SOX2 in Mesenchymal-Epithelial Transition Figure 2. Silencing SOX2 induced a MET process in colorectal cancer cells. (A) Western blot analysis of expression of E-cadherin and vimentin in SW620-mock and SW620-shRNA-SOX2 cells. GAPDH was used as a loading control. The right panel showed the right panel quantified by the Quantity One software (Bio-Rad). GAPDH was used for normalization. (B) Immunofluorescence images of SW620-mock and SW620-shRNA-SOX2 cells stained with antibodies against E-cadherin, vimentin and SOX2. Secondary antibody was conjugated with FITC and Cy3. Cell nuclei were stained with Hoechest 33258. (C) Semi-quantitative RT-PCR analysis of EMT-related regulatory factors. GAPDH was used as a loading control. doi:10.1371/journal.pone.0041335.g002 increased dramatically (Figure S1B). However, the increase expression of vimentin seemed not enough to produce the phenotype at the cellular level as we did not observe the corresponding morphological changes related to EMT (data not shown). Inhibition of SOX2 in SW620 Cells Leads to Reduction in ß-catenin/TCF/LEF Signaling EMT is marked by nuclear translocation of ß-catenin and the activation of WNT/ß-catenin signaling pathway [21–24]. We stained SOX2 knock down and MOCK knock down colorectal cancer cells with ß-catenin antibody and found that there is an accumulation of ß-catenin in the cell membrane relative to cytoplasm and nucleus in the SOX2 knocked-down cells compared to the MOCK control (Figure 3A). A Western blot analysis further confirmed the reduced cytoplasmic and nuclear expression of ß-catenin in the SOX2 knocked-down cells compared to the MOCK control (Figure 3B). As upon transloca- tion to the nucleus, ß-catenin serves as an activator of T-cell factor (TCF) and activates transcription of downstream targets in the canonical WNT pathway [25], we next measured the LEF/TCF activity using LEF/TCF reporter luciferase assay in the SOX2 knock down and MOCK knock down cells. We found that silencing SOX2 led to about 3-fold reduction of ß-catenin/TCF/ LEF signaling activity (Figure 3C). Interestingly, the expression of TCF itself was also down regulated in the SOX2 knock down SW620 cells (Figure 3D), so were the expression of TCF specific target genes such as c-Myc and cyclin D1 (Figure 3E, F). Thus, we demonstrated that SOX2 knock down resulted in reduced ß- catenin nuclear translocation and reduced canonical WNT pathway activities. SOX2 affects cell migration/invasion via down regulation of MMP2 and its expression predicts liver metastasis and lymph node metastasis of CRC patients We next assessed the SOX2’s effect on cell migration and invasion using an established stable SW620 colorectal cancer cells expressing shRNA-SOX2 (SW620shRNA-SOX2) using transwell migration and invasion assays (Corning Inc., USA). We found that SOX2 knock down reduced the ability of cell migration by about 8 times (Figure 4A), and the ability of cell invasion by about 6 times (Figure 4B), compared with MOCK knock down cells. To understand if the gelatinases (matrix metalloproteinases MMP2 and MMP9) were involved in the SOX2 mediated invasion process, we measured MMP2 and MMP9 activities using gelatin zymography [26] and found that the activity of the MMP2, but not of the MMP9, was significantly decreased in SOX2 knock down cells comparing with MOCK knock down cells (Figure 4C). After showing that knock down SOX2 reduced cell migration and invasion in vitro, we conducted an in vivo analysis. We compared the abilities of the SOX2 knock down and the mock knock down cells to form distant metastasis in mouse models. 56106 cells were injected respectively into the tail veins of 6-week- old female nude mice to introduce cells into the circulatory blood system. Mice were sacrificed on day 30 and lung and liver metastasis were examined using HE staining (Figure 5, A, B, C, D). 6 of 6 (100%) and 4 of 6 (60%) mice carrying the mock knock down cells had extensive lung and liver metastasis respectively. In contrast, only 3 of 7 (43%) and 2 of 7 (28.5%) mice injected with PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e41335