**4.2 HA regulation of a pro-inflammatory environment by non-immune cells**

### **4.2.1 Breast cancer cells and their contribution to a pro-inflammatory environment**

BCA cells secrete a variety of cytokines and chemokines which promote tumour progression. Studies by Tafani *et al*. (2010), showed that MCF-7 cells upregulate proinflammatory gene transcription and translation *in vitro*, and a pro-inflammatory gene expression profile can be seen in human BCA tumours even in the absence of an immune infiltrate. This illustrates that BCA cells themselves contribute to the pro-inflammatory/protumourigenic TME. One or both of HER2 and ERα, which are often expressed on BCA cells, promote the expression and secretion of CXCL8 (IL-8) through the PI3K and ERK pathways. CXCL8 is a pro-angiogenic chemokine and secretion of CXCL8 by the MCF7 BCA line (which express both HER2 and ERα) is additive upon stimulation of both of these receptors (Haim *et al*., 2008). The pro-inflammatory chemokines CCL2 and CCL5 are also secreted by BCA cells (Ben-Baruch, 2003) and expression and secretion of all three chemokines requires HA fragment/CD44 interactions on TAMs, tumour associated fibroblasts (TAFs) and BCA tumour cells. Both CCL2 and CCL5 are monocyte-recruiting chemokines and their expression in BCA tumours is correlated with poor prognosis, and in the case of CCL2, proangiogenesis factors and vascular invasion (Soria and Ben-Baruch, 2008). TNF-α secretion by TAMs activates a positive feedback loop in BCA tumour cells, stimulating further secretion of growth promoting chemokines (Ben-Baruch *et al*., 2003). Eck *et al* (2009) also showed that conditioned media from BCA cells stimulates the expression of pro-inflammatory genes in normal mammary fibroblasts, polarizing them towards a TAF phenotype. Furthermore, TAF migration is increased, along with the secretion of MMP-1 and CXCR4 (IL-1/SDF-1 receptor), both of which are important factors in BCA progression (Eck *et al*., 2009).

Hyaluronan Associated Inflammation and Microenvironment

wound assays (Itano *et al*., 2002).

**5.2 Angiogenesis** 

(Tafani *et al*., 2010).

Remodelling Influences Breast Cancer Progression 221

proliferation, migration and spreading of human dermal fibroblasts *in vitro*. HA seems to regulate motility via a variety of mechanisms that include indirect and direct effects on the migrating cell population. An example of an indirect effect was provided by a study of the role of HA on fibroblast migration using a porcine skin wound model. The wound matrix, which contained HA, promoted cell migration and recruitment of fibroblasts. This was shown to be in part due to wounding produced HA, which promotes collagen fibril formation, thus indirectly affecting cell motility (Docherty *et al*., 1989). Direct effects of HA on cell motility can result from its structural properties and from its ability to activate motogenic signalling cascades such as ERK1,2 and PI3 kinase. Both of these effects have been related to an association of HA with cell surface receptors such as CD44 and RHAMM. For example, extracellular HA accumulation induces penetration of stromal cells by increasing turgidity and hydration or disrupting cell-to-cell junctions. These effects may be a result of interactions with CD44 and RHAMM (Itano *et al*., 2008). HA fragments bind to CD44 and/or RHAMM to induce activation of MAPK (ERK1,2) that results in enhanced BCA cell migration and invasion (Hamilton *et al*., 2007). Moreover, upon HA-mediated activation of PI3 kinase, increased HAS2 production induces faster migration in scratch

Hypoxic conditions within tumours require neovascularisation of the microenvironment for the tumour to continue to grow and metastasize. Hypoxia, a condition often found within the TME, induces the activation, as seen by nuclear translocation, of either or both of NFκB and HIF-1α. This effect has been shown both *in vitro* in MCF-7 BCA cells, and *in vivo* (Tafani *et al.,* 2010). Invasion, migration, and proliferation of endothelial cells, as well as tissue remodelling, are essential processes during angiogenesis, which directly and indirectly help to promote tumour growth and metastasis. Necrotic cells, which have died as a result of hypoxia, also release chemokines that recruit macrophages and a pro-inflammatory response conducive to tissue remodelling. Hypoxia may produce ROS which in turn cause HA fragmentation and Noble *et al.* (1996) showed that NFκB transcription in macrophages is activated by HA fragments (Noble *et al*., 1996). Later, Rockey *et al.* (1998) were the first to show in hepatocytes that HA activation of NFκB induces NOS2 production, which can be synergistically increased in the presence of cytokines such as IFN-γ (Rockey *et al*., 1998). It has since been shown that HA fragments activate the NFκB pathway through TLR4 in both DC and macrophages (Termeer *et al*., 2002). Hypoxia induced activation of HIF-1α and NFκB induces pro-inflammatory gene expression and both mRNA and protein levels of inflammatory mediators such as RAGE, PTX3, NOS2, COX2, and CXCR4 are increased. Increased expression of CXCR4, which is the receptor for SDF-1, is seen on MCF-7 cells subjected to hypoxic conditions (Tafani *et al*., 2010). This increases the migratory and invasive capacity of these cells, which are usually non-invasive. In these same studies it was found that nuclear translocation of NFκB is at least partly dependent on HIF-1α, indicating that it may be under hypoxic regulation, as inhibition of HIF-1α decreases nuclear localisation of NFκB, and in turn RAGE and P2X7R expression, inhibiting cell invasion

In general, high MW HA inhibits angiogenesis while fragments promote angiogenesis. Overexpression of HA and HYALs has been linked to an increase in angiogenesis in several types of cancers including breast (Tan *et al*., 2010), bladder (Lokeshwar *et al.*, 2000, Golshani
