Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer

Abstract

Introduction: Ductal carcinoma in situ (DCIS) is a non-invasive lesion of the breast that is frequently detected by mammography and subsequently removed by surgery. However, it is estimated that about half of the detected lesions would never have progressed into invasive cancer. Identifying DCIS and invasive cancer specific epigenetic lesions and understanding how these epigenetic changes are involved in triggering tumour progression is important for a better understanding of which lesions are at risk of becoming invasive.

Methods: Quantitative DNA methylation analysis of ABCB1, CDKN2A/p16INK4a, ESR1, FOXC1, GSTP1, IGF2, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A was performed by pyrosequencing in a series of 27 pure DCIS, 28 small invasive ductal carcinomas (IDCs), 34 IDCs with a DCIS component and 5 normal breast tissue samples. FOXC1, ABCB1, PPP2R2B and PTEN were analyzed in 23 additional normal breast tissue samples. Real-Time PCR expression analysis was performed for FOXC1.

Results: Aberrant DNA methylation was observed in all three diagnosis groups for the following genes: ABCB1, FOXC1, GSTP1, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A. For most of these genes, methylation was already present at the DCIS level with the same frequency as within IDCs. For FOXC1 significant differences in methylation levels were observed between normal breast tissue and invasive tumours (P < 0.001). The average DNA methylation levels were significantly higher in the pure IDCs and IDCs with DCIS compared to pure DCIS (P = 0.007 and P = 0.001, respectively). Real-time PCR analysis of FOXC1 expression from 25 DCIS, 23 IDCs and 28 normal tissue samples showed lower gene expression levels of FOXC1 in both methylated and unmethylated tumours compared to normal tissue (P < 0.001). DNA methylation levels of FOXC1, GSTP1, ABCB1 and RASSF1A were higher in oestrogen receptor (ER) positive vs. ER negative tumours; whereas methylation levels of FOXC1, ABCB1, PPP2R2B and PTEN were lower in tumours with a TP53 mutation.

Conclusions: Quantitative methylation analysis identified ABCB1, FOXC1, PPP2R2B and PTEN as novel genes to be methylated in DCIS. In particular, FOXC1 showed a significant increase in the methylation frequency in invasive tumours. Low FOXC1 gene expression in both methylated and unmethylated DCIS and IDCs indicates that the loss of its expression is an early event during breast cancer progression.

Figure 1

Methylation overview. A: Bar chart displaying promoter methylation frequencies across the three diagnosis groups. Methylation frequency is defined as the number of methylated samples within each category. The average values of methylation for all CpGs were calculated for each sample and each gene. A sample was scored as hypermethylated if the measured methylation values were two times above the standard deviation of the mean of the normal controls, and conversely, as hypomethylated if methylation values were below two times the standard deviation of the mean of the normal control tissues. DCIS = light grey, pure invasive = dark grey, and mixed = black. B: Methylation overview per gene across the three diagnosis groups. Black boxes indicate methylated and white boxes indicate unmethylated samples. For the imprinted gene IGF2; white boxes indicate the expected allele-specific methylation, black boxes indicate hypermethylation, and grey boxes indicate hypomethylation.

Figure 2

Newly identified aberrantly methylated genes in DCIS. Differences in the average DNA methylation (%) between normal and DCIS tissue for the newly identified methylated genes in DCIS; PPP2R2B, ABCB1, FOXC1 and PTEN. The average DNA methylation (%) value is the average value of methylation for all CpGs calculated for each sample. Abbreviations: N = normal tissue, D = DCIS.

Figure 3

Differential FOXC1 methylation across diagnosis groups and subsequent validation by qRT-PCR. A: Differences in FOXC1 average DNA methylation (%) between normal breast tissue and the different diagnosis groups. The FOXC1 average DNA methylation (%) value is the average value of methylation for all CpGs calculated for each tumour sample. B: Differences in relative expression levels of FOXC1 as measured by qRT-PCR in normal breast tissue versus methylated and unmethylated tumours. Expression of FOXC1 was measured relative to the expression of the reference gene PGK1. Black horizontal bars represent median value for each diagnosis group.

Figure 4

Association between clinicopathological factors and DNA methylation. A: FOXC1, GSTP1, ABCB1 and RASSF1A were significantly differentially methylated between ER-positive and ER-negative tumours. B: FOXC1, ABCB1, PPP2R2B and PTEN were significantly differentially methylated between TP53 wild type and mutated tumours. C: ABCB1 was significantly differentially methylated between Ki67-negative and Ki67-positive tumours and GSTP1 was significantly differentially methylated between PR-positive and PR-negative tumours. All types of lesions were combined for these analyses. All P-values were obtained by using a false discovery rate <5%.