ezrin complex in cancer metastasis

Critical Reviews in Oncology/Hematology 46 (2003) 165 /186 www.elsevier.com/locate/critrevonc

The role of the CD44/ezrin complex in cancer metastasis Tracey A. Martin *, Gregory Harrison, Robert E. Mansel, Wen G. Jiang Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, S. Wales CF14 4XN, UK Accepted 8 November 2002

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. CD44: a cell surface receptor with diverse roles . . . . . . . . . . . . . . . . . . . . . 1.2. Ezrin: a structural and regulatory linking molecule of the ERM family . . . . . . . .

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2.

CD44 and ezrin structures . . . . . . . . . . . . . . . 2.1. The structure of CD44 . . . . . . . . . . . . . . 2.2. The structure of ezrin: the ERM protein family 2.3. ERM family binding partners . . . . . . . . . .

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3.

Expression and distribution of CD44 and REM family in normal and cancer cells . . . . . 3.1. Expression of CD44 in normal and cancer cells . . . . . . . . . . . . . . . . . . . . . 3.2. Expression of ezrin in cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

170 170 170

4.

Regulation and activation of CD44 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Regulation of expression and activation of CD44 . . . . . . . . . . . . . . . . . . . . 4.2. Regulation/activation of ezrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Interaction between CD44 and ezrin family members

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Physiological functions of CD44 and ERM family . . . . . . . . . . . 6.1. Function of CD44 in normal cells . . . . . . . . . . . . . . . . 6.1.1. Extracellular interactions between CD44 and its ligands 6.1.2. Intracellular interactions between CD44, ezrin and other 6.1.3. Other ligands for CD44 . . . . . . . . . . . . . . . . . . 6.2. Function of ezrin in normal cells . . . . . . . . . . . . . . . . . 6.2.1. Membrane attachment: molecular interactions . . . . . 6.2.2. Cytoskeletal association . . . . . . . . . . . . . . . . . . 6.2.3. Signal transductions for CD44/ezrin complex . . . . . .

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Expression of CD44 and ERM family members and the roles in cancer cells . . . . 7.1. Expression of CD44 in cancer cells . . . . . . . . . . . . . . . . . . . . . . . 7.2. Expression and possible functions of ezrin in cancer cells . . . . . . . . . . 7.3. Co-expression of CD44 and the ERM family members in cancer cells . . .

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8.

The clinical aspect of CD44, its variants and the ezrin family members in human cancers . . 8.1. The expression of CD44 and its variants in human cancers and their prognostic value 8.1.1. Colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.2. Gynaecological cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.3. Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.4. Gastric cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.5. Prostate cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.6. Other cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Soluble CD44 and its variants in cancer . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. The values of evaluating CD44 and its ligand hyluronan in cancer . . . . . . . . . .

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1040-8428/03/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1040-8428(02)00172-5

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* Corresponding author. Tel.:
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/44-29-207-61623; http://www.uwcm.ac.uk/mrg. E-mail address: [email protected] (T.A. Martin).

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T.A. Martin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 165 /186 8.4.

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Perspectives and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8.5. 8.6. 8.7. 9.

Levels and cellular location of ezrin in cancer . . . . . . . . . . . . . . . . 8.4.1. Ezrin and ERM family members in cancer . . . . . . . . . . . . . 8.4.2. CD44-ezrin complex in cancer . . . . . . . . . . . . . . . . . . . . CD44 and the spread of cancer cells . . . . . . . . . . . . . . . . . . . . . CD44 may be useful in predicting the therapeutic response in cancer . . . Hypermethylation of CD44 gene promotor region and low-level expression in prostate and ovarian cancers . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract CD44 is a cell adhesion molecule that was traditionally known as ‘homing receptor’. This molecule is known to interact with the ezrin family (ERM family) members and form a complex that plays diverse roles within both normal and abnormal cells, particularly cancer cells. CD44 and ezrin and their respective complex have properties suggesting that they may be important in the process of tumour /endothelium interactions, cell migrations, cell adhesion, tumour progression and metastasis. This article reviews the role of CD44, ezrin family and the CD44/ezrin complex in cancer cells and their clinical impact in patients with cancer. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CD44; Ezrin; Hyaluranon; Cell adhesion; Cancer metastasis

1. Introduction Metastasis of cancer cells proceeds via a number of steps: tumour cells must first invade the surrounding stroma by detaching from the tumour mass and becoming motile; angiogenesis must be initialised for transport of nutrients to and removal of waste products from the tumour site [1]. The blood vessels within the tumour then provide a route for detached tumour cells to enter the circulatory system and metastasise to distant sites [2,3]. As most tumour cells are surrounded by stroma, interaction between stromal cells and the malignant cells are extremely important in the development of tumour metastasis [4]. To metastasise, the detached tumour cells must enter the blood circulation, survive the immune system and arrive at a distant site. Here they interact with the endothelial cells by undergoing biochemical interactions (mediated by carbohydrate /carbohydrate locking reactions, which occur weakly but quickly), develop adhesion to the endothelial cell to form stronger bonds, thus penetrating the endothelium and the basement membrane. The new tumour can them proliferate. During the process of metastasis, molecules that may assist cancer cells to locate the favourable organ for metastasis, to interact with endothelial cells, and to migrate through the endothelium and to subsequently penetrate structures including matrix and vascular endothelium, would greatly facilitate the metastatic process [5,6]. Two proteins found in a number of cell types have properties suggesting that they may be important in the process of tumour metastasis; CD44 a cell /cell adhesion molecule

and ezrin, a cytoskeleton protein. Both have diverse roles within both normal and abnormal cells. 1.1. CD44: a cell surface receptor with diverse roles Cell surface receptors play an important role in the interactions between cells as well as between the cell and the extracellular matrix (ECM). Specific cell surface glycoproteins termed cell adhesion molecules, in addition to their role in cell/cell or cell /matrix interactions, are involved in more complex interactions that include cell motility and migration, differentiation, signal transduction, and gene transcription. One such cell adhesion molecule, CD44 (Ly-24, lymphocyte homing receptor (gp90Hermes), phagocytic glycoprotein (Pgp-1), extracellular matrix receptor III (ECMRIII), and hyaluronate receptors (H-CAM-homing cellular adhesion molecule) and HUTCH-1), has gained increasing important with respect to tumour cell invasion and metastasis. Originally identified on haematopoietic cells, CD44 is a family of transmembrane glycoprotein molecules that has been discovered on many different cell types from several mammal species. However, the adhesion molecule was thought not to have an intracellular anchor to the cytoskeleton, until recently that the ezrin family is found to play the role as CD44 anchorage molecule in the cell. 1.2. Ezrin: a structural and regulatory linking molecule of the ERM family Structural support for the plasma membrane of eukaryotic cells is provided by the cortical cytoskeleton,

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which is also involved in endocytosis, exocytosis and transmembrane signalling pathways [7]. The cortical actin cytoskeleton participates in many varied processes involving the plasma membrane, necessitating the involvement of molecular components that have considerable plasticity in action [8]. Regulation of such processes is dependent on their function. A group of proteins termed the ERM proteins (ezrin/radixin/moesin) have been shown to perform structural and regulatory roles in the assembly and stabilisation of plasma membrane domains [7]. Ezrin and related molecules are concentrated at surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane. Actin binding proteins allow cross-linking of actin filaments and regulation of actin filaments prior to cell motility. ERM proteins are believed to as membrane organisers and linkers between plasma membrane molecules such as CD44 and ICAM-2 and the cytoskeleton [9,10]. There is now compelling scientific and clinical evidence that adhesion molecules and the ezrin family are important structures in controlling cell functions such as adhesion as well as controlling the progressive nature of cancer cells.

