- Email: [email protected]
Immunohistochemical assessment of hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase IX (CA IX) in ameloblastomas
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
Contents lists available at ScienceDirect
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology journal homepage: www.elsevier.com/locate/jomsmp
Oral Pathology/Original Research
Immunohistochemical assessment of hypoxia-inducible factor-1␣ (HIF-1␣) and carbonic anhydrase IX (CA IX) in ameloblastomas Yoshiki Arima a,b , Mariko Oikawa b,∗ , Yoshinaka Shimizu b , Seishi Echigo a , Tetsu Takahashi a , Hiroyuki Kumamoto b a Division of Oral and Maxillofacial Surgery, Department of Oral Medicine and Surgery, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan b Division of Oral Pathology, Department of Oral Medicine and Surgery, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
a r t i c l e
i n f o
Article history: Received 23 August 2016 Received in revised form 5 October 2017 Accepted 13 October 2017 Available online 27 March 2018 Keywords: Ameloblastoma Hypoxia HIF-1␣ CA IX Angiogenesis
a b s t r a c t Objective: To investigate roles of hypoxia-related proteins in odontogenic tumors, we analyzed the immunohistochemical expression of hypoxia-inducible factor-1␣ (HIF-1␣) and carbonic anhydrase IX (CA IX) and compared with angiogenesis. Methods: 10 dental follicles and 67 ameloblastomas were immunohistochemically examined with antibodies against HIF-1␣, CA IX, and CD34. Results: Immunohistochemical reactivity for HIF-1␣ and CA IX was detected in odontogenic epithelial cells, as well as in several macrophages and fibroblasts, in dental follicles and ameloblastomas. HIF1␣ was positive in 8 of 10 dental follicles, 45 of 48 primary ameloblastomas, and all 19 recurrent ameloblastomas. Increased HIF-1␣ reactivity was often found in keratinizing cells in acanthomatous ameloblastomas. Immunoreactivity for CA IX was detected in all samples of dental follicles and primary and recurrent ameloblastomas. CA IX reactivity was significantly higher in ameloblastomas than in dental follicles, in solid ameloblastomas than in unicystic ameloblastomas, and in follicular ameloblastomas than in plexiform ameloblastomas. Acanthomatous ameloblastomas showed increased CA IX reactivity around keratinizing areas, while granular cell ameloblastomas showed increased reactivity in peripheral non-granular cells. Microvessel density (MVD) of CD34-positive capillaries in solid ameloblastomas was significantly higher than in unicystic ameloblastomas. MVD tended to be greater in follicular ameloblastomas than in plexiform ameloblastomas, in granular cell ameloblastomas than in other subtypes of ameloblastomas, and in mural unicystic ameloblastomas than in other types of unicystic ameloblastomas. Conclusions: Our data suggest that hypoxia-related molecules and angiogenesis play a role in tumorigenesis, tissue structuring, cell differentiation, and prognosis of ameloblastomas in the intraosseous microenvironment. © 2018 Asian AOMS, ASOMP, JSOP, JSOMS, JSOM, and JAMI. Published by Elsevier Ltd. All rights reserved.夽
1. Introduction Ameloblastoma is the most frequently encountered epithelial odontogenic tumor arising in the jawbones, and is characterized by benign but locally invasive behavior with a high rate of recurrence. Therefore, clinical long-term follow-up is essential, and the required treatment includes excision with an adequate margin of uninvolved tissues, similar to that for malignant tumors [1,2]. This tumor is usually solid and unicystic. Histologically, solid ameloblas-
∗ Corresponding author. E-mail address: [email protected] (M. Oikawa).
tomas show follicular and plexiform proliferation patterns with or without acanthomatous and granular cell changes, and unicystic ameloblastomas have luminal, intraluminal, and mural variants [1]. In some respects, these epithelial odontogenic tumors histologically resemble physiological structures, such as enamel organ or dental lamina; however, the detailed mechanisms of oncogenesis and cytodifferentiation remain unknown. Hypoxia is thought to trigger a more aggressive tumor phenotype by inducing genomic instability, loss of apoptotic potential, and angiogenesis, which is the sprouting of new capillaries from a pre-existing vascular bed [3–5]. It plays a significant role in tumor recurrence, metastasis, and poor response to treatment, including radiotherapy, chemotherapy, and antiangiogenic treatment [5,6].
https://doi.org/10.1016/j.ajoms.2017.10.002 2212-5558/© 2018 Asian AOMS, ASOMP, JSOP, JSOMS, JSOM, and JAMI. Published by Elsevier Ltd. All rights reserved.夽
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
Recently, molecules involved in the hypoxic response of tumor cells were identified as endogenous hypoxia markers using noninvasive and cost-effective methods to determine the condition of tumor hypoxia [7–10]. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric protein consisting of an alpha and beta subset, in which the ␣ subset mediates HIF-1 function as a transcription factor in response to cellular hypoxia [11]. Expression of HIF-1␣ correlates with advanced tumor stage and metastasis, leading to poor outcomes of common human malignancies, such as carcinomas of the breast, brain, lung, prostate, and head and neck [8–10,12–16]. Carbonic anhydrase IX (CA IX) is one of the HIF-1␣-dependent enzymes most consistently upregulated under hypoxic conditions. Since there is an oxygen-dependent step that inhibits HIF-1␣ transactivation of target genes, it would be anticipated that CA IX protein levels, like other target gene products, would show a better correlation with low oxygen levels than would HIF-1␣ itself [4,7,17]. The effect of CA IX on the tumor microenvironment is characterized by the regulation of pH [18]. Recently, some human tumors have been reported to express CA IX, which is proposed to influence the clinical features of these tumors [4,5,7–9,16]. Thus, HIF-1␣ and CA IX play a role in angiogenesis, apoptosis, invasion, and metastasis of malignant tumors [8,9]. Our previous studies have confirmed the presence of angiogenic factors, vascular endothelial growth factor (VEGF), thymidine phosphorylase, and angiopoietins, and apoptosis-inducing molecules, p53, Bax, BH3-only proteins, and TNF family molecules, in ameloblastic tumors [19–24]. However, hypoxia-related factors in epithelial odontogenic tumors remains poorly understood. In the present study, HIF-1␣ and CA IX expression, as well as CD34 as a marker of microvessel density (MVD), were immunohistochemically examined in ameloblastomas as well as in dental follicles to clarify the role of these hypoxic agents in epithelial odontogenic tumors.
2. Material and methods The study protocol was reviewed and approved by the Research Ethics Committee of Tohoku University Graduate School of Dentistry (No. 2014-1-854).
2.1. Tissue preparation Specimens were surgically removed from 63 patients with ameloblastoma at Tohoku University Hospital and affiliated hospitals. The specimens were fixed in 10% buffered formalin for one to several days and were embedded in paraffin. The tissue blocks were sliced into 3 m-thick sections for routine histological and subsequent immunohistochemical examinations. Tissue sections were stained with hematoxylin and eosin for histological diagnosis according to the World Health Organization histological classification of odontogenic tumors [1]. The specimens consisted of 48 primary ameloblastomas and 19 recurrent ameloblastomas. Of the primary ameloblastomas, 37 were solid, and 11 were unicystic. The solid ameloblastomas were divided into 19 follicular and 18 plexiform types, including 11 acanthomatous and three granular cell subtypes. The unicystic ameloblastomas were divided into five luminal, three intraluminal, and three mural types. All recurrent ameloblastomas were solid and were divided into 13 follicular and 6 plexiform types, including 10 acanthomatous and 1 granular cell subtypes. Specimens of 10 dental follicles of the mandibular third molars were similarly prepared and compared with the ameloblastomas.
