**3.1 p130Cas in ErbB2 dependent transformation**

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

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-

p130Cas and p140Cap as the Bad and Good Guys in

Breast Cancer Cell Progression to an Invasive Phenotype 411

mammary cells MCF10A.B2, the concomitant activation of ErbB2 and p130Cas overexpression provides invasive properties (Figure 5). Consistently, p130Cas drives N202-1A cells *in vivo* lung metastases formation. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel

therapeutic target in ErbB2 positive human breast cancers (Cabodi *et al*., 2010c).

Fig. 4. p130Cas is required for *in vivo* ErbB2 tumorigenesis. Intra-nipple injection was performed in BalbC-NeuT female mice. Control (Ctr siRNA) or p130Cas stabilised siRNA (p130Cas siRNA) were injected once a week for 5 weeks starting from week 12. Left: Whole mount analyses of fixed mammary gland at week 18. The gland is composed of a tree-like structure of branching ducts. Small lesions that have histologic aspects of a solid carcinoma are visible. Black arrows indicate the lymph node. Ctr siRNA picture shows larger lesions on the right of the lymph node. Right: The histogram shows the mean tumour volume measured from two independent experiments with 8 mice per group. \*p<0.0329 (two-tailed

Fig. 5. p130Cas triggers acina invasion of ErbB2 transformed MCF10 cells. p130Cas overexpressing or Mock ErbB2 transformed MCF10 cells were plated on a Matrigel/collagen 1:1 matrix and left un-stimulated or activated for ErbB2 by treating with the small molecule AP1510. 3D invasive protrusions are present only in p130Cas over-expressing and ErbB2

activated acinar structures. The figure is modified from Tornillo *et* al., 2010.

P value). The figure is modified from Cabodi *et al*., 2010c.

transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth and *in vivo* growth of orthotopic N202-1A tumours. Moreover intra-nipple injection of p130Casstabilized siRNAs in the mammary gland of MMTV-HER2-Neu mice decreases the growth of spontaneous tumours (Figure 4) (Cabodi *et al*., 2010c).

Fig. 3. Kinetics of tumor occurrence in p130Cas/HER2-Neu and HER2-Neu mice. A) Tumor formation in p130Cas/HER2-Neu (gray line and black circles ) and HER2-Neu (black line and empty squares) mice. Twenty mice were analyzed for each group. The difference of occurrence between the two groups is statistically significant, P < 0.001. B) Independent epithelial cell culture were derived from four distinct tumors excised from p130Cas/HER2-Neu and HER2-Neu mice. Western blot analysis of protein extracts was done with the indicated antibodies and representative results are shown. MW, molecular weight markers. The figure is modified from Cabodi *et al*., 2006.

To precisely underline the mechanism implicated in p130Cas/ErbB2-mediated transformation, cultures of MECs grown on three dimensional matrix**,** that share several properties with breast epithelial acini were evaluated. These in vitro three-dimensional acini-like structures provide a developmental context and serve as an important tool to study the biological effects of oncogenic signals. Most oncogenic signals that promote proliferative signals have the ability disrupt acini organization with oncogene-specific features. For instance, activation of ErbB2 induces formation of abnormal non invasive structures consisting of individual units (Muthuswamy *et al.*, 2001). Interestingly, in human

transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth and *in vivo* growth of orthotopic N202-1A tumours. Moreover intra-nipple injection of p130Casstabilized siRNAs in the mammary gland of MMTV-HER2-Neu mice decreases the growth

Fig. 3. Kinetics of tumor occurrence in p130Cas/HER2-Neu and HER2-Neu mice.

weight markers. The figure is modified from Cabodi *et al*., 2006.

A) Tumor formation in p130Cas/HER2-Neu (gray line and black circles ) and HER2-Neu (black line and empty squares) mice. Twenty mice were analyzed for each group. The difference of occurrence between the two groups is statistically significant, P < 0.001. B) Independent epithelial cell culture were derived from four distinct tumors excised from p130Cas/HER2-Neu and HER2-Neu mice. Western blot analysis of protein extracts was done with the indicated antibodies and representative results are shown. MW, molecular

To precisely underline the mechanism implicated in p130Cas/ErbB2-mediated transformation, cultures of MECs grown on three dimensional matrix**,** that share several properties with breast epithelial acini were evaluated. These in vitro three-dimensional acini-like structures provide a developmental context and serve as an important tool to study the biological effects of oncogenic signals. Most oncogenic signals that promote proliferative signals have the ability disrupt acini organization with oncogene-specific features. For instance, activation of ErbB2 induces formation of abnormal non invasive structures consisting of individual units (Muthuswamy *et al.*, 2001). Interestingly, in human

of spontaneous tumours (Figure 4) (Cabodi *et al*., 2010c).

mammary cells MCF10A.B2, the concomitant activation of ErbB2 and p130Cas overexpression provides invasive properties (Figure 5). Consistently, p130Cas drives N202-1A cells *in vivo* lung metastases formation. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers (Cabodi *et al*., 2010c).

