B146
Loss of TGFβigh3 from gastric cancer associated myofibroblasts results in increased cancer cell migration
Christopher Holmberg1, Cedric Duval1, Roz Jenkins1, Peter Hegyi2, Gyorgy Lazar2, Islay Steele1, Graham Dockray1, Andrea Varro1
1School of Biomedical Sciences, University of Liverpool, UK, 2University of Szeged, Szeged, Hungary
Background
The contribution of stroma to the development and progression of many epithelial cancers is increasingly recognised, but the relevant mechanisms remain uncertain. We have studied the mechanisms by which a key stromal cell type, the myofibroblast, influences migration of gastric cancer cells.
Method
Cancer-derived myofibroblasts and their counterparts from adjacent normal tissue were prepared from 11 patients with gastric cancer. Cells were characterised by positive staining for α-smooth muscle actin and vimentin, and negative staining for cytokeratin and desmin. Quantitative proteomics methods (SILAC, iTRAQ) were used to define myofibroblast secretomes. Cell migration was studied using Boyden chambers.
Results
Both methods identified TGFβigh3, a 75kD TGFβ-induced protein, in myofibroblast cell media and together with Western blotting indicated either decreased abundance or degradation in media from 9 of 11 tumour-derived samples compared with cells from adjacent normal tissue. There was similar abundance of TGFβigh3 in cell extracts of tumour-derived and normal myofibroblasts. There was increased uPA activity in the media of cancer-derived myofibroblasts. Recombinant uPA, via conversion of plasminogen to plasmin, cleaved TGFβigh3 to yield fragments of 55 and 27kD. The latter corresponds to cleavage within a FAS1 domain reported to be essential in cell adhesion. Myofibroblast migration was stimulated by IGF-II and uPA, inhibited by recombinant TGFβigh3, and enhanced by TGFβigh3 knock-down using siRNA. Media from cancer-derived myofibroblasts significantly increased the migration of gastric adenocarcinoma (AGS) cells compared with media from control myofibroblasts and this was also reduced by exogenous TGFβigh3.
Conclusion
Increased proteolytic activity in the secretome of gastric cancer-derived myofibroblasts promotes cancer cell migration as a consequence of cleavage of the extracellular matrix protein TGFβigh3.
