SOX2 in Mesenchymal-Epithelial Transition Figure 3. SOX2 is involved in the WNT signaling pathway in colorectal cancer cells. (A) Immunofluorescence images of SW620-mock and SW620-shRNA-SOX2 cells stained with anti-beta-catenin antibody. Secondary antibody was conjugated with CY3. Cell nuclei were stained with Hoechest 33258. The top 6 panels are in 1006 magnification and the lower 6 panels are in 4006 magnification. (B) Western blot analysis of beta- catenin expression using cytoplasmic and nuclear fractions of SW620-mock and SW620-shRNA-SOX2 cells. Alpha-tubulin and Lamin B were used as loading controls for the cytoplasmic and nuclear fractions respectively. The right panel showed the band intensities (left panel) quantified by the Quantity One software (Bio-Rad). (C) LEF1/TCF1-luciferase activity in SW620-mock and SW620-shRNA-SOX2 cells. (D) Quantitative real time PCR (upper panel) and western blot (lower panel) analyses of LEF1 in SW620-mock and SW620-shRNA-SOX2 cells. GAPDH was used as a loading control. (E) Quantitative real time PCR analysis of c-Myc gene in SW620-mock and SW620-shRNA-SOX2 cells. (F) Quantitative real time PCR analysis of cyclin D1 expression in SW620-mock and SW620-shRNA-SOX2 cells. doi:10.1371/journal.pone.0041335.g003 the SOX2 knock down cells had lung liver metastasis respectively. A statistical analysis (Chi-square test using log likelihood ratio) showed that the P values were 0.012 combing both lung and liver tumor metastatic events in mice. In addition, the numbers of tumor loci are about 6 times higher (3.361.75 vs. 0.5760.79) in metastases with the mock knock down cells compared to the SOX2 knock down cells. These data suggested that SOX2 knock down reduced the ability of colorectal cancer cells to form metastasis. With the above in intro and in vivo data showing SOX2’s role in invasion and metastasis in colorectal cancer cells, we asked the question whether SOX2 expression in primary human colorectal cancer tissues could predict tumor metastasis in human CRC patients. We examined SOX2 expression by IHC of paraffin- embedded tumor tissues from 44 CRC patients and found that ,20% of patients (9/44) stained strongly with the SOX2 antibody (Figure 6A, B, C, D, E, F). In these SOX2 positive CRC tumors, the percentage of liver and lymph node metastasis were 77.8% and 88.8% (7/9 and 8/9 cases) respectively. However, in the SOX2 negative CRC tumors, the percentage of liver and lymph node metastasis were 40.0% and 51.4% (14/35 and 18/35 cases) respectively. The difference is statistically significant (P values of 0.029 and 0.039 respectively for lymph node and liver metastasis, Chi-square test using log likelihood ratio, two tails) (Figure 6G). We did not find any correlation between the SOX2 staining and the location of CRC to the right or left colon. However, we found that SOX2 staining correlated with advanced T3–T4 tumor stages, which are defined by tumor invasion through the muscularis propria into the pericolorectal tissues (T3) or by tumor penetration to the surface of the visceral peritoneum or to other organs or structures (T4) (T1–T2 tumors vs. T3–T4 tumors, P = 0.0006, Chi-square test using log likelihood ratio) (Figure 6G). Discussion SOX2’s role in the Epithelial-Mesenchymal Transition (EMT) process in colorectal cancers We demonstrated for the first time a connection between SOX2 and the Epithelial-Mesenchymal Transition (EMT) process [its reciprocal process is the Mesenchymal-Epithelial Transition (MET) process] [27]. During EMT, cells undergo morphological changes from the epithelial polarized morphology to the mesen- chymal fibroblastoid morphology [27,28]. After EMT process, epithelial cells lose cell-cell or cell-substrate contacts, and gain increased migratory capabilities. EMT is often marked by the loss of cell-cell adhesion molecules (e.g. E-cadherin), down-regulation of epithelial differentiation markers including cytokeratins and E- cadherin [27,28]. In the meanwhile, mesenchymal markers such as vimentin, fibronectin and N-cadherin are up regulated [27,28]. Our data on the morphological and molecular changes between SOX2 knock down and mock control cells reflected the corresponding MET process (the reversal process of the EMT process) (Figure 2), suggesting SOX2 is involved in the EMT process. PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e41335