Abstract 1130: AKT-mediated phosphorylation is responsible for TWIST1-mediated tumor growth and metastasis

Abstract

Abstract Cancer cells show epithelial-mesenchymal transition (EMT) during cell migration, invasion and dissemination. Although it has been shown by numerous publications that the evolutionary conserved basic helix-loop-helix (b-HLH) transcription factor TWIST1 plays pivotal role during EMT the molecular mechanism responsible for TWIST1 function is not fully understood. Here we show that Twist1 binds to and phosphorylated by AKT/protein kinase-B at S42, T121 and S123. While conversion of S42, T121 and S123 to phosphorylation-mimicking Aspartic or glutamic acids created active Twist1, Alanin mutants of the same sites diminished the DNA-binding and transctivating functions of Twist1. In line with this, Glutamic Acids mutants suppressed the expression of E-Cadherin, whose expression is negatively regulated by active TWIST1, Alanin mutants induced the expression of E-Cadherin. Similarly, we tested the impact of above mentioned mutants on cell migration and proliferation. Our results demonstrated that while Glutamic acid mutants accelerated, Alanin mutants suppressed the migration and proliferation of 293T cells. Our in vitro results prompted us to test our mutants under in vivo conditions using breast cancer as a model. We transfected non-metastatic mouse mammary tumor cell line, 67NR, with Glutamic acid mutants, and metastatic mouse mammary tumor cell line, 4T1, with Alanin mutants of mouse TWIST1 and selected the Neo-resistant populations. We injected 1 million 67NR cells over-expressing wild type or Glutamic acid mutants of Twist1, and 100.000 4T1 cells over-expressing wild type or Alanin mutants of Twist1 into the breast tissue of female inbred BalBC mice, and animals were monitored for metastasis. At the end of the 4th week, we sacrifised and examined the animals for tumor growth and metastasis. Although 67NR cells over-expressing wild type Twist1 did not show any metastasis, cells over-expressing 1E (S42E) and 2E (S42E, T121E) mutants of Twist1 showed 15-20 macroscopic metastasis to liver and Lungs. Paralel to this, 4T1 cells expressing 1A (S42A) and 2A (S42A, T121A) mutants of TWIST1 showed no macroscopic metastasis. Our results clearly indicate that phosphorylation of S42 and T121 by AKT is essential for TWIST1-mediated tumor growth and metastasis. Citation Format: Osman N. Ozes, Suray Pehivanoglu, Gokhan Ertosun, Gokhan Gorgisen, Nuray Erin, Duygu Unal, Gamze Tanriover. AKT-mediated phosphorylation is responsible for TWIST1-mediated tumor growth and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1130. doi:10.1158/1538-7445.AM2014-1130