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Heparanase gene and protein expression in ameloblastoma: possible role in local invasion of tumor cells
Oral Oncology (2005) 41, 542–548
http://intl.elsevierhealth.com/journals/oron/
Heparanase gene and protein expression in ameloblastoma: possible role in local invasion of tumor cells Hitoshi Nagatsuka a,*, Phuu Pwint Han a, Hidetsugu Tsujigiwa a, Chong Huat Siar b, Mehmet Gunduz a, Toshio Sugahara c, Akira Sasaki d, Motowo Nakajima e, Yoshio Naomoto f, Noriyuki Nagai a a
Department of Oral Pathology and Medicine, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8525, Japan b Department of Oral Pathology, Oral Medicine and Periodontology, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia c Department of Oral and Maxillofacial Reconstructive Surgery, Graduate School of Medicine and Dentistry, Okayama University, Shikata-cho, Okayama 700-8525, Japan d Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Dentistry, Okayama University, Shikata-cho, Okayama 700-8525, Japan e Tsukuba Research Institute, Novartis Pharma KK Tsukuba, Japan f Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Graduate School of Medicine and Dentistry, Okayama University, Shikata-cho, Okayama 700-8558, Japan Received 13 December 2004; accepted 4 January 2005
KEYWORDS
Summary Ameloblastoma is the most common odontogenic neoplasm, particularized by its local invasiveness. Heparanase is the endo-glucuronidase enzyme that specifically cleaves heparan sulfate, the important modulator of extracellular matrix, and related to invasion of tumor cells. In this study, we addressed to show the gene expression and localization of heparanase in ameloblastoma. Immunohistochemistry and in situ hybridization of heparanase were carried out in 23 ameloblastomas. Strong expression of heparanase at both mRNA and protein levels was detected in all ameloblastomas studied. Small tumor nests and budding epithelial branches showed stronger staining pattern and the stromal tissues at the immediate vicinity of the tumor nests with strong heparanase expression were loose and edematous. Cystic areas and squamous metaplastic areas of the tumor showed intense staining with heparanase antibody proposing the implication of heparanase in these
Ameloblastoma; Heparanase; Heparan sulfate; Local invasion
* Corresponding author. Tel.: +81 86 235 6652; fax: +81 86 235 6654. E-mail address: [email protected] (H. Nagatsuka).
1368-8375/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2005.01.004
Heparanase gene and protein expression in ameloblastoma
543
processes. These results suggest the possible contribution of heparanase in the local invasiveness and secondary morphologic changes of ameloblastoma. c 2005 Elsevier Ltd. All rights reserved.
Introduction Ameloblastoma is the most common odontogenic tumor characterized by its histological resemblance to the enamel organ of the developing tooth germ. The tumor is composed of islands or strands of epithelial cells arranged by peripheral palisaded columnar to cuboidal cells referred to as ameloblast- or preameloblast-like cells and the central polyhedral cells with reticular pattern bearing a resemblance to stellate reticulum of the enamel organ.1,2 There are two main types of ameloblastoma namely follicular and plexiform types. Follicular ameloblastoma is composed of epithelial islands of peripheral ameloblast-like cells with reverse nuclear polarity and central stellate reticulum-like cells. Cystic degeneration is seen due to degeneration of the stellate reticulum-like cells in the center. In the case of plexiform type, the strands of odontogenic epithelial cells form reticular pattern with buddings instead of isolated follicles and the cyst formation is the result of the stromal degeneration.1,2 Although it is classified as the benign odontogenic neoplasm, ameloblastoma is locally invasive and destructive tumor of the jawbone. Frequent recurrence of the tumor necessitates wide surgical resection for treatment. In some cases, recurrence is inevitable even after radical surgery.3 Many researches tried to uncover the cause of this local invasiveness of ameloblastoma but it is still an enigma and has not yet clearly identified. Cellular invasion requires break down of the basement membrane and the surrounding extracellular matrix followed by the growth and proliferation of cells. The invasive ability of ameloblastoma is also thought to be related to the high proliferation rate of the tumor cells and/or the release of biologically active molecules produced by the tumor cells.4 Recently, heparanase, the mammalian endo-glucuronidase enzyme, which selectively cleaves extracellular matrix molecule, heparan sulfate (HS) at specific sites have been well reported to play important roles in degradation and remodeling of the extracellular matrix (ECM) in normal condition and dissemination and invasion of cells associated with inflammation and cancer metastasis.5,6 The target molecule of heparanase, heparan sul-
fate is present in the extracellular matrix (ECM) in the form of heparan sulfate proteoglycans (HSPGs). HSPGs have certain important functional roles in ECM assembly and integrity. Their HS chains also sequester and interact with many biologically active molecules such as growth factors, cytokines, chemokines and cell adhesion molecules.7,8 It is an interesting point to know whether the tumor cells of ameloblastoma express and produce heparanase, as this can be one of the important determinants of its local invasiveness. In this study, we tried to analyze the role of heparanase in the local invasiveness of ameloblastoma at the protein as well as the gene expression level in paraffinembedded sections of human ameloblastoma specimens by immunohistochemistry and in situ hybridization procedures.
