Hyaluronan promotes the malignant phenotype

Bryan P. Toole

Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111, USA

Accepted on December 21, 2001;

Abstract

Hyaluronan is a high-molecular-weight, negatively charged polysaccharide with unusual physical and interactive properties. Hyaluronan is localized in the extracellular matrix, at the cell surface, and inside cells. Its tissue distribution is ubiquitous, but it is particularly concentrated in pericellular matrices surrounding proliferating and migrating cells. Hyaluronan contributes to cell behavior in at least three ways. Its unique physical properties influence the biomechanical properties of extracellular and pericellular matrices; it is a template for assembly of other pericellular macromolecules; and it interacts directly with cell surface receptors that transduce intracellular signals. Experimental studies in animal models have documented a crucial role for hyaluronan in tumor growth and metastasis. Cellular manipulations have shown that hyaluronan promotes anchorage-independent growth and invasiveness, hallmarks of the malignant phenotype.

Key words: anchorage-independent growth/CD44/hyaluronan/RHAMM/ tumor invasion

Introduction

Hyaluronan (also called hyaluronic acid or hyaluronate) is a linear glycosaminoglycan composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine:[ß1,4-GlcUA-ß1,3-GlcNAc-]n. It usually contains 2000–25,000 disaccharides, exhibits varying molecular weights ranging from 105 to 107 Da, and has an extended length of up to 25 µm, depending on tissue source and physiological conditions. In free solution under physiological conditions of pH and ionic strength, it forms a stiffened and expanded random coil. It can occupy a very large solvent domain, but at relatively high concentration individual molecules entangle and form continuous networks. Because, in tissues, hyaluronan interacts with numerous binding proteins (known as hyaladherins) (Toole, 1990Go), its conformation is likely to alter dramatically from that measured under free solution conditions. Hyaluronan is distributed ubiquitously in vertebrate tissues, both in the embryo and in the adult. It is also produced by some bacteria and viruses but is apparently absent in invertebrate metazoans.

The hyaluronan synthases (identified as Has1, Has2, and Has3) are integral plasma membrane proteins whose active sites are located at the intracellular face of the membrane (Weigel et al., 1997Go; DeAngelis, 1999Go). Newly synthesized hyaluronan is extruded directly onto the cell surface; it is either retained there by sustained attachment to the synthase or by interactions with receptors, or it is released into pericellular and extracellular matrices. Hyaluronan is also found in several intracellular compartments. Regulation of targeting to these various locations is not understood at this time.

Hyaluronan has multiple physiological and cellular roles that arise from its unique biophysical and interactive properties. Its charge characteristics and polymeric properties contribute in several ways to tissue homeostasis and biomechanics. Its interactions with other extracellular macromolecules (especially aggregating proteoglycans) are crucial to the assembly and integrity of extracellular and pericellular matrices. Its interactions with cell surface receptors, such as CD44 and RHAMM, influence cell behavior in a variety of morphogenetic and physiological systems. Both RHAMM and CD44 exist as multiple, alternatively spliced forms that vary in their physiological functions. RHAMM is present both on the cell surface and intracellularly and most likely has different functions at these sites.

Several recent reviews discuss the properties and normal functions of hyaluronan and its binding partners in more detail (Toole, 2000Go, 2001; Lee and Spicer, 2000Go; Day and Prestwich, 2001Go; Tammi et al., 2001Go; Turley et al., 2001Go). There is also an excellent and extensive series of reviews on the Web (see "Science of Hyaluronan Today" at http://www.glycoforum.gr.jp).

Numerous studies performed over the past three decades have demonstrated a correlation between levels of hyaluronan production and malignancy in several types of tumor, both in animal models and in human patients (Toole et al., 1979Go, 2001; Knudson et al., 1989Go; Knudson, 1996Go). In the case of human breast, ovarian, and colon carcinomas, a high level of hyaluronan associated with cancer cells themselves or with the tumor stroma is a reliable prognostic indicator of patient morbidity (Ropponen et al., 1998Go; Anttila et al., 2000Go; Auvinen et al., 2000Go). This review summarizes some of the experimental evidence demonstrating the important role of hyaluronan in tumor progression and probing the possible underlying mechanisms whereby hyaluronan influences the malignant phenotype.

