Yonsei Med J.  2017 Jul;58(4):749-755. 10.3349/ymj.2017.58.4.749.

Lack of Aberrant Methylation in an Adjacent Area of Left-Sided Colorectal Cancer

• Sambuudash O
• Kim HS
• Cho MY

• KMID: 2419080
• DOI: http://doi.org/10.3349/ymj.2017.58.4.749

Abstract

PURPOSE
The molecular nature and the rate-limiting step of epigenetic field defects in the evolution of left-sided colorectal cancer (LCA) remain uncertain.
MATERIALS AND METHODS
The methylation status of 27 candidate field defect markers, six classic CpG island methylator phenotype (CIMP) markers, and LINE-1 were determined in LCA and adjacent normal mucosas (ADJs) from 33 LCA patients and in left normal colorectal mucosa (LNM) from 33 age- and sex-matched controls. Hotspot mutation analyses in KRAS codons 12 and 13 and BRAF V600E were performed by genomic PCR and pyrosequencing using DNA extracted from endoscopically biopsied tissues.
RESULTS
Among the 27 candidate genes tested, we confirmed 15 differentially methylated genes in cancer (15 DMGs; ER, SFRP1, MYOD1, MGMT, CD8a, SPOCK2, ABHD9, BNIP3, IGFBP3, WIF1, MAL, GDNF, ALX4, DOK5, and SLC16A12) in comparison to ADJ samples. We further compared the methylation status of 15 DMGs of ADJs to LNM and found only methylation levels of SLC16A12 in ADJs of LCA patients to be significantly higher than that in LNM (17.3% vs. 11.5%, p=0.002). Based on the CIMP, no significant differences in methylation levels of the 15 DMGs were found between ADJs in CIMP positive LCA cases and those without CIMP. In mutation analyses, no mutation was found in ADJs, while significant KRAS mutations (6/33, 18%) were noted in LCA samples.
CONCLUSION
Epigenetic field defect marked by aberrant methylation is uncommon in normal-appearing ADJs of LCA, indicating the critical rate-limiting change of methylation is likely to occur with morphological alterations in the evolution of LCA.

Keyword

Methylation; colorectal neoplasms; CpG islands; phenotype; field

MeSH Terms

Age Factors
Biomarkers, Tumor/genetics
Case-Control Studies
Colorectal Neoplasms/*genetics/pathology
CpG Islands/genetics
DNA Methylation/*genetics
Female
Humans
Male
Middle Aged
Mutation/genetics
Phenotype
Polymerase Chain Reaction
Promoter Regions, Genetic/genetics
Proto-Oncogene Proteins B-raf/genetics
Proto-Oncogene Proteins p21(ras)/genetics
Biomarkers, Tumor
Proto-Oncogene Proteins B-raf
Proto-Oncogene Proteins p21(ras)

Reference

1. Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer. 1953; 6:963–968. PMID: 13094644.

2. Braakhuis BJ, Tabor MP, Kummer JA, Leemans CR, Brakenhoff RH. A genetic explanation of Slaughter's concept of field cancerization: evidence and clinical implications. Cancer Res. 2003; 63:1727–1730. PMID: 12702551.

3. Grady WM. Epigenetic events in the colorectum and in colon cancer. Biochem Soc Trans. 2005; 33(Pt 4):684–688. PMID: 16042574.

4. Luo Y, Yu M, Grady WM. Field cancerization in the colon: a role for aberrant DNA methylation? Gastroenterol Rep (Oxf). 2014; 2:16–20. PMID: 24760232.

5. Yi JM, Kim TO. Epigenetic alterations in inflammatory bowel disease and cancer. Intest Res. 2015; 13:112–121. PMID: 25931995.

6. Baba Y, Ishimoto T, Kurashige J, Iwatsuki M, Sakamoto Y, Yoshida N, et al. Epigenetic field cancerization in gastrointestinal cancers. Cancer Lett. 2016; 375:360–366. PMID: 26971491.

7. Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet. 2000; 16:168–174. PMID: 10729832.

8. Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet. 2006; 7:21–33. PMID: 16369569.

9. Rashid A, Shen L, Morris JS, Issa JP, Hamilton SR. CpG island methylation in colorectal adenomas. Am J Pathol. 2001; 159:1129–1135. PMID: 11549606.

10. Li H, Myeroff L, Smiraglia D, Romero MF, Pretlow TP, Kasturi L, et al. SLC5A8, a sodium transporter, is a tumor suppressor gene silenced by methylation in human colon aberrant crypt foci and cancers. Proc Natl Acad Sci U S A. 2003; 100:8412–8417. PMID: 12829793.

11. Kondo Y, Issa JP. Epigenetic changes in colorectal cancer. Cancer Metastasis Rev. 2004; 23:29–39. PMID: 15000147.

12. Shen L, Kondo Y, Rosner GL, Xiao L, Hernandez NS, Vilaythong J, et al. MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst. 2005; 97:1330–1338. PMID: 16174854.

13. Esteller M. Epigenetic lesions causing genetic lesions in human cancer: promoter hypermethylation of DNA repair genes. Eur J Cancer. 2000; 36:2294–2300. PMID: 11094302.

14. Issa JP. CpG island methylator phenotype in cancer. Nat Rev Cancer. 2004; 4:988–993. PMID: 15573120.

15. Issa JP, Ahuja N, Toyota M, Bronner MP, Brentnall TA. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res. 2001; 61:3573–3577. PMID: 11325821.

16. Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994; 22:2990–2997. PMID: 8065911.

17. Colella S, Shen L, Baggerly KA, Issa JP, Krahe R. Sensitive and quantitative universal pyrosequencing methylation analysis of CpG sites. Biotechniques. 2003; 35:146–150. PMID: 12866414.

18. Estécio MR, Gharibyan V, Shen L, Ibrahim AE, Doshi K, He R, et al. LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability. PLoS One. 2007; 2:e399. PMID: 17476321.

19. Ogino S, Kawasaki T, Brahmandam M, Yan L, Cantor M, Namgyal C, et al. Sensitive sequencing method for KRAS mutation detection by pyrosequencing. J Mol Diagn. 2005; 7:413–421. PMID: 16049314.

20. Shen L, Toyota M, Kondo Y, Lin E, Zhang L, Guo Y, et al. Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer. Proc Natl Acad Sci U S A. 2007; 104:18654–18659. PMID: 18003927.

21. Estécio MR, Yan PS, Ibrahim AE, Tellez CS, Shen L, Huang TH, et al. High-throughput methylation profiling by MCA coupled to CpG island microarray. Genome Res. 2007; 17:1529–1536. PMID: 17785535.

22. Chung W, Kwabi-Addo B, Ittmann M, Jelinek J, Shen L, Yu Y, et al. Identification of novel tumor markers in prostate, colon and breast cancer by unbiased methylation profiling. PLoS One. 2008; 3:e2079. PMID: 18446232.

23. Yamashita S, Tsujino Y, Moriguchi K, Tatematsu M, Ushijima T. Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2'-deoxycytidine treatment and oligonucleotide microarray. Cancer Sci. 2006; 97:64–71. PMID: 16367923.

24. Hasegawa S, Furukawa Y, Li M, Satoh S, Kato T, Watanabe T, et al. Genome-wide analysis of gene expression in intestinal-type gastric cancers using a complementary DNA microarray representing 23,040 genes. Cancer Res. 2002; 62:7012–7017. PMID: 12460921.

25. Deng D, El-Rifai W, Ji J, Zhu B, Trampont P, Li J, et al. Hypermethylation of metallothionein-3 CpG island in gastric carcinoma. Carcinogenesis. 2003; 24:25–29. PMID: 12538345.

26. Mikata R, Yokosuka O, Fukai K, Imazeki F, Arai M, Tada M, et al. Analysis of genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine in gastric cancer cell lines. Int J Cancer. 2006; 119:1616–1622. PMID: 16671088.

27. Chung SK, Lee MG, Ryu BK, Lee JH, Han J, Byun DS, et al. Frequent alteration of XAF1 in human colorectal cancers: implication for tumor cell resistance to apoptotic stresses. Gastroenterology. 2007; 132:2459–2477. PMID: 17570219.

28. Ebert MP, Model F, Mooney S, Hale K, Lograsso J, Tonnes-Priddy L, et al. Aristaless-like homeobox-4 gene methylation is a potential marker for colorectal adenocarcinomas. Gastroenterology. 2006; 131:1418–1430. PMID: 17101318.

29. Watanabe Y, Toyota M, Kondo Y, Suzuki H, Imai T, Ohe-Toyota M, et al. PRDM5 identified as a target of epigenetic silencing in colorectal and gastric cancer. Clin Cancer Res. 2007; 13:4786–4794. PMID: 17699856.

30. Issa JP, Ottaviano YL, Celano P, Hamilton SR, Davidson NE, Baylin SB. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet. 1994; 7:536–540. PMID: 7951326.

31. Goel A, Nagasaka T, Arnold CN, Inoue T, Hamilton C, Niedzwiecki D, et al. The CpG island methylator phenotype and chromosomal instability are inversely correlated in sporadic colorectal cancer. Gastroenterology. 2007; 132:127–138. PMID: 17087942.

32. Boland CR, Shin SK, Goel A. Promoter methylation in the genesis of gastrointestinal cancer. Yonsei Med J. 2009; 50:309–321. PMID: 19568590.

33. Zou H, Harrington JJ, Shire AM, Rego RL, Wang L, Campbell ME, et al. Highly methylated genes in colorectal neoplasia: implications for screening. Cancer Epidemiol Biomarkers Prev. 2007; 16:2686–2696. PMID: 18086775.

34. Chan AO, Broaddus RR, Houlihan PS, Issa JP, Hamilton SR, Rashid A. CpG island methylation in aberrant crypt foci of the colorectum. Am J Pathol. 2002; 160:1823–1830. PMID: 12000733.

35. Jothy S, Slesak B, Harłozin´ska A, Lapin´ska J, Adamiak J, Rabczyn´ski J. Field effect of human colon carcinoma on normal mucosa: relevance of carcinoembryonic antigen expression. Tumour Biol. 1996; 17:58–64. PMID: 7501974.

36. Nguyen C, Liang G, Nguyen TT, Tsao-Wei D, Groshen S, Lübbert M, et al. Susceptibility of nonpromoter CpG islands to de novo methylation in normal and neoplastic cells. J Natl Cancer Inst. 2001; 93:1465–1472. PMID: 11584062.

37. Ushijima T, Nakajima T, Maekita T. DNA methylation as a marker for the past and future. J Gastroenterol. 2006; 41:401–407. PMID: 16799880.

38. Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene. 2002; 21:5400–5413. PMID: 12154403.

39. Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, et al. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 2000; 28:E32. PMID: 10734209.