**1. Introduction**

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

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#### **1.1 Global incidence of breast cancer**

Worldwide, breast cancer remains a leading cause of death amongst women. Annually, it is estimated that breast cancer is diagnosed in over a million women (Kasler *et al*., 2009) with over 450,000 deaths worldwide (Tirona *et al*., 2010). The incidence of the disease is highest in economically-developed countries, with lower rates in developing countries. Despite continual advances in breast cancer care which have led to reduced mortality, however, the incidence of the disease is still rising. The decrease in breast cancer-specific mortality has been attributed to improvements in screening techniques which permit earlier detection, surgical and radiotherapy interventions, better understanding of disease pathogenesis and utilization of traditional chemotherapies in a more efficacious manner. Consequently, early stage breast cancer is now a curable disease while advanced breast cancer remains a significant clinical problem.

Breast cancer is a heterogeneous disease encompassing many subtypes, which differ both in terms of their molecular backgrounds and clinical prognosis. These breast cancer subtypes range from pre-invasive early stage disease to advanced invasive disease. The simplest classifications of disease subdivide breast cancer into pre-invasive and invasive forms; with the pre-invasive forms being ductal carcinoma *in situ* (DCIS) and lobular carcinoma *in situ* (LCIS). Carcinoma *in situ* is proliferation of cancer cells within the epithelial tissue without invasion of the surrounding stromal tissue (Bland & Copeland, 1998). DCIS arises in the terminal ductal lobular units (TDLU) and in extra-lobular ducts while LCIS occurs in the breast lobules, and is recognisable histopathologically by the presence of populations of aberrant cells with small nuclei (Hanby & Hughes, 2008). Invasive breast cancers are subclassified into invasive ductal breast cancer, invasive lobular breast cancer, inflammatory breast cancer and Paget's disease. Invasive ductal carcinoma (IDC) is the most common form of invasive breast cancer, accounting for around 85% of all cases.

DCIS is frequently considered as an obligate precursor to IDC, progressing from lower to higher grades and then onto invasive cancer with progressive accumulation of genomic changes (Farabegoli *et al*., 2002). However it has alternately been suggested that there exist genetically-distinct subgroups of DCIS, only some of which have the potential to progress to invasion (Shackney & Silverman, 2003). Long-term natural history studies of DCIS have provided supportive evidence for both possibilities (Page *et al*., 1995; Collins *et al*., 2005; Sanders *et al*., 2005). Despite such controversies, the large extent to which the genome is

Junctional Adhesion Molecules (JAMs)- New Players in Breast Cancer? 489

oncogenes and tumour suppressor genes (along with subsequent interactions between defective genes and the breast microenvironment) alter not just cell proliferation, but also differentiation, survival and genome stability (Hahn & Weinberg, 2002) of breast cells,

Much evidence supports the contention that the pathogenesis of breast cancer is influenced by complex interactions between ductal epithelial cells and the cells that compose the tumour microenvironment (Weaver *et al*., 1996; Polyak & Hu, 2005; Hu *et al*., 2008). The next section will focus on the cells of the microenvironment with respect to normal breast tissue

Fig. 1. Structure of the breast showing lobules and lactiferous ducts terminating at the nipple

leading to abnormal cell growth and potentially cancer.

structure and also their possible involvement in breast tumourigenesis.

altered in DCIS strongly suggests that genomic instability precedes phenotypic evidence of invasion (Hwang *et al*., 2004). This serves to underline the fact that malignant transformation in a heterogeneous disease like breast cancer is a dynamic process evolving through multiple multi-step pathway models.

Many factors are thought to be responsible for the development of breast cancer. Genetic factors play a vital role in the predisposition to breast cancer, with mutations of *BRCA1*  and *BRCA2* genes accounting for 5–10% of breast cancer cases and being responsible for 80% of inherited breast cancers (Nathanson *et al*., 2001). On a more complex level, much insight has been gained from the genetic profiling of thousands of tumours to generate gene signatures of prognostic value (Sorlie *et al*., 2001; van 't Veer *et al*., 2002; van de Vijver *et al*., 2002), which have spurred the development of commercially-available diagnostic tests. The importance of reproductive factors in the aetiology of breast cancer is also well recognised with early onset of menarche, nulliparity, late menopause, endogenous and exogenous hormones representing the main risk factors (Reeves *et al*., 2000; Key *et al*., 2001; Howell & Evans, 2011). Several other studies have reported an increased risk of breast cancer with lack of physical activity (especially in pre menopausal women), as well as increasing age and obesity (Clarke *et al*., 2006; Walker & Martin, 2007; Harrison *et al*., 2009; Rod *et al*., 2009; Awatef *et al*., 2011). These risk factors accentuate the abnormal growth control of cells by increasing the circulating levels of oestrogen thereby promoting tumourigenesis within the breast microenvironment. A proper understanding of the breast cancer microenvironment is essential for understanding breast cancer, and will be explored in detail in the next sections.