Hyaluronan influences cell behavior by several different mechanisms

There are three fundamentally different ways in which hyaluronan can influence normal and abnormal cell behavior. First, due to its unique biophysical properties, free hyaluronan has a profound effect on the biomechanical properties of extracellular and pericellular matrices in which cells reside. Second, hyaluronan forms a repetitive template for specific interactions with other pericellular macromolecules, thus contributing to the assembly, structural integrity, and physiological properties of these matrices. Third, hyaluronan interacts with cell surface receptors that transduce intracellular signals and influence cellular form and behavior. A recent study of Has2-knockout mice (Camenisch et al., 2000Go) highlights the various ways hyaluronan contributes to tissue structure and cell behavior: (1) extracellular matrices in Has2-null mice are more compact, i.e., less hydrated, than normal; (2) the organization of pericellular matrix components, especially versican, is altered; and (3) major defects occur in morphogenetic events, especially those involved in development of the cardiovascular system. With respect to the latter, cell migration and epithelial-mesenchymal transformation required for development of cardiac cushion tissues are inhibited. This is due at least in part to the absence of hyaluronan–cell interactions that regulate intracellular Ras signaling (Camenisch et al., 2000Go).

Numerous studies of embryonic development and adult tissue remodeling have demonstrated that extracellular matrices surrounding proliferating and migrating cells are enriched in hyaluronan and are often highly hydrated. Hyaluronan has the ability to trap large amounts of water within its structure and to exert swelling pressure as a result of compressing hyaluronan molecules into a volume less than that occupied when fully extended. Thus, an early interpretation of the association of changes in hyaluronan concentration or organization with dynamic cell behavior was that hyaluronan creates fluid, malleable matrices in which cells can readily change shape during mitosis or penetrate tissues during migration (Toole, 1991Go, 2000). In agreement with this idea, it was shown in a recent study that addition of hyaluronan promotes glioblastoma cell migration within a fibrin gel by causing increased hydration and thus gel porosity (Hayen et al., 1999Go). It also appears that extrusion of hyaluronan onto the cell surface at mitosis may create a hydrated microenvironment that promotes partial detachment and rounding of dividing cells. Hyaluronan synthase activity fluctuates with the cell cycle, peaking at mitosis, and inhibition of hyaluronan synthesis leads to cell cycle arrest at mitosis, just before cell rounding and detachment (Brecht et al., 1986Go). Also, hyaluronan-dependent pericellular matrix forms around dividing cells immediately preceding mitosis; removal of this matrix by competitive displacement with hyaluronan oligosaccharides inhibits cell division (Evanko et al., 1999Go). A similar hyaluronan-dependent matrix assembles around migrating cells, especially at the leading and trailing edges, and displacement of this matrix with hyaluronan oligomers reduces the rate of movement (Banerjee and Toole, 1992Go; Evanko et al., 1999Go).

These observations suggest that the biomechanical properties of pericellular hyaluronan may facilitate regional detachment of a cell from its substratum, a necessary step in both mitosis and migration, by creating a hydrated zone around the cell. This hydrated zone, or pericellular matrix, would separate the cell from physical barriers to penetration or shape changes required for division or movement. However, to create this hydrated pericellular matrix, hyaluronan requires cooperative interactions with other matrix molecules, especially "aggregating" proteoglycans, such as aggrecan (Knudson and Knudson, 2001Go) or versican (Evanko et al., 1999Go; Camenisch et al., 2000Go). Interaction with these proteoglycans causes hyaluronan to take on a more linear conformation that extends out from the cell surface, where it is tethered either by binding to CD44 or by transmembrane interaction of "nascent" hyaluronan with hyaluronan synthase (Lee et al., 1993Go; Toole, 2001Go). The resulting pericellular matrix also incorporates other agents, such as the hyaladherins, TSG-6, and inter-{alpha}-trypsin inhibitor (Carrette et al., 2001Go; Mukhopadhyay et al., 2001Go), which may contribute to the physical properties of the matrix or to cell surface interactions that lead to downstream cellular events.

In addition to its roles in the biomechanical and physiological properties of matrices, hyaluronan also interacts directly with the cell to influence intracellular signaling. Hyaluronan interacts with the plasma membrane in at least two ways, that is, by sustained attachment to hyaluronan synthase on the cytoplasmic side of the plasma membrane or by binding to cell such surface receptors as CD44 and RHAMM. As mentioned, hyaluronan synthase activity and extrusion of hyaluronan onto the cell surface peak at mitosis. In addition, however, hyaluronan is enriched within the cytoplasm of proliferating cells, especially around and between chromosomes prior to mitosis (Evanko and Wight, 1999Go). Also, hyaluronan internalized by ras-transformed cells is distributed to several intracellular sites and promotes cell motility (Collis et al., 1998Go). Several intracellular hyaladherins that interact with kinases important in regulation of cell behavior have been characterized (Grammatikakis et al., 1995Go; Entwistle et al., 1996Go; Huang et al., 2000Go; Turley et al., 2001Go). Thus it has been proposed that intracellular hyaluronan interactions may be involved in coordination of hyaluronan synthase activity, intracellular hyaluronan concentrations, and cell behavior.