Materials and methods Surgical materials Thirty blocks of ameloblastoma (total 23 cases) embedded in paraffin were selected from the surgical pathology unit of the Department of Oral Pathology and Medicine, Graduate School of Medicine and Dentistry of Okayama University, Japan. The samples were fixed in 10% neutral buffered formaline, decalcified with 10% formic acid if necessary and routinely processed and embedded in paraffin. Histological diagnosis of ameloblastoma was done by routine hematoxylin and eosin staining slides, according to WHO histological typing odontogenic tumors. It included 12 follicular and 11 plexiform ameloblastomas. Serial 4 lm sections were cut and used for the immunohistochemistry and in situ hybridization procedures.
Antibodies Mouse monoclonal antibody against human heparanase, which can recognize both 65 kDa proform and 50 kDa mature form of the Human heparanase, as reported previously was used.9 For antibody detection, Mouse IgG ABC kit (Vectastain Elite ABC kits, Vector Laboratories, USA) was used and
544 the immunoreaction was visualized by DAB (Histofine DAB substrate; Nichirei, Japan).
Probe Digoxigenin 11-UTP-labelled single-stranded RNA probes were prepared using DIG labeling Kit (Roche Diagnostic GmbH, Penzberg, Germany) according to the manufacturer’s instructions. For generation of single stranded heparanase RNA probe, a 571-bp fragment of human heparanase cDNA (bases 261– 832 of the total cDNA) [Gene Bank Accession No. AF144325] was produced by RT-PCR, subcloned into pCR21 (Invitrogen) and amplified by PCR.
Immunohistochemistry Immunohistochemical staining was performed using 4 lm sections mounted on silanized slides. Briefly, sections were deparaffinized in a series of xylene for 15 min and rehydrated in graded ethanol solutions. Endogenous peroxidase activity was blocked by incubating the sections in 0.3% H2O2 in methanol for 30 min. Antigen retrieval on paraffin sections was achieved by microwaving the sections immersed in 10 mM citrate buffer solution for 1 min. After blocking of non-specific reactivity with horse normal serum (Mouse IgG ABC kit) for 20 min at room temperature, the sections were incubated overnight with anti-heparanase antibody (1:500 dilution) at 4 °C. The tagging of primary antibody was achieved by subsequent application of biotinylated anti-mouse IgG antibody and avidin–biotin complexes (Mouse IgG ABC kit). Visualization of immunohistochemical reaction was performed by developing the enzyme complex with DAB/H2O2 solution (Histofine DAB substrate; Nichirei, Japan) and counterstained with Mayer’s hematoxylin. Negative control sections were incubated with omission of the primary antibody under the same protocol. In each lesion, assessment of heparanase was based on granular staining pattern in the cytoplasm and/or nucleus of the tumor cells.
In situ hybridization For in situ hybridization, the 4 lm sections were deparaffinized in xylene, rehydrated in ethanol and incubated with 3 mg/ml of proteinase K (Roche Diagnostics) in 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA for 15 min at 37 °C. Acetylation of the sections was performed by incubating with freshly prepared 0.25% acidic anhydride in 0.1 M triethanolamine– HCl buffer (pH 8.0) for 10 min at room temperature. Hybridization solution contains 50% deionized form-
H. Nagatsuka et al. amide, 10% dextran sulfate, 1x Dehardt’s solution, 600 mM NaCl, 0.25% SDS, 250 mg per ml of Escherichia coli tRNA (proteinases treated) 10 mM dithiothreitol, and 0.1–2.0 mg/ml of digoxigenin-UTP labeled RNA probe. The probe was placed on the sections and covered by parafilm and incubated at 50 °C overnight in a moist chamber. After hybridization, the slides were washed with a series of SSC at 50 °C and then incubated with 1.5% blocking reagent in DIG1 buffer for 60 min. Anti-digoxigenin-AP Fab fragment (1:800) (Roche Diagnostics) in DIG 1 buffer was applied to the sections and incubated for 1 h at room temperature. Coloring solution containing 337.5 lg/ ml of nitro blue tetrazolium and 165 lg/ml of 5-bromo-4-chloro-3-indolyl phosphate in DIG 3 buffer (100 mM Tris–HCL, pH 9.5, 100 mM NaCl, 50 mM MgCl2) was mounted on the sections and incubated at 37 °C until the signal–noise ratio was maximum. The slides were mounted with promount after counterstaining with methyl green. The negative controls included hybridization with sense (mRNA) probe.
Results We noticed that heparanase enzyme was strongly expressed in ameloblastoma as all tumor samples studied showed positive staining in both mRNA and protein level. Generally the protein localization reflects the mRNA expression pattern. No significant difference of heparanase in the gene expression as well as protein localization pattern was noted between the subtypes of ameloblastoma. The detail pattern of expression and localization of heparanase were discussed separately.
Expression of heparanase mRNA in ameloblastoma The mRNA for heparanase was detected in both peripheral and central cells of the tumor islands. The peripheral ameloblasts-like cells especially showed stronger expression although the central stellate reticulum-like cells also showed some degree of staining (Fig. 1A). Especially, the basal end of the peripheral columnar cells of follicular ameloblastoma showed stronger staining. In plexiform type, where there is no prominent stellate cells, the staining intensity was approximately the same within the epithelial strands except for the budding areas (Fig. 2A). The expression was especially strong in the invasive budding areas and smaller tumor islands at the invasive fronts and the peripheral cells of the cystic formation areas (Fig. 3A). Squamous metaplastic areas within
Heparanase gene and protein expression in ameloblastoma
545
Figure 1 Heparanase expression in follicular-type ameloblastoma (A) in situ hybridization for heparanase mRNA: mRNA signal was stronger in the peripheral palisading cells especially at the apical ends (B) immunoperoxidase stain for anti-heparanase antibody: localization of protein reflected the mRNA expression. The peripheral cells of the nest showed intense granular cytoplasmic staining but the central stellate cells showed weak staining.
