**1.1 The breast microenvironment**

The breast is an organ composed predominantly of glandular, fatty, and fibrous tissues. Glandular tissue is composed of ducts lined by luminal epithelial cells that secrete milk, and is surrounded by a layer of myoepithelial cells that contract to release milk. Myoepithelial cells produce proteases, growth factors and growth factor receptors that contribute to remodelling during breast tissue expansion. Each duct is enclosed by a laminin-rich basement membrane and embedded in extracellular matrix (ECM). Mammary gland ECM and is a mixture of fibrillar proteins such as collagens, laminins, fibronectin, and polysaccharides such as heparin sulphate, chondroitin sulphate and hyaluronan (HA). These collectively provide the mechanical and structural support required for maintaining mammary tissue architecture and for storage of the soluble regulatory molecules needed for tissue homeostasis, plasticity, and remodelling. ECM promotes both the differentiated, homeostatic integrity of mammary tissue and is also a key determinant in branching morphogenesis, response-to-injury and pathological processes such as neoplastic disease. The importance of the ECM in determining homeostatic vs. tumourigenic events was originally demonstrated three decades ago by Beatrice Mintz, who showed that marked embryonic carcinoma cells injected into blastocysts do not give rise to tumours but instead contribute to normal tissue architecture. The same cells injected into adult mice develop into tumours (Mintz and Illmensee, 1975). Components of the microenvironment that support tumour progression have since been identified. For example, chick embryos infected with Rous Sarcoma virus express the oncogene v-src in every cell but tumours develop only at sites of wounding due to the accumulation of TGF-β1 (Weigelt and Bissell, 2008).

Hyaluronan Associated Inflammation and Microenvironment

Toole and Slomiany, 2008, Veiseh and Turley, 2011).

Fig. 2. HA structure and molecular weight ranges.

**2.2 HA synthesis and tumourigenesis** 

Remodelling Influences Breast Cancer Progression 211

(wound and disease) functions of HA (Itano *et al.*, 2008, Jiang *et al.*, 2007, Veiseh and Turley, 2011). BCA cells are particularly adept at producing and responding to HA fragments. BCA cells produce increased levels of HA by increasing HA synthase expression, rapidly fragmenting HA as a result of increased Reactive Oxygen Species (ROS) production, and increasing hyaluronidase expression and release, and increasing expression and display of HA receptors to elevate the response to these fragments (Simpson and Lokeshwar, 2008,

HA is synthesized by three HAS isoforms, HAS1-3, which are located on different chromosomes but share from 57 to 80% sequence homology (Weigel *et al*., 1997, Lokeshwar and Selzer, 2008, Stern, 2008). The mature enzymes are multi-pass integral proteins, which are primarily located in the plasma membrane and catalyze polymerization of HA from the uridine diphosphate (UDP) sugars uridine diphosphate glucuronic acid (UDP-Glc-UA) and uridine diphosphate *N*-acetylglucosamine (UDP-GlcNAC). Synthesis and secretion of HA occur concurrently, allowing for the rapid production and release of large polymers into the ECM (Weigel *et al*., 1997). There is some evidence that HASs are resident in endosomes, ER and the perinuclear membrane although whether or not these produce intracellular HA is not yet clear (Karousou *et al*., 2010, Vigetti *et al*., 2010). HAS1 and 2 are widely expressed throughout the embryo while HAS3 expression is more restricted, for example, to developing tooth-forming neural crest cells and hair follicles. Genetic deletion of HAS2 is embryonic lethal in mice due to severe defects in cardiac tissue development, whereas targeted disruption of the HAS1 or 3 alleles results in fertile viable animals with only minor

Fig. 1. Breast tumour microenvironment

Conversely, breast tumour cells can be reverted by blocking signalling through ECM receptors, including integrins (Turley *et al*., 2008) and HA receptors such as RHAMM (Hall *et al*., 1995). These and other studies have revealed a key role of ECM in initiating and sustaining breast cancer and introduced the novel concept that transformation can be a plastic rather than irreversible process. Specifically, increased HA accumulation in tumour cells or stroma is associated with poor outcome in Breast Cancer (BCA) (Tammi *et al.*, 2008). These studies predict that HA is an important component of ECM that determines a homeostatic vs. tumourigenesis "switch".
