**1. Introduction**

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

Zinzalla, G.; Haque, M.R.; Basu, B.P.; Anderson, J.; Kaye, S.L.; Haider, S.; Hasan, F.;

signalling. *Bioorg. Med. Chem. Lett.,* 20, 7029-7032.

Antonow, D.; Essex, S.; Rahman, K.M.; Palmer, J.; Morgenstern, D.; Wilderspin, A.F.; Neidle, S. & Thurston, D.E. (2010) A novel small-molecule inhibitor of IL-6

> Breast cancer is an aggressive malignancy affecting a large woman population. Even though important progress have been made in providing new therapies to treat this neoplasia, our knowledge on the mechanisms underlying the transformation of breast epithelial cells in tumor cells is still superficial. The neoplastic phenotype results from the alteration of multiple cellular signaling mechanisms controlling proliferation, survival and invasiveness. Moreover, the prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. However the mechanisms by which epithelial tumor cells escape from the primary tumor and colonize a distant site are not entirely understood. In this chapter we will discuss recent data on the relevance of p130Cas and p140Cap adaptor molecules in breast cancer signalling related to the acquirement on invasive properties. Due to the presence of adaptor modules, these proteins create signalling platforms proximal to plasma membrane cell surface receptors, such as integrins and growth factor receptors. p130Cas and p140Cap exert opposite regulation on cell signalling. Indeed p130Cas has been shown to increase survival, proliferation and migration of normal and transformed cells either in response to cell matrix adhesion or to hormones and growth factors. Moreover, p130Cas has been recently linked to resistance to breast cancer treatments, revealing its potential use as a novel therapeutic target. Instead, p140Cap behaves as a potent negative regulator of signalling pathways leading to cancer cell proliferation and migration. In this chapter, we will discuss the increasing evidence that highlight the importance of these adaptor proteins in breast cancer.

> It is well established that to migrate and to invade a cell needs to detach from its neighbors, i.e. adjacent cells in an epithelium, to extend lamellipodia and filopodia from the leading edge and to create new dynamic adhesions, which form and rapidly disassemble at the base of protrusions (Mitra *et al.*, 2005; Ridley *et al.*, 2003). Cell invasion also requires the release or activation of proteases that degrade the extracellular matrix (ECM) and allows cells to sort out from the basal lamina invading surrounding tissues (Eliceiri *et al.*, 2002). Under physiological conditions cell motility and invasion are tightly controlled by a complex interplay among cell-cell, cell matrix and growth factors receptors resulting in the maintenance of the architectural integrity of human tissues. This subtle regulation is lost in

p130Cas and p140Cap as the Bad and Good Guys in

Fig. 1. p130Cas and p140Cap structure.

Cortactin and Csk have yet to be defined.

**2.2 p130Cas in human breast cancer** 

mediate Crk binding.

*et al.*, 2000).

Breast Cancer Cell Progression to an Invasive Phenotype 405

Fak, PYK2/RAFTK, phosphatases like PTP-PEST, PTP1B, and effectors as C3G and CIZ (Sakai *et al.*, 1994; Tikhmyanova *et al.*). The substrate domain, upon Src family kinases activation, is tyrosine phosphorylated and exposes additional binding sites for SH2 containing proteins such as the Crk adaptors (Salgia *et al.*, 1996)**,** while the serine rich region represents a docking site for other partners such as 14-3-3 and Grb2. Lastly, the C-terminus contains a polyproline-rich region responsible for the binding of the Src family kinase, PI3K, Bcar3/AND-34, Chat-H and ubiquitin ligases such as AIP4, APC/C and CDH1, as well as a binding site for the adaptor protein p140Cap (Bouton *et al.*, 2001; Cabodi *et al.*, 2004; O'Neill

A) p130Cas consists of an N-terminal SH3 domain, a substrate domain (SD), a serine rich region (SRR), and a C-terminal domain (CT). The main interactors are indicated. In particular, many proteins associate to the N-terminal domain and the Src family kinases (SFKs) bind the CT domain. The 15 YxxP motifs are phosphorylated by Src family kinases to

B) p140Cap consists of an N-terminal tyrosine–rich region (Tyr-rich), an actin binding domain (ABD), a proline rich domain (Pro1), a coil-coiled region (C1-C2), two domains rich in charged amino acids (CH1, CH2) and a C-terminal proline rich domain (Pro2). Src, p130Cas, EB3 and Vinexin bind to the Pro2 domain of p140Cap. The binding regions of

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).

human tumours leading to uncontrolled dissemination of cancer cells into the body (Berx *et al.*, 2007; Cavallaro and Christofori, 2004; Giancotti, 2003; Guo and Giancotti, 2004),

At least three major classes of membrane proteins are involved in these events, namely, the E-cadherin, the Receptor tyrosine kinases (RPTKs), and the integrin receptors. The cell-cell adhesion receptor E-cadherin is the major membrane protein involved in binding between neighbouring cells in adherens junctions. As a practical consequence of its adhesive functions, E-cadherin has also been shown to prevent EGFR activation and downstream signalling, leading to negative regulation of proliferation (Berx and Van Roy, 2001; Gutkind, 2000; Perrais *et al.*, 2007; Qian *et al.*, 2004). E-cadherin is frequently down-regulated or lost in epithelial tumours, and its loss correlates with increased cancer cell invasiveness ((Peinado *et al.*, 2007; Reynolds and Carnahan, 2004);.

Integrins are cell surface heterodimeric receptors for the ECM formed by the non covalent association of alpha and beta subunits (Hynes, 2004). Integrins specifically localize to focal adhesions, which are sites of close apposition with the ECM where actin filaments are anchored to the plasma membrane. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors or growth factor and cytokine receptors, thus regulating integrin ability to transduce signals inside the cells, the so called "outside-in signalling" ( Cabodi *et al*., 2010). A growing body of evidence shows that integrins, RPTKs and cytokine receptors have no longer to be considered as individual receptors, but rather as joint modules in which attachment to the matrix confers positional control to respond to soluble growth factors (Cabodi *et al*., 2010b; Cabodi *et al.*, 2008; Desgrosellier *et al.*, 2009; Streuli, 2009; Uberti *et al.*). In the case of the EGF receptor (EGFR), beta1 integrin is both sufficient to partially activate the receptor itself and required for the full activation of the EGFR in response to EGF (Morello *et al.*; Moro *et al.*, 1998)). Integrin-dependent EGFR trans-activation accounts for a specific repertoire of mechanisms, namely cell survival and actin cytoskeleton organization involved in cell migration.

In this chapter we will focus on p140Cap and p130Cas adaptors as major regulators of cell migration and invasion (Cabodi *et al.*). Owing to their modular structure, both proteins can undergo tyrosine phosphorylation and association with effector proteins, leading to the assembly of molecular platforms that regulate the variety of signalling events originating from the complex cross-talk among integrins, E-cadherin and RPTKs.
