**2.2 HA synthesis and tumourigenesis**

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

Hyaluronan Associated Inflammation and Microenvironment

**2.3 HA fragmentation and its role in tumourigenesis** 

**signalling pathways by HA fragments** 

**3.1 CD44** 

Remodelling Influences Breast Cancer Progression 213

ERK1,2 in response to treatment with Heregulin (Bourguignon *et al*., 2007) and monoubquitination of K190 on HAS2 rapidly inactivates this enzyme (Karousou *et al*., 2010).

In addition to HAS1-3 expression, the amount and polymer size of HA are also affected by reactive oxygen species (ROS) and secreted hyaluronidases (HYALs), which fragment HA to various sizes. Significant levels of ROS can be generated during times of oxidative stress and these are considered critical in cancer initiation, promotion and progression (Karihtala *et al*., 2007). ROS are produced in response to extracellular stimuli such as bacterial infections and environmental toxins, but can also be produced by cellular metabolism (Yu *et al*., 2011). Five HYALs fragment HA: HYAL-1-3, PH-20 and HYAL-5. The HYALs differ in their cellular location and enzymatic properties. HYAL-1 and 2 are the major HYALs produced by somatic tissues whereas HYAL-3 is expressed mostly in bone marrow and testes. Both PH-20 and HYAL-5 expression are normally restricted to testes but PH20 is aberrantly expressed in BCA (Stern, 2008). HYAL-1 and 2 cooperate to degrade HMW HA in a coordinated fashion. HYAL-2, which is GPI anchored to the cell surface, degrades extracellular HA to fragments of 20 kDa, which are then taken up into endocytic vesicles. HYAL-1 present in the lysosome further degrades intracellular HA into tetrasaccharides (Tammi *et al*., 2001, Stern, 2008, Simpson and Lokeshwar, 2008). Coordinated breakdown of HA by HYALs increases the rate of HA metabolism and this appears to be an important factor in tumourigenesis (Veiseh and Turley, 2011). For example, co-expression of HAS3 and HYAL-1 increases the aggressiveness and spread of prostate cancer cells compared to expression of either alone (Bharadwaj *et al*., 2009). In BCA, HYAL-1 and HYAL-2 are often coordinately overexpressed compared to non-malignant breast tissue. Knockdown of HYAL-1, which is overexpressed in MDA-MB-231 and MCF-7 BCA lines, reduces tumour xenograft size (Tan *et al*., 2010).

**3. HA receptors detect oligosaccharides and fragments: Control of key** 

CD44 is a class I transmembrane receptor, which binds to HA via a link domain and is expressed by a variety of cells, including fibroblasts, endothelial and epithelial cells, smooth muscle, and haematopoietic cells. A vital role of CD44 is recruiting cells, including immune cells and fibroblasts, to sites of inflammation through HA-mediated signalling. Under homeostatic conditions, CD44 is in a low HA binding state, but during injury and tumourigenesis its binding affinity is increased and it mediates the inflammatory and tissue repair responses (Thorne *et al*., 2004, Naor *et al.*, 2008). CD44 is expressed as many different isoforms due to extensive splicing in a region proximal to the transmembrane domain (Thorne *et al*., 2004). The smallest CD44 isoform, CD44s (standard form), skips this variable region. The role of CD44s and variants in BCA progression is still controversial. For example, CD44s expression in CD44low MCF-7 human BCA cells results in xenograft metastasis to the liver (Ouhtit *et al*., 2007) while CD44-/- mice develop more lung metastases than wildtype animals in response to polyomavirus middle T (Lopez *et al*., 2005). Importantly, a recent study by Brown *et al*. (2011) demonstrated that CD44s expression is elevated and required for epithelial-mesenchymal transition of immortalized human mammary epithelial cells and for recurrence of HER2/*neu* induced murine mammary tumours (Lopez *et al*., 2005). HA synthesis is elevated in CD44+ BCAs

aberrations in tooth and follicle development (Weigel and DeAngelis, 2007). It is not fully understood why only HAS2 is absolutely required for organogenesis, but it has been suggested that it produces high molecular weight tissue HA while the other HASs produce the smaller HA sizes (Itano *et al*., 1999). There are differences in the mechanisms by which HAS isoform expression and enzyme activity are regulated that may be relevant to their functions and essential or non-essential roles in organogenesis (Tammi *et al*., 2008).

