Hyaluronidase-2 Overexpression Accelerates Intracerebral but Not Subcutaneous Tumor Formation of Murine Astrocytoma Cells1

[CANCER RESEARCH 59, 6246–6250, December 15, 1999] Hyaluronidase-2 Overexpression Accelerates Intracerebral but not Subcutaneous Tumor Formation of Murine Astrocytoma Cells1

Ulrike Novak,2 Stanley S. Stylli, Andrew H. Kaye, and Guenter Lepperdinger Departments of Surgery [U. N., S. S. S., A. H. K.] and Neurosurgery [S. S. S., A. H. K.], The Royal Melbourne Hospital, University of Melbourne, Parkville, 3050 Victoria, Australia, and Institute of Molecular Biology, Austrian Academy of Sciences, A-5020 Salzburg, Austria [G. L.]

ABSTRACT tion products of hyaluronan with intermediate size have biological activity (21–23). In addition, they are good substrates for other hyal- Gliomas are highly invasive, invariably fatal intracerebral tumors. It uronidases that degrade these fragments further (19). On the other seems that receptors for hyaluronan are required for the invasive process. hand, small oligosaccharides have been described as having angio- Hyaluronan is a major component of the extracellular matrix in the brain, and all of the gliomas express CD44, the principal receptor for hyaluro- genic activity (24, 25). nan. To investigate the role of lysosomal hyaluronidases on tumor invasion The hyaluronidases HYAL1, HYAL2, and HYAL3 have been we overexpressed hyaluronidase-2 (HYAL2) in murine astrocytoma cells. cloned recently (19, 20, 26). They are related to the sperm-derived We found that high expression of HYAL2 accelerated intracerebral tumor hyaluronidase, PH-20 (or SPAM1), which is an enzyme bound to the growth dramatically, whereas the same cells formed s.c. tumors within the cell surface by a glycosylphosphatidylinositol (GPI)-anchor (27). The same time as the parental cells. The brain tumors were highly vascularized Mr 64,000 form of PH-20 has a neutral pH optimum for catalytic and more invasive than the control tumors. It seems that the interactions activity, whereas the Mr 53,000 form has an acidic pH optimum (28). of the HYAL2-expressing tumor cells with the hyaluronan-containing Although HYAL1 and HYAL2 are expressed ubiquitously, PH-20 is extracellular matrix in the brain mediate these effects, whereas the same expressed only in the testes and in some tumors (19, 29, 30). The cells in a s.c. environment, which lacks the high hyaluronan level, behave newly described HYAL3 is expressed predominantly in the liver and like the parental cells. the brain, but presently there is no information about the functional activity of HYAL3 (31). INTRODUCTION Our experiments were aimed at the elucidation of the role of High-grade gliomas are highly invasive brain tumors that are in- HYAL2 in glioma growth and invasion in cells that express high variably fatal. The factors controlling the invasive behavior of these levels of the hyaluronan receptor, CD44. The results indicate that tumors are poorly understood. There is, however, good evidence that HYAL2 very potently activates tumor growth and the invasion of interactions between hyaluronan, a highly polymerized glycosamin- intracerebral tumors but has no such activity in s.c. tumors formed by oglycan, which is abundantly present in the brain, and receptors of the same cells. hyaluronan play an important role in the invasiveness of the tumor (1–5). There are several hyaluronan-binding proteins in the extracellular matrix of the brain, including link protein and other members of MATERIALS AND METHODS 3 a family of related proteins such as BEHAB /brevican, neurocan, Cells. SMA560 cells, derived from a spontaneous murine astrocytoma, versican, the glial hyaluronate-binding protein (GHAP), and hyalu- kindly provided by Dr. D. Ashley (Royal Children’s Hospital, Melbourne, ronectin, both of the latter proteins derived by proteolytic degradation Australia), were cultured in DMEM, supplemented