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Current Drug Targets - Cardiovascular & Haematological Disorders, 2005, 5, 3-14

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Hyaluronan and Hyaluronan Synthases: Potential Therapeutic Targets in Cancer Sophia Adamia*, Christopher A. Maxwell and Linda M. Pilarski Department of Oncology, University of Alberta & Cross Cancer Institute, Edmonton, AB, Canada. Abstract: Current models of oncogenesis describe cancer as a progression of genetic mutations in a tumor cell mass. However, tumors are more than a clonal expansion of malignant cells. Tumors are heterogeneous, with a complex 3D structure, analogous to organs comprised of different tissues. In a tumor mass, the component cell types interact with each other and with their microenvironment by exchanging information through cell-cell interactions and/or through interactions with the extracellular matrix (ECM). These synergetic interactions facilitate tumor progression. Furthermore, tumor invasion and metastatic development are accomplished through the breakdown of ECM. Disruption of ECM promotes abnormal inter- and/or intracellular signaling, leading to dysregulation of cell proliferation, growth and cytoskeleton reorganization. The disruption of the ECM in turn promotes the overproduction of growth factors, which induce elevated epithelial cell proliferation and other abnormalities including carcinogenesis. In this review we will demonstrate that hyaluronan (HA), a core component of ECM, contributes to certain types of cancer development. Additional to extracellular HA, intracellular and nuclear forms of HA have been detected. Intracellular HA is involved in cell signaling, whereas nuclear HA could promote chromatin condensation and thus facilitate mitosis. HA molecules are synthesized by hyaluronan synthases (HASs)—HAS1, HAS2 and HAS3 enzymes. Dysregulation of HAS genes results in abnormal production of HA and promotion of abnormal biological processes such as transformation and metastasis. The function of HASs appears to be cell and tissue specific. HAS1 maintains a low, basal level of HA. HAS2 is involved in embryonic and cardiac cushion morphogenesis and subsequent development through cell migration and invasion. HAS2 stimulates cell proliferation and angiogenesis. HAS3 appears to favor the malignant phenotype in many types of malignancies. However, the exact function of HAS isoenzymes and their role in cell signaling remains to be elucidated. A better understanding of HA and HASs may facilitate the design of novel therapeutic strategies to counter presumptive cancer-promoting effects of microenvironmental components.

INTRODUCTION Current models of carcinogenesis describe cancer as a progression of genetic mutations in a tumor cell mass. These models have contributed to the discoveries of many tumor suppressor genes and potential oncogenes [1]. Solid tumors, however, are more than clonal expansions of tumor cells; tumors are heterogeneous and have a complex structure. Furthermore, Bissell describes a tumor as a unique “organ” formed by “tissues” [2]. The cells composing these tissues interact with each other and with other types of cells and exchange information through cell-cell interactions or through interactions with cytokines and the extracellular matrix (ECM) [2]. These combined interactions facilitate tumor progression. A number of authors have suggested that the ECM plays a significant role in tumor growth, progression, and metastatic development [2]. Tumor invasion and metastatic development are mediated through the breakdown of the ECM. Disruption of the ECM promotes abnormal interand/or intracellular signaling leading to abnormal cell proliferation, cell growth, cytoskeleton reorganization, and alteration of other cell functions [3]. HA is involved in many biological processes including malignant cell migration [4, 5]. Disruption of the ECM occurs in the case of sustained *Address correspondence to this author at the Department of Oncology, University of Alberta & Cross Cancer Institute Edmonton, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2; Tel: 780-432-8924; Fax: 780-432-8928; E-mail: [email protected] 1568-0061/05 $50.00+.00

inflammation; as a result, constitutive activation of stromal fibroblasts leads to upregulation of enzymes such as metalloproteinases (MMPs), ultimately promoting the overproduction of growth factors. These factors endorse aberrant signaling and consequently induce elevated epithelial cell proliferation and promote other abnormalities [2, 3]. In addition, disruption of the ECM is a hallmark of cancer and this process, in particular, plays a significant role in tumor progression. The structural integrity of extracellular and pericellular matrices, which are composed of glycoproteins, proteoglycans (PG), cytokines and growth factors, are retained through the interaction between the negatively charged polysaccharide, HA, and link proteins and/or PG. HA is composed of repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine. Despite its simple chemical composition HA has been linked with many biological processes including space filling, lubrication, embryogenesis, cell adhesion and motility, cell growth and differentiation and angiogenesis [5-10]. HA facilitates its functions through interactions with the HA receptors RHAMM (Receptor for HA Mediated Motility) and CD44, as well as with other proteins, including hyaladherins and PG such as aggrecan, versican, neurocan, brevican, which serve as linker proteins. HA-PG interactions have significant structural importance in retaining of the ECM and thus facilitating tissue homeostasis. The many functions of HA are also regulated through its interactions with the HA binding proteins, the “hyaladherins” such as © 2005 Bentham Science Publishers Ltd.

