DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines

Inappropriate gene silencing resulting from aberrant DNA methylation significantly contributes to neoplastic transformation, tumorigenesis, and tumor progression [1, 2], contributing to some of the hallmarks of cancer [3]. While abnormal DNA methylation affecting a variety of genes occurs in nearly every type of cancer that has been evaluated, some tumors exhibit aberrant concurrent hypermethylation of numerous genes, a phenomenon known as the CpG island methylator phenotype (CIMP). CIMP was first described in a distinct subset of human colorectal carcinomas that displayed high rates of concordant methylation of specific genes [4]. Subsequently, CIMP has been described in other human neoplasms, including tumors of the ovary [5], bladder [6], prostate [6], stomach [7], liver [8], pancreas [9], esophagus [10], and kidney [11], as well as neuroblastomas [12], and leukemias and lymphomas [13, 14]. While tissue type is important in determining which genes are targeted for methylation in a given neoplasm, CIMP-positive tumors in each of these tissue types exhibit gene silencing that is due to cancer-specific (rather than age-specific) hypermethylation of epigenetically-regulated genes. Definitive evidence for a hypermethylation defect (similar to CIMP) among human breast cancers has not emerged, and some investigators have suggested that such a hypermethylator phenotype does not occur in breast tumors [15]. Nevertheless numerous epigenetically-regulated genes are known to be directly silenced by DNA methylation in breast cancer including cell cycle control genes (APC, RASSF1, RB, TFAP2A), steroid receptor genes (ESR1, PGR, RARα), tumor suppressor genes (BRCA1, CDKN2A, CST6), and metastasis-associated genes (CDH1, CEACAM6, PCDHGB6), among others [16‐19].

In the current study, we analyzed 12 breast cancer cell lines for differential expression of 64 methylation-sensitive genes, to determine if subsets of breast cancer cell lines methylate genes at disparate frequencies, and subsequently confirmed that lack of gene expression was attributable to methylation-dependent silencing. Unsupervised cluster analysis of gene expression patterns reveals two distinct groups of breast cancer cell lines that possess different methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A), whereas the low-frequency methylator cell lines typically lack methylation of these genes. Analysis of the enzymes responsible for human DNA methylation reveals aberrant DNMT3b protein expression and elevated total DNA methyltransferase activity in hypermethylator cell lines. These observations combine to suggest the existence of a distinct subset of human breast cancer cell lines that possess novel biological properties related to dysregulation of the methylation machinery resulting in the acquisition of a hypermethylator phenotype.

The CpG island methylator phenotype (CIMP) was first used to describe a distinct subset of colorectal tumors that display high rates of concordant methylation of specific genes [4]. Subsequently, similar epimutational phenomena have been described in a wide range of neoplasms [5‐12, 14, 20]. The results of the present study suggest that a subset of human breast cancer cell lines express a hypermethylator phenotype that is characterized by concurrent methylation-dependent silencing of a number of genes, including a specific set of genes with excellent predictive power (CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A) that are involved in a wide range of neoplastic processes. CEACAM6 is a tumor-related gene that is involved in adhesion, migration, invasion, metastasis, apoptosis, and chemoresistance [21, 22], although the implications of its loss in breast cancers is not well understood. Cystatin M (CST6) is a recognized breast cancer tumor suppressor gene [23] that was recently reported to be silenced due to promoter hypermethylation in numerous breast cancer cell lines, as well as primary breast tumors [24, 25]. E-cadherin (CDH1) is a well-known suppressor of invasion/metastasis that functions in the maintenance of cell-cell adhesion [26]. CDH1 and ESR1 are frequently concurrently methylated in breast tumors [19], a relationship also discernible in the present study. The nuclear hormone receptor ESR1, which is silenced by methylation in the majority of estrogen-negative breast tumors [19], may be the foremost important methylation-sensitive gene in breast carcinogenesis, holding important implications for sensitivity to hormone therapy and clinical outcome. Much less well understood is the role of ion transport gene SCNN1A in breast carcinogenesis, although its epigenetic regulation in MCF7 cells has previously been noted [24]. LCN2 is involved in invasion and metastasis [27], and its expression has been linked to poor prognosis in ER/PR-negative breast tumors [28, 29]. Thus, methylation-sensitive genes function in various aspects of the normal biology of the breast epithelium. Therefore, concurrent methylation-dependent silencing of multiple genes in neoplastic breast epithelium (as observed in hypermethylator cell lines) is likely to significantly contribute to tumor biology and behavior.

A previous study that examined methylation patterns of primary breast tumors in search of a hypermethylator phenotype found frequent but essentially equally distributed methylation events at 12 genes among different histologic subsets of neoplasms [15]. These authors concluded that a CpG island methylator phenotype does not occur in breast cancer [15]. The difference in conclusions about the existence of a hypermethylator phenotype in breast cancer between the current study and the earlier report [15] is likely attributable to the number and choice of genes examined in the two studies, as well as the analysis of primary breast tumors versus established cancer cell lines. The previous study did not examine many of the genes that we found to be highly predictive of a hypermethylator phenotype (CEACAM6, CST6, LCN2, and SCNN1A), but did include several genes (including GSTP1, RARβ, RB, and others) which were less useful for predicting the hypermethylator phenotype. Thus, our results are consistent with the previous findings: when the genes are analyzed by Bae et al [15], no distinct hypermethylator phenotype is detectible. It is only through a survey of numerous methylation-sensitive genes that evidence for a hypermethylator phenotype emerges. Additionally, we examined not only genes with conventionally defined CpG islands, but also those with atypical CpG features (such as CEACAM6), which have only recently been reported as epigenetically-regulated [24]. Thus, we use the term "hypermethylator phenotype" rather than "CpG island methylator phenotype" to describe the hypermethylation defect in breast cancer cell lines, since the targets of aberrant methylation are not restricted to genes with large CpG islands.

