Abstract 1969: PKCα-induced Twist1 phosphorylation is a novel regulator of Twist1 stabilization

Abstract

Abstract Background: Epithelial - mesenchymal transition (EMT) is a pre-requisite for cancer metastasis. Twist-1 is a transcription factor with a central role in the process of EMT and therefore metastasis formation. Using ovarian cancer models, our group has previously shown that although the stationary epithelial phenotype express Twist1 mRNA, this phenotype is enforced by mechanisms that constitutively ubiquitinates and degrades the Twist1 protein. The objective of this study is to identify molecular mechanisms that promote Twist1 protein stabilization and therefore confer a mesenchymal and mobile phenotype. Materials and methods: Novel phosphorylation sites on Twist1 protein, which are adjacent to ubiquitination sites, were identified using NetPhosK and Ubpred. Further analysis demonstrates that most of these sites contain the consensus sequence for PKCα. Constitutively active PKCα (PKCαcat) was ectopically expressed in HEK293T cells and patient-derived epithelial ovarian cancer cell lines. Protein expression and phosphorylation status were determined using western blot analysis using either whole cell lysates or cellular fractions. Protein-protein interaction was determined by immunoprecipiation. The specific Twist1 domain required for PKCα interaction was determined by co-transfection with Twist1 deletion mutants. Results: Ectopic expression of PKCαcat in HEK293T cells and ovarian cancer cells resulted in increased levels of Twist1 protein compared to empty vector control. In ovarian cancer cells, PKCcat-induced upregulation of Twist1 protein is not associated with an increase in Twist1 mRNA but instead is associated with Twist1 phosphorylation, decreased ubiquitination, and enhanced stabilization. We identify the twist box (WR) domain of Twist1 as a pre-requisite for PKCα binding and PKCαcat-induced Twist1 stabilization. Moreover, we identify TGFβ1 as a potent activator of endogenous PKCα in epithelial ovarian cancer cells. TGFβ1 (1 ng/ml) is able to: promote the activation and membranal translocation of PKCα; increase Twist1 protein levels; and induce spheroid formation and EMT in epithelial ovarian cancer cells. Interestingly, TGFβ1 had no effect on Twist1 mRNA suggesting that this mechanism is independent of the classical TGFβ-Smad pathway. Conclusions: We demonstrate for the first time a novel TGFβ-PKCα signaling pathway that specifically targets Twist1 protein for phosphorylation and stabilization resulting in EMT. This mechanism is independent of the classical TGFβ-Smad pathway that controls EMT via transcriptional regulation. Since TGFβ is a pleiotropic cytokine that can affect multiple cell types, the identification of PKCα as a novel target in ovarian cancer cells may aid in the development of better therapeutic modalities that can prevent EMT and curtail metastasis formation. Citation Format: Roslyn Tedja, Ayesha B. Alvero, Carlos Cardenas, Mary Pitruzzello, Gang Yin, Yang Yang-Hartwich, Cai Roberts, Carlotta Glackin, Gil G. Mor. PKCα-induced Twist1 phosphorylation is a novel regulator of Twist1 stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1969. doi:10.1158/1538-7445.AM2017-1969