Skip to main content
SpringerLink
Log in

Chromosomal Instability Associated with Global DNA Hypomethylation is Associated with the Initiation and Progression of Esophageal Squamous Cell Carcinoma

  • Translational Research and Biomarkers
  • Published:
Annals of Surgical Oncology Aims and scope Submit manuscript

Abstract

Background

Global DNA hypomethylation is associated with increased chromosomal instability and plays an important role in tumorigenesis. The methylation status of the long interspersed nuclear element-1 (LINE-1) element is a useful surrogate marker for global DNA methylation. Although LINE-1 hypomethylation is recognized as a poor prognostic marker, the correlation of LINE-1 methylation level with tumor suppressor gene mutation, chromosomal instability, and clinical significance in esophageal squamous cell carcinoma (ESCC) remains unclear.

Methods

Using resected tumor tissues and the corresponding normal esophageal mucosa from 105 patients with ESCC, bisulfite pyrosequencing analysis was performed to quantify the LINE-1 methylation levels. p53 mutations in exons two to ten were detected by polymerase chain reaction direct sequencing. Chromosomal instability was assessed by single nucleotide polymorphism array comparative genomic hybridization analysis.

Results

The LINE-1 methylation level of ESCC was significantly lower than matched normal mucosa. LINE-1 methylation levels of normal mucosa from the esophagus had a significant inverse correlation with both cigarette smoking and alcohol consumption of the study subjects. LINE-1 hypomethylation of ESCC was significantly associated with lymph node metastasis, lymphovascular invasion, the frequency of p53 mutation and poor survivability. The LINE-1 methylation levels in ESCC had a significant inverse association with the percentage of copy number alterations in the whole genome, mirroring chromosomal instability.

Conclusions

Our results suggested that whole genome hypomethylation caused by chronic inflammation could initiate carcinogenesis of esophageal squamous cells through chromosomal instability. In addition, chromosomal instability associated with the global hypomethylation might correlate highly with the progression of ESCC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Allum WH, Stenning SP, Bancewicz J, et al. Long-term results of a randomized trial of surgery with or without preoperative chemotherapy in esophageal cancer. J Clin Oncol. 2009;27:5062–7.

    Article  PubMed  Google Scholar 

  2. Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med. 2003;349:2241–52.

    Article  CAS  PubMed  Google Scholar 

  3. Morita M, Yoshida R, Ikeda K, et al. Advances in esophageal cancer surgery in Japan: an analysis of 1000 consecutive patients treated at a single institute. Surgery. 2008;143:499–508.

    Article  PubMed  Google Scholar 

  4. Morita M, Kumashiro R, Kubo N, et al. Alcohol drinking, cigarette smoking, and the development of squamous cell carcinoma of the esophagus: epidemiology, clinical findings, and prevention. Int J Clin Oncol. 2010;15:126–34.

    Article  PubMed  Google Scholar 

  5. Toh Y, Oki E, Ohgaki K, et al. Alcohol drinking, cigarette smoking, and the development of squamous cell carcinoma of the esophagus: molecular mechanisms of carcinogenesis. Int J Clin Oncol. 2010;15:135–44.

    Article  CAS  PubMed  Google Scholar 

  6. Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene. 2002;21:5400–13.

    Article  CAS  PubMed  Google Scholar 

  7. Gaudet F, Hodgson JG, Eden A, et al. Induction of tumors in mice by genomic hypomethylation. Science. 2003;300:489–92.

    Article  CAS  PubMed  Google Scholar 

  8. Holm TM, Jackson-Grusby L, Brambrink T, et al. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell. 2005;8:275–85.

    Article  CAS  PubMed  Google Scholar 

  9. 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.

    Article  CAS  PubMed  Google Scholar 

  10. Nishida N, Kudo M, Nishimura T, et al. Unique association between global DNA hypomethylation and chromosomal alterations in human hepatocellular carcinoma. PLoS One. 2013;8:e72312.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10:691–703.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Yang AS, Estecio MR, Doshi K, et al. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 2004;32:e38.

    Article  PubMed Central  PubMed  Google Scholar 

  13. Ikeda K, Shiraishi K, Eguchi A, et al. Long interspersed nucleotide element 1 hypomethylation is associated with poor prognosis of lung adenocarcinoma. Ann Thorac Surg. 2013;96:1790–4.

    Article  PubMed  Google Scholar 

  14. Igarashi S, Suzuki H, Niinuma T, et al. A novel correlation between LINE-1 hypomethylation and the malignancy of gastrointestinal stromal tumors. Clin Cancer Res. 2010;16:5114–23.

    Article  CAS  PubMed  Google Scholar 

  15. 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;100:1734–8.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. 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;257:449–55.

    Article  PubMed  Google Scholar 

  17. Shigaki H, Baba Y, Watanabe M, et al. LINE-1 hypomethylation in noncancerous esophageal mucosae is associated with smoking history. Ann Surg Oncol. 2012;19:4238–43.