The results of the current study suggest that the mechanism that accounts for the hypermethylator phenotype in human breast cancer cell lines is elevated DNMT activity secondary to overexpression of DNMT3b. DNMT3b protein is significantly elevated in hypermethylator cell lines, and these cells exhibit aberrantly increased DNMT activity and correspondingly high rates of methylation-dependent gene silencing compared to both low-frequency methylator cells and non-neoplastic counterparts. These results are in agreement with those of other recent studies, in which aberrant DNMT3b overexpression was implicated in the methylation abnormalities of breast cancers [30] and other cancers [31]. Tumor cells exhibiting DNMT3b overexpression are likely to exhibit methylation-based aberrant gene expression; one study showed that breast tumors that overexpress DNMT3b are more likely to be ESR1-negative, display increased proliferation, and be associated with poor patient prognosis [30]. Thus, it seems reasonable to expect that aberrant expression of DNMT3b protein may produce significant differences in tumor biology for breast tumors of the hypermethylator phenotype. In addition to the six hypermethylator cell lines which had elevated DNMT3b protein and total DNMT activity, one low-frequency methylator cell line (ZR-75-1) exhibited a similar hypermethylation defect. However ZR-75-1 cells retain expression of a number of epigenetically-regulated genes, making it functionally similar to other low-frequency methylator cell lines. A number of explanations may account for this apparent discrepancy: ZR-75-1 cells may methylate other epigenetically-regulated genes which were not surveyed in the present study; alternatively ZR-75-1 cells may possess the same functional defect in the DNMT machinery as cells of the hypermethylator phenotype but express additional repressor proteins which block the methylation capacity of the overabundant DNMT3b protein. Additional studies will be required to resolve these possibilities. The detection of a hypermethylator phenotype in breast cancer cell lines constitutes a first step towards determining if a hypermethylation defect can be identified in primary breast neoplasms in vivo. If a subset of primary breast cancers express a hypermethylator phenotype, we would predict these tumors to differentially express other important characteristics related to tumor biology/behavior and patient outcome. This is the case in colorectal cancer, where CIMP status is associated with various clinical features [32‐34]. Likewise, CIMP-positive neuroblastomas, esophageal tumors, and leukemias tend to have poorer prognosis and are associated with significantly higher relapse and mortality rates [12, 35, 36].

Our findings suggest that breast cancer cell lines that express the hypermethylation defect correspond to estrogen-receptor negative tumors, suggesting that the hypermethylator phenotype cosegregates with a subset of breast cancers (ER-negative) that tend to have poor prognosis [37]. A number of molecular subtypes of breast cancer have been described (including luminal A, luminal B, HER2+ and basal-like), and these different subtypes correlate with important differences in tumor biology, clinical behavior, and patient survival. Luminal A and luminal B tumors are ER-positive and respond better to treatment, resulting in better long-term patient outcome compared to the ER-negative basal-like and HER2+ subtypes [38]. Our microarray data mining analysis of primary breast cancer gene expression suggests that the hypermethylation defect observed in breast cancer cell lines can also be identified in primary tumors. Preliminary investigation of a limited dataset (n = 88 tumors) identified a strong cluster of tumors that express the hypermethylator signature (Figure 6), with low levels of expression of the six genes of interest (CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A). All of the tumors in this cluster were classified as basal-like, and 75% of the basal-like tumors in the dataset expressed the hypermethylation signature. This observation suggests that the hypermethylator defect represents a biological property of basal-like breast cancers. Basal-like breast tumors make up ~25% of all breast cancers but contribute disproportionately to breast cancer deaths as they tend to display more aggressive tumor characteristics such as increased size, rapid tumor growth, increased rate of metastasis, higher incidence of relapse, and lower overall patient survival [39, 40]. In has also been observed that this subtype of breast cancer is overrepresented in young, African-American women [41]. These tumors lack expression of the hormone growth factor receptor genes (ER and PR) that are targeted by some drug regimens, eliminating options for targeted therapy. While further studies are needed to understand fully the relationship between basal-like breast cancers and the hypermethylator phenotype, recognition of this fundamental biological property of the basal-like breast cancers may present new molecular targets for development of novel treatment strategies.

Unraveling the complexities of this hypermethylation defect in neoplastic breast disease holds important implications for cancer diagnosis, identification of new targets for therapy, and development of new strategies for clinical management. Since overexpression of DNMT is thought to be an early event in carcinogenesis [42‐44], elevated DNMT3b protein (which characterizes the hypermethylator phenotype in vitro) may constitute an important biomarker for early detection in patients developing breast tumors of the hypermethylator phenotype. Furthermore, the various proteins and enzymes of the DNA methylation machinery may represent novel targets for breast cancer therapy. It follows that patients with breast cancer of the hypermethylator phenotype may benefit significantly from a targeted demethylation treatment as an adjunct to standard chemotherapeutic regimens. Epigenetic chemosensitization has been shown to improve the efficacy of standard chemotherapeutics against tumor cells with known methylation defects [45, 46], and evidence suggests that chemotherapeutic resistance can be overcome with demethylating treatment in certain cases [47]. While more research needs to be done to fully understand the clinicopathological implications of the hypermethylator phenotype in primary breast tumors, the existence of a subset of breast cancer cells with aberrant DNA methylation and other epimutations that are potentially reversible holds promise for better diagnosis and improved treatment.