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Supplementary Methods, Figures 1-5 from Phosphorylation of Serine 68 of Twist1 by MAPKs Stabilizes Twist1 Protein and Promotes Breast Cancer Cell Invasiveness

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posted on 2023-03-30, 21:35 authored by Jun Hong, Jian Zhou, Junjiang Fu, Tao He, Jun Qin, Li Wang, Lan Liao, Jianming Xu
Supplementary Methods, Figures 1-5 from Phosphorylation of Serine 68 of Twist1 by MAPKs Stabilizes Twist1 Protein and Promotes Breast Cancer Cell Invasiveness

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ARTICLE ABSTRACT

Twist1, a basic helix–loop–helix transcription factor, promotes breast tumor cell epithelial–mesenchymal transition (EMT), invasiveness, and metastasis. However, the mechanisms responsible for regulating Twist1 stability are unknown in these cells. We identified the serine 68 (Ser 68) as a major phosphorylation site of Twist1 by mass spectrometry and with specific antibodies. This Ser 68 is phosphorylated by p38, c-Jun N-terminal kinases (JNK), and extracellular signal-regulated kinases1/2 in vitro, and its phosphorylation levels positively correlate with Twist1 protein levels in human embryonic kidney 293 and breast cancer cells. Prevention of Ser 68 phosphorylation by an alanine (A) mutation (Ser 68A) dramatically accelerates Twist1 ubiquitination and degradation. Furthermore, activation of mitogen-activated protein kinases (MAPK) by an active Ras protein or TGF-β treatment significantly increases Ser 68 phosphorylation and Twist1 protein levels without altering Twist1 mRNA expression, whereas blocking of MAPK activities by either specific inhibitors or dominant negative inhibitory mutants effectively reduces the levels of both induced and uninduced Ser 68 phosphorylation and Twist protein. Accordingly, the mammary epithelial cells expressing Twist1 exhibit much higher degrees of EMT and invasiveness on stimulation with TGF-β or the active Ras and paclitaxel resistance compared with the same cells expressing the Ser 68A-Twist1 mutant. Importantly, the levels of Ser 68 phosphorylation in the invasive human breast ductal carcinomas positively correlate with the levels of Twist1 protein and JNK activity and are significantly higher in progesterone receptor–negative and HER2-positive breast cancers. These findings suggest that activation of MAPKs by tyrosine kinase receptors and Ras signaling pathways may substantially promote breast tumor cell EMT and metastasis via phoshorylation and stabilization of Twist1. Cancer Res; 71(11); 3980–90. ©2011 AACR.

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