Role of Versican, Hyaluronan and CD44 in Ovarian Cancer Metastasis

There is increasing evidence to suggest that extracellular matrix (ECM) components play an active role in tumor progression and are an important determinant for the growth and progression of solid tumors. Tumor cells interfere with the normal programming of ECM biosynthesis and can extensively modify the structure and composition of the matrix. In ovarian cancer alterations in the extracellular environment are critical for tumor initiation and progression and intra-peritoneal dissemination. ECM molecules including versican and hyaluronan (HA) which interacts with the HA receptor, CD44, have been shown to play critical roles in ovarian cancer metastasis. This review focuses on versican, HA, and CD44 and their potential as therapeutic targets for ovarian cancer.


Introduction
Ovarian cancer is the most lethal gynecological cancer and ranks as the fifth most common cause of cancer-related death in women in the Western world [1,2]. Over 70% of women present with advanced disease and despite aggressive treatment, the 5 year survival rate of patients with ovarian carcinoma is only 30-50%. This poor prognosis results from late diagnosis and ineffective therapy for advanced stage disease. A major challenge for ovarian cancer research remains the identification of tumor markers to aid in early diagnosis and the identification of novel therapeutic strategies for advanced disease. Significant improvements in ovarian cancer survival will require the development of more effective molecularly targeted diagnostics and/or therapeutics.

Ovarian Cancer Metastasis
Ovarian carcinomas are believed to spread by detaching from the surface of the ovary and attaching to and invading the peritoneum which lines the organs of the abdominal cavity. Involvement of the peritoneum predicates an adverse situation for the patient which impacts significantly on prognosis. This is evidenced by the fact that stage I patients have 5 and 10 year survival rates of over 90%, whereas patients with stages III disease have a 5 year survival of only about 30% [3,4]. Ovarian cancer cells are anatomically placed to metastasize into the peritoneal cavity. However it is not clear whether the anatomical location is solely responsible for intra-peritoneal spread of ovarian carcinomas or whether other factors may foster the implantation of cancer cells onto the peritoneal lining. Peritoneal cells are known to express and secrete several extracellular matrix (ECM) proteins, adhesion molecules and many other factors that provide an important microenvironment to aid the implantation of ovarian cancer cells [5][6][7][8][9][10][11]. Alterations in the extracellular microenvironment surrounding ovarian cancer cells are therefore believed to be critical for tumor initiation, progression and peritoneal tumor spread. Indeed, it has been demonstrated that peritoneal cells produce both extracellular matrix molecule, hyaluronan (HA) and the HA receptor, CD44 [12][13][14], and can promote ovarian cancer motility by increasing their HA production in co-culture with ovarian cancer cells [14].

Role of the Extracellular Matrix in Ovarian Cancer Metastasis
The extracellular matrix (ECM) is a highly organized three-dimensional structure with many physiological and pathological roles. The ECM, in addition to maintaining tissue integrity, also regulates cell migration, cellular differentiation, and proliferation and also provides a reservoir of cytokines and growth factors. Tumor cells interfere with the normal programming of ECM biosynthesis and can extensively modify the structure and composition of the matrix [15]. There is increasing evidence to suggest that ECM components play an active role in tumor progression and are important determinants for the growth and progression of solid tumors [16]. The extensive remodeling of the normal ECM in tumors can proceed through the degradation of pre-existing ECM molecules and/or by the synthesis of ECM components, which in many cases are not present in the ECM of normal tissues. Altered expression of several ECM molecules including versican, hyaluronan (HA) and CD44 have been described in ovarian cancer and impact on ovarian cancer outcome [17]. Table 1 summarizes the studies which have investigated the expression of HA, the HA degrading enzymes hyaluronidases, CD44 and versican in ovarian cancer progression and their functional role on ovarian cancer cells.  [42] CD44-HA interactions promotes cell migration via HER2 activation and beta-catenin nuclear translocation [43] Heregulin mediated ErbB2-ERK signaling activates HAS and CD44 dependent ovarian tumor growth and migration [44] Co-expression of CD44 and multiple drug resistance proteins MDR1 and MDP2 correlates with ovarian cancer progression [45]

