**2. Cell-cell adhesion and the functional roles of JAMs in epithelial/endothelial cells**

#### **2.1 Introduction to cell-cell adhesion complexes and JAMs**

Cells within the breast tumour microenvironment physically interact with each other and with the extracellular matrix through a range of cell adhesion proteins. Cell adhesion proteins play fundamental roles in normal physiology (such as the control of cell polarity and epithelial barrier function), but their dysregulation has been shown to participate in

Junctional Adhesion Molecules (JAMs)- New Players in Breast Cancer? 493

JAM proteins have been implicated in a diverse array of physiological functions involving cell–cell adhesion/barrier function (Liang *et al*., 2000; Liu *et al*., 2000; Mandell *et al*., 2004), leukocyte migration (Martin-Padura *et al*., 1998; Palmeri *et al*., 2000; Johnson-Leger *et al*., 2002; Ostermann *et al*., 2002), platelet activation (Kornecki *et al*., 1990; Naik *et al*., 1995; Gupta *et al*., 2000; Ozaki *et al*., 2000; Sobocka *et al*., 2000; Naik *et al*., 2001; Babinska *et al*., 2002; Babinska *et al*., 2002) and angiogenesis (Naik *et al*., 2003; Naik *et al*., 2003). These functions

**2.2 JAM proteins regulate epithelial/endothelial cell–cell adhesion and barrier function**  JAM proteins are well-known to be important for cell-cell adhesion in both epithelial and endothelial cells (for review see Mandell & Parkos, 2005), but emerging evidence supports the possibility that they also regulate cell-matrix adhesion complexes. Interestingly, JAM-A knockdown in endothelial cells and MCF7 breast cancer cells has been shown to reduce adhesion to fibronectin and vitronectin (McSherry *et al*., 2011; Naik & Naik, 2006), while JAM-C overexpression in endothelial cells reportedly decreases attachment to fibronectin, vitronectin, and laminin (Li *et al*., 2009). This apparent incongruity may relate to the fact that JAM-A may activate β1 integrins (McSherry *et al*., 2011), while JAM-C has conversely been described to inactivate β1 integrins (Li *et al*., 2009). An inverse relationship between JAMs – A and –C has also been observed in terms of tight junction function, with JAM-A promoting tight junction sealing while phosphorylated JAM-C increases paracellular leakiness due to its redistribution away from TJs (Li *et al*., 2009). Furthermore, adhesion of the lung carcinoma cell line NCI-H522 to endothelial cells was significantly blocked by soluble JAM-

The contribution of JAM proteins to cell-cell adhesion and the assembly of epithelial/endothelial TJs relates to their ability to promote the localization of ZO-1, AF-6, CASK and occludin at points of cell-cell contact. Evidence suggests that both homophilic and heterophilic interactions, as well as an intact PDZ binding motif, are important for such protein functions of JAMs. Accordingly, JAMs have been shown to physically interact with the PDZ proteins, ZO-1 (Bazzoni *et al*., 2000; Ebnet *et al*., 2000), AF-6 (Ebnet *et al*., 2000), CASK (Martinez-Estrada *et al*., 2001), PAR-3 (Ebnet *et al*., 2001; Itoh *et al*., 2001) and MUPP-1 (Hamazaki *et al*., 2002); which are involved in actin cytoskeletal rearrangement (Fanning *et al*., 2002), cell signalling (McSherry *et al*., 2011; Boettner *et al*., 2000) and the control of cell polarity. However JAMs can also bind to non-PDZ proteins such as cingulin (Bazzoni *et al*., 2000), and indirectly bind occludin (Bazzoni *et al*., 2000) and claudin 1 via their interactions with ZO-1 (Hamazaki *et al*., 2002). Although the manner in which JAMs interact with some of these proteins is incompletely understood, it appears that homo-dimerisation of JAM proteins is important for regulating some key downstream functions. This has been illustrated by the fact that dimerisation-blocking anti-JAM-A antibodies (Liu *et al*., 2000) and soluble Fc–JAM-A (Liang *et al*., 2000) delay the recovery of electrical resistance (a marker of

TJ function) in epithelial cells following transient depletion of extracellular calcium.

In general cell adhesion and cell migration are inversely related, and serve to control important physiological functions and pathophysiological events. However, in the case of JAM family members, close functional associations with cell polarity proteins may act as a switch between increased adhesion (predisposing to slow, directional migration) and decreased

**2.3 JAM proteins regulate epithelial/endothelial migration** 

will be further discussed in the next sections.

