Annals of Surgical Oncology

, Volume 22, Issue 4, pp 1280–1287 | Cite as

LINE-1 Methylation Level and Patient Prognosis in a Database of 208 Hepatocellular Carcinomas

  • Kazuto Harada
  • Yoshifumi Baba
  • Takatsugu Ishimoto
  • Akira Chikamoto
  • Keisuke Kosumi
  • Hiromitsu Hayashi
  • Hidetoshi Nitta
  • Daisuke Hashimoto
  • Toru Beppu
  • Hideo Baba
Gastrointestinal Oncology

Abstract

Background

The level of long interspersed nucleotide element-1 (LINE-1) methylation has become regarded as a surrogate marker of global DNA methylation. Previously, we demonstrated that LINE-1 hypomethylation might contribute to the acquisition of aggressive tumor behavior through genomic gains of oncogenes such as cyclin-dependent kinase 6 (CDK6) in esophageal squamous cell carcinoma. However, the relationship between LINE-1 hypomethylation and clinical outcome in hepatocellular carcinoma (HCC) remains unclear.

Methods

LINE-1 methylation level in 208 samples of curatively resected HCCs was measured by pyrosequencing assay, and the prognostic value of LINE-1 methylation level in HCC was examined.

Results

LINE-1 methylation levels in the 208 HCC patients investigated were distributed as follows: mean 64.7; median 64.6; standard deviation (SD) 13.6; range 21.5–99.1; interquartile range 62.9–66.6. Univariate Cox regression analysis revealed a significantly higher cancer recurrence rate in the low-methylation-level group than in the high-methylation-level group (hazard ratio 1.58; 95 % CI 1.05–2.47; p = 0.028). Interestingly, the influence of LINE-1 hypomethylation on patient outcome was modified by hepatitis virus infection (p of interaction = 0.023); LINE-1 hypomethylation was associated with a higher cancer recurrence rate in patients without hepatitis virus infection (log-rank p = 0.0047). CDK6 messenger RNA expression levels were inversely associated with LINE-1 methylation levels (p = 0.0075; R = −0.37).

Conclusions

Genome-wide DNA hypomethylation, as measured by LINE-1 levels, might be associated with poor disease-free survival in HCC patients, suggesting a potential role for LINE-1 methylation level as a biomarker for identifying patients who will experience an unfavorable clinical outcome.

Notes

Acknowledgments

Author contributions: conception and design: Kazuto Harada, Yoshifumi Baba, Toru Beppu, and Hideo Baba; acquisition of data: Kazuto Harada, Yoshifumi Baba, and Toru Beppu; analysis and interpretation of data: Kazuto Harada and Yoshifumi Baba; manuscript writing: Kazuto Harada, Yoshifumi Baba, and Hideo Baba. All authors approved the final manuscript.

Conflict of interest

No conflicts of interest exist.

Supplementary material

10434_2014_4134_MOESM1_ESM.tif (768 kb)
Kaplan–Meier curves for the study group (red) and excluded group (blue). The left and right panels show theoverall survival rate and the disease-free survival rate, respectively (TIFF 768 kb)
10434_2014_4134_MOESM2_ESM.tif (2 mb)
The pyrosequencing assay used to measure the LINE-1 methylation level. The overall LINE-1methylationlevel is the average proportion of C (%) at the 4 CpG sites. The percentages of C at each CpG site afterbisulfite conversion, used to compute the methylation level at each CpG site, are given in blue font. Upperand lower panels show the results for LINE-1 hypermethylated tumor (methylation level, 81.5%) and LINE-1hypomethylated tumor (methylation level, 48.3%), respectively (TIFF 2064 kb)
10434_2014_4134_MOESM3_ESM.docx (25 kb)
Supplementary material 3 (DOCX 25 kb)
10434_2014_4134_MOESM4_ESM.docx (25 kb)
Supplementary material 4 (DOCX 24 kb)