295
2.2. Immunohistochemistry for HIF-1˛, CA IX, and CD34 expression Tissue sections were deparaffinized and immersed in methanol with 0.3% hydrogen peroxide. Sections were heated in 0.01 M citrate buffer (pH 6.0; for HIF-1␣) or 1 mM EDTA buffer (pH 9.0; for CA IX) for 10 min by autoclaving (121 ◦ C, 2 atm). Next, the sections were incubated with primary antibodies at 4 ◦ C overnight. The antibodies used were as follows: rabbit anti-HIF-1␣ monoclonal antibody (Abcam, Cambridge, UK; isotype IgG; diluted at 1:100), rabbit anti-CA IX polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; purified IgG; diluted at 1:50), and mouse anti-CD34 monoclonal antibody (Nichirei, Tokyo, Japan; isotype IgG1; prediluted). The sections were allowed to react with peroxidase-conjugated anti-rabbit IgG (for HIF-1␣ and CA IX) or anti-mouse IgG (for CD34) polyclonal antibodies (Histofine Simple Stain MAX-PO; Nichirei, Tokyo, Japan) for 45 min, and reaction products were visualized by immersing the sections in 0.03% diaminobenzidine solution containing 2 mM hydrogen peroxide for 3–5 min. Nuclei were lightly stained with Mayer’s hematoxylin. For control studies of the antibodies, the serial sections were treated with phosphate-buffered saline, normal rabbit IgG, and mouse antidesmin monoclonal antibody (Nichirei; isotype IgG1) instead of the primary antibodies; these were confirmed to be unstained. 2.3. Evaluation of immunostaining and statistical analysis Immunohistochemical reactivity for HIF-1␣ was evaluated and classified into three groups: (−) negative in epithelial or tumor cells, (±) weakly positive (less than 30% of epithelial or tumor cells), and (+) moderately to strongly positive (more than 30% of epithelial or tumor cells) [14]. Immunohistochemical reactivity for CA IX was evaluated and classified into two groups: (+) diffusely positive in most epithelial or tumor cells, and (++) diffusely positive in most epithelial or tumor cells with increased reactivity in those epithelial or tumor cells near the basement membrane. Immunoreactivity for CD34 was conducted to evaluate MVD in the dental follicles and ameloblastomas. After scanning 5 areas showing the highest neovascularization (vascular hotspots) in normal mesenchymal tissues or tumor stromal tissues at 20-fold magnification, CD34-positive vessels were counted in the hotspots at 200-fold magnification, and the average count was recorded as MVD for each case [19,25,26]. Based on the criteria of Weidner et al. [27], a highlighted endothelial cell or a cell cluster clearly separate from adjacent microvessels, tumor cells, and other connective tissue elements was regarded as a distinct countable microvessel. A lumen was not required, nor was the presence of red blood cells. Single cell sprouts were included in the counts. The statistical significance of the differences in the percentage of cases with various HIF-1␣ and CA IX reactivity levels, as well as mean MVD, was determined by the Mann-Whitney U-test for differences between two groups or the Kruskal-Wallis test for differences among three or more groups. P-values <0.05 were considered to indicate statistical significance. 3. Results The results of immunohistochemical studies of HIF-1␣ and CA IX expression and MVD in dental follicles and ameloblastomas are summarized in Table 1. Immunohistochemical reactivity for HIF-1␣ was detected in the cytoplasm and nuclei of odontogenic epithelial cells as well as in several macrophages and fibroblasts in the dental follicles and ameloblastomas (Fig. 1). Dental lamina showed HIF-1␣ reactivity in 8 of 10 dental follicles (Fig. 1A, Table 1). In primary ameloblastomas, tumor cells showed HIF-1␣
296
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
Table 1 Immunohistochemical reactivity for HIF-1␣ and CA IX, as well as MVD in dental follicles and ameloblastomas.
HIF-1␣ expression: (−) negative in epithelial or tumor cells, (±) weakly (less than 30% of epithelial or tumor cells) positive, (+) moderately to strongly (more than 30% of epithelial or tumor cells) positive. CA IX expression: (+) diffusely positive in most epithelial or tumor cells, (++) diffusely positive in most epithelial or tumor cells with increased reactivity in epithelial or neoplastic cells near the basement membrane. Values in parentheses denote percentage values.HIF-1␣: hypoxia-inducible factor-1␣; CA IX: carbonic anhydrase IX; MVD: microvessel density.
reactivity in 45 of 48 cases, including 35 of 37 solid ameloblastomas and 10 of 11 unicystic ameloblastomas (Fig. 1B–D, Table 1). Solid ameloblastomas showed HIF-1␣ expression in all 19 follicular cases and 16 of 18 plexiform cases, and all acanthomatous and granular cell ameloblastomas were reactive (Fig. 1B, C, Table 1). Increased HIF-1␣ reactivity was often found in keratinizing cells of acanthomatous ameloblastomas (Fig. 1C). Tumor cell in unicystic ameloblastomas showed HIF-1␣ reactivity in all 5 luminal, all 3 intraluminal, and 2 of 3 mural types (Fig. 1D, Table 1). While 35 of 37 primary solid ameloblastomas were reactive with HIF-1␣, HIF1␣ reactivity was noted in all 19 recurrent solid ameloblastomas (Table 1). HIF-1␣ reactivity did not differ significantly among these odontogenic lesions or ameloblastomas variants. Immunoreactivity for CA IX was detected in the cell membrane and cytoplasm of odontogenic epithelial cells as well as some macrophages and fibroblasts in dental follicles and ameloblastomas (Fig. 2). CA IX reactivity was detected in dental lamina of dental follicles (Fig. 2A). Ameloblastomas showed CA IX expression in most tumor cells (Fig. 2B-F). Solid ameloblastomas often showed increased CA IX reactivity in peripheral columnar or cuboidal tumor cells as compared with central polyhedral cells (Fig. 2B, E). CA IX expression in ameloblastomas was significantly higher than that in dental follicles (P < 0.05, Table 1), and immunoreactivity for CA IX was higher in solid ameloblastomas than in unicystic ameloblastomas (P < 0.01, Table 1). Follicular ameloblastomas exhibited
statistically higher CA IX expression than plexiform ameloblastomas (P < 0.01, Table 1). Acanthomatous ameloblastomas showed increased reactivity around keratinizing areas (Fig. 2D), while granular cell ameloblastomas showed decreased reactivity in granular tumor cells (Fig. 2E, Table 1). Unicystic ameloblastomas showed CA IX reactivity in ameloblastomatous epithelium that lined, protruded into, and infiltrated cyst walls (Fig. 2F, Table 1). Difference of CA IX reactivity between primary and recurrent ameloblastomas did not achieve statistical significance (Table 1). Immunohistochemical staining for CD34 was detected in the cytoplasm of endothelial cells in both normal and tumor odontogenic tissues (Fig. 3). In dental follicles, CD34-positive microvessels were observed in areas near dental lamina (Fig. 3A). Ameloblastomas showed CD34 reactivity in microvessels of tumor stroma (Figs. 3B-D), and MVD in solid ameloblastomas was significantly higher than that in unicystic ameloblastomas (P < 0.05; Table 1). Follicular ameloblastomas showed CD34-positive vessels in many small vessels (Fig. 3B), whereas CD34-positive vessels in plexiform ameloblastomas were scattered and dilated (Fig. 3C, Table 1). In granular cell ameloblastomas, numerous CD34-positive microvessels were found as compared with other cellular variants (Table 1). The distribution of CD34 reactivity in the unicystic ameloblastomas was similar to that in solid ameloblastomas (Fig. 3D). In mural type unicystic ameloblastomas, many CD34-positive microvessels were recognized as compared to luminal or intraluminal types (Table 1).