Fig. 4. p130Cas is required for *in vivo* ErbB2 tumorigenesis. Intra-nipple injection was performed in BalbC-NeuT female mice. Control (Ctr siRNA) or p130Cas stabilised siRNA (p130Cas siRNA) were injected once a week for 5 weeks starting from week 12. Left: Whole mount analyses of fixed mammary gland at week 18. The gland is composed of a tree-like structure of branching ducts. Small lesions that have histologic aspects of a solid carcinoma are visible. Black arrows indicate the lymph node. Ctr siRNA picture shows larger lesions on the right of the lymph node. Right: The histogram shows the mean tumour volume measured from two independent experiments with 8 mice per group. \*p<0.0329 (two-tailed P value). The figure is modified from Cabodi *et al*., 2010c.

Fig. 5. p130Cas triggers acina invasion of ErbB2 transformed MCF10 cells. p130Cas overexpressing or Mock ErbB2 transformed MCF10 cells were plated on a Matrigel/collagen 1:1 matrix and left un-stimulated or activated for ErbB2 by treating with the small molecule AP1510. 3D invasive protrusions are present only in p130Cas over-expressing and ErbB2 activated acinar structures. The figure is modified from Tornillo *et* al., 2010.

p130Cas and p140Cap as the Bad and Good Guys in

2000; Di Stefano *et al.*, 2004).

brain (Collins *et al.*, 2005).

**4.2 p140Cap interacting proteins** 

Breast Cancer Cell Progression to an Invasive Phenotype 413

p130Cas by affinity cromatography and MALDI-Mass spectrometry in epithelial cells (Di Stefano, 2004). p140Cap is a multisite docking protein, composed by a putative N-terminal mirystilation site, a tyrosine-rich domain, two prolin-rich regions, a coil-coiled domain, two regions rich in charged amino acids and a putative actin binding site (Figure 1)(Chin *et al.*,

p140Cap is mainly expressed in brain, testis and epithelial rich tissues such as mammary gland, lung, colon and kidney (Chin *et al.*, 2000; Di Stefano *et al.*, 2004; Ito *et al.*, 2008). The protein is present at least in two N-terminal alternative and two C-terminal different isoforms. The presence of many conserved sequence motifs that could undergo extensive post-translational modification, mostly tyrosine and serine phosphorylation, led to predict that p140Cap could promotes protein–protein interactions, leading to the formation of multiprotein complexes. Indeed p140Cap is tyrosine phosphorylated in epithelial cells upon integrin-mediated adhesion and EGF receptor activation (Di Stefano *et al.*, 2004). In addition, global phospho-proteomic analysis of human brain extracts revealed that p140Cap is phosphorylated on serine 859 in the context of the sequence 857RGS\*DELTVPR866 (DeGiorgis *et al.*, 2005). The same sequence has also been found phosphorylated in mouse

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

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

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).

p140Cap ability to regulate Src activation and downstream signaling (see below).

family proteins in tumour cells remains to be established.

We further analysed the molecular mechanisms through which p130Cas controls ErbB2 dependent invasion in three-dimensional cultures of mammary epithelial cells. Concomitant p130Cas over-expression and ErbB2 activation enhance PI3K/Akt and Erk1/2 MAPK signalling pathways and promote invasion of mammary acini. By using pharmacological inhibitors, we demonstrate that both signaling cascades are required for the invasive behaviour of p130Cas over-expressing and ErbB2 activated acini. Erk1/2 MAPK and PI3K/Akt signaling triggers invasion involving mTOR/p70S6K and Rac1 activation, respectively (Figure 6). Moreover, in silico analyses indicate that p130Cas expression in ErbB2 positive human breast cancers significantly correlates with higher risk to develop distant metastasis, thus underlying the value of the p130Cas/ErbB2 synergism in regulating breast cancer invasion. In conclusion, high levels of p130Cas favour progression of ErbB2 transformed cells towards an invasive phenotype (Tornillo *et al.,* 2010).

Fig. 6. Scheme illustrating the signaling pathways leading to 3D invasion of ErbB2 transformed MCF10 over-expressing p130Cas.