Figure 2 Heparanase expression in plexiform ameloblastoma (A) in situ hybridization for heparanase mRNA and (B) immunoperoxidase stain for anti-heparanase antibody: mRNA and protein are almost evenly distributed among the tumor cells. The staining intensity is more prominent in the budding areas of the strands. Note the loose myxoid nature of the stroma tissue surrounding the tumor strands.
Figure 3 Localization of heparanase in the cystic degeneration area (A,B) and central squamous metaplastic area (C,D) of the ameloblastic islands. Strong expression is seen in the tumor cells surrounding the central cystic area (A). Heparanase mRNA is expressed by the surrounding tumor cells but not in the squamous cells (C). Note the strong immunostaining and extracellular localization of heparanase in cystic spaces and the central squamous cells. Nuclear staining by anti-heparanase antibody is also evident (B,D).
546 the tumor nests showed very weak mRNA staining intensity (Fig. 3C). Expression of heparanase mRNA was also detected in the inflammatory cells, stromal fibroblasts and endothelial cells of the blood vessels near the tumor nests.
Distribution of Heparanase protein in ameloblastoma Generally, the localization of heparanase protein was closely related to that of the mRNA expression. Positive staining was recognized as the granular cytoplasmic as well as nuclear staining pattern. Stronger staining pattern was observed in the basal ameloblast-like cells especially at the clear vacuolar structures near the basement membrane in follicular ameloblastoma. The central stellate reticulum-like cells were also positive but with a weaker degree (Fig. 1B). In plexiform ameloblastoma the tumor cells showed uniform staining intensity throughout the tumor strands (Fig. 2B). Stronger heparanase localization was observed in the small tumor cell nests of follicular ameloblastoma and invasive buddings of plexiform ameloblastoma. The stromal fibroblasts near the tumor cells nests also showed weak staining pattern but the staining is stronger in inflammatory cells and endothelial cells of small blood capillaries. The stromal tissue near the tumor cells with strong heparanase immunostaining, were more myxoid or loose compare with other areas. We noticed that squamous metaplastic areas showed strong immunostaining for heparanase antibody despite there was weak mRNA expression within these squamous cells by in situ hybridization (Fig. 3). Extracellular staining for heparanase was only seen in cystic degeneration areas and some of the central squamous metaplastic areas (Fig. 3B and D) but not in the intercellular spaces of stellate reticulum-like cells.
Discussion Ameloblastoma is a benign odontogenic neoplasm of epithelial origin.1,2 Although it is of benign in nature, local recurrence rate is quite high.3 This is thought to be responsible by the local invasive and proliferative abilities of the tumor cells.4 Heparanase is an endo-b-D-glucuronidase that specifically cleaves the carbohydrate chains of heparan sulfate proteoglycan (HSPG).10 This enzyme has been recognized in a variety of normal and malignant cells and various tumors.9 HSPG are distributed rampantly at cell surfaces such as syndecans and in extracellular matrix including basement
H. Nagatsuka et al. membrane such as perlecan, which interact with various signaling molecules, cell adhesion molecules and other extracellular matrix and basement membrane molecules.11,12 Moreover, HS chains of these HSPGs sequester a multitude of bioactive proteins such as growth factors, morphogens, cytokines, chemokines, enzymes, and cell adhesion molecules and serve as the reservoir for these molecules.13 Recent studies reported that degradation of heparan sulfate chains by heparanase promote tumor invasion and metastasis by two different mechanisms; (1) by destroying cell–cell and cell– matrix attachment thus enabling the tumor cells to readily penetrate the ECM or BM and (2) by releasing the bioactive molecules attached to HS resulting profound effects on cellular function and proliferation.5–8 Normal oral epithelium only showed very weak heparanase level and the expression increased with inflammatory, dysplastic or malignant change of the epithelium.14 In this study, the results showed that there was increased expression of heparanase in all ameloblastoma samples studied irrespective of the type either follicular or plexiform. The peripheral basal cells of the tumor islands and strands seemed to be the main producers of heparanase particularly in the invasive or branching areas. Although inflammatory cells showed strong heparanase expression, expression in the stromal compartment is minimal compared to the epithelial nests. Moreover, the strong immunostaining of heparanase in tumor cells was seen in association with the surrounding loose connective tissue stroma, showing the invasive property of ameloblastoma is somewhat concerned with increased heparanase expression of the tumor cells. These findings are also consistent with the previous reports as pathological increase in heparanase mRNA expression and protein levels has been reported in many types of malignant tumors including gastric, esophageal, pancreatic, colon, bladder and hepatocellular carcinomas and melanoma and this increase in expression is well related with tumor invasion, metastasis and prognosis.9,15–22 The cystic degeneration area and central squamous metaplastic areas were strongly positive for heparanase protein indicating that heparanase may have roles in these processes. Heparanase may contribute both directly and indirectly to the invasion and cystic degeneration process of ameloblastoma. Directly, heparanase can weaken the extracellular matrix integrity by digesting the HS chains of the extracellular matrix HSPGs resulting in loss of cell–cell and cell–matrix attachment promoting cystic degeneration and invasion.23 In