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
297
Fig. 1. Immunohistochemical expression of hypoxia-inducible factor-1␣. (A) Dental follicle showing no expression ( × 400). (B) Plexiform ameloblastoma showing strong expression in most tumor cells ( × 200). (C) Acanthomatous ameloblastoma showing expression in most tumor cells with increased reactivity in keratinizing cells (arrow) ( × 400). (D) Unicystic ameloblastoma (mural type) showing expression in ameloblastomatous epithelium lining and infiltrating the cyst wall ( × 200).
MVDs in primary and recurrent ameloblastomas were not statistically different (Table 1). 4. Discussion Hypoxia induces transcription of physiologically important genes, including erythropoietin and VEGF. HIF-1␣ is a transcription factor responsive to hypoxia, and expression of HIF-1␣ is regulated by cellular oxygen level alterations [28]. Under normoxic conditions, HIF-1␣ is a short lived-protein because of its continuous proteolysis via the ubiquitin-proteasome pathway. In contrast, reduced oxygen availability induces HIF-1␣ accumulation through the inhibition of its degradation [29,30]. CAs are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide, participating in calcification, acid-base balance, bone resorption, and formation of the aqueous humor [31]. CAs include at least 15 types of isozymes, among which CA IX can be activated via the HIF-1 pathway under hypoxic conditions. CA IX maintains pH and adapts cellular components to the hypoxic microenvironment, thus ensuring the continued proliferation of cells in hypoxic milieus [32,33]. Recently, HIF-1␣ and CA IX expression has been reported in several normal human tissues [3,6,34]. Tooth germs have been shown to express HIF-1␣, suggesting its ability to regulate the intraosseous microenvironment and to promote odontogenic cell survival in hypoxic conditions [35]. The present study showed expression of HIF-1␣ and CA IX in epithelial and mesenchymal odontogenic cells of human dental follicle tissues. Our previous study showed VEGF expression in tooth development processes, which was anticipated given the hypoxic conditions of these processes [19]. These findings suggest that angiogenesis during tooth formation might be regulated under a certain level of hypoxia. Expression of HIF-1␣ has been found in many human tumors, including breast, brain, renal, and head and neck carcinomas, and has been correlated with more aggressive tumor grades, tumor invasion, metastatic potential, resistance to radiation therapy, and
poorer prognoses [7–10,12,15]. Cell lines genetically manipulated to knock down HIF-1˛ have shown decreased cell growth in vitro [36]. In the present study, immunohistochemical reactivity for HIF1␣ was found mainly in tumor cells of ameloblastomas, often strongly in keratinizing areas, suggesting that the tumor tissues were exposed to hypoxic conditions similarly to dental follicle tissues. Our previous study revealed VEGF expression was higher in ameloblastic tumors than in tooth germs [19], whereas the present study did not detect differences between non-neoplastic and neoplastic odontogenic tissues. Furthermore, follicular ameloblastomas tended to show slightly higher expression of HIF-1␣ than plexiform ameloblastomas, and cases without cellular variation tended to exhibit low HIF-1␣ expression compared with acanthomatous and granular cell subtypes. These features suggest that tissue structuring and/or cell differentiation of ameloblastomas may be affected by HIF-1␣ expression. Furthermore, immunohistochemical analysis revealed that recurrent ameloblastomas showed slightly higher HIF-1␣ expression than primary ameloblastomas, suggesting that a hypoxic microenvironment may be related to prognosis in ameloblastomas after surgical treatment. CA IX expression has been found in many malignancies, including those arising in the brain, head and neck, kidney, pancreas, uterine cervix, and other organs. This suggests that CA IX expression correlates with oncogenesis, tumor progression, and outcome [4,5,7,8,34,35]. In the present study, CA IX expression was predominantly found in tumor cells of ameloblastomas, similarly to HIF-1␣ expression, and was often marked in peripheral areas of tumor tissues. Expression levels of CA IX were significantly higher in ameloblastomas than in dental follicles, suggesting that HIF-1␣ signaling may play a role in increased proliferation and tumorigenesis of the odontogenic epithelium. Moreover, solid ameloblastomas showed significantly higher expression of CA IX than unicystic ameloblastomas, and follicular ameloblastomas exhibited significantly higher CA IX reactivity than plexiform ameloblastomas. These features suggest that the tissue architecture of ameloblas-
298
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
Fig. 2. Immunohistochemical expression of carbonic anhydrase IX. (A) Dental follicle showing expression in dental lamina ( × 400). (B) Follicular ameloblastoma showing strong expression in many peripheral columnar cells and moderate to strong reactivity in central polyhedral cells ( × 200). (C) Plexiform ameloblastoma showing diffusely expression in most tumor cells ( × 200). (D) Acanthomatous ameloblastoma showing expression in most tumor cells and increased reactivity around a keratinizing area (arrows) ( × 200). (E) Granular cell ameloblastoma showing expression in most tumor cells and increased reactivity in peripheral non-granular cells ( × 100). (F) Unicystic ameloblastoma (intraluminal type) showing diffusely expression in ameloblastomatous epithelium lining and protruding into the cyst wall ( × 40).
tomas might reflect their hypoxic microenvironment. Besides, we found increased CA IX reactivity around keratinizing cells as well as high HIF-1␣ reactivity in keratinizing areas in acanthomatous ameloblastomas, and non-granular tumor cells exhibited strong CA IX expression in granular cell ameloblastomas. These features suggest that CA IX may be associated with specific cytodifferentiation of ameloblastoma cells. Expression of HIF-1␣ and CA IX in acanthomatous ameloblastomas is considered to indicate close correlation between hypoxic states and keratinization in the epithelial odontogenic tumors. In normal physiological conditions, angiogenesis is tightly regulated by various positive effectors and natural inhibitors [37]. The hypoxic induction of angiogenesis is a hallmark of pathological processes, such as wound healing and solid tumor formation, and is strongly correlated with the disrupted circulation and rapid growth characteristics of those processes. In these hypoxic areas, VEGF is a powerful hypoxia-induced mitogen for vascular endothelial cell growth and plays a critical role in the development of vessels [4,38]. In our previous study, VEGF up-regulation and increased MVD were detected in ameloblastic tumors, suggesting that VEGF
production by odontogenic epithelial cells and its related angiogenesis were associated with pathophysiology of the neoplastic lesions [19]. Previous immunohistochemical studies have shown association of HIF-1␣ expression with increased MVD in lung and urothelial carcinoma [38,39] as well as a relationship between CA IX expression and high MVD in pancreatic ductal adenocarcinoma [40]. In the present study, immunohistochemical evaluation of MVD revealed no significant difference in CD34 expression between dental follicles and ameloblastomas, nor between primary and recurrence ameloblastomas. MVD was significantly higher in solid ameloblastomas than in unicystic ameloblastomas. Furthermore, MVD tended to be greater in follicular ameloblastomas than in plexiform ameloblastomas, in granular cell ameloblastomas than in other subtypes of ameloblastomas, and in mural type unicystic ameloblastomas than in other types of unicystic ameloblastomas. Alterations of these MVD values were generally linked to expression of HIF-1␣ and CA IX, suggesting that angiogenesis in a variety of tissue structures and tumor cell differentiation of ameloblastomas might be affected by the hypoxic agents in the neoplasm microenvironment.
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
299
Fig. 3. Immunohistochemical expression of CD34. (A) Dental follicle showing expression in microvessels of the connective tissue ( × 400). (B) Follicular ameloblastoma showing expression in small vessels in the tumor stroma ( × 200). (C) Plexiform ameloblastoma showing expression in dilated vessels in the tumor stroma ( × 200). (D) Unicystic ameloblastoma (mural type) showing expression in small vessels in the tumor stroma ( × 200).