Both PI3K/Akt and Erk1/2 pathways are activated during invasion triggered by ErbB2 transformation of p130Cas over-expressing MEC. ErbB2/p130Cas/Erk1/2 MAPK signalling pathway preferentially targets mTOR/p70S6K, whereas the ErbB2/p130Cas/PI3K/Akt cascade triggers Rac1 activation. Both signaling pathways are required for mammary epithelia invasion in 3D suggesting that they cooperate in the regulation of different processes that ultimately lead to cell invasion. The figure is modified from Tornillo *et* al., 2010.

### **4. p140Cap adaptor protein**

#### **4.1 p140Cap structure and phosphorylation**

The human p140Cap (Cas associated protein) is codified by the gene Srcin1, previously known as SNIP, P140 or p140Cap. The Srcin1 gene is conserved in human, mouse, rat, dog, cow, and zebrafish and in human is localized on Chromosome 17 q21.1.

The p140Cap protein was originally identified in rat brain as SNIP, a Synaptosomeassociated protein SNAP-25b-interacting protein implicated in regulated exocytosis (Chin *et al.*, 2000). The name p140Cap derives from its identification as a protein associated to

We further analysed the molecular mechanisms through which p130Cas controls ErbB2 dependent invasion in three-dimensional cultures of mammary epithelial cells. Concomitant p130Cas over-expression and ErbB2 activation enhance PI3K/Akt and Erk1/2 MAPK signalling pathways and promote invasion of mammary acini. By using pharmacological inhibitors, we demonstrate that both signaling cascades are required for the invasive behaviour of p130Cas over-expressing and ErbB2 activated acini. Erk1/2 MAPK and PI3K/Akt signaling triggers invasion involving mTOR/p70S6K and Rac1 activation, respectively (Figure 6). Moreover, in silico analyses indicate that p130Cas expression in ErbB2 positive human breast cancers significantly correlates with higher risk to develop distant metastasis, thus underlying the value of the p130Cas/ErbB2 synergism in regulating breast cancer invasion. In conclusion, high levels of p130Cas favour progression of ErbB2-

transformed cells towards an invasive phenotype (Tornillo *et al.,* 2010).

Fig. 6. Scheme illustrating the signaling pathways leading to 3D invasion of ErbB2

Both PI3K/Akt and Erk1/2 pathways are activated during invasion triggered by ErbB2 transformation of p130Cas over-expressing MEC. ErbB2/p130Cas/Erk1/2 MAPK signalling pathway preferentially targets mTOR/p70S6K, whereas the ErbB2/p130Cas/PI3K/Akt cascade triggers Rac1 activation. Both signaling pathways are required for mammary epithelia invasion in 3D suggesting that they cooperate in the regulation of different processes that ultimately lead to cell invasion. The figure is modified from Tornillo *et* al.,

The human p140Cap (Cas associated protein) is codified by the gene Srcin1, previously known as SNIP, P140 or p140Cap. The Srcin1 gene is conserved in human, mouse, rat, dog,

The p140Cap protein was originally identified in rat brain as SNIP, a Synaptosomeassociated protein SNAP-25b-interacting protein implicated in regulated exocytosis (Chin *et al.*, 2000). The name p140Cap derives from its identification as a protein associated to

cow, and zebrafish and in human is localized on Chromosome 17 q21.1.

transformed MCF10 over-expressing p130Cas.

**4.1 p140Cap structure and phosphorylation** 

**4. p140Cap adaptor protein** 

2010.

p130Cas by affinity cromatography and MALDI-Mass spectrometry in epithelial cells (Di Stefano, 2004). p140Cap is a multisite docking protein, composed by a putative N-terminal mirystilation site, a tyrosine-rich domain, two prolin-rich regions, a coil-coiled domain, two regions rich in charged amino acids and a putative actin binding site (Figure 1)(Chin *et al.*, 2000; Di Stefano *et al.*, 2004).

p140Cap is mainly expressed in brain, testis and epithelial rich tissues such as mammary gland, lung, colon and kidney (Chin *et al.*, 2000; Di Stefano *et al.*, 2004; Ito *et al.*, 2008). The protein is present at least in two N-terminal alternative and two C-terminal different isoforms. The presence of many conserved sequence motifs that could undergo extensive post-translational modification, mostly tyrosine and serine phosphorylation, led to predict that p140Cap could promotes protein–protein interactions, leading to the formation of multiprotein complexes. Indeed p140Cap is tyrosine phosphorylated in epithelial cells upon integrin-mediated adhesion and EGF receptor activation (Di Stefano *et al.*, 2004). In addition, global phospho-proteomic analysis of human brain extracts revealed that p140Cap is phosphorylated on serine 859 in the context of the sequence 857RGS\*DELTVPR866 (DeGiorgis *et al.*, 2005). The same sequence has also been found phosphorylated in mouse brain (Collins *et al.*, 2005).
