Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression

Oncogene volume 35, pages 3565–3576 (2016)Cite this article

Subjects

Abstract

Previous studies have indicated the important roles of MYCN in tumorigenesis and progression of neuroblastoma (NB), the most common extracranial solid tumor derived from neural crest in childhood. However, the regulatory mechanisms of MYCN expression in NB still remain largely unknown. In this study, through mining public microarray databases and analyzing the cis-regulatory elements and chromatin immunoprecipitation data sets, we identified CCCTC-binding factor (CTCF) as a crucial transcription factor facilitating the MYCN expression in NB. RNA immunoprecipitation, RNA electrophoretic mobility shift assay, RNA pull down and in vitro binding assay indicated the physical interaction between CTCF and MYCN opposite strand (MYCNOS), a natural noncoding RNA surrounding the MYNC promoter. Gain- and loss-of-function studies revealed that MYCNOS facilitated the recruitment of CTCF to its binding sites within the MYCN promoter to induce chromatin remodeling, resulting in enhanced MYCN levels and altered downstream gene expression, in cultured NB cell lines. CTCF cooperated with MYCNOS to suppress the differentiation and promote the growth, invasion and metastasis of NB cells in vitro and in vivo. In clinical NB tissues and cell lines, CTCF and MYCNOS were upregulated and positively correlated with MYCN expression. CTCF was an independent prognostic factor for unfavorable outcome of NB, and patients with high MYCNOS expression had lower survival probability. Taken together, these results demonstrate that CTCF cooperates with noncoding RNA MYCNOS to exhibit oncogenic activity that affects the aggressiveness and progression of NB through transcriptional upregulation of MYCN.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Westermann F, Schwab M . Genetic parameters of neuroblastomas. Cancer Lett 2002; 184: 127–147.

    Article  CAS  PubMed  Google Scholar 

  2. Schwab M, Alitalo K, Klempnauer KH, Varmus HE, Bishop JM, Gilbert F et al. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour. Nature 1983; 305: 245–248.

    Article  CAS  PubMed  Google Scholar 

  3. Weiss WA, Aldape K, Mohapatra G, Feuerstein BG, Bishop JM . Targeted expression of MYCN causes neuroblastoma in transgenic mice. EMBO J 1997; 16: 2985–2995.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lutz W, Stöhr M, Schürmann J, Wenzel A, Löhr A, Schwab M . Conditional expression of N-myc in human neuroblastoma cells increases expression of alpha-prothymosin and ornithine decarboxylase and accelerates progression into S-phase early after mitogenic stimulation of quiescent cells. Oncogene 1996; 13: 803–812.

    CAS  PubMed  Google Scholar 

  5. Tanaka N, Fukuzawa M . MYCN downregulates integrin alpha1 to promote invasion of human neuroblastoma cells. Int J Oncol 2008; 33: 815–821.

    CAS  PubMed  Google Scholar 

  6. Tee AE, Ling D, Nelson C, Atmadibrata B, Dinger ME, Xu N et al. The histone demethylase JMJD1A induces cell migration and invasion by up-regulating the expression of the long noncoding RNA MALAT1. Oncotarget 2014; 5: 1793–1804.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Thomas WD, Raif A, Hansford L, Marshall G . N-myc transcription molecule and oncoprotein. Int J Biochem Cell Biol 2004; 36: 771–775.

    Article  CAS  PubMed  Google Scholar 

  8. Inge TH, Casson LK, Priebe W, Trent JO, Georgeson KE, Miller DM et al. Importance of Sp1 consensus motifs in the MYCN promoter. Surgery 2002; 132: 232–238.

    Article  PubMed  Google Scholar 

  9. Nutt SL, Morrison AM, Dörfler P, Rolink A, Busslinger M . Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments. EMBO J 1998; 17: 2319–2333.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhang X, Xing G, Saunders GF . Proto-oncogene N-myc promoter is down regulated by the Wilms' tumor suppressor gene WT1. Anticancer Res 1999; 19: 1641–1648.

    CAS  PubMed  Google Scholar 

  11. Kramps C, Strieder V, Sapetschnig A, Suske G, Lutz W . E2F and Sp1/Sp3 synergize but are not sufficient to activate the MYCN gene in neuroblastomas. J Biol Chem 2004; 279: 5110–5117.

