Galectin-3 facilitates cell motility in gastric cancer by up-regulating protease-activated receptor-1 (PAR-1) and matrix metalloproteinase-1 (MMP-1)

Abstract

Background: Galectin-3 is known to regulate cancer metastasis. However, the underlying mechanism has not been defined. Through the DNA microarray studies after galectin-3 silencing, we demonstrated here that galectin-3 plays a key role in up-regulating the expressions of protease-activated receptor-1 (PAR-1) and matrix metalloproteinase-1 (MMP-1) PAR-1 thereby promoting gastric cancer metastasis.

Methodology/principal findings: We examined the expression levels of Galectin-3, PAR-1, and MMP-1 in gastric cancer patient tissues and also the effects of silencing these proteins with specific siRNAs and of over-expressing them using specific lenti-viral constructs. We also employed zebrafish embryo model for analysis of in vivo gastric cancer cell invasion. These studies demonstrated that: a) galectin-3 silencing decreases the expression of PAR-1. b) galectin-3 over-expression increases cell migration and invasion and this increase can be reversed by PAR-1 silencing, indicating that galectin-3 increases cell migration and invasion via PAR-1 up-regulation. c) galectin-3 directly interacts with AP-1 transcriptional factor, and this complex binds to PAR-1 promoter and drives PAR-1 transcription. d) galectin-3 also amplifies phospho-paxillin, a PAR-1 downstream target, by increasing MMP-1 expression. MMP-1 silencing blocks phospho-paxillin amplification and cell invasion caused by galectin-3 over-expression. e) Silencing of either galectin-3, PAR-1 or MMP-1 significantly reduced cell migration into the vessels in zebrafish embryo model. f) Galectin-3, PAR-1, and MMP-1 are highly expressed and co-localized in malignant tissues from gastric cancer patients.

Conclusions/significance: Galectin-3 plays the key role of activating cell surface receptor through production of protease and boosts gastric cancer metastasis. Galectin-3 has the potential to serve as a useful pharmacological target for prevention of gastric cancer metastasis.

Figure 1. Knock down studies showing that galectin-3 regulates PAR-1 expression and gastric cancer cell migration and invasion.

A, mRNA and protein expression levels after transfection of human gastric cancer MKN-28 cells with 20 nM scRNA, or siRNAs of galectin-3 or PAR-1. Total RNA and protein obtained after transfection for 48 hr. Cells were harvested and analyzed by RT-PCR and western blotting. β-actin was used as a loading control. B, Protein levels of galectin-3 and PAR-1 by western blotting after transfection with pcDNA3.1/NT-GFP-Galectin-3 and vector control of pcDNA3.1/NT-GFP in SNU-638 cells. C, Cell migration assay performed of galectin-3 or PAR-1 in silenced MKN-28 cells. The results were present as a histogram (* p<0.001 vs. Cont group), and cell photos of cell migration assays. D, Histogram was present that cell invasion assays of galectin-3 or PAR-1 in silenced MKN-28 cells (* p<0.001 vs. Cont group).

Figure 2. Galectin-3 enhances gastric cancer cell migration/invasion by increasing PAR-1 expression.

A, mRNA and protein levels of galectin-3 and PAR-1 detected by RT-PCR and western blot analysis in SNU-638 cells, infected with lenti-virus containing LacZ or galectin-3, and then transfected with PAR-1 siRNA or scRNA for a negative control. β-actin was used as a loading control. B and C, Migration (B) and invasion (C) assays were conducted of SNU-638 cells infected with lenti-virus containing LacZ or galectin-3 cells, and then transfected with PAR-1 siRNA or scRNA for a negative control. Results was shown as histogram (* p<0.001 vs. Cont group). D, Schematic model of PAR-1 promoter with AP-1 binding site, Primers used for ChIP assay were prepared to detect AP-1 binding site (−463∼−474) from −666 to −307. E, Galectin-3 interacts with c-Jun and fra-1 (as, an AP-1 complex [20]) in MKN-28 cells. Immunoprecipitation was performed as described in “Material and Methods”, and then galectin-3, c-Jun and fra-1 detected by western blot analysis. Whole cell lysates (WCLs) were used as a positive control. F, Analysis of chromatin immunoprecipitation assay using antibodies to galectin-3, c-Jun and Fra-1 in MKN-28 cells transfected with scRNA and galectin-3 siRNA. PCR primer for the PAR-1 gene promoter was used to detect promoter fragment in immunoprecipitates. Input lane, total genomic DNA used as control for the PCR reaction. G, Luciferase activity of AP-1 in lacZ and galectin-3 over-expressing cells. Luciferase assay was performed using AP-1 expression luciferase vector transfection to lacZ and galectin-3 over-expressing cells (* p<0.001 vs. Cont). β-galactoside was used as negative control.

