**3.2 JAM proteins regulate trafficking of leukocytes**

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

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

**3. Functional regulation of cells in the breast cancer microenvironment by** 

As already alluded to, JAM proteins are highly expressed on endothelial cells and have been crucially implicated in the control of barrier function and cell motility. In the context of cancer, however, endothelial cells assume a new importance via the development of neovascularisation sites to support growing tumours (Hanahan & Folkman, 1996). This section will review the evidence currently linking JAM proteins to angiogenesis as a

Angiogenesis in response to enhanced growth factor signalling is of particular relevance in tumour microenvironments. A body of work from Naik *et al* has convincingly shown an important role for JAM-A in angiogenesis induced by basic fibroblast growth factor (bFGF). Specifically, bFGF signalling facilitates the disassembly of an inhibitory complex between JAM-A and αvβ3 integrin, permitting JAM-A-dependent activation of MAP kinase which leads to endothelial tube formation, a surrogate for angiogenesis (Naik *et al*., 2003). JAM-A has also been shown to activate extracellular signal-related kinase (ERK) signalling in response to bFGF, facilitating endothelial migration (Naik *et al*., 2003) in a matrix-specific context (Naik & Naik, 2006). *In vivo*, JAM-A expression has been linked with the very early stages of murine embryonic vasculature development (Parris *et al*., 2005), and although deletion of JAM-A appears to be dispensable for vascular tree development, homozygous JAM-null mice were found to be incapable of supporting FGF-2-induced angiogenesis in isolated aortic ring assays (Cooke *et al*., 2006). In the context of tumour neovascularisation, others have reported reduced angiogenesis in a model of pancreatic carcinoma in JAM-A-

Other JAM family members appear to contribute similarly to angiogenesis; with functional blockade of JAM-C being shown to decrease aortic ring angiogenesis and block angiogenesis in hypoxic vessels of the murine retina (Lamagna *et al*., 2005; Orlova *et al*., 2006). Furthermore, soluble JAM-C shed into the serum of patients with inflammatory conditions (presumably following cleavage by ADAM enzymes) was noted to induce endothelial tube formation in a Matrigel model (Rabquer *et al*., 2010). An interesting dichotomy, however, is that amplification of JAM-B in a trisomy-21 mouse model of Down's syndrome has been linked with reductions in VEGF-induced angiogenesis and thus anti-tumour effects in a

Taken together, these studies illustrate that by influencing angiogenic functions in endothelial cells, JAMs may indirectly influence the ability of tumours to survive and progress. While there appears to be a consensus that JAMs –A and –C activate signalling cascades that promote angiogenesis, it is possible that clear roles for the other family members in the regulation of angiogenesis will also emerge in time. It is tempting to speculate that pharmacological antagonism of JAMs will show promise as an option for

**3.1 JAM proteins regulate endothelial angiogenesis** 

contributory mechanism to cancer progression.

null mice (Murakami *et al*., 2010).

lung carcinoma model in these mice (Reynolds *et al*., 2010).

promotes self-renewal.

**JAMs** 

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 proliferation.

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

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

Junctional Adhesion Molecules (JAMs)- New Players in Breast Cancer? 499

pharmacological targeting of JAM molecules could prove particularly useful in cancers that overexpress one or more JAMs. This naturally pre-supposes that JAMs are causally involved in the disease process rather than simply acting as passive biomarkers, a fact that remains to be solidified. However, irrespective of the last caveat, another facet worth exploring is the potential of targeting JAMs to promote drug delivery. Since tight junctions (TJs) as a whole are primary regulators of paracellular transport across epithelial cells (Gonzalez-Mariscal *et al*., 2005), successful drug delivery may require modulation of TJ proteins to allow drug molecules to pass (Matsuhisa *et al*., 2009). However disruption of TJ proteins for drug delivery purposes is a double-edged sword, given the risk of disrupting homeostatic mechanisms of polarity, differentiation and migration which are tightly regulated by TJs in

normal tissues and whose dysregulation may themselves promote tumourigenesis.

settings remains an open question.

future.

**5. Conclusion** 

than an aspiration.

As yet, there are no cancer therapies on the market which specifically target tight junctions. However several tight junction proteins have been described as receptors for specific molecules or organisms, and as such, these might provide valid and novel targets for drug delivery. A particular precedent exists with the claudin family of TJ proteins; Claudins-3 and -4 having been suggested as drug delivery targets since they act as the receptor for *Clostridium perfringens* enterotoxin (CPE). The ability of CPE to rapidly and specifically lyse cells expressing claudin-3 or -4 could potentially be exploited in the treatment of breast cancers over-expressing these proteins (Katahira *et al*., 1997; Morin, 2005; Santin *et al*., 2007; Santin *et al*., 2007). Sub-lytic doses of CPE could alternatively be used to compromise TJs thus enhancing the influx of drug molecules across the epithelium. This could be of particular benefit in accessing hypoxic tumour cores, around which the tumour cells may be very tightly packed and thus relatively inaccessible to chemotherapeutic drugs. To date CPE administration has been shown to reduce growth of claudin-4 overexpressing pancreatic tumour cells (Michl *et al*., 2001; Michl *et al*., 2003), but their potential use in other cancer

How JAM molecules might be therapeutically targeted also remains an unanswered question, but one could predict value in using monoclonal antibodies or small molecule inhibitors to block the signalling functions which contribute to processes such as migration and angiogenesis. However, to date, the role of JAMs as chemotherapeutic targets (or even prognostic/predictive biomarkers) in the clinical setting of breast cancer has yet to be elucidated and validated. Following the lead of JAM-A as a potential biomarker and therapeutic target for breast cancer (McSherry *et al*., 2009; Gotte *et al.,* 2010; McSherry *et al.*, 2011; Murakami *et al.,* 2011), we speculate that this will be a lucrative area of research in the

To conclude, breast cancer remains a leading cause of cancer worldwide (Jemal *et al*., 2008), and the search for new targets of prognostic and therapeutic relevance will continue particularly in this era where semi-personalised medicine is becoming more of a likelihood

This chapter has attempted to summarize the known roles of the JAM family in controlling cell adhesion, polarity and barrier function, and their emerging roles in controlling functional behaviours within cells of the breast tumour microenvironment which promote cancer progression. Finally, it introduced the topic of JAM as a potential drug target in breast cancer; whether to directly influence JAM-dependent oncogenic signalling or indeed to interfere with cell-cell adhesion for the purposes of enhancing drug delivery. Continued
