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Elevated Hyaluronan Concentration without Hyaluronidase Activation in Malignant Epithelial Ovarian Tumors¹

Departments of Pathology and Forensic Medicine [E. L. J. H., M. A., K. R., V-M. K.], Anatomy [A. K., R. T., M. T.], and Gynecology and Obstetrics [M. A., J. P., M. Y.], University of Kuopio and Kuopio University Hospital, 70211 Kuopio, Finland, and Departments of Gynecology and Obstetrics [A. T. K.] and Pathology [M. J.], Jyväskylä Central Hospital, 40620 Jyväskylä, Finland

	ABSTRACT

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The concentration and histological distribution of hyaluronan, a tumor promoting extracellular matrix polysaccharide, and the activity of hyaluronidase, a potential source of angiogenic hyaluronan oligosaccharides, were analyzed in malignant epithelial (n = 24), borderline (n = 8), benign epithelial (n = 20), functional cyst (n = 21), and normal (n = 5) tissue samples of human ovary. Hyaluronan concentration increased specifically in cancers (P = 0.001), particularly in grade 3 tumors (>49-fold) and in metastases (>89-fold). Hyaluronan staining in the tissues correlated with hyaluronan concentration (P = 0.002). Hyaluronidase activity slightly decreased from semimalignant through low grade to high grade tumors (P = 0.041). Therefore, hyaluronan accumulation, but not hyaluronidase activation, is associated with the aggressiveness of ovarian epithelial cancer.

	Introduction

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Ovarian cancer is one of the most common causes of cancer-related deaths in women. Approximately 90% of all ovarian cancers are epithelial and of these 80% represent the serous histological subtype. Because early-stage ovarian cancer produces no symptoms, the majority of patients continue to present with advanced disease and poor prognosis.

Hyaluronan, an extracellular matrix polysaccharide expressed in connective, neural, and epithelial tissues, is required for a number of homeostatic processes, including morphogenesis and wound healing (1) . Increased hyaluronan concentrations are found in several human cancers (2 , 3) and may contribute to tumor growth and invasiveness by providing an expanded, loose matrix for cancer cells, protecting the tumor from immune reactions, stimulating migration, and increasing cell proliferation at a point where cells enter into the S phase of the cell cycle (4, 5, 6) .

Hyaluronidases, a group of hyaluronan-cleaving endoenzymes, produce low molecular weight hyaluronan fragments that may enhance tumor growth by stimulating angiogenesis and malignant neovascularization (7) . Increased hyaluronidase expression has been reported in colon (8) , prostate (9) , and bladder cancers (10) , as well as in breast tumor metastases (11) .

There are no previous biochemical assays on hyaluronan and hyaluronidase levels in ovarian tissue, but histological analysis of epithelial ovarian cancers has suggested accumulation of hyaluronan in the cancer stroma and in some tumor cells (12) . In this study, we analyzed hyaluronan concentration and hyaluronidase activity by sensitive ELISA-like assays in the extracts of normal ovary, various epithelial ovarian tumors, and ovarian cancer metastases and correlated their levels with the stage, grade, and histological subtype of the malignant tumors. The localization and intensity of hyaluronan was concomitantly analyzed by a biotinylated hyaluronan-specific probe.

	Materials and Methods

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Patients.
This study consisted of ovarian tissue specimens (n = 87) from 78 patients, divided into 5 groups: malignant epithelial (n = 24); borderline (n = 8); benign epithelial (n = 20); functional cysts (n = 21); and normal ovary (from patients operated for uterine diseases; n = 5). Metastasis specimens were obtained from 9 patients. Malignant tumors were also divided into different histological subtypes (serous: n = 11; mucinous: n = 3; clear cell: n = 2; endometrioid: n = 5; undifferentiated: n = 3). Ovarian cancers were staged according to FIGO (Ref. 13 ; Table 1 ). The mean age of the patients was 57 ± 18 years (range, 33–80 years). The ethical committee of the Kuopio University hospital approved the study protocol.

Tissue Samples.
Each tissue sample was divided into two parts: the first was fixed in 10% buffered formalin and embedded in paraffin, and the second (0.5–2 g) was snap frozen and stored at -70°C. The frozen part was later homogenized in 1 mM sodium EDTA containing 1 mM benzamidine-HCl, 1 mM saccharic acid-1,4-lactone, 1 mM ß-mercaptoethanol, 1 mM iodoacetate, and 0.5% Triton X-100. The homogenates were clarified by centrifugation at 4°C (1,000 x g for 15 min and 10,000 x g for 30 min). The extracts were stored at -70°C until assayed.