C (Santoso *et al*., 2005).

tumour cell migration, invasion and adhesion (for review, see Brennan *et al.,*2010). Adhesion proteins rarely exist in isolation from each other on the cell membrane, rather they form components of multi-cellular adhesion complexes containing a network of adhesion, scaffolding and signalling proteins. Breast epithelial cells express various types of adhesion complexes, namely hemidesmosomes and focal adhesions at the cell-matrix interface, with tight junctions, adherens junctions, desmosomes and gap junctions at the cell-cell interface. Collectively, adhesion complexes are composed of integral membrane proteins and cytoplasmic scaffolding proteins that organise signalling complexes and anchor cell-cell contacts to intermediate filaments (at desmosomes and hemidesmosomes) or to actin filaments (at adherens junctions, tight junctions and focal adhesions).

Tight junctions (TJs) play a vital role in regulating the paracellular flux of ions, small molecules and inflammatory cells as well as defining distinctly-polarized membrane domains and facilitating bi-directional signalling between the intracellular and extracellular compartments. These functions of the TJ are regulated by the balance of three different types of integral membrane proteins; (1) Occludins and Tricellulin, (2) Claudins and (3) Immunoglobulin Superfamily (IgSF) members. Of most interest in this chapter is the Junctional Adhesion Molecule (JAM) subfamily of the IgSF, and its potential contribution to cancer initiation and progression.

The JAM family consists of 5 proteins (JAM-A, -B, -C, -4, -L) which are major components of TJs in endothelial and epithelial cells in a variety of vertebrate and invertebrate tissues (Martin-Padura *et al*., 1998; Liang *et al*., 2000; Liu *et al*., 2000; Arrate *et al*., 2001; Aurrand-Lions *et al*., 2001; Itoh *et al*., 2001; Hirabayashi *et al*., 2003; Tajima *et al*., 2003). JAM proteins are also expressed on the surface of haematopoetic cells such as platelets, neutrophils, monocytes, lymphocytes, leukocytes and erythrocytes; in addition to connective tissue cells such as fibroblasts and smooth muscle cells (Azari *et al*., 2010; Kornecki *et al*., 1990; Naik *et al*., 1995; Malergue *et al*., 1998; Williams *et al*., 1999; Cunningham *et al*., 2000; Palmeri *et al*., 2000; Arrate *et al*., 2001; Aurrand-Lions *et al*., 2001; Moog-Lutz *et al*., 2003; Morris *et al*., 2006). JAMs are type I transmembrane proteins consisting of an N-terminal signal peptide, an extracellular domain (consisting of two immunoglobulin-like domains), a single membranespanning domain and a short cytoplasmic tail (Martin-Padura *et al*., 1998; Liu *et al*., 2000; Sobocka *et al*., 2000; Aurrand-Lions *et al*., 2001; Naik *et al*., 2001; Santoso *et al*., 2002). The cytoplasmic tail is thought to play a major role in the assembly of adhesion signalling complexes, since it has been reported to bind to PDZ domain-containing scaffold proteins such as ZO-1 (Bazzoni *et al*., 2000; Ebnet *et al*., 2000), AF-6 (Ebnet *et al*., 2000) and MUPP1 (Hamazaki *et al*., 2002).

JAMs -A, -B and -C exhibit a short cytoplasmic tail of 45–50 residues that ends with a type II PDZ binding motif, while JAM-4 and JAM-L have longer cytoplasmic tails (of 105 and 98 residues respectively). JAM-4 and JAM-L differ in that the cytoplasmic tail of the former ends in a canonical type I PDZ binding motif, while that of the latter lacks a PDZ-binding motif (Mandell & Parkos, 2005). The cytoplasmic tails of JAM proteins also contain consensus phosphorylation sites that may serve as substrates for protein kinase C, protein kinase A and Casein Kinase II (Naik *et al*., 1995; Cunningham *et al*., 2000; Ozaki *et al*., 2000; Sobocka *et al*., 2000; Arrate *et al*., 2001; Naik *et al*., 2001). Indeed, evidence suggests that specific phosphorylation sites may be critical for targeting of JAMs to intercellular junctions (Ozaki *et al*., 2000; Ebnet *et al*., 2003).

tumour cell migration, invasion and adhesion (for review, see Brennan *et al.,*2010). Adhesion proteins rarely exist in isolation from each other on the cell membrane, rather they form components of multi-cellular adhesion complexes containing a network of adhesion, scaffolding and signalling proteins. Breast epithelial cells express various types of adhesion complexes, namely hemidesmosomes and focal adhesions at the cell-matrix interface, with tight junctions, adherens junctions, desmosomes and gap junctions at the cell-cell interface. Collectively, adhesion complexes are composed of integral membrane proteins and cytoplasmic scaffolding proteins that organise signalling complexes and anchor cell-cell contacts to intermediate filaments (at desmosomes and hemidesmosomes) or to actin