Heparanase gene and protein expression in ameloblastoma another way, it has been reported that the HS can serve as the extracellular docking molecules for matrix metalloproteinases (MMPs)24 and the release of MMPs by heparanase digestion is also a possible mechanism.4 However, we assume that the increased mRNA and protein expression of heparanase in ameloblastoma might in part be the physiologic response as it was reported that perlecan protein was produced and secreted by the tumor cells of the ameloblastoma25 and heparanase is necessary for the natural turn over of heparan sulfate proteoglycans.26 This may explain that the invasiveness of ameloblastoma is only local but not distant metastasis. Another fact is, our previous studies and other study observed that the localization of basement membrane molecules like type IV collagen, laminin were seen mainly as continuous linear pattern, around the tumor nests of ameloblastoma except for some areas of discontinuity.27–29 These findings also indirectly explain that the increased heparanase expression in ameloblastoma is somewhat regulated and responsible for local invasiveness only as the BMs of the tumor nests are mainly intact. Very recently, it has been reported that nuclear heparanase expression is associated with the differentiation of tumor cells in esophageal carcinoma.17 Likewise, heparanase may be associated with the squamous differentiation of central metaplastic tumor cells in ameloblastoma as they showed very strong immunostaining to anti-heparanase antibody. Another possibility is, heparanase, which is a very stable enzyme, may be secreted and accumulated in the central areas of the tumor nests after production from the tumor cells. It is also an interesting finding as in oral squamous cell carcinoma, well-differentiated tumor cells showed weaker heparanase staining compared to the less-differentiated counterparts.14 Although we could not discuss about the exact mechanism and action of heparanase in ameloblastoma at this time, this is the first study to report that ameloblastoma showed increase heparanase expression at the mRNA as well as the protein level, which may be one of the important determinants of its local invasiveness and so further studies of the detail mechanism and function of heparanase in this tumor is encouraged.
Acknowledgement This study was supported by Grant-in-Aid for Scientific Research (A) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 1520960).
547
References 1. Kramer IRH, Pindborg JJ, Shear M. Histological typing of odontogenic tumors. 2nd ed. Berlin: Springer; 1992., p 11–2. 2. Kramer IR, Pindborg JJ, Shear M. The WHO histological typing of odontogenic tumors—A commentary on the Second Edition. Cancer 1992;70:2894–988. 3. Reichart PA, Philipsen HP, Sonner S. Ameloblastoma: biological profile of 3677 cases. Eur J Cancer B Oral Oncol 1995;31B(2):86–99. 4. Pinheiro JJ, Freitas VM, Moretti AI, Jorge AG, Jaeger RG. Local invasiveness of ameloblastoma. Role played by matrix metalloproteinases and proliferative activity. Histopathology 2004;45(1):65–72. 5. Parish CR, Freeman C, Hulett MD. Heparanase: a key enzyme involved in cell invasion. Biochim et Biophys Acta 2001;1471(3):M99–108. 6. Vlodavsky I, Goldshmidt O, Zcharia E, Atzmon R, RanginiGuatta Z, et al. Mammalian heparanase: involvement in cancer metastasis, angiogenesis and normal development. Semin Cancer Biol 2002;12(2):121–9. 7. Vlodavsky I, Friedmann Y. Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis. J Clin Invest 2001;108(3):341–7. 8. Vlodavsky I, Miao HQ, Medalion B, Danagher P, Ron D. Involvement of heparan sulfate and related molecules in sequestration and growth promoting activity of fibroblast growth factor. Cancer Metastasis Rev 1996;15(2): 177–86. 9. Takaoka M, Naomoto Y, Ohkawa T, Uetsuka H, Shirakawa Y, Nagatsuka H, et al. Heparanase expression correlates with invasion and poor prognosis in gastric cancers. Lab Invest 2003;83(5):613–22. 10. Nakajima M, Irimura T, Nicolson GL. Heparanases and tumor metastasis. J Cell Biochem 1988;36(2):157–67. 11. Carey DJ. Syndecans: multifunctional cell-surface co-receptors. Biochem J 1997;327(Pt 1):1–16. 12. Erickson AC, Couchman JR. Still more complexity in mammalian basement membranes. J Histochem Cytochem 2000;48(10):1291–306. 13. Bernfield M, Gotte M, Park PW, et al. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 1999;68:729–77. 14. Nagatsuka H, Siar CH, Tsujigiwa H, Han PP, Gunduz M, Nagai N, et al. Heparanase mRNA gene and protein expression in normal, dysplastic and neoplastic human oral mucosa: evidence for its role in oral cancer development and progression, under review. 