Skip to main content

A Versatile Assay for Detection of Aberrant DNA Methylation in Bladder Cancer

Part of the Methods in Molecular Biology book series (MIMB,volume 1655)

Abstract

Urothelial carcinoma of the bladder is one of the most common malignancies in the industrialized world, mainly caused by smoking and occupational exposure to chemicals. The favorable prognosis of early stage bladder cancer underscores the importance of early detection for the treatment of this disease. The high recurrence rate of this malignancy also highlights the need for close post-diagnosis monitoring of bladder cancer patients. As for other malignancies, aberrant DNA methylation has been shown to play a crucial role in the initiation and progression of bladder cancer, and thus holds great promise as a diagnostic and prognostic biological marker. Here, we describe a protocol for a versatile DNA methylation enrichment method, the Methylated CpG Island Recovery Assay (MIRA), which enables analysis of the DNA methylation status in individual genes or across the entire genome. MIRA is based on the ability of the methyl-binding domain (MBD) proteins, the MBD2B/MBD3L1 complex, to specifically bind methylated CpG dinucleotides. This easy-to-perform method can be used to analyze the methylome of bladder cancer or urothelial cells shed in the urine to elucidate the evolution of bladder carcinogenesis and/or identify epigenetic signatures of chemicals known to cause this malignancy.

Key words

  • Aromatic amines
  • Biomarkers
  • Epigenetics
  • Methyl-binding domain (MBD) proteins
  • Tobacco smoke
  • Urine

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

Fig. 1

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Stewart BW, Wild CP (eds) (2014) World Cancer Report 2014. International Agency for Research on Cancer (IARC), Lyon, France

    Google Scholar 

  2. Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F (2016) Bladder cancer incidence and mortality: a global overview and recent trends. Eur Urol 71(1):96–108. doi:10.1016/j.eururo.2016.06.010

    CrossRef  PubMed  Google Scholar 

  3. Kamat AM, Hahn NM, Efstathiou JA, Lerner SP, Malmstrom PU, Choi W, Guo CC, Lotan Y, Kassouf W (2016) Bladder cancer. Lancet 388(10061):2796–2810. doi:10.1016/S0140-6736(16)30512-8

    CrossRef  PubMed  Google Scholar 

  4. Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer J Clin 66(1):7–30

    CrossRef  PubMed  Google Scholar 

  5. Carradori S, Cristini C, Secci D, Gulia C, Gentile V, Di Pierro GB (2012) Current and emerging strategies in bladder cancer. Anticancer Agents Med Chem 12(6):589–603

    CAS  CrossRef  PubMed  Google Scholar 

  6. Besaratinia A, Cockburn M, Tommasi S (2013) Alterations of DNA methylome in human bladder cancer. Epigenetics 8(10):1013–1022

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Griffiths TR, on behalf of Action on Bladder Cancer (2013) Current perspectives in bladder cancer management. Int J Clin Pract 67(5):435–448

    CAS  CrossRef  PubMed  Google Scholar 

  8. Miremami J, Kyprianou N (2014) The promise of novel molecular markers in bladder cancer. Int J Mol Sci 15(12):23897–23908

    CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Netto GJ (2012) Molecular biomarkers in urothelial carcinoma of the bladder: are we there yet? Nat Rev Urol 9(1):41–51

    CAS  CrossRef  Google Scholar 

  10. Boffetta P (2008) Tobacco smoking and risk of bladder cancer. Scand J Urol Nephrol Suppl 218:45–54

    CrossRef  Google Scholar 

  11. Besaratinia A, Tommasi S (2013) Genotoxicity of tobacco smoke-derived aromatic amines and bladder cancer: current state of knowledge and future research directions. FASEB J 27(6):2090–2100

    CAS  CrossRef  PubMed  Google Scholar 

  12. Kiriluk KJ, Prasad SM, Patel AR, Steinberg GD, Smith ND (2012) Bladder cancer risk from occupational and environmental exposures. Urol Oncol 30(2):199–211

    CAS  CrossRef  PubMed  Google Scholar 

  13. U.S. Department of Health and Human Services (2014) The health consequences of smoking – 50 years of progress. A report of the Surgeon General. U.S. Department of Health and Human Services. Public Health Service. Office of the Surgeon General. Rockville, MD

    Google Scholar 

  14. Freedman ND, Silverman DT, Hollenbeck AR, Schatzkin A, Abnet CC (2011) Association between smoking and risk of bladder cancer among men and women. JAMA 306(7):737–745

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  15. Internationa Agency for Research on Cancer (IARC) (2010) Some aromatic amines, organic dyes, and related exposures. In: IARC monographs on the evaluation of carcinogenic risk to humans, vol 99. Lyon, France

