Dynamic changes in E-cadherin gene promoter methylation during metastatic progression in papillary thyroid cancer

Thyroid cancer cells undergo epithelial to mesenchymal transition (EMT) in the invasive front of tumor. To determine whether the mesenchymal feature of invasive cancer cells is maintained in metastatic sites, we examined E-cadherin methylation and E-cadherin expression in 66 papillary thyroid cancer (PTC) samples and in 34 corresponding lymph node metastases (LNM). Relationships between E-cadherin and cell motility were evaluated using thyroid cancer cell lines. Hypermethylation of the E-cadherin gene promoter was detected in 39.3% of the PTCs, and loss of E-cadherin expression correlated with lymphocytic infiltration, extrathyroidal invasion and the presence of metastases. Comparing primary PTCs to the corresponding LNM, E-cadherin methylation status was identical in 60% of the cases. The switch in E-cadherin promoter status from unmethylated in PTCs to hypermethylated in LNM was detected in 5%; and from hypermethylatated in PTCs to unmethylated in LNM in 35%. Loss of epigenetic silencing in LNM was associated with a gain of E-cadherin expression. Hypermethylation of the E-cadherin gene promoter was detected in thyroid cancer cell lines with mesenchymal-like morphology. Loss of E-cadherin expression in these cells correlated with high migratory ability. Inhibition of RAS/ERK or P13K/AKT signaling decreased the migratory ability of these cells but did not induce E-cadherin expression. In the cells with epithelial-like morphology, treatments with phorbol-ester or tumor necrosis factor (TNF)-alpha resulted in translocation of membranous E-cadherin to the cytoplasm and induction of migration. E-cadherin promoter methylation status and E-cadherin expression were not affected by TNF. Demethylating agents induced apoptosis in the mesenchymal-like cells but had no effect on E-cadherin expression or on migratory ability. Together, dynamic changes in E-cadherin methylation occur during metastatic progression in thyroid cancer. Epigenetic mechanisms and TNF-inducible signaling independently contribute to the regulation of E-cadherin expression and localization. These mechanisms may play a role in the induction of EMT in primary tumors and in the conversion from the mesenchymal to the epithelial phenotype in metastases.