SOX2 in Mesenchymal-Epithelial Transition Figure 4. SOX2 is involved in cell migration and invasion in colorectal cancer cells. (A) Left panel, photographs of SW620-shRNA-SOX2 and SW620-mock cells using the Transwell migration assays; right panel, quantification data (mean 6 SD) of the cells that migrated to the lower chambers. (B) Photographs (left panel) and the quantification results (mean 6 SD) (right panel) of the cells that migrated to the lower chambers for SW620-shRNA-SOX2 and SW620-mock cells using the Transwell cell invasion assays. (C) Gelatin zymography assay for gelatinases (matrix metalloproteinases MMP2 and MMP9) in SW620-shRNA-SOX2 and SW620-mock cells. doi:10.1371/journal.pone.0041335.g004 SOX2’s role in the Epithelial-Mesenchymal Transition (EMT) is mediated through the WNT pathway The EMT is also marked by nuclear relocalization of ß-catenin [29]. We also observed that relocalization of ß-catenin from cytoplasmic/nuclear to membrane after SOX2 knock down. ß- catenin could form complexes in different cellular compartments including the adhesion complex at the plasma membrane and the signaling complex in the nucleus [30] (Figure 2A–B). Our data suggests a model for SOX2’s role regulating the balance between ß-catenin in the adhesion complex at the plasma membrane and in the signaling complex in the nucleus EMT process: SOX2 could reduce the expression of E-cadherin at plasma membranes, thus reducing the binding capacity of the adhesion complex for ß- catenin. In the meantime, the WNT signaling components in the nucleus recruit more ß-catenin, activates WNT signaling and its downstream targets. The WNT pathway is widely regarded as the crucial pathway for colorectal carcinogenesis [31–33]. Our data also suggested that SOX2 activates WNT signaling activity as demonstrated by reduced LEF/TCF activity and reduced expression of well-known downstream target gene cyclin-D1 and c-Myc in SOX2-knock down cells (Figure 3). Our data in CRC is consistent with the report by Chen et al. who showed that SOX2 physically interacts with ß-catenin in human breast cancer cells [34]. The relationship between other SOX family proteins and ß- catenin has also been reported. For example, Zorn et al. showed that in Xenopus, two SOX family members (XSox17 alpha and beta, and XSox3) physically bind to beta-catenin and inhibit WNT pathway activity [35]. Zhang et al. showed that in Xenopus, XSox3 did not physically interact with beta-catenin, but could regulate Xnr5, a beta-catenin/VegT-regulated early zygotic gene, thereby indirectly involved in the WNT signaling [36]. The existence of a link between EMT and SOX2 is not surprising, as EMT is key developmental program in embryo development [37] and SOX2 plays a critical role in the early embryonic development and formation of tissues and organs [6]. Furthermore, the EMT process is often activated during cancer invasion and metastasis [37] and SOX2 is also over expressed in many types of cancers [15,20,34,38,39]. With multiple attempts, we over-express SOX2 (initially as SOX2-GFP construct, and later as SOX2 construct alone) in the previously SOX2 knock down CRC cells, and showed that we achieved SOX2 overexpression in the SOX2 knock down cells PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e41335