**5. Conclusions**

416 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

to breast cancer resistance to hormonal therapies. Thus these data provide evidence that p140Cap behaves mechanistically as a tumour suppressor molecule in breast and colon

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.*,

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

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).

cancer cells, with a broad effect on cell proliferation and tumorigenesis.

2007).

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

surface and the ability to scatter in response to EGF (Damiano *et al.,* 2010)

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

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.

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

evaluated by Student's t-test: Ctr EGF vs p140 EGF (\*p<0.05).

As outlined in this chapter p130Cas and p140Cap adaptor proteins represent key elements in the control of cell migration and invasion in breast cancer cells. Interestingly, in breast cancers, p130Cas results frequently over-expressed, while p140Cap is not expressed in the more aggressive human breast cancers. Interestingly, Src kinase is a common target of these two proteins. However, even though both p130Cas and p140Cap have been described to bind to Src, they exert opposite roles on Src activity. Indeed p130Cas enhances and sustains Src activity, while p140Cap is a negative regulator of Src kinase. Therefore, it is likely that Src activity is finely tuned by p130Cas and p140Cap relative expression in cells in which they are co-expressed. As a consequence in breast tumors their reciprocal levels of expression might profoundly influence the ability of cancer cells to acquire invasive properties. Although still limited, the analysis of human breast tumors suggests that an overbalance towards p130Cas over-expression might represent a negative prognostic marker in human breast cancer specimens, indicating progression to a more aggressive phenotype.
