Aberrant methylation of the maspin promoter is an early event in human breast cancer

Abstract

The maspin gene functions as a tumor suppressor in human breasts, and its expression is frequently lost during breast cancer progression. In vitro models of human breast cancer indicate that the loss of maspin expression is closely linked to aberrant methylation of the maspin promoter. We conducted a study on 30 archival ductal carcinoma in situ (DCIS) specimens to determine if aberrant methylation of the maspin promoter occurred in vivo, and whether it occurred early in breast cancer evolution. Healthy tissue obtained from reduction mammoplasty was used as normal control. Results from immunohistochemical analysis indicate that maspin expression is lost in a substantial fraction of DCIS specimens (57%). Bisulfite sequencing of DNA isolated from laser capture-microdissected normal and neoplastic ducts showed that loss of maspin expression was often, but not always, linked to aberrant methylation of the maspin promoter, suggesting that other mechanisms, in addition to aberrant methylation, participate and/or cooperate to silence maspin gene expression. Taken together, these results indicate that aberrant methylation of the maspin promoter is an early event in human breast cancer.

Figure 1

(A) Immunohistochemical analysis of maspin expression in human mammary tissue from patient 5. On the right side of the photomicrograph is a normal duct showing maspin-positive ductal epithelial cells. On the left side of the photomicrograph, a DCIS duct where the ductal epithelial cells have become maspinnegative can be seen. (B) H&E staining of an adjacent section from patient 5.

Figure 2

Maspin immunohistochemistry and corresponding H&E staining for representative normal and DCIS specimens. (A and B) Maspin expression in normal breast tissue from a healthy individual (patient A). (C and D) Maspin expression in adjacent normal ducts taken from DCIS patient 12. (E–L) Maspin expression of neoplastic ducts in DCIS patients 2, 5, 9, and 19. Maspin promoter methylation patterns of these samples are shown in the histograms in Figure 4.

Figure 3

LCM of neoplastic ductal epithelial tissue. Top panel shows a DCIS from patient 9 before LCM of neoplastic cells, whereas the middle panel shows the same tissue after LCM of the neoplastic cells. The bottom pane shows the captured cells that were analyzed for maspin promoter methylation using bisulfite sequencing.

Figure 4

Cytosine methylation status of the maspin promoter of the respective DCIS specimens shown in Figure 2. The methylation status of individual CpG sites was determined by comparison of the bisulfite sequence obtained with the known maspin sequence. The y-axis represents the percent methylation at the seven CpG sites in the region analyzed; the x-axis represents the nucleotide position relative to the maspin transcription start site, based on RefSeq data. Percent methylation of each site was determined by dividing the number of methylated CpG sites at a specific site by the total number of clones analyzed.

Figure 5

(A) Immunohistochemical analysis of maspin shows differential subcellular localization and intratumoral heterogeneity of maspin expression in DCIS patient 3. (B) H&E staining of an adjacent section from patient 3.

Figure 6

Methylation heterogeneity in phenotypically heterogeneous tumor samples. Location and methylation status of seven CpG sites in the maspin promoter are shown. Each circle represents a CpG site. Open circles are unmethylated CpG sites and filled circles are methylated CpG sites. Each row of circles represents the methylation results obtained from an individual clone. Each column indicates the location of the CpG site in the maspin promoter relative to transcription start.