**4.5 p140Cap affects cell proliferation and in vivo tumour growth of breast cancer cells**

The ability of p140Cap to regulate Src and Ras pathways profoundly affects cell proliferation. Elevated expression of p140Cap in both breast and colon cancer cells inhibits in vitro proliferation, but does not affect cell survival (Damiano *et al.*; Di Stefano *et al.*, 2007). Interestingly, p140Cap over-expression impairs colony formation in soft agar, while its silencing leads to a significantly increased number of colonies, demonstrating that p140Cap, likely through the regulation of integrin signalling, controls anchorage-independent growth (Di Stefano *et al.*, 2007). *In vivo* xenografts of breast and colon cancer cells show that cells expressing high levels of p140Cap are impaired in tumour formation. Consistently, p140Cap silencing in carcinoma cells dramatically increases *in vivo* tumour formation. Strikingly, p140Cap knock-down is sufficient for *in vivo* growth of MCF7 cells even in the absence of estrogen pellets, a condition in which control cells are unable to grow. These last findings also rise the possibility that p140Cap may regulate estrogen receptor signalling, contributing

p130Cas and p140Cap as the Bad and Good Guys in

*al.*, 2011).

phenotype.

**7. References** 

**6. Acknowledgement** 

Torino. S. Cabodi and P. Defilippi are co-last authors.

cancer. *Breast Cancer Res* 2: 211-6.

tumorigenesis. *Proc Natl Acad Sci U S A* 97: 3444-9.

**5. Conclusions** 

Breast Cancer Cell Progression to an Invasive Phenotype 417

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* 

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

This work was supported by EU FP7 Metafight project, AIRC, MUR (PRIN), Regione Piemonte – Progetti Sanità, Oncoprot, PiSTEM, Druidi and CIPE, Compagnia San Paolo,

Andrechek ER, Hardy WR, Siegel PM, Rudnicki MA, Cardiff RD, Muller WJ (2000).

Andrechek ER, Muller WJ (2000). Tyrosine kinase signalling in breast cancer: tyrosine

Amplification of the neu/erbB-2 oncogene in a mouse model of mammary

kinase-mediated signal transduction in transgenic mouse models of human breast

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