Promoter CpG island hypermethylation during breast cancer progression

Abstract

This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions [flat epithelial atypia (FEA), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS)] to invasive ductal carcinoma (IDC). We performed MethyLight analysis for the methylation status of 57 promoter CpG island loci in 20 IDCs and their paired normal breast tissues. After selecting 15 CpG island loci showing breast cancer-specific DNA methylation, another set of normal breast tissue (n = 10), ADH/FEA (n = 30), DCIS (n = 35), and IDC (n = 30) of the breast were analyzed for these loci. We found six new methylation markers of breast cancer, namely DLEC1, GRIN2B, HOXA1, MT1G, SFRP4, and TMEFF2, in addition to APC, GSTP1, HOXA10, IGF2, RARB, RASSF1A, RUNX3, SCGB3A1 (HIN-1), and SFRP1. The number of methylated genes increased stepwise from normal breast to ADH/FEA and DCIS, while IDC did not differ from DCIS. Methylation levels and frequencies of APC, DLEC1, HOXA1, and RASSF1A promoter CpG islands were significantly higher in ADH/FEA than in normal breast tissue. GRIN2B, GSTP1, HOXA1, RARB, RUNX3, SFRP1, and TMEFF2 showed higher methylation levels and frequencies in DCIS than in ADH/FEA. DICS and IDC did not differ in the methylation levels or frequencies for most CpG island loci except SFRP1 and HOXA10. Our findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and was similar in IDC and DCIS, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression.

Distinct patterns of promoter CpG island methylation of breast cancer subtypes are associated with stem cell phenotypes

“…MethyLight reaction primers and probes for the 15-CpG island loci (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF20) were used as described previously. 13 Briefly, MethyLight PCR was performed in 30-ml volumes containing 0.2 mM deoxyribonucleotide triphosphates, 0.3 mM forward and reverse primers, 0.05 mM probe, 3.5 mM MgCl 2 , 0.05% gelatin, 0.01% Tween-20, 1 Â PCR buffer, and 0.5 U of AmpliTaq Gold polymerase. The amplification conditions for all primers consisted of 95 1C for 10 min, followed by 50 cycles of 95 1C for 20 s, and 59 1C for 40 s. ALU repeats were used as an internal reference to normalize input DNA and to generate a standard curve.…”

“…13 The different samples varied in levels of methylation of the 15 genes, as shown in the methylation map of the PMR values obtained for each CpG island locus (Figure 1). First, we compared the number of methylated CpG islands according to subtype.…”

“…8,9 In breast cancer, promoter CpG island hypermethylation has been described for genes involved in all aspects of cellular function. 7 There are reports that promoter CpG island hypermethylation is associated with various histopathologic characteristics of breast cancers, including histologic type, 10,11 tumor grade, 12,13 hormone receptor, and HER2/neu status. [14][15][16][17] Array-based comprehensive DNA methylation profiling has shown that breast cancer molecular subtypes have their own methylation profiles.…”

“…13 We investigated in particular whether the distinct patterns of CpG island methylation in each subtype reflected their cancer stem cell phenotypes as represented by CD44 þ /CD24À phenotype and ALDH1 expression. Formalin-fixed, paraffin-embedded archival tissue from 129 patients who underwent surgical resection for invasive breast cancer between 2004 and 2009 were selected from the files of the Department of Pathology, Seoul National University Bundang Hospital, based on immunohistochemical findings of ER, PR, HER2, Ki-67, cytokeratin 5/6 and EGFR, and breast cancer subtype defined by immunohistochemical profile.…”

“…In addition, we included immunohistochemical and methylation data for 50 cases of invasive breast cancer from a previous study. 13 All patients were female, with a mean age of 51 (range, 20-85 years). Clinicopathologic information was obtained by reviewing pathology reports and hematoxylin and eosin-stained sections.…”