Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis.

The tissue inhibitor of metalloproteinase-3 (TIMP3) is the only member of the TIMP family that binds to the extracellular matrix and suppresses cancer cell growth, angiogenesis, migration, and invasion. However, whether the abnormal expression and promoter methylation of TIMP3 facilitates oral cancer metastasis remain unclear. In this study, the DNA methylation levels of TIMP3 CpG islands were assessed through pyrosequencing. Artificial modulation of TIMP3 was performed to explore the role of TIMP3 in tumor metastasis in vitro and in vivo. Our results showed that the suppression of TIMP3 transcription by DNA methylation involves the inhibition of the binding of the transcription factor Sp1 to the TIMP3 promoter as well as the upregulation of DNMT1 and DNMT3B. Functional analyses revealed that TIMP3 overexpression reduced migration and invasion abilities in oral cancer cells and inhibited lymph node metastasis in vivo. Moreover, TIMP3 regulated epithelial-mesenchymal transition by increasing the expression of the epithelial markers and reducing the expression of the mesenchymal markers. In conclusion, our findings suggested that the suppression of TIMP3 by DNA methylation contributes to oral cancer metastasis.