Gelsolin Induces Cancer Invasion via uPA Cascade hour serum-free conditioned media of cells (right). (B) Knockdown of gelsolin (KD) reduced the secretion of uPA. Gelsolin-overexpressing HCT116, wildtype HCT116, DLD-1 and Caco-2 cells were treated with gelsolin siRNA or control siRNA for 24 hours prior to culture under serum-free conditions for a further 48 hours. The levels of secreted uPA were detected using ELISA (left). Zymographic analysis indicated that uPA activity in the 3-hour conditioned media of the colorectal tumor cells was reduced 48 hours after gelsolin siRNA knockdown treatment (right). (C) Gelsolin expression affects TNF-a-stimulated uPA secretion in colorectal cancer cells. DLD-1 and Caco-2 cells were treated with gelsolin siRNA (KD) or control siRNA (Cont) for at least 24 hours and serum-starved overnight prior to incubation with 5 ng/mL (DLD-1) and 10 ng/mL (Caco-2) TNF-a for 24 hours. The levels of secreted uPA in the supernatant of cultured cells were detected using ELISA. uPA secretion was effectively stimulated by TNF-a in DLD-1 and Caco-2. However, siRNA knockdown of gelsolin significantly attentuated TNFa-stimulated uPA levels in the colorectal cancer cell lines. (D) Inhibition of uPA cascade attenuates the invasion of gelsolin-overexpressing HCT116 cells through matrigel. Gelsolin-overexpressing cells were treated with either 50 mM amiloride, 200 mg/mL of function-blocking anti-uPA or 80 mg/mL of anti-uPAR antibodies and examined for changes in invasive potential through matrigel. DMSO treatment and mouse IgG antibody at 200 mg/mL were used as controls to amiloride and the function-blocking antibodies and respectively. Untreated vector control HCT116 was included and normalized to DMSO vehicle control. The anti-uPA and anti-uPAR as well as amiloride treatments significantly attenuated invasion of gelsolin-overexpressing HCT116 cells, indicating that the enhanced invasiveness induced by gelsolin is mediated through the uPA cascade. All data shown are the mean 6 standard error of triplicate (ELISA) and duplicate (invasion assay) measurements and are representative of at least two independent experiments. P,0.05 (student’s t test). doi:10.1371/journal.pone.0043594.g005 effect of gelsolin siRNA knockdown on invasion through matrigel may be partially attributed to a reduced migratory capacity of cells. However, the pro-invasive role of gelsolin via matrix degradation was reaffirmed by zymographic analyses, which enabled the detection of proteolytic activity of secreted proteases. Our work shows that, in addition to previous work highlighting gelsolin’s roles in driving lamellipodia protrusion and turnover in migration [43], gelsolin also confers invasive properties via its ability to modulate the expression of well-known mediators of tumor invasion - genes involved in the uPA cascade which degrade the ECM. The uPA cascade is initiated when secreted uPA from cancer cells [44] or stromal components [45] binds to its receptor uPAR and converts the inactive plasminogen to active plasmin by proteolytic cleavage. Plasmin promotes remodeling of ECM by direct proteolysis, or indirectly through activation of MMPs [46,47]. The uPA system is implicated as a major factor leading to aggressive tumor behaviour, as it promotes invasion and metastasis in several tumor types including colorectal cancer [48,49]. uPA and uPAR activities contribute to proteolysis of ECM at the invasive front of tumors [50], and elevated levels in colorectal tumors correlate with tumor progression and poor survival [51,52]. Our findings revealed the ability of gelsolin to modulate uPA gene expression and secretion. The roles of cytoskeletal proteins in regulation of gene expression is an emerging field of interest, as it is now clear that the nuclear cytoskeleton participates in several transcriptional regulatory processes [53]. Indeed, actin-binding proteins, including gelsolin and its family members have prominent roles in mediating nuclear receptor-directed transcrip- tion [54]. Gelsolin was previously reported to physically interact with androgen receptor (AR) and enhance AR transcriptional activity, [55]. More recently, gelsolin was found to be a key determinant for the assembly and/or stability of estrogen receptor nucleus complexes [56]. Interestingly, the nuclear import of CapG, another gelsolin family member, is suggested to interfere with chromosome condensation [53] and promote invasion into collagen [57]. It is unknown if gelsolin also regulates non-nuclear receptor-dependent transcription, though its presence in the nuclei of colorectal tumor tissues suggests wider roles for nuclear- associated gelsolin. As yet, it is unclear how gelsolin regulates uPA gene expression - this would be an interesting avenue for further exploration into gelsolin’s roles in transcriptional regulation of invasion genes. Besides modulating the mRNA levels of uPA, we also found that gelsolin regulates uPA secretion by colorectal cancer cells. Increased circulating levels of plasma uPA have previously been associated with advanced cancers including colorectal [58] and prostate cancers [59]. Very recently gelsolin was reported to regulate insulin exocytosis through its direct interaction with syntaxin 4, a plasma membrane protein which mediates docking of transport vesicles [60]. The expression of other actin-associated proteins, such as cortactin and LIMK1, has also been reported to correlate with the secretion of matrix proteases including MMPs [25,26,61]. LIMK1 in particular, which regulates the activities of actin-depolymerizing factor (ADF/cofilin), has also been shown to interact with membrane type 1 (MT1)-MMP and regulate its vesicular trafficking [25]. It is possible that gelsolin may also participate in the transport of vesicles containing uPA or other proteases through its influence on F-actin dynamics which is essential for vesicular trafficking. Future studies on the role of gelsolin in the secretory pathway of proteases such as uPA would yield a clearer picture of the integration of the distinct but synergistic cellular processes mediated by gelsolin to promote invasion. Nevertheless, the revelation of gelsolin’s function in regulating uPA expression and secretion in colorectal cancer invasion provides further insight into the mechanisms behind gelsolin’s oncogenic role, and lends support to the multiple roles of actin cytoskeletal proteins in promoting cancer cell dissemination. In the current study we also investigated the contribution of a number of MMPs in the gelsolin-mediated invasion in colorectal cancer cells. Inhibition of the gelatinase MMP-2 alone led to a slight decrease in invasion but not the inhibition of MMP-3, -7, - 8 and -9 in gelsolin-overexpressing cells. Treatment with GM6001 which inhibits MMP-1 in addition to MMP-2, MMP-3, MMP-8 and MMP-9 reduced invasion to a small extent but the results were not statistically significant. Interestingly proMMP-2 was reported to be activated by plasmin [47,62], which may increase the pool of active MMP-2. Although plasmin can also activate the other gelatinase proMMP-9, it is pertinent to add that plasmin is not an efficient activator of proMMP-9 [46]. Nevertheless, the balance between MMPs and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs), is critical in determining the net effect on matrix degradation. Our unpublished data indicated that TIMP-2 was increased in gelsolin-overexpressing HCT116 cells, with the direct implication being that the matrix- degrading effects of MMPs can be counter-balanced. Of note, literature also reveals paradoxical, pro-tumor roles of TIMPs in cancer biology [63]. Due to the complexity of the biological activities of MMPs and TIMPs, it is not within the scope of this study to look into the interactions between MMPs and TIMPs in detail. Moreover the evidence we presented in this manuscript suggests that the uPA cascade is a major downstream pathway by which gelsolin induce matrix degradation. Together with the well-established functions of gelsolin in cytoskeletal dynamics, our findings implicate gelsolin as a regula- tory determinant of the uPA cascade, with significant impact on colorectal cancer invasion. The potential contributions of gelsolin PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43594