15. Endo K, Maejara U, Baba H, Tokunaga E, Koga T, Ikeda Y, et al. Heparanase gene expression and metastatic potential in human gastric cancer. Anticancer Res 2001;21(5): 3365–9. 16. Mikami S, Ohashi K, Usui Y, Nemoto T, Katsube K, Yanagishita M, et al. Loss of syndecans-1 and increased expression of heparanase in invasive esophageal carcinomas. Jpn J Cancer Res 2001;91(10):1062–73. 17. Ohkawa T, Nomoto Y, Takaoka M, Nobuhisa T, Uetsuka H, Nagatsuka H, et al. Localization of heparanase in esophageal cancer cells: respective roles in prognosis and differentiation. Lab Invest 2004;84(10):1289–304. 18. Koliopanos A, Friess H, Kleeff J, Shi X, Liao Q, Pecker I, et al. Heparanase expression in primary and metastastic pancreatic cancer. Cancer Res 2001;61(12):4655–9. 19. Friedmann Y, Vlodavsky I, Aingorn H, Aviv A, Peretz T, Pecker I, et al. Expression of heparanase in normal,
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H. Nagatsuka et al. dysplastic, and neoplastic human colonic mucosa and stroma. Evidence for its role in colonic tumorigenesis. Am J Pathol 2000;157(4):1167–75. Gohji K, Okamoto M, Kitazawa S, Toyoshima M, Dong J, Katsuoka Y, et al. Heparanase protein and gene expression in bladder cancer. J Urol 2001;166(4):1286–90. El-Assal ON, Yamanoi A, Ono T, Kohno H, Nagasue N. The clinicopathological significance of heparanase and basic fibroblast growth factor expressions in hepatocellular carcinoma. Clin Caner Res 2001;7(5):1299–305. Jin L, Nakajima M, Nicolson GL. Immunochemical localization of heparanase in mouse and human melanomas. Int J Cancer 1990;45(6):1088–95. Reiland J, Sanderson RD, Waguespack M, Barker SA, Long R, Carson DD, et al. Heparanase degrades syndecans-1 and perlecan heparan sulfate functional implications for tumor cell invasion. J Biol Chem 2004;279(9):8047–55. Yu WH, Woessner Jr JF. Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7). J Biol Chem 2000;275(6):4183–91.
25. Ida-Yonemochi H, Ikarashi T, Nagata M, Hoshina H, Takagi T, Saku T. The basement membrane-type heparan sulfate proteoglycan (perlecan) in ameloblastomas: its intercellular localization in stellate reticulum-like foci and biosynthesis by tumor cells in culture. Virchows Arch 2002;441(2): 165–73. 26. Bame KJ. Heparanases: endoglycosidases that degrade heparan sulfate proteoglycans. Glycobiology 2001;11(6): 91R–8R. 27. Nagatsuka H, Siar CH, Nakano K, Tsujigiwa H, Gunduz M, Choufuku H, et al. Differential expression of collagen IV alpha1 to alpha6 chains in basement membranes of benign and malignant odontogenic tumors. Virchows Arch 2002; 441(4):392–9. 28. Nakano K, Siar CH, Nagai N, Nagatsuka H, Tsujigiwa H, Gunduz M, et al. Distribution of basement membrane type IV collagen alpha chains in ameloblastoma: an immunofluorescence study. J Oral Pathol Med 2002;31(8):494–9. 29. Sauk JJ. Basement membrane confinement of epithelial tumor islands in benign and malignant ameloblastomas. J Oral Pathol 1985;14(4):307–14.
http://intl.elsevierhealth.com/journals/oron/
Heparanase gene and protein expression in ameloblastoma: possible role in local invasion of tumor cells Hitoshi Nagatsuka a,*, Phuu Pwint Han a, Hidetsugu Tsujigiwa a, Chong Huat Siar b, Mehmet Gunduz a, Toshio Sugahara c, Akira Sasaki d, Motowo Nakajima e, Yoshio Naomoto f, Noriyuki Nagai a a
Department of Oral Pathology and Medicine, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8525, Japan b Department of Oral Pathology, Oral Medicine and Periodontology, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia c Department of Oral and Maxillofacial Reconstructive Surgery, Graduate School of Medicine and Dentistry, Okayama University, Shikata-cho, Okayama 700-8525, Japan d Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Dentistry, Okayama University, Shikata-cho, Okayama 700-8525, Japan e Tsukuba Research Institute, Novartis Pharma KK Tsukuba, Japan f Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Graduate School of Medicine and Dentistry, Okayama University, Shikata-cho, Okayama 700-8558, Japan Received 13 December 2004; accepted 4 January 2005
KEYWORDS
Summary Ameloblastoma is the most common odontogenic neoplasm, particularized by its local invasiveness. Heparanase is the endo-glucuronidase enzyme that specifically cleaves heparan sulfate, the important modulator of extracellular matrix, and related to invasion of tumor cells. In this study, we addressed to show the gene expression and localization of heparanase in ameloblastoma. Immunohistochemistry and in situ hybridization of heparanase were carried out in 23 ameloblastomas. Strong expression of heparanase at both mRNA and protein levels was detected in all ameloblastomas studied. Small tumor nests and budding epithelial branches showed stronger staining pattern and the stromal tissues at the immediate vicinity of the tumor nests with strong heparanase expression were loose and edematous. Cystic areas and squamous metaplastic areas of the tumor showed intense staining with heparanase antibody proposing the implication of heparanase in these
Ameloblastoma; Heparanase; Heparan sulfate; Local invasion
* Corresponding author. Tel.: +81 86 235 6652; fax: +81 86 235 6654. E-mail address: [email protected] (H. Nagatsuka).