    Article  CAS  PubMed  Google Scholar 

  12. Suenaga Y, Kaneko Y, Matsumoto D, Hossain MS, Ozaki T, Nakagawara A . Positive auto-regulation of MYCN in human neuroblastoma. Biochem Biophys Res Commun 2009; 390: 21–26.

    Article  CAS  PubMed  Google Scholar 

  13. Yu M, Ohira M, Li Y, Niizuma H, Oo ML, Zhu Y et al. High expression of ncRAN, a novel non-coding RNA mapped to chromosome 17q25.1, is associated with poor prognosis in neuroblastoma. Int J Oncol 2009; 34: 931–938.

    CAS  PubMed  Google Scholar 

  14. Pandey GK, Mitra S, Subhash S, Hertwig F, Kanduri M, Mishra K et al. The risk-associated long noncoding RNA NBAT-1 controls neuroblastoma progression by regulating cell proliferation and neuronal differentiation. Cancer Cell 2014; 26: 722–737.

    Article  CAS  PubMed  Google Scholar 

  15. Vance KW, Sansom SN, Lee S, Chalei V, Kong L, Cooper SE et al. The long non-coding RNA Paupar regulates the expression of both local and distal genes. EMBO J 2014; 33: 296–311.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Liu PY, Erriquez D, Marshall GM, Tee AE, Polly P, Wong M et al. Effects of a novel long noncoding RNA, lncUSMycN, on N-Myc expression and neuroblastoma progression. J Natl Cancer Inst 2014, e-pub ahead of print 6 June 2014 doi:10.1093/jnci/dju113.

  17. Armstrong B, Krystal G . Isolation and characterization of complementary DNA for N-cym, a gene encoded by the DNA strand opposite to N-myc. Cell Growth Differ 1992; 3: 385–390.

    CAS  PubMed  Google Scholar 

  18. Jacobs J, van Bokhoven H, van Leeuwen F, Hulsbergen-van de Kaa CA, de Vries I, Adema G et al. Regulation of MYCN expression in human neuroblastoma cells. BMC Cancer 2009; 9: 239.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Suenaga Y, Islam SMR, Alagu J, Kaneko Y, Kato M, Tanaka Y et al. NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3β resulting in the stabilization of MYCN in human neuroblastomas. PLoS Genet 2014; 10: e1003996.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Yang JH, Li JH, Jiang S, Zhou H, Qu LH . ChIPBase: a database for decoding the transcriptional regulation of long non-coding RNA and microRNA genes from ChIP-Seq data. Nucleic Acids Res 2013; 41: D177–D187.

    Article  CAS  PubMed  Google Scholar 

  21. Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A et al. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 2005; 21: 2933–2942.

    Article  CAS  PubMed  Google Scholar 

  22. Hasan MK, Nafady A, Takatori A, Kishida S, Ohira M, Suenaga Y et al. ALK is a MYCN target gene and regulates cell migration and invasion in neuroblastoma. Sci Rep 2013; 3: 3450.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Henriksen J, Haug B, Buechner J, Tomte E, Lokke C, Flaegstad T et al. Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma. BMC Dev Biol 2011; 11: 1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Michel AM, Fox G, M Kiran A, De Bo C, O'Connor PB, Heaphy SM et al. GWIPS-viz: development of a ribo-seq genome browser. Nucleic Acids Res 2014; 42: D859–D864.

    Article  CAS  PubMed  Google Scholar 

  25. Wang X, Arai S, Song X, Reichart D, Du K, Pascual G et al. Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature 2008; 454: 126–130.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sun S, Del Rosario Brian C, Szanto A, Ogawa Y, Jeon Y, Lee Jeannie T . Jpx RNA activates Xist by evicting CTCF. Cell 2013; 153: 1537–1551.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Saldaña-Meyer R, González-Buendía E, Guerrero G, Narendra V, Bonasio R, Recillas-Targa F et al. CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53. Genes Dev 2014; 28: 723–734.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Lovén J, Zinin N, Wahlström T, Müller I, Brodin P, Fredlund E et al. MYCN-regulated microRNAs repress estrogen receptor-alpha (ESR1) expression and neuronal differentiation in human neuroblastoma. Proc Natl Acad Sci USA 2010; 107: 1553–1558.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Zhang H, Qi M, Li S, Qi T, Mei H, Huang K et al. microRNA-9 targets matrix metalloproteinase 14 to inhibit invasion, metastasis, and angiogenesis of neuroblastoma cells. Mol Cancer Ther 2012; 11: 1454–1466.