In a more conventional manner, binding of hyaluronan to its cell surface receptors leads to many downstream signaling events, the precise nature of which depends on cell type and physiology (Herrlich et al., 2000Go; Turley et al., 2001Go). This very active area of research, as it relates to the malignant phenotype, is discussed in the following sections.

Experimental manipulation of hyaluronan in vivo influences tumor progression

Experimental evidence has been obtained in animal models that directly implicates hyaluronan in tumor progression. Several approaches have been used, including manipulation of levels of hyaluronan and perturbation of endogenous hyaluronan interactions.

Experimental overexpression of the hyaluronan synthase, Has2, in HT1080 human fibrosarcoma cells gives rise to elevated hyaluronan production and causes increased tumor growth in vivo (Kosaki et al., 1999Go). Similar results were obtained on overexpression of Has3 in TSU human prostate tumor cells (Liu et al., 2001aGo). In another study, mouse mammary carcinoma cell lines were selected for high and low hyaluronan production and tested for growth and metastasis in vivo. In this case no effect on growth was observed, but decreased formation of metastatic nodules in the lung after intravenous injection occurred in lines selected for low hyaluronan production; the metastatic potential of these cells was rescued by increasing hyaluronan production via transfection with Has1 (Itano et al., 1999Go).

Three approaches that have been used to manipulate endogenous hyaluronan–protein interactions are overexpression of soluble hyaladherins, administration of hyaluronan oligosaccharides, and treatment with antibodies that block hyaluronan–CD44 binding. Soluble hyaladherins competitively displace hyaluronan from its endogenous cell surface receptors, for example, CD44 or RHAMM, thus inhibiting putative downstream events. Several studies have demonstrated inhibition of tumor progression by treatment with soluble forms of CD44 (e.g., Sy et al., 1992Go; Bartolazzi et al., 1994Go). Overexpression of soluble CD44 in mouse mammary carcinoma cells or in human malignant melanoma cells leads to inhibition in vivo of growth, local invasion, and metastasis (Yu et al., 1997Go; Yu and Stamenkovic, 1999Go; Peterson et al., 2000Go; Ahrens et al., 2001bGo). No significant effects were obtained in these studies if the soluble CD44 was mutated such that hyaluronan binding was eliminated. Soluble RHAMM, another hyaladherin, also inhibits metastasis (Mohapatra et al., 1996Go) and a hyaluronan-binding complex from cartilage, containing link protein and fragments of aggrecan, inhibits both tumor growth and metastasis (Liu et al., 2001bGo). Hyaluronan oligomers compete for endogenous polymeric hyaluronan–receptor interactions, thus resulting in low-valency, low-affinity binding rather than polyvalent, high-affinity interactions with receptors (Underhill et al., 1983Go). Oligomers containing 6–18 sugar residues are effectively monovalent in their interaction with CD44 (Lesley et al., 2000Go). Thus displacement of endogenous polymeric hyaluronan with oligomers of this size could potentially lead to the loss of hyaluronan-induced signaling. In similar fashion to soluble hyaluronan-binding proteins, these oligomers inhibit growth of several tumor types in vivo (Zeng et al., 1998Go; Ghatak et al., unpublished data). Likewise, treatment with antibodies that block hyaluronan binding to CD44 inhibit tumor growth and invasion (Guo et al., 1994Go; Zahalka et al., 1995Go).

