Matrix metalloproteinase 9 inhibits the motility of highly aggressive HSC-3 oral squamous cell carcinoma cells

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.Peer reviewe

Caveolin-1 accumulation in the tongue cancer tumor microenvironment is significantly associated with poor prognosis : an in-vivo and in-vitro study

Peer reviewe

Tumour Microenvironments Induce Expression of Urokinase Plasminogen Activator Receptor (uPAR) and Concomitant Activation of Gelatinolytic Enzymes

Peer reviewe

Tumour microenvironment induced uPAR protein expression in tongue tumours.

<p>Tumour growth pattern and uPAR protein levels in tongue tumours generated from the EV1, EV2, uPAR1 and uPAR2 cells. <b>A–B:</b> Representative images depicting the tumour growth pattern at the tumour-stroma interface in hematoxylin/eosin stained EV1 (<b>A</b>) and uPAR1 (<b>B</b>) tumours. Images were recorded at 10x magnification. <b>C–D:</b> Representative images depicting the IHC uPAR staining of the EV1 (<b>C</b>) or uPAR1 tumours (<b>D</b>). Images were recorded at 4x magnification. <b>E–H:</b> The images show high power magnification (20x magnifications) of the EV1 (<b>E</b>), uPAR1 (<b>F</b>), EV2 (<b>G</b>) and uPAR2 (<b>H</b>) tumours IHC stained for uPAR protein. Positive uPAR staining is seen as brown colour, and counterstaining was done with haematoxylin. <b>I:</b> The average staining index (SI) of the uPAR staining in the tumours. Maximum obtainable score is 9. The error bars shows the +SEM. N = number of tumours; EV1, N = 8/10; EV2, N = 5/10; uPAR1, N = 4/10; uPAR2 N = 9/10. One-way ANOVA; **p<0.01, *p<0.05. T = Tumours, S = Stroma.</p

Expression of murine <i>Plaur</i> in AT84 cells.

<p><i>In vitro</i> characterization of AT84 cells stably transfected with either empty vector (EV) or a vector containing cDNA encoding murine uPAR (<i>Plaur</i>). <b>A:</b> Western blot analysis of whole cell lysates using a polyclonal anti-murine uPAR antibody (AF534). A total of 7.5 ng of recombinant murine uPAR (rmuPAR) was loaded as a positive control. Re-probing for β-actin was used as a loading control. <b>B:</b> Western blot analysis of cellular membrane fractions using a polyclonal anti-murine uPAR antibody (AF534). Total protein was measured per sample and 53.5 µg of protein was loaded per lane. <b>A and B:</b> Images were cropped, as no additional bands were detectable. <b>C:</b> FACS analysis of non-permeabilized cells using a polyclonal anti-murine uPAR antibody (AF534). Alexa Fluor 488 anti-goat secondary antibody (A11055) was used as the secondary antibody. The quantified mean Alexa 488 fluorescence signal per cell line is presented in the panel to the right. <b>D and E:</b> Relative <i>Plaur</i> mRNA (uPAR) (<b>D</b>) or <i>Plau</i> mRNA (uPA) (<b>E</b>) expression levels as analysed using RT-qPCR. All expression levels were normalized to the expression of the reference genes <i>Trfc</i> and β-<i>actin</i>. Error bars represent the standard error of mean (+SEM) and N = 3. One-way ANOVA; *p<0.05. <b>F:</b> Plasminogen-gelatin (upper panel) and gelatin (lower panel) zymography analysis of conditioned medium of cells cultured for 24 hours in SFM. HMW-uPA and mPLM (mouse plasmin) were loaded as positive controls. The images were cropped to size. <b>G:</b> Relative <i>Plasminogen</i> mRNA (Plg) expression levels as analysed using RT-qPCR. Error bars represent standard error of mean (+SEM) and N = 3.</p

Knock-down of uPAR expression in AT84 cells.

<p>shRNA knock-down of <i>Plaur</i> in AT84 cells. <b>A:</b> Flow chart showing the generation of the single cell clones. <b>B:</b> Western blot analysis of whole cell lysates from the single cell clones stably transfected with either shRNA-constructs (shRNA 3 = sh3, shRNA 4 = sh4 or shRNA 5 = sh5) targeting <i>Plaur</i> or constructs containing non-target shRNA (NT) or the empty vector (EV). uPAR was detected using a polyclonal anti-murine uPAR antibody (AF534). Re-probing for β-actin was used as a loading control. Images were cropped, as no additional bands were detected in the blot.</p

Gelatinolytic activity is enhanced in tumours expressing high levels of uPAR.

<p>ZBF-fixed tongue tumours were sectioned and analysed for the presence of gelatinolytic activity using DQ-gelatin <i>in situ</i> zymography. Gelatinolytic activity is seen as green fluorescence. <b>A:</b> Representative confocal images of tongue tumours generated from the uPAR1-NT cells (left panel) and EV1-sh3 cells (right panel). <b>B:</b> Quantification of fluorescence intensity (analysed using Volocity as described in materials and methods) for a minimum of 5 images per tumour, presented as mean values. Three individual uPAR1-NT tumours (No.1–No.3) and three EV1-sh tumours (No.1–No.3) were analysed. Error bars shows the standard deviation (+SD) between the five images analysed. Dark grey bars represents gelatinolytic activity in the tumour sections, light grey bars represents gelatinolytic activity in tumour sections treated with the metalloproteinase inhibitor EDTA. Mann-Whitney rank sum test; ***p<0.001, **p<0.01, *p<0.05.</p

Leiomyoma stroma is a strong inducer of uPAR expression.

<p>Representative images of low- (EV1-sh3) and high- (uPAR1-NT) uPAR-expressing cells invading the <i>ex vivo</i> leiomyoma tissue. Cells were incubated for 7 and 14 days, as indicated. The tissue was IHC stained for uPAR. Positive uPAR staining is seen as brown colour, counterstained with haematoxylin. Images were recorded at 10x magnification.</p