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Current Drug Targets - Cardiovas. & Haemat. Dis., 2005, Vol. 5, No. 1

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Hyaluronan binding protein 1 (HABP1), which plays a significant role in fertilization. Majumdar and colleagues suggest that HABP1 is a cytoplasmic substrate for MAP kinase, and HABP1 translocation into the nucleus is accomplished by MAPK itself [11]. However, this study also suggests that activation of ERK is a requirement for the translocation of HABP1 into the nucleus. It is important to note that sequence analysis revealed that this protein is identical to P32 which was co-purified with alternative splicing factor SF2 [12-14]. This finding suggests the potential role of HABP1 in splicing machinery. Recently Spicer et al. reported the existence of the HA binding link protein gene family (HAPLN) [15]. The authors suggest that the functions of these proteins are analogous to those of cartilage link proteins, but the exact function of this protein family has yet to be elucidated. The data accumulated in the literature clearly suggest the multifunctional role and significance of the HA molecules in the biology of vertebrates and human. Thus, upregulation and/or downregulation of HA production can lead to abnormal cell behavior.

McBride et al. showed that HA matrices around adherent fibrosarcoma cells act as protective barriers and prevent lysis by cytolytic lymphocytes which are specific to the antigens expressed by the malignant cells [32]. Hyaluronidase treatment of fibrosarcoma cells resulted in the disruption of HA matrix around the fibrosarcoma cell plasma membrane, and also enhanced the cytotoxic action of cytolytic lymphocytes against them [32].

HA also plays a significant role in maintaining the luminal capillary, arteriole and venule glycocalyces as a barrier [16]. The glycocalyces, in turn, prevent penetration of large macromolecules through the membranes [16]. The glycocalyx, which is a very complex structure, is a dynamic component of endothelial cell function. Dysregulation of endothelial cell matrix formation may promote abnormal angiogenesis, potentially leading to tumor progression. Dramatically increased HA-rich matrix formation has been observed around proliferating and migrating cells during morphogenesis, regeneration and healing. High amounts of HA molecules are synthesized 1) prior to mesenchymal cell differentiation and throughout embryonic development, where the condensation and differentiation of the mesenchymal cells are accompanied by the spatial distribution of HA in the different regions of the limb bud [17-19], 2) During brain development around proliferating and migrating neuronal cells [20], and 3) during formation of heart valves when cushion cells migrate from the endocardium to the myocardium [10]. HA creates hydrated pathways, thus facilitating free movement of the cells in this microenvironment. HA molecules are conducive to cell proliferation and migration, preventing differentiation of cells until a sufficient number and appropriate positioning of cells is established, which is essential for the formation of tissues and/or organs [21]. In addition, the formation of hydrated pathways by HA molecules, is closely associated with the surface of different types of cells, and these associations promote cell adhesion, an important process in tumor progression [22, 23]. An important characteristic of HA is its ability to stimulate malignant cell migration [24-28]. Histological studies of various tumors, using an HA binding protein as a probe, revealed HA as a central component of the stroma, which surrounds and supports the tumor [29]. A high level of stromal HA is a strong predictor for the survival of patients with breast and ovarian cancer [29, 30]. In addition, ectopic production of HA by malignant cells themselves has been detected in patients with breast, colon carcinomas, stomach, bladder and ovarian cancers [29-31].