Role of HA in Ovarian Cancer
HA is a large polymer which extrudes into the extracellular space and is made up of repeating disaccharides of N-acetyl glucosamine and D-glucuronic acid with a varying molecular weight and size depending on the tissue in which it is found [51]. HA is synthesized at the surface of cells by one of three HA synthases (HAS 1, 2, or 3) and plays a role in various cellular functions including adhesion, motility and differentiation [52]. The physiological function of HA varies greatly depending on HA size, and the presence or absence of HA binding proteins and cell surface receptors [53,54] Many tumors are enriched in HA [53,55]. HA levels can be increased within the tumor cells themselves or within the tumoral stroma [20,56]. Increased HA has been shown to be closely correlated with the degree of invasiveness and metastatic potential in ovarian cancer tumor models [19,22]. Accumulation of high levels of HA have been found to be associated with reduced levels of the hyaluronidase HYAL1 [28]. High levels of stromal HA are associated with a poorer prognosis in patients with breast cancer, non-small-cell lung cancer, colon cancer, prostate cancer and ovarian cancer [20, [55][56][57][58][59][60]. In ovarian cancer patients, high HAS1 levels in ovarian cancer cells but not HAS2 or HAS3 are associated with reduced overall survival [24]. This is consistent with other reports describing high levels of stromal HA and HAS1 to be independent predictors of overall survival [20,24]. Our recent studies have confirmed the presence of high HA levels in the peritumoral stroma of serous ovarian carcinomas which correlate with CD44 and versican expression ( Figure 1).

CD44-A Key Receptor for HA
The CD44 glycoprotein is an acidic molecule whose charge is largely determined by sialic acid. CD44 is a multi-structural cell surface receptor which binds HA with a particularly high affinity [61,62]. It belongs to a family of transmembrane glycoproteins which contain a variable extracellular domain, a 23-amino acid transmembrane domain, and a 70-amino acid cytoplasmic domain [63]. Up to ten different CD44 isoforms have been documented due to differential splicing of the 10 variant exons [64,65]. The most common isoform is the standard CD44 (CD44s), in which exon 5 is spliced directly to exon 16 resulting in an approximately 85 kDa glycoprotein.

The Role of CD44 and Its Interactions with HA in Cancer
The binding of HA to CD44 triggers direct cross-signaling between different signaling pathways including HER2, c-src kinase and ERK [21,33,37,43,44,66] and it is this function which is thought to be involved in increased motility, adhesion, and invasion of cancer cells as well as tumor growth, Differences between the studies could be attributed to technical factors like the use of different antibodies and different detection methods. Furthermore, the cohorts of ovarian cancer patients examined in the various studies were highly heterogeneous and composed of patients with variable tumor types, stages and treatment regimes. Our recent studies have confirmed the presence of high CD44 immunostaining particularly in the peritumoral stroma of serous ovarian carcinomas which was associated with high HA and versican expression ( Figure 1).
In ovarian cancer, versican has been shown to be present in both cancer cells and the peritumoral stroma and increased expression relates with tumor progression and poorer survival outcome [46,49,88]. High expression of versican in peritumoral stroma was associated with advanced stage, large residual disease, serous histological type, and reduced 5-year survival [46]. Increased expression of versican was also identified as a key protein involved in ovarian cancer metastasis [48]. Elevated levels of versican have been observed in primary ovarian tumors and secondary metastases when compared with normal ovaries [47]. Immunohistochemistry studies in our laboratory have confirmed that versican is increased in the stromal compartment of serous ovarian carcinoma compared to the stroma of normal ovaries or benign ovarian tumors ( Figure 1).

Role of Versican and HA in Promoting Cancer Cell Motility and Invasion
HA becomes deposited in the tissue spaces immediately surrounding invasive tumors and together with other ECM components, forms a protective pericellular coat around the cells [89]. The assembly of pericellular matrix rich in HA and versican is a prerequisite for proliferation and migration of mesenchymal cells [90] and has recently been shown to promote the motility of prostate cancer cells [91]. Our most recent data also shows that motile SKOV-3 ovarian cancer cells expressing CD44 also form a HA/versican pericellular matrix [26] ( Figure 2). Furthermore, we demonstrated that versican treatment can induce ovarian cancer cell invasion through an ECM barrier [26]. These findings are also supported by a recent study demonstrating that versican-treated ovarian cancer cells have increased invasive potential [88]. Our results suggest that formation of a CD44/HA/versican macromolecular complex promotes the motility and invasion of ovarian cancer cells. The dependence of CD44 for the versican and HA-mediated effects was confirmed by the inhibition of pericellular matrix formation as well as versican mediated motility and invasion of ovarian cancer cells following treatment with CD44 neutralizing antibody [26].

