Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells Qiong Li a,1 , Mingfu Wu a,1 , Hui Wang b , Gang Xu a , Tao Zhu a , Yongtao Zhang a , Ping Liu a , Anping Song a , Chen Gang a , Zhiqiang Han a , Jianfeng Zhou a , Li Meng a , Yunpin Lu a , Shixuan Wang a, * , Ding Ma a, * a Cancer Biology Research Center, Tongji Hospital, Huazhong University of Science and Technology, 1095 Jiefang Anv, Wuhan, Hubei 430030, PR China b Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China Received 20 August 2007; received in revised form 2 November 2007; accepted 5 November 2007 Abstract Ezrin primarily acts as a linker between the plasma membrane and the cytoskeleton and is a key component in tumor metastasis. In the present study, RNA interference (RNAi) using ezrin small hairpin RNAs (ezrin shRNAs) was used to define the roles of ezrin in the regulation of malignant behaviors of human breast cancer. The highly metastatic human breast cancer cell MDA-MB-231, in which ezrin mRNA and protein levels are the highest, was selected as a cell model in vitro. In addition, we also found that ezrin expression was up-regulated and its immuno-staining trans-located from cell membrane to cyto- plasm, whereas E-cadherin expression decreased and showed the same cell distribution as ezrin in lymphatic metastases of human breast carcinomas. After repression of ezrin by more than 85% of G3PDH and 75% of b-actin in mRNA and protein levels was maintained in the stable expressing ezrin shRNAs MDA-MB-231 cell clones, the abilities of cell motility and inva- siveness were obviously inhibited with a 4-fold and 2-fold, respectively, and the altered cell polarity was observed. Western blot analyses further revealed that the silencing of ezrin induced an increased E-cadherin expression and a decreased phos- phorylation of b-catenin by inhibiting phosphorylation levels of c-src. These data indicate that ezrin overexpression positively correlated with metastatic potentials of human breast cancer cells, especially lymphatic system metastasis. Decreased ezrin expression by shRNA reversed metastatic behaviors of human breast cancer cells by inducing c-src-mediated E-cadherin expression, suggesting that ezrin may have potential values in assessing lymphatic metastasis of human breast cancers. Ó 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Ezrin; Breast cancer; RNAi; Metastasis 1. Introduction Breast cancer is one of the most common female cancers in the world. The poor prognosis of patients with breast cancer is related to tumor metastasis [1,2]. Despite significant advances in the treatment of primary human breast cancer such as surgery, 0304-3835/$ - see front matter Ó 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.11.018 Abbreviations: ERM, ezrin–radirin–moesin; shRNA, small hair- pin RNA; PI3K, PI3-kinase; GFP, green fluorescent protein; R- NAi, RNA interference. * Corresponding authors. Fax: +86 27 83662681 (S. Wang), +86 27 83662779 (D. Ma). E-mail addresses: sxwang@tjh.tjmu.edu.cn (S. Wang), dma@ tjh.tjmu.edu.cn (D. Ma). 1 These authors contributed equally to this work. Available online at www.sciencedirect.com Cancer Letters 261 (2008) 55–63 www.elsevier.com/locate/canlet