Accurate prognosis and predictive factors are necessary for the optimum management of patients with cancer and are especially important in breast cancer, because of its widely varying outcomes and the availability of potentially beneficial systemic adjuvant therapies. The definitive assessment of the clinical value of a predictive factor is a long process , but the use of an unbiased genome-wide approach has permitted the rapid identification of a number of genes that strongly predict a poor clinical outcome . The identification of mutations and/or epigenetic alterations in cancer may be useful to develop novel, more effective biomarkers and therapies in breast and colon cancer . In breast cancer, promoter hypermethylation has been reported for various genes that cover most cell functions [11, 15, 17, 23, 24].
Estrogen receptor status is an important factor in the diagnosis and prognosis of breast cancer. A previous study by our group found a significant difference in serum values of ESR1 and 14-3-3-σ gene promoters between breast cancer patients and healthy controls . The present study also found a significant difference in serum methylated 14-3-3-σ gene promoter between metastatic breast cancer patients and healthy controls. In contrast, however, ESR1 appeared to be unmethylated in the present patients with metastatic breast cancer. Theoretically, ESR1 is considered to be preferentially methylated in tumors because its inactivation confers a selective clonal advantage. It is possible that the specific environmental and nutritional setting of breast cancer metastases produces changes in this epigenetic alteration. However, further in-depth study is required to explain this intriguing finding. Reports of differences in methylation pattern between primary and metastatic breast cancer  may indicate that therapeutic targets in primary breast cancer are not be the same as targets in metastatic sites.
14-3-3 proteins are crucial in a wide variety of cell responses, including DNA damage checkpoints and apoptosis . Disruption of the G2-M checkpoint also appears to contribute to the change in the sensitivity of cells to chemo- and radiotherapy [27, 28]. 14-3-3 sigma sequesters the cdc2-cyclin B1 complex in the cytoplasm, resulting in G2 arrest. Among the genes involved in the G2-M checkpoint, 14-3-3σ, a transcriptional target of p53, is frequently silenced by DNA methylation of the 14-3-3σ gene promoter or by induction of estrogen-responsive ubiquitin ligase that specifically targets 14-3-3σ for proteosomal degradation [23, 29]. The inactivation and reduced expression of 14-3-3σ have been reported in various cancers, including breast cancer [16, 26, 30]. To date, the sigma isoform of 14-3-3 proteins has been the isoform most directly implicated in carcinogenesis and is recognized as a tumor-suppressor gene . Although the molecular basis for the tumor-suppressor function of 14-3-3σ is unknown , it has been suggested that 14-3-3σ is a critical regulator of G2-M . It has also been demonstrated that endogenous 14-3-3σ preferentially forms homodimers in cells . Knocking out 14-3-3σ in cancer cells leads to mitotic catastrophe and cell death from DNA damage due to the absence of G2-M arrest . Moreover, the highly conserved human 14-3-3 gene family encodes proteins with either tumor-promoting or tumor-suppressing activities, suggesting that the cellular balance among different 14-3-3 isoforms is crucial for the proper functioning of cells . 14-3-3 proteins have been found in primary breast cancer, enhancing its biological activity . The structure of the p53 C-terminus bound to the adaptor protein 14-3-3 has been recently described, providing a rationale for the observed stabilizing effect of 14-3-3 binding . Consistent with these data, the G2-M checkpoint is impaired in cancer cell lines that show methylation of 14-3-3 σ, while restoration of the expression of these genes using 5-aza-dC restores G2-M arrest induced by DNA damage . This molecule also contributes to mitotic catastrophe in carcinoma cells treated with chemotherapy agents .
Results of a recent study  showed that 14-3-3 and HSP70 expression may be useful as biomarkers and targets for the diagnosis and treatment of human triple-negative breast cancer. Breast cancer metastasis is the main cause of treatment failure, and the goal of adjuvant therapy is to eliminate disseminated tumor cells after complete removal of the tumor. However no tool is available to monitor its efficacy . Response to adjuvant treatment is usually evaluated retrospectively based on recurrence and survival rates. Therefore, the identification of metastasis biomarkers at an early stage may contribute to the early diagnosis and treatment of breast cancer patients. There is increasing recognition of the importance of epigenetic changes in the metastatic process. Cells may acquire an epi-genotype that allows them to disseminate from the primary tumor mass or survive and proliferate at a secondary tissue site . Overall, these results offer evidence of a difference in protein profile between metastatic and primary breast cancer.
The expression profile of the metastatic tumor is known to differ between primary tumor and heterogeneous metastasis . The present findings show that breast cancer methylation profiling might yield biomarkers for the diagnosis and treatment efficacy of breast cancer metastasis. Thus, we found that ROC analysis of serum levels of 14-3-3-σ methylated gene-promoter discriminated between healthy individuals and metastatic breast cancer patients with a sensitivity of 81% (95% CI: 74.0 - 86.8) and a specificity of 96.2% (95% CI: 80.45 - 99.9), making this biomarker a candidate for use in metastasis screening in the follow-up of treated breast cancer patients. It would be of special interest to investigate whether the elevated post-surgical serum 14-3-3-σ levels in some of the present patients (DFBC group) and in our previous study  indicate a risk of metastatic cancer or tumor recurrence. Although this type of investigation requires a prolonged follow-up , identification of this biomarker as a risk factor and its correlation with other clinical factors may lead to improvements in breast cancer treatment.
14-3-3-σ proteins are known to be crucial in a wide variety of cell responses, including cell cycle progression, DNA damage checkpoints, and apoptosis , and 14-3-3-σ hypermethylation is a significant event in primary breast cancer . However, its impact on tumor progression and its potential as a predictive factor remain unknown. Because 14-3-3-σ proteins regulate normal cell processes, the loss of their expression (mainly by hypermethylation of 14-3-3-σ gene promoter) may be implicated in breast cancer progression [31, 42]. This hypothesis is supported by our data, since hypermethylation of 14-3-3-σ was significantly associated with the response to metastatic breast cancer treatment. We calculated BRR values for the "continuous decline" and "rise-and-fall" groups (values plotted in Figure 5A). Analysis by unpaired t-test with Welch's correction showed a significant difference in mean values between these groups (P = 0.021). Subsequent ROC analysis, considering the "continuous decline group" as controls and the rise-and-fall group as cases (Figure 5B), showed a sensitivity of 75% (95% CI: 47.6 - 86.7) and a specificity of 66.7% (95% CI: 41.0 - 86.7) to discriminate between the groups for a cut-off level of BRR = 2.39.
The area under the ROC curve (Z = 0.804 ± 0.074) indicates that this test is a good approach to post-treatment prognosis and supports the very recent idea that 14-3-3 proteins may be related to breast cancer metastasis and evolution . However, a small amount of 14-3-3-σ methylated was detected in sera from healthy controls , which may be explained by presence of occult benign breast disease, although other possible sources of this DNA include: normal tissues, which show higher methylation values with increasing age; leukocytes , or breast benign disease . The above findings raise some questions about the utilization of 14-3-3-σ gene promoter in DNA extracted from serum for metastasis screening in the follow-up of treated breast cancer patients. Further research is warranted to elucidate this issue and to establish the impact of 14-3-3σ on tumor progression and its potential to predict the response to treatment of metastatic breast cancer.