Although the cited data point clearly to a crucial role for hyaluronan in tumor progression in vivo, other data suggest that this involvement is complex. For example, methods used to perturb hyaluronan interactions, that is, overexpression of soluble hyaladherins or treatment with hyaluronan oligomers, could have manifold biochemical effects with differing downstream consequences. These include competition for binding of endogenous hyaluronan to cell surface receptors, as discussed (Ahrens et al., 2001bGo), reduced hyaluronan endocytosis and degradation (Yu et al., 1997Go), interference with CD44 clustering in the plasma membrane (Yu and Stamenkovic, 1999Go), and disruption of interactions within the pericellular matrix (Yu et al., 1992Go; Knudson et al., 1999Go; Knudson et al., 2000Go). Also, it is possible that, in addition to interfering with downstream effects of hyaluronan polymer interactions, hyaluronan oligomers may interact with cells in a manner that directly induces signaling. Direct signaling by oligomers and small polymers of hyaluronan has been demonstrated with endothelial cells (Deed et al., 1997Go; Slevin et al., 1998Go), macrophages (Horton et al., 1998Go), and other cell types (Turley et al., 2001Go). In the case of endothelial cells, such signaling is mediated by RHAMM and CD44 (Lokeshwar and Selzer, 2000Go; Savani et al., 2001Go) and leads to increased angiogenesis (West et al., 1985Go; West and Kumar, 1989bGo; Montesano et al., 1996Go). A related finding is that tumor cells often exhibit elevated levels not only of hyaluronan itself but also of hyaluronidase and the ability to internalize and degrade hyaluronan (Culty et al., 1994Go; Liu et al., 1996Go; Yu et al., 1997Go; Lokeshwar et al., 2001Go). It has been concluded from these studies that angiogenic hyaluronan oligomers are produced in aggressive tumors and would be expected to promote tumor progression (Liu et al., 1996Go; Lokeshwar et al., 2001Go). However, administration of such oligomers in vivo inhibits tumor growth (Zeng et al., 1998Go; Ghatak et al., unpublished data), and at least one of the several hyaluronidase genes corresponds to a previously mapped tumor suppressor (Csoka et al., 1998Go). Further study is necessary to clarify these apparent contradictions.

Hyaluronan stimulates intracellular signaling pathways that promote anchorage-independent growth and invasiveness of tumor cells

Recent investigations indicate that hyaluronan is critically involved in anchorage-independent survival and growth, one of the fundamental attributes of the malignant phenotype. Overexpression of Has2 in HT1080 human fibrosarcoma cells (Kosaki et al., 1999Go) or Mero-25 mesothelioma cells (Li and Heldin, 2001Go) has been shown to stimulate anchorage-independent growth in soft agar. Other studies have demonstrated that perturbation of endogenous hyaluronan interactions, either by overexpression of soluble CD44 (Peterson et al., 2000Go) or by addition of hyaluronan oligomers (Ghatak et al., unpublished data), inhibits anchorage-independent growth of several tumor cell types, including breast carcinoma, colon carcinoma, and glioma cells. Such perturbations lead to cell cycle arrest or apoptosis of the tumor cells, both in vivo (Yu et al., 1997Go; Peterson et al., 2000Go) and under anchorage-independent conditions in vitro (Ghatak et al., unpublished data). The underlying mechanism whereby hyaluronan oligomers inhibit anchorage-independent growth is suppression of the phosphoinositol-3-kinase/Akt survival pathway (Ghatak et al., unpublished data). This is in agreement with other studies showing involvement of this pathway in signaling events arising from interactions of hyaluronan with CD44 (Kamikura et al., 2000Go; Sohara et al., 2001Go).

Although the evidence suggests a positive role for hyaluronan in tumor cell proliferation, in vitro studies of anchorage-dependent growth have produced variable results. One reason for this is that in many cases the effect of hyaluronan on cell proliferation in routine culture is highly dependent on concentration and molecular weight. High concentrations of high-molecular-weight hyaluronan are usually inhibitory, whereas low concentrations of lower-molecular-weight hyaluronan can be neutral or stimulatory (Goldberg and Toole, 1987Go); the molecular basis of these different effects is not known. A recent study has shown that hyaluronan-dependent interaction of CD44 with the tumor suppressor merlin mediates contact inhibition of growth of several cell types (Morrison et al., 2001Go). Apparently, however, many tumor cells have lost this inhibitory mechanism because hyaluronan–CD44 interactions clearly promote proliferation in vitro and growth in vivo in several tumor cell types, such as melanoma (Bartolazzi et al., 1994Go; Ahrens et al., 2001aGo,b), mammary carcinoma (Peterson et al., 2000Go), and ovarian carcinoma (Bourguignon et al., 1997Go). In the case of ovarian tumor cells, hyaluronan-stimulated proliferation has been shown to involve interaction of CD44 with and activation of the HER2/neu oncogene (Bourguignon et al., 1997Go). Also, hyaluronan may influence growth by activating Ras signaling through interactions with CD44 and RHAMM (Zhang et al., 1998Go; Camenisch et al., 2000Go; Fitzgerald et al., 2000Go).