2. CD44 and ezrin structures

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domain between exons 5 and 16 of the RNA transcript, corresponding to a position between aa 202 and 203. The short hydrophobic transmembrane domain contains 23 aa and is encoded by exon 18. The 70 aa cytoplasmic region (residues 272 /341) is encoded by part of exon 18 (three aa) and by exons 20. Exons 19 and 20 can be alternatively spliced, with the expression of exon 19 resulting in a short, truncated form (additional three aa) or exon 20 that results in the much more prevalent long form by the inclusion of the C-terminal exon (additional 67 aa). It is unclear, whether the shorttail form of CD44 is expressed on the cell surface, and if it has any functional role. Both the cytoplasmic and the amino-terminal domains are highly conserved (80 /90%) between species, implying these domains mediates one or more essential functions. The aa sequence of standard isoform of CD44 predicts a polypeptide that has a theoretical molecular mass of 37 kDa, however, the estimated molecular mass of the protein by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is 80 /95 kDa. This is due to extensive post-translational modification resulting in the attachment of numerous carbohydrates to N- and O-linked glycosylation sites of the extracellular domain [12 /15]. The degree of glycosylation will vary among different cell types and can give rise to CD44 molecules with a molecular mass ranging from 80 to 250 kDa.

2.1. The structure of CD44 2.2. The structure of ezrin: the ERM protein family In humans, the CD44 family is encoded by a single gene located on chromosome 11p13 and comprises at least 20 exons [11]. Exons 1 /5, 16/18 and 20, are spliced together to form a CD44 transcript known as the standard isoform (abbreviated to CD44s) or haematopoietic isoform of CD44 (CD44H). At least ten exons can be alternatively spliced and inserted into the standard isoform (CD44s) at an insertion site between exons 5 and 16 to give rise to variant isoforms of CD44. Thus, exons 6 /15 are variant exons and are typically identified as v1 /v10. The standard isoform of human CD44 (CD44s) is a type 1 transmembrane molecule composed of 341 amino acids (aa) and can be subdivided into three major domains; a 70 aa C-terminal cytoplasmic domain, a 23 aa transmembrane domain, and a 248 aa N-terminal extracellular domain (see Fig. 1). The extracellular domain can be subdivided further into two distinct regions; the N-terminal extracellular domain is encoded by exons l /5 and is highly conserved (85%) between mammalian species. The membrane proximal region of the extracellular domain is encoded by exons 16 and 17 as well as part of exon 5 (85 aa) is less well conserved (approximately 35/50%). The ten alternatively spliced exons v1/v10, encoding up to 381 aa, can be inserted at a single site in the membrane proximal extracellular

The prototype member of the ERM protein family is ezrin (also known as cytovillin, p81, 80k and Villin-2), which was first characterised as a component of microvilli [16]. Radixin, moesin and merlin (moesin /ezrin / radixin-like protein also known as schwannomin or neurofibromatosis 2 protein) are the other members of this highly conserved group [10]. Also a member is DAL-1, which possesses a unique carboxy-domain with an expression that is greatly reduced in NSCLC tumours, where it may function as a tumour suppressor gene [17]. Homologous proteins exist in many invertebrate species, including Drosophila , Caenorhabditis elegans , sea urchins and parasites [18]. The ERM family belongs to a superfamily of proteins whose prototypes are talin and band 4.1R (erythrocyte membrane /cytoskeleton linker protein band 4.1R or EPB41), two proteins whose roles in membrane/cytoskeleton interactions are well documented [19]. 4.1R exists in a number of isoforms generated from tissue-specific and developmental-stage-specific splicing events [20]. Other proteins in the band 4.1 superfamily include PTPH1 and PTBMEG [21]. The ERM family has high similarity with 70% identity. They are composed of three main domains (Fig. 2): the highly conserved globular aa-terminal (85%

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Fig. 1. (A) CD44 genomic structure with block size representing the number of aa encoded by each exon. (B) Illustration of CD44 protein structure showing extracellular and cytoplasmic domains.

identity) that is membrane binding; an extended ahelical domain; and a positively charged carboxyterminal actin-binding domain [21,23]. Ezrin is able to exist in a dormant conformation that requires activation to expose otherwise masked association sites. The

carboxy-terminal domain of ezrin is able to bind to the aa-terminal domain in an intramolecular association that inactivates the protein from binding to other molecules [21] (Fig. 3). Indeed, the amino-terminal residues (296 aa) enable ezrin to associate with the

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Fig. 2. Schematic showing the domain structure of ezrin (from [7,16]).

carboxy-terminal domain (107 residues) of any ERM member [7]. It is suggested that the amino-terminal residues bind directly or indirectly to the plasma membrane, with the carboxy-terminal residues binding laterally to actin [24]. 2.3. ERM family binding partners The amino-terminal domain of ezrin is also able to bind to cytoplasmic proteins such as EBP-50, a PDZ protein [21]. Ezrin has also been shown to anchor cyclic

AMP-dependent protein kinase [17]. Ezrin has also been reported to indirectly bind to Na
H
exchanger 3 (NHE3) via the cytoplasmic phosphoprotein called EBP50 or NHERF [25]. NHERF is a scaffold protein that indirectly recruits PKA to NHE-3 by binding to ezrin [26]. Ezrin is also believed to mediate extension of cellular projections in the presence of signals elicited by invading micro-organisms such as Shigella flexneri [27] leading to bacterial engulfment by a macropinocytic process.

Fig. 3. Associations of ezrin. (A) Ezrin is able to exist in a dormant conformation that requires activation to expose the otherwise masked association sites. Self-association of ezrin can occurs in tandem with the binding of the amino-terminal of one protein to the carboxy-terminal of another. (B) Extended ezrin molecules lie on the actin filament with only the carboxy-terminus binding the actin. Ezrin can also form parallel dimers that interact with actin filaments via actin binding sites. (C) Two classes of binding site to membrane proteins coexist within the N-ERMAD: direct binding using full length ezrin and indirect binding via an adapter molecule (using unfolded, activated ezrin). (D) Phosphorylation appears to regulate the binding of ezrin to actin, and it can be induced by numerous growth factors; phosphorylation also appears to open the conformationally masked carboxyterminal actin-binding site, which can be mediated via several alternative mechanisms.

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Heiska et al. [28] looked at the co-localisation of ezrin in microvilli projection finding that it co-localised with ICAM-1 and ICAM-2. CD43 and ERM proteins localise at the uropods if tumour-infiltrating T-lymphocytes [29]. Ezrin binds to cell adhesion molecules such as CD43, CD44, ICAM-1, ICAM-2, all of which are implicated in cell migration and metastasis. Many types of tumour express ezrin but little is understood of its role in metastasis [30].

3. Expression and distribution of CD44 and REM family in normal and cancer cells 3.1. Expression of CD44 in normal and cancer cells CD44 is expressed on a variety of cells including most hematopoietic cells: T-lymphocytes, B-cells, monocytes, granulocytes, erythrocytes, many epithelial cell types; keratinocytes, chondrocytes, mesothelial and some endothelial and neural cells [14,31/34]. The two most important isoforms are the standard isoform CD44s, which is expressed mainly on leukocytes and haemopoietic cells but also on fibroblasts and CD44E (R1), which is associated with actively dividing epithelial cells (e.g. keratinocytes) [12,35 /39]. These two isoforms differ in that CD44s can bind to HA and mediate lymphocyte homing, while CD44E can perform neither of these functions [37,39,40]. The expression of variant isoforms of larger size appear to be highly restricted in their expression on normal tissues, and some have been found only on malignant cells and tumour-derived cell lines. Individual cells can simultaneously express more than one CD44 isoform. Haemopoietic cells are characterised by a high expression of CD44; their membrane contains 105 /106 molecules, mainly of the standard type. Almost all cells of connective tissue, such as fibroblasts, endothelial cells, and macrophages contain large amounts of CD44s. 3.2. Expression of ezrin in cells Ezrin is found in most cell types, although it has been found to have an organ specific level of expression. Ezrin is expressed at very high levels in the small intestine, stomach, lung, pancreas and kidney; at intermediate levels in the spleen, thymus, lymph nodes and bone marrow; at very low levels in the heart, brain, testis and muscles [30]. No clear functional differences are documented between the different members of the ERM protein family. They do, however, show a divergent distribution pattern between cell types. Ezrin is found within the apical domain of polarised cells, a region characterised

