The expression patterns and correlations of claudin-6, methy-CpG binding protein 2, DNA methyltransferase 1, histone deacetylase 1, acetyl-histone H3 and acetyl-histone H4 and their clinicopathological significance in breast invasive ductal carcinomas

Breast cancer is the most common cancer in women worldwide and the leading cause of death among women with cancer [1]. It has been estimated that 230, 480 women would be diagnosed with and 39, 520 women would die of cancer of the breast in 2011. http://​seer.​cancer.​gov/​statfacts/​html/​breast.​html.

It is widely accepted that the loss of cell-to-cell adhesion in tumor-derived epithelium is necessary for the invasion of surrounding stromal elements and subsequent tumor metastasis [2]. The cell-to-cell adhesion of epithelial cells is primarily mediated through two types of junctions: adherens junctions and tight junctions. Tight junctions consist of three parts, depending on their distribution within the junction: transmembrane proteins (which include occludins, the claudin family and junctional adhesion molecules), cytoplasmic plaques (the zonula occludens family), and associated/regulatory proteins (Rho-subfamily proteins) [3].

The claudin family includes at least 27 members [4, 5]. Claudin-6 contains four transmembrane domains, similar to other members of the claudin family [6]. Recent studies have demonstrated that epigenetic mechanisms are essential for claudin regulation [7‐9]. The most studied epigenetic regulators are DNA methylation and histone modification [10]. MeCP2 is a methyl-CpG binding protein that represses gene transcription. DNA methyltransferases are crucial enzymes for hypermethylation of tumor suppressor genes [11]. DNA methyltransferase 1 (DNMT1) is the best known and studied member of the DNMT family [12]. Moreover, DNA methylation occurs in a complex chromatin network and is influenced by modifications in histone structure [13, 14]. Histone deacetylase 1 (HDAC1), a zinc-dependent deacetylase, is a member of class I histone deacetylases, it is deregulated in many cancers and plays a crucial role in cell cycle progression and proliferation [15]. However, the expression levels of histone deacetylase 1 in breast invasive ductal carcinoma do not appear to have been studied.

We hypothesized that alterations in the expression levels of several epigenetic regulators, such as MeCP2, DNMT1, HDAC1, H3Ac and H4Ac, are responsible for the loss of claudin-6 in breast invasive ductal carcinomas. Here, we have analysed 100 breast invasive ductal carcinomas and 22 mammary gland fibroadenomas by immunohistochemistry. Our results demonstrate decreased claudin-6 expression in 75% of our cases of invasive breast carcinomas. Claudin-6 expression was found to be independent of the tumor subtype but was inversely correlated with lymph node metastasis.

In a previous study, we showed that the protein and gene expression of claudin-6 was low or undetectable in human and rat mammary cancer cell lines [21], and the cell growth, migration and invasion were inhibited by overexpression of claudin-6 in breast cancer MCF-7 cells. These results suggested that claudin-6 is a tumor repressor that inhibits malignant progression of breast cancer [22]. Claudin-6 expression is inactivated by aberrant CpG island DNA hypermethylation in its promoter region in the breast cancer cell MCF-7 [23], suggesting that DNA methylation may have an important role in regulation of claudin-6 expression in breast cancer. The most studied epigenetic regulators are DNA methylation and histone modification [10]. DNA methylation can cause gene silencing by interfering with the interactions between transcriptional activators and target-binding sites on genomic DNA, condensing the chromatin to alter DNA accessibility, and recruiting methyl-CpG binding proteins, which mediate downstream biological effects [24]. However, whether or not methylation associated proteins and histone modification proteins act to repress claudin-6 expression and how they influence the clinicopathological characteristics of IDC tumors is not yet understood. Consequently, our main goal in this study was to analyze the importance of MeCP2, DNMT1, HDAC1, H3Ac and H4Ac in the down-regulation of claudin-6 in breast IDC.

