Gelsolin Induces Cancer Invasion via uPA Cascade invasion through matrigel-coated transwells were ascertained over 48 hours, and was observed to be increased compared to vector control cells. (E) siRNA downregulation of gelsolin abrogates invasion. Gelsolin knockdown significantly reduced invasion of HCT116 and gelsolin-overexpressing HCT116 through matrigel. A requirement for gelsolin in invasion was also observed in E1 and DLD-1 cells. All data shown are the mean 6 standard error of duplicate measurements and are representative of at least three independent experiments. *P,0.05. doi:10.1371/journal.pone.0043594.g003 Gelsolin Modulates the Expression of Invasion-associated Urokinase-type Plasminogen Activator (uPA) Among the invasion-associated genes identified to be induced by more than 2-fold in gelsolin-overexpressing cells is the serine protease, urokinase-type plasminogen activator (uPA), which is involved in the plasminogen activation cascade that results in the activation of a broad-spectrum protease plasmin. Although microarray analysis detected less than 2-fold changes in the levels of other uPA cascade genes, we included these in real-time PCR analysis as these comprise important components for regulating uPA-dependent matrix degradation. Besides uPA, the following genes were screened for mRNA expression: the uPA receptor uPAR, and known inhibitors of the uPA cascade including plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2) and a2-antiplasmin (a2-AP) [34]. Real-time PCR showed that increased levels of gelsolin in HCT116 cells upregulated the expression of uPA and uPAR, and reduced the levels of the inhibitors PAI-2 and a2-AP (Figure 4A). Consistent with observations in the overexpression studies, mRNA levels of uPA were also reduced by siRNA knockdown of gelsolin in wild- type HCT116, DLD-1 and Caco-2 cells (Figure 4B). The mRNA levels of uPAR were reduced by gelsolin siRNA-treated HCT116 and Caco-2 cells but not in DLD-1 cells. Our data indicates that uPA levels are altered by changes in gelsolin expression, and the uPA cascade is a possible mechanism through which gelsolin mediates invasion. Gelsolin Increases uPA Secretion by Colorectal Tumor Cells and Promotes Invasion through the uPA Cascade In the plasminogen activator cascade, the secretion of uPA is required to activate the proenzyme plasminogen to active plasmin in the extracellular environment. We investigated the levels of secreted uPA protein in the conditioned media of gelsolin- overexpressing colorectal cancer cells obtained after 48 hours of serum-deprivation, using enzyme-linked immunosorbent assays (ELISA). Consistent with the increase in uPA gene expression, we also detected significantly increased uPA secretion in gelsolin- overexpressing HCT116 compared to control cells (Figure 5A). We examined the effects of gelsolin-induced uPA expression on matrix degradation using zymographic analysis. Conditioned media of the cells were loaded and electrophoresed in zymo- graphic gels containing plasminogen and fibrinogen for analysis of uPA activity. Gelsolin-overexpressing HCT116 cells displayed greater uPA activity in comparison to vector control and wildtype HCT116, as evident from the enhanced lysis zones (Figure 5A). The increased proteolytic ability of gelsolin-overexpressing cells, as ascertained by our zymographic analysis, corroborates with earlier observations from the transwell invasion assay which showed that gelsolin conferred invasiveness in colorectal cancer cells (Figure 3D–E). Conversely, siRNA knockdown of gelsolin in the colorectal tumor cell lines HCT116, DLD-1 and Caco-2 significantly reduced the level of secreted uPA and its proteolytic activity (Figure 5B). Our data suggests that gelsolin modulates uPA activity on the extracellular matrix by influencing uPA expression and its secretion. The cytokine, tumor necrosis factor alpha (TNF-a), is produced by several types of tumors and has been reported to stimulate uPA production [35] and enhance invasion [36]. As shown in Figure 5C, treatment with TNF-a for 24 hours enhanced uPA secretion in DLD-1 and Caco-2 cells. However when gelsolin expression was reduced by siRNA knockdown, the increases in uPA secretion induced by TNF-a were significantly attenuated to levels similar to that in control cells. Our data shows that TNF- a induced uPA secretion is attenuated in the absence of gelsolin. However, it is currently unclear how gelsolin modulates uPA secretion in response to TNF-a stimulation. The involvement of the uPA cascade in the increased invasiveness of gelsolin-overexpressors was further confirmed by inhibiting uPA function and examining the effects on invasion through matrigel. The highly invasive gelsolin-overexpressing HCT116 cells were treated with function-blocking antibodies against either uPA or uPAR, or by treatment with the uPA inhibitor amiloride [37]. Control experiments were conducted using isotype-control mouse IgG and vehicle control dimethyl sulfoxide (DMSO), respectively. Blockade of the uPA cascade using either anti-uPA or anti-uPAR antibody, or amiloride significantly attenuated the invasiveness of gelsolin-overexpressing HCT116 cells across matrigel, by 40% to 55% (Figure 5D). Our data indicated that gelsolin enhances invasion through the uPA activator cascade, by promoting uPA secretion which can enhance matrix degradation. Apart from members of the uPA cascade, the genes of various other proteases or related proteins such as metalloproteinase-7 (MMP7) and TIMP2 were also modulated by gelsolin over- expression in HCT116 (Table S1). Since tumor cells may secrete different proteases during the course of dissemination, we in- vestigated whether the gelsolin-mediated invasion may involve, besides uPA, members of the MMP family. Gelsolin-overexpres- sing cells were subjected to similar transwell invasion assays following treatment with either the synthetic pan-MMP inhibitor GM6001 (which blocks MMP-1, -2, -3, -8 and -9 activities) or other specific inhibitors to MMP-2, MMP-2/29, MMP-3, MMP- 7 and MMP-8. Pan-MMP inhibition using GM6001, as well as MMP-2 inhibition reduced the invasiveness of cells by about 20%, although the results were not statistically significant (Figure S6). The simultaneous inhibition of MMP-2/MMP-9 showed similar effect to inhibition of MMP-2 alone, suggesting that MMP-9 played little or no role in the invasion of gelsolin-overexpressing cells. There was however no significant increase in the MMP-2 activity in gelsolin-overexpressing cells compared to vector control and HCT116 based on gelatin zymography (Data not shown). Inhibition of MMP-3, MMP-7 and MMP-8 had no effect on invasion (Figure S6). Taken together our data indicated that in addition to its pro-migratory role, gelsolin promotes invasion in the colorectal carcinoma cells predominantly via increased expression and secretion of uPA. Discussion In this study, we show that invading populations of tumor cells enriched in gelsolin are found in both primary as well as metastatic human colorectal cancers. This is consistent with earlier observa- tions of prominent gelsolin expression along the invasive front of liver metastases, in contrast to its low expression in primary colorectal adenocarcinomas [28]. The significance of ‘‘leading cells’’ at the invasive front of tumors in promoting tumor spread PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43594