Tight junctions (TJs) play a vital role in regulating the paracellular flux of ions, small molecules and inflammatory cells as well as defining distinctly-polarized membrane domains and facilitating bi-directional signalling between the intracellular and extracellular compartments. These functions of the TJ are regulated by the balance of three different types of integral membrane proteins; (1) Occludins and Tricellulin, (2) Claudins and (3) Immunoglobulin Superfamily (IgSF) members. Of most interest in this chapter is the Junctional Adhesion Molecule (JAM) subfamily of the IgSF, and its potential contribution to

The JAM family consists of 5 proteins (JAM-A, -B, -C, -4, -L) which are major components of TJs in endothelial and epithelial cells in a variety of vertebrate and invertebrate tissues (Martin-Padura *et al*., 1998; Liang *et al*., 2000; Liu *et al*., 2000; Arrate *et al*., 2001; Aurrand-Lions *et al*., 2001; Itoh *et al*., 2001; Hirabayashi *et al*., 2003; Tajima *et al*., 2003). JAM proteins are also expressed on the surface of haematopoetic cells such as platelets, neutrophils, monocytes, lymphocytes, leukocytes and erythrocytes; in addition to connective tissue cells such as fibroblasts and smooth muscle cells (Azari *et al*., 2010; Kornecki *et al*., 1990; Naik *et al*., 1995; Malergue *et al*., 1998; Williams *et al*., 1999; Cunningham *et al*., 2000; Palmeri *et al*., 2000; Arrate *et al*., 2001; Aurrand-Lions *et al*., 2001; Moog-Lutz *et al*., 2003; Morris *et al*., 2006). JAMs are type I transmembrane proteins consisting of an N-terminal signal peptide, an extracellular domain (consisting of two immunoglobulin-like domains), a single membranespanning domain and a short cytoplasmic tail (Martin-Padura *et al*., 1998; Liu *et al*., 2000; Sobocka *et al*., 2000; Aurrand-Lions *et al*., 2001; Naik *et al*., 2001; Santoso *et al*., 2002). The cytoplasmic tail is thought to play a major role in the assembly of adhesion signalling complexes, since it has been reported to bind to PDZ domain-containing scaffold proteins such as ZO-1 (Bazzoni *et al*., 2000; Ebnet *et al*., 2000), AF-6 (Ebnet *et al*., 2000) and MUPP1

JAMs -A, -B and -C exhibit a short cytoplasmic tail of 45–50 residues that ends with a type II PDZ binding motif, while JAM-4 and JAM-L have longer cytoplasmic tails (of 105 and 98 residues respectively). JAM-4 and JAM-L differ in that the cytoplasmic tail of the former ends in a canonical type I PDZ binding motif, while that of the latter lacks a PDZ-binding motif (Mandell & Parkos, 2005). The cytoplasmic tails of JAM proteins also contain consensus phosphorylation sites that may serve as substrates for protein kinase C, protein kinase A and Casein Kinase II (Naik *et al*., 1995; Cunningham *et al*., 2000; Ozaki *et al*., 2000; Sobocka *et al*., 2000; Arrate *et al*., 2001; Naik *et al*., 2001). Indeed, evidence suggests that specific phosphorylation sites may be critical for targeting of JAMs to intercellular junctions

filaments (at adherens junctions, tight junctions and focal adhesions).

cancer initiation and progression.

(Hamazaki *et al*., 2002).

(Ozaki *et al*., 2000; Ebnet *et al*., 2003).

JAM proteins have been implicated in a diverse array of physiological functions involving cell–cell adhesion/barrier function (Liang *et al*., 2000; Liu *et al*., 2000; Mandell *et al*., 2004), leukocyte migration (Martin-Padura *et al*., 1998; Palmeri *et al*., 2000; Johnson-Leger *et al*., 2002; Ostermann *et al*., 2002), platelet activation (Kornecki *et al*., 1990; Naik *et al*., 1995; Gupta *et al*., 2000; Ozaki *et al*., 2000; Sobocka *et al*., 2000; Naik *et al*., 2001; Babinska *et al*., 2002; Babinska *et al*., 2002) and angiogenesis (Naik *et al*., 2003; Naik *et al*., 2003). These functions will be further discussed in the next sections.