ELISA-like Assay for Hyaluronan.
A sandwich-type ELISA-like assay (15) was modified to determine the concentration of hyaluronan. The hyaluronan-specific probe, HABC,³ was prepared from bovine articular cartilage as described previously (16) .

Maxisorp 96-well plates (Nunc, Roskilde, Denmark) were coated with 2 µg/ml HABC in 50 mM sodium carbonate buffer (pH 9.5). After incubation overnight at 4°C, the plates were washed with 0.5% Tween-PBS and blocked with 1% BSA-PBS. Standard hyaluronan (1–50 ng/ml, ProVisc; Alcon, Fort Worth, TX) and tissue extracts in 1% BSA-PBS were applied to the plates and incubated for 1 h at 37°C. The plates were washed with 0.5% Tween-PBS and incubated with 1 µg/ml bHABC for 1 h at 37°C and washed again. Horseradish peroxidase-streptavidin (Vector Laboratories, Inc., Burlingame, CA) diluted 1:20,000 with PBS was added to the wells, incubated at 37°C for 1 h, and washed with 0.5% Tween-PBS. The substrate solution was prepared just before use by dissolving a 30 mg o-phenylenediamine dihydrochloride tablet (Sigma, St. Louis, MO) in 30 ml of 0.1 M citrate-phosphate buffer (pH 5.0) and adding 30 µl of 30% H₂O₂. The plates were incubated with the substrate at 37°C until sufficient color developed (15 min), and the reaction was stopped with 8 N H₂SO₄. The absorbances were read at 490 nm, and a linear standard curve was used to calculate hyaluronan concentrations normalized to the content of extracted protein (17) and tissue wet weight. The coefficient of variation for repeated assays on the same homogenates was 10.7% and that for replicates 5.2%.

Preparation of Biotinylated Hyaluronan.
Biotin was cross-linked to hyaluronan as previously described (18) , with some modifications. Two g of hyaluronan solution (0.05 mM disaccharide units, ProVisc; Alcon) were diluted into 20 ml with distilled water by mixing at room temperature for 2 h and at 4°C for 48 h. After the addition of a 40-fold M excess of adipid dihydrazide (Sigma), pH was adjusted to 4.75 with HCl and agitated with 0.05 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma) for 1 h at room temperature. Then pH was adjusted to 5.5 with HCl/NaOH, the mixture extensively dialyzed against water at 4°C, and 720 µl of 1 M NaHCO₃ were added. Two ml portions of the mixture were stirred with 25 mg of N-hydroxysulfosuccimimido biotin (Pierce, Rockford, IL) overnight at room temperature, diluted with 20 ml of distilled water, extensively dialyzed against distilled water at 4°C, and stored at -20°C.

Plate Coating with Hyaluronan.
The activity of hyaluronidase in tissue extracts was determined according to the principle described previously (19) . Covalink 96-well plates (Nunc) were coated with 100 µl of biotinylated hyaluronan (0.5 µg/ml) and 50 µl of EDC (5.8 mg/ml; Sigma) in 0.1 M 4-morpholinepropanesulfonic acid buffer (pH 6.0) for 2 h at room temperature. The plates with the cross-linked hyaluronan coat were washed in 0.05% Tween-PBS, then in 4 M GuCl in 0.5% Triton-X-100, 100 mM acetate (pH 6.0), with 0.05% Tween-PBS, and finally in PBS, followed by blocking in 1% BSA-PBS for 1 h at 37°C.

Hyaluronidase Assay.
Aliquots of the tissue extracts and 0.001–10 units of hyaluronidase standards [Bovine Testes, type IV-S, H-3884 (pH 6.0); Sigma] were diluted in the incubation buffers [0.1 M Na-acetate (pH 6.0) for standards and 0.2 M NaCl in 0.1 M formate (pH 3.7 and pH 7.0) for tissue extracts] and kept in the hyaluronan coated wells at 37°C for 2 h. The standards contained the same concentrations of protease inhibitors as the samples. The wells were washed with 0.05% Tween-PBS, and the biotinylated hyaluronan remaining in the wells was quantitated using the avidin-biotin detection system as above. The hyaluronidase activity (mU) of each tissue extract was calculated by using a logarithmic standard curve, and the results were normalized to protein concentration as above. The coefficient of variation for repeated assays on the same homogenates was 11.0% and that for intra-assays 5.9%.

