Exploring DNA methylation changes in promoter, intragenic, and intergenic regions as early and late events in breast cancer formation.
BACKGROUND
Breast cancer formation is associated with frequent changes in DNA methylation but the extent of very early alterations in DNA methylation and the biological significance of cancer-associated epigenetic changes need further elucidation.


METHODS
Pyrosequencing was done on bisulfite-treated DNA from formalin-fixed, paraffin-embedded sections containing invasive tumor and paired samples of histologically normal tissue adjacent to the cancers as well as control reduction mammoplasty samples from unaffected women. The DNA regions studied were promoters (BRCA1, CD44, ESR1, GSTM2, GSTP1, MAGEA1, MSI1, NFE2L3, RASSF1A, RUNX3, SIX3 and TFF1), far-upstream regions (EN1, PAX3, PITX2, and SGK1), introns (APC, EGFR, LHX2, RFX1 and SOX9) and the LINE-1 and satellite 2 DNA repeats. These choices were based upon previous literature or publicly available DNA methylome profiles. The percent methylation was averaged across neighboring CpG sites.


RESULTS
Most of the assayed gene regions displayed hypermethylation in cancer vs. adjacent tissue but the TFF1 and MAGEA1 regions were significantly hypomethylated (p ≤0.001). Importantly, six of the 16 regions examined in a large collection of patients (105 - 129) and in 15-18 reduction mammoplasty samples were already aberrantly methylated in adjacent, histologically normal tissue vs. non-cancerous mammoplasty samples (p ≤0.01). In addition, examination of transcriptome and DNA methylation databases indicated that methylation at three non-promoter regions (far-upstream EN1 and PITX2 and intronic LHX2) was associated with higher gene expression, unlike the inverse associations between cancer DNA hypermethylation and cancer-altered gene expression usually reported. These three non-promoter regions also exhibited normal tissue-specific hypermethylation positively associated with differentiation-related gene expression (in muscle progenitor cells vs. many other types of normal cells). The importance of considering the exact DNA region analyzed and the gene structure was further illustrated by bioinformatic analysis of an alternative promoter/intron gene region for APC.


CONCLUSIONS
We confirmed the frequent DNA methylation changes in invasive breast cancer at a variety of genome locations and found evidence for an extensive field effect in breast cancer. In addition, we illustrate the power of combining publicly available whole-genome databases with a candidate gene approach to study cancer epigenetics.
Division of Epidemiology and Biostatistics, University of Illinois-Chicago, School of Public Health, M/C 923, Chicago, IL, 60612, USA. garthr@uic.edu.
Garth H
GH
Rauscher
Division of Epidemiology and Biostatistics, University of Illinois-Chicago, School of Public Health, M/C 923, Chicago, IL, 60612, USA. jkreso2@uic.edu.
Jacob K
JK
Kresovich
EpigenDx, Inc., Hopkinton, MA, USA. mpoulin@epigendx.com.
Matthew
M
Poulin
EpigenDx, Inc., Hopkinton, MA, USA. lyan@epigendx.com.
Liying
L
Yan
Department of Pathology, University of Illinois-Chicago, Chicago, IL, USA. vmacias@uic.edu.
Virgilia
V
Macias
Department of Pathology, University of Illinois-Chicago, Chicago, IL, USA. amahmo4@uic.edu.
Abeer M
AM
Mahmoud
Division of Epidemiology and Biostatistics, University of Illinois-Chicago, School of Public Health, M/C 923, Chicago, IL, 60612, USA. ualale2@uic.edu.
Umaima
U
Al-Alem
Department of Pathology, University of Illinois-Chicago, Chicago, IL, USA. aballa@uic.edu.
Andre
A
Kajdacsy-Balla
Department of Pathology, University of Illinois-Chicago, Chicago, IL, USA. ewiley@uic.edu.
Elizabeth L
EL
Wiley
Department of Biopharmaceutical Sciences, University of Illinois-Chicago, Chicago, IL, USA. dtonetti@uic.edu.
Debra
D
Tonetti
Human Genetics Program, Tulane Cancer Center, and Center for Bioinformatics and Genomics, Tulane University Health Sciences Center, 1430 Tulane Ave., New Orleans, LA, 70112, USA. ehrlich@tulane.edu.
Melanie
M
Ehrlich
D000328:Adult; D000368:Aged; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D018899:CpG Islands; D019175:DNA Methylation; D021901:DNA, Intergenic; D030541:Databases, Genetic; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006657:Histones / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA
D021901:DNA, Intergenic; D006657:Histones