Relative levels of DSC3 mRNA expression from a panel of normal tissue RNA were determined by quantitative real time RT-PCR analysis. DSC3 mRNA levels were normalized to the ubiquitously expressed GAPDH gene, then expression values reported relative to the primary HMEC line expression (Fig. ). DSC3 expression was limited to certain epithelial cell types, including those of the airway, breast, skin, prostate, and mouth. DSC3 was undetectable in the following non-epithelial cell types: skin fibroblasts, lymphocytes, bone marrow, heart, and kidney. Therefore, expression analysis of DSC3 shows a significant cell type specific pattern of expression, which is limited to cells of epithelial origin, including breast epithelium.
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.
To confirm and extend previous studies [6
], we analyzed 32 frozen breast cancer specimens from patients who underwent lumpectomy or mastectomy randomly obtained from patients who underwent surgery at the University Medical Center in Tucson, AZ. Our data set comprised 32 specimens: 24 invasive ductal carcinomas (IDC), two of which are metastatic IDCs isolated from patients' lymph nodes, seven invasive lobular carcinomas (ILCs), and one mucoid ductal carcinoma. Incidentally, we received two independent tumors from one diseased breast both of which were IDC specimens. From these specimens, total RNA was collected and DSC3
expression was analyzed by quantitative real time RT-PCR with expression levels of the tumor samples being normalized to HMECs. DSCs are expressed in both 'a' and 'b' isoforms as a result of alternate splicing of exon 16. The ABI probe used in these studies spans exon1 and exon2, which are both conserved in the DSC3a
isoforms, allowing us to analyze both isoforms in the specimens tested. DSC3
expression is reduced to less than 10% of the expression seen in HMEC in 18 of 24 (75%) of the IDCs, 5 of 7 (71%) ILCs, and in the mucinous carcinoma (Table ). The 10% cutoff was chosen to address the potential of reduced expression of DSC3
due to contaminating stromal, non-epithelial elements. The majority of specimens analyzed consisted of 50% tumor based on pathology examination. Thus, the greatly reduced expression of DSC3
is a common event in primary breast tumor specimens.
Summary of DSC3 expression and methylation state in primary breast tumors
We next examined the cytosine methylation profiles of the breast tumor specimens to see if loss of expression correlates with cytosine methylation of the promoter region (Table , Fig. ). The DSC3
promoter region meets the criteria of a CpG island based on size, GC content, CpG dinucleotide frequency, as well as its location with respect to the transcriptional unit (Fig. ). We used sodium bisulfite genomic sequencing to assess the cytosine methylation status of 24 CpG dinucleotides within the DSC3
promoter region upstream of the DSC3
transcriptional start site. The region analyzed consists of the p53 binding site, the minimal promoter region, and 75 to 100 bases immediately 5' of the minimal promoter region [6
]. Ten to twelve cloned PCR products were sequenced to determine the percent methylation of the 24 CpG sites in the 5' promoter region. Of the 18 IDC samples that showed a loss of DSC3
expression, 10 (56%) of these specimens contained methylated cytosines within the CpG island. In the eight remaining IDC specimens that lack DSC3
expression we predict that other mechanisms of silencing such as mutation to p53 or loss of other transcription factors are participating in DSC3
gene silencing. Of the five ILC specimens that lacked DSC3
, two (40%) were shown to contain methylated CpG islands. The one mucinous carcinoma specimen analyzed showed a loss of DSC3
expression with a concomitant increase in cytosine methylation. In addition, we analyzed two benign fibrocystic disease specimens and in both cases we saw no methylation of the CpG island and DSC3
gene expression in one of two specimens analyzed. At the very 5' region we saw CpG sites that show methylation variable positions in many of the DSC
-positive specimens; we interpret these CpGs to likely be demarcating the edge of the CpG island. Indeed, the first four 5' sites are outside of the minimal promoter region and are likely to be at the edge of the functional CpG island where methylation is more variable [37
]. Nonetheless, methylation of the DSC3
promoter correlates with a lack of expression of DSC3
in a significant proportion of the primary tumor specimens examined.
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 ). (b) Summary of 5-methylcytosine levels obtained by sodium (more ...)
