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

Aslaug Aa Muggerud,Florence Busato,Jovana Jovanovic,Fredrik Wärnberg,Hiroko Solvang,Ida Bukholm,Vessela N Kristensen,Jo Anders Rønneberg,Jörg Tost,Anne-Lise Børresen-Dale,Johan Botling,Therese Sørlie

doi:10.1186/bcr2466

Abstract

IntroductionDuctal 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.MethodsQuantitative 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.ResultsAberrant 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.ConclusionsQuantitative 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.

Highlights

Ductal carcinoma in situ (DCIS) is a non-invasive lesion of the breast that is frequently detected by mammography and subsequently removed by surgery
Such genomic aberrations lead to altered gene expression, and comprehensive gene expression studies comparing DCIS and invasive ductal cancer (IDC) have identified stage-specific markers ([4,5,6] and Muggerud et al, submitted) along with a gene expression classifier which differed between DCIS and invasive breast cancer [7]
Aberrant DNA methylation was observed in all three diagnosis groups for the following genes: ABCB1 (40.7% vs. 39.3% vs. 44.1%), FOXC1 (22.2% vs. 53.6% vs. 67.6%), GSTP1 (22.2% vs. 14.3% vs. 26.5%), MGMT (3.7% vs. 3.6% vs. 5.9%), MLH1 (7.4% vs. 3.6% vs. 2.9%), PPP2R2B (55.0% vs. 78.6% vs. 70.6%), CDKN2A/p16INK4a (0% vs. 10.7% vs. 5.9%), PTEN (18.5% vs. 14.3% vs. 23.5%) and RASSF1A (85.2% vs. 82.1% vs. 85.3%)

Summary

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. The multistep model of breast cancer progression suggests a transition from normal epithelium to invasive carcinoma via intraductal hyperplasia and in situ carcinoma [1]. These presumptive precursor lesions are currently defined by their histological features. Copy number alterations are already present in DCIS but their frequency tends to increase in IDCs [3] Such genomic aberrations lead to altered gene expression, and comprehensive gene expression studies comparing DCIS and IDCs have identified stage-specific markers ([4,5,6] and Muggerud et al, submitted) along with a gene expression classifier which differed between DCIS and invasive breast cancer [7]. The frequency of TP53 mutations in DCIS is similar to what is observed in invasive tumours and in situ and invasive components from the same tumour exhibit the same mutations, indicating the same cellular origin of the two components [8,9,10]

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