with 10% fetal bovine of versican (6–11). In addition there are several cell-associated hya- serum. To generate cell lines that express human HYAL2, the cells were luronan-binding proteins including: (a) CD44, the predominant hya- lipofected with a linearized HYAL2 expression vector, pCATCH (a gift from luronan receptor; (b) BEHAB/brevican; (c) a COOH-terminal frag- Dr. C. Hovens, University of Melbourne, Melbourne, Australia) using the ment of BEHAB/brevican; and (d) RHAMM/IHABP (12–16). These Lipofectamine Reagent (Life Technologies, Rockville, MD) together with cell-associated hyaluronan receptors have been reported to mediate pPGK-Puro (a gift from Dr. S. Cory, Walter and Eliza Hall Institute, Mel- cell migration in response to hyaluronan during fetal development and bourne, Australia). After selection with puromycin for 2 weeks, individual colonies were isolated and expanded. during tumor formation (3–5, 17). Tumorigenicity of Cells after Intracerebral or s.c. Injection. For in vivo Hyaluronan can be taken up by cells via CD44 interactions and studies, the cells were injected into adult (8–12-week-old) syngeneic VM/Dk transported into lysosomes, in which lysosomal hyaluronidases cause mice. Intracerebral inoculations of 105 cells in 10 ␮l of PBS were given its degradation (18, 19). HYAL2 degrades hyaluronan from Mr through the coronal suture approximately 2 mm from the midline with a ϳ ϳ 8,000,000 to Mr 20,000, or about 100 sugar moieties (19). The 27-gauge needle fitted with a plastic sleeve, which allowed penetration into the hyaluronidase, found in the serum HYAL1, similar to the sperm- frontal cortex to a depth of 3 mm. The mice were monitored daily, and, when derived hyaluronidase, PH-20/SPAM1, cleaves hyaluronan into small they showed neurological symptoms, they were monitored every 2 h. All of the oligosaccharides of two to three tandem repeats of D-glucuronic acid mice in one cohort were killed when they became moribund, which occurred and N-acetyl-glucosamine (20). There is some evidence that degrada- within 12 h of the first appearance of symptoms. The brains were excised, cut in half through the inoculation site, and embedded in Jung tissue-embedding medium (Leica Instruments, Nussloch, Germany). s.c. inoculations of 106 cells Received 6/7/99; accepted 10/19/99. in 100 ␮l of PBS were given in the flank of the mice. The s.c. tumors were 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 measured in three dimensions daily. The animals were killed as soon as the 18 U.S.C. Section 1734 solely to indicate this fact. tumors had a volume exceeding 1 cm3, in accordance with our local animal 1 This work was supported by grants from the Friends of the Royal Melbourne Hospital ethics regulations. The s.c. tumors were excised and frozen in Jung tissue- Neuroscience Foundation, the Clive and Vera Ramaciotti Foundation, and the Neurosur- embedding medium. Frozen sections were cut at a thickness of 7 ␮m and gical Research Foundation, Adelaide. 2 To whom requests for reprints should be addressed, at University of Melbourne, collected on 3-aminopropyltriethoxysilane-coated slides. The sections were Royal Melbourne Hospital, Department of Surgery, Parkville, 3050, Australia. Phone: then either stained with H&E or used for immunohistochemical analyses. 61-3-9342-7703; Fax: 61-3-9347-6488; E-mail: [email protected]. Northern Blot Analysis of mRNA. RNA was prepared from cells or 3 The abbreviations used are: BEHAB, brain-enriched hyaluronan-binding; tissues following standard procedures (32). Total RNA was separated on 1% HYAL1, hyaluronidase-1; HYAL2, hyaluronidase-2; HYAL3, hyaluronidase-3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PECAM, platelet endothelial agarose gels, blotted onto Hybond-N membranes (Amersham, Amersham, cell adhesion molecule. United Kingdom) and hybridized with 32P-labeled cDNA probe, generated by 6246