Conflict of interest statement The authors declare that they have no conflicts of interest to declare. References [1] Gardner DG, Heikinheimo K, Shear M, Philipsen HP, Coleman H. Ameloblastomas. In: Barnes L, Eveson JW, Reichart PA, Sidransky D, editors. WHO classification of tumours. Pathology and genetics. Head and neck tumours. Lyon: IARC Press; 2005. p. 296–300. [2] Robinson RA, Vincent SD. AFIP atlas of tumor pathology. Tumors and cysts of the jaws. Silver Spring : ARP Press; 2012. p. 45–64. [3] Höckel M, Vaupel P. Biological consequences of tumor hypoxia. Semin Oncol 2001;28:36–41. [4] Said HM, Hagemann C, Staab A. Stojic J, Kühnel S, Vince GH, et al Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1␣ in human glioma in vitro and in vivo. Radiotherapy Oncol 2007;83:398–405. [5] Erpolat OP, Gocun PU, Akmansu M, Ozgun G, Akyol G. Hypoxia-related molecules HIF-1␣, CA9, and osteopontin: predictors of survival in patients with high-grade glioma. Strahlenther Onkol 2013;189:147–54. [6] Le QT, Courter D. Clinical biomarkers for hypoxia targeting. Cancer Metastasis Rev 2008;27:351–62. [7] Vordermark D, Brown JM. Endogenous markers of tumor hypoxia. Strahlenther Onkol 2003;179:801–11. [8] Le QT, Kong C, Lavori PW, O’byrne K, Erler JT, Huang X, et al. Expression and prognostic significance of a panel of tissue hypoxia markers in head and neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys 2007;69:167–75. [9] Jensen RL. Brain tumor hypoxia: tumorigenesis, angiogenesis, imaging, pseudoprogression, and as a therapeutic target. J Neurooncol 2009;92:317–35. [10] Sreekanthreddy P, Srinivasan H, Kumar DM, Nijaguna MB, Sridevi S, Vrinda M, et al. Identification of potential serum biomarkers of glioblastoma: serum osteopontin levels correlate with poor prognosis. Cancer Epidemiol Biomarkers Prev 2010;19:1409–22. [11] Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995;92:5510–4. [12] Fillies T, Werkmeister R, van Diest PJ, Brandt B, Joos U, Buerger H. HIF1-alpha overexpression indicates a good prognosis in early stage squamous cell carcinomas of the oral floor. BMC Cancer 2005;5:84. [13] Brennan PA, Mackenzie N, Quintero M. Hypoxia-inducible factor 1alpha in oral cancer. J Oral Pathol Med 2005;34:385–9.
[14] Birner P, Schindl M, Obermair A, Plank C, Breitenecker G, Oberhuber G. Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res 2000;60:4693–6. [15] Bos R, van der Groep P, Greijer AE, Shvarts A, Meijer S, Pinedo HM, et al. Levels of hypoxia-inducible factor 1alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer 2003;97:1573–81. [16] Schilling D, Bayer C, Emmerich K, Molls M, Vaupel P, Huber RM, et al. Basal HIF-1␣ expression levels are not predictive for radiocensitivity of human cancer cell lines. Strahlenther Onkol 2012;188:353–8. [17] Lando D, Peer DJ, Whelan DA, Gorman JJ, Whitelaw ML. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 2002;295:858–61. [18] Potter C, Harris AL. Hypoxia inducible carbonic anhydrase IX, marker of tumor hypoxia, survival pathway and therapy target. ABBV Cell Cycle 2004;3:164–7. [19] Kumamoto H, Ohki K, Ooya K. Association between vascular endothelial growth factor (VEGF) expression and tumor angiogenesis in ameloblastomas. J Oral Pathol Med 2002;31:28–34. [20] Kumamoto H, Izutsu T, Ohki K, Takahashi N. Ooya K. p53 gene status and expression of p53, MDM2, and p14 proteins in ameloblastomas. J Oral Pathol Med 2004;33:292–9. [21] Kumamoto H, Ooya K. Immunohistochemical analysis of bcl-2 family proteins in benign and malignant ameloblastomas. J Oral Pathol Med 1999;28:343–9. [22] Kumamoto H, Ooya K. Immunohistochemical detection of BH3-only proteins in ameloblastic tumors. Oral Dis 2008;14:550–5. [23] Kumamoto H, Ooya K. Expression of tumor necrosis factor alpha, TNF-related apoptosis-inducing ligand, and their associated molecules in ameloblastomas. J Oral Pathol Med 2005;34:287–94. [24] Kumamoto H, Ooya K. Immunohistochemical detection of platelet-derived endothelial cell growth factor/thymidine phosphorylase and angiopoietins in ameloblastic tumors. J Oral Pathol Med 2006;35:606–12. [25] Takahashi Y, Kiatadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 1995;55:3964–8. [26] Vermeulen PB, Gasparini G, Fox SB, Toi M, Martin L, McCulloch P, et al. Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation. Eur J Cancer 1996;32A:2474–84. [27] Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma. N Engl J Med 1991;324:1–8. [28] Krieg M, Haas R, Brauch H, Acker T, Flamme I, Plate KH. Up-regulation of hypoxia-inducible factors HIF-1␣ and HIF-2␣ under normoxic conditions in
300
[29]
[30]
[31]
[32]
[33] [34]
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300 renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function. Oncogene 2000;19:5435–43. Salceda S, Caro J. Hypoxia-inducible factor 1␣ (HIF-1␣) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 1997;272:22642–7. Zhou J, Hara K, Inoue M, Hamada S, Yasuda H, Moriyama H, et al. Regulation of hypoxia-inducible factor 1 by glucose availability under hypoxic conditions. Kobe J Med Sci 2008;53:283–96. Opavsky´ R, Pastoreková S, Zelník V, Gibadulinová A, Stanbridge EJ, Závada J, et al. Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships. Genomics 1996;33:480–7. Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, et al. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrase in human cancer. Am J Pathol 2001;158:905–19. Li Y, Dong M, Sheng W, Huang L. Roles of carbonic anhydrase IX in development of pancreatic cancer. Pathol Oncol 2016;22:277–86. Luong-Player A, Liu H, Wang HL, Lin F. Immunohistochemical reevaluation of carbonic anhydrase IX (CA IX) expression in tumors and normal tissues. Am J Clin Pathol 2014;141:219–25.
[35] Guo L, Li J, Qiao X, Yu M, Tang W, Wang H, et al. Comparison of odontogenic differentiation of human dental follicle cells and human dental papilla cells. PLoS One 2013;8:e62332. [36] Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J 1998;17:3005–15. [37] Schlaeppi JM, Wood JM. Targeting vascular endothelial growth factor (VEGF) for anti-tumor therapy, by anti-VEGF neutralizing monoclonal antibodies or by VEGF receptor tyrosine-kinase inhibitors. Cancer Metastasis Rev 1999;18:473–81. [38] Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H, Talks K, Pezzella F, et al. Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival. Br J Cancer 2001;85:881–90. [39] Chai CY, Chen WT, Hung WC, Kang WY, Huang YC, Su YC, et al. Hypoxia-inducible factor-1␣ expression correlates with focal macrophage infiltration, angiogenesis and unfavourable prognosis in urothelial carcinoma. J Clin Pathol 2008;61:658–64. [40] Couvelard A, O’Toole D, Leek R, Turley H, Sauvanet A, Degott C, et al. Expression of hypoxia-inducible factors is correlated with the presence of a fibrotic focus and angiogenesis in pancreatic ductal adenocarcinomas. Histopathology 2005;46:668–76.