To further characterize in vitro
models for studying the epigenetic state of the DSC3
promoter we extended prior studies [6
] and analyzed 14 human breast tumor cell lines for DSC3
expression by quantitative real time RT-PCR. Tumor expression levels were normalized to GAPDH
, and expression was then compared to HMECs. Normalized expression levels are shown in Fig. . In the breast tumor cell lines tested, 11 of 14 (79%) showed a loss of DSC3
expression, whereas HS578T, MDA-MB-468, and UACC3199 showed moderate expression levels. Of note, three of the breast tumor cell lines tested, BT549, MDA-MB-231, and MDA-MB-157, are in agreement with earlier findings [29
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 (more ...)
To determine if loss of mRNA expression correlated with a decrease in protein levels, we conducted western blot analysis of DSC3 in a select group of cell lines. Chosen for analysis were the MDA-MB-157, MDA-MB-231, UACC1179, HS578T, and BT549 breast tumor cell lines, as well as the immortalized but non-tumorigenic breast epithelial cell line MCF10A. HaCaT cells, which are a spontaneously immortalized human keratinocyte cell line, served as a positive control for DSC3 expression [40
]. The lack of mRNA expression resulted in a marked reduction of DSC3 protein expression in the cell lines tested (Fig. ). The HS578T cell line, which showed a 7% expression of DSC3
mRNA, did not produce any detectable protein expression, which is likely below the limit of detection for the western blot conducted. MCF10A cells, which express DSC3
mRNA, showed protein expression comparable to the HaCaT cells; however, no protein bands were present in any of the tumor cell lines examined. Therefore, the lack of DSC3
mRNA expression results in a significant loss of DSC3 protein expression in breast tumor cell lines.
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.
To determine if DSC3
expression is lost in association with aberrant methylation of the DSC3
promoter we used sodium bisulfite genomic sequencing to assess the cytosine methylation status of the DSC3
promoter region. Again, 10 to 12 cloned PCR products were sequenced to determine the percent methylation of the 24 CpG sites in the 5' promoter region. The DSC3 promoter region was relatively unmethylated in the DSC3
-positive, HMECs, and MCF10A cells (Fig. ). In the DSC3
-negative cell lines MB231, UACC1179, and BT549, there is a strong correlation between cytosine methylation of the promoter region and lack of expression. Interestingly, in the two remaining DSC3
-negative cell lines, UACC2087 and MB-453, loss of expression does not correlate with cytosine methylation of its promoter region, which suggests that other mechanisms of gene silencing are present in these cell lines. These results are similar to the conditions found in the clinical specimens where DSC3
is silenced due to cytosine methylation of its promoter region in 41% of specimens analyzed. Notably, as each of these cell lines contain mutant p53, the loss of this transcription factor is likely participating in the silencing of DSC3
]. Finally, in the two remaining tumor cell lines that express DSC3
we saw little or no methylation of the promoter region. Therefore, the lack of DSC3
expression in these breast tumor specimens is due in part to both epigenetic and genetic mechanisms of gene silencing.
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 (more ...)
Another facet of epigenetic regulation causally linked to aberrant cytosine methylation is localized changes to chromatin architecture. Generally, methylated and silenced regions are associated with a 'closed' chromatin structure whereas unmethylated and transcriptionally competent regions are associated with an 'open' chromatin structure. We therefore analyzed the chromatin structure of the DSC3
CpG island region by measuring the accessibility of MspI to its cognate binding site (CCGG) (Fig. ) using a quantitative real-time, linker-mediated PCR approach [6
]. The PCR for this assay involved a hemi-nested amplification approach, with one primer being specific to the ligated linker and two gene specific primers. The first round of PCR used the linker specific primer and a downstream gene specific primer, while the second round used a portion of the first round product and a gene specific primer 3' to that of the first round gene specific primer to increase specificity of the reaction. Using this technique, we showed a 6.5 to 8.5 cycle difference, which translates to a 90 to 362-fold decrease in chromatin accessibility between the two tumor cell lines tested in comparison to MCF10A cells (Fig. ). Therefore, DSC3
gene silencing is linked to aberrant cytosine methylation and a closed chromatin structure.
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, (more ...)