immortalized human breast epithelial MCF10A cell line induced changes suggestive of epithelial-mesenchymal transformation [9]. However, there is also evidence that the re-emergence of gelsolin in tumors may promote aggressive behavior, as progressively malignant stages in cancer are associated with high gelsolin expression. Increased gelsolin expression was found to correlate with lymphatic invasion in small cell lung cancer [10] and higher tumor grade in renal cell carcinoma [11]. Intriguingly, in urothelial and oral carcinomas, gelsolin exhibited a biphasic expression profile, being down-regulated in premalignant lesions but increased in higher grade lesions [12,13]. Furthermore, the co- expression of gelsolin with erb-B2 and epidermal growth factor receptor (EGFR) is a predictor of poor prognosis in breast cancer [14]. It is likely that the role of gelsolin differs during the course of tumor progression, and in more advanced disease gelsolin may cooperate with other oncogenic factors to accelerate progression. Several in vivo and in vitro studies clearly indicate that gelsolin is crucial for migration and invasion in several cell types, including migration of untransformed cells such as fibroblasts and neutrophils [15,16], as well as invasion of transformed immortalized human and canine kidney cells [17] and tumor cells including breast and prostate cancer cells across collagen and matrigel matrices [18,19]. Gelsolin’s pro-migratory effects have been attributed to its actin-severing actions [16,20]. However, its pro-invasive activities in epithelial cancers are unclear, and may involve a combination of mechanisms including migration and interactions with signaling proteins. Gelsolin has been shown to be a downstream effector of signaling pathways mediating invasion, including Ras and Rac GTPases, as well as phosphotidylinositol 3-kinase (PI3K) [17,20]. Gelsolin also facilitates osteoclast podosome formation [21], and associates with the oncogenic tyrosine kinase Src in these structures [22]. Podosomes are rich in actin and mediate dynamic cell-matrix adhesion and ECM remodeling [23,24]. Although there is now a pool of convincing evidence linking gelsolin to invasion [17,18,19], there is little insight (beyond gelsolin’s role in actin dynamics) on the mechanisms downstream of gelsolin leading to invasion. Previous studies have correlated the expression of actin-associated proteins such as cortactin and Lim Kinase-1 (LIMK1) with protease secretion [25,26], and it is unknown whether gelsolin also modulates the proteolytic machinery to induce invasion. This study aims to address the gap in knowledge between gelsolin and the matrix degradation process during cancer cell invasion. We investigated the influence of gelsolin on colorectal tumor cell dissemination and the mechanisms underlying its pro-invasive activity. Immunohistochemical (IHC) analysis showed prominent gelsolin expression along the tumor borders of both primary human colon tumors and liver metastases. The effects of gelsolin in human colorectal tumor cells were examined by inducing gelsolin over- expression as well as silencing with siRNA. Microarray analysis and quantitative PCR in these models indicated that gelsolin modulates the expression of several invasion-related genes in the urokinase-type plasminogen activator (uPA) cascade, resulting in activation of plasmin, a potent matrix degradation protease [27]. uPA and its receptor uPAR were further determined to be crucial for gelsolin- dependent invasion in colorectal tumor cells. Our work thus elucidates a novel role for gelsolin in colorectal tumor dissemination, by modulation of the uPA cascade which is crucial for invasion. Results Gelsolin Expression is Prominent at the Invasive Front of Colorectal Tumors We analyzed the expression of gelsolin by IHC in 24 primary colorectal tumors and 26 colorectal liver metastases as well as 15 Gelsolin Induces Cancer Invasion via uPA Cascade normal tissues from the surgical margins of clearance. Gelsolin expression in tumor tissues as well as the adjacent normal tissues was scored for intensity of staining (scale 0–3) and proportion of tumor positivity (scale 0–3). Primary antibody exclusion as well as mouse IgG were included as negative controls (Figure S1). In the adjacent normal colonic mucosa, the expression was prominent at the surface epithelium which comprises absorptive cells but weakly expressed in goblet cells (Figure S2). In positively-stained mucosal cells, gelsolin was present in the cytoplasm and nuclear staining was generally observed in a small proportion of cells. Gelsolin was highly expressed in myocytes of the muscularis propria and in vessel walls, consistent with previous findings [28,29], as well as lymphoid cells. We found gelsolin expression to be heterogeneously expressed in the matched primary tumors and liver metastases, with regions of low and high expression seen within a tumor. Gelsolin was detectable in the cytoplasm as well as the nuclei of tumor cells (Figure 1). Emerging evidence supports the importance of identifying changes within specific tumor populations, such as those at the infiltrating borders which are involved in tumor invasion and metastasis [30]. We therefore analyzed the pattern of gelsolin expression at the tumor borders compared to the tumor bulk, as these populations are potentially disseminative. In order to define the infiltrative tumor borders, adjacent sections of liver metastases were also stained with the pan-cytokeratin stain, AE1/ 3, which identifies tumor cells of epithelial origins. Gelsolin expression was pronounced along the tumor borders compared to tumor bulk in both primary tumors and liver metastases (Figure 2). In liver metastases, gelsolin expression was significantly higher in the tumor borders compared to the main tumor bulk (p = 0.0075, Mann-Whitney test). Interestingly we also observed high gelsolin expression in infiltrating clusters of less-differentiated cells, some of which appeared to be breaking away from well-formed glandular structures, supporting the hypothesis that gelsolin is involved in colorectal tumor cell invasion and dissemination. Our data suggests that gelsolin is further upregulated along the metastatic tumor borders and may promote secondary spread of colorectal cancer cells within the primary and secondary host tissues. Gelsolin expression was also determined in a panel of commer- cially-available colorectal tumor cell lines derived from primary tumors (HCT116, HT29, WiDr, RCM-1, RKO, Caco-2, SW837, SW480, SW403, DLD-1, LS513) as well as those obtained from metastatic lymph node (SW620) and ascites (COLO201 and COLO205). Among the commercially-available cell lines, the ascites-derived COLO201 and COLO205 and the primary tumor cell lines DLD-1 and LS513 expressed the highest levels of gelsolin (Figure S3). Gelsolin Promotes Migration and Invasion of Colorectal Tumor Cells The oncogenic potential of gelsolin in colorectal tumor cells was investigated by modulating gelsolin levels using gelsolin over- expression and siRNA knockdown. Using the technique of in-vivo passaging through athymic nude mice, we had previously established the highly metastatic colon carcinoma E1 cell line from the poorly-metastatic HCT116 cell line [31]. HCT116 was observed to express intermediate levels of gelsolin in a panel of colorectal carcinoma cell lines investigated (Figure S3). Consistent with our hypothesis that there exists a correlation between metastatic potential and gelsolin levels, E1, the metastatic variant, expressed increased gelsolin levels compared to HCT116 (Figure 3A). To further investigate this, we overexpressed gelsolin in HCT116 cells and performed functional assays. Overexpression of gelsolin in the HCT116 was induced by stable transfection with PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43594