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

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.

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

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

Junctional Adhesion Molecules (JAMs)- New Players in Breast Cancer? 495

invasion through the regulation of integrin expression and activation (McSherry *et al*., 2011;

In breast cancer, the formation of metastases at distant sites is the leading cause of cancerrelated death. In order for breast cancer cells to metastasize, they must first migrate out of the primary tumour before ever reaching a distant organ and potentially proliferating into a secondary tumour. While JAMs are already known to regulate migration, the possibility that they are also involved in the regulation of proliferation will be referred to in section 3.3 of

All together these data highlight the role of JAM family members in controlling the balance between cell adhesion and migration. Although much remains to be understood about the exact role of JAMs in breast cancer cell migration, the classic description of tumours as "wounds which do not heal" (Riss *et al*., 2006) suggests that the migratory mechanisms employed by JAMs in physiological responses (such as wound healing) may also be utilised

In previous sections we discussed the biphasic role of JAM family members in regulating cell adhesion and migration. In this section we will outline the emerging contribution of the JAM family to cellular differentiation. Cell differentiation in the context of normal tissue usually involves the transition from an undifferentiated stem/progenitor cell to a

JAM-A, JAM-B, JAM-C and JAM-4 have been found to be highly expressed on hematopoietic stem cells (HSCs) in the bone marrow, with their expression decreasing during the acquisition of a more differentiated state (Nagamatsu *et al*., 2006; Sakaguchi *et al*., 2006; Sugano *et al*., 2008; Praetor *et al*., 2009). Furthermore JAM-A expression has been reported to be high on undifferentiated HC11 mammary epithelial cells relative to differentiated cells (Perotti *et al*., 2009). In support of a potential association between high JAM-A and poor differentiation status, high JAM-A gene or protein expression has been associated with a poorer grade of differentiation in tissues from patients with invasive breast cancer (McSherry *et al*., 2009). Conversely, JAM-A has been found to mediate the differentiation of CD34+ progenitor cells to endothelial progenitor cells and to facilitate CD34+ cell-induced re-endothelialization *in vitro* (Stellos *et al*., 2010). This suggests that JAM-A is required for circulating CD34+ progenitor cells to recognise a site of injury, differentiate into endothelial cells and proliferate to repair the injured endothelium. In addition, JAM-A is reportedly upregulated during the differentiation of pancreatic AR42J cells (Yoshikumi *et al*., 2008), while JAM-A mRNA and protein levels have been shown to be increased during differentiation of human monocytic cell THP-1 into mature dendritic cells (Ogasawara *et al*., 2009). JAM-L is also induced during differentiation of myeloid leukaemia cells, with expression of JAM-L in myeloid leukaemia cells resulting in enhanced cell adhesion to endothelial cells (Moog-Lutz *et al*., 2003). This upregulation of JAM-A during differentiation is reportedly followed by increased expression of the polarity proteins par3 and PKCλ (Yoshikumi *et al*., 2008), which have been previously shown to affect cell polarity and migration. While these data suggest conflicting roles for JAMs in stem cell populations versus their role in differentiation, at this early stage the exact role(s) of JAMs in stem cell renewal or differentiation can only be speculated upon. Fundamentally, it is also unknown whether the expression of JAMs is actively required or

Naik & Naik, 2006; Li *et al*., 2009; McSherry *et al*., 2009).

by cancer cells to promote tumour progression or survival.

**2.4 Potential role of JAM proteins in epithelial/endothelial differentiation** 

terminally-differentiated cell such as an epithelial, muscle or nerve cell.

this chapter.

adhesion (predisposing to faster, more random motility). For example, JAM-A re-expression in JAM-A-/- mouse endothelial cells has been shown to reduce the occurrence of spontaneous and random motility. This ability of JAM-A to influence the polarised movement of cells was reliant on its interaction with polarity proteins through its PDZ binding motif (Bazzoni & Dejana, 2004). JAM-A deletion mutants lacking their PDZ-binding residues have been shown to have increased availability of Par3 (Ebnet *et al*., 2001), resulting in PKCζ inactivation and the loss of contact-dependent inhibition of cell motility (Mishima *et al*., 2002; Bazzoni & Dejana, 2004). These data show that loss of functional JAM-A results in faster random motility with reduced cell-cell contact inhibition of migration. Interestingly, JAM-C redistribution away from TJs stimulates β1 and β3 integrin activation, resulting in increased cell migration and adhesion (Aurrand-Lions *et al*., 2001). Furthermore, JAM-A and JAM-4 have been found to induce the formation of actin-based membrane protrusions, an essential part of cell migration, in endothelial and COS-7 cells (Mori *et al*., 2004). Together these data suggest loss of JAM-A promotes random motility, while JAM-A, JAM-C and JAM-4 promote directional cell migration through their effects on integrin function and cytoskeletal reorganization.

