Abstract 4235: Phosphorylation of Serine 861 in the ECT2 guanine nucleotide exchange factor activates Rho family GTPases and is a biomarker for several tumor types

Abstract

Abstract The Rho family of GTPases, which cycle between inactive GDP-bound and active GTP-bound states, have high activity in many cancers. One mechanism for their activation is by guanine nucleotide exchange factors (GEFs), which stimulate the replacement of GDP-bound Rho with GTP-bound Rho. In this study, we report phosphorylation of Epithelial Cell Transforming 2 (ECT2), which is a Rho family-specific GEF, is a biomarker in several tumor types, based on bioinformatic analysis and biological assays. Our analysis of the NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) database found that Serine 861 (S861) in ECT2 is more highly phosphorylated in several tumor types compared to its phosphorylation in the relevant normal tissue. S861 phosphorylation is 2.1-fold and 2.4-fold higher in colon cancer and ovarian cancer, respectively, and high S861 phosphorylation in breast cancer is correlated with a poorer prognosis. Phosphorylation of S861 is especially high in triple-negative breast cancer, as it is 2.3-fold higher in this type compared with the other types of breast cancer. Increased phosphorylation of S861 is associated increased ECT2 mRNA and protein levels in colon cancer and breast cancer. In experimental studies, siRNA knockdown of endogenous ECT2 in two triple-negative breast cancer lines resulted in lower levels of RhoA-GTP, Rac-GTP, and Cdc42-GTP, and led to reduced cell migration, cell proliferation, and anchorage-independent growth, suggesting that ECT2 is a key regulator of cellular signaling and oncogenic growth in the triple-negative lines. Conversely, ectopic expression of wild-type ECT2 induced increased levels of RhoA-GTP, Rac-GTP, and Cdc42-GTP and promoted migration, proliferation, and anchorage-independent growth. Compared with wild type ECT2, ectopic expression of ECT2 carrying a phospho-defective (S861A) or phospho-mimic (S861D) mutation, respectively, reduced or enhanced ECT2 GEF and biological activities. Our results indicate that phosphorylation of S861 enhances the pro-oncogenic activity of ECT2, which is correlated with the increased phosphorylation of S861 in the three cancer types, the increased expression of ECT2 in two of these cancer types, and the prognostic significance of S861 phosphorylation in breast cancer. * Dunrui Wang and Xiaolan Qian are Equal contributors Citation Format: Dunrui Wang, Xiaolan Qian, Sarah Eng, Shivani Nellore, Alex G. Papageorge, Douglas R. Lowy. Phosphorylation of Serine 861 in the ECT2 guanine nucleotide exchange factor activates Rho family GTPases and is a biomarker for several tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4235.