Considering all above-mentioned functions of HA, it seems likely that the impact of HA on malignant growth is multi-factorial. HA molecules present in solid tumors could support integrity of the tumor tissue and prevent malignant cell differentiation. Alternatively, HA could facilitate detachment of malignant cells from the tumor mass and therefore promote spread of the malignant cells. Furthermore, partial degradation of HA molecules promotes angiogenesis, a vital requirement for tumor growth [33, 34]. The precise mechanisms that mediate HA-dependent malignant cell migration remain unclear. HA may contribute to malignant cell migration by increasing tissue hydration and providing a supportive environment for malignant cell migration similar to embryonic cell movement [35]. Thus, controlling HA production, which is produced by the malignant cells, may modulate migration of malignant cells and may promote dormancy of the tumor by inhibiting malignant spread. Alternatively, HA may exert an anti-tumor effect when administered exogenously. In a xenotransplant model of human breast cancer, intratumoral injection of HA was shown to inhibit tumor growth and promote tumor regression [36]. HA reverses myeloid differentiation blockage in acute myeloid leukemia cells [37]. For astrocytoma cells, HA was found to decrease invasion [38]. Finally HA oligomers inhibit anchorage independent growth of tumor cells [39]. Thus, these observations suggest the therapeutic use of HA to inhibit tumor growth and spread. HYALURONAN RECEPTORS RHAMM AND CD44 Two HA specific receptors: RHAMM and CD44 have been identified thus far in humans. Based on the localization of RHAMM to both the cell surface and cell interior, and the HA- regulated redistribution between sites, RHAMM has been termed an “itinerant” receptor [5, 40-44]. RHAMM is an acidic coiled-coil protein. On malignant cells, surface RHAMM binds HA, while intracellular forms interact with microtubules, centrosomes and the mitotic spindle [5, 40, 4446]. HA binding by basal levels of surface RHAMM likely stimulates redistribution of intracellular RHAMM to the cell surface, indicative of a possible link between surfacelocalized and intracellular pools of RHAMM, regulated by interactions with HA [5, 40, 43-46]. RHAMM has recently been identified as a centrosomal protein that mediates the stability of the mitotic spindle [46]. Deletion constructs that lack the HA binding domains are unable to associate with the centrosome [46]. Thus, the domains of RHAMM that bind HA and function on the cell surface, are also responsible for the centrosomal localization of RHAMM, indicating that this region of RHAMM mediates multiple functional roles, and that interactions with HA may have multiple functional

Hyaluronan and Hyaluronan Synthases


outcomes. Both surface and intracellular forms of RHAMM efficiently bind HA [40]. This means that inside the cell, cytoplasmic HA may interfere/compete with normal cytoskeletal associations of intracellular RHAMM, and if present in excess on the cell surface may lead to deregulation of proliferation and cell behavior.

have arisen by a gene duplication event [53]. In transfectants, each isoenzyme of the HAS protein synthesizes different sizes of HA molecules with different functions [53, 54].

RHAMM appears to play a role in B lymphoid malignancies, breast, stomach and colorectal cancers [5, 44, 45, 47-49]. In multiple myeloma, a cancer of the bone marrow, overexpressed RHAMM is the only oncogenic abnormality consistently expressed in all MM patients analyzed [47]. CD44 appears to be a major binding site of HA for many cell types. Ten splice variants of CD44 have been identified thus far. CD44 containing various exons have been implicated in many types of cancers. However there have been no studies to show that expression of CD44 is absolutely necessary for HA mediated metastatic development. HAS proteins themselves in their structure include HA binding sites and thus can retain a HA matrix around the cells not expressing CD44 receptors. This also suggests that CD44 mediated tumor progression is more complex and extends beyond CD44-HA interactions. For lymphocytes, CD44 is usually abundantly expressed as compared to RHAMM, which is usually expressed at relatively low density. On the cell surface, both RHAMM and CD44 contribute to HA binding by human lymphocytes but only RHAMM appears to be a significant contributor to HA dependent motility [5, 40]. HYALURONAN SYNTHASES AND THEIR ROLE IN CANCER One of the essential steps in studying the biology of HA is to investigate the hyaluronan synthases (HASs). HA molecules are synthesized at the inner face of the cell plasma membrane by HASs and are immediately extruded into the ECM or distributed intracellularly [10]. HASs are integral plasma membrane proteins with a number of significant functions [50, 51]. The topological structure of the HASs, proposed by Heldermon et al. includes four transmembrane domains, two extracellular loops, two membrane-associated regions, an intracellular central loop, and intracellular amino and carboxyl terminus [52]. Examination of the hydrophobic domains of HAS isoenzymes has revealed the existence of essential cysteine (Cys) residues in the amino acid sequences of the central domain [52]. Modification of these Cys residues changes the enzymatic activities of the HAS proteins. A significant number of protein kinase C (PKC) phosphorylation sites are predicted to occur within the intracellular loop of the HAS protein suggesting that HAS activation is perhaps regulated by direct phosphorylation [52]. Three isoenzymes of HAS-- HAS1, HAS2, and HAS3-have been detected in humans thus far. The related but separate genes of the HASs, which share at least one or two exon-intron boundaries and 55-71% amino acid sequence identity, are located on different chromosomes (hCh19HAS1, hCh8-HAS2, hCh16-HAS3) and encode three different proteins with distinct enzymatic properties [53, 54]. These similarities in gene structure suggest that these genes might