As noted, another mechanism whereby hyaluronan may influence tumor growth is via stimulation of angiogenesis, a process that, again, is likely to be dependent on molecular weight and concentration (West and Kumar, 1989aGo; Banerjee and Toole, 1992Go). Recent studies have indicated that interactions of hyaluronan with both cell surface RHAMM and CD44 are involved in endothelial cell proliferation, migration, and assembly of endothelial tubules (Savani et al., 2001Go) and that hyaluronan stimulates FAK and ERK kinase activities in these cells (Lokeshwar and Selzer, 2000Go).

A large body of work indicates that hyaluronan–CD44 and hyaluronan–RHAMM interactions promote tumor cell movement and invasion as well as growth. Interactions with src kinases, Rac and Ras signaling pathways, and cytoskeletal components have been implicated in the effects of hyaluronan. In breast carcinoma cells, hyaluronan-stimulated interaction of CD44 with Tiam1 leads to Rac1 signaling. This results in cytoskeletal rearrangements, membrane ruffling and increased cell migration (Bourguignon et al., 2000Go). Local application of hyaluronan to the surface of mammary epithelial cells also causes Rac1 activation and lamellipodia formation via hyaluronan-CD44 interaction (Oliferenko et al., 2000Go). However, a puzzling aspect of the latter study was the observation that unsaturated hyaluronan disaccharides, which presumably do not react with CD44 (Underhill et al., 1983Go; Lesley et al., 2000Go), also elicited this effect. Stimulation of ovarian carcinoma cell migration by hyaluronan involves interaction of CD44 with and activation of src kinase. This leads to phosphorylation of cortactin, rearrangement of actin filaments, and stimulation of migration (Bourguignon et al., 2001Go). Hyaluronan–RHAMM interaction induces transient phosphorylation of p125FAK in concert with turnover of focal adhesions in ras-transformed cells, thus leading to initiation of locomotion. Suppression of hyaluronan–RHAMM interaction inhibits cell locomotion (Hall et al., 1994Go, 1995). These signaling events lead to alterations in the cytoskeleton required for migration and invasion. The cytoplasmic tail of CD44 has been shown to interact with ankyrin (Zhu and Bourguignon, 2000Go) and the ezrin-radixin-moesin family (Morrison et al., 2001Go; Yonemura et al., 1998Go; Zohar et al., 2000Go) and both types of interaction promote migration. RHAMM also interacts directly with the cytoskeleton (Entwistle et al., 1996Go; Assmann et al., 1999Go; Turley et al., 2001Go).

Another event that promotes migration is shedding of CD44 from the cell surface, presumably allowing the cell to deadhere from hyaluronan during movement. CD44 shedding is mediated by a membrane-associated protease (Okamoto et al., 1999Go), most likely membrane-type 1-matrix metalloproteinase (Kajita et al., 2001Go). As a result of cleavage, the cytoplasmic region of CD44 is targeted to the nucleus, where it stimulates transcription of several genes, including CD44 itself (Okamoto et al., 2001Go). In accord with the studies of signaling discussed previously, cleavage of CD44 is regulated by Ras and Rho family signaling and requires PI3-kinase activity (Kawano et al., 2000Go). A crucial event in metastasis is invasion of extracellular matrices, a process that involves not only migratory activity but also remodeling of the matrix. Soluble CD44 (Yu et al., 1997Go; Yu and Stamenkovic, 1999Go; Peterson et al., 2000Go), antibody to CD44 (Koochekpour et al., 1995Go), and hyaluronan oligomers (Ward et al., unpublished data) inhibit tumor cell invasion of extracellular matrix, implicating hyaluronan in this process. An underlying mechanism, demonstrated in mammary carcinoma cells, is hyaluronan-mediated clustering of CD44 that leads to docking of matrix metalloproteinase-9 (gelatinase B) and, consequently, increased invasiveness (Yu and Stamenkovic, 1999Go). Docking of gelatinase B to CD44 also mediates processing of TGF-beta and stimulation of angiogenesis (Yu and Stamenkovic, 2000Go).

In conclusion, it is apparent that hyaluronan contributes in many ways to hallmark properties of the malignant phenotype, especially anchorage-independent growth and invasiveness. However, numerous questions remain unanswered. What is the basis for the different effects of various sized preparations of hyaluronan? Why do hyaluronan oligomers stimulate angiogenesis but inhibit tumor growth? What is the significance of active hyaluronan internalization by tumor cells? What are the interrelations between hyaluronan-induced signaling and integrin or growth factor signaling with respect to tumor progression? Are the Has genes oncogenes? Is hyaluronan involved in tumor initiation as well as progression? In what ways can manipulation of hyaluronan-induced events be applied therapeutically?

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