**2.2 p130Cas in human breast cancer**

Although several reports highlight the relevance of p130Cas in tumour cell lines and animal models, investigation of its expression in biopsies of different human malignancies using immunohistochemistry, is still limited. However, it is noteworthy that a significant subset of human breast cancers where both ErbB2 and p130Cas are over-expressed are associated with increased proliferation and low prognosis (Cabodi *et al.*, 2006). In estrogen receptor (ER)-positive human breast tumours, p130Cas over-expression correlates with intrinsic resistance to tamoxifen treatment, high risk of relapse and loss of oestrogen-receptor in a large subset of human breast cancer samples, indicating that elevated BCAR1 might be a prognostic marker for breast tumours (Dorssers *et al.*, 2001; van der Flier *et al.*, 2001).

p130Cas and p140Cap as the Bad and Good Guys in

cells.

Breast Cancer Cell Progression to an Invasive Phenotype 407

Fig. 2. p130Cas and p140Cap signalling involved in migration and invasion of breast cancer

Upon extracellular matrix binding or growth factors stimulation, integrins and Receptor Protein Tyrosine Kinases (RPTK) represent the major upstream regulators of p130Cas and p140Cap, mainly through the regulation of Src kinase activity. Once tyrosine phosphorylated by Src, p130Cas recruits proteins that activate downstream pathways, resulting in actin cytoskeleton re-organization, increased cell motility and migration. p130Cas by acting on metalloproteinases (MMPs) promoter is also required for the invasive program. Upon cell matrix adhesion or mitogen stimulus, p140Cap inhibits Src kinase activity and p130Cas tyrosine phosphorylation and p130Cas/Crk complex formation. As a consequence, the effect of p130Cas on actin cytoskeleton re-organization is impaired and cell migration and invasion are inhibited. (Di Stefano et al., 2007) Moreover, by inactivating Src, p140Cap also regulates the epidermal growth factor receptor (EGFR) pathway through Ecadherin-dependent inactivation of EGFR signalling. p140Cap by interacting with Ecadherin and EGFR at the cell membrane, immobilizes E-Cadherin at the cell membrane

thus preventing cell migration and invasion. (Damiano et al., 2010)

Therefore, at least in two classes of breast cancer that account for more than 90% of breast tumors, p130Cas over-expression is revealing its potential as prognostic factor in terms of therapy and disease progression.

#### **2.3 p130Cas tyrosine phosphorylation in cell migration and invasion**

p130Cas represents a nodal signalling platform on which integrin and RPTKs signalling convey. Integrins, RPTKs and oestrogen receptor (ER) are major upstream regulators of p130Cas, mainly through the activation of Src and Fak kinases, leading to p130Cas tyrosine phosphorylation on the C-terminal binding site YDYVHL (Figure 1) (Cabodi *et al.*). Moreover, physical stretching of p130Cas induces a conformational change that enables Srcfamily kinase-dependent p130Cas tyrosine phosphorylation. These findings point out a function for p130Cas as a sensor that integrates mechanical forces coming from the extracellular environment into intracellular signals leading to actin cytoskeleton reorganization (Kostic and Sheetz, 2006; Sawada *et al.*, 2006). The role of p130Cas in cell migration was initially inferred by studies performed on mouse embryo fibroblasts (MEFs) derived from p130Cas knock-out mice. p130Cas null MEFs show defects in stress fibre formation and cell spreading, impaired actin bundling and cell migration (Honda *et al.*, 1998), that were restored by full-length p130Cas expression. The tyrosine phosphorylation of the substrate domain of p130Cas provides binding sites for Crk proteins that in turn associates with DOCK180, a guanine nucleotide exchange factor that switches the small GTPase Rac1 from a GDP-bound inactive to a GTP-bound active state at lamellipodia and filopodia adhesion sites (Figure 2) (Kiyokawa *et al.*, 1998; Klemke *et al.*, 1998). This drives localized Rac activation, membrane ruffling and actin cytoskeleton remodelling, focal adhesion turnover, pseudopodia formation and extension. In addition, ARP2/3 and PAK kinase activation enhance cell migration (Heasman and Ridley, 2008). Uncoupling of p130Cas/Crk negatively regulates cell migration. Indeed, the non-receptor tyrosine kinase Abl phosphorylates Crk-II on tyrosine 221, inducing intramolecular folding that prevents binding of the C-terminal Crk-II SH2 domain to the phosphorylated p130Cas substrate domain, leading to decreased cell movement (Holcomb *et al.*, 2006; Kobashigawa *et al.*, 2007). Additional molecules that play important roles in modulating tyrosine phosphorylation of p130Cas leading to cell migration are the zyxin/Ajuba family of LIM proteins. These proteins bind to actin cytoskeleton and are implicated in cell motility. Ajuba allows p130Cas localization to nascent adhesive sites in migrating cells thereby leading to the activation of the small GTPase Rac, whereas Zyxin interacts with the SH3 domain of p130Cas and with a nucleocytoplasmic transcription factor, CIZ/NMP4/ZNF384 (Janssen and Marynen, 2006). Recent data also show that p130Cas activates several GTPases other than Rac. The association between p130Cas and And-34, an NSP family member, which acts as a GTP exchange factor for Ral, Rap1 and R-Ras enhances Src activation and cell migration, likely through a Rap1-dependent mechanism (Figure 2)(Riggins *et al.*, 2003). p130Cas tyrosine phosphorylation upon integrin or growth factor receptor activation has also been linked to cell invasion and it has been reported that the SH3 domain of p130Cas is also required for this process. Indeed, Focal adhesion kinase (Fak)-null cells are not invasive when transformed by v-Src, but they acquire invasive properties upon over-expression of p130Cas SH3 domain, indicating that this domain is required for rescue of v-Src cell invasion. In this context, the formation of Src/p130Cas/Crk/DOCK180 complex increases Rac1 and JNK activities and MMP-9 expression, leading to an invasive cell phenotype (Hsia *et al.*, 2003).

