**4.2 p140Cap interacting proteins**

Since its discovery, many proteins have been shown to bind directly or to associate in molecular complexes with p140Cap. In normal epithelial cells, p140Cap was found associated to the adaptor protein p130Cas. Although *in vitro* binding studies indicate that p140Cap and p130Cas are not directly linked, their association is mediated by the last 217 amino acids of the p140Cap C-terminal region and the p130Cas region encompassing amino acids 544-678. Through the same C-terminal region, p140Cap binds directly to the SH3 domain of the Src kinase. Moreover in MCF7 cells p140Cap has been shown by Far Western Blotting to bind directly the kinase C-terminal Src kinase (Csk), a potent negative regulator of Src (Di Stefano *et al.*, 2007). The physiological significance of p140Cap interaction with Src and Csk relates to p140Cap ability to regulate Src activation and downstream signaling (see below).

By two hybrid screen in human brain, the C-terminal motif of p140Cap has also been found to associate with the SH3 domain of Vinexin (Ito *et al.*, 2008), belonging to a family composed of vinexin, c-Cbl associated protein/ponsin, and Arg-binding protein 2 (Kioka *et al.*, 2002; Matsuyama *et al.*, 2005). In non-neuronal cells, Vinexin is localized at focal adhesions and shown to be involved in growth factor- and integrin-mediated signal transduction, actin cytoskeletal organization, cell spreading, motility, and growth (Kioka *et al.*, 2002). Always in brain, p140Cap directly associates with all the members of the microtubule plus-end tracking protein EB family through a short 92 amino acid C-terminal region, likely through a positively charged S/P-rich region (Jaworski *et al.*, 2009). The p140Cap interaction with Vinexin and EB family proteins in tumour cells remains to be established.

Finally, in breast cancer cells, p140Cap has also been shown to bind with Cortactin (Damiano *et al.*, 2011). Cortactin is a major substrate of Src kinase and localizes to cortical actin structures where it regulates early cell migration and invasion by controlling actin assembly (Weed *et al.*, 2000; Wu and Parsons, 1993; Wu *et al.*, 1991). p140Cap/Cortactin association requires the second proline-rich domain of p140Cap and the Cortactin SH3 domain, suggesting a direct interaction between the two proteins. p140Cap binding to Cortactin controls invasion properties of breast cancer cells (Damiano *et al.*, 2011).

p130Cas and p140Cap as the Bad and Good Guys in

sustained Rac activation (Di Stefano *et al.*, 2007).

2010).

Breast Cancer Cell Progression to an Invasive Phenotype 415

The major function of the p140Cap adaptor is its ability to regulate Src kinase activation. In particular, in breast cancer cells, upon cell-matrix adhesion or EGF stimulation, p140Cap activates the Csk kinase, that phosphorylates the negative regulatory tyrosine 530 on the Cterminal domain of Src (Latour and Veillette, 2001) , resulting in inhibition of Src kinase. Consistently p140Cap silencing increases Src activation, leading to a fine tuning of integrin and growth factor receptor signalling (Figure 2) (Damiano *et al.,* 2010; Di Stefano *et al.*, 2007) As a consequence, in breast cancer cells expressing high levels of p140Cap, upon integrinmediated adhesion, the association between Src and Fak is impaired as well as integrindependent p130Cas phosphorylation (Figure 2). As described above p130Cas phosphorylation leads to the assembly of a p130Cas-Crk signalling complex that drives for cell migration and invasion through activation of Rac. Therefore elevated levels of p140Cap severely impair integrin-dependent Rac activity, while its down-regulation induces a

In MCF7 breast cancer cells, p140Cap functionally interacts with E-cadherin and EGFR at the cell membrane, behaving as a new player in E-cadherin-dependent down-regulation of EGFR signalling. Indeed p140Cap-dependent inhibition of Src kinase activity results in Ecadherin immobilization at the cell membrane (Damiano *et al.,* 2010). E-cadherin is known to inhibit EGFR, either by interaction through the extracellular domains or by a beta catenindependent mechanism (Perrais *et al.*, 2007; Qian *et al.*, 2004; Takahashi and Suzuki, 1996). Consistently, EGFR activation, association and phosphorylation of Grb2 and Shc and Ras/Erk1/2 MAPK activities are profoundly impaired by p140Cap over-expression and enhanced by its silencing (Damiano *et al.,* 2010). Interestingly, rescue of Src activity and of Ecadherin mobility is sufficient to recover EGFR phosphorylation, but not Ras and Erk1/2 activation, that require an active RasV12, suggesting that p140Cap might regulate the Ras pathway through an additional mechanism. Therefore, in MCF7 cancer cells, p140Cap regulates EGFR signalling with dual mechanisms, involving both an E-cadherin-dependent inactivation of EGFR and a Ras-dependent inhibition of Erk1/2 activity (Damiano *et al.,* 

Moreover, p140Cap expression also inhibits EGFR, Src and Erk phosphorylation in the highly aggressive MTLn3-EGFR breast cancer cells. Interestingly, in these cells, p140Cap

**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

affects also Cortactin phosphorylation in response to EGF (Damiano *et al.*, 2011).

**4.4 p140Cap modulates Src activity and EGFR signalling in breast cancer cells** 

In conclusion, p140Cap is involved in direct interactions with several proteins (Figure 1). The p140Cap binding partners are mainly implicated in membrane fusion and actin cytoskeleton remodelling. p140Cap association to p130Cas, Src, Cortactin and the presence of a putative actin binding domain in the p140Cap sequence, suggest that p140Cap could be an actin binding protein. Indeed, p140Cap has been described to co-localize with actin stress fibers and cortical actin both in epithelial and in neuroectodermal cells (Chin *et al.*, 2000; Di Stefano *et al.*, 2004; Jaworski *et al.*, 2009).