Figure 3. Galectin-3 mediated MMP-1 expression promotes gastric cancer cell invasion through PAR-1 activation.

A, galectin-3, PAR-1, MMP-1 and MMP-9 mRNA and galectin-3, PAR-1, MMP-1, phospho-Paxillin (pY181) and paxillin protein levels after transfection with scRNA or each siRNAs of galectin-3, PAR-1, MMP-1 in MKN-28 cells. Total RNA and protein obtained after transfection for 48 hr and cells were harvested and analyzed by RT-PCR and western blot. MMP-9 activity was assayed by gelatin zymography using the medium of MKN-28 cells. B, mRNA expression levels of galectin-3, PAR-1 and MMP-1 by RT-PCR; protein expression levels of galectin-3, PAR-1, MMP-1, phospho-paxillin (pY181) and paxillin by western blot analysis in SNU-638 cells, which were infected with lenti-virus containing LacZ or galectin-3, and then transfected with MMP-1 siRNA or scRNA for a negative control. C, Invasion assay of SNU-638 cells infected with lenti-virus containing LacZ or galectin-3 cells, and then transfected with MMP-1 siRNA or scRNA for a negative control. The data are presented as histogram (* p<0.001 vs. Cont group).

Figure 4. Over-expressed MMP-1 influences gastric cancer cell invasion and schematic model of galectin-3 regulate gastric cancer cell invasion and migration.

A, protein expression of galectin-3, PAR-1, MMP-1, phospho-Paxillin (pY181) and paxillin levels were measured by western blot analysis, after infection with lenti-viral construct containing pLECE3 (vector only) and pLECE3-MMP-1 in AGS cells, transfected with galectin-3 or PAR-1 siRNAs. B, Cell invasion performed by the above cells present as a histogram (* p<0.001 vs. Cont group). C, Galecitn-3 enhances PAR-1 expression via binding with AP-1 transcription factor, also, galectin-3 regulation of MMP-1 expression. MMP-1 increase causes dual effects in gastric cancer invasion; 1) cleavage of PAR-1 tethered ligand and PAR-1 activation 2) degradation of extracellular matrices (ECM).

Figure 5. Studies on the migration of gastric cancer cells in the zebrafish model.

A, At 53 hours post fertilization (hpf), the transplanted AGS cells which transfected galectin-3, PAR-1, MMP-1 siRNA and scRNA (red) were located in the center of yolk sac of living transgenic zebrafish in which embryonic vessels are visualized with green fluorescence at 4 hours post transplantation (hpt); up to 50 hpt were clearly detected only cancer cells. B, The number of migrated cells per embryo were counted, and shown as histogram. These data were derived from three replicated experiments. Scale bars, 200 µm and 50 µm in last panels. C and D, Increased expression of galectin-3, PAR-1 and MMP-1 in gastric cancer patients. C, Expression and localization of galectin-3, PAR-1 and MMP-1 in malignant tissues from gastric cancer patients using immunohistochemical staing (brown) with H&E by fluorescence microscopy. Magnification: (upper panel)×200; (lower panel)×400. D, mRNA of galectin-3, PAR-1 and MMP-1 levels in tissues of gastric cancer patients detected by RT-PCR (Figure S3). Malignant and normal tissues were obtained from 20 gastric patients subjected to RT-PCR, quantified and analyzed by NIH image analyzer. Higher expression levels of galectin-3, PAR-1 and MMP-1 in malignant tissues are shown as percentage increases over their levels in normal tissues.