Contents lists available at ScienceDirect
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology journal homepage: www.elsevier.com/locate/jomsmp
Oral Pathology/Original Research
Immunohistochemical assessment of hypoxia-inducible factor-1␣ (HIF-1␣) and carbonic anhydrase IX (CA IX) in ameloblastomas Yoshiki Arima a,b , Mariko Oikawa b,∗ , Yoshinaka Shimizu b , Seishi Echigo a , Tetsu Takahashi a , Hiroyuki Kumamoto b a Division of Oral and Maxillofacial Surgery, Department of Oral Medicine and Surgery, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan b Division of Oral Pathology, Department of Oral Medicine and Surgery, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
a r t i c l e
i n f o
Article history: Received 23 August 2016 Received in revised form 5 October 2017 Accepted 13 October 2017 Available online 27 March 2018 Keywords: Ameloblastoma Hypoxia HIF-1␣ CA IX Angiogenesis
a b s t r a c t Objective: To investigate roles of hypoxia-related proteins in odontogenic tumors, we analyzed the immunohistochemical expression of hypoxia-inducible factor-1␣ (HIF-1␣) and carbonic anhydrase IX (CA IX) and compared with angiogenesis. Methods: 10 dental follicles and 67 ameloblastomas were immunohistochemically examined with antibodies against HIF-1␣, CA IX, and CD34. Results: Immunohistochemical reactivity for HIF-1␣ and CA IX was detected in odontogenic epithelial cells, as well as in several macrophages and fibroblasts, in dental follicles and ameloblastomas. HIF1␣ was positive in 8 of 10 dental follicles, 45 of 48 primary ameloblastomas, and all 19 recurrent ameloblastomas. Increased HIF-1␣ reactivity was often found in keratinizing cells in acanthomatous ameloblastomas. Immunoreactivity for CA IX was detected in all samples of dental follicles and primary and recurrent ameloblastomas. CA IX reactivity was significantly higher in ameloblastomas than in dental follicles, in solid ameloblastomas than in unicystic ameloblastomas, and in follicular ameloblastomas than in plexiform ameloblastomas. Acanthomatous ameloblastomas showed increased CA IX reactivity around keratinizing areas, while granular cell ameloblastomas showed increased reactivity in peripheral non-granular cells. Microvessel density (MVD) of CD34-positive capillaries in solid ameloblastomas was significantly higher than in unicystic ameloblastomas. MVD tended to be greater in follicular ameloblastomas than in plexiform ameloblastomas, in granular cell ameloblastomas than in other subtypes of ameloblastomas, and in mural unicystic ameloblastomas than in other types of unicystic ameloblastomas. Conclusions: Our data suggest that hypoxia-related molecules and angiogenesis play a role in tumorigenesis, tissue structuring, cell differentiation, and prognosis of ameloblastomas in the intraosseous microenvironment. © 2018 Asian AOMS, ASOMP, JSOP, JSOMS, JSOM, and JAMI. Published by Elsevier Ltd. All rights reserved.夽
1. Introduction Ameloblastoma is the most frequently encountered epithelial odontogenic tumor arising in the jawbones, and is characterized by benign but locally invasive behavior with a high rate of recurrence. Therefore, clinical long-term follow-up is essential, and the required treatment includes excision with an adequate margin of uninvolved tissues, similar to that for malignant tumors [1,2]. This tumor is usually solid and unicystic. Histologically, solid ameloblas-
∗ Corresponding author. E-mail address: [email protected] (M. Oikawa).
tomas show follicular and plexiform proliferation patterns with or without acanthomatous and granular cell changes, and unicystic ameloblastomas have luminal, intraluminal, and mural variants [1]. In some respects, these epithelial odontogenic tumors histologically resemble physiological structures, such as enamel organ or dental lamina; however, the detailed mechanisms of oncogenesis and cytodifferentiation remain unknown. Hypoxia is thought to trigger a more aggressive tumor phenotype by inducing genomic instability, loss of apoptotic potential, and angiogenesis, which is the sprouting of new capillaries from a pre-existing vascular bed [3–5]. It plays a significant role in tumor recurrence, metastasis, and poor response to treatment, including radiotherapy, chemotherapy, and antiangiogenic treatment [5,6].
https://doi.org/10.1016/j.ajoms.2017.10.002 2212-5558/© 2018 Asian AOMS, ASOMP, JSOP, JSOMS, JSOM, and JAMI. Published by Elsevier Ltd. All rights reserved.夽
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
Recently, molecules involved in the hypoxic response of tumor cells were identified as endogenous hypoxia markers using noninvasive and cost-effective methods to determine the condition of tumor hypoxia [7–10]. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric protein consisting of an alpha and beta subset, in which the ␣ subset mediates HIF-1 function as a transcription factor in response to cellular hypoxia [11]. Expression of HIF-1␣ correlates with advanced tumor stage and metastasis, leading to poor outcomes of common human malignancies, such as carcinomas of the breast, brain, lung, prostate, and head and neck [8–10,12–16]. Carbonic anhydrase IX (CA IX) is one of the HIF-1␣-dependent enzymes most consistently upregulated under hypoxic conditions. Since there is an oxygen-dependent step that inhibits HIF-1␣ transactivation of target genes, it would be anticipated that CA IX protein levels, like other target gene products, would show a better correlation with low oxygen levels than would HIF-1␣ itself [4,7,17]. The effect of CA IX on the tumor microenvironment is characterized by the regulation of pH [18]. Recently, some human tumors have been reported to express CA IX, which is proposed to influence the clinical features of these tumors [4,5,7–9,16]. Thus, HIF-1␣ and CA IX play a role in angiogenesis, apoptosis, invasion, and metastasis of malignant tumors [8,9]. Our previous studies have confirmed the presence of angiogenic factors, vascular endothelial growth factor (VEGF), thymidine phosphorylase, and angiopoietins, and apoptosis-inducing molecules, p53, Bax, BH3-only proteins, and TNF family molecules, in ameloblastic tumors [19–24]. However, hypoxia-related factors in epithelial odontogenic tumors remains poorly understood. In the present study, HIF-1␣ and CA IX expression, as well as CD34 as a marker of microvessel density (MVD), were immunohistochemically examined in ameloblastomas as well as in dental follicles to clarify the role of these hypoxic agents in epithelial odontogenic tumors.
2. Material and methods The study protocol was reviewed and approved by the Research Ethics Committee of Tohoku University Graduate School of Dentistry (No. 2014-1-854).
2.1. Tissue preparation Specimens were surgically removed from 63 patients with ameloblastoma at Tohoku University Hospital and affiliated hospitals. The specimens were fixed in 10% buffered formalin for one to several days and were embedded in paraffin. The tissue blocks were sliced into 3 m-thick sections for routine histological and subsequent immunohistochemical examinations. Tissue sections were stained with hematoxylin and eosin for histological diagnosis according to the World Health Organization histological classification of odontogenic tumors [1]. The specimens consisted of 48 primary ameloblastomas and 19 recurrent ameloblastomas. Of the primary ameloblastomas, 37 were solid, and 11 were unicystic. The solid ameloblastomas were divided into 19 follicular and 18 plexiform types, including 11 acanthomatous and three granular cell subtypes. The unicystic ameloblastomas were divided into five luminal, three intraluminal, and three mural types. All recurrent ameloblastomas were solid and were divided into 13 follicular and 6 plexiform types, including 10 acanthomatous and 1 granular cell subtypes. Specimens of 10 dental follicles of the mandibular third molars were similarly prepared and compared with the ameloblastomas.