    Article  PubMed  Google Scholar 

  18. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature. 2000;408:307–10.

    Article  CAS  PubMed  Google Scholar 

  19. Levine AJ, Oren M. The first 30 years of p53: growing ever more complex. Nat Rev Cancer. 2009;9:749–58.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Egashira A, Morita M, Yoshida R, et al. Loss of p53 in esophageal squamous cell carcinoma and the correlation with survival: analyses of gene mutations, protein expression, and loss of heterozygosity in Japanese patients. J Surg Oncol. 2011;104:169–75.

    Article  CAS  PubMed  Google Scholar 

  21. Olive KP, Tuveson DA, Ruhe ZC, et al. Mutant p53 gain of function in two mouse models of Li–Fraumeni syndrome. Cell. 2004;119:847–60.

    Article  CAS  PubMed  Google Scholar 

  22. Liu DP, Song H, Xu Y. A common gain of function of p53 cancer mutants in inducing genetic instability. Oncogene. 2010;29:949–56.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Muller PA, Vousden KH. p53 mutations in cancer. Nat Cell Biol. 2013;15:2–8.

    Article  CAS  PubMed  Google Scholar 

  24. Saeki H, Kitao H, Yoshinaga K, et al. Copy-neutral loss of heterozygosity at the p53 locus in carcinogenesis of esophageal squamous cell carcinomas associated with p53 mutations. Clin Cancer Res. 2011;17:1731–40.

    Article  CAS  PubMed  Google Scholar 

  25. Hu N, Clifford RJ, Yang HH, et al. Genome wide analysis of DNA copy number neutral loss of heterozygosity (CNNLOH) and its relation to gene expression in esophageal squamous cell carcinoma. BMC Genomics. 2010;11:576.

    Article  PubMed Central  PubMed  Google Scholar 

  26. Egashira A, Morita M, Kakeji Y, et al. p53 gene mutations in esophageal squamous cell carcinoma and their relevance to etiology and pathogenesis: results in Japan and comparisons with other countries. Cancer Sci. 2007;98:1152–6.

    Article  CAS  PubMed  Google Scholar 

  27. Florea AM. DNA methylation pyrosequencing assay is applicable for the assessment of epigenetic active environmental or clinical relevant chemicals. Biomed Res Int. 2013;2013:486072.

    Article  PubMed Central  PubMed  Google Scholar 

  28. Liu Z, Li A, Schulz V, et al. MixHMM: inferring copy number variation and allelic imbalance using SNP arrays and tumor samples mixed with stromal cells. PLoS One. 2010;5:e10909.

    Article  PubMed Central  PubMed  Google Scholar 

  29. Gasche JA, Hoffmann J, Boland CR, Goel A. Interleukin-6 promotes tumorigenesis by altering DNA methylation in oral cancer cells. Int J Cancer. 2011;129:1053–63.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Alvarez H, Opalinska J, Zhou L, et al. Widespread hypomethylation occurs early and synergizes with gene amplification during esophageal carcinogenesis. PLoS Genet. 2011;7:e1001356.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Saeki H, Kimura Y, Ito S, et al. Biologic and clinical significance of squamous epithelial dysplasia of the esophagus. Surgery. 2002;131:S22–7.

    Article  PubMed  Google Scholar 

  32. Bass AJ, Watanabe H, Mermel CH, et al. SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas. Nat Genet. 2009;41:1238–42.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Han X, Fang X, Lou X, et al. Silencing SOX2 induced mesenchymal–epithelial transition and its expression predicts liver and lymph node metastasis of CRC patients. PLoS One. 2012;7:e41335.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Hibi K, Trink B, Patturajan M, et al. AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci USA. 2000;97:5462–7.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Ramsey MR, Wilson C, Ory B, et al. FGFR2 signaling underlies p63 oncogenic function in squamous cell carcinoma. J Clin Investig. 2013;123:3525–38.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Takahashi Y, Ishii G, Taira T, et al. Fibrous stroma is associated with poorer prognosis in lung squamous cell carcinoma patients. J Thorac Oncol. 2011;6:1460–7.

    Article  PubMed  Google Scholar 

  37. Cai C, Rajaram M, Zhou X, et al. Activation of multiple cancer pathways and tumor maintenance function of the 3q amplified oncogene FNDC3B. Cell Cycle. 2012;11:1773–81.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Wang MT, Chen G, An SJ, et al. Prognostic significance of cyclinD1 amplification and the co-alteration of cyclinD1/pRb/ppRb in patients with esophageal squamous cell carcinoma. Dis Esophagus. 2012;25:664–70.

    Article  PubMed  Google Scholar 

  39. Miyawaki Y, Kawachi H, Ooi A, et al. Genomic copy-number alterations of MYC and FHIT genes are associated with survival in esophageal squamous-cell carcinoma. Cancer Sci. 2012;103:1558–66.