In the context of cancer, knockdown of JAM-A has been shown to enhance invasiveness of the breast cancer cell lines MDA-MB-231 and T47D, and the renal cancer cell line RCC4 (Naik *et al*., 2008; Gutwein *et al*., 2009). Conversely, the overexpression of JAM-A in MDA-MB-231 cells reportedly inhibits both migration and invasion through collagen gels (Naik *et al*., 2008), suggesting that loss of JAM-A expression increases cancer cell dissemination and invasion. However, the specific contribution of JAM-A to breast cancer progression remains controversial. McSherry *et al* showed a significant association between *high* JAM-A gene or protein expression and poor survival in 2 large cohorts of patients with invasive breast cancer, and concurrently a decrease in the migratory abilities of high JAM-A-expressing MCF-7 cells upon knockdown or functional inhibition of JAM-A (McSherry *et al*., 2009). Reduced motility after JAM-A loss was subsequently linked to reduced interactions between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2, resulting in reduced activity of Rap1 GTPase (McSherry *et al*., 2011), a known activator of β1-integrins (Sebzda *et al*., 2002) and a regulator of breast tumourigenesis (Itoh *et al*., 2007). Complementary evidence in a recent publication by Gotte *et al.* has also supported the theory that JAM-A overexpression is of more functional relevance in breast cancer than JAM-A loss, since over-expression of micro RNA (miR)-145 in breast cancer cells led to a decrease in cellular migration and invasion via downregulation of JAM-A expression (Gotte *et al*., 2010). Still more recently (during the proofing stage of this chapter), additional histopathological evidence has been provided for a link between JAM-A over-expression and poor prognosis in breast cancer patients (Murakami *et al.,* 2011). This, along with the finding that JAM-A promotes the survival of mammary cancer cells (Murakami *et al.,* 2011), strongly suggests that JAM-A depletion or antagonism could offer promise in reducing breast tumour progression. Furthermore, depletion of JAM-A has been found to inhibit bFGF-induced migration of human umbilical vein endothelial cells (HUVEC) on vitronectin, through effects on integrin function (Naik & Naik, 2006). In other cell systems, silencing of the JAM-A gene has been shown to block the migration of inflamed smooth muscle cells (Azari *et al*., 2010) and to increase the random motility of dendritic cells (Cera *et al*., 2004). JAM-A has also been shown to be required for neutrophil directional motility (Corada *et al*., 2005), and to promote neutrophil chemotaxis by controlling integrin internalization and recycling (Cera *et al*., 2009). Thus while the area remains controversial, increasing evidence is suggesting that JAMs promote migration and

adhesion (predisposing to faster, more random motility). For example, JAM-A re-expression in JAM-A-/- mouse endothelial cells has been shown to reduce the occurrence of spontaneous and random motility. This ability of JAM-A to influence the polarised movement of cells was reliant on its interaction with polarity proteins through its PDZ binding motif (Bazzoni & Dejana, 2004). JAM-A deletion mutants lacking their PDZ-binding residues have been shown to have increased availability of Par3 (Ebnet *et al*., 2001), resulting in PKCζ inactivation and the loss of contact-dependent inhibition of cell motility (Mishima *et al*., 2002; Bazzoni & Dejana, 2004). These data show that loss of functional JAM-A results in faster random motility with reduced cell-cell contact inhibition of migration. Interestingly, JAM-C redistribution away from TJs stimulates β1 and β3 integrin activation, resulting in increased cell migration and adhesion (Aurrand-Lions *et al*., 2001). Furthermore, JAM-A and JAM-4 have been found to induce the formation of actin-based membrane protrusions, an essential part of cell migration, in endothelial and COS-7 cells (Mori *et al*., 2004). Together these data suggest loss of JAM-A promotes random motility, while JAM-A, JAM-C and JAM-4 promote directional cell

migration through their effects on integrin function and cytoskeletal reorganization.