    Google Scholar 

  16. Scelo G, Brennan P (2007) The epidemiology of bladder and kidney cancer. Nat Clin Pract Urol 4(4):205–217

    CrossRef  PubMed  Google Scholar 

  17. Stern MC, Lin J, Figueroa JD, Kelsey KT, Kiltie AE, Yuan JM, Matullo G, Fletcher T, Benhamou S, Taylor JA, Placidi D, Zhang ZF, Steineck G, Rothman N, Kogevinas M, Silverman D, Malats N, Chanock S, Wu X, Karagas MR, Andrew AS, Nelson HH, Bishop DT, Sak SC, Choudhury A, Barrett JH, Elliot F, Corral R, Joshi AD, Gago-Dominguez M, Cortessis VK, Xiang YB, Gao YT, Vineis P, Sacerdote C, Guarrera S, Polidoro S, Allione A, Gurzau E, Koppova K, Kumar R, Rudnai P, Porru S, Carta A, Campagna M, Arici C, Park SS, Garcia-Closas M, International Consortium of Bladder C (2009) Polymorphisms in DNA repair genes, smoking, and bladder cancer risk: findings from the international consortium of bladder cancer. Cancer Res 69(17):6857–6864

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  18. Jirtle RL, Skinner MK (2007) Environmental epigenomics and disease susceptibility. Nat Rev Genet 8(4):253–262

    CAS  CrossRef  PubMed  Google Scholar 

  19. Cortessis VK, Thomas DC, Levine AJ, Breton CV, Mack TM, Siegmund KD, Haile RW, Laird PW (2012) Environmental epigenetics: prospects for studying epigenetic mediation of exposure-response relationships. Hum Genet 131(10):1565–1589

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  20. Schulz WA, Goering W (2016) DNA methylation in urothelial carcinoma. Epigenomics 8(10):1415–1428. doi:10.2217/epi-2016-0064

    CAS  CrossRef  PubMed  Google Scholar 

  21. Sandoval J, Esteller M (2012) Cancer epigenomics: beyond genomics. Curr Opin Genet Dev 22(1):50–55

    CAS  CrossRef  PubMed  Google Scholar 

  22. You JS, Jones PA (2012) Cancer genetics and epigenetics: two sides of the same coin? Cancer Cell 22(1):9–20

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  23. Feinberg AP, Koldobskiy MA, Gondor A (2016) Epigenetic modulators, modifiers and mediators in cancer aetiology and progression. Nat Rev Genet 17(5):284–299

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  24. Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9(6):465–476

    CAS  CrossRef  PubMed  Google Scholar 

  25. Kulis M, Esteller M (2010) DNA methylation and cancer. Adv Genet 70:27–56

    PubMed  Google Scholar 

  26. Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13(7):484–492

    CAS  CrossRef  PubMed  Google Scholar 

  27. Besaratinia A, Tommasi S (2014) Epigenetics of human melanoma: promises and challenges. J Mol Cell Biol 6(5):356–367

    CAS  CrossRef  PubMed  Google Scholar 

  28. Beekman R, Kulis M, Martín-Subero JI (2016) The DNA methylomes of cancer. In: Fraga M, Fernandez AF (eds) Epigenomics in health and disease Elsevier Inc, pp 183–207

    CrossRef  Google Scholar 

  29. Verma M (2015) The role of Epigenomics in the study of cancer biomarkers and in the development of diagnostic tools. Adv Exp Med Biol 867:59–80

    CAS  CrossRef  PubMed  Google Scholar 

  30. Rodriguez-Paredes M, Esteller M (2011) Cancer epigenetics reaches mainstream oncology. Nat Med 17(3):330–339

    CAS  CrossRef  PubMed  Google Scholar 

  31. Costa-Pinheiro P, Montezuma D, Henrique R, Jeronimo C (2015) Diagnostic and prognostic epigenetic biomarkers in cancer. Epigenomics 7(6):1003–1015

    CAS  CrossRef  PubMed  Google Scholar 

  32. Chihara Y, Kanai Y, Fujimoto H, Sugano K, Kawashima K, Liang G, Jones PA, Fujimoto K, Kuniyasu H, Hirao Y (2013) Diagnostic markers of urothelial cancer based on DNA methylation analysis. BMC Cancer 13:275

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  33. Harb-de la Rosa A, Acker M, Kumar RA, Manoharan M (2015) Epigenetics application in the diagnosis and treatment of bladder cancer. Can J Urol 22(5):7947–7951

    PubMed  Google Scholar 

  34. Wu P, Cao Z, Wu S (2016) New progress of epigenetic biomarkers in urological cancer. Dis Markers 2016:9864047. doi:10.1155/2016/9864047