1368-8375/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2005.01.004
Heparanase gene and protein expression in ameloblastoma
543
processes. These results suggest the possible contribution of heparanase in the local invasiveness and secondary morphologic changes of ameloblastoma. c 2005 Elsevier Ltd. All rights reserved.
Introduction Ameloblastoma is the most common odontogenic tumor characterized by its histological resemblance to the enamel organ of the developing tooth germ. The tumor is composed of islands or strands of epithelial cells arranged by peripheral palisaded columnar to cuboidal cells referred to as ameloblast- or preameloblast-like cells and the central polyhedral cells with reticular pattern bearing a resemblance to stellate reticulum of the enamel organ.1,2 There are two main types of ameloblastoma namely follicular and plexiform types. Follicular ameloblastoma is composed of epithelial islands of peripheral ameloblast-like cells with reverse nuclear polarity and central stellate reticulum-like cells. Cystic degeneration is seen due to degeneration of the stellate reticulum-like cells in the center. In the case of plexiform type, the strands of odontogenic epithelial cells form reticular pattern with buddings instead of isolated follicles and the cyst formation is the result of the stromal degeneration.1,2 Although it is classified as the benign odontogenic neoplasm, ameloblastoma is locally invasive and destructive tumor of the jawbone. Frequent recurrence of the tumor necessitates wide surgical resection for treatment. In some cases, recurrence is inevitable even after radical surgery.3 Many researches tried to uncover the cause of this local invasiveness of ameloblastoma but it is still an enigma and has not yet clearly identified. Cellular invasion requires break down of the basement membrane and the surrounding extracellular matrix followed by the growth and proliferation of cells. The invasive ability of ameloblastoma is also thought to be related to the high proliferation rate of the tumor cells and/or the release of biologically active molecules produced by the tumor cells.4 Recently, heparanase, the mammalian endo-glucuronidase enzyme, which selectively cleaves extracellular matrix molecule, heparan sulfate (HS) at specific sites have been well reported to play important roles in degradation and remodeling of the extracellular matrix (ECM) in normal condition and dissemination and invasion of cells associated with inflammation and cancer metastasis.5,6 The target molecule of heparanase, heparan sul-
fate is present in the extracellular matrix (ECM) in the form of heparan sulfate proteoglycans (HSPGs). HSPGs have certain important functional roles in ECM assembly and integrity. Their HS chains also sequester and interact with many biologically active molecules such as growth factors, cytokines, chemokines and cell adhesion molecules.7,8 It is an interesting point to know whether the tumor cells of ameloblastoma express and produce heparanase, as this can be one of the important determinants of its local invasiveness. In this study, we tried to analyze the role of heparanase in the local invasiveness of ameloblastoma at the protein as well as the gene expression level in paraffinembedded sections of human ameloblastoma specimens by immunohistochemistry and in situ hybridization procedures.
Materials and methods Surgical materials Thirty blocks of ameloblastoma (total 23 cases) embedded in paraffin were selected from the surgical pathology unit of the Department of Oral Pathology and Medicine, Graduate School of Medicine and Dentistry of Okayama University, Japan. The samples were fixed in 10% neutral buffered formaline, decalcified with 10% formic acid if necessary and routinely processed and embedded in paraffin. Histological diagnosis of ameloblastoma was done by routine hematoxylin and eosin staining slides, according to WHO histological typing odontogenic tumors. It included 12 follicular and 11 plexiform ameloblastomas. Serial 4 lm sections were cut and used for the immunohistochemistry and in situ hybridization procedures.
Antibodies Mouse monoclonal antibody against human heparanase, which can recognize both 65 kDa proform and 50 kDa mature form of the Human heparanase, as reported previously was used.9 For antibody detection, Mouse IgG ABC kit (Vectastain Elite ABC kits, Vector Laboratories, USA) was used and
544 the immunoreaction was visualized by DAB (Histofine DAB substrate; Nichirei, Japan).
Probe Digoxigenin 11-UTP-labelled single-stranded RNA probes were prepared using DIG labeling Kit (Roche Diagnostic GmbH, Penzberg, Germany) according to the manufacturer’s instructions. For generation of single stranded heparanase RNA probe, a 571-bp fragment of human heparanase cDNA (bases 261– 832 of the total cDNA) [Gene Bank Accession No. AF144325] was produced by RT-PCR, subcloned into pCR21 (Invitrogen) and amplified by PCR.
Immunohistochemistry Immunohistochemical staining was performed using 4 lm sections mounted on silanized slides. Briefly, sections were deparaffinized in a series of xylene for 15 min and rehydrated in graded ethanol solutions. Endogenous peroxidase activity was blocked by incubating the sections in 0.3% H2O2 in methanol for 30 min. Antigen retrieval on paraffin sections was achieved by microwaving the sections immersed in 10 mM citrate buffer solution for 1 min. After blocking of non-specific reactivity with horse normal serum (Mouse IgG ABC kit) for 20 min at room temperature, the sections were incubated overnight with anti-heparanase antibody (1:500 dilution) at 4 °C. The tagging of primary antibody was achieved by subsequent application of biotinylated anti-mouse IgG antibody and avidin–biotin complexes (Mouse IgG ABC kit). Visualization of immunohistochemical reaction was performed by developing the enzyme complex with DAB/H2O2 solution (Histofine DAB substrate; Nichirei, Japan) and counterstained with Mayer’s hematoxylin. Negative control sections were incubated with omission of the primary antibody under the same protocol. In each lesion, assessment of heparanase was based on granular staining pattern in the cytoplasm and/or nucleus of the tumor cells.