In the context of cancer, knockdown of JAM-A has been shown to enhance invasiveness of the breast cancer cell lines MDA-MB-231 and T47D, and the renal cancer cell line RCC4 (Naik *et al*., 2008; Gutwein *et al*., 2009). Conversely, the overexpression of JAM-A in MDA-MB-231 cells reportedly inhibits both migration and invasion through collagen gels (Naik *et al*., 2008), suggesting that loss of JAM-A expression increases cancer cell dissemination and invasion. However, the specific contribution of JAM-A to breast cancer progression remains controversial. McSherry *et al* showed a significant association between *high* JAM-A gene or protein expression and poor survival in 2 large cohorts of patients with invasive breast cancer, and concurrently a decrease in the migratory abilities of high JAM-A-expressing MCF-7 cells upon knockdown or functional inhibition of JAM-A (McSherry *et al*., 2009). Reduced motility after JAM-A loss was subsequently linked to reduced interactions between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2, resulting in reduced activity of Rap1 GTPase (McSherry *et al*., 2011), a known activator of β1-integrins (Sebzda *et al*., 2002) and a regulator of breast tumourigenesis (Itoh *et al*., 2007). Complementary evidence in a recent publication by Gotte *et al.* has also supported the theory that JAM-A overexpression is of more functional relevance in breast cancer than JAM-A loss, since over-expression of micro RNA (miR)-145 in breast cancer cells led to a decrease in cellular migration and invasion via downregulation of JAM-A expression (Gotte *et al*., 2010). Still more recently (during the proofing stage of this chapter), additional histopathological evidence has been provided for a link between JAM-A over-expression and poor prognosis in breast cancer patients (Murakami *et al.,* 2011). This, along with the finding that JAM-A promotes the survival of mammary cancer cells (Murakami *et al.,* 2011), strongly suggests that JAM-A depletion or antagonism could offer promise in reducing breast tumour progression. Furthermore, depletion of JAM-A has been found to inhibit bFGF-induced migration of human umbilical vein endothelial cells (HUVEC) on vitronectin, through effects on integrin function (Naik & Naik, 2006). In other cell systems, silencing of the JAM-A gene has been shown to block the migration of inflamed smooth muscle cells (Azari *et al*., 2010) and to increase the random motility of dendritic cells (Cera *et al*., 2004). JAM-A has also been shown to be required for neutrophil directional motility (Corada *et al*., 2005), and to promote neutrophil chemotaxis by controlling integrin internalization and recycling (Cera *et al*., 2009). Thus while the area remains controversial, increasing evidence is suggesting that JAMs promote migration and invasion through the regulation of integrin expression and activation (McSherry *et al*., 2011; Naik & Naik, 2006; Li *et al*., 2009; McSherry *et al*., 2009).

In breast cancer, the formation of metastases at distant sites is the leading cause of cancerrelated death. In order for breast cancer cells to metastasize, they must first migrate out of the primary tumour before ever reaching a distant organ and potentially proliferating into a secondary tumour. While JAMs are already known to regulate migration, the possibility that they are also involved in the regulation of proliferation will be referred to in section 3.3 of this chapter.

All together these data highlight the role of JAM family members in controlling the balance between cell adhesion and migration. Although much remains to be understood about the exact role of JAMs in breast cancer cell migration, the classic description of tumours as "wounds which do not heal" (Riss *et al*., 2006) suggests that the migratory mechanisms employed by JAMs in physiological responses (such as wound healing) may also be utilised by cancer cells to promote tumour progression or survival.

#### **2.4 Potential role of JAM proteins in epithelial/endothelial differentiation**

In previous sections we discussed the biphasic role of JAM family members in regulating cell adhesion and migration. In this section we will outline the emerging contribution of the JAM family to cellular differentiation. Cell differentiation in the context of normal tissue usually involves the transition from an undifferentiated stem/progenitor cell to a terminally-differentiated cell such as an epithelial, muscle or nerve cell.

JAM-A, JAM-B, JAM-C and JAM-4 have been found to be highly expressed on hematopoietic stem cells (HSCs) in the bone marrow, with their expression decreasing during the acquisition of a more differentiated state (Nagamatsu *et al*., 2006; Sakaguchi *et al*., 2006; Sugano *et al*., 2008; Praetor *et al*., 2009). Furthermore JAM-A expression has been reported to be high on undifferentiated HC11 mammary epithelial cells relative to differentiated cells (Perotti *et al*., 2009). In support of a potential association between high JAM-A and poor differentiation status, high JAM-A gene or protein expression has been associated with a poorer grade of differentiation in tissues from patients with invasive breast cancer (McSherry *et al*., 2009). Conversely, JAM-A has been found to mediate the differentiation of CD34+ progenitor cells to endothelial progenitor cells and to facilitate CD34+ cell-induced re-endothelialization *in vitro* (Stellos *et al*., 2010). This suggests that JAM-A is required for circulating CD34+ progenitor cells to recognise a site of injury, differentiate into endothelial cells and proliferate to repair the injured endothelium. In addition, JAM-A is reportedly upregulated during the differentiation of pancreatic AR42J cells (Yoshikumi *et al*., 2008), while JAM-A mRNA and protein levels have been shown to be increased during differentiation of human monocytic cell THP-1 into mature dendritic cells (Ogasawara *et al*., 2009). JAM-L is also induced during differentiation of myeloid leukaemia cells, with expression of JAM-L in myeloid leukaemia cells resulting in enhanced cell adhesion to endothelial cells (Moog-Lutz *et al*., 2003). This upregulation of JAM-A during differentiation is reportedly followed by increased expression of the polarity proteins par3 and PKCλ (Yoshikumi *et al*., 2008), which have been previously shown to affect cell polarity and migration. While these data suggest conflicting roles for JAMs in stem cell populations versus their role in differentiation, at this early stage the exact role(s) of JAMs in stem cell renewal or differentiation can only be speculated upon. Fundamentally, it is also unknown whether the expression of JAMs is actively required or