We wanted to find the normal mammary gland tissue as a normal control, but it is rarely available for research in China. In this study, 15 (15%) of breast cancer cases had tumor free mammary gland tissue adjacent to the tumor samples. Since we regarded this as too small an amount to be used as a control, we then chose tissue from fibroadenomas for this purpose. Hua et al. studied that the expression of fibroblast activation protein-alpha (FAP-α) and Calponin was a novel marker for pathologically diagnosing whether DCIS had microinvasion, and FAP-α promoted the formation of microemboli, which facilitated the metastasis of breast cancer [25]. We also compared the expression of proteins we studied in tissue adjacent to tumors and in fibroadenomas. There was no difference in the expression of proteins between normal tissues adjacent to the 15 tumors, and breast fibroadenomas (P > 0.05; Table 3). Consequently chose the fibroadenomas as a control tissue.

In this study, we have shown that the expression of claudin-6 was observed in both membrane and cytoplasm/membrane in the FA and IDC of the breast, similar to previous studies on the claudin-6 in atypical teratoid/rhabdoid tumors [26], claudin-1, and claudin-7 in colonic and renal carcinomas, respectively [27, 28]. This observation suggests that in breast IDCs and FAs, claudin-6 has an abnormal cellular localization, and was not restricted to cell-cell boundaries. Subsequent evaluation by immuno- histochemistry of 25 (25%) IDC samples and 20 (91%) FA samples showing positive immunoreactivity for claudin-6, showed that the expression of claudin-6 was significantly reduced in breast IDC tissues (P < 0.001). This result indicates that the reduced expression of claudin-6 may be an important molecular event in the development of breast cancers. Next, we investigated the correlation between claudin-6 expression and the clinicopathological characteristics of breast IDCs. We found that the expression of claudin-6 was negatively correlated with lymphatic metastasis of breast IDCs. This observation suggested low claudin-6 expression might facilitate lymph node invasion by tumor cells, and distant metastasis. On the one hand, the down-regulation of claudin-6 might result in abnormal proliferation, poor differentiation and decrease apoptosis, and played a role in mammary epithelial cell malignant transformation in the breast cancers; On the other hand, the down-regulation of claudin-6 may also lead to dysfunction of tight junctions, resulting in loss of cell-cell adhesion and polarity, causing tumor cells invasion and metastasis.

We have previously investigated the possibility that the expression of claudin-6 was negatively correlated with the hypermethylation promoter of claudin-6 in breast cancer tissues (data unpublished), suggesting that the down-regulation of claudin-6 is associated with its DNA methylation. DNA methylation is mediated by DNA methyltransferases (DNMTs) that catalyze the transfer of the methyl group from S-adenosyl L-methionine (SAM) to the cytosine in CpG dinucleotide [29]. Maintenance of methylation patterns is mediated by DNMT1. Altered levels of DNMT1 expression have frequently been associated with several types of tumors. DNMT1 expression was upregulated in pancreatic cancer cells, possibly associated with the progression of disease symptoms in pancreatic carcinoma [18]. In the present study, DNMT1 was more highly expressed in breast IDCs than nonmalignant tissues, but positive expression of DNMT1 was not associated with any clinical parameters. The Spearman's correlation test showed that claudin-6 expression was not correlated with the DNMT1 expression, but there were 56 cases of IDC in which the expression of claudin-6 and DNMT1 showed a reverse trend. The results show that the increased expression of DNMT1 plays an important role in the progression of breast IDC and that claudin-6 is partly inhibited by DNMT1.

MeCP2, the most studied member of the methyl-CpG binding domain proteins (MBDs), binds methylated DNA, and also serves as a transcriptional repressor [30]. Therefore, we considered whether claudin-6 expression might be related to the expression of MeCP2 in breast IDCs. Our results show that MeCP2 protein expression was statistically significantly higher in breast IDC specimens than in non-neoplastic lesions, although MeCP2 was not associated with any clinical parameters. The expression of claudin-6 was not significantly correlated with the expression of MeCP2, just as Wojdacz' s research, the methylation of breast cancer related genes (BRCA1, APC and RASSF1A) in peripheral blood DNA did not directly link to somatic methylation of the same genes in tumor DNA [31]. But there also were 74 cases of breast IDC in which the expression of claudin-6 and MeCP2 showed a reverse trend, suggesting that the increased expression of MeCP2 also played an important role in the progression of breast IDC. Similarly, MeCP2 mRNA expression levels have be shown to be increased in breast cancer specimens [32]. In addition to binding methylated DNA sequences, MeCP2 contains a C-terminal transcriptional repression domain which has been identified as the region involved in the gene repression activity [33]. MeCP2 binds methylation of the breast cancer 1 gene (BRCA1) and MAGE-A promoter, and results in tumor suppression [34, 35]. Our group has previously found that MeCP2 can bind the promoter of claudin-6 in breast cancer cell line MCF-7 (data unpublished), A number of studies have shown that MeCP2 is responsible for the initial recruitment of HDAC1, Sin3A and HDAC2, forming a tumor suppressor complex and were essential for MeCP2-mediated tumor suppression [36, 37].