Staining of Hyaluronan.
Deparaffinized 5-µm sections were rehydrated, washed with 0.1 M sodium PB (pH 7.4), treated with 1% hydrogen peroxide for 5 min to inactivate peroxidases, and blocked with 1% BSA in PB. The sections were incubated in bHABC (2.5 µg/ml, diluted in 1% BSA) overnight at 4°C, washed with PB, and treated with avidin-biotin-peroxidase (ABC Vectastain Elite kit; Vector Laboratories). The sections were washed with PB, and the color was developed with 0.05% diaminobenzidine tetrahydrochloride (Sigma) and 0.03% hydrogen peroxide in PB. The slides were counterstained with Mayer’s hematoxylin. Staining specificity was controlled by predigesting some of the sections with Streptomyces hyaluronidase in the presence of protease inhibitors before staining or by preincubating the bHABC probe with hyaluronan oligosaccharides (16) .

All samples were analyzed by two observers (E. L. J. H., V-M. K.) unaware of the clinical data. The hyaluronan score consisted of the intensity and fraction of hyaluronan positivity (stroma and epithelial cells) in the whole tumor section according to our previous study (12) .

Statistical Methods.
Hyaluronan concentrations and hyaluronidase activities were measured in triplicates. Because the levels of hyaluronan and hyaluronidase did not always conform to normal distribution, nonparametric tests were used. Comparisons between the patient groups were made with the Kruskal-Wallis test. The difference between a primary tumor and its metastasis was analyzed using the Wilcoxon’s signed ranks test. Correlation between hyaluronan concentrations and hyaluronidase activities was done with the Pearson correlation test.

	Results

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Hyaluronan Concentration.
Hyaluronan concentration in normal ovarian tissue was 1.6 ng/mg (median), and comparable values were detected in specimens taken from benign epithelial tumors (2.7 ng/mg), functional cysts (3.1 ng/mg), and borderline tumors (4.0 ng/mg). In contrast, half of the malignant epithelial tumors contained high amounts of hyaluronan, leading to a median hyaluronan content of 13.7 ng/mg in this group (Fig. 1) . The median hyaluronan concentration in the malignant tissues was thus 2–7 times higher than in the borderline, benign, and normal tissues (P = 0.001; Kruskal-Wallis test). The concentration of hyaluronan did not significantly differ between the FIGO stages I–IV (P = 0.65). The different histological subtypes of the malignant tumors showed no statistically significant difference between each other (P = 0.064), although there was a tendency that undifferentiated tumors had elevated, whereas those of the mucinous type had decreased hyaluronan levels. However, the grade of the malignant tumor showed a strong correlation with hyaluronan concentration. The median hyaluronan level in grade 3 tumors was 196.6 ng/mg, a value significantly higher than 5.3 ng/mg in grade 2 and 3.6 ng/mg in grade 1 tumors (P = 0.009). The median hyaluronan level in the metastases was even higher, 355.4 ng/mg. In six of the nine metastases, hyaluronan concentration was higher than in the primary tumor, although there was no statistically significant correlation between hyaluronan concentrations in the primary tumors and metastases.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Hyaluronan concentration and hyaluronidase activity in ovarian tissue extracts. Hyaluronan levels and hyaluronidase activities were measured as described in "Materials and Methods" and normalized per mg soluble protein in the extract. The scatter diagrams indicate the values of the individual samples. The median of each group is shown as a horizontal bar. Within the malignant group, grades 1, 2, and 3 tumors are shown , , and , respectively.

Hyaluronan Staining.
The intensity of the hyaluronan signal was higher in the intra- and peritumoral stroma of malignant tumors as compared with normal ovarian tissue and all other tissue lesions (P < 0.000; Fig. 2 ). Epithelial cells of the normal ovary were hyaluronan negative, but hyaluronan positive cancer cells were found in 48% of the malignant tumor tissues. The histochemical score of hyaluronan staining correlated with hyaluronan concentrations measured by the ELISA-like assay (n = 78, r = 0.3, P = 0.002; Pearson correlation test).

View larger version (127K): [in this window] [in a new window]   Fig. 2. Histochemical staining for hyaluronan in ovarian tissues. The sections were processed as described in "Materials and Methods." An example of the biological heterogeneity among the tumors is demonstrated by the serous cancers in a and b, with a low stromal hyaluronan intensity (arrow) and hyaluronan-positive cells (arrowhead) in a, and high stromal hyaluronan staining (arrow) but completely hyaluronan-negative carcinoma cells (arrowhead) in b (bar, 20 µm). A serous borderline tumor is shown in c (bar, 20 µm); a benign serous tumor in d (bar, 50 µm); a simple cyst in e (bar, 50 µm); and a normal ovary in f (bar, 50 µm).