#### **1.2 Breast structure and breast cancer microenvironment**

The breasts are modified sweat glands with a specialized function to produce milk. In the adult, the mature breast extends from the second ribs to the seventh rib and from the lateral border of the sternum to the midaxillary line and projects into the axilla at the axillary tail of Spence (Monkhouse, 2007). The breast is located within the superficial fascia of the anterior thoracic wall and is made up of 15-20 lobes of glandular tissue (Bland & Copeland, 1998). Fibrous connective tissue forms the framework that supports the lobes and adipose tissue which fills the space between the lobes. Each lobe of the mammary gland terminates in a lactiferous duct which opens onto the nipple and is lined with breast epithelial tissue. These ducts have a sinus at the base beneath the areola called the lactiferous sinus (Figure 1).

Breast cancers are characterised by abnormal proliferation of breast epithelial cells and mostly originate in milk ducts (Sainsbury *et al*., 2000). Normal milk ducts consist of an outer myoepithelial cell layer and an inner luminal epithelial layer. Myoepithelial cells, which are of ectodermal origin, lie between the surface epithelial cells and the basal lamina. Both the epithelial and myoepithelial cells of the breast duct lie on a basement membrane composed of extracellular matrix factors secreted by those cells (Figure 2). The basement membrane is important for defining the barriers of the normal duct, and thus alterations in the basement membrane have been implicated in abnormal cell differentiation and the formation of metastases (Kleinman *et al*., 2001).

Proliferation of cells within the breast ducts is controlled by growth-promoting protooncogenes and growth-inhibiting tumour suppressor genes. In most cases, normal cells divide as many times as needed and then stop. Carcinogenic mutations in either (or both)

altered in DCIS strongly suggests that genomic instability precedes phenotypic evidence of invasion (Hwang *et al*., 2004). This serves to underline the fact that malignant transformation in a heterogeneous disease like breast cancer is a dynamic process evolving through

Many factors are thought to be responsible for the development of breast cancer. Genetic factors play a vital role in the predisposition to breast cancer, with mutations of *BRCA1*  and *BRCA2* genes accounting for 5–10% of breast cancer cases and being responsible for 80% of inherited breast cancers (Nathanson *et al*., 2001). On a more complex level, much insight has been gained from the genetic profiling of thousands of tumours to generate gene signatures of prognostic value (Sorlie *et al*., 2001; van 't Veer *et al*., 2002; van de Vijver *et al*., 2002), which have spurred the development of commercially-available diagnostic tests. The importance of reproductive factors in the aetiology of breast cancer is also well recognised with early onset of menarche, nulliparity, late menopause, endogenous and exogenous hormones representing the main risk factors (Reeves *et al*., 2000; Key *et al*., 2001; Howell & Evans, 2011). Several other studies have reported an increased risk of breast cancer with lack of physical activity (especially in pre menopausal women), as well as increasing age and obesity (Clarke *et al*., 2006; Walker & Martin, 2007; Harrison *et al*., 2009; Rod *et al*., 2009; Awatef *et al*., 2011). These risk factors accentuate the abnormal growth control of cells by increasing the circulating levels of oestrogen thereby promoting tumourigenesis within the breast microenvironment. A proper understanding of the breast cancer microenvironment is essential for understanding breast cancer, and

The breasts are modified sweat glands with a specialized function to produce milk. In the adult, the mature breast extends from the second ribs to the seventh rib and from the lateral border of the sternum to the midaxillary line and projects into the axilla at the axillary tail of Spence (Monkhouse, 2007). The breast is located within the superficial fascia of the anterior thoracic wall and is made up of 15-20 lobes of glandular tissue (Bland & Copeland, 1998). Fibrous connective tissue forms the framework that supports the lobes and adipose tissue which fills the space between the lobes. Each lobe of the mammary gland terminates in a lactiferous duct which opens onto the nipple and is lined with breast epithelial tissue. These ducts have a sinus at the base beneath the areola called

Breast cancers are characterised by abnormal proliferation of breast epithelial cells and mostly originate in milk ducts (Sainsbury *et al*., 2000). Normal milk ducts consist of an outer myoepithelial cell layer and an inner luminal epithelial layer. Myoepithelial cells, which are of ectodermal origin, lie between the surface epithelial cells and the basal lamina. Both the epithelial and myoepithelial cells of the breast duct lie on a basement membrane composed of extracellular matrix factors secreted by those cells (Figure 2). The basement membrane is important for defining the barriers of the normal duct, and thus alterations in the basement membrane have been implicated in abnormal cell differentiation and the formation of

Proliferation of cells within the breast ducts is controlled by growth-promoting protooncogenes and growth-inhibiting tumour suppressor genes. In most cases, normal cells divide as many times as needed and then stop. Carcinogenic mutations in either (or both)

multiple multi-step pathway models.

will be explored in detail in the next sections.

the lactiferous sinus (Figure 1).

metastases (Kleinman *et al*., 2001).