high levels (2s-2 and 2s-7) and one cell line that expresses low levels (2s-6) of HYAL2 mRNA were selected for all of the subsequent studies (Fig. 1). The cells had the same morphology as the parental SMA560 cells. However, unlike the parental cells, the HYAL2- expressing cells tended to form foci on the culture dish and easily lifted off the dish within 3 days of incubation, never forming confluent monolayers of cells (Fig. 2). All of the cell lines had the same doubling time of about 13 h when grown in the absence of hyaluronan, which increased slightly in the presence of 80 ␮g/ml hyaluronan (data not shown). Overexpression of HYAL2 Causes Accelerated Intracerebral Tumor Growth but Has No Effect on s.c. Tumor Growth. Inocu- lation of syngeneic VM/Dk mice intracerebrally with 105 SMA560 cells resulted in tumor formation within 18 days and gave a range of tumor sizes, with most mice harboring medium to large size tumors (Table 1). 2s-6 cells, which express low levels of HYAL2 mRNA, formed tumors faster, requiring only 13 days for 8 of 10 mice to develop large tumors. However, intracerebral inoculation of mice with 2s-2 or 2s-7 cells resulted in rapid tumor formation, giving 10 of 10 large tumors within 10 days using 2s-2 cells and 7 of 9 large tumors Fig. 1. HYAL2 mRNA expression in SMA560-derived cell lines. Total RNA from the in 9 days using 2s-7 cells (Fig. 3; Table 1). The differences in survival parental SMA560 cells and from three HYAL2-transfected clones was separated on an agarose gel, blotted, and probed with HYAL2 cDNA. After exposure to X-ray film, the time of the animals were highly significant. All of the tumors resulting blot was reprobed with GAPDH cDNA. from inoculation with HYAL2-expressing cells expressed CD44 similar to the parental SMA560 cells (Fig. 3 and data not shown). In random primer extension as described previously (32). After exposure to X-ray addition, the tumors derived from 2s-2 and 2s-7 cells were more film, the membranes were boiled in 1% SDS and were reprobed. invasive than the tumors resulting from the parental SMA560 cells Immunohistochemistry. Cells grown on glass chamber slides (Nunc, (Fig. 4). Roskilde, Denmark) and frozen tissue sections of 7 ␮m, were air-dried for 1 h, fixed in cold acetone for 10 min, and incubated with antibodies to murine s.c. inoculation of mice with the HYAL2-expressing and the pa- CD44 (a gift from Dr. R. Bischof, University of Melbourne, Melbourne, rental cell lines resulted in tumor formation as well, with the first Australia) and murine PECAM (PharMingen, San Diego, CA) following tumors being measurable within 7 days. However, the s.c. tumor standard procedures. After incubation with horseradish peroxidase-conjugated growth rate was comparable using the parental SMA560 cells or the secondary antibodies (DAKO, Botany, Australia), the cells and sections were 2s-2 and 2s-7 cells, which both express HYAL2 at high levels (Fig. 5). incubated with 3,3Ј-diaminobenzidine tetrahydrochloride, dihydrate (DAB). Increased Vascularization in HYAL2-overexpressing Intracra- After several washes in water, the sections were lightly counterstained with nial Tumors. The 2s-2- and 2s-7-cell-derived intracerebral tumors hematoxylin. The antibody binding was detected by microscopy. were red in color and contained small necrotic areas, whereas SMA560- and 2s-6 cell-derived tumors were faint pink in color and RESULTS did not contain necrotic areas (Fig. 6a). Staining of intracerebral In vitro Characteristics of HYAL2-expressing Cells. Two cell tumor-derived sections with the endothelial cell-specific anti-PECAM lines derived from the murine astrocytoma SMA560 cells that express antibodies showed that 2s-2 and 2s-7 cells formed highly vascularized

Fig. 2. Photomicrograph of SMA560 cells and HYAL2-expressing clones. Cells were grown on plastic and photographed. a, SMA560; b, 2s-6; c, 2s-2; d, 2s-7.