References

  1. 1.
    Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90.CrossRefPubMedGoogle Scholar
  2. 2.
    Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol. 2006;45:529–538.CrossRefPubMedGoogle Scholar
  3. 3.
    Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet. 2012;379:1245–1255.CrossRefPubMedGoogle Scholar
  4. 4.
    Feng GS. Conflicting roles of molecules in hepatocarcinogenesis: paradigm or paradox. Cancer cell. 2012;21:150–154.CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Iakova P, Timchenko L, Timchenko NA. Intracellular signaling and hepatocellular carcinoma. Semin Cancer Biol. 2011;21:28–34.CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Herath NI, Leggett BA, MacDonald GA. Review of genetic and epigenetic alterations in hepatocarcinogenesis. J Gastroenterol Hepatol. 2006;21:15–21.CrossRefPubMedGoogle Scholar
  7. 7.
    Nishida N, Goel A. Genetic and epigenetic signatures in human hepatocellular carcinoma: a systematic review. Curr Genomics. 2011;12:130–137.CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Calvisi DF, Ladu S, Gorden A, et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest. 2007;117:2713–2722.CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Pogribny IP, Rusyn I. Role of epigenetic aberrations in the development and progression of human hepatocellular carcinoma. Cancer Lett. 2014;342:223–230.CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Berman BP, Weisenberger DJ, Aman JF, et al. Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat Genet. 2012;44:40–46.CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Gaudet F, Hodgson JG, Eden A, et al. Induction of tumors in mice by genomic hypomethylation. Science. 2003;300:489–492.CrossRefPubMedGoogle Scholar
  12. 12.
    Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002;3:415–428.CrossRefPubMedGoogle Scholar
  13. 13.
    Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10:691–703.CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Irahara N, Nosho K, Baba Y, et al. Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells. J Mol Diagn. 2010;12:177–183.CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Ogino S, Kawasaki T, Nosho K, et al. LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer. 2008;122:2767–2773.CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 2004;32:e38.CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Ogino S, Nosho K, Kirkner GJ, et al. A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J Natl Cancer Inst. 2008;100:1734–1738.CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Iwagami S, Baba Y, Watanabe M, et al. LINE-1 hypomethylation is associated with a poor prognosis among patients with curatively resected esophageal squamous cell carcinoma. Ann Surg. 2013;257:449–455.CrossRefPubMedGoogle Scholar
  19. 19.
    Shigaki H, Baba Y, Watanabe M, et al. LINE-1 hypomethylation in gastric cancer, detected by bisulfite pyrosequencing, is associated with poor prognosis. Gastric Cancer. 2013;16:480–487.CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Pattamadilok J, Huapai N, Rattanatanyong P, et al. LINE-1 hypomethylation level as a potential prognostic factor for epithelial ovarian cancer. Int J Gynecol Cancer. 2008;18:711–717.CrossRefPubMedGoogle Scholar
  21. 21.
    Baba Y, Watanabe M, Murata A, et al. LINE-1 hypomethylation, DNA copy number alterations, and CDK6 amplification in esophageal squamous cell carcinoma. Clin Cancer Res. 2014;20:1114–1124.CrossRefPubMedGoogle Scholar
  22. 22.
    Sobin LH, Gospodarowicz MK, Wittekind C, International Union against Cancer. TNM classification of malignant tumours. 7th ed. Chichester; Hoboken, NJ: Wiley-Blackwell; 2010.Google Scholar
  23. 23.
    McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM. Reporting recommendations for tumor marker prognostic studies (REMARK). J Natl Cancer Inst. 2005;97:1180–1184.CrossRefPubMedGoogle Scholar
  24. 24.
    Baba Y, Huttenhower C, Nosho K, et al. Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors. Mol Cancer. 2010;9:125.CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Iwagami S, Baba Y, Watanabe M, et al. Pyrosequencing assay to measure LINE-1 methylation level in esophageal squamous cell carcinoma. Ann Surg Oncol. 2012;19:2726–2732.CrossRefPubMedGoogle Scholar
  26. 26.
    Kinoshita H, Okabe H, Beppu T, et al. CYLD downregulation is correlated with tumor development in patients with hepatocellular carcinoma. Mol Clin Oncol. 2013;1:309–314.PubMedCentralPubMedGoogle Scholar
  27. 27.
    Ichida F, Tsuji T, Omata M, et al. New Inuyama classification: new criteria for histological assessment of chronic hepatitis. Int Hepatol Commun. 1996;6:112–119.CrossRefGoogle Scholar
  28. 28.
    Ohka F, Natsume A, Motomura K, et al. The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma. PloS one. 2011;6:e23332.CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Sigalotti L, Fratta E, Bidoli E, et al. Methylation levels of the “long interspersed nucleotide element-1” repetitive sequences predict survival of melanoma patients. J Transl Med. 2011;9:78.CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Roman-Gomez J, Jimenez-Velasco A, Agirre X, et al. Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia. Oncogene. 2005;24:7213–7223.CrossRefPubMedGoogle Scholar