Histone deacetylases are modification enzymes that catalyze the removal of acetyl molecules from lysines to balance the activities of histone acetyl-transferases [38]. Previous studies have shown that HDAC1 was overexpressed in many cancers, including gastric [39], colorectal [40] and pancreatic [41] carcinomas. To investigate the interactions between claudin-6 and HDAC1, H3Ac and H4Ac, we evaluated the expression patterns and the relation among the HDAC1, H3Ac and H4Ac in the breast IDC. In our series, HDAC1 expression was increased in IDC specimens relative to breast FA specimens. Interestingly, our data suggested that up-regulation of HDAC1 favored tumor cell metastasis, as evidenced by the fact that those tumors showing increased HDAC1 expression correlated with poor differentiation, older age, lymph node metastasis. Therefore, our data indicate that higher expression levels of HDAC1 are correlated with more aggressive tumors. This finding was not consistent with the observations of Krusche et al [42], who showed that HDAC1 expression predicted a better prognosis. The reasons for these results is unknown and the clinical significance of HDAC1 expression at the protein level in breast IDC also needs to be confirmed in a larger patient cohort. The Spearmen's relation test showed the expression of HDAC1 was not correlated with the expression of claudin-6, but there also were 64 cases of breast IDC in which the expression of claudin-6 and HDAC1 showed a reverse trend, suggesting that there was a partial role of increased expression HDAC1 in repressing the expression of claudin-6. Histone modifications (e.g. acetylation, methylation) are important regulators of transcriptional activities; therefore, we evaluated the total histone H3 acetylation (H3Ac) and histone H4 acetylation (H4Ac) by immunohistochemistry. In the present study, H3Ac expression levels were markedly increased in IDC specimens compared to breast FA specimens. H3Ac levels were increased in tumors < 5 cm in size and cancers of clinical stage I and II. H4Ac protein expression was high in IDC. However, we found no statistically significant correlations between H4Ac and clinicopathological parameters. We did not find statistically significant correlation between claudin-6 and H3Ac, H4Ac. These results therefore suggest that DNA methylation and histone modification play only a partial role in inhibition of claudin-6 expression. MeCP2, DNMT1, HDAC1, H3Ac and H4Ac might form a repressor complex, and inhibit the expression of claudin-6. Whether or not such complex might be responsible for the down-regulation of claudin-6 observed in our cases would require further investigation. Interestingly, Spearman's correlation test showed the expression of MeCP2 was positively correlated with the expression of H3Ac and H3Ac expression positively correlated with the expression of H4Ac. These results indicate that the expression of DNA methylation associated gene (MeCP2) may influence the expression of histone acetylation (H3Ac and H4Ac) in breast IDC and that the expressions of MeCP2, H3Ac and H4Ac play an important role in the generation of breast IDC.

We would have liked to include a survival analysis of the IDCs, but the breast cancer cases were mostly from the period 2009-2010, only two or three years after surgery. The 5-year survival rate seems to be more meaningful for assessment than nodal metastasis. This research is being conducted now and we hope to be able to publish the data in the future.

In summary, our data indicates that claudin-6 is down-regulation in breast IDC and that it is mediated by molecular mechanisms other than aberrant expression of the methylation associated proteins (MeCP2, DNMT1) and histone modification associated proteins (HDAC1, H3Ac, H4Ac). The expression of MeCP2 is positively correlated with the expression of H3Ac and H4Ac. Our data suggests that down-regulation of claudin-6 is an important factor influencing lymphatic metastasis; whereas up-regulation of HDAC1 is associated with tumor progression and invasiveness in breast IDC.