In situ hybridization For in situ hybridization, the 4 lm sections were deparaffinized in xylene, rehydrated in ethanol and incubated with 3 mg/ml of proteinase K (Roche Diagnostics) in 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA for 15 min at 37 °C. Acetylation of the sections was performed by incubating with freshly prepared 0.25% acidic anhydride in 0.1 M triethanolamine– HCl buffer (pH 8.0) for 10 min at room temperature. Hybridization solution contains 50% deionized form-
H. Nagatsuka et al. amide, 10% dextran sulfate, 1x Dehardt’s solution, 600 mM NaCl, 0.25% SDS, 250 mg per ml of Escherichia coli tRNA (proteinases treated) 10 mM dithiothreitol, and 0.1–2.0 mg/ml of digoxigenin-UTP labeled RNA probe. The probe was placed on the sections and covered by parafilm and incubated at 50 °C overnight in a moist chamber. After hybridization, the slides were washed with a series of SSC at 50 °C and then incubated with 1.5% blocking reagent in DIG1 buffer for 60 min. Anti-digoxigenin-AP Fab fragment (1:800) (Roche Diagnostics) in DIG 1 buffer was applied to the sections and incubated for 1 h at room temperature. Coloring solution containing 337.5 lg/ ml of nitro blue tetrazolium and 165 lg/ml of 5-bromo-4-chloro-3-indolyl phosphate in DIG 3 buffer (100 mM Tris–HCL, pH 9.5, 100 mM NaCl, 50 mM MgCl2) was mounted on the sections and incubated at 37 °C until the signal–noise ratio was maximum. The slides were mounted with promount after counterstaining with methyl green. The negative controls included hybridization with sense (mRNA) probe.
Results We noticed that heparanase enzyme was strongly expressed in ameloblastoma as all tumor samples studied showed positive staining in both mRNA and protein level. Generally the protein localization reflects the mRNA expression pattern. No significant difference of heparanase in the gene expression as well as protein localization pattern was noted between the subtypes of ameloblastoma. The detail pattern of expression and localization of heparanase were discussed separately.
Expression of heparanase mRNA in ameloblastoma The mRNA for heparanase was detected in both peripheral and central cells of the tumor islands. The peripheral ameloblasts-like cells especially showed stronger expression although the central stellate reticulum-like cells also showed some degree of staining (Fig. 1A). Especially, the basal end of the peripheral columnar cells of follicular ameloblastoma showed stronger staining. In plexiform type, where there is no prominent stellate cells, the staining intensity was approximately the same within the epithelial strands except for the budding areas (Fig. 2A). The expression was especially strong in the invasive budding areas and smaller tumor islands at the invasive fronts and the peripheral cells of the cystic formation areas (Fig. 3A). Squamous metaplastic areas within
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Figure 1 Heparanase expression in follicular-type ameloblastoma (A) in situ hybridization for heparanase mRNA: mRNA signal was stronger in the peripheral palisading cells especially at the apical ends (B) immunoperoxidase stain for anti-heparanase antibody: localization of protein reflected the mRNA expression. The peripheral cells of the nest showed intense granular cytoplasmic staining but the central stellate cells showed weak staining.
Figure 2 Heparanase expression in plexiform ameloblastoma (A) in situ hybridization for heparanase mRNA and (B) immunoperoxidase stain for anti-heparanase antibody: mRNA and protein are almost evenly distributed among the tumor cells. The staining intensity is more prominent in the budding areas of the strands. Note the loose myxoid nature of the stroma tissue surrounding the tumor strands.
Figure 3 Localization of heparanase in the cystic degeneration area (A,B) and central squamous metaplastic area (C,D) of the ameloblastic islands. Strong expression is seen in the tumor cells surrounding the central cystic area (A). Heparanase mRNA is expressed by the surrounding tumor cells but not in the squamous cells (C). Note the strong immunostaining and extracellular localization of heparanase in cystic spaces and the central squamous cells. Nuclear staining by anti-heparanase antibody is also evident (B,D).
546 the tumor nests showed very weak mRNA staining intensity (Fig. 3C). Expression of heparanase mRNA was also detected in the inflammatory cells, stromal fibroblasts and endothelial cells of the blood vessels near the tumor nests.
Distribution of Heparanase protein in ameloblastoma Generally, the localization of heparanase protein was closely related to that of the mRNA expression. Positive staining was recognized as the granular cytoplasmic as well as nuclear staining pattern. Stronger staining pattern was observed in the basal ameloblast-like cells especially at the clear vacuolar structures near the basement membrane in follicular ameloblastoma. The central stellate reticulum-like cells were also positive but with a weaker degree (Fig. 1B). In plexiform ameloblastoma the tumor cells showed uniform staining intensity throughout the tumor strands (Fig. 2B). Stronger heparanase localization was observed in the small tumor cell nests of follicular ameloblastoma and invasive buddings of plexiform ameloblastoma. The stromal fibroblasts near the tumor cells nests also showed weak staining pattern but the staining is stronger in inflammatory cells and endothelial cells of small blood capillaries. The stromal tissue near the tumor cells with strong heparanase immunostaining, were more myxoid or loose compare with other areas. We noticed that squamous metaplastic areas showed strong immunostaining for heparanase antibody despite there was weak mRNA expression within these squamous cells by in situ hybridization (Fig. 3). Extracellular staining for heparanase was only seen in cystic degeneration areas and some of the central squamous metaplastic areas (Fig. 3B and D) but not in the intercellular spaces of stellate reticulum-like cells.