    Article  CAS  PubMed  Google Scholar 

  40. Hu N, Wang C, Su H, et al. High frequency of CDKN2A alterations in esophageal squamous cell carcinoma from a high-risk Chinese population. Genes Chromosomes Cancer. 2004;39:205–16.

    Article  CAS  PubMed  Google Scholar 

  41. Walsh CS, Ogawa S, Scoles DR, et al. Genome-wide loss of heterozygosity and uniparental disomy in BRCA1/2-associated ovarian carcinomas. Clin Cancer Res. 2008;14:7645–51.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Plo I, Nakatake M, Malivert L, et al. JAK2 stimulates homologous recombination and genetic instability: potential implication in the heterogeneity of myeloproliferative disorders. Blood. 2008;112:1402–12.

    Article  CAS  PubMed  Google Scholar 

  43. Pickering CR, Zhang J, Yoo SY, et al. Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers. Cancer Discov. 2013;3:770–81.

    Article  CAS  PubMed  Google Scholar 

Download references

Conflict of interest

The authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Hiroyuki Kawano MD, Hiroshi Saeki MD, PhD, Yasuo Tsuda MD, Hajime Otsu MD, Koji Ando MD, PhD, Shuhei Ito MD, PhD, Akinori Egashira MD, PhD, Eiji Oki MD, PhD, Masaru Morita MD, PhD & Yoshihiko Maehara MD, PhD

  2. Department of Molecular Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Hiroyuki Kitao PhD

  3. Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Yoshinao Oda MD, PhD

Authors
  1. Hiroyuki Kawano MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroshi Saeki MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroyuki Kitao PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yasuo Tsuda MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hajime Otsu MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Koji Ando MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shuhei Ito MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Akinori Egashira MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eiji Oki MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Masaru Morita MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yoshinao Oda MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yoshihiko Maehara MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiroshi Saeki MD, PhD.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 59 kb)

Supplementary material 2 (DOC 73 kb)

Supplementary material 3 (DOC 44 kb)

Supplementary Fig. 1

Pyrosequencing to measure LINE-1 methylation level. a Representative LINE-1 hypomethylated tumor. b Representative LINE-1 hypermethylated noncancerous tissue. The percent (in gray shade) is the proportion of C at each CpG site after bisulfite conversion, and the methylation levels of each CpG sites are estimated by the proportion of C (%). The overall LINE-1 methylation level is calculated as the average of the proportions of C (%) at the three CpG sites (TIFF 1521 kb)

Supplementary Fig. 2

Two representative cases of SNP-CGH analysis. Two transformed parameters, the log-normalized intensity ratio (log R ratio) and B allele frequency, were plotted along the entire genome for all SNPs on the array in single sample analysis mode. a Data from one tissue sample with a p53 wild-type (case 10 in Fig. 3) that was in the hypermethylation group, showed a handful of CNAs, mirroring a % defect score <10 %. This case showed a little deflection in the log R ratio, and the heterozygotes were clustered around +0.5 in the B allele frequency in almost all chromosomes. b Data from another sample with a p53 mutation (case 4 in Fig. 3) that was in the hypomethylation group. This sample showed an abundance of CNAs, mirroring a % defect score >20 %. Drastic chromosomal alterations were seen in multiple chromosomes, including chromosome 17. Given that tumor tissue samples were contaminated with stromal cells, an accurate percentage of CNLOH could not be computed. However, we assigned most of the CNAs in ESCC as CNLOH, because many regions of CNAs showed both the log R ratio normal distribution and allelic imbalance (TIFF 1521 kb)

Supplementary Fig. 3

CNAs of some cancer-related genes in ESCC. a SNP-CGH of chromosome 11. This panel shows the amplification of the CCND1 locus in the log R ratio, the heterozygous state split into two clusters in the B allele frequency. CCND1 showed high-level amplification (>four copy gains). b SNP-CGH of chromosome nine. This panel shows the deletion of CDKN2A/p16 locus in the log R ratio, the heterozygous state split into two clusters in the B allele frequency. c SNP-CGH of chromosome 17. There was no deflection in the log R ratio, and the heterozygous state split into two clusters in the B allele frequency. (TIFF 1521 kb)

Supplementary Fig. 4

Arm-level CNAs in ESCC. The arm-level CNAs of highly involved chromosome arms are shown. Almost all p53 mutant ESCCs were accompanied by arm-level CNAs (TIFF 1521 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kawano, H., Saeki, H., Kitao, H. et al. Chromosomal Instability Associated with Global DNA Hypomethylation is Associated with the Initiation and Progression of Esophageal Squamous Cell Carcinoma. Ann Surg Oncol 21 (Suppl 4), 696–702 (2014). https://doi.org/10.1245/s10434-014-3818-z

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1245/s10434-014-3818-z

Keywords

Search

Navigation