Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin’s influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites

A CRITICAL ROLE FOR GELSOLIN IN COLORECTAL CANCER CELL DISSEMINATION

Ph.DDOCTOR OF PHILOSOPH

Gelsolin increases uPA secretion of colorectal tumor cells and enhances invasion via the urokinase-type plasminogen (uPA) cascade.

<p>(<b>A</b>) Increased gelsolin in HCT116 cells augmented the secretion and activity of uPA. The levels of secreted uPA by cells cultured for 48 hours in serum-free conditions were detected in the supernatant using ELISA. Gelsolin-overexpressing HCT116 cells secreted significantly higher uPA levels compared to control cells (<i>left</i>), which correlated with higher uPA enzymatic activity, as evident from uPA zymographic analysis using 16-hour serum-free conditioned media of cells (<i>right</i>). (<b>B</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 (<i>left</i>). 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 (<i>right</i>). (<b>C</b>) Gelsolin expression affects TNF-α-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-α 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-α in DLD-1 and Caco-2. However, siRNA knockdown of gelsolin significantly attentuated TNFα-stimulated uPA levels in the colorectal cancer cell lines. (<b>D</b>) Inhibition of uPA cascade attenuates the invasion of gelsolin-overexpressing HCT116 cells through matrigel. Gelsolin-overexpressing cells were treated with either 50 µM amiloride, 200 µg/mL of function-blocking anti-uPA or 80 µg/mL of anti-uPAR antibodies and examined for changes in invasive potential through matrigel. DMSO treatment and mouse IgG antibody at 200 µg/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 ± 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).</p

Gelsolin promotes migration and invasion of colorectal cancer cells.

<p>(<b>A</b>) Endogenous gelsolin level was increased in an <i>in vivo</i>-derived metastatic variant of HCT116, E1, which was described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043594#pone.0043594-Tay1" target="_blank">[31]</a>. (<b>B</b>) Gelsolin overexpression plasmid was constructed by cloning human cytoplasmic gelsolin cDNA into pIRES2-EGFP vector (<i>left</i>). HCT116 cells were either transfected with gelsolin-pIRES2-EGFP plasmid or empty pIRES2-EGFP plasmid to establish stable gelsolin-overexpressing cell lines and vector control cell lines respectively. Western blot analysis confirmed increased gelsolin expression in HCT116 cell lines stably-transfected with the gelsolin-overexpression plasmid compared to those transfected with control plasmid. (<b>C</b>) Western blot showing transient gelsolin siRNA knockdown (KD) at 3 days in wild-type HCT116 and gelsolin-overexpressing HCT116 cells, E1, as well as other colorectal cancer cell lines DLD-1 and Caco-2 cells. Control cells (Cont) were treated with control siRNA. (<b>D</b>) Increased gelsolin in HCT116 enhanced tumor cell migration and invasion. The migration of gelsolin-overexpressing HCT116 cells through uncoated transwells and invasion through matrigel-coated transwells were ascertained over 48 hours, and was observed to be increased compared to vector control cells. (<b>E</b>) 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 ± standard error of duplicate measurements and are representative of at least three independent experiments. *P<0.05.</p

Gelsolin immunohistochemistry in human colorectal carcinoma tissues.

<p>Gelsolin is heterogeneously expressed in a number of primary tumors (A) and liver metastases (B), with islands of low (<i>arrowed in blue</i>) and high (<i>arrowed in red</i>) expression observed within a tumor. Gelsolin expression is mainly cytoplasmic but occasionally, nuclear (C) and perinuclear (D) staining are detected (<i>arrowed</i>). Gelsolin is strongly expressed in a mucinous adenocarcinoma (E) and in stroma (A, B).</p

Gelsolin expression is prominent at the invasive front of human colorectal cancer tumors.

<p>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 µm.</p

La mise en scène d'un drame intérieur dans le poème "Sur le péché originel" d'Avit de Vienne

This article highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse gastric cancer

Genetic Polymorphism Characteristics of <i>Brucella canis</i> Isolated in China

<div><p>In China, brucellosis is an endemic disease typically caused by <i>Brucella melitensis</i> infection (biovars 1 and 3). <i>Brucella canis</i> infection in dogs has not traditionally recognized as a major problem. In recent years however, brucellosis resulting from <i>Brucella canis</i> infection has also been reported, suggesting that infections from this species may be increasing. Data concerning the epidemiology of brucellosis resulting from <i>Brucella canis</i> infection is limited. Therefore, the purpose of this study was to assess the diversity among Chinese <i>Brucella canis</i> strains for epidemiological purposes. First, we employed a 16-marker VNTR assay (<i>Brucella</i> MLVA-16) to assess the diversity and epidemiological relationship of 29 <i>Brucella canis</i> isolates from diverse locations throughout China with 38 isolates from other countries. MLVA-16 analysis separated the 67 <i>Brucella canis</i> isolates into 57 genotypes that grouped into five clusters with genetic similarity coefficients ranging from 67.73 to 100%. Moreover, this analysis revealed a new genotype (2-3-9-11-3-1-5-1:118), which was present in two isolates recovered from Guangxi in 1986 and 1987. Second, multiplex PCR and sequencing analysis were used to determine whether the 29 Chinese <i>Brucella canis</i> isolates had the characteristic BMEI1435 gene deletion. Only two isolates had this deletion. Third, amplification of the <i>omp25</i> gene revealed that 26 isolates from China had a T545C mutation. Collectively, this study reveals that considerable diversity exists among <i>Brucella canis</i> isolates in China and provides resources for studying the genetic variation and microevolution of <i>Brucella</i>.</p></div