by the presence of microvilli. All the ERM proteins are found in the brush border of kidney proximal tubule epithelium; however, other epithelia preferentially express membrane, such as bile duct epithelia. ERM proteins are found concentrated in the actin rich surface structures such as microvilli, membrane ruffles and filopodia. Ezrin especially has been detected not only in ruffles membranes, but also at the leading edges of spreading cells, and is therefore, associated with changes in cell motility [41]. Ezrin is especially found in the cytoplasmic aspect of microvilli in human placental syncytiophoblasts and mouse mesothelia [7]. Ezrin plays an important role in the membrane translocation mechanism of gastric parietal cells [42]. Ezrin is also localised at specific membrane sites of the intestinal brush border microvilli, dorsal microvilli and membrane ruffles. Isolated ezrin exists in different forms depending on the cellular source. Ezrin from gastric mucosa contains several isoforms, possibly the result of differences in phosphorylation [43]. Indirect binding of ezrin to actin has been reported at the barbed end of actin [44], whereas the direct binding of ezrin to actin has been observed at filament sites [43]. It has been concluded from this the existence of different physiologically significant modes of binding [18]. Ezrin has been shown to be located at cleavage furrows in cytogenesis and within the cell-to-cell junctions themselves, indicating just where the cytoskeletal network is being remodelled together with the modification of cell /cell and cell /matrix adhesion molecules: a further implication of its role as a regulator of cell morphology and cell attachment [41]. Ezrin (and moesin) have been found to be co-localised with ICAM-3 and the adhesion molecule PSGL-1 in the uropods of stimulated neutrophils [45]. CD43 also interacts with ezrin in T-lymphocytes, where it regulates ezrin distribution to the uropods at cell /cell contacts [29]. Hypoxic keratinocytes exhibit increases expression and redistribution of ezrin associated with lamellipoida [46]. Some evidence suggests that ICAM-2 recruits ezrin into the uropodia [47]. Interestingly, it has been shown that the existence of an amino-terminal cleavage form of ezrin exists. This form is found localised in the nucleus, with its over expression inducing cytoskeletal changes [48].

4. Regulation and activation of CD44 The expression and location of CD44 is well studied. A number of protein factors, primarily cytokines are known to regulate the level and location of CD44. Table 1 summarises cytokines that are known to have an effect on this molecule.

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Table 1 Effects of cytokines on CD44 Cytokine/growth factor

Effect on CD44 expression

IL-4

Enhanced expression of CD44 variants; v3, v6, v9 IL-4 (IL13) upregulate CD44 Upregulated expression in B cell precursors from patients with acute lymphoblastic leukemia (ALL) Stimulates HA binding Downregulation of CD44 Upregulation of CD44v10 Upregulation of CD44 variants and enhanced adhesion No effect No effect Stimulates HA binding Upregulated CD44s in cancer cells Upregulation of CD44 variants and enhanced adhesion Increased expression of CD44 on endothelial cells

IL-7 Oncostatin IFN-g TNF-a TGF-b

IL-8 HGF

4.1. Regulation of expression and activation of CD44 Regulation of CD44 binding to its primary ligand, HA, is relatively well understood, but the regulation of interactions with other ligands is not so well characterised. The mechanisms employed to regulate CD44 expression and functions are diverse, and include phosphorylation of the cytoplasmic domain, interaction with the cytoskeleton, alternative splicing, glycosylation, availability of the ligand, and CD44 shedding from the cell surface. 4.2. Regulation/activation of ezrin Soluble ERM proteins in the cytoplasm are dormant in terms of their cross-linking activity through mutual intramolecular and/or intermolecular association of their amino- and carboxy-terminal halves. Cellular signals are thought to activate the dormant ezrin by exposing the halves to allow interaction with integral membrane proteins and actin filaments [9]. The amino- and carboxy-terminal domains of ezrin interact synergistically in a salt-dependent manner to trigger the self-association of ezrin (Fig. 3). Such selfassociation properties could represent a way in which the number of ezrin molecules bound at a specific membrane site could be regulated [42]. The small GTP-binding protein rhoA is involved in the regulation of membrane/membrane interactions and ERM /actin cytoskeleton association [21]. ERM proteins are, therefore, regulated by an intramolecular association of the FERM and carboxyterminal tail domains that masks their binding sites (Fig. 3). The FERM domain has three compact lobes including an integrated PTB/PH/EVH1 fold with the carboxy-terminal segment bound as an extended peptide

Tumour cell

Refs.

Colon carcinoma T-lymphoma

[49] [50] [51]

Lung epithelial Pancreatic Colon carcinoma Colon carcinoma Pancreatic Pancreatic Lung epithelial Gastric carcinoma Colon carcinoma

[52] [53] [54] [55] [53] [53] [52] [56,57] [55] [41]

masking a large surface area of the FERM domain [58]. Such as extensive binding phenomenon indicates an unusual mechanism for producing varying levels of activation, depending on which signals were sent [58]. Phosphorylation appears to regulate the binding of membrane to actin, and it can be induced by numerous growth factors [18]; phosphorylation also appears to open the conformationally masked carboxy-terminal actin-binding site [59] that can be mediated via several alternative mechanisms. EGF is able to phosphorylate membrane (at Tyr 145 and 353) in A431 cancer cells, with the membrane being redistributed to the microvilli [60]; this involves the formation of membrane dimers and oligomers. Ezrin functions as the anchoring protein for cAMPdependent protein kinase during cell regulation [61]. Treatment of HT115 colon cancer cells with hepatocyte growth factor/scatter factor (HGF/SF) induces tyrosine phosphorylation of membrane and its redistribution from the cytosol to areas of ruffled membrane [5]. Ezrin may also be regulated by phosphatidylinositol 4,5biphosphate (PIP2), increasing membrane binding to CD44, ICAM’s -1 and -2 [62]. Rho acts as an upstream factor regulating ERM activation [18].

5. Interaction between CD44 and ezrin family members Function of ERM proteins is critical to maintaining cell adhesion. CD44 was the first cell surface protein to which ERM proteins were demonstrated to interact [62,63] and in vitro binding analysis revealed that the cytoplasmic domain of CD44 binds directly to the amino-terminal half of ERM proteins [63]. Binding of ERM’s to CD44 is regulated by the GTP-binding protein Rho and by PIP2 that bind to the amino-