Involvement of HA and CD44 in the Adhesion of Ovarian Cancer Cells to Peritoneal Cells
Ovarian cancer cell adhesion to mesothelial cell monolayers is mediated at least in part by the interaction between HA and CD44 [6,92]. It has also been suggested that ovarian cancer cell interactions with mesothelial cell HA may also mediate tumour metastasis [93]. The addition of anti-CD44 antibodies has been reported to significantly decrease adhesion of ovarian cancer cells to HA [6]. In vivo studies have suggested that CD44s is required for human ovarian cancer cell adhesion to mesothelial cell surface HA [12,29]. Our own in vitro studies have shown that HA increases the adhesion of CD44 expressing ovarian cancer cells to peritoneal cells [26].
Overall the combined data supports a role for versican together with HA and CD44 in a number of the key steps needed for ovarian cancer metastasis. Our working model proposes that versican from the peritumoral stroma binds HA in the ECM (Figure 3). The formation of a stabilized HA/versican pericellular matrix surrounding ovarian cancer cells, protects the ovarian cancer cells against the mechanical forces in the peritoneal cavity and enables strong ovarian cancer cell adhesion to CD44 expressed by peritoneal cells. This provides the basis for subsequent ovarian cancer dissemination throughout the abdominal cavity.

HA Therapies
Because of the ability to bind cell surface CD44, which is upregulated in many cancer types, HA has been conjugated or co-admininistered with traditional chemotherapy drugs to increase the direct targeting of the drug to the cancer cells. More pronounced cytotoxic effects were observed when HA was conjugated to paclitaxel on CD44-overexpressing breast cancer cells compared to CD44-deficient cells, suggesting that HA-conjugation can be potentially utilized as tumor-targeted therapy [94]. Conjugated HA-cisplatin has also been shown to be effective in targeting breast cancer cells in an orthotopic mouse model [95].
In ovarian cancer cells, paclitaxel-HA conjugate interacted with CD44, entered the cells through a receptor-mediated mechanism, and exerted a concentration-dependent inhibitory effect on tumor cell growth [96]. Furthermore, after intra-peritoneal administration in mice HA bioconjugate distributed uniformly within the peritoneal cavity, was well tolerated, and not associated with local toxicity [96]. Blood levels of paclitaxel in mice treated with HA-conjugated paclitaxel were also much higher and persisted longer than those obtained with the unconjugated paclitaxel [96]. Paclitaxel-HA resulted in a 2.5-fold increase in therapeutic activity when compared with paclitaxel alone [96] and decreased tumor burden in ovarian cancer xenograft mice [97].
HA has also been investigated as a potential conjugate with other types of cancer therapies besides chemotherapy drugs. When polyethylenimine (PEI) DNA particles are conjugated with HA they showed more specific targeting of breast cancer cells with high CD44 expression [98]. A siRNA/PEI-HA complex exhibited higher CD44 gene silencing efficiency in melanoma cells compared to siRNA/PEI complex alone [99] Similarly, liposomes-protamine-HA nanoparticles used for systemic delivery of siRNA into melanoma cells had a broader effective therapeutic dose range than nanoparticles without HA [100].
Methods of eliminating HA in tumors using hyaluronidase enzymes to degrade the HA have also been investigated. Pilot clinical studies with a bovine hyaluronidase preparation examined whether this enzyme could increase anticancer drug action in combination therapies [101]. However, despite early signs of clinical activity, systemic administration of the bovine hyaluronidase elicited allergic reactions which, combined with a poor plasma half-life, limited clinical efficacy to local-regional chemotherapy in bladder carcinoma [102]. A pegylated variant of human hyaluronidase PH20, PEGPH20 with an increased half life has recently been evaluated in prostate cancer xenograft models [103]. In PC3 prostate tumors, i.v. administration of PEGPH20 depleted tumor HA and significantly inhibited tumor growth. PEGPH20 treatment also enhanced both docetaxel and liposomal doxorubicin activity in PC3 tumors [103]. More recent studies have also demonstrated that mice bearing tumors treated with an oncolytic adenovirus expressing PH20 showed more anti tumor efficacy than mice treated with the parental virus alone [104]. These results suggest that PH20 may represent a potentially innovative treatment approach against tumors which produce high levels of HA which includes the majority of ovarian cancers. The company Halozyme has recently commenced a Phase 1 clinical trial which will evaluate a range of doses of PEGPH20 in advanced cancer patients [105].
Thus, HA presents a novel and promising candidate for increasing efficacy and reducing toxicity of cancer therapies. HA conjugates also have a potential role in the development of more effective intra-peritoneal treatment regimes in ovarian cancer [96].

