Intermediate filament dynamics and breast cancer: aberrant promoter methylation of the Synemin gene is associated with early tumor relapse.

Synemin (SYNM) is a type IV intermediate filament that has recently been shown to interact with the LIM domain protein zyxin, thereby possibly modulating cell adhesion and cell motility. Owing to this multiplicity of potential functions relevant to cancer development, we initiated a study to decipher SYNM expression and regulation in benign human breast tissue and breast cancer. Dot blot array analysis showed significant SYNM mRNA downregulation in 86% (n=100, P<0.001) of breast cancers compared with their normal tissue counterparts, a result that was confirmed by real-time PCR analysis (n=36, P<0.0001). Immunohistochemistry analysis showed abundant SYNM protein expression in healthy myoepithelial breast cells, whereas SYNM expression loss was evident in 57% (n=37, P<0.001) of breast cancer specimens. Next, we analyzed methylation of the SYNM promoter to clarify whether the SYNM gene can be silenced by epigenetic means. Indeed, methylation-specific PCR analysis showed tumor-specific SYNM promoter methylation in 27% (n=195) of breast cancers. As expected, SYNM promoter methylation was tightly associated (P<0.0001) with SYNM expression loss. In-depth analysis of the SYNM promoter by pyrosequencing showed extensive CpG methylation of DNA elements supposed to regulate gene transcription. Demethylating treatment of SYNM methylated breast cancer cell lines with 5-aza-2-deoxycytidine clearly reestablished the SYNM expression. Statistical analysis of the patient cohort showed a close association between SYNM promoter methylation and unfavorable recurrence-free survival (hazard ratio=2.941, P=0.0282). Furthermore, SYNM methylation positively correlated with lymph node metastases (P=0.0177) and advanced tumor grade (P=0.0275), suggesting that SYNM methylation is associated with aggressive forms of breast cancer. This is the first study on the epigenetic regulation of the SYNM gene in a cancer entity. We provide first hints that SYNM could represent a novel putative breast tumor suppressor gene that is prone to epigenetic silencing. SYNM promoter methylation may become a useful predictive biomarker to stratify breast cancer patients' risk for tumor relapse.