**4.6 p140Cap affects in vitro motility and invasion of breast cancer cells**

As expected for the major role of Src in actin cytoskeleton dynamics and cell migration, high levels of p140Cap impair spreading and extension of lamellipodia and filopodia on extracellular matrix proteins of breast cancer cells. In addition, p140Cap over-expression also inhibits migration on fibronectin-coated transwells and invasion in Matrigel. Consistently, p140Cap silencing induces an increase in cell spreading in the early phases of cell adhesion, a fibroblastic-like shape and increased motility and invasion. Cells expressing a truncated form of p140Cap, lacking the Src-binding domain, restores integrin-dependent Src and Rac activation and are capable of migrating and invading properly (Di Stefano *et al.*, 2007).

In addition, p140Cap specifically interferes with invasive and migratory properties of cancer cells blocking E-cadherin/EGFR cross-talk in both breast and colon cancer cells. The ability of p140Cap to immobilize E-cadherin at the cell surface strengthenes cell-cell adhesion and inhibition of cell scatter in response to EGF. Rescue of Src activity by the expression of a kinase-defective Csk mutant or by Csk silencing, recover E-cadherin mobility at the cell surface and the ability to scatter in response to EGF (Damiano *et al.,* 2010)

Moreover, we recently identified p140Cap as a critical regulator of *in vitro* cell motility and invasion and *in vivo* metastasis formation of highly metastatic MTLn3-EGFR breast cancer cells. Our data show that increasing p140Cap expression in the highly aggressive MTLn3- EGFR cells results in an 80% decrease in *in vivo* lung metastasis formation (Figure 8).

The figure is modified from Damiano *et al.*, 2011.

Fig. 8. p140Cap over-expression inhibits spontaneous lung metastasis formation. A) 5x105 Ctr and p140 cells were injected subcutaneously in Rag2−/<sup>−</sup> <sup>γ</sup>c−/− mice. Right panels: after sacrificing the mice, lungs were coloured with ink, metastasis were counted and the number of metastasis reported in the y axis of the histogram. Statistical significances were evaluated by Student's t-test: Ctr EGF vs p140 EGF (\*p<0.05).

B) Upper panels: two representative pictures of lung metastases visualized with the FLI (GFP detection) after spontaneous metastasis assay with the MTLn3-EGFR Ctr and p140 cells. Lower panels: two representative pictures of the lungs coloured with ink are shown. Consistently, p140Cap over-expressing MTLn3-EGFR cells show also reduced anchorageindependent cell growth, which is an *in vitro* characteristic that predicts the *in vivo* metastatic potential of many tumour cells. Furthermore, detailed *in vitro* analysis of cell migratory and invasive abilities showed that p140Cap over-expressing cells have an impaired capacity to migrate in response to EGF. Remarkably, p140Cap over-expressing cells display an increased number and area of focal adhesions, which correlate with the presence of actin stress fibers consistent with a less dynamic turnover of adhesive structures. Cortactin tyrosine phosphorylation has been shown to regulate MTLn3 cells invadopodia assembly and maturation (Oser *et al.*, 2009). Our results show that in p140Cap overexpressing cells cortactin phosphorylation in response to EGF is decreased. Indeed, the expression of the phosphomimetic cortactin mutant is sufficient to completely rescue the defects in migration and invasion of MTLn3-EGFR p140Cap over-expressing cells. Taken together, these data demonstrate that p140Cap suppresses the invasive properties of highly metastatic breast carcinoma cells by inhibiting cortactin-dependent cell motility (Damiano *et al.*, 2011).