CD44 Therapies
CD44 is also promising target against cancer. Methods which have been used to block the action of CD44 include, neutralizing antibodies, siRNA, antisense RNA, cDNA vaccination as well as the using the naturally occurring CD44-inhibitor silibinin. Silibinin, a natural drug extracted from common thistles, which inhibits CD44 promoter activity and expression, is known to have anti-tumor properties in breast cancer [106], non small cell lung cancer [107], and colorectal carcinoma [108]. It has also been shown to decrease motility and invasion in prostate cancer cells and to decrease adhesion of prostate cancer cells to HA and fibronectin [109]. CD44 cDNA vaccination has been shown to decrease tumor mass and metastatic potential in experimental mammary tumors in mice [110]. In addition, transfection of CD44 antisense RNA into a highly metastatic mammary tumor cell line disrupted expression of CD44 in the tumor cells and reduced their ability to establish local tumors as well as metastatic colonies in the lung [110]. Treatment of gemcitabine resistant pancreatic cancer cells with CD44 siRNA inhibited their proliferative activity [111]. Furthermore, in vivo targeting of CD44 by siRNA resulted in anti-tumor activity in both colon cancer and leukemia xenografts [112,113]. In hepatocellular carcinomas, CD44 antisense oligonucleotide significantly down-regulated CD44 expression, induced apoptosis, decreased tumorigenesis and invasion, and increased the cancer cell sensitivity to chemotherapy drugs [114]. In ovarian cancer cells, in vitro adhesion, invasion, and resistance to apoptosis were inhibited by CD44 siRNA [115]. Tumor growth and peritoneal dissemination of human ovarian cancer xenografts in nude mice were also decreased.
Another very promising therapeutic approach is the use of anti-CD44 antibodies such as IM7 which resulted in a greater than 50% decrease of HA production in glioma cells and enhanced glioma apoptosis [116]. Treatment of chondrosarcoma cells with IM7 resulted in a significant decrease in cell viability but did not reduce cell viability in normal human chondrocytes [117]. Another antibody against CD44, called P245, inhibited growth of breast cancer xenograft in mice [118]. Affify et al. showed that anti-CD44s could inhibit breast cancer cell adhesion, motility and invasion, whilst anti-CD44v6 inhibited cell motility [119]. Furthermore, Guo et al. showed that treatment with an anti-CD44 monoclonal antibodies inhibited the formation of melanoma metastases [120] and decreased the invasive ability of breast cancer cells [121]. Treatment of prostate cancer cells with a neutralising CD44 antibody decreased cancer cell adhesion to human bone endothelial cells, a primary site for prostate cancer cell metastasis [122]. In ovarian cancer, Casey et al. showed that their CD44 monoclonal antibody did not inhibit ovarian cancer cell invasion but inhibited motility [93], Furthermore, Strobel et al. showed a decrease in the number of total peritoneal ovarian cancer metastases in mice when treated with a anti-CD44 antibody [32]. However, despite promising in vitro studies, clinical trials with CD44 neutralizing antibodies have been terminated due to unacceptable levels of toxicity [123]. However, with numerous studies finding that disruption of the HA-CD44 interaction decreases the proliferative and metastatic behavior of tumor cells, CD44 still remains a valid target for anti-cancer therapy. Non toxic alternative therapies need to be developed for future trials.

