SOX2 in Mesenchymal-Epithelial Transition Figure 5. SOX2 knock down reduced in vivo metastasis of colorectal cancer cells in mice. (A) HE staining of mice lung tissue containing metastasis. (B) HE staining of mice liver tissue containing metastasis. (C) HE staining of mice lung tissue without metastasis. (D) HE staining of mice liver tissue without metastasis. (E) SOX2 staining of the lung metastasis with the arrow indicating strong staining of SOX2 in the invasive frontal regions. (F). SOX2 staining of the mouse lung tissue without metastasis. All images are in 1006. doi:10.1371/journal.pone.0041335.g005 comparing to mock control (Data not show). However, we were not able to recue or reverse the MET process, or initiate an EMT process. We also overexpressed the SOX2 in the SOX2-negative SW480 CRC cells (Fig. S1A) and showed that vimentin expression increased dramatically (Figure S1B). However, the increase expression of vimentin did not generate the corresponding morphological changes related to EMT (data not shown). This observation, although to our surprise, might suggest that SOX2 interacts or works with other factors in the process. The discrepancy between the effect of gene knock down and over expression have prior incidences. For example, Burbridge et al. knock down of Dcdc2 resulted in hippocampal malformations and a bimodal migration pattern of the transfected neurons [40]. However, they showed that the treatment of shRNA-transfected neurons with the DCDC2 overexpression construct failed to rescue the migration phenotype [40]. Retinoid X receptor alpha- (RXRalpha-null) mutants exhibit hypoplasia of their ventric- ular myocardium and die at the fetal stage [41]. However, Subbarayan et al. showed that RXRa overexpression in cardio- myocytes causes dilated cardiomyopathy but fails to rescue myocardial hypoplasia in RXRa-null fetuses [41]. Another example involves perlecan (Pln), which is a major heparan sulfate proteoglycan (HSPG) of extracellular matrices [42]. Savore et al. showed that addition of exogenous Pln does not rescue growth responses to FGF-2 in Pln knockdown C4-2B prostate cancer cells [42]. We showed that SOX2 silencing increased the expression of E- cadherin (Figure 2A). However, the regulating mechanism remains unknown. Snail and Slug are both transcriptional factors found to Figure 6. Immunohistochemical (IHC) staining of SOX2 in CRC tissues. (A–D) IHC images of SOX2-positve colorectal cancer tissues. Arrows indicate patches of strong SOX2 staining could indicate active tumor invasion fronts of the tumor capsules. (E–F) IHC images of SOX2- negative colorectal cancer tissues. All images are in 1006. (G) Statistical analysis of the relationship of SOX2 IHC staining with tumor stages, lymph node or liver metastases. doi:10.1371/journal.pone.0041335.g006 bind to the promoter regions of E-cadherin and repress E-cadherin expression during the EMT process [43–45]. In our analysis, we found that Snail, Slug and Zeb1 were down regulated after SOX2 silencing induced MET process. This suggested that SOX2 might induce the EMT process by acting through Snail, Slug or Zeb1. However, whether SOX2 directly or indirectly modulate these regulatory factors remains to be investigated. Knock down SOX2 reduced cell migration in vitro and invasion in mouse models and its expression predicts liver metastasis and lymph node metastasis of CRC patients We further demonstrated that silencing SOX2 could significant decrease the ability of cell migration and invasion in vitro (Figure 4A–B) and reduce the ability of tumor metastasis in vivo (Figure 5). Matrix metalloproteinases (MMPs) are involved in PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e41335