Research article
Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer
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* Corresponding author: Vessela N Kristensen vessela.kristensen@medisin.uio.no
- † Equal contributors
1 Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Montebello, Oslo, N-0310, Norway
2 Faculty of Medicine, Division The Norwegian Radium Hospital, University of Oslo, Montebello, Oslo, N-0310, Norway
3 Department of Surgery, Uppsala University Hospital, Dag Hammarskjölds väg 20, Uppsala, SE-751 85, Sweden
4 Department of Genetics and Pathology, Uppsala University Hospital, Dag Hammarskjölds väg 20, Uppsala, SE-751 85, Sweden
5 Institute for Clinical Epidemiology and Molecular Biology (EpiGen), Faculty of Medicine, Division Akershus University Hospital, University of Oslo, Sykehusveien 27, Nordbyhagen, N-1474, Norway
6 Department of Surgery, Akershus University Hospital, Sykehusveien 27, Nordbyhagen, N-1474, Norway
7 Faculty of Medicine, Division Akershus University Hospital, Sykehusveien 27, Nordbyhagen, N-1474, Norway
8 Biomedical Research Group, Department of Informatics, University of Oslo, P.O. Box 1080 Blindern, Oslo, N-0316, Norway
9 Laboratory for Epigenetics, Centre National de Génotypage, CEA-Institut de Génomique, 2 rue Gaston Crèmieux, 91000 Evry, France
10 Department of Biostatistics, Institute of Basic Medical Science, University of Oslo, P.O. Box 1122 Blindern, Oslo, N-0317, Norway
Breast Cancer Research 2010, 12:R3 doi:10.1186/bcr2466
Published: 7 January 2010Abstract
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.