Breast carcinogenesis is a multistep process involving genetic and epigenetic changes. Tumor tissues are frequently characterized by gene-specific hypermethylation and global DNA hypomethylation. Aberrant DNA methylation levels have, however, not only been found in tumors, but also in tumor-surrounding tissue appearing histologically normal. This phenomenon is called field cancerization. Knowledge of the existence of a cancer field and its spread are of clinical relevance. If the tissue showing pre-neoplastic lesions is not removed by surgery, it may develop into invasive carcinoma.
We investigated the prevalence of gene-specific and global DNA methylation changes in tumor-adjacent and tumor-distant tissues in comparison to tumor tissues from the same breast cancer patients (n = 18) and normal breast tissues from healthy women (n = 4). Methylation-sensitive high resolution melting (MS-HRM) analysis was applied to determine methylation levels in the promoters of APC, BRCA1, CDKN2A (p16), ESR1, HER2/neu and PTEN, in CDKN2A exon 2 and in LINE-1, as indicator for the global DNA methylation extent. The methylation status of the ESR2 promoter was determined by pyrosequencing.
Tumor-adjacent and tumor-distant tissues frequently showed pre-neoplastic gene-specific and global DNA methylation changes. The APC promoter (p = 0.003) and exon 2 of CDKN2A (p < 0.001) were significantly higher methylated in tumors than in normal breast tissues from healthy women. For both regions, significant differences were also found between tumor and tumor-adjacent tissues (p = 0.001 and p < 0.001, respectively) and tumor and tumor-distant tissues (p = 0.001 and p < 0.001, respectively) from breast cancer patients. In addition, tumor-adjacent (p = 0.002) and tumor-distant tissues (p = 0.005) showed significantly higher methylation levels of CDKN2A exon 2 than normal breast tissues serving as control. Significant correlations were found between the proliferative activity and the methylation status of CDKN2A exon 2 in tumor (r = −0.485, p = 0.041) and tumor-distant tissues (r = −0.498, p = 0.036).
From our results we can conclude that methylation changes in CDKN2A exon 2 are associated with breast carcinogenesis. Further investigations are, however, necessary to confirm that hypermethylation of CDKN2A exon 2 is associated with tumor proliferative activity.
Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490(7418):61–70. CrossRef
Esteller M. Epigenetic gene silencing in cancer: the DNA hypermethylome. Hum Mol Genet. 2007;16(1):R50–9.
Chai H, Brown RE. Field effect in cancer-an update. Ann Clin Lab Sci. 2009;39(4):331–7. PubMed
Lewis CM, Cler LR, Bu DW, Zöchbauer-Müller S, Milchgrub S, Naftalis EZ, et al. Promoter hypermethylation in benign breast epithelium in relation to predicted breast cancer risk. Clin Cancer Res. 2005;11(1):166–72. PubMed
Heaphy CM, Griffith JK, Bisoffi M. Mammary field cancerization: molecular evidence and clinical importance. Breast Cancer Res Tr. 2009;118(2):229–39. CrossRef
Hoque MO, Prencipe M, Poeta ML, Barbano R, Valori VM, Copetti M, et al. Changes in CpG islands promoter methylation patterns during ductal breast carcinoma progression. Cancer Epidemiol Biomark Prev. 2009;18(10):2694–700. CrossRef
Xu X, Gammon MD, Zhang Y, Cho YH, Wetmur JG, Bradshaw PT, et al. Gene promoter methylation is associated with increased mortality among women with breast cancer. Breast Cancer Res Tr. 2010;121(3):685–92. CrossRef
Spitzwieser M, Pirker C, Koblmüller B, Pfeiler G, Hacker S, Berger W et al. Promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in human cancer cell lines, multidrug-resistant cell models and tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients. Oncotarget. 2016;7(45):73347–69.
Wong EM, Southey MC, Fox SB, Brown MA, Dowty JG, Jenkins MA, et al. Constitutional methylation of the BRCA1 promoter is specifically associated with BRCA1 mutation-associated pathology in early-onset breast cancer. Cancer Prev Res. 2011;4(1):23–33. CrossRef
Huang KT, Dobrovic A, Yan M, Karim RZ, Lee CS, Lakhani SR, et al. DNA methylation profiling of phyllodes and fibroadenoma tumours of the breast. Breast Cancer Res Tr. 2010;124(2):555–65. CrossRef
Tserga A, Michalopoulos NV, Levidou G, Korkolopoulou P, Zografos G, Patsouris E, et al. Association of aberrant DNA methylation with clinicopathological features in breast cancer. Oncol Rep. 2012;27(5):1630–8. PubMed
National Center for Biotechnology Information (NCBI). http://www.ncbi.nlm.nih.gov/nuccore/. Accessed 21 Mar 2016.
Transcriptional Regulatory Element Database (TRED). http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=searchPromForm. Accessed 21 Mar 2016.