    Article  PubMed  Google Scholar 

  30. Zhang H, Pu J, Qi T, Qi M, Yang C, Li S et al. MicroRNA-145 inhibits the growth, invasion, metastasis and angiogenesis of neuroblastoma cells through targeting hypoxia-inducible factor 2 alpha. Oncogene 2014; 33: 387–397.

    Article  PubMed  Google Scholar 

  31. Li D, Mei H, Qi M, Yang D, Zhao X, Xiang X et al. FOXD3 is a novel tumor suppressor that affects growth, invasion, metastasis and angiogenesis of neuroblastoma. Oncotarget 2013; 4: 2021–2044.

    PubMed  PubMed Central  Google Scholar 

  32. Pession A, Tonelli R . The MYCN oncogene as a specific and selective drug target for peripheral and central nervous system tumors. Curr Cancer Drug Targets 2005; 5: 273–283.

    Article  CAS  PubMed  Google Scholar 

  33. Bell E, Chen L, Liu T, Marshall GM, Lunec J, Tweddle DA . MYCN oncoprotein targets and their therapeutic potential. Cancer Lett 2010; 293: 144–157.

    Article  CAS  PubMed  Google Scholar 

  34. Lin CY, Lovén J, Rahl PB, Paranal RM, Burge CB, Bradner JE et al. Transcriptional amplification in tumor cells with elevated c-Myc. Cell 2012; 151: 56–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chipumuro E, Marco E, Christensen CL, Kwiatkowski N, Zhang T, Hatheway CM et al. CDK7 inhibition suppresses super-enhancer-linked oncogenic transcription in MYCN- driven cancer. Cell 2014; 159: 1126–1139.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Filippova GN, Fagerlie S, Klenova EM, Myers C, Dehner Y, Goodwin G et al. An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. Mol Cell Biol 1996; 16: 2802–2813.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Phillips JE, Corces VG . CTCF: master weaver of the genome. Cell 2009; 137: 1194–1211.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Rao SS, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Robinson JT et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 2014; 159: 1665–1680.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. De La Rosa-Velázquez IA, Rincón-Arano H, Benítez-Bribiesca L, Recillas-Targa F . Epigenetic regulation of the human retinoblastoma tumor suppressor gene promoter by CTCF. Cancer Res 2007; 67: 2577–2585.

    Article  PubMed  Google Scholar 

  40. Witcher M, Emerson BM . Epigenetic silencing of the p16(INK4a) tumor suppressor is associated with loss of CTCF binding and a chromatin boundary. Mol Cell 2009; 34: 271–284.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Soto-Reyes E, Recillas-Targa F . Epigenetic regulation of the human p53 gene promoter by the CTCF transcription factor in transformed cell lines. Oncogene 2010; 29: 2217–2227.

    Article  CAS  PubMed  Google Scholar 

  42. Docquier F, Farrar D, D'Arcy V, Chernukhin I, Robinson AF, Loukinov D et al. Heightened expression of CTCF in breast cancer cells is associated with resistance to apoptosis. Cancer Res 2005; 65: 5112–5122.

    Article  CAS  PubMed  Google Scholar 

  43. Huang K, Jia J, Wu C, Yao M, Li M, Jin J et al. Ribosomal RNA gene transcription mediated by the master genome regulator protein CCCTC-binding factor (CTCF) is negatively regulated by the condensin complex. J Biol Chem 2013; 288: 26067–26077.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Chernukhin IV, Shamsuddin S, Robinson AF, Carne AF, Paul A, El-Kady AI et al. Physical and functional interaction between two pluripotent proteins, the Y-box DNA/RNA-binding factor, YB-1, and the multivalent zinc finger factor, CTCF. J Biol Chem 2000; 275: 29915–29921.

    Article  CAS  PubMed  Google Scholar 

  45. Shoji W, Suenaga Y, Kaneko Y, Islam SM, Alagu J, Yokoi S et al. NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells. Biochem Biophys Res Commun 2015; 461: 501–506.