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terminal of membrane [64]. In immunofluorescence experiments CD44 was shown to co-localise with the members of ERM family in hamster and mouse cells [65]. Both standard and variant isoforms of CD44 such as CD44E isoform and v9 /v10 containing isoforms associate with ERM proteins, the affinity of the variant isoforms being substantially greater. Since the cytoplasmic domains of variant and standard of CD44 are identical, it is thought possible that the tendency of variant CD44 isoforms to oligomerise results in a more stable complex with ERM proteins than does CD44s. Alternatively, it is possible that the binding of some unknown v9, v10 ligand could transmit a signal that alters the conformation of the CD44 cytoplasmic domain so as to increase its affinity for ERM family members. The region responsible for ERM protein binding in the CD44 was narrowed down to aa residues 289/300 of the membrane-proximal region of the cytoplasmic domain. Within this region are two clusters of positively charged aa (R292RR294 and K298KK300) shown to be essential for the binding of moesin and ezrin. Mutations within either cluster of basic residues resulted in impaired ERM binding. These two binding-binding sites are completely (100%) conserved between species and are found in all of the alternatively spliced CD44 isoforms. The cluster Lys298 /Lys300 in CD44 was also identified as an ERM-binding region in CD43 and ICAM-2 [63]. The presence of the second ERM-binding site in CD44 may be unique to CD44 and could reflect the importance of CD44 and its interaction with the ERM proteins. In vitro binding assays by Tsukita and co-workers have shown that the binding affinity of ERM proteins to the CD44 cytoplasmic domain is weak at physiological ionic strength conditions compared with CD44 and ankyrin binding (with a high affinity Kd
/1/2 nM). It appears that ERM proteins must be activated in order to function as cross-linkers between the membrane proteins and actin filaments. The mechanism by which this activation occurs has not been fully determined, but it does involve both tyrosine and serine/threonine phosphorylation plus the presence of PIP2 and small GTP-binding proteins. The presence of phosphatidylinositol 4,5-bisphosphate binding at the amino-terminal portion of ERM proteins alters their conformation and so increasing the affinity of binding of ERM proteins to the CD44 cytoplasmic domain. The differential binding affinities between CD44 isoforms and various cytoskeletal proteins (e.g. ankyrin and/or ERM) may influence selective signalling pathways leading to the onset of different CD44-mediated functions. Liu and colleagues found Jurkat transfectants bearing CD44 proteins lacking either the RRR or KKK motifs were unable to bind high levels of HA [66]. However, other investigators reported ERM proteins are not

required for ligand binding as the basic aa can be deleted without affecting binding to HA. It is, therefore, more likely that the ERM proteins mediate events downstream of ligand binding such as stable cell adhesion or cell movement. In addition, mutation of the basic clusters, individually or in combination, had no effect on CD44 localisation to membrane projections or ruffles. In conclusion, Although CD44 and ERM proteins can associate neither plays a primary role in determining the localisation of the other.

6. Physiological functions of CD44 and ERM family 6.1. Function of CD44 in normal cells The functional diversity of CD44 glycoproteins is the result of alternative splicing as well as of differential glycosylation and glycanation. Many of the functional roles are related to the binding of HA and to a lesser extent, other ligands of CD44. CD44 has functional roles in lymphocyte homing and adhesion during haematopoiesis, lymphocyte and monocyte activation, cell migration. CD44 has been shown to mediate cell aggregation of macrophages, lymphocytes, and fibroblasts [14,67 /70] and the adhesion of stromal cells and lymphoid precursor cells in the bone marrow [71]. HA accumulates in angiogenesis, wound healing, cell proliferation, cell motility, cell migration, and regulation of immune function in inflammation [72,73]. 6.1.1. Extracellular interactions between CD44 and its ligands According to several reports, the cytoplasmic domain of CD44 interacts with cytoskeletal components such as actin, ankyrin, or members of the ezrin/radixin/moesin family [65,69,74 /80]. The interaction between the cytoplasmic domain of CD44 cytoplasmic tail and cytoskeletal proteins may be regulated by Kinase C-mediated phosphorylation [77,81], palmitoylation [76], and GTP binding [80,82]. The CD44 family is a member of a family of cell adhesion molecules, termed the hyaladherins [83,84]. Hyaladherins, all share a limited (ca. 30%) sequence homology and act mainly as a receptors for hyaluronic acid (HA), which is an abundant extracellular polysaccharide found in mammalian ECM of many tissues. HA (hyaluronate, hyaluronan) is a linear, polymeric glycosaminoglycan (GAG) composed of repeating disaccharide units of D-glucuronic acid and N -acetyl-Dglucosamine. Unlike other GAGs of the ECM, HA is not sulphated and associates non-covalently with a number of HA-binding proteins. HA plays an important role in several important physiological functions such as providing cellular sup-

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port, maintaining homeostasis of water and plasma proteins in the intercellular matrix and promoting/ inhibiting mitosis). In addition, HA regulates cell /cell adhesion as well as cell growth and differentiation [74]. Thus differences in the HA binding state of CD44 are cell specific and have been shown to be related to posttranslational modification patterns. 6.1.2. Intracellular interactions between CD44, ezrin and other molecules The interaction of CD44 with the cytoskeleton was initially suggested by the close correspondence between the distribution of CD44 on the cell surface and actin filaments beneath the plasma membrane [85,86]. There is evidence that CD44 may be a link between the ECM and the membrane cytoskeleton via binding of its cytoplasmic domain to ankyrin [76,80,87]. The cytoskeletal protein, ankyrin, originally was determined to cross-link the band 3 membrane protein to the cytoskeleton in erythrocytes. Ankyrin is known to bind to a number of plasma membrane-associated proteins including two other members of the anion exchange gene family, Na
/K
-ATPase, the amiloride-sensitive Na
channel, the voltage-dependent Na
/ channel, Ca2
channels and the adhesion molecule, CD44. It has been suggested that the binding of ankyrin to certain membrane-associated molecules may be needed for signal transduction leading to a variety of cellular functions. 6.1.3. Other ligands for CD44 The polymorphic nature of CD44 may account for its ability to bind many ligands and the multi-functional nature of this molecule. It is thought there CD44 may interact with yet to be identified ligands [88,89]. Although CD44 is the principal HA receptor, it can bind to other ligands, including osteopontin (OPN), collagen, fibronectin, and laminin [79,90/97]. In addition, CD44 has been reported to recognise a number of other non-ECM related ligands including addressin [39], serglycin [98,99], and the major histocompatibility complex (MHC) class II invariant chain (Ii) [100]. 6.2. Function of ezrin in normal cells ERM proteins play a role in the formation of microvilli, cell adhesion sites, lamellipodia formation and contractile rings during cytokinesis [101]. The ERM protein family is involved in the interaction of the cell cytoskeleton with the plasma membrane, during signal transduction and growth control [42]. They, therefore, play a key role in the control of cell morphology [41]. ERM proteins are thought to function as general crosslinkers between plasma membranes and actin filaments in the cytoplasm [102]. In most cultured cells, ERM proteins are co-expressed and concentrated just beneath

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specialised domains of plasma membranes such as microvilli and cell-to-cell or cell-to-substrate adhesion sites where the actin filaments are densely associated. This expression is specifically regulated in differentiated tissues. As full length native membrane has a masked actin-binding site (as well as a masked amino-terminal site) by intra and/or intermolecular head-to-tail self association (Fig. 3), a signal is required to release the masking mechanism in order for membrane to function as a cross-linker beneath the plasma membrane [63,102]. Phosphorylation and phosphinositide turn over are supposed to be involved in this activation, together with Rho regulation. RhoA is found co-localised with membrane at early membrane ruffles of endothelial cells spread on collagen (but not on fibronectin). RhoA is translocated to and associated with cortical actin in focal contact domains at these membrane ruffles and at lamellipodia of spreading/migrating endothelial cells [103]. RhoA is known to trigger actin reorganisation and has been shown to mediate the formation of focal adhesions and stress fibres in quiescent fibroblasts. The activated membrane is directly involved in the morphogenesis of the free surface domain of plasma membranes, especially in the orientation of microvilli [9]. In the initial stages of anoxic injury and the apoptotic process, microvilli mostly disappear from the surface of the cell. In both cases, membrane and other ERM proteins are translocated from the microvilli to the cytoplasm with concomitant dephosphorylation [9]. Ezrin interacts with a number of cell surface adhesion molecules (Table 2) suggesting its involvement as a regulator of cell adhesion events [41].

