Silencing SOX2 Induced Mesenchymal-Epithelial Transition and Its Expression Predicts Liver and Lymph Node Metastasis of CRC Patients Xu Han1, Xuefeng Fang 4 , Xiaoyan Lou 1 , Dasong Hua1, Wenchao Ding 1 , Gregory Foltz2, Leroy Hood 3 , Ying Yuan4*, Biaoyang Lin1,2,5* 1 Systems Biology Division, Zhejiang–California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, China, 2 Swedish Neuroscience Institute, Swedish Medical Center, Seattle, Washington, United States of America, 3 The Institute for Systems Biology, Seattle, Washington, United States of America, 4 Department of Medical Oncology, The 2nd Hospital of Zhejiang University Medical College, Hangzhou, Zhejiang, China, 5 Department of Urology, University of Washington, Seattle, Washington, United States of America Abstract SOX2 is an important stem cell marker and plays important roles in development and carcinogenesis. However, the role of SOX2 in Epithelial-Mesenchymal Transition has not been investigated. We demonstrated, for the first time, that SOX2 is involved in the Epithelial-Mesenchymal Transition (EMT) process as knock downof SOX2 in colorectal cancer (CRC) SW620 cells induced a Mesenchymal-Epithelial Transition (MET) process with recognized changes in the expression of key genes involved in the EMT process including E-cadherin and vimentin. In addition, we provided a link between SOX2 activity and the WNT pathway by showing that knock down of SOX2 reduced the WNT pathway activity in colorectal cancer (CRC) cells. We further demonstrated that SOX2 is involved in cell migration and invasion in vitro and in metastasis in vivo for CRC cells, and that the process might be mediated through the MMP2 activity. Finally, an IHC analysis of 44 cases of colorectal cancer patients suggested that SOX2 is a prognosis marker for metastasis of colorectal cancers. Citation: Han X, Fang X, Lou X, Hua D, Ding W, et al. (2012) Silencing SOX2 Induced Mesenchymal-Epithelial Transition and Its Expression Predicts Liver and Lymph Node Metastasis of CRC Patients. PLoS ONE 7(8): e41335. doi:10.1371/journal.pone.0041335 Editor: Michael Klymkowsky, University of Colorado Boulder, United States of America Received December 2, 2011; Accepted June 25, 2012; Published August 17, 2012 Copyright: 2012 Han et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by grants to BL from the Ministry of Science and Technology of China [grant 2006AA02A303, 2006AA02Z4A2, 2006DFA32950 and 2007DFC30360], and grant to XF from the National Natural Science Foundation of China (No. 81101580). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yuanying1999@zju.edu.cn (YY); biaoylin@gmail.com (BL) Introduction The epithelial to mesenchymal transition (EMT) is well- coordinated process during embryonic development as well as progression of cancers including colorectal cancers [1–4]. Epithe- lial cells gain polarity and motility during EMT, which are necessary for tumor invasion and metastasis in different types of epithelial carcinomas [3,5]. For example, colorectal cancer (CRC) cells at the invasive front usually acquire mesenchymal properties including highly migratory, poorly differentiated, hyperprolifera- tive, and loss of cell-cell contact–mediated growth inhibition [4]. SOX2 is one of the key members of the SOX family gene and plays critical role in embryonic stem cells [6] and in induced pluripotent stem cells [7–10]. It is also involved in invasion and metastasis of pancreatic carcinoma [11], and in carcinogenesis of gastric [12], breast [13], pancreatic cancers [11], and osteosarco- mas [14] and glioma [15,16]. Furthermore, SOX2 also maintains self-renewal of cancer stem cells [14] or is activated in cancer stem cells [17]. An intriguing question to ask is whether cancer cells in epithelial-to-mesenchymal transition and tumor-propagating–can- cer stem cells are distinct, overlapping or same populations [18]. Mani et al. reported that induction of EMT in human mammary epithelial cells (HMLEs) resulted in the gain of epithelial stem cell properties in HMLEs [19]. In this work, we asked the question whether the key stem cell gene SOX2 plays a role in the EMT process. We used colorectal cancer as a.model to address the question. As a result, we demonstrated that SOX2 knock down in colorectal cell (CRC) SW620 induced a Mesenchymal-Epithelial Transition (MET) process, with characteristic morphological changes from spindle and fibroblastoid shape to cobblestone-like cell shape, and with associated changes in expression of key genes involved in the MET process including E-cadherin and vimentin. In addition, MMP2 activity and the WNT pathway activity were decreased significantly in the SOX2 knock down colorectal cells. We further showed that knocking down SOX2 could inhibit cell mobility and invasion in vitro and suppress metastasis in vivo for CRC cells. Finally, we showed that elevated expression of SOX2 is significantly correlated with metastases in CRCs. Our manuscript describes, for the first time, a novel role of SOX2 in regulating the EMT process in cancers. Materials and Methods Cell culture The human colorectal cell line SW620 was a kindly gift from The Second Affiliated Hospital, Zhejiang University School of Medicine. The stable transfected cells, SW620mock and PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e41335