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Table 1 Intracerebral tumor formation the gene loci for HYAL1, HYAL2, and HYAL3, is often associated The data represent one experiment. Each experiment was terminated, and the cohort with small cell lung cancer (30, 31, 33). Therefore, this chromosomal was culled when the first mice showed neurological symptoms. Each experiment was repeated several times with varying numbers of animals and was found to be reproducible. location was thought to encode a tumor suppressor. However, in most The survival time of the animals in each group was compared with the control group of instances, these deletions are very large, and many other genes could 2 mice inoculated with SMA560 cells and was calculated using the ␹ test. be on the same deleted region of the chromosome. Similarly, in a SMA560 6 of 10 large, 2 medium, and 2 small tumors after 18 days murine model, two congenic strains of mice that differ only in the Ͻ 2s-2 10 of 10 large tumors after 10 days, P 0.0001 HYAL1 locus on chromosome 9 were used as recipients for tumor 2s-6 8 of 10 large, 1 medium, and 1 no tumor after 13 days, P ϭ 0.001 2s-7 7 of 9 large tumors, 1 small, and 1 no tumor after 9 days, P ϭ 0.001 cells. The mice expressing higher levels of hyaluronidase activity were found to be more resistant to tumor development (34). However, at least one of the cell lines used for the inoculations, B16F10, expressed very high levels of hyaluronidase activity, and the results may, thus, be difficult to explain. On the other hand, it has been suggested that hyaluronidases may be involved in breast tumor me- tastasis formation because metastases express about four times higher levels of hyaluronidase as the primary tumor (35). In addition, it has been proposed that elevated urinary hyaluronidase levels can be used as diagnostic markers for high-grade bladder cancer because of a strict correlation of hyaluronidase levels with tumor grade (36). The aber- rant expression of the gene, located on chromosome 7, encoding the testicular hyaluronidase, PH-20/SPAM1, has been reported to occur in high-grade gliomas, breast cancers, and colon cancers but not in low-grade tumors of the same organs (21). A third chromosomal

Fig. 3. Photomicrograph of 2s-2-derived brain tumor. The brain of a 2s-2 tumor- bearing animal was sectioned, and consecutive sections were stained with anti-CD44 antibodies (a) or with H&E (b). tumors (Fig. 6b), whereas 2s-6 cells formed tumors that had the same density of blood vessels as the tumors formed by the parental SMA560 cells. Expression of HYAL2 in Human Brain Tumor Specimens. HYAL2 is not expressed in normal adult brain, but is expressed in fetal brain (19, 33). However, because our experiments indicated that Fig. 4. Increased invasiveness in HYAL2-expressing tumors. Representative sections HYAL2 overexpression in murine glioma cells enhances tumorige- of a SMA560-derived tumor (a) and a 2s-2-derived tumor (b) were stained with H&E. nicity, we analyzed several primary human brain tumor specimens by Northern blot hybridization. One ganglioglioma (667), one glioblastoma multiforme (674), and two meningioma specimens (611 and 673) were found to express HYAL2. However, three anaplastic astrocytomas (228, 433, and 446) and one glioblastoma multiforme (675) were found not to express HYAL2 (Fig. 7). This result indicates that HYAL2 expression may occur in human brain tumors, although it does not seem to correlate well with invasiveness or stage.

DISCUSSION Our results indicate that overexpression of HYAL2 increases the tumorigenicity of the murine astrocytoma cells, SMA560, after inoculation of the brain but not after s.c. inoculation. Furthermore, the HYAL2 overexpressing brain tumors are very well vascularized. Hyaluronidase genes have been proposed to be a family of tumor suppressor genes or genes that may facilitate tumor growth and Fig. 5. HYAL2-expressing cells and parental SMA560 cells form s.c. tumors within the same time period. Cells (106) of each cell line—SMA560, 2s-2, and 2s-7—were injected invasiveness, but evidence in most publications has been circumstan- s.c. into syngeneic mice. The size of the tumors was measured daily. The results shown tial. A deletion of part of the human chromosome 3, which contains represent ten mice. Bar, SE. 6248