6.2.1. Membrane attachment: molecular interactions Ezrin is involved in the redistribution and binding of intercellular adhesion molecules, together with organising the cell membrane structures [18]. Although membrane is a known binding partner for the transmembrane protein CD44, it is also co-localised with CD43 and ICAM-3 in some cell types [63,102] that are concentrated at microvilli. In vitro assays have indicated a direct association between the amino-terminal domain of membrane, and the cytoplasmic domain of syndecan-2 [21]. Syndecan-2 belongs to the family of heparin sulphate proteoglycans known to associate with the actin cytoskeleton, so transducing signals from the ECM. Ezrin /syndecan-2 binding is believed to be regulated by rhoA. Upon phosphorylation (by HCl secretion), membrane binds more tightly to the membrane in gastric parietal cells, showing, together with its specific location and abundance in these cells, that membrane is a key regulatory element of membrane translocation in parietal cells [42].

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174 Table 2 Interactions of ezrin Location

Interacting molecule

Function

Cytoplasm

Actin

C-terminal binding to activated (non-soluble forms of) ezrin. Within the [18,21,102,103] F-actin binding site, a conserved Thr residue at 566 is a target for phosphorylation by Rho-kinase (Rho-K) and protein kinase theta (PKC-u). Phosphorylation at 566 stops N- and C-termini interaction C-terminal interaction C- to N-terminal binding by monomeric folding of polymeric associations N-terminal binding to activated ezrin [21,102]

Cell membrane

Tubulin Ezrin EBP50 (PDZ-containing phosphoprotein) E3KARP (PDZ-containing protein) MBS (myosin binding subunit of myosin phosphatase) Dbl (GDP/GTP exchange factor) Regulatory RII subunit of protein kinase A (PKA) RhoGDi (Rho GDP dissociation inhibitor) CD44 (Hyaluronan receptor)

ICAM-1, ICAM-2, ICAM-3 (intercellular adhesion molecules)

CD43 (leukosialin) CD46

Refs.

N-terminal binding to activated ezrin N-terminal binding to dormant ezrin

[104]

C-terminal binding to dormant ezrin a-Helical domain binding to dormant ezrin. Bound via the AKAP site at residues 379 /439 Amino-terminal binding to activated ezrin

[105]

[30,62,105,106]

N-terminal binding to dormant ezrin. The interaction is facilitated [9,10,30,62] in vitro by phosphatidylinositol 4,5-biphosphate, for which there is a binding site in the N-ERMAD N-terminal binding to dormant ezrin. Believed to be a primary [9,45,62,107] membrane anchoring site for ezrin on certain cell lineages (e.g. lymphoid). Direct binding appears to involve integral membrane proteins with a single membrane-spanning domain and adhesive properties N-terminal binding to dormant ezrin [29,102] N-terminal binding

6.2.2. Cytoskeletal association Eukaryotic cells express a number of actin-binding proteins such as membrane, allowing a network of cross-linked actin filaments to be formed for mechanical rigidity and regulation the rapid turnover of actin filaments necessary for many cell motility processes [108]. The carboxy-terminal domain of membrane associates with actin filaments and the amino-terminal domain is able to associate with G-actin [21]. Such associations indicate that ERM proteins link receptors to the actin cytoskeleton. Integrity of the cytoskeleton is essential for CD95induced apoptosis through a mediated /mediated association between CD95 and the actin cytoskeleton [109].

6.2.3. Signal transductions for CD44/ezrin complex Ezrin is believed to be involved in the intracellular signal transduction that is related to changes in cell motility as it is thought to be a substrate for tyrosine kinases and the cross-linker molecule at the inner surface of the cell membrane [30]. Phosphatidylinositol 4,5-biphosphate induces interactions between mediated and ICAM-1, and enhances interactions between mediated and ICAM-2. It also enhances the interaction between mediated and CD44 through the regulation of the small GFP-linking protein Rho [30].

7. Expression of CD44 and ERM family members and the roles in cancer cells 7.1. Expression of CD44 in cancer cells The expression of variant isoforms of larger size appear to be highly restricted in their expression on normal tissues, and some have been found only on malignant cells and tumour-derived cell lines. CD44 is known to have a function in cell migration and metastasis. A number of studies indicate that multiple CD44 variant (CD44v) isoforms are expressed on the surface of tumour cells and may correlate with metastatic behaviour, Table 3. In addition, it supports the migration of invasive tumours and induces intracellular signalling [71,78,125 /131]. 7.2. Expression and possible functions of ezrin in cancer cells Disruption of actin filaments and a decrease in focal adhesion are common features following transformation of cells by various oncogenes [101]. That these changes in microfilament structure are related to anchoragedependent growth and cellular tumourigenicity, suggests fundamental roles for actin filaments in oncogenic transformation. Changes in actin filament structure are mirrored by decreases in expression of cytoskeletalassociated proteins [132] that lead to a transformed cell

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Table 3 CD44 expression in tumour types Human tumours

Change in CD44 expression

Acute myeloid leukemia Colorectal carcinoma Gastric carcinoma HCC Non-small cell lung carcinomas Melanoma Multiple myeloma Nodular sclerosing Hodgkin’s disease 7 Non-Hodgkin’s lymphoma Oesophageal squamous cell carcinoma Oral squamous cell carcinoma Pancreatic adenocarcinoma Primary pancreatic cancer Thyroid carcinoma Urothelial carcinoma Uterine cervical carcinoma

2 CD44v6 Correlates with poor prognosis [103] CD44 v3 Correlates with poor prognosis [104] 3 CD44v5, v6, v9 Upregulated during disease progression [105,106] 4 Upregulation of CD44s and v5, v6, v7 /8, v10 Correlates with poor prognosis [107] Upregulation of CD44v6 Correlates with metastases and poor prognosis [108,109] 5 CD44v3 Correlates with metastases [110] CD44v9 Upregulated during disease progression [111] 6 CD44v10 Upregulated during disease progression [112] 8 CD44v6 Correlates with poor prognosis [113,114] 9 V2 Correlates with poor prognosis [115] Downregulation of CD44v4, v5, v9 Correlates with metastases and poor prognosis [116,117] CD44v6 Correlates with poor prognosis [118] CD44v2 and v6 Correlates with poor prognosis [119] Downregulation of CD44s Correlates with poor prognosis [120] CD44v6 [121,122] CD44v6, v7 /8 Correlates with poor prognosis [123,124]

type. Some studies have implicated ERM proteins in this transformation as they have been found to be overexpressed in metastatic cell lines [133] such as changes in invasion of endrometrial cancer cells [134]. High expression of mediated in many cultured cells causes cell transformation and is associated with cell proliferation [135]. Many tumour types express ezrin, but little is known about its function. However, the Neurofibromatosis 2 (NF2) tumour suppressor protein Merlin (Shwannomin) has been found to be a homologue of the ERM proteins, with an NH2-terminal domain very similar to that of binding. Merlin is structurally related to mediated and is involved in the tumourigenesis of NF2-associated and sporadic schwannomas and meningiomas, but the tumour-suppressor mechanism is poorly understood [22]. Interestingly, merlin is also able to self-associate and binds to mediated by hetertypic binding between the amino- and carboxy-terminii. ERM proteins are found to interact with the TSC1 tumour-suppressor hamartin [136] where misregulation of cell /cell and cell /matrix interactions through the loss of this interaction contributes to the development of hamatomas in individuals carrying TSC1 mutations. This illustrates the regulation of cell adhesion by Rhomediated signalling pathways may constitute a ratelimiting step in tumour formation. Ezrin is required for ROCK-mediated transformation by the Net and Dbl oncogenes (RhoA GEFs). Rock phosphorylates mediated (at threonin 567); mutation of this site (to alanine) can inhibit ROCK-induced relocalisation of mediated to actin-containing structures, and also inhibit RhoA-mediated contractivity and focal adhesion formation [137].

