**3.2 RHAMM/HMMR**

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

compared to CD44- and both CD44+ and HER2+ BCAs are amongst the most aggressive and invasive subtypes of BCA with poor prognosis. Expression of variant exons, in particular exon v6, is associated with increased *in vitro* cell migration and invasion of human BCA cells (Herrera-Gayol and Jothy, 1999). Although CD44v6 expression has been correlated with multiple clinicopathological features (primary tumour size, axillary nodal status, histological grade and pTNM stage) it is not an independent prognostic factor (Ma *et al*., 2005). A study by Rys *et al*. (2003) found a correlation between the expression of CD44 v3 and the presence of BCA metastasis. Additionally, high CD44s expression correlates with increased disease free survival in node negative invasive BCA (Diaz *et al*., 2005). The controversies surrounding CD44 and its role in BCA progression may be caused by a limited number of patient samples in some of these studies, heterogeneity of BCA, and CD44 expression by cancer stem cells. The latter, in particular, has raised much recent interest in CD44 since several groups have identified CD44 as a potential marker for BCA stem cells. This is a highly tumourigenic population of cancer cells that, although only representing a small percentage of cells in the tumour, are thought to be responsible for tumour recurrence, metastasis and treatment failure. Aggressive BCA and BCA tumour progenitor cells have enhanced CD44 expression, associated with an increase in

In BCA cells, HA triggers CD44 interactions with a variety of signalling mediators involved in cell proliferation, migration and chemo-resistance. Ankyrin is a membrane-associated component of the cytoskeleton that is involved in regulation of cytoskeleton turnover and IP3 receptor-mediated regulation of intracellular Ca2+. CD44-HA interactions induce CD44 ankyrin coupling and modify receptor-dependent Ca2+ mobilization (Bourguignon *et al*., 2008). CD44 also localizes ankyrin and IP3 receptor to lipid rafts, which are cholesterol and caveolin rich signalling microdomains in the plasma membrane (Fig. 3). The Rho GTPases, RhoA, Rac and CDC42, are key regulators of cell migration and HA stimulates RhoA in BCA cells. RhoA activity is regulated by RhoGEF, a guanine nucleotide exchange factor that forms a complex with CD44 in BCA cells. One of the downstream RhoA targets, ROK, phosphorylates the cytoplasmic domain of CD44 thereby increasing CD44-ankyrin interactions. Other targets of ROK are myosin phosphatase and myosin light chain, two important mediators of actin-myosin dependent membrane ruffling required for cell migration. HA also activates the PI3 kinase/AKT pathway: Gab-1 phosphorylation by ROK stimulates PI3 kinase and AKT activation, leading to increased cell proliferation, invasion and cytokine production (Bourguignon *et al*., 2008). Additionally, ROK phosphorylates and activates NHE1, a Na+-H+ exchanger, causing intracellular and extracellular acidification leading to HYAL-2 driven HA degradation, ECM breakdown and tumour progression. CD44-HA interactions stimulate signalling through Rac1, another RhoGTPase, via the GEF Tiam1. In MDA-MB-231 cells, CD44-HA interactions also activate c-Src kinase resulting in activation and nuclear translocation of the transcription factor Twist, miR-10b expression and down-regulation of the tumour suppressor gene HOXD10 (Bourguignon *et al*., 2010 Toole, 2004). CD44 undergoes sequential proteolytic cleavages resulting in the release of its ectodomain from the cell surface and formation of a CD44 intracellular domain fragment, which is translocated to the nucleus, acting as a transcription co-regulator (Nagano and Saya, 2004). CD44 ectodomain cleavage is mediated by MT1-MMP and is stimulated by multiple factors, including HA fragments and TGF-β (Kuo *et al*., 2009, Sugahara *et al*., 2006)

HA synthesis and CD44-HA binding affinity (Heldin *et al*., 2008).

which, contribute to tumour cell migration and invasion (Fig. 3).

Receptor for HA Mediated Motility (RHAMM/HMMR) belongs to a group of proteins that are found intracellularly as well as extracellularly. RHAMM does not contain a transmembrane domain or classical export signal and is likely exported through an unconventional mechanism that does not involve the Golgi/ER. RHAMM is expressed as multiple isoforms and one of these, an N-terminal truncation that lacks the first 163 aa residues, is transforming in mesenchymal cells (Hall *et al*., 1995). On the cell surface, RHAMM interacts with HA and forms complexes with transmembrane receptors such as CD44, PDGFR, and RON (Maxwell *et al.*, 2008). Interestingly, CD44 surface display is reduced in mesenchymal cells isolated from RHAMM-/- mice, demonstrating functional interplay between these two HA receptors (Tolg *et al*., 2006). RHAMM is elevated in most types of cancer in particular breast, ovarian, and prostate cancer, as well as in MM, AML and CML. In BCA, RHAMM is a tumour marker, novel susceptibility factor and prognostic factor for poor outcome (Maxwell *et al*., 2008). Consistent with these clinical correlations, RHAMM has tumourigenic properties in experimental systems that have been linked to its ability to bind HA. In BCA cells, RHAMM/CD44/HA complexes sustain phosphorylation and activation of the Ras/MAPK (ERK1,2) signalling pathway, leading to BCA progression and constitutively high rates of motility and invasion (Hamilton *et al*., 2007). The relationship between RHAMM and ERK1,2 activation has recently been confirmed in BCA samples where concomitant upregulation of phosphorylated ERK1,2 and RHAMM in tumour samples correlates with a high tumour grade (Ward C., in preparation). Intracellularly, RHAMM binds directly to tubulin and is involved in regulation of microtubule stability and turnover as a result of its association with ERK1,2. In mesenchymal cells, the absence of RHAMM increases microtubule stability resulting in reduced cell migration and aberrant mitotic spindle formation (Tolg *et al*., 2010, Groen *et al.*, 2004). RHAMM interacts directly with ERK1, inferring that RHAMM may act as a scaffolding protein that directs ERK1 to its substrates including microtubule associated proteins that regulate microtubule stability (Tolg *et al*., 2010). Interestingly, RHAMM expression is downregulated by p53, an important tumour suppressor gene, suggesting that RHAMM may be involved in p53 loss-induced tumour progression (Buganim and Rotter, 2008, Godar and Weinberg, 2008, Sohr and Engeland, 2008). RHAMM also acts on the BRCA1, pathway and may play an important role in BCA tumours arising from loss or inactivation of BRCA1 (Joukov *et al.,* 2006)