295
2.2. Immunohistochemistry for HIF-1˛, CA IX, and CD34 expression Tissue sections were deparaffinized and immersed in methanol with 0.3% hydrogen peroxide. Sections were heated in 0.01 M citrate buffer (pH 6.0; for HIF-1␣) or 1 mM EDTA buffer (pH 9.0; for CA IX) for 10 min by autoclaving (121 ◦ C, 2 atm). Next, the sections were incubated with primary antibodies at 4 ◦ C overnight. The antibodies used were as follows: rabbit anti-HIF-1␣ monoclonal antibody (Abcam, Cambridge, UK; isotype IgG; diluted at 1:100), rabbit anti-CA IX polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; purified IgG; diluted at 1:50), and mouse anti-CD34 monoclonal antibody (Nichirei, Tokyo, Japan; isotype IgG1; prediluted). The sections were allowed to react with peroxidase-conjugated anti-rabbit IgG (for HIF-1␣ and CA IX) or anti-mouse IgG (for CD34) polyclonal antibodies (Histofine Simple Stain MAX-PO; Nichirei, Tokyo, Japan) for 45 min, and reaction products were visualized by immersing the sections in 0.03% diaminobenzidine solution containing 2 mM hydrogen peroxide for 3–5 min. Nuclei were lightly stained with Mayer’s hematoxylin. For control studies of the antibodies, the serial sections were treated with phosphate-buffered saline, normal rabbit IgG, and mouse antidesmin monoclonal antibody (Nichirei; isotype IgG1) instead of the primary antibodies; these were confirmed to be unstained. 2.3. Evaluation of immunostaining and statistical analysis Immunohistochemical reactivity for HIF-1␣ was evaluated and classified into three groups: (−) negative in epithelial or tumor cells, (±) weakly positive (less than 30% of epithelial or tumor cells), and (+) moderately to strongly positive (more than 30% of epithelial or tumor cells) [14]. Immunohistochemical reactivity for CA IX was evaluated and classified into two groups: (+) diffusely positive in most epithelial or tumor cells, and (++) diffusely positive in most epithelial or tumor cells with increased reactivity in those epithelial or tumor cells near the basement membrane. Immunoreactivity for CD34 was conducted to evaluate MVD in the dental follicles and ameloblastomas. After scanning 5 areas showing the highest neovascularization (vascular hotspots) in normal mesenchymal tissues or tumor stromal tissues at 20-fold magnification, CD34-positive vessels were counted in the hotspots at 200-fold magnification, and the average count was recorded as MVD for each case [19,25,26]. Based on the criteria of Weidner et al. [27], a highlighted endothelial cell or a cell cluster clearly separate from adjacent microvessels, tumor cells, and other connective tissue elements was regarded as a distinct countable microvessel. A lumen was not required, nor was the presence of red blood cells. Single cell sprouts were included in the counts. The statistical significance of the differences in the percentage of cases with various HIF-1␣ and CA IX reactivity levels, as well as mean MVD, was determined by the Mann-Whitney U-test for differences between two groups or the Kruskal-Wallis test for differences among three or more groups. P-values <0.05 were considered to indicate statistical significance. 3. Results The results of immunohistochemical studies of HIF-1␣ and CA IX expression and MVD in dental follicles and ameloblastomas are summarized in Table 1. Immunohistochemical reactivity for HIF-1␣ was detected in the cytoplasm and nuclei of odontogenic epithelial cells as well as in several macrophages and fibroblasts in the dental follicles and ameloblastomas (Fig. 1). Dental lamina showed HIF-1␣ reactivity in 8 of 10 dental follicles (Fig. 1A, Table 1). In primary ameloblastomas, tumor cells showed HIF-1␣
296
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
Table 1 Immunohistochemical reactivity for HIF-1␣ and CA IX, as well as MVD in dental follicles and ameloblastomas.
HIF-1␣ expression: (−) negative in epithelial or tumor cells, (±) weakly (less than 30% of epithelial or tumor cells) positive, (+) moderately to strongly (more than 30% of epithelial or tumor cells) positive. CA IX expression: (+) diffusely positive in most epithelial or tumor cells, (++) diffusely positive in most epithelial or tumor cells with increased reactivity in epithelial or neoplastic cells near the basement membrane. Values in parentheses denote percentage values.HIF-1␣: hypoxia-inducible factor-1␣; CA IX: carbonic anhydrase IX; MVD: microvessel density.
reactivity in 45 of 48 cases, including 35 of 37 solid ameloblastomas and 10 of 11 unicystic ameloblastomas (Fig. 1B–D, Table 1). Solid ameloblastomas showed HIF-1␣ expression in all 19 follicular cases and 16 of 18 plexiform cases, and all acanthomatous and granular cell ameloblastomas were reactive (Fig. 1B, C, Table 1). Increased HIF-1␣ reactivity was often found in keratinizing cells of acanthomatous ameloblastomas (Fig. 1C). Tumor cell in unicystic ameloblastomas showed HIF-1␣ reactivity in all 5 luminal, all 3 intraluminal, and 2 of 3 mural types (Fig. 1D, Table 1). While 35 of 37 primary solid ameloblastomas were reactive with HIF-1␣, HIF1␣ reactivity was noted in all 19 recurrent solid ameloblastomas (Table 1). HIF-1␣ reactivity did not differ significantly among these odontogenic lesions or ameloblastomas variants. Immunoreactivity for CA IX was detected in the cell membrane and cytoplasm of odontogenic epithelial cells as well as some macrophages and fibroblasts in dental follicles and ameloblastomas (Fig. 2). CA IX reactivity was detected in dental lamina of dental follicles (Fig. 2A). Ameloblastomas showed CA IX expression in most tumor cells (Fig. 2B-F). Solid ameloblastomas often showed increased CA IX reactivity in peripheral columnar or cuboidal tumor cells as compared with central polyhedral cells (Fig. 2B, E). CA IX expression in ameloblastomas was significantly higher than that in dental follicles (P < 0.05, Table 1), and immunoreactivity for CA IX was higher in solid ameloblastomas than in unicystic ameloblastomas (P < 0.01, Table 1). Follicular ameloblastomas exhibited
statistically higher CA IX expression than plexiform ameloblastomas (P < 0.01, Table 1). Acanthomatous ameloblastomas showed increased reactivity around keratinizing areas (Fig. 2D), while granular cell ameloblastomas showed decreased reactivity in granular tumor cells (Fig. 2E, Table 1). Unicystic ameloblastomas showed CA IX reactivity in ameloblastomatous epithelium that lined, protruded into, and infiltrated cyst walls (Fig. 2F, Table 1). Difference of CA IX reactivity between primary and recurrent ameloblastomas did not achieve statistical significance (Table 1). Immunohistochemical staining for CD34 was detected in the cytoplasm of endothelial cells in both normal and tumor odontogenic tissues (Fig. 3). In dental follicles, CD34-positive microvessels were observed in areas near dental lamina (Fig. 3A). Ameloblastomas showed CD34 reactivity in microvessels of tumor stroma (Figs. 3B-D), and MVD in solid ameloblastomas was significantly higher than that in unicystic ameloblastomas (P < 0.05; Table 1). Follicular ameloblastomas showed CD34-positive vessels in many small vessels (Fig. 3B), whereas CD34-positive vessels in plexiform ameloblastomas were scattered and dilated (Fig. 3C, Table 1). In granular cell ameloblastomas, numerous CD34-positive microvessels were found as compared with other cellular variants (Table 1). The distribution of CD34 reactivity in the unicystic ameloblastomas was similar to that in solid ameloblastomas (Fig. 3D). In mural type unicystic ameloblastomas, many CD34-positive microvessels were recognized as compared to luminal or intraluminal types (Table 1).