Association in tumour progression

Ezrin has been suggested as a mediator of cell motility. Stimulation of epithelial tumour cells with the potent cytokine HGF/SF, results in the tyrosine phosphorylation of mediated, together with its translocation from a generalised cytoplasmic area to the ruffled regions of the cell membrane [5]. Work by Hiscox and Jiang [41] also suggests that mediated is essential for the maintenance of cell /cell adhesion and that it is inhibitory towards cell/matrix adhesion in human colonic epithelial cells. Ezrin can also be co-precipitated with Ecadherin and b-catenin, two key proteins involved in cell adhesion functions. Ezrin has been found to be crucial to HGF/SF-mediated morphogenesis on polarised kidney derived epithelial cells (LLC-PK1). Ezrin is a substrate for the tyrosine kinase HGF/SF receptor both in vitro and in vivo. HGF/SF stimulation causes enrichment of mediated recovered in the detergentinsoluble cytoskeleton fraction [19]. The truncated form of mediated, however, impairs the cells morphogenic and motogenic response to HGF/SF, suggesting a dominant-negative mechanism of action. The binding of mediated to cell surface adhesion molecules raises the possibility that not is it involved in cell migration, but that it may be involved in metastasis, as high levels of CD44 are associated with invasive and metastatic behaviour of tumour cells [30]. The relationship between mediated and tumourigenesis is also suggested by its shared homology with the NF2 gene product. It should be noted that pancreatic cell lines with very high levels of mediated and a great metastatic potential [30]. High levels of mediated has also been detected in the stromal cells of hemangioblastomas, tumours which are associated with mutations in the von Hippel Landau tumour suppressor gene [138]. Transfected mediated

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redistributes ICAM-2 to newly formed uropods on the surface of tumour cells, which thus become susceptible to lysis by NK cells probably due to the concentration of ICAM-2 [139]. 7.3. Co-expression of CD44 and the ERM family members in cancer cells CD44 and ezrin are both upregulated in fibroblasts transfected by vfos; they co-localise to membrane ruffles and microvilli of A431 cells after EGF treatment [140]. The ERM family member moesin has been shown to be directly associated with the cytoplasmic domain of CD44 in epithelial skin cells; there was, however, a loss of this associated in basal skin carcinoma, Bowen’s Disease and extramammary Paget’s disease suggesting that changes in expression are associated with a transformation to a cancerous phenotype [141]. This distribution is also true of moesin and CD44 in melanocytic tumours [142]. Neurofibromatosis 2 (NF2) protein that shares the ERM domain structure is seen as a linker between the cytoskeleton and CD44 [10]. The hepatitis B virus X protein (HBx) is thought to contribute to the acquisition of metastatic properties of hepatocellular carcinoma (HCC) by inducing the redistribution to pseudopodial tips of ERM family proteins in a Rho- and Rac-dependent manner, increasing the association of CD44 with moesin [143]. CD44 has been shown to act as a tumour promoter by recruiting ezrin to its cytoplasmic tail and so linking to the cytoskeleton, as occurs under the influence of growth factors, resulting in proliferation and invasiveness [144]. Conversely, in certain tumours, such as neuroblastomas and prostate cancer, loss of CD44 confers transformation to the invasive pheonotype due to loss of contact inhibition through loss of binding to ERM proteins. OPN, a secreted glycoproetin and ligand for CD44 has been shown to co-localise with both CD44 and ezrin in foetal fibroblasts, periodontal ligament cells, activated macrophages, and metastatic breast cancer cells. This co-localisation was prominent at the leading edge of migrating fibroblasts and suggests that OPN exists as an integral component of the hyaluronan/CD44 /ERM attachment complex involved in metastatic cells [145].

8. The clinical aspect of CD44, its variants and the ezrin family members in human cancers The biological role of CD44 and its variants in cancer cells has promoted a number of clinical studies to assess the clinical usefulness of the molecule in predicting outcome, prognosis, and response to treatment, in a variety of human cancers. Furthermore, there are studies that examined the impact of ligand for CD44,

hyluronan in cancer. Although the information is overwhelming, it is difficult to draw a clear conclusion from these studies, due to the number of CD44 variants, the location of the variants (either in cancer cells, matrix or stromal cells), the presence of soluble forms of the molecule, and what’s more different tumour types displaying different connections. 8.1. The expression of CD44 and its variants in human cancers and their prognostic value Most widely studied CD44 in clinical cancers are probably standard CD44 (CD44), CD44v6, CD44v3. 8.1.1. Colorectal cancer In colorectal cancer, CD44v6 was detected in almost all of the adenoma or carcinoma tissues, but only in 38% of metastatic lesions (liver or lymph nodes) [146]. The staining of CD44v6 was weaker in tumour and particularly in metastasis lesion, compared with benign lesion. Eighty five percent colon cancer cells expressed CD44 and 77% expressed CD44v6 [147]. In a study with 93 colorectal cancers, Jungling and colleague [148] failed to demonstrate a link between CD44V6 and clinical or histological grades. The study also failed to show a correlation between the variant and survival. About 37.8% (42/111) of colon cancer tissues showed a high level staining of CD44, and 62.2% exhibited low or no staining of the molecule [149]. However, there is no association between the staining of CD44 with grade, invasion, metastasis, survival and nodal involvement. In 61 colorectal cancer tissues, 83.7% of cases that have no node metastasis expressed CD44. However, only 11.1% of patients who have lymph node metastasis expressed this molecule, indicating the predicative role of CD44 in the lymphatic spread of colon cancer [150]. 8.1.2. Gynaecological cancers In a multiple centre study of 99 patients with vulvar carcinoma, 39.4% of the patients stained CD44v6, and the level of staining was correlated with poor diseasefree survival and overall survival. This correlation is independent upon other prognostic factors. In contrast, CD44v3, which was found in 33.3% of the patients bore no relation to prognosis [151]. In squamous cell carcinoma of vulva, CD44v9 was downregulated [152]. The level was particularly low in those who had recurrent disease and died of the same cancer. The level of CD44v10 expression was also found to be associated with recurrence. The same group has also reported that CD44v4 existed at a low level in vulva SCC cells, and most obvious reduction was seen in tumours from those patients who died of the disease [153]. In contrast, CD44v5 and v7 did not correlate with survival. A further observation is the presence of CD44v3 in nodal

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metastatic lesions (100%), compare with a very low level (3.3%) of CD44v3 in normal lymph nodes. In ovarian cancer, 71% primary tumours stained CD44v4, however, only 22% of metastatic tumour stained the variant [154]. The same was seen with CD44v6 (67 vs. 22%, respectively). CD44 was seen in a similar proportion in both primary and metastatic tumours (86 /87%). The authors suggest a role of CD44v4 and v6 in the recurrence and metastatic disease in this tumour type. In 56 ovarian carcinoma, 39% of tumour stained CD44 [155]. The presence of CD44 was significantly correlated with survival, but not with grade or the stage of tumours. A study of Sancho-Torres and colleagues [156] has examined the expression of variant forms of CD44 in 22 clear cell carcinoma of ovary. While a significant proportion of the tumour tissues express CD44v3, v5, v7 and v10, metastasis lesions were seen with significant reduction of these variants. The presence of CD44v10 appears to be linked to the death of patients, i.e. 71% of patients with this variant died of the cancer. 8.1.3. Breast cancer In breast cancer CD44v6 and CD44 was not related to clinical parameters such as size, grade, nodal status, angiogenesis or prognosis [157]. In breast cancer (n / 115), CD44v5 was found in 56% of primary tumour and in 94% of nodal metastasis, CD44v6 in 24 and 92%, CD44v7/8 in 15 and 89%, respectively [158,159]. Although the presence of these three variants shown a correlation with disease free survival, they are not independent indicators for clinical outcomes. 8.1.4. Gastric cancer In gastric cancer, 72% of 198 cases express CD44 and 55% expressed CD44v3 [160]. CD44, but not CD44v3 is connected with invasiveness and HA level. However, none of the two forms have exhibited relation to clinical survival and histological setting. 8.1.5. Prostate cancer Although CD44, CD44v6 and v9 were found to frequently expressed in prostate cancer, they do not appear to have any prognositic value [161]. 8.1.6. Other cancers CD44v6 was found to be at a higher proportion in metastatic lesion in lymph nodes from head and neck SCC than in the primary tumours from which metastasis derived [162]. In a retrospective study of 25 patient with oral/oropharynx squamous cell carcinoma, level of staining of CD44 in cancer cells was found to be correlated with prognosis, however, in a positive correlation, the higher the level, the longer the survival and visa versa [163]. Variants forms of CD44 and binding may have added value here.

