functions. Endocrinology 2010, 151:939-947.
• [23]Florl AR, Schulz WA: Chromosomal instability in bladder cancer. Arch Toxicol 2008, 82:173-182.
• [24]Goebell PJ, Knowles MA: Bladder cancer or bladder cancers? Genetically distinct malignant conditions of the urothelium. Urol Oncol 2010, 28:409-428.
• [25]Blaveri E, Brewer JL, Roydasgupta R, Fridlyand J, DeVries S, Koppie T, Pejavar S, Mehta K, Carroll P, Simko JP, Waldman FM: Bladder cancer stage and outcome by array-based comparative genomic hybridization. Clin Cancer Res 2005, 11:7012-7022.
• [26]Hurst CD, Fiegler H, Carr P, Williams S, Carter NP, Knowles MA: High-resolution analysis of genomic copy number alterations in bladder cancer by microarray-based comparative genomic hybridization. Oncogene 2004, 23:2250-2263.
• [27]Knowles MA: Identification of novel bladder tumour suppressor genes. Electrophoresis 1999, 20:269-279.
• [28]Strefford JC, Lillington DM, Steggall M, Lane TM, Nouri AM, Young BD, Oliver RT: Novel chromosome findings in bladder cancer cell lines detected with multiplex fluorescence in situ hybridization. Cancer Genet Cytogenet 2002, 135:139-146.
• [29]Ying L, Huang Y, Chen H, Wang Y, Xia L, Chen Y, Liu Y, Qiu F: Downregulated MEG3 activates autophagy and increases cell proliferation in bladder cancer. Mol Biosyst 2013, 9:407-411.
• [30]Huang J, Zhang X, Zhang M, Zhu JD, Zhang YL, Liu GZ, Yu J, Cui Y, Yang PY, Wang ZQ, Han ZG: Up-regulation of DLK1 as an imprinted gene could contribute to human hepatocellular carcinoma. Carcinogenesis 2007, 28:1094-1103.
• [31]Zhang J, Finney RP, Rowe W, Edmonson M, Yang SH, Dracheva T, Jen J, Struewing JP, Buetow KH: Systematic analysis of genetic alterations in tumors using Cancer Genome WorkBench (CGWB). Genome Res 2007, 17:1111-1117.
• [32]Chang WY, Cairns P, Schoenberg MP, Polascik TJ, Sidransky D: Novel suppressor loci on chromosome 14q in primary bladder cancer. Cancer Res 1995, 55:3246-3249.
• [33]Xi L, Fondufe-Mittendorf Y, Xia L, Flatow J, Widom J, Wang JP: Predicting nucleosome positioning using a duration Hidden Markov Model. BMC Bioinformatics 2010, 11:346. BioMed Central Full Text
• [34]Turanyi E, Dezso K, Paku S, Nagy P: DLK is a novel immunohistochemical marker for adrenal gland tumors. Virchows Arch 2009, 455:295-299.
• [35]Zhang X, Zhou Y, Klibanski A: Isolation and characterization of novel pituitary tumor related genes: A cDNA representational difference approach. Mol Cell Endocrinol 2010, 326:40-47.
• [36]Kapoor-Vazirani P, Kagey JD, Powell DR, Vertino PM: Role of hMOF-dependent histone H4 lysine 16 acetylation in the maintenance of TMS1/ASC gene activity. Cancer Res 2008, 68:6810-6821.
• [37]Shahbazian MD, Grunstein M: Functions of site-specific histone acetylation and deacetylation. Annu Rev Biochem 2007, 76:75-100.
• [38]Fraga MF, Esteller M: Towards the human cancer epigenome: a first draft of histone modifications. Cell Cycle 2005, 4:1377-1381.
• [39]Kelly TK, Liu Y, Lay FD, Liang G, Berman BP, Jones PA: Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res 2012, 22:2497-2506.
• [40]Portella G, Battistini F, Orozco M: Understanding the connection between epigenetic DNA methylation and nucleosome positioning from computer simulations. PLoS Comput Biol 2013, 9:e1003354.
• [41]Perez A, Castellazzi CL, Battistini F, Collinet K, Flores O, Deniz O, Ruiz ML, Torrents D, Eritja R, Soler-López M, Orozco M: Impact of methylation on the physical properties of DNA. Biophys J 2012, 102:2140-2148.
• [42]Bock M, Hinley J, Schmitt C, Wahlicht T, Kramer S, Southgate J: Identification of ELF3 as an early transcriptional regulator of human urothelium. Dev Biol 2014, 386:321-330.
• [43]Chapman EJ, Kelly G, Knowles MA: Genes involved in differentiation, stem cell renewal, and tumorigenesis are modulated in telomerase-immortalized human urothelial cells. Mol Cancer Res 2008, 6:1154-1168.
• [44]Gejman R, Batista DL, Zhong Y, Zhou Y, Zhang X, Swearingen B, Stratakis CA, Hedley-Whyte ET, Klibanski A: Selective loss of MEG3 expression and intergenic differentially methylated region hypermethylation in the MEG3/DLK1 locus in human clinically nonfunctioning pituitary adenomas. J Clin Endocrinol Metab 2008, 93:4119-4125.
• [45]Pantoja C, de Los RL, Matheu A, Antequera F, Serrano M: Inactivation of imprinted genes induced by cellular stress and tumorigenesis. Cancer Res 2005, 65:26-33.
• [46]Zhou Y, Zhang X, Klibanski A: MEG3 noncoding RNA: a tumor suppressor. J Mol Endocrinol 2012, 48:R45-R53.
• [47]Dokun OY, Florl AR, Seifert HH, Wolff I, Schulz WA: Relationship of SNCG, S100A4, S100A9 and LCN2 gene expression and DNA methylation in bladder cancer. Int J Cancer 2008, 123:2798-2807.
• [48]Swiatkowski S, Seifert HH, Steinhoff C, Prior A, Thievessen I, Schliess F, Schulz WA: Activities of MAP-kinase pathways in normal uroepithelial cells and urothelial carcinoma cell lines. Exp Cell Res 2003, 282:48-57.
• [49]Tanaka M, Koul D, Davies MA, Liebert M, Steck PA, Grossman HB: MMAC1/PTEN inhibits cell growth and induces chemosensitivity to doxorubicin in human bladder cancer cells. Oncogene 2000, 19:5406-5412.
• [50]Seifert HH, Meyer A, Cronauer MV, Hatina J, Muller M, Rieder H, Hoffmann MJ, Ackermann R, Schulz WA: A new and reliable culture system for superficial low-grade urothelial carcinoma of the bladder. World J Urol 2007, 25:297-302.
• [51]Ribarska T, Ingenwerth M, Goering W, Engers R, Schulz WA: Epigenetic inactivation of the placentally imprinted tumor suppressor gene TFPI2 in prostate carcinoma. Cancer Genomics Proteomics 2010, 7:51-60.