  31. 31.
    Cho NY, Kim BH, Choi M, et al. Hypermethylation of CpG island loci and hypomethylation of LINE-1 and Alu repeats in prostate adenocarcinoma and their relationship to clinicopathological features. J Pathol. 2007;211:269–277.CrossRefPubMedGoogle Scholar
  32. 32.
    Saito K, Kawakami K, Matsumoto I, Oda M, Watanabe G, Minamoto T. Long interspersed nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non-small cell lung cancer. Clin Cancer Res. 2010;16:2418–2426.CrossRefPubMedGoogle Scholar
  33. 33.
    Esteller M. Epigenetics in cancer. N Engl J Med. 2008;358:1148–1159.CrossRefPubMedGoogle Scholar
  34. 34.
    Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation. Science. 2003;300:455.CrossRefPubMedGoogle Scholar
  35. 35.
    Holm TM, Jackson-Grusby L, Brambrink T, Yamada Y, Rideout WM 3rd, Jaenisch R. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell. 2005;8:275–285.CrossRefPubMedGoogle Scholar
  36. 36.
    Karpf AR, Matsui S. Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells. Cancer Res. 2005;65:8635–8639.CrossRefPubMedGoogle Scholar
  37. 37.
    Suzuki K, Suzuki I, Leodolter A, et al. Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. Cancer Cell. 2006;9:199–207.CrossRefPubMedGoogle Scholar
  38. 38.
    Cruickshanks HA, Tufarelli C. Isolation of cancer-specific chimeric transcripts induced by hypomethylation of the LINE-1 antisense promoter. Genomics. 2009;94:397–406.CrossRefPubMedGoogle Scholar
  39. 39.
    Howard G, Eiges R, Gaudet F, Jaenisch R, Eden A. Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice. Oncogene. 2008;27:404–408.CrossRefPubMedGoogle Scholar
  40. 40.
    Schulz WA. L1 retrotransposons in human cancers. J Biomed Biotechnol. 2006;2006:83672.CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Shukla R, Upton KR, Munoz-Lopez M, et al. Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell. 2013;153:101–111.CrossRefPubMedCentralPubMedGoogle Scholar
  42. 42.
    Weber B, Kimhi S, Howard G, Eden A, Lyko F. Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription. Oncogene. 2010;29:5775–5784.CrossRefPubMedGoogle Scholar
  43. 43.
    Zhu C, Utsunomiya T, Ikemoto T, et al. Hypomethylation of long interspersed nuclear element-1 (LINE-1) is associated with poor prognosis via activation of c-MET in hepatocellular carcinoma. Ann Surg Oncol. Epub 4 Jul 2014.Google Scholar
  44. 44.
    Berdasco M, Esteller M. Aberrant epigenetic landscape in cancer: how cellular identity goes awry. Dev Cell. 2010;19:698–711.CrossRefPubMedGoogle Scholar
  45. 45.
    Bjornsson HT, Brown LJ, Fallin MD, et al. Epigenetic specificity of loss of imprinting of the IGF2 gene in Wilms tumors. J Natl Cancer Inst. 2007;99:1270–1273.CrossRefPubMedGoogle Scholar
  46. 46.
    Cheah MS, Wallace CD, Hoffman RM. Hypomethylation of DNA in human cancer cells: a site-specific change in the c-myc oncogene. J Natl Cancer Inst. 1984;73:1057–1065.PubMedGoogle Scholar
  47. 47.
    Shahrzad S, Bertrand K, Minhas K, Coomber BL. Induction of DNA hypomethylation by tumor hypoxia. Epigenetics. 2007;2:119-125.CrossRefPubMedGoogle Scholar
  48. 48.
    Li J, Xu Y, Long XD, et al. Cbx4 governs HIF-1alpha to potentiate angiogenesis of hepatocellular carcinoma by its SUMO E3 ligase activity. Cancer Cell. 2014;25:118–131.CrossRefPubMedGoogle Scholar
  49. 49.
    Musgrove EA, Caldon CE, Barraclough J, Stone A, Sutherland RL. Cyclin D as a therapeutic target in cancer. Nat Rev Cancer. 2011;11:558–572.CrossRefPubMedGoogle Scholar
  50. 50.
    Aravalli RN, Steer CJ, Cressman EN. Molecular mechanisms of hepatocellular carcinoma. Hepatology. 2008;48:2047–2063.CrossRefPubMedGoogle Scholar
  51. 51.
    Zhang C, Xu Y, Zhao J, et al. Elevated expression of the stem cell marker CD133 associated with Line-1 demethylation in hepatocellular carcinoma. Ann Surg Oncol. 2011;18:2373–2380.CrossRefPubMedGoogle Scholar
  52. 52.
    Choi GH, Kim DH, Kang CM, et al. Prognostic factors and optimal treatment strategy for intrahepatic nodular recurrence after curative resection of hepatocellular carcinoma. Ann Surg Oncol. 2008;15:618–629.CrossRefPubMedGoogle Scholar
  53. 53.
    Poon RT, Fan ST, Lo CM, Liu CL, Wong J. Intrahepatic recurrence after curative resection of hepatocellular carcinoma: long-term results of treatment and prognostic factors. Ann Surg. 1999;229:216–222.CrossRefPubMedCentralPubMedGoogle Scholar
  54. 54.
    Sasaki Y, Imaoka S, Masutani S, et al. Influence of coexisting cirrhosis on long-term prognosis after surgery in patients with hepatocellular carcinoma. Surgery. 1992;112:515–521.PubMedGoogle Scholar
  55. 55.
    Shirabe K, Shimada M, Kajiyama K, et al. Clinicopathologic features of patients with hepatocellular carcinoma surviving >10 years after hepatic resection. Cancer. 1998;83:2312–2316.CrossRefPubMedGoogle Scholar

Copyright information

© Society of Surgical Oncology 2014

Authors and Affiliations

  • Kazuto Harada
    • 1
  • Yoshifumi Baba
    • 1
  • Takatsugu Ishimoto
    • 1
  • Akira Chikamoto
    • 1
  • Keisuke Kosumi
    • 1
  • Hiromitsu Hayashi
    • 1
  • Hidetoshi Nitta
    • 1
  • Daisuke Hashimoto
    • 1
  • Toru Beppu
    • 1
  • Hideo Baba
    • 1
  1. 1.Department of Gastroenterological Surgery, Graduate School of Medical ScienceKumamoto UniversityKumamotoJapan

Personalised recommendations