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

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.

Association of aberrant DNA methylation with clinicopathological features in breast cancer

“…Our findings fall in the middle of the reported range (33.3%) in accordance with several previous studies (19,27,(31)(32)(33). As far as the two major histological subtypes are concerned, a similar frequency of methylation was detected as previously reported (6,29,34). Interestingly, a statistically-significant relationship between RASSF1A promoter methylation and advanced T-category was found in accordance to Karray-Chouayekh et al (35) suggesting its involvement as a late event in breast tumourigenesis.…”

“…As far as invasive ductal breast carcinomas are concerned, methylation up to 32% of the cases has been reported (28,34,36) in accordance with our findings. Finally, in invasive lobular breast carcinomas, for which there are currently no data, we detected methylation in a subset of the examined cases (11.1%).…”

Dysregulation of microRNA‐204 mediates migration and invasion of endometrial cancer by regulating FOXC1

“…26 Muggerud et al also reported that FOXC1 knockdown suppressed cell proliferation, migration and invasion in breast cancer cells. 27 Taube et al reported that FOXC1 involvement in an epithelial to mesenchymal transition signature that correlates strongly with poor outcomes in breast cancer. 28 These reports are consistent with our results that FOXC1 may play a role in endometrial cancer migration and invasion.…”

Ductal carcinoma in situ of the breast: the importance of morphologic and molecular interactions

“…These findings suggest that most of the dramatic epigenetic changes take place during the transition from normal epithelium to DCIS and that aberrant methylation may not contribute to the development of invasion but rather play an important role in early breast carcinogenesis [58][59][60]. Recent studies, some of which have used a genome-wide methylation screen approach have described a small set of genes such as APC, CACNA1A, CDH1, FOXC1, HOXA10, MGMT, SFPR1, TFAP2A, and TWIST1 that exhibit differences in either frequency or density of methylation between DCIS and invasive carcinoma [61][62][63][64][65][66][67][68][69][70].…”