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
297
Fig. 1. Immunohistochemical expression of hypoxia-inducible factor-1␣. (A) Dental follicle showing no expression ( × 400). (B) Plexiform ameloblastoma showing strong expression in most tumor cells ( × 200). (C) Acanthomatous ameloblastoma showing expression in most tumor cells with increased reactivity in keratinizing cells (arrow) ( × 400). (D) Unicystic ameloblastoma (mural type) showing expression in ameloblastomatous epithelium lining and infiltrating the cyst wall ( × 200).
MVDs in primary and recurrent ameloblastomas were not statistically different (Table 1). 4. Discussion Hypoxia induces transcription of physiologically important genes, including erythropoietin and VEGF. HIF-1␣ is a transcription factor responsive to hypoxia, and expression of HIF-1␣ is regulated by cellular oxygen level alterations [28]. Under normoxic conditions, HIF-1␣ is a short lived-protein because of its continuous proteolysis via the ubiquitin-proteasome pathway. In contrast, reduced oxygen availability induces HIF-1␣ accumulation through the inhibition of its degradation [29,30]. CAs are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide, participating in calcification, acid-base balance, bone resorption, and formation of the aqueous humor [31]. CAs include at least 15 types of isozymes, among which CA IX can be activated via the HIF-1 pathway under hypoxic conditions. CA IX maintains pH and adapts cellular components to the hypoxic microenvironment, thus ensuring the continued proliferation of cells in hypoxic milieus [32,33]. Recently, HIF-1␣ and CA IX expression has been reported in several normal human tissues [3,6,34]. Tooth germs have been shown to express HIF-1␣, suggesting its ability to regulate the intraosseous microenvironment and to promote odontogenic cell survival in hypoxic conditions [35]. The present study showed expression of HIF-1␣ and CA IX in epithelial and mesenchymal odontogenic cells of human dental follicle tissues. Our previous study showed VEGF expression in tooth development processes, which was anticipated given the hypoxic conditions of these processes [19]. These findings suggest that angiogenesis during tooth formation might be regulated under a certain level of hypoxia. Expression of HIF-1␣ has been found in many human tumors, including breast, brain, renal, and head and neck carcinomas, and has been correlated with more aggressive tumor grades, tumor invasion, metastatic potential, resistance to radiation therapy, and
poorer prognoses [7–10,12,15]. Cell lines genetically manipulated to knock down HIF-1˛ have shown decreased cell growth in vitro [36]. In the present study, immunohistochemical reactivity for HIF1␣ was found mainly in tumor cells of ameloblastomas, often strongly in keratinizing areas, suggesting that the tumor tissues were exposed to hypoxic conditions similarly to dental follicle tissues. Our previous study revealed VEGF expression was higher in ameloblastic tumors than in tooth germs [19], whereas the present study did not detect differences between non-neoplastic and neoplastic odontogenic tissues. Furthermore, follicular ameloblastomas tended to show slightly higher expression of HIF-1␣ than plexiform ameloblastomas, and cases without cellular variation tended to exhibit low HIF-1␣ expression compared with acanthomatous and granular cell subtypes. These features suggest that tissue structuring and/or cell differentiation of ameloblastomas may be affected by HIF-1␣ expression. Furthermore, immunohistochemical analysis revealed that recurrent ameloblastomas showed slightly higher HIF-1␣ expression than primary ameloblastomas, suggesting that a hypoxic microenvironment may be related to prognosis in ameloblastomas after surgical treatment. CA IX expression has been found in many malignancies, including those arising in the brain, head and neck, kidney, pancreas, uterine cervix, and other organs. This suggests that CA IX expression correlates with oncogenesis, tumor progression, and outcome [4,5,7,8,34,35]. In the present study, CA IX expression was predominantly found in tumor cells of ameloblastomas, similarly to HIF-1␣ expression, and was often marked in peripheral areas of tumor tissues. Expression levels of CA IX were significantly higher in ameloblastomas than in dental follicles, suggesting that HIF-1␣ signaling may play a role in increased proliferation and tumorigenesis of the odontogenic epithelium. Moreover, solid ameloblastomas showed significantly higher expression of CA IX than unicystic ameloblastomas, and follicular ameloblastomas exhibited significantly higher CA IX reactivity than plexiform ameloblastomas. These features suggest that the tissue architecture of ameloblas-
298
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
Fig. 2. Immunohistochemical expression of carbonic anhydrase IX. (A) Dental follicle showing expression in dental lamina ( × 400). (B) Follicular ameloblastoma showing strong expression in many peripheral columnar cells and moderate to strong reactivity in central polyhedral cells ( × 200). (C) Plexiform ameloblastoma showing diffusely expression in most tumor cells ( × 200). (D) Acanthomatous ameloblastoma showing expression in most tumor cells and increased reactivity around a keratinizing area (arrows) ( × 200). (E) Granular cell ameloblastoma showing expression in most tumor cells and increased reactivity in peripheral non-granular cells ( × 100). (F) Unicystic ameloblastoma (intraluminal type) showing diffusely expression in ameloblastomatous epithelium lining and protruding into the cyst wall ( × 40).
tomas might reflect their hypoxic microenvironment. Besides, we found increased CA IX reactivity around keratinizing cells as well as high HIF-1␣ reactivity in keratinizing areas in acanthomatous ameloblastomas, and non-granular tumor cells exhibited strong CA IX expression in granular cell ameloblastomas. These features suggest that CA IX may be associated with specific cytodifferentiation of ameloblastoma cells. Expression of HIF-1␣ and CA IX in acanthomatous ameloblastomas is considered to indicate close correlation between hypoxic states and keratinization in the epithelial odontogenic tumors. In normal physiological conditions, angiogenesis is tightly regulated by various positive effectors and natural inhibitors [37]. The hypoxic induction of angiogenesis is a hallmark of pathological processes, such as wound healing and solid tumor formation, and is strongly correlated with the disrupted circulation and rapid growth characteristics of those processes. In these hypoxic areas, VEGF is a powerful hypoxia-induced mitogen for vascular endothelial cell growth and plays a critical role in the development of vessels [4,38]. In our previous study, VEGF up-regulation and increased MVD were detected in ameloblastic tumors, suggesting that VEGF
production by odontogenic epithelial cells and its related angiogenesis were associated with pathophysiology of the neoplastic lesions [19]. Previous immunohistochemical studies have shown association of HIF-1␣ expression with increased MVD in lung and urothelial carcinoma [38,39] as well as a relationship between CA IX expression and high MVD in pancreatic ductal adenocarcinoma [40]. In the present study, immunohistochemical evaluation of MVD revealed no significant difference in CD34 expression between dental follicles and ameloblastomas, nor between primary and recurrence ameloblastomas. MVD was significantly higher in solid ameloblastomas than in unicystic ameloblastomas. Furthermore, MVD tended to be greater in follicular ameloblastomas than in plexiform ameloblastomas, in granular cell ameloblastomas than in other subtypes of ameloblastomas, and in mural type unicystic ameloblastomas than in other types of unicystic ameloblastomas. Alterations of these MVD values were generally linked to expression of HIF-1␣ and CA IX, suggesting that angiogenesis in a variety of tissue structures and tumor cell differentiation of ameloblastomas might be affected by the hypoxic agents in the neoplasm microenvironment.
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300
299
Fig. 3. Immunohistochemical expression of CD34. (A) Dental follicle showing expression in microvessels of the connective tissue ( × 400). (B) Follicular ameloblastoma showing expression in small vessels in the tumor stroma ( × 200). (C) Plexiform ameloblastoma showing expression in dilated vessels in the tumor stroma ( × 200). (D) Unicystic ameloblastoma (mural type) showing expression in small vessels in the tumor stroma ( × 200).