    PubMed  PubMed Central  Google Scholar 

  35. Chung W, Bondaruk J, Jelinek J, Lotan Y, Liang S, Czerniak B, Issa JP (2011) Detection of bladder cancer using novel DNA methylation biomarkers in urine sediments. Cancer Epidemiol Biomarkers Prev 20(7):1483–1491

    Google Scholar 

  36. Sapre N, Anderson PD, Costello AJ, Hovens CM, Corcoran NM (2014) Gene-based urinary biomarkers for bladder cancer: an unfulfilled promise? Urol Oncol 32(1):48.e49–48.e17

    CrossRef  Google Scholar 

  37. Laird PW (2010) Principles and challenges of genomewide DNA methylation analysis. Nat Rev Genet 11(3):191–203

    CAS  CrossRef  PubMed  Google Scholar 

  38. Fouse SD, Nagarajan RO, Costello JF (2010) Genome-scale DNA methylation analysis. Epigenomics 2(1):105–117

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  39. Taylor KH, Shi H, Caldwell CW (2010) Next generation sequencing: advances in characterizing the methylome. Genes 1(2):143–165

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  40. Olkhov-Mitsel E, Bapat B (2012) Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers. Cancer Med 1(2):237–260

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  41. Tommasi S, Karm DL, Wu X, Yen Y, Pfeifer GP (2009) Methylation of homeobox genes is a frequent and early epigenetic event in breast cancer. Breast Cancer Res 11(1):R14

    CrossRef  PubMed  PubMed Central  Google Scholar 

  42. Mitchell N, Deangelis JT, Tollefsbol TO (2011) Methylated-CpG Island recovery assay. Methods Mol Biol 791:125–133

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  43. Choi JH, Li Y, Guo J, Pei L, Rauch TA, Kramer RS, Macmil SL, Wiley GB, Bennett LB, Schnabel JL, Taylor KH, Kim S, Xu D, Sreekumar A, Pfeifer GP, Roe BA, Caldwell CW, Bhalla KN, Shi H (2010) Genome-wide DNA methylation maps in follicular lymphoma cells determined by methylation-enriched bisulfite sequencing. PLoS One 5(9):e13020

    CrossRef  PubMed  PubMed Central  Google Scholar 

  44. Almamun M, Levinson BT, Gater ST, Schnabel RD, Arthur GL, Davis JW, Taylor KH (2014) Genome-wide DNA methylation analysis in precursor B-cells. Epigenetics 9(12):1588–1595

    CrossRef  PubMed  PubMed Central  Google Scholar 

  45. Green BB, McKay SD, Kerr DE (2015) Age dependent changes in the LPS induced transcriptome of bovine dermal fibroblasts occurs without major changes in the methylome. BMC Genomics 16:30

    CrossRef  PubMed  PubMed Central  Google Scholar 

  46. Tommasi S, Kim SI, Zhong X, Wu X, Pfeifer GP, Besaratinia A (2010) Investigating the epigenetic effects of a prototype smoke-derived carcinogen in human cells. PLoS One 5(5):e10594

    CrossRef  PubMed  PubMed Central  Google Scholar 

  47. Tommasi S, Zheng A, Weninger A, Bates SE, Li XA, Wu X, Hollstein M, Besaratinia A (2013) Mammalian cells acquire epigenetic hallmarks of human cancer during immortalization. Nucleic Acids Res 41(1):182–195

    CAS  CrossRef  PubMed  Google Scholar 

  48. Tommasi S, Zheng A, Yoon JI, Li AX, Wu X, Besaratinia A (2012) Whole DNA methylome profiling in mice exposed to secondhand smoke. Epigenetics 7(11):1302–1314

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  49. Tommasi S, Zheng A, Yoon JI, Besaratinia A (2014) Epigenetic targeting of the Nanog pathway and signaling networks during chemical carcinogenesis. Carcinogenesis 35(8):1726–1736

    CAS  CrossRef  PubMed  Google Scholar 

  50. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

Download references

Acknowledgments

ST and AB declare no conflicts of interest.

Work of the authors is funded by grants from the National Institute of Dental and Craniofacial Research of the National Institutes of Health (1R01DE026043-01) to AB and from the University of California Tobacco-Related Disease Research Program (TRDRP-25IP-0001) to ST.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stella Tommasi .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Tommasi, S., Besaratinia, A. (2018). A Versatile Assay for Detection of Aberrant DNA Methylation in Bladder Cancer. In: Schulz, W., Hoffmann, M., Niegisch, G. (eds) Urothelial Carcinoma. Methods in Molecular Biology, vol 1655. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7234-0_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7234-0_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7233-3

  • Online ISBN: 978-1-4939-7234-0

  • eBook Packages: Springer Protocols