TES was epigenetically silenced and suppressed the epithelial-mesenchymal transition in breast cancer

The TES gene was frequently lost in breast cancer, which could inhibit tumor invasion and the formation of distant metastasis. However, the underlying mechanisms remain unknown yet. In the present study, we aimed to investigate how TES was silenced and its roles in EMT--the key step for tumor metastasis. Real-time polymerase chain reaction (PCR) and Western blot were used to detect the mRNA and protein expression of target genes; the status of TES promoter was determined by methylation-specific PCR and subsequently, DNA sequencing. Overexpression or downregulation of TES was achieved by pcDNA3.1-TES or shRNA-TES transfection. Cellular adhesion and migration were investigated by the adhesion and Transwell assays. Morphological changes of breast cancer cells were observed under the optical microscope. The Rho A activity was measured using a commercial kit, and its roles in TES-manipulated EMT were determined by real-time PCR and Western blot. The 42.3% (33/78) breast cancer tissues presented hypermethylation of the TES gene, whereas only 2 (2.6%) non-malignant cases were hypermethylated (P<0.001). Moreover, TES hypermethylation was significantly correlated with larger tumor diameter (P=0.03) and lympho node metastasis (P=0.024). In primary cultured breast cancer cells, the demethylation treatment using 5-aza-dC notably restored the expression of TES. In vitro, overexpression of TES enhanced cellular adhesion inhibited migration and suppressed EMT, while downregulation of TES impaired cellular adhesion, promoted migration, and enhanced EMT. TES overexpression also activated the Rho A signal, which is a critical factor for the effects of TES on the EMT procedure. We firstly proved that frequent loss of TES in breast cancer was caused by promoter hypermethylation, which was correlated with poor prognosis. In vitro, TES enhanced cellular adhesion, suppressed tumor migration, and inhibited EMT. Moreover, the Rho A pathway was critical for the effects of TES on EMT, which can be blocked by the Rho A inhibitor. Therefore, we propose restoration of TES as a potent strategy for breast cancer therapy.