	Discussion

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The high concentration of hyaluronan probably involves up-regulation of the hyaluronan synthase activity in the tumor, most likely in the stromal cells, but this remains to be confirmed. Changes in the clearance of hyaluronan may also be involved such as reduced cellular uptake by CD44 for lysosomal degradation and enhanced trapping in the matrix by hyaluronan binding molecules like versican. Our unpublished data indicate that the expression of CD44 is reduced in high-grade ovarian cancers, but the lack of inverse correlation between the levels of stromal hyaluronan and CD44 on the malignant cells suggests that CD44 down-regulation does not explain hyaluronan accumulation. Our unpublished data also suggest that many of the hyaluronan-rich ovarian tumors show an elevated immunohistochemical signal for versican, probably another member in the set of tumor promoting matrix molecules (20) .

The hyaluronidase activity was higher in the borderline and benign epithelial ovarian tumors, as compared with controls and functional cysts, whereas it remained low in the malignant epithelial tumors. Furthermore, within the group of malignant tumors, there was a tendency (Fig. 1) to lower hyaluronidase activity in grades 2–3 tumors than in grade 1, suggesting that a malignant transformation in the ovarian epithelium may associate with an unchanged or even reduced hyaluronidase activity. These findings are in contrast with those in the breast (11) , bladder (10) , and prostate (9) cancer, showing elevated hyaluronidase activity, and with the increased hyaluronidase activity found in the higher grades of those tumors (9 , 10) . In addition, hyaluronidase activity was higher in the metastases of the breast (11) and prostate (9) tumors, as compared with the primary tumor, whereas in the present ovarian tumor material, no obvious difference was noted between the primary tumor and its metastasis. A higher level of hyaluronidase, through the production hyaluronan oligosaccharides and their angiogenic activity, was suggested to contribute to the progression of prostate (3) and bladder (10) tumors. The present results suggest that such mechanisms are not likely to operate in the majority of epithelial ovarian cancers.

Because a high concentration of hyaluronan is associated with the progression of many malignant epithelial tumors, including those of the ovary, one could expect that hyaluronidases inhibit tumor growth and that a deprivation of hyaluronidase activity enhances hyaluronan accumulation and tumor spreading (21) . Indeed, HYAL1 gene maps within a candidate tumor suppressor gene locus (22) , and loss of one allele occurs in many oral, head and neck, and lung carcinomas, with mutations and other inactivation mechanisms described for the remaining allele (21) . Our finding presented here warrants additional studies on the possible tumor suppressor gene activity of hyaluronidases in epithelial ovarian cancer.

However, the issue remains quite complicated because the present and previous assays on tissue hyaluronidases (3 , 10) show little activity above pH 4, suggesting that hyaluronidases in both normal and malignant ovarian tissues are mostly functional in the lysosomes and unable to degrade extracellular hyaluronan. Nevertheless, it was recently shown that ectopic overexpression of HYAL2 in Xenopus depletes tissue hyaluronan (23) , perhaps, in part, by its contribution to the cellular uptake process (21) . Considering the role of hyaluronidases in cancer, it is important to note that the biological response to hyaluronan fragments is specific to each cell type and that the size distribution of the fragments determines the signals. Furthermore, the hyaluronidase proteins may have functions in malignant transformation other than the hyaluronan lyase activity assayed in the present work (21 , 24) .

In conclusion, epithelial ovarian cancers, unlike benign tumors, cysts, and normal ovary, have elevated hyaluronan concentrations, which are proportional to the histological grade of the tumor. In contrast, hyaluronidase activity is low in most of the malignant tumors as compared with the benign and borderline lesions, and the lowest values are found in the high-grade (2, 3) tumors. The present data confirm the adverse role of hyaluronan accumulation and show that epithelial ovarian cancers, unlike some other malignancies, do not show a concurrent increase of hyaluronan lyase activity with tumor progression. In fact, additional studies are warranted to check whether a loss of a hyaluronidase promotes these malignancies.

	ACKNOWLEDGMENTS

 
We thank Dr. Paraskevi Heldin (University of Uppsala) for help in the development of the hyaluronan assay.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by grants from the Finnish Cancer Foundation (to R. T., V-M. K.), Foundation of Finnish Cancer Institutes (to R. T.), EVO Funds of Kuopio University Hospital (to M. A., M. T., V-M. K.), and Academy of Finland Grant Nos. 40807 and 54062 (to M. T.).

² To whom requests for reprints should be addressed, at Department of Pathology and Forensic Medicine, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland. Phone: 358-17-162740; Fax 358-17-162753.

³ The abbreviations used are: HABC, hyaluronan binding link protein complex; bHABC, biotinylated HABC; FIGO, International Federation of Gynecologists and Obstetrics.

Received 6/10/02. Accepted 9/26/02.

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