Therefore, at least in two classes of breast cancer that account for more than 90% of breast tumors, p130Cas over-expression is revealing its potential as prognostic factor in terms of

p130Cas represents a nodal signalling platform on which integrin and RPTKs signalling convey. Integrins, RPTKs and oestrogen receptor (ER) are major upstream regulators of p130Cas, mainly through the activation of Src and Fak kinases, leading to p130Cas tyrosine phosphorylation on the C-terminal binding site YDYVHL (Figure 1) (Cabodi *et al.*). Moreover, physical stretching of p130Cas induces a conformational change that enables Srcfamily kinase-dependent p130Cas tyrosine phosphorylation. These findings point out a function for p130Cas as a sensor that integrates mechanical forces coming from the extracellular environment into intracellular signals leading to actin cytoskeleton reorganization (Kostic and Sheetz, 2006; Sawada *et al.*, 2006). The role of p130Cas in cell migration was initially inferred by studies performed on mouse embryo fibroblasts (MEFs) derived from p130Cas knock-out mice. p130Cas null MEFs show defects in stress fibre formation and cell spreading, impaired actin bundling and cell migration (Honda *et al.*, 1998), that were restored by full-length p130Cas expression. The tyrosine phosphorylation of the substrate domain of p130Cas provides binding sites for Crk proteins that in turn associates with DOCK180, a guanine nucleotide exchange factor that switches the small GTPase Rac1 from a GDP-bound inactive to a GTP-bound active state at lamellipodia and filopodia adhesion sites (Figure 2) (Kiyokawa *et al.*, 1998; Klemke *et al.*, 1998). This drives localized Rac activation, membrane ruffling and actin cytoskeleton remodelling, focal adhesion turnover, pseudopodia formation and extension. In addition, ARP2/3 and PAK kinase activation enhance cell migration (Heasman and Ridley, 2008). Uncoupling of p130Cas/Crk negatively regulates cell migration. Indeed, the non-receptor tyrosine kinase Abl phosphorylates Crk-II on tyrosine 221, inducing intramolecular folding that prevents binding of the C-terminal Crk-II SH2 domain to the phosphorylated p130Cas substrate domain, leading to decreased cell movement (Holcomb *et al.*, 2006; Kobashigawa *et al.*, 2007). Additional molecules that play important roles in modulating tyrosine phosphorylation of p130Cas leading to cell migration are the zyxin/Ajuba family of LIM proteins. These proteins bind to actin cytoskeleton and are implicated in cell motility. Ajuba allows p130Cas localization to nascent adhesive sites in migrating cells thereby leading to the activation of the small GTPase Rac, whereas Zyxin interacts with the SH3 domain of p130Cas and with a nucleocytoplasmic transcription factor, CIZ/NMP4/ZNF384 (Janssen and Marynen, 2006). Recent data also show that p130Cas activates several GTPases other than Rac. The association between p130Cas and And-34, an NSP family member, which acts as a GTP exchange factor for Ral, Rap1 and R-Ras enhances Src activation and cell migration, likely through a Rap1-dependent mechanism (Figure 2)(Riggins *et al.*, 2003). p130Cas tyrosine phosphorylation upon integrin or growth factor receptor activation has also been linked to cell invasion and it has been reported that the SH3 domain of p130Cas is also required for this process. Indeed, Focal adhesion kinase (Fak)-null cells are not invasive when transformed by v-Src, but they acquire invasive properties upon over-expression of p130Cas SH3 domain, indicating that this domain is required for rescue of v-Src cell invasion. In this context, the formation of Src/p130Cas/Crk/DOCK180 complex increases Rac1 and JNK activities and MMP-9

**2.3 p130Cas tyrosine phosphorylation in cell migration and invasion** 

expression, leading to an invasive cell phenotype (Hsia *et al.*, 2003).

therapy and disease progression.

Fig. 2. p130Cas and p140Cap signalling involved in migration and invasion of breast cancer cells.