Fig. 6. Increased vascularization of intracerebral tumors. a, photographs of coronally cut brains of mice bearing tumors derived from SMA560 or 2s-2 cells. b, immunohistochemical staining of intracerebral tumors derived from SMA560 cells, 2s-2, 2s-6, and 2s-7 cells with anti-PECAM antibodies.

location mapping to 10q24.1-q24.3 and encoding a hyaluronidase Although the serum hyaluronidase HYAL1 and other lysosomal expressed in meningiomas has been described recently (37). hyaluronidases that require an acidic pH for enzymatic activity are The extracellular matrix of the brain is rich in hyaluronan, and found in a secreted form, they are inactive in the extracellular envi- high-grade human gliomas contain elevated levels of hyaluronan and ronment because the pH is close to neutral. This would imply that only the hyaluronan receptor, CD44, which is required for the uptake of hyaluronan, which is taken up by the cell and translocated into the hyaluronan (2, 38). In addition, the levels of hyaluronectin, a hyalu- lysosomal compartment, can be degraded by these enzymes. Muta- ronan-binding protein that is proposed to counteract the effects of tions in HYAL1 that led to the inactivation of the protein were found hyaluronan fragments, are drastically reduced in high-grade gliomas to be the cause of a lysosomal storage disorder, mucopolysacchari- (11). Hyaluronan fragments are described to be angiogenic, but the dosis IX (31). As SMA560 cells express high levels of CD44, uptake molecular mechanisms mediating this effect are not understood as yet of hyaluronan from the extracellular matrix seems to be possible in (24, 25). these cells. The parental SMA560 cells seem not to contain hyaluronidase activity as assayed by zymography gels (data not shown). It is interesting to speculate, whether the rather large hyaluronan fragments that are generated by HYAL2 have biological activity or whether further degradation by low levels of other hyaluronidases took place in the HYAL2-expressing SMA560 cell clones. The increased vascularization of the tumors may have been caused by smaller hyaluronan fragments or may be due to increased microne- crosis within the tumor. Necrosis is known to stimulate the synthesis of vascular endothelial growth factor, VEGF, which in turn activates Fig. 7. HYAL2 expression in human brain tumors. Total RNA of human brain tumors neovascularization (39). was separated on formaldehyde-containing agarose gels, blotted, and probed with The tumor growth rate of the HYAL2-expressing cells was en- HYAL2, then reprobed with GAPDH. RNA specimens 228, 433, 446 were derived from anaplastic astrocytomas, 674 and 675 from glioblastoma multiforme, 667 from a ganglio- hanced only in the brain and not if grown s.c. This may reflect the glioma, and 611 and 673 from meningiomas. different extracellular environment, with higher levels of hyaluronan, 6249

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1999 American Association for Cancer Research. HYAL2 ACCELERATES INTRACEREBRAL GLIOMA GROWTH found in the brain. Although the skin itself is very rich in hyaluronan, 16. Hofmann, M., Fieber, C., Assmann, V., Gottlicher, M., Sleeman, J., Plug, R., the s.c. space may well have no, or very low, levels of hyaluronan. Howells, N., von Stein, O., Ponta, H., and Herrlich P. Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein. J. Cell Sci., 27: 1673–1684, Similarly, the vascularization level, which may depend on small 1998. hyaluronan fragments being generated, is only increased in the intrac- 17. Jaworski, D. M., Kelly, G. M., Piepmeier, J. M., and Hockfield, S. BEHAB (brain erebral tumors. enriched hyaluronan binding) is expressed in surgical samples of glioma and in intracranial grafts of invasive glioma cell lines. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1999 American Association for Cancer Research. Hyaluronidase-2 Overexpression Accelerates Intracerebral but not Subcutaneous Tumor Formation of Murine Astrocytoma Cells

Ulrike Novak, Stanley S. Stylli, Andrew H. Kaye, et al.

Cancer Res 1999;59:6246-6250.

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