**1.2 Breast structure and breast cancer microenvironment** 

oncogenes and tumour suppressor genes (along with subsequent interactions between defective genes and the breast microenvironment) alter not just cell proliferation, but also differentiation, survival and genome stability (Hahn & Weinberg, 2002) of breast cells, leading to abnormal cell growth and potentially cancer.

Much evidence supports the contention that the pathogenesis of breast cancer is influenced by complex interactions between ductal epithelial cells and the cells that compose the tumour microenvironment (Weaver *et al*., 1996; Polyak & Hu, 2005; Hu *et al*., 2008). The next section will focus on the cells of the microenvironment with respect to normal breast tissue structure and also their possible involvement in breast tumourigenesis.

Fig. 1. Structure of the breast showing lobules and lactiferous ducts terminating at the nipple

Junctional Adhesion Molecules (JAMs)- New Players in Breast Cancer? 491

potential involvement of fibroblasts in promoting tumour progression both at genomic and transcriptomic levels, with reports of altered genetic signatures between normal and tumour-associated fibroblasts supporting a complex role for fibroblasts in influencing

Macrophages within the breast cancer microenvironment have been shown to enhance tumour growth through the secretion of pro-angiogenic factors like vascular endothelial growth factor (VEGF); (Murdoch *et al*., 2004; Lamagna *et al*., 2005 ; Lewis & Hughes, 2007). They have also been implicated in promoting a metastatic phenotype, via the secretion of pro-migratory factors such as EGF (Wyckoff *et al*., 2004) which enhance cellular dissemination from a primary tumour. Accordingly, the enhanced physical juxtaposition of macrophages, tumour cells and endothelial cells has been proposed as a new prognostic histopathological marker associated with increased risk of metastases in human breast

Endothelial cells which line the blood vessels are derived from angioblasts forming the vascular network. Enhanced vessel density occurring as a result of tumour-associated angiogenesis is a major contributor to both the survival of primary breast tumours (via the delivery of systemic growth factors) and the risk of metastasis (via increased access of disseminated tumour cells to a circulatory source). Expression of pro-angiogenic factors such as VEGF has been shown to increase in haematological malignancies (Fiedler *et al*., 1997; Molica *et al*., 1999) in addition to solid tumours including breast, renal, ovarian, gastric and lung cancer (Patel *et al*., 2009; Burger, 2011; Gou *et al*., 2011; Sharma *et al*., 2011). VEGF promotes neovascularisation via mitogenic and pro-migratory effects on endothelial cells

Finally, myoepithelial cells are known to play a role in the formation of the basement membrane and thereby assist in maintaining polarity of the breast ductal epithelium. They also interact with epithelial cells to regulate the cell cycle and suppress breast cancer cell growth, invasion and angiogenesis (Weaver *et al*., 1996; Alpaugh *et al*., 2000; Barsky, 2003). Tumour and non-tumour primary myoepithelial cells have been described to differ in functional properties relating to the secretion of extracellular matrix components such as laminin-1 (Gudjonsson *et al*., 2002), and accordingly myoepithelial cells reportedly lose their established tumour-suppressive properties during tumour progression (Polyak & Hu, 2005). Taken together, the many cell types within the breast tumour microenvironment can both individually and coordinately regulate several functions relevant to tumour progression. In order to better understand their relative contributions to breast cancer, it is necessary to dissect the signals that regulate their own functions. Since adhesive functions are central to the behaviour of all of these cell types, the remainder of this chapter will focus on their potential regulation by a family of adhesion proteins termed the Junction Adhesion Molecules (JAMs), whose role in breast cancer initiation and progression is just emerging.

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

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

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

tumour progression (Hu *et al.,* 2005; Hu *et al.,* 2008; Ma *et al*., 2009).

cancer (Robinson *et al*., 2009).

(Asahara *et al*., 1999).

**cells** 

Fig. 2. Diagram of a normal breast duct depicting cells of the microenvironment.