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CD44 expression was detected in 88% of 67 cases of patients with Barrett’s-associated adenocarcinoma [164]. An increase in the expression of CD44 was significantly associated with shortened patient survival. In adenocarcinoma of Barrett’s oesophagus, reduction in immunoreactive CD44 and CD44v4 was correlated with shorter survival [165]. It seems that CD44v4 in this tumour type is an independent prognositic factor. In a series of 107 HCC, 34% exhibited staining for CD44, 49% for CD44v5, 27% for CD44v6, 38% for CD44v7/8, and 24% for CD44v10 [107]. CD44v6 showed a strong correlation with vascular invasion by tumour cells. Presence of each of these variants was linked to a shorter survival and presence of more than one of these variants was associated with a poor overall survival. In extrahepatic bile duct carcinoma and ampulary carcinoma, expression of CD44v6 was found in approximate a third of tumours (n/36) [166]. Interestingly, 60.9% of patients whose tumour are lack of CD44v6 had lymph node metastasis, compared with 15.4% with CD44v6 and with node metastasis. In these patients lack of both CD44 and CD44v6 are correlated with a poor prognosis. 8.2. Soluble CD44 and its variants in cancer As already discussed in early section, CD44 is generally localised in the membrane fraction. The molecule is prone to be modified by other enzymes; in this case, CD44 is known to be proteolytically cleaved, probably as the results of the action of proteolytic enzymes, such as metalloproteinases (MMPs) [167]. The rapid loss of CD44 from cell membrane is probably mediated by the action of rho family. The cleaved product is normally shed into extracellular space and in clinical situation, appeared as a soluble fragment of CD44 and its variants in the blood. Investigation into these soluble CD44s has also provided very used information. The soluble form of CD44 (sCD44) and CD44v6 (sCD44v6) was found to be significantly raised in the sera of patients with metastatic breast cancer, compared with non-metastatic and normal sera [168]. The level of sCD44v6 was correlated with the involvement of liver and the number of organs bearing metastatic lesions. However, the same was not found in other tumour types, i.e. head and neck squamous cell carcinoma [169], in which soluble CD44v6 in the serum of patients with HNSCC exhibited at the same level as in normal control subjects. The authors conclude that sCD44v6 in the sera of HNSCC patients is largely derived from normal epithelial cells. Soluble CD44v5 was found to elevate in sera of patients with breast cancer and higher proportion of patients with metastatic disease exhibited a marked

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increase in the sera level [170]. This large study found sCD44v5 is a poor marker for predicting malignancies, metastasis and clinical outcome. 8.3. The values of evaluating CD44 and its ligand hyluronan in cancer Hyluronan, the ligand for CD44, have been assessed in connection with CD44, in cancer cells, normal epithelial cells, stromal cells and stromal matrix. The expression pattern between this variance may be of important value in predicting the behaviour of a tumour. To evaluate the aspect of tumour cell hyaluronan in cancer development, Ropponen et al. [171] assessed 202 colon cancers with a mean follow-up of 14 years. It was shown that over 93% of tumours are positive for HA, with the stronger staining seen in Duke’s C and D tumours. A significant correlation was seen between a poor survival and a high proportion of positive cells and higher level of HA. In breast cancer, HA was found to exist in moderate of high level in peritumoural stroma [172]. Normal ductal epithelium do not express HA, however, over half of the tumours exhibited HA staining. Both stroma- and cancer cell-related HA were correlated with lymph node involvement and a poor survival. It has been reported that only 20% of follicular carcinoma cells of the thyroid are positive for HA staining, compared with 47% in papillary and /90% normal thyroid follicle epithelial cells. The level of HA staining appears to be correlated with CD44, CD44V3 and v6 [173]. It was interesting to note that high level of stromal HA, but not cancer cell HA staining, is associated with the occurrence of distant metastasis and mortality. HA staining in cancer cells was associated with perineural infiltration of the tumour and high Gleason score [174]. It was interesting to note an inverse relationship between the expression of HA and CD44 in cancer cells. The level of cancer cell CD44 and stroma HA was also inversely correlated. Tumour stroma staining of HA was found to be related to metastatic disease and perineural infiltration [174]. High level of stroma and cancer HA was associated with poor clinical outcome. The location of CD44 appears to be an important aspect in predicting clinical outcomes [175]. In this study with 133 colorectal cancers, CD44 was seen in 90% of stromal matrix and 98% of stromal cells. In clear contrast, CD44v6 was detected in only 12% of the stromal matrix and 17% of stromal cells. This significant reduction in matrix related CD44 is correlated with an increase in death rate. A further interesting observation with HA is that it may be of diagnostic value in bladder. In a study of 513 urine samples from patients with bladder cancer, the

urinary HA levels were elevated (2.5 /6.5-fold) in bladder cancer patients (1173.79/173.4; n /261) as compared with normals (246.19/38.5; n/41). The HA test showed 83.1% sensitivity, 90.1% specificity and 86.5% accuracy in detecting bladder cancer, regardless of the tumour grade. Thus, it appears that high stromal HA and high CD44 in cancer cells may be an important pattern that determine the invasive and metastatic potential of cancer cells. 8.4. Levels and cellular location of ezrin in cancer In contrast to CD44, studies on binding and ERM family members in cancer are rather limited. This is partly due to the fact that the full functions of the molecules are still not entirely clear, and that the connection between CD44 and binding has only been recently established. Laboratory studies have shown a possible link between targeting binding and reduction of cancer cell migration and motility [41,176]. However, limited reported already indicated that ERM family members might play an important part in cancer, in particular, when considered together with CD44. 8.4.1. Ezrin and ERM family members in cancer In a study with uterine endometrioid adenocarcinoma (n /20), the protein level and cellular location of binding was compared with normal endometrium and endometrial hyperplasia [177]. Protein level of binding was found to be significantly higher in cancerous tissues than normal tissues. When subcellular fractions were separated examined, it was revealed that in non-tumour tissues, binding was mainly seen in the cytosolic fraction, whereas it detection in both membrane and cytosolic fractions in tumour tissues. Interestingly, binding was seen mainly in the membrane region of metastatic cancer cells, but mainly in cytosols in normal and hyperplasia cells, thus indicating the importance of the subcellular location of binding in cancer and particularly in the metastatic spread of cancer. Ezrin was overexpressed in ovarian epithelial carcinoma (n/ 25). The highest level of staining of binding was seen in metastatic lesions [178]. This is probably reflecting the in vitro observation that binding on cell surface in endometrial cancer cells is required during the invasive process [158]. In human colon cancer, binding was found to be expressed at a lower level in cancer tissues compared with normal tissues [41]. The most striking difference is the location of binding, which is located in the membrane fraction in normal colon mucosa, but became cytosolic in tumour tissues. Recently, a new protein that with the ERM proteins, known as NHE-RF, was found to be highly expression in hyperproliferative tumour tissues [179].

