Cancer du sein et micro-environnement tumoral: rôle de la protéase ...

using cath-D-/- MEF transfected with human cath-D ( cath-D-/- MEF-cath-D), cath-D-/- MEFtransfected with proteolytically-inactive cath-D ( cath-D-/- MEF- D231N cath-D), and HMFcells, which express LRP1 (Fig. 5 and Fig. 6).Our findings demonstrate that LRP1 silencing in cath-D-/- MEF-cath-D cells usingsiRNA3 (Fig. 5A, panel e) and siRNA4 (Fig. Sup. 2, panel e) reduced their cath-Ddependentoutgrowth (Fig. 5A, panels a and c; Fig. Sup. 2, panels a and c),compared to cells transfected with Luc siRNA (Fig. 5A, panels b and d; Fig. Sup. 2,panels b and d). Similar results were observed when LRP1 expression was inhibitedin cath-D-/- MEF- D231N cath-D cells, indicating a mechanism independent of cath-Dproteolytic activity (Fig. 5B). These findings indicate that cath-D does indeed requireLRP1 expression to promote fibroblast invasive outgrowth.To gain further insight into whether LRP1 mediates the paracrine action ofsecreted pro-cath-D on fibroblast outgrowth, we performed 3D co-culture assays ofLuc- and LRP1-silenced HMF fibroblasts (Fig. 6, panel e) and control- or cath-Dsecreting3Y1-Ad12 cancer cell lines (Fig. 6, panel f). The co-culture assay revealedthat only pro-cath-D-secreting 3Y1-Ad12 cells stimulated the outgrowth of HMFfibroblasts (Fig. 6, panel b), in contrast to 3Y1-Ad12 control cells (Fig. 6, panel a).Our results further showed that silencing LRP1 expression in HMF fibroblasts usingsiRNA1 (Fig. 6, panel e) blocked their mitogenic response to secreted pro-cath-D(Fig. 6, panel d). Similar results were obtained with LRP1 siRNA2 (Fig. Sup. 3). Incontrol experiments, we verified that the viability of HMF cells remained unaffected byLRP1-silencing (data not shown). These experiments demonstrate that the paracrinestimulation of cath-D-induced fibroblast outgrowth requires LRP1 expression.DISCUSSION13