    Article  CAS  PubMed  Google Scholar 

  46. Kaneko Y, Suenaga Y, Islam SM, Matsumoto D, Nakamura Y, Ohira M et al. Functional interplay between MYCN, NCYM, and OCT4 promotes aggressiveness of human neuroblastomas. Cancer Sci 2015; 106: 840–847.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Jiang G, Zheng L, Pu J, Mei H, Zhao J, Huang K et al. Small RNAs targeting transcription start site induce heparanase silencing through interference with transcription initiation in human cancer cells. PLoS One 2012; 7: e31379.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Li D, Mei H, Pu J, Xiang X, Zhao X, Qu H et al. Intelectin 1 suppresses the growth, invasion and metastasis of neuroblastoma cells through up-regulation of N-myc downstream regulated gene 2. Mol Cancer 2015; 14: 47.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Xiang X, Zhao X, Qu H, Li D, Yang D, Pu J et al. Hepatocyte nuclear factor 4 alpha promotes the invasion, metastasis and angiogenesis of neuroblastoma cells via targeting matrix metalloproteinase 14. Cancer Lett 2015; 359: 187–197.

    Article  CAS  PubMed  Google Scholar 

  50. Qu H, Zheng L, Pu J, Mei H, Xiang X, Zhao X et al. miRNA-558 promotes tumorigenesis and aggressiveness of neuroblastoma cells through activating the transcription of heparanase. Human Mol Genet 2015; 24: 2539–2551.

    Article  CAS  Google Scholar 

  51. Sivak LE, Tai KF, Smith RS, Dillon PA, Brodeur GM, Carroll WL . Autoregulation of the human N-myc oncogene is disrupted in amplified but not single-copy neuroblastoma cell lines. Oncogene 1997; 15: 1937–1946.

    Article  CAS  PubMed  Google Scholar 

  52. Zheng L, Qi T, Yang D, Qi M, Li D, Xiang X et al. microRNA-9 suppresses the proliferation, invasion and metastasis of gastric cancer cells through targeting cyclin D1 and Ets1. PLoS One 2013; 8: e55719.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zheng L, Pu J, Qi T, Qi M, Li D, Xiang X et al. miRNA-145 targets v-ets erythroblastosis virus E26 oncogene homolog 1 to suppress the invasion, metastasis, and angiogenesis of gastric cancer cells. Mol Cancer Res 2013; 11: 182–193.

    Article  CAS  PubMed  Google Scholar 

  54. Xiang JF, Yin QF, Chen T, Zhang Y, Zhang XO, Wu Z et al. Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus. Cell Res 2014; 24: 513–531.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Zheng L, Li D, Xiang X, Tong L, Qi M, Pu J et al. Methyl jasmonate abolishes the migration, invasion and angiogenesis of gastric cancer cells through down-regulation of matrix metalloproteinase 14. BMC Cancer 2013; 13: 74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Zheng L, Jiang G, Mei H, Pu J, Dong J, Hou X et al. Small RNA interference-mediated gene silencing of heparanase abolishes the invasion, metastasis and angiogenesis of gastric cancer cells. BMC Cancer 2010; 10: 33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to Drs William L Carroll and Danny Reinberg for providing vectors. This work was supported by the National Natural Science Foundation of China (81272779, 81372667, 81372401, 81472363, 81402301, 81402408, 81572423), Fundamental Research Funds for the Central Universities (2012QN224, 2013ZHYX003, 01-18-530112, 01-18-530115) and Natural Science Foundation of Hubei Province (2014CFA012).

Author information

Author notes

  1. X Zhao, D Li and K Huang: These three authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China

    X Zhao, D Li, J Pu, H Mei, D Yang, X Xiang, H Qu & Q Tong

  2. Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China

    K Huang, L Zheng & Q Tong

  3. Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China

    K Huang

  4. Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China

    L Zheng

Authors
  1. X Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J Pu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H Mei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. X Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. H Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. K Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. L Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Q Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to L Zheng or Q Tong.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, X., Li, D., Pu, J. et al. CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression. Oncogene 35, 3565–3576 (2016). https://doi.org/10.1038/onc.2015.422

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2015.422

This article is cited by

Search

Advanced search

Quick links