Junctional Adhesion Molecules (JAMs)- New Players in Breast Cancer? 497

blocking tumour progression, similar to the VEGF-A-neutralizing antibody bevacizumab

In addition to the potential regulatory roles of JAM proteins on the vascular endothelium, effects exerted on JAM-expressing leukocytes within the breast tumour microenvironment may also have relevance to cancer progression. For instance, JAMs are known to play important roles in the transendothelial migration of monocytes, which differentiate into macrophages once in the breast tissue. Accordingly, a function-blocking monoclonal antibody directed against JAM-A (BV11) has been described to inhibit spontaneous and chemokine-induced monocyte transmigration both *in vitro* and *in vivo* (Martin-Padura *et al*., 1998). Furthermore, treatment of mice with a monoclonal antibody directed against JAM-C has been shown to reduce macrophage infiltration into a murine lung tumour model (Lamagna *et al*., 2005), and to promote reverse transmigration of monocytes back into the bloodstream from inflamed tissue sites (Bradfield *et al*., 2007). Given the existence of a breast tumour-promoting paracrine loop between epidermal growth factor secreted by macrophages and colony-stimulating factor-1 secreted by tumour cells (Goswami *et al*., 2005), this implies that JAM-based regulation of monocyte transmigration could have a profound and self-amplifying influence on macrophage trafficking and tumour

In the context of leukocytes other than monocytes/macrophages, many studies have implicated JAMs in the functional control of neutrophil transmigration across both epithelial (Zen *et al*., 2004; Zen *et al*., 2005) and endothelial (Sircar *et al*., 2007; Woodfin *et al*., 2007) barriers. As yet nothing is known about JAM-dependent events that might control neutrophil trafficking or activation within the breast tissue, despite the fact that neutrophils accumulate in highly aggressive inflammatory breast cancers. In other tissues, JAM-A has been shown to be required for efficient infiltration of neutrophils into the inflamed peritoneum or into the heart upon ischemia–reperfusion injury; as evidenced by increased adhesion and impaired transmigration in JAM-A-deficient mice (Corada *et al*., 2005). Interestingly, in this model JAM-A expression on the neutrophil appears to be more important than that on the endothelium; since selective loss of endothelial JAM-A did not phenocopy the transmigration deficits (Corada *et al*., 2005). In addition, soluble JAM-A shed from cultured endothelial cells has been shown to reduce *in vitro* transendothelial migration

of neutrophils and to decrease neutrophil infiltration *in vivo* (Koenen *et al*., 2009).

Recent evidence also proves that family members other than JAM-A can participate in leukocyte trafficking, with JAM-C over-expressing mice exhibiting an increased accumulation of leukocytes into inflammatory sites or during ischaemia/reperfusion injury, while JAM-C neutralization or loss reduces leukocyte recruitment in models of lung, kidney or muscular inflammation (Aurrand-Lions *et al*., 2005; Scheiermann *et al*., 2009). Finally leukocytic expression of JAM-L has been shown to promote attachment to endothelium (Luissint *et al*., 2008), and functional inhibition of JAM-B is reported to decrease migration of peripheral blood lymphocytes across cultured human umbilical vein endothelial cells

Collectively these data highlight an important role for JAMs in the migration of immune cells across endothelia, a mechanism that could be hijacked by JAM-overexpressing cancer

(avastin) (Van Meter & Kim, 2010).

(HUVECs) (Johnson-Leger *et al*., 2002).

cells as they leave the breast and invade into blood vessels.

proliferation.

**3.2 JAM proteins regulate trafficking of leukocytes** 

passively upregulated in stem cell populations. However, based on the increased expression of JAM-A in poorly-differentiated breast cancers (McSherry *et al*., 2009) and the emerging role of JAM-A in regulating proliferation and apoptosis (Azari *et al*., 2010; Nava *et al*., 2011; Naik *et al*., 2003; Murakami *et al*., 2011), it will be interesting to determine if JAM-A is upregulated on cancer stem cell populations and whether its expression promotes self-renewal.