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In a study using differential display and quantitative PCR analysis of mRNA, ridixin, binding and moesin were found to be expressed at a lower level in lung adenocarcinoma [180]. At protein level, ridixin and moesin were found to exist at a very low level in cancer cells. However, binding in these cancer cells display a most interesting location abnormality, invasive cancer cells and disorganised cancer cells exhibited a high level of cytosolic or diffused staining of binding. The new member of the ERM family, known as DAL1 (differentially expressed in adenocarcinoma of the lung), was reduced in over half of the non-small cell lung cancer, compared with normal tissues [17]. The clinical impact of this new molecule is yet to be determined. In ER-positive breast cancer (n/29), moesin mRNA level was reduced, compared with ER negative tumours [181]. The same over-expression was seen in ER-negative breast cancer cell lines. Interestingly, CD44v6 also associated with hormone dependence in breast cancer [157]. It may indicate that moesin and, indeed, the ERM family members may play a different role in the development and progression of breast cancer. In experimental models, mutation of NF2, a member of the ERM family, was linked to the development of metastasis at a very high rate [182,183]. This together with clinical observations probably supports a role of ERM family members as tumour suppressors [41]. 8.4.2. CD44-ezrin complex in cancer OPN is reported to be an integral part of CD44/ezrin complex [145]. OPN is known to associate with tumour progression [184,185] where malignant secretion of OPN and CD44v are linked, causing migration of tumour cells to specific sites of metastasis formation. From these limited studies on binding, one probably can argue that the overall alteration of the levels of binding in cancer is less significant. However, the location changes, i.e. from membrane fraction to cytosolic fraction and from cell adhesion area to other cellular locations, represent the most obvious change. This may indicate that relocation of binding from one part of the cell may result in CD44 co-translocated to different regions to exert different functions.

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further investigation. The former can be probably seen from as study in prostate cancer, in that the presence of CD44 in prostate cancer tissues was correlated with the occurrence of circulating prostate tumour cells in patients (pre-operative sampling) [186]. 8.6. CD44 may be useful in predicting the therapeutic response in cancer Soluble form of CD44v6 was significantly higher in the sera of patients with metastatic breast cancer [187]. Interestingly, higher levels of sCD44v6 are associated with the non-responsiveness of second line hormone and chemotherapy. The predicative value of CD44v6 in response to neoadjuvant chemotherapy in cervical cancer has also been reported [188]. In non-Hodgkin’s lymphoma, high level of soluble CD44 in sera was associated with poor response to therapies [189]. 8.7. Hypermethylation of CD44 gene promotor region and low-level expression of CD44 in prostate and ovarian cancers The promoter region of the CD44 gene was found to be methylated in prostate cancer and in particular metastatic prostate cancer [190]. This also reflected a reduction of mRNA and protein for CD44 in this circumstance. The hypermethylation of CD44 gene promotor region suppresses transcriptionally the expression of CD44 in various cells. This hypermethylation in prostate cancer was found to be significantly correlated the disease progression and metastatic diseases [191]. This hypermethylation may reflect the low level staining of CD44 and CD44v6 in prostate cancer [192], in that low level of CD44 and CD44v6 are associated with the occurrence of metastasis and high Gleason score, and poor prognosis in these patients. In ovarian cancer, CD44 was found to be expressed at low level and almost complete loss in metastatic nodes and malignant ascites [193]. The loss of CD44 was associated with stage, survival, and dissemination of cancer cells.

8.5. CD44 and the spread of cancer cells 9. Perspectives and conclusions As discussed in Section 1.1, CD44 and its variants are correlated with the disease progression and metastasis in selective tumours. Furthermore, the level of CD44 ligand, hyluronan, in the stromal matrix, together with the level of CD44 in cancer cells may determine the invasive nature of a cancer cells. Clearly, the pattern of CD44 in endothelial cells will also be a factor that determines the fate of cancer cells in the circulation, as tumour cells will utilise these molecules to locate the site for extravasation. This latter aspect clearly requires

From the evidence presented thus far, it is evident that CD44 and its adhesion complex play a very important role in the migration, invasiveness and metastatic behaviour of cancer cells. It is one of the key components in determine the tumour /endothelial interaction and tumour /stromal interactions. From clinical studies, it is also clear that expression and aberrant expression of CD44 is widely detected in a variety of human tumour and it bearing important link with clinical out comes.

T.A. Martin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 165 /186

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The following summarises the importance of the CD44/ ezrin complex in cancer: 1) 2) 3)

4) 5) 6) 7)

8)

9)

10)

11)

CD44 is a key cell adhesion structure in cancer cells. CD44 interacts with binding and ERM family members, via which it exerts its functions. CD44 is together with binding is important aspect to determine the migration and motility function of a cancer cell. Abnormalities in CD44 and/or binding will result abnormal cell functions. Endothelial CD44 is important during cancer extravasation. CD44 and its ligand are regulators of angiogenesis in cancer. CD44 and its variants are aberrantly expressed in human cancer and have prognostic value in human cancer. There is aberrant expression and mutation of ezrin and its family members in cancer. However, the abnormal location of these molecules may be important. Proteolytic cleavage of CD44 and its variants frequently occur in human cancer and the soluble fragments of these molecules are important prognostic factors. In selected tumours, such as prostate and ovarian cancer, the hypermethylation of the promotor region of CD44 gene may be responsible for the low level expression. The combined expression pattern of CD44 and its ligand, hyluronan, is important indicator of the invasive and metastatic nature of a tumour.

While there is a good body of literature on CD44 in human cancer, there is very little data to enable us to examine the full aspect of binding and its family in this case. It is also clear that CD44, its variants and binding are unlikely to be stand-alone prognostic and predictive factor in clinical outcome. A combination of these molecules with other established marker may have better prognostic value. There is little study to explore if targeting CD44 would be of therapeutic value. Given the link between soluble CD44 and its variants in the circulation of patients with cancer, targeting these cleaved products may be a worthwhile approach.

Reviewers Jeffrey S. Ross, Department of Pathology, MC 81, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA.

Professor Fred T. Bosman, Institut de Pathologie, University Hospital of Lausanne (CHUV), Rue du Bugnon, CH-1011 Lausanne, Switzerland. Professor Frederick Naftolin, Department of Obstetrics and Gynecology, Yale University, 333 Cedar Street, FMB 331, New Haven, CT 06520-8063, USA.

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Biographies Following her first degree in Microbiology with Genetics, Dr Tracey Martin began her research career with a Ph.D. in microbial molecular ecology at Cardiff University, Wales. Following this, she became a research fellow at the University of Wales College of Medicine, where she still works. Her main interests are changes in tight junction functions between normal and cancerous cells, and the interaction of cancer cells in cell-adhesion and cell-signaling, especially when they are related to angiogenesis. Following a first degree in Biotechnology at Cardiff University, Gregory Harrison is currently undertaking a Ph.D. at MRG, prior to which he completed a M.Phil. in the Department of Medical Biochemistry and Immunology at UWCM. His research interests include the role of CD44/ezrin complex in prostate tumour metastasis and the relationship between this complex and the E-cadherin/catenin complex. Professor Robert E. Mansel , has been the Chairman of the Department of surgery at the University of Wales

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College of Medicine since 1992. Prior to this post he was Professor of Surgery at the University of Manchester and the Christie Hospital Cancer Centre in Manchester. He is currently Chairman of the British Association of Surgical Oncology Breast Group. His research interests cover benign breast disease, high risk family history and the management of the axilla in breast cancer. He is the principal investigator of the UK ALMANAC Study of Sentinel Node Biopsy in Breast Cancer. Dr Wen G. Jiang , is a Senior Lecturer in the University Department of Surgery, University of Wales

College of Medicine (UWCM) in Cardiff, UK. Previously he was a Senior Research Fellow in University of Wales College of Medicine. His main research interest is the molecular and cellular mechanism of cancer metastasis and methods to inhibit cancer invasion and metastasis. He is particularly interested in the role cell adhesion molecules and hepatocyte growth factor/scatter factor in cancer metastasis and angiogenesis.