Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer.
BACKGROUND
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.
Muggerud
Aslaug Aa
AA
Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Montebello, Oslo, N-0310, Norway.
Rønneberg
Jo Anders
JA
Wärnberg
Fredrik
F
Botling
Johan
J
Busato
Florence
F
Jovanovic
Jovana
J
Solvang
Hiroko
H
Bukholm
Ida
I
Børresen-Dale
Anne-Lise
AL
Kristensen
Vessela N
VN
Sørlie
Therese
T
Tost
Jörg
J
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000235:genetics* / Q000473:pathology; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D051858:Forkhead Transcription Factors / Q000235:genetics*; D016158:Genes, p53; D006801:Humans; D009154:Mutation; D009361:Neoplasm Invasiveness; D009419:Nerve Tissue Proteins / Q000235:genetics*; D051059:PTEN Phosphohydrolase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D054648:Protein Phosphatase 2 / Q000235:genetics*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C112743:FOXC1 protein, human; D051858:Forkhead Transcription Factors; D009419:Nerve Tissue Proteins; C505936:PPP2R2B protein, human; D054648:Protein Phosphatase 2; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human