Gelsolin Induces Cancer Invasion via uPA Cascade Figure 2. Gelsolin expression is prominent at the invasive front of human colorectal cancer tumors. Gelsolin expression is high along the tumor periphery in primary tumors and liver metastases. The tumor periphery is outlined as red dotted line. A magnified view of a region of primary tumor edge (boxed) is shown on the bottom panel. The invading liver metastases shown are confirmed by cytokeratin stain, AE1/3, on an adjacent slide (bottom middle panel). Increased gelsolin expression was also detected in less-differentiated tumor cells which appeared to be breaking away from well-formed glandular structures (arrowed, top right panel). Gelsolin expression were scored and represented in the scatter dot plot (bottom). The median scores are represented by the red horizontal bars. Mann-whitney test was to compare the gelsolin expression score between the main tumor bulk and their periphery. Bar: 50 mm. doi:10.1371/journal.pone.0043594.g002 the pIRES2-EGFP plasmid encoding human cytoplasmic gelsolin cDNA (Figure 3B; refer to Figure S4 for the cloned sequence of gelsolin cDNA). Control HCT116 cells were generated by transfection with the empty pIRES2-EGFP plasmid. In addition gelsolin expression was reduced using siRNA knockdown in several colorectal tumor cell lines (HCT116 and its metastatic variant E1, gelsolin-overexpressing HCT116, DLD-1 and Caco- 2), and compared to cells transfected with control siRNA. As shown in Figure 3C, gelsolin was greatly reduced after gelsolin siRNA treatment in these cell lines. The effects of gelsolin overexpression and knockdown in colorectal tumor cells were determined as described in the sections below. Gelsolin overexpression in HCT116 augmented migration through uncoated transwells and invasion through matrigel-coated transwells (Figure 3D). The invasiveness of the stably-transfected gelsolin-overexpressing HCT116 cells as well as wild-type HCT116 cells was significantly decreased by siRNA knockdown of gelsolin expression, indicating a reliance on gelsolin for invasion (Figure 3E). The pro-invasive role of gelsolin was consistently demonstrated in E1 and DLD-1 cells, where reduction of gelsolin by siRNA significantly attenuated invasion. Our observations indicate that gelsolin confers invasive capacity in colorectal cancer cells, which are consistent with the reported effects of gelsolin in other types of cells [17,32]. Gelsolin Modulates the Expression of Genes Important for Tumor Dissemination Although gelsolin appears to be necessary for invasive behavior, little is known about the mechanisms by which it enhances invasion. This may partly be attributable to its ability to enhance cellular motility through its influence on cytoskeletal dynamics such as when lamellipodia are formed [33]. However, invasion is a complex process dependent on multiple contributory factors besides motility [5]. To further elucidate the downstream mechanisms by which gelsolin induces invasion in colorectal tumor cells, microarray analysis was performed to screen for potential genes that are differentially expressed when gelsolin levels are increased. The gene expression profiles of four stable gelsolin-overexpressing HCT116 clones were compared with two vector control HCT116 clones. The gelsolin- overexpressing clones expressed a consistent pattern of differential gene expression, suggesting that gelsolin induced specific changes in gene expression of HCT116 cells (Figure S5A). In total, we identified 469 genes with an average alteration in expression level of at least two- fold. Using the Gene Ontology functional annotation under DAVID Bioinformation Resources, genes were classified into their respective biological processes, including cell differentiation, cell motility and regulation of cell adhesion (Figure S5B). The representative table of genes modulated by gelsolin is supplied in Table S1. PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43594