Epigenetic silencing of DSC3 is a common event in human breast cancer

Abstract

Introduction: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens.

Methods: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines.

Results: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure.

Conclusion: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

Figure 1

DSC3 expression is restricted to a subset of normal human epithelial cell types. DSC3 expression relative to human mammary epithelium cells (HMECs) was assessed by real-time quantitative RT-PCR; GAPDH expression was used to normalize the data.

Figure 2

The DSC3 promoter is aberrantly methylated in primary breast tumor samples. (a) Diagram of the DSC3 promoter region analyzed (with the minimal promoter region demarcated as described in [36]). (b) Summary of 5-methylcytosine levels obtained by sodium bisulfite genomic sequencing of the DSC3 promoter. Ten to twelve cloned PCR products were sequenced to determine the percent methylation of the 24 CpG sites in the region analyzed. Cytosine methylation frequency histograms are shown for normal HMECs and eight primary tumor specimens. The y-axis is percent cytosine methylation and the x-axis is the nucleotide position relative to the transcription start site.

Figure 3

DSC3 gene expression is silenced or greatly reduced in a high percentage of breast tumor cell lines. DSC3 expression relative to human mammary epithelium cells (HMECs) was assessed by real-time quantitative RT-PCR; GAPDH expression was used to normalize the data.

Figure 4

DSC3 protein is not expressed in breast tumor cells with undetectable DSC3 mRNA levels. Protein expression was analyzed by western blot analysis. MCF10A and HaCaT cells were used as positive controls for DSC3 expression.

Figure 5

The DSC3 promoter is aberrantly methylated in breast tumor cell lines. Ten to twelve cloned PCR products were sequenced to determine the percent methylation of the 24 CpG sites in the region analyzed. Cytosine methylation frequency histograms are shown for human mammary epithelium cells (HMECs) and the immortalized non-tumorigenic MCF10A cells, and seven human breast cancer cell lines examined. The y-axis is percent cytosine methylation and the x-axis is the nucleotide position relative to the transcription start site.

Figure 6

Hypermethylated DSC3 promoter regions are inaccessible to in vivo MspI endonuclease digestion. Intact nuclei were isolated from MDA-MB-231 and UACC1179 cells and digested in vivo with MspI. Isolated DNA was ligated with a linker specific to the MspI ends, and hemi-nested, linker mediated PCR was conducted with two rounds of PCR with two gene specific primers. Increased amounts of PCR product reveal the presence of accessible chromatin. Inset within the graph is the average calculated fold decrease and standard deviation when MDA-MB-231 and UACC1179 cells are compared to MCF10A. The graph shown is representative of three independent replicates.