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HAS3, which synthesizes shorter forms of HA molecules (0.12-1x106), is thought to be more active than HAS1 and HAS2, both of which produce longer HA molecules (3.9x106) [54]. It has not yet been conclusively established whether endogenous HAS isozymes synthesize different sizes of HA molecules. Within in vivo systems, it is expected that the existence of various sizes of HA molecules can be the result of either upregulation of hyaluronidases, which degrade the long chain of HA, or the existence of novel variants of HAS which produce smaller size of HA. Differential expression of HAS genes has been detected in human adult tissue by a northern analysis [53]. HAS1 and HAS3 transcripts were highly expressed in the heart, liver, skeletal muscles, prostate and ovary, while HAS2 transcripts were predominantly expressed in the heart and small intestine [53]. Overexpression of HAS proteins and subsequent overproduction of HA molecules promotes growth and/or metastatic development in fibrosarcoma, prostate and mammary carcinoma [55-59]. However, the removal of the HA matrix from a migratory cell membrane inhibits cell movement as has been demonstrated by Banerjee et al . and Evanko et al. [9, 60]. HAS proteins, particularly the HAS2 isoenzyme, which is involved embryonic and cardiac cushion morphogenesis and subsequent development through cell migration and invasion, appear to facilitate abnormal cell proliferation and the activation of cell signaling cascades that stimulate angiogenesis and may promote tumor progression [6, 33]. HAS, through the synthesis of HA, could facilitate tumor progression through reorganization of the cytoskeleton, including lamellipodial formation which is a prerequisite for cell spreading and metastatic development. It is also important to note here the importance of focal adhesion formation and cell spreading in metastatic development. All these processes are accomplished by complex signaling pathways. We do not intend to describe these signaling pathways in detail. However, some examples of theses signaling pathways are as follows: overexpression of HAS2 genes in fibroblasts appeared sufficient to induce activation of the HA specific cell surface receptor CD44 [61]. Nevertheless, HA produced by overexpressed HAS2 could interact not only with CD44 but with cell surface RHAMM. HA-CD44 and/or HA-RHAMM interactions have been shown to trigger the activation of the MAPK and PI3 kinase pathways, resulting in active cytoskeleton rearrangement and lamellipodia formation [61, 62]. Moreover, overexpression of HAS2 enhances the anchorage-independent growth and tumorogenicity of human fibrosarcoma cells [63]. Also, Hall et al. showed that src regulates actin cytoskeleton by a pathway that is triggered by interactions between extracellular HA and its receptor RHAMM [64]. In this paper, the authors indicate that src-regulated transient phosphorylation of FAK exerts control over cytoskeletal organization. Interestingly, HA overproduction by cushion cells not only provides a substrate for cardiac cell migration but also

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influences the transformation of these cells into a motile phenotype, suggesting a significant role in oncogenesis [10, 65]. HA is produced in large quantities by cells undergoing mitosis. It facilitates cell rounding and is involved in the post-mitotic separation of daughter cells [60, 66]. The activation of HAS enzymes appears to be essential for these events. The expression of the antisense of HAS2 and/or HAS3 in the aggressive prostate adenocarcinoma PC3MLN4 cell line inhibited tumor growth [67]. This finding suggests that overproduction of HA is required for tumor progression, and that elevated production of HA by prostate stroma and malignant cells is a negative prognostic factor [67]. Overexpression of HAS2 and HAS3 promotes anchorage-independent growth and tumorigenicity in immuno-compromised mice [63, 67, 68]. Compared to HAS1 and HAS3, the HAS2 gene is readily regulated in response to mechanical injury in human peritoneal mesonthelial cells in vitro and in dermal fibroblasts and osteoblasts in response to glucocorticoids [69-71]. In addition, HAS3 expression has been correlated with increased blood vessel formation [67]. In contrast, Simson et al. showed that decreased vessel formation occurs in tumors as a result of HAS2 overexpression [72]. These divergent outcomes may reflect the synergistic activation of hyaluronidase (HAase) in response to the overexpression of HAS enzymes (Fig. 1).

likely requires significant changes in the cell or tissue in response to external or internal stimuli.

Overexpression of HAS1 or HAS2, both of which appear to synthesize high molecular weight HA, may activate hyaluronidase, which degrades HA molecules and which is upregulated or downregulated during the progression of human cancer [6]. Shorter forms of HA resulting from HA degradation have been implicated in angiogenesis [6, 33, 34]. In conclusion, HAS1 and/or HAS3 gene regulation most