BCA cells use several mechanisms to rapidly control the synthesis and release of HA, thereby modifying their ECM, including substrate availability, gene expression, posttranslational control of enzyme activity, and differential response to cytokines and ECM signalling. The availability of UDP sugars can profoundly influence the yield of HAS enzymes (Kakizaki *et al*., 2004). This has been demonstrated by the use of 4- Methylumbelliferone (4-MU), which depletes intracellular levels of UDP-Glc-UA (Kakizaki *et al*., 2004) by serving as a glucuronidation substrate. It blocks HA production and reduces BCA tumourigenicty.

The genomic plasticity and instability of cancer cells often leads to chromosomal aberrations that can result in both de-regulation of gene expression and allele duplication. Chromatin breakpoint analysis using a BCA line revealed significant chromosomal rearrangements close to the HAS2 gene. These result in de-regulation of HAS2 expression and significantly higher HAS2 mRNA levels in transformed cells compared to normal breast cells (Unger *et al*., 2009). Detailed *in vitro* and *in vivo* studies of BCA lines and xenografts have provided numerous insights into the effects of genetically modifying HAS expression levels on HA concentration within the tumour and peri-tumoural stroma. Antisense inhibition of HAS2 in MDA-MB-231 BCA cells delays proliferation via a transient arrest of the cell cycle (Udabage *et al*., 2005). Knockdown of HAS expression also results in significant alterations in genes associated with HA metabolism. CD44 and HYAL1 expression are both down-regulated in response to antisense inhibition of HAS2. *In vivo*, MDA-MB-231 cells expressing antisense HAS2 do not form tumours in nude mice after 12 weeks, whereas the parental cell line readily establishes both primary and secondary tumours during this time. This clearly implicates tumour cell HA as a significant driver of BCA formation. Elevated HA accumulation within BCA peritumoural stroma is also a prognostic factor and appears to promote a microenvironment suitable for BCA growth. For example, HAS2-/- fibroblasts transplanted with BCA cells into the fat pads of NOD/SCID mice fail to recruit macrophages and promote angiogenesis to the same extent as HAS2+/+ fibroblasts. This defect results in decreased tumour volume (Kobayashi *et al*., 2010).

The expression of all three HASs is controlled by growth factors and cytokines. However, there appear to be subtle differences in the response of each isoform that depend upon the cell type. For example, PDGF and TGFβ induce HAS2 expression in fibroblasts but HAS1 or 3 expression in synoviocytes and keratinocytes, respectively (Karousou *et al*., 2010). H-Ras transformation increases only HAS2 expression in 3Y-1 tumour cells, while transformation with v-src or v-fos increases both HAS1 and HAS2 expression in the same cells (Itano *et al*., 2004). Posttranslational modification of HAS, including phosphorylation by PKC, PKA, and the ERK/ErbB2 MAPK pathways (Goentzel *et al*., 2006, Itano and Kimata, 2008) as well as mono-ubiquitination (Karousou *et al*., 2010) also affects HAS activity. HAS3 serine phosphorylation is enhanced upon treatment with a PKC activator (Goentzel *et al*., 2006). All three HAS isoforms expressed by SKOV3 ovarian cancer cell line are phosphorylated by ERK1,2 in response to treatment with Heregulin (Bourguignon *et al*., 2007) and monoubquitination of K190 on HAS2 rapidly inactivates this enzyme (Karousou *et al*., 2010).