Conflict of interest statement The authors declare that they have no conflicts of interest to declare. References [1] Gardner DG, Heikinheimo K, Shear M, Philipsen HP, Coleman H. Ameloblastomas. In: Barnes L, Eveson JW, Reichart PA, Sidransky D, editors. WHO classification of tumours. Pathology and genetics. Head and neck tumours. Lyon: IARC Press; 2005. p. 296–300. [2] Robinson RA, Vincent SD. AFIP atlas of tumor pathology. Tumors and cysts of the jaws. Silver Spring : ARP Press; 2012. p. 45–64. [3] Höckel M, Vaupel P. Biological consequences of tumor hypoxia. Semin Oncol 2001;28:36–41. [4] Said HM, Hagemann C, Staab A. Stojic J, Kühnel S, Vince GH, et al Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1␣ in human glioma in vitro and in vivo. Radiotherapy Oncol 2007;83:398–405. [5] Erpolat OP, Gocun PU, Akmansu M, Ozgun G, Akyol G. Hypoxia-related molecules HIF-1␣, CA9, and osteopontin: predictors of survival in patients with high-grade glioma. Strahlenther Onkol 2013;189:147–54. [6] Le QT, Courter D. Clinical biomarkers for hypoxia targeting. Cancer Metastasis Rev 2008;27:351–62. [7] Vordermark D, Brown JM. Endogenous markers of tumor hypoxia. Strahlenther Onkol 2003;179:801–11. [8] Le QT, Kong C, Lavori PW, O’byrne K, Erler JT, Huang X, et al. Expression and prognostic significance of a panel of tissue hypoxia markers in head and neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys 2007;69:167–75. [9] Jensen RL. Brain tumor hypoxia: tumorigenesis, angiogenesis, imaging, pseudoprogression, and as a therapeutic target. J Neurooncol 2009;92:317–35. [10] Sreekanthreddy P, Srinivasan H, Kumar DM, Nijaguna MB, Sridevi S, Vrinda M, et al. Identification of potential serum biomarkers of glioblastoma: serum osteopontin levels correlate with poor prognosis. Cancer Epidemiol Biomarkers Prev 2010;19:1409–22. [11] Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995;92:5510–4. [12] Fillies T, Werkmeister R, van Diest PJ, Brandt B, Joos U, Buerger H. HIF1-alpha overexpression indicates a good prognosis in early stage squamous cell carcinomas of the oral floor. BMC Cancer 2005;5:84. [13] Brennan PA, Mackenzie N, Quintero M. Hypoxia-inducible factor 1alpha in oral cancer. J Oral Pathol Med 2005;34:385–9.
[14] Birner P, Schindl M, Obermair A, Plank C, Breitenecker G, Oberhuber G. Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res 2000;60:4693–6. [15] Bos R, van der Groep P, Greijer AE, Shvarts A, Meijer S, Pinedo HM, et al. Levels of hypoxia-inducible factor 1alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer 2003;97:1573–81. [16] Schilling D, Bayer C, Emmerich K, Molls M, Vaupel P, Huber RM, et al. Basal HIF-1␣ expression levels are not predictive for radiocensitivity of human cancer cell lines. Strahlenther Onkol 2012;188:353–8. [17] Lando D, Peer DJ, Whelan DA, Gorman JJ, Whitelaw ML. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 2002;295:858–61. [18] Potter C, Harris AL. Hypoxia inducible carbonic anhydrase IX, marker of tumor hypoxia, survival pathway and therapy target. ABBV Cell Cycle 2004;3:164–7. [19] Kumamoto H, Ohki K, Ooya K. Association between vascular endothelial growth factor (VEGF) expression and tumor angiogenesis in ameloblastomas. J Oral Pathol Med 2002;31:28–34. [20] Kumamoto H, Izutsu T, Ohki K, Takahashi N. Ooya K. p53 gene status and expression of p53, MDM2, and p14 proteins in ameloblastomas. J Oral Pathol Med 2004;33:292–9. [21] Kumamoto H, Ooya K. Immunohistochemical analysis of bcl-2 family proteins in benign and malignant ameloblastomas. J Oral Pathol Med 1999;28:343–9. [22] Kumamoto H, Ooya K. Immunohistochemical detection of BH3-only proteins in ameloblastic tumors. Oral Dis 2008;14:550–5. [23] Kumamoto H, Ooya K. Expression of tumor necrosis factor alpha, TNF-related apoptosis-inducing ligand, and their associated molecules in ameloblastomas. J Oral Pathol Med 2005;34:287–94. [24] Kumamoto H, Ooya K. Immunohistochemical detection of platelet-derived endothelial cell growth factor/thymidine phosphorylase and angiopoietins in ameloblastic tumors. J Oral Pathol Med 2006;35:606–12. [25] Takahashi Y, Kiatadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 1995;55:3964–8. [26] Vermeulen PB, Gasparini G, Fox SB, Toi M, Martin L, McCulloch P, et al. Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation. Eur J Cancer 1996;32A:2474–84. [27] Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma. N Engl J Med 1991;324:1–8. [28] Krieg M, Haas R, Brauch H, Acker T, Flamme I, Plate KH. Up-regulation of hypoxia-inducible factors HIF-1␣ and HIF-2␣ under normoxic conditions in
300
[29]
[30]
[31]
[32]
[33] [34]
Y. Arima et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 30 (2018) 294–300 renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function. Oncogene 2000;19:5435–43. Salceda S, Caro J. Hypoxia-inducible factor 1␣ (HIF-1␣) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 1997;272:22642–7. Zhou J, Hara K, Inoue M, Hamada S, Yasuda H, Moriyama H, et al. Regulation of hypoxia-inducible factor 1 by glucose availability under hypoxic conditions. Kobe J Med Sci 2008;53:283–96. Opavsky´ R, Pastoreková S, Zelník V, Gibadulinová A, Stanbridge EJ, Závada J, et al. Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships. Genomics 1996;33:480–7. Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, et al. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrase in human cancer. Am J Pathol 2001;158:905–19. Li Y, Dong M, Sheng W, Huang L. Roles of carbonic anhydrase IX in development of pancreatic cancer. Pathol Oncol 2016;22:277–86. Luong-Player A, Liu H, Wang HL, Lin F. Immunohistochemical reevaluation of carbonic anhydrase IX (CA IX) expression in tumors and normal tissues. Am J Clin Pathol 2014;141:219–25.
[35] Guo L, Li J, Qiao X, Yu M, Tang W, Wang H, et al. Comparison of odontogenic differentiation of human dental follicle cells and human dental papilla cells. PLoS One 2013;8:e62332. [36] Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J 1998;17:3005–15. [37] Schlaeppi JM, Wood JM. Targeting vascular endothelial growth factor (VEGF) for anti-tumor therapy, by anti-VEGF neutralizing monoclonal antibodies or by VEGF receptor tyrosine-kinase inhibitors. Cancer Metastasis Rev 1999;18:473–81. [38] Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H, Talks K, Pezzella F, et al. Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival. Br J Cancer 2001;85:881–90. [39] Chai CY, Chen WT, Hung WC, Kang WY, Huang YC, Su YC, et al. Hypoxia-inducible factor-1␣ expression correlates with focal macrophage infiltration, angiogenesis and unfavourable prognosis in urothelial carcinoma. J Clin Pathol 2008;61:658–64. [40] Couvelard A, O’Toole D, Leek R, Turley H, Sauvanet A, Degott C, et al. Expression of hypoxia-inducible factors is correlated with the presence of a fibrotic focus and angiogenesis in pancreatic ductal adenocarcinomas. Histopathology 2005