Upon extracellular matrix binding or growth factors stimulation, integrins and Receptor Protein Tyrosine Kinases (RPTK) represent the major upstream regulators of p130Cas and p140Cap, mainly through the regulation of Src kinase activity. Once tyrosine phosphorylated by Src, p130Cas recruits proteins that activate downstream pathways, resulting in actin cytoskeleton re-organization, increased cell motility and migration. p130Cas by acting on metalloproteinases (MMPs) promoter is also required for the invasive program. Upon cell matrix adhesion or mitogen stimulus, p140Cap inhibits Src kinase activity and p130Cas tyrosine phosphorylation and p130Cas/Crk complex formation. As a consequence, the effect of p130Cas on actin cytoskeleton re-organization is impaired and cell migration and invasion are inhibited. (Di Stefano et al., 2007) Moreover, by inactivating Src, p140Cap also regulates the epidermal growth factor receptor (EGFR) pathway through Ecadherin-dependent inactivation of EGFR signalling. p140Cap by interacting with Ecadherin and EGFR at the cell membrane, immobilizes E-Cadherin at the cell membrane thus preventing cell migration and invasion. (Damiano et al., 2010)

p130Cas and p140Cap as the Bad and Good Guys in

monoclonal antibody Herceptin.

induced cell migration.

**3.1 p130Cas in ErbB2 dependent transformation** 

putative target for clinical therapy (Cabodi *et al.*, 2006).

Breast Cancer Cell Progression to an Invasive Phenotype 409

also as a predictive marker, given that its elevated expression predicts tamoxifen resistance of the primary tumor and the response to anti-HER2 targeted therapy such as the

Further understanding of the mechanisms by which ErbB2 leads to tumorigenesis in the mammary gland comes from studies of ErbB2 mouse models. Expression of Neu mutation that promotes spontaneous receptor dimerization (NeuT), under the MMTV promoter, or more recently under the ErbB2 endogenous promoter (ErbB2/KI model), leads to the formation of mammary adenocarcinomas (Andrechek *et al.*, 2000; Muller *et al.*, 1998). Interestingly, the expression of the ErbB2 protooncogene in a MMTV-transgenic mice show late tumor latency with a low penetrance of lung metastasis, suggesting that gene amplification of the wild type receptor may be the main mechanism implicated in ErbB2 mediated tumorigenesis. Indeed, elevated protein and mRNA ErbB2 levels in the ErbB2/KI model also correlated with selective genomic amplification of the activated ErbB2 allele (Andrechek and Muller, 2000; Hodgson *et al.*, 2005; Montagna *et al.*, 2002). One of the most significant effects associated with ErbB2 activation is enhanced and sustained signal transduction cascades leading to the regulation a variety of cellular processes, including proliferation, apoptosis, cell polarity, migration and invasion (Feigin and Muthuswamy, 2009). Activation of specific ErbB homo- or heterodimer pairs leads to initiation of the mitogen activated protein kinase (MAPK) cascade, activation of phospholipase C gamma (PLCγ) and phosphatidylinositol 3 kinase (PI3K), as well as induction of the small GTPases Rho, Rac and Cdc42, among many other effectors (Hynes and MacDonald, 2009; Kurebayashi, 2001). Several reports have demonstrated a role for these pathways in ErbB-

In the context of ErbB2 positive breast cancer, previous studies generated by our group placed p130Cas as an important regulator of ErbB2-dependent tumorigenesis. To investigate the mechanisms through which p130Cas is linked to tumorigenesis, we generated mouse mammary tumor virus (MMTV)-p130Cas mice overexpressing p130Cas in the mammary gland. MMTVp130Cas transgenic mice are characterized by extensive mammary epithelial hyperplasia during development and pregnancy and by delayed involution at the end of lactation. These phenotypes are associated with activation of Src kinase, Erk1/2 MAPK, and Akt pathways, leading to an increased rate of proliferation and a decreased apoptosis. A double-transgenic line derived from crossing MMTV-p130Cas with MMTV-HER2-Neu mice expressing the activated form of the HER2-Neu oncogene develops multifocal mammary tumors with a significantly shorter latency than the HER2-Neu parental strain alone (Figure 3). MECs isolated from tumors of double-transgenic mice display increased tyrosine phosphorylation, c-Src, and Akt activation compared with cells derived from HER2-Neu tumors. In addition, p130Cas down-regulation by RNA interference increases apoptosis in HER2-Neu-expressing cells, indicating that p130Cas regulates cell survival. These findings provide evidences for a role of p130Cas as a positive regulator of both proliferation and survival in normal and transformed mammary epithelial cells. Its overexpression contributes to HER2-Neu-induced breast tumorigenesis, thus identifying this protein as a

More recent studies further assessed the functional role of p130Cas in ErbB2-dependent breast tumorigenesis by its silencing in breast cancer cells derived from mouse mammary tumours over-expressing ErbB2 (N202-1A cells), and by its re-expression in ErbB2-