Discussion Ameloblastoma is a benign odontogenic neoplasm of epithelial origin.1,2 Although it is of benign in nature, local recurrence rate is quite high.3 This is thought to be responsible by the local invasive and proliferative abilities of the tumor cells.4 Heparanase is an endo-b-D-glucuronidase that specifically cleaves the carbohydrate chains of heparan sulfate proteoglycan (HSPG).10 This enzyme has been recognized in a variety of normal and malignant cells and various tumors.9 HSPG are distributed rampantly at cell surfaces such as syndecans and in extracellular matrix including basement
H. Nagatsuka et al. membrane such as perlecan, which interact with various signaling molecules, cell adhesion molecules and other extracellular matrix and basement membrane molecules.11,12 Moreover, HS chains of these HSPGs sequester a multitude of bioactive proteins such as growth factors, morphogens, cytokines, chemokines, enzymes, and cell adhesion molecules and serve as the reservoir for these molecules.13 Recent studies reported that degradation of heparan sulfate chains by heparanase promote tumor invasion and metastasis by two different mechanisms; (1) by destroying cell–cell and cell– matrix attachment thus enabling the tumor cells to readily penetrate the ECM or BM and (2) by releasing the bioactive molecules attached to HS resulting profound effects on cellular function and proliferation.5–8 Normal oral epithelium only showed very weak heparanase level and the expression increased with inflammatory, dysplastic or malignant change of the epithelium.14 In this study, the results showed that there was increased expression of heparanase in all ameloblastoma samples studied irrespective of the type either follicular or plexiform. The peripheral basal cells of the tumor islands and strands seemed to be the main producers of heparanase particularly in the invasive or branching areas. Although inflammatory cells showed strong heparanase expression, expression in the stromal compartment is minimal compared to the epithelial nests. Moreover, the strong immunostaining of heparanase in tumor cells was seen in association with the surrounding loose connective tissue stroma, showing the invasive property of ameloblastoma is somewhat concerned with increased heparanase expression of the tumor cells. These findings are also consistent with the previous reports as pathological increase in heparanase mRNA expression and protein levels has been reported in many types of malignant tumors including gastric, esophageal, pancreatic, colon, bladder and hepatocellular carcinomas and melanoma and this increase in expression is well related with tumor invasion, metastasis and prognosis.9,15–22 The cystic degeneration area and central squamous metaplastic areas were strongly positive for heparanase protein indicating that heparanase may have roles in these processes. Heparanase may contribute both directly and indirectly to the invasion and cystic degeneration process of ameloblastoma. Directly, heparanase can weaken the extracellular matrix integrity by digesting the HS chains of the extracellular matrix HSPGs resulting in loss of cell–cell and cell–matrix attachment promoting cystic degeneration and invasion.23 In
Heparanase gene and protein expression in ameloblastoma another way, it has been reported that the HS can serve as the extracellular docking molecules for matrix metalloproteinases (MMPs)24 and the release of MMPs by heparanase digestion is also a possible mechanism.4 However, we assume that the increased mRNA and protein expression of heparanase in ameloblastoma might in part be the physiologic response as it was reported that perlecan protein was produced and secreted by the tumor cells of the ameloblastoma25 and heparanase is necessary for the natural turn over of heparan sulfate proteoglycans.26 This may explain that the invasiveness of ameloblastoma is only local but not distant metastasis. Another fact is, our previous studies and other study observed that the localization of basement membrane molecules like type IV collagen, laminin were seen mainly as continuous linear pattern, around the tumor nests of ameloblastoma except for some areas of discontinuity.27–29 These findings also indirectly explain that the increased heparanase expression in ameloblastoma is somewhat regulated and responsible for local invasiveness only as the BMs of the tumor nests are mainly intact. Very recently, it has been reported that nuclear heparanase expression is associated with the differentiation of tumor cells in esophageal carcinoma.17 Likewise, heparanase may be associated with the squamous differentiation of central metaplastic tumor cells in ameloblastoma as they showed very strong immunostaining to anti-heparanase antibody. Another possibility is, heparanase, which is a very stable enzyme, may be secreted and accumulated in the central areas of the tumor nests after production from the tumor cells. It is also an interesting finding as in oral squamous cell carcinoma, well-differentiated tumor cells showed weaker heparanase staining compared to the less-differentiated counterparts.14 Although we could not discuss about the exact mechanism and action of heparanase in ameloblastoma at this time, this is the first study to report that ameloblastoma showed increase heparanase expression at the mRNA as well as the protein level, which may be one of the important determinants of its local invasiveness and so further studies of the detail mechanism and function of heparanase in this tumor is encouraged.
Acknowledgement This study was supported by Grant-in-Aid for Scientific Research (A) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 1520960).
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