Eukaryotic Promoter Database (EPD). http://epd.vital-it.ch/human/human_database.php. Accessed 30/05/2016.
Office of Pesticide Programs, U.S. Environmental Protection Agency Washington, DC. Assigning values to non-detected/non-quantified pesticide residues in human health food exposure assessments 2000. https://archive.epa.gov/pesticides/trac/web/pdf/trac3b012.pdf. Accessed 30 May 2016.
Woodcock DM, Linsenmeyer ME, Doherty JP, Warren WD. DNA methylation in the promoter region of the p16 ( CDKN2/ MTS-1/ INK4A) gene in human breast tumours. Brit J Can. 1999;79(2):251–6. CrossRef
Tao MH, Shields PG, Nie J, Millen A, Ambrosone CB, Edge SB, et al. DNA hypermethylation and clinicopathological features in breast cancer: the western New York exposures and breast cancer (WEB) study. Breast Cancer Res Tr. 2009;114(3):559–68. CrossRef
Xu X, Gammon MD, Zhang Y, Bestor TH, Zeisel SH, Wetmur JG, et al. BRCA1 promoter methylation is associated with increased mortality among women with breast cancer. Breast Cancer Res Tr. 2009;115(2):397–404. CrossRef
Wei M, Xu J, Dignam J, Nanda R, Sveen L, Fackenthal J et al. Estrogen receptor alpha, BRCA1, and FANCF promoter methylation occur in distinct subsets of sporadic breast cancers. Breast Cancer Res Tr. 2008;111(1):113-120.
Esteller M, Sparks A, Toyota M, Sanchez-Cespedes M, Capella G, Peinado MA, et al. Analysis of adenomatous polyposis coli promoter hypermethylation in human cancer. Cancer Res. 2000;60(16):4366–71. PubMed
Virmani AK, Rathi A, Sathyanarayana UG, Padar A, Huang CX, Cunnigham HT, et al. Aberrant methylation of the adenomatous polyposis coli ( APC) gene promoter 1A in breast and lung carcinomas. Clin Cancer Res. 2001;7(7):1998–2004. PubMed
Jin Z, Tamura G, Tsuchiya T, Sakata K, Kashiwaba M, Osakabe M, et al. Adenomatous polyposis coli (APC) gene promoter hypermethylation in primary breast cancers. Brit J Can. 2001;85(1):69–73. CrossRef
Sarrío D, Moreno-Bueno G, Hardisson D, Sánchez-Estévez C, Guo M, Herman JG, et al. Epigenetic and genetic alterations of APC and CDH1 genes in lobular breast cancer: relationships with abnormal E-cadherin and catenin expression and microsatellite instability. Int J Cancer. 2003;106(2):208–15. CrossRefPubMed
García JM, Silva J, Peña C, García V, Rodríguez R, Cruz MA, et al. Promoter methylation of the PTEN gene is a common molecular change in breast cancer. Gene Chromosome Canc. 2004;41(2):117–24. CrossRef
Martínez-Galán J, Torres-Torres B, Núñez MI, López-Peñalver J, Del Moral R, Ruiz De Almodóvar JM, et al. ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients. BMC Cancer. 2014;14:59. CrossRefPubMedPubMedCentral
Medina-Jaime AD, Reyes-Vargas F, Martinez-Gaytan V, Zambrano-Galvan G, Portillo-Delcampo E, Burciaga-Nava JA, et al. ESR1 and PGR gene promoter methylation and correlations with estrogen and progesterone receptors in ductal and lobular breast cancer. Asian Pac J Cancer Prev. 2014;15(7):3041–4. CrossRefPubMed
Rody A, Holtrich U, Solbach C, Kourtis K, von Minckwitz G, Engels K, et al. Methylation of estrogen receptor beta promoter correlates with loss of ER-beta expression in mammary carcinoma and is an early indication marker in premalignant lesions. Endocr-Relat Cancer. 2005;12(4):903–16. CrossRefPubMed
van Hoesel AQ, van de Velde CJ, Kuppen PJ, Liefers GJ, Putter H, Sato Y, et al. Hypomethylation of LINE-1 in primary tumor has poor prognosis in young breast cancer patients: a retrospective cohort study. Breast Cancer Res Tr. 2012;134(3):1103–14. CrossRef
Branham MT, Marzese DM, Laurito SR, Gago FE, Orozco JI, Tello OM, et al. Methylation profile of triple-negative breast carcinomas. Oncogene. 2012;1:e17. CrossRef
Cho YH, Shen J, Gammon MD, Zhang YJ, Wang Q, Gonzalez K, et al. Prognostic significance of gene-specific promoter hypermethylation in breast cancer patients. Breast Cancer Res Tr. 2012;131(1):197–205. CrossRef
- Hypermethylation of CDKN2A exon 2 in tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II