Investigation of activation mechanism and natural inhibitors of TWIST1 transcription factor

Epithelial-Mesenchymal Transition (EMT) is a major mechanism that causes cancer metastasis. In this process, proteins of extracellular matrix (ECM) are either degraded or their expression is supressed resulting in mobilization of tumor cells. Depending on tumor dissemination, cancer associated mortality rates increases with EMT. The majority of deaths (about 90%) associated with cancer are due to the metastasis. Recently discovered transcription factor TWIST, a transcription factor with bHLH motif, is tightly associated with EMT because of its ability to repress transcription of E-Cadherin, a major constituent of ECM. Therefore, increased expression of TWIST1 contribute to EMT and cancer metastasis. Although, we know the involvement of TWIST1 in EMT, little is known about the mechanism of activation of TWIST1. By searching the amino acid sequence of TWIST1 we have found 3 conserved AKT/PKB phosphorylation sites in the consensus RxRxxS/T motif. These sequences are Ser42, Thr121, and Ser123 in human TWIST1. In our study, we showed that AKT is activated by tissue specific growth factors and phosphorylate TWIST1 at indicated sites. To further analyse the significance of phosphorylation of these sites, we converted above residues to unphosphorylatable Alanine or phosphorylation mimicking negatively charged Glutamic acid. Alanine mutants of TWIST1 turn out to be inactive whereas Glutamic acid mutants are active. Our results indicate that TWIST1 is phosphorylated by AKT and this phosphorylation is required for its activation. As anticipated, activated TWIST1 respresses the transcription of E-Cadherin while inducing the expresssion of N-cadherin and Vimentin. In conclusion, TWIST1 activation requires phosphorylation by AKT and this is necessary for TWIST1-regulated EMT.