Versican Therapies
Versican levels in the tumor environment can be reduced by inhibition of its production. This has been achieved by treatment with genistein, a tyrosine kinase inhibitor which has been shown to inhibit versican synthesis in vascular smooth muscle cells (SMCs) and malignant mesothelioma cell lines [124,125]. Versican production could also be blocked by the leukotriene receptor antagonist, montelukast, in both bronchial and arterial SMCs [126]. No studies to date have yet investigated whether these inhibitors are effective in inhibiting the effects of versican in cancer models.
More recent studies have demonstrated that drugs used in the treatment of asthma including formoterol, a long acting β 2 -adrenergic agonist, and budesonide, a glucocorticoid steroid, could decrease protein levels of a number of proteoglycans including versican in airway SMCs and human lung fibroblasts [127,128]. Combination treatment with budesonide and formoterol was significantly superior in reducing versican levels than either drug alone [128]. These studies suggest that the changes in versican deposition which occur in cancer tissues, as well as the asthmatic airway and atherosclerotic lesions, could be inhibited by a number of asthma drugs. This, combined with their safe use in humans, makes them potential therapeutic agents against ovarian cancer.
There is increasing evidence that ADAMTS proteases play an important role in the cleavage of versican, and the local accumulation of versican fragments generated by ADAMTS digestion may also promote cancer cell motility and invasion. The broad spectrum MMP inhibitor, GM6001 (Galardin) which inhibits the activity of MMPs and ADAMTS proteases has been shown to inhibit cancer cell invasion and metastasis in several model systems [93,[129][130][131]. One of the more interesting MMP inhibitors is present in green tea, which is made from the leaves of Camellia sinensis and contains catechin gallate esters. Catechin gallate esters have been shown to selectively inhibit ADAMTS-1,-4, and -5 and inhibit aggrecan processing in cartilage tissue [132]. The ability of GM6001 and catechin gallate esters to inhibit versican cleavage and versican-induced motility and metastasis of ovarian cancer cells has not been investigated yet. Manipulation of the versican catabolic pathways may provide novel therapeutic targets against cancer invasion and metastasis.
The formation of a pericellular matrix rich in HA and versican by vascular SMCs and tumor cells can be inhibited following treatment with HA oligomers [26, 90,133]. Disruption of the HA-CD44 interaction with HA oligomers has been shown to markedly inhibit the growth of melanoma cells [134]. Additionally, both link-TSG6 and G1 domain of aggrecan were shown to bind to polymeric HA and these interactions could be competed with HA-8 and HA-10 oligomers, respectively [135], making it likely that HA oligomers will also block the interaction of versican with HA. Treatment with HA-8 oligomers inhibited the formation of pericellular matrix by osteosarcoma cells and reduced HA accumulation in local tumors, tumor growth, and the formation of distant lung metastases. Furthermore, in osteosarcoma cell lines, both motility and invasiveness were inhibited by the use of HA oligomers [136]. Additionally, colorectal carcinoma growth was reduced both in vitro and in vivo after treatment with HA oligomers [137]. Our own recent work has shown that HA oligomers can reduce adhesion of ovarian cancer cells to peritoneal cells, and block ovarian cancer motility and invasion induced by versican and HA treatment [26].
HA oligomers have also been shown to reverse chemotherapy resistance in some cancer cell lines [138,139]. Adriamycin resistance of leukemic cells was effectively reversed by HA-4 oligomers by increasing the intracellular accumulation of adriamycin [138], whilst in malignant peripheral nerve sheath tumors (MPNST), HA oligomers resulted in the disassembly of CD44-transporter complexes and induced internalization of CD44 [139]. Furthermore, in vivo systemic administration of HA oligomers inhibited the growth of MPNST and ovarian cancer xenografts [139,140]. These findings suggest that the potent anti-tumor effects of HA oligomers are due in part to the blocking of the formation of HA-rich cell-associated matrices. The use of HA oligomers is a potentially attractive reagent to block versican HA interactions as well as local tumor invasion in ovarian cancer but need further in vivo investigation.

Conclusions
Tumor cells interfere with the normal programming of ECM biosynthesis and can extensively modify the structure and composition of the matrix. The peritumoral stroma surrounding ovarian cancer cells is enriched in HA, versican and CD44 all of which can promote ovarian cancer metastatic behavior. These molecules hold promise as therapeutic targets in ovarian cancer and warrant further evaluation.