In mammary carcinoma cells, transfection with HAS1 transcripts resulted in the formation of increased metastases as compared to that in controls [57]. However, little is known about the role of HAS1 in various types of cancers, and regulation of this gene in response to external stimuli is not clear because of characteristics of this gene. The lifetime of HAS1 transcripts may be short and/or HAS1 may be expressed at low levels, compromising its detection by standard conventional gel electrophoresis. HYALURONAN AND HYALURONAN SYNTHASES IN MULTIPLE MYELOMA Dahl et al. demonstrated that abnormally high or very low levels of HA in the serum of patients with multiple myeloma (MM) correlate with dramatically reduced median survival of these patients [73]. Interestingly, Dahl et al. found an inverse correlation between Ig production and HA levels in the IgG and IgA myeloma patients. This phenomenon was interpreted as providing evidence for the existence of unoccupied HA specific receptors on the surface of malignant MM plasma cells. The author suggested that these unoccupied receptors of HA can bind HA molecules synthesized by cells other than malignant plasma cells residing in the bone marrow and this caused depletion of serum HA in MM patients. The authors also suggest that the low level of HA is due to the absence of autocrine mechanisms of HA production by MM cells in addition to the internalization of HA receptors by an excess of circulating HA. Based on these considerations, Dahl et al. suggested that the level of HA alone measured in the serum

Fig. (1). Synergistic regulation of HAS and HAase, and their effects on HA production.



of patients with MM does not provide the significant prognostic information [73].

multidrug resistance [84]. Baumgartner et al. showed that hyaluronidase treatment improved the effects of various chemotherapeutic agents [85]. MM B cells expressing HAS1 are highly drug resistant [4, 86]. Thus, synthesis of extracellular HA by malignant MM B cells may impact disease biology by contributing to drug resistance of these cells.

In addition to extracellular HA molecules which are extruded into the extracellular compartment, an intracellular HA has been detected in the cytoplasm and the nucleus of various tissues such as the brain, liver, and arteries, as well as in cumulus cells and oocytes [74-80]. This intracellular HA may regulate gene transcription and/or the cell cycle by binding to the cell cycle control protein CDC37 since this protein includes putative HA binding motifs. In addition intracellular HA could contribute the activation of the erk kinase pathway via the intracellular form of RHAMM. The source of intracellular HA is unclear. However, our own work investigating the role of HAS in myeloma patients, revealed three aberrant novel variants of HAS1 which may synthesize intracellular HA. These novel variants of HAS1 (HAS1Va, HAS1Vb and HAS1Vc) are also detected in malignant B lineage cells from patients with Waldenstrom’s macroglobulinemia (WM) (Adamia et al. in revision, Blood, 2004) [81].

Since expression of HAS1 and its novel variants correlated with poor survival of MM patients, manipulation of these genes could be beneficial for these patients. Also, the methods developed for MM patients can be applied to patients with other hematological malignancies. The manipulation of HAS genes and the production of HA molecules can be accomplished through introduction of antisense copies of these genes into cells. Nishada et al. in their studies conducted using human articular chondrocytes showed that transfection of these cells with HAS2 antisense oligonucleotides abolished expression of the HAS2 gene and, subsequently, the production of HA molecules [87]. Inhibition of HAS genes can also be accomplished by small inhibitory RNA (siRNA) which could be administered therapeutically. Hyaluronidase, an enzyme which degrades HA, can also be used as an option for the treatment of patients. However, this effective approach should be used very carefully since HAase is known to degrade HA and produce fragment, which contribute to angiogenesis.

We have shown that HAS1 and HAS2 isozymes, which are undetectable in B cells from healthy individuals, are overexpressed in malignant B and plasma cells from patients with MM or with WM (Table 1) [81]. In addition, both cancers are characterized by expression of novel HAS1 variants (Table 1) (Adamia et al. in revision, Blood, 2004).

HYALURONAN AND HYALURONAN SYNTHASES IN MITOSIS

The screening of PB samples obtained from 70 MM patients demonstrated that the expression of HAS1Vb variants correlates with the shorter survival of these patients (Adamia et al. in revision, Blood, 2004). More than 50% of the analyzed MM patients expressed novel HAS1 variants. Based on results obtained thus far, we speculate that HAS1 variants may contribute to intracellular pools of cytoplasmic and/or nuclear HA [60, 75, 82, 83]. In WM patients, we investigated the expression frequency of these variants at the single cell level [81]. We found that the majority of individual malignant cells from WM patients expressed novel HAS1 variants. Thus, in addition to the serum levels of secreted HA, the expression patterns of HAS genes and the novel HAS variants can be significant facilitators of malignant growth and progression in MM and WM.

HAS isoenzyme activity fluctuates throughout the cell cycle and reaches maximum levels at mitosis [66]. As a result, HA levels are greatly increased around dividing cells [66]. An increased HA matrix around the cell plasma membrane promotes cell rounding and detachment resulting in cell progression into mitosis [88]. In addition to extracellular HA, an intracellular HA has been detected throughout cell division [89]. These investigations suggest an intracellular and pericellular role of HA during mitosis. Abnormal expression of the HA molecules either inside or outside the cell during mitosis may promote abnormal cell proliferation and as a result, tumor progression. Furthermore, Kosaki et al. demonstrated that in fibrosarcoma cells overexpression of HAS and subsequently, overproduction of HA promotes anchorage independent growth in culture, which is one of the characteristics of tumorigenicity in vivo [63].

Recently, it has been demonstrated that interaction between endogenous HA and malignant cells regulates their Table 1.

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Expression Profiles of HAS Genes in MM and WM Patients

Cell type

HAS1 variants

HAS1

HAS2

HAS3

MM B

+++

+++

__

+++

MM PC

--/+

__

+++

+++

WM PB B

+++

+++

__

+++

WM BM B

+++

+++

__

+++

Healthy Donors PB B

__

__

__

+++

In the table “+ + +” indicate high level expression of HAS genes. PB = peripheral blood, BM = bone marrow, PC = CD138+ plasma cells, B = CD19+ and/or CD20+ B lymphocytes. Purified PC and B cells were obtained by flow cytometric sorting prior to analysis.

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Intracellular localization of HA has been detected in nucleoli and the nuclear periphery [60]. Evanko et al. showed localization of intracellular HA during mitosis at the metaphase plate [60]. Furthermore, intracellular HA was detected around chromosomes during their rearrangement and separation in anaphase. In concert with previous findings, we speculate that HA and HASs could play a significant role in mitosis. Additionally, Evanko et al. based on their studies in combination with previous findings, suggested a possible role for nuclear HA in ribosomal production and trafficking, or mRNA processing [60].

mammary carcinoma and melanoma cells leads to docking of gelatinase B, an enzyme which promotes tumor invasion and angiogenesis [108, 109]. CD44 also mediates a variety of intracellular signaling cascades and interacts with cytoskeletal proteins, thus promoting specific signaling pathways that are essential for the normal functioning of the cells. However, aberrant activation of these signaling pathways can lead to malignant behavior.

The HA binding receptor RHAMM, in addition to its role as a cell surface receptor for HA, is a centrosomal protein that maintains stability of the mitotic spindle. Depletion of intracellular RHAMM leads to the formation of tri- or tetrapolar mitotic spindles leading to dysregulation of mitosis [46]. Overexpression of exogenous RHAMM induces aberrant spindle architecture as well. Thus, increased RHAMM expression may induce extensive genetic instability in cancers, particularly in MM. Interestingly, the HA binding domain of RHAMM has significant homology to a basic leucine zipper motif (bzip) of human kinesin-like protein 2 (HKlp2) [46]. This bzip motif of Hklp2 interacts with targeting protein of Xklp2 (TPX2) to localize Hklp2 to microtubule minus ends in mitosis [90]. Work in our lab demonstrates that a significant pool of RHAMM interacts with the TPX2 in a mitosis specific manner. Recent investigations into the mechanisms of spindle assembly during meiosis and mitosis demonstrates that TPX2 is a vital regulator of bipolar spindle assembly and directly regulates the localization and activation of Aurora A kinase [91-96]. Aurora A kinase, in turn, has been implicated in tumorigenesis, centrosomal amplification, genetic instability as well as direct interactions with p53 and RASGAP/survivin ternary complexes [97-102]. The COOHterminal bzip motif of RHAMM overlaps with the HA binding domain and likely facilitates mitosis specific RHAMM-TPX2 interactions. HYALURONAN AND INTRACELLULAR SIGNALING All cell types, including tumor cells, display a variety of surface receptors which interact with components of the ECM. These interactions influence cell shape and behavior, including cell response to growth factors. Depending on the composition of the ECM, the same growth factors can induce cell proliferation, differentiation, growth arrest or apoptosis. Aberration of these processes can lead to tumor development via altered intracellular signaling. HA activates intracellular signaling through the activation of molecules such as MAPK, PKC, non-receptor tyrosine kinase src, Her2/neu receptor and focal adhesion kinase- FAK [8, 29, 62, 64, 103106]. It is well known that HA stimulated cell migration and signaling are accomplished through the interactions mediated by the HA cell surface receptors, CD44 and RHAMM, both of which promote tumor progression [65]. As mentioned earlier, the interaction of HA with CD44 has been implicated in cell proliferation, migration and angiogenesis [43, 49, 65, 107]. For example, CD44 clustering on the surface of

HA activated CD44 directly interacts with p185HER2 and c-src, and regulates components of the cytoskeleton such as actin filaments and microtubules [8, 110, 111]. These interactions mediate cytoskeleton-regulated cell migration. The cytoplasmic domain of CD44v10 is a substrate for PKC. The phosphorylation of CDv10 by PKC enhances interaction of CD44v10 with cytoskeletal proteins and as a result promotes rearrangement of cell cytoskeleton. Moreover, another variant of CD44, CD44v3, is predominantly detected in highly malignant breast carcinoma tissue samples [112114]. CD44v3 serves as a substrate for Roh-A activated ROK which phosphorylates the cytoplamic domain of CD44v3. Next, CD44v3 interacts with the cytoskeletal protein ankyrin, promoting malignant cell migration in patients with breast tumors [115]. Furthermore, ROK is involved in cross-talk between Ras and Roh signaling pathways, thus leading to cellular transformation which favors malignant cell proliferation and motility [115]. Additionally, CD44-HA interaction promotes intracellular Ca2+ mobilization which activates various signaling pathways including Ca2+ dependent IP 3 and Ca2+/calmodulin dependent signaling pathways. Ca2+/calmodulin, in concert with Roh-A and Rac1, regulates cytoskeletal function through phosphorylation of many important cytoskeleton regulating proteins such as the myosin light chain. The activated myosin light chain has been shown to promote endothelial cell migration and thus play important role during angiogenesis [115]. Most likely, CD44-HA mediated endothelial cell migration, and consequently spread of malignancies induced by the accumulation of intracellular Ca +2, can be manipulated using Ca2+ chelators and/or calmodulin inhibitors. In addition, intracellular Ca2+ could activate inducible nitric oxide synthase resulting in accumulation nitric oxide (NO) in the cells. Thus, it is feasible to control the level of NO and its downstream effects on the cells through manipulation of HA production. HA-RHAMM interaction activates several intracellular proteins through tyrosine phosphorylation (FAK phosphorylation) leading activation signaling pathways that regulate cytoskeletal modifications [42, 47, 105, 116]. Furthermore, the phosphorylation/dephosphorylation of pp125FAK in addition to the activation of proto-oncogene pp60c-src modulates cytoskeletal reorganization which promotes actin filament and cell-matrix adhesion structure rearrangements in the cell [64, 103, 117, 118]. Both of these processes play significant role in cell locomotion [64, 103, 117, 118]. RHAMM also activates p42/44 extracellular-regulated kinases (ERKs), which are invoked during receptor tyrosine kinase signaling [45, 118, 119]. RHAMM is involved in cell signaling and migration via interactions with HA,



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Fig. (2). Intracellular HA may facilitate aberrant mitosis. A model for HA and HAS facilitated oncogenesis. A) The HA binding domain of RHAMM overlaps the dynein-dynactin binding domain. B) RHAMM binds to microtubules via dynein-dynactin and helps to maintain spindle pole integrity. C) intracellular HA, produced by novel variants of HAS1 may disrupt RHAMM-dynein-dynactin interactions thereby promoting aberrant mitosis.

microtubules, and calmodulin [64, 103, 117, 118]. In H-rastransformed fibrosarcoma cells inhibition of RHAMM receptor results in cell cycle arrest in the G2/M phase through suppression of cdc2/cyclin B1 expression [120]. Thus, RHAMM appears to be key regulator regulator of cell growth and plays significant role in metastic development. HA stimulated cell migration and signaling are also accomplished through HA interaction with its receptor RHAMM. The coiled-coil protein RHAMM is distributed in

different compartments of the cell, including cell surface, nucleus and cytoplasm [43, 111]. Overexpression of RHAMM promotes malignant cell migration and leads to metastatic development and tumor growth [116]. RHAMM is implicated in the Ras signaling pathway [116]. Two splice variants of RHAMM have been identified: RHAMM-exon4 and RHAMM-exon13, both of which are overexpressed in MM and other B lymphocyte malignancies suggesting a significant role for RHAMM in mediating the spread of malignant cells throughout the bone marrow of patients with MM [47]. In

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addition, this study also points out the possible role of RHAMM in the spread of other B cell malignancies such as lymphoma and CLL. Overexpression of RHAMM has been implicated in motility of B-CLL cells [5]. Assmann et al. first demonstrated that intracellular RHAMM interacts with microtubules and microfilaments and may mediate cell locomotion through these interactions [45]. The same study showed that the RHAMM protein has the capacity to bind to the regulatory protein calmodulin in Ca2+ dependent manner. RHAMM-calmodulin interactions may be controlled by activation or deactivation of HAS proteins, since it is known that increased production of HA resulting from activation of HASs increases concentration of intracellular Ca 2+ [52]. This work supplements a large body of published evidence implicating RHAMM and HA in cancer [5, 40, 41, 44]. Thus RHAMM and HASs may act in concert to promote tumor progression through cytoskeletal rearrangement. Inhibition of HAS proteins may be an effective means to downregulate RHAMM mediated malignant cell migration. In addition, dowregulation of members of the family of HAS1, particularly novel variants of HAS1, may deplete the intracellular pool of HA and consequently may promote aberrant mitosis through modifiying the function of RHAMM that is overexpressed in MM [46, 47]. As mentioned earlier, the C-terminal bzip motif of RHAMM, a dyeinin-dynactin binding centrosomal targeting domain, overlaps with the HA binding domain and likely facilitates mitosis specific RHAMM-TPX2 interactions (Fig. 2). RHAMM-intracellular HA interactions may augment RHAMM-TPX2 interactions and regulate downstream Aurora kinase activities with consequent dysregulation of mitosis, genetic stability and tumorigenesis (Fig. 2). Maxwell et al . in his study demonstrated that disruption of the interaction between RHAMM and dyenin-dynactin leads to a multiple spindle formation, which in turn promotes misseggregation of chromosomes promoting aberrant mitosis (Fig. 2). This phenomenon was demonstrated though blocking intracellular RHAMM by intracellular injection of anti-RHAMM antibody. In vivo intracellular HA produced by one or more novel variants of HAS1 may compete with microtubule for the HA binding domain of RHAMM and disrupts RHAMM-microtubule interaction [46]. Thus, HAS1 and its variants, in concert with RHAMM, may contribute to genetic instability in MM. Therapeutic depletion of HAS1 and its variants may restore genetic stability. Considering various new possibilities that inhibit activity of HAS1 genes, it is intriguing to propose here that the turning off of HAS1 genes at the mRNA level will be ideal option for abrogating expression of HAS1 genes. The HAS1 transcript in its 3’ UTR region includes adenylate- uridylaterich elements (AREs) (ARED 2.0 database- http://rc.kfshrc. edu.sa/ared/). These elements recruit ARE binding proteins (AUBPs), which in turn bind to an exosome. Then, AUBP/ exosome complex promotes activation of a ribonuclease and mediates the rapid turnover of mRNAs [121-123]. Each mRNA has specific patterns for the distribution of the ARE motifs that are regulated by the three following elements: the first is the ARE motif composed of AUUUA

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stretches, which is a consensus sequence of a given mRNA located in cis; the second is the AU- binding proteins (AUBP), which act as a co-regulators and the third is the exosome (multi-subunit protein complex) in trans, which promotes degradation of mRNA [123]. The identification of ARE motif distribution patterns in the 3’UTR of HAS1 in concert with the analysis of the secondary structure of HAS1 genes will facilitate the identification of sub-regions located near the ARE motifs of the HAS1 gene. These sub-regions of the HAS1 can be targeted by antisense oligodeoxynucleotides (ODNs) or by synthetic ribozymes. Therefore, the ARE of HAS1 will become more accessible to the endogenous complex proteins, which modulate HAS1 function. Furthermore, HAS1 will be more susceptible to ribonuclease H, enzyme that degrades HAS1 mRNA transcripts. Thus, even though the biochemical mechanism that regulates ARE-dependent degradation is not completely known, ARE can be used as effective therapeutic targets. This model suggests usage of the endogenous protein mobilization to abrogate the expression of HAS1and its novel variants. CONCLUSION Despite intensive studies conducted thus far, the complete function of HA molecules in eukaryotes is not fully understood. Nevertheless, HA appears to be vitally important since many inherited diseases detected thus far are accompanied by aberrant HA production and turnover of this polysaccharide. As summarized above, HA-cell interactions have a major impact on normal and malignant cell behavior. Taken together, the body of work describing the impact of HA and HASs in cancer and other diseases makes it abundantly clear that the HA and the HAS isozymes should be important targets for novel strategies to eradicate and/or control malignant growth and progression. ACKNOWLEDGEMENTS This work was supported by CA80963 from the US National Cancer Institute. SA is supported by awards from the Alberta Heritage Foundation for Medical Research (AHFMR), Alberta Cancer Board and the Department of Oncology Myeloma Studentship. CAM is supported by AHFMR and the National Science and Engineering Research Council. ABBREVIATIONS ECM

=

Extracellular Matrix

HA

=

Hyaluronan

MMP

=

Metalloproteinases

PG

=

Proteoglycans

RHAMM =

Receptor for HA Mediated Motility

HAS

=

Hyaluronan Synthase

Cys

=

Cysteine

PKC

=

Protein Kinase C

MM

=

Multiple Myeloma


PB

=

Peripheral Blood

WM

=

Waldenstrom’s Macroglobulinemia

NO

=

Nitric Oxide

FAK

=

Focal Adhesion Kinase

Current Drug Targets - Cardiovas. & Haemat. Dis., 2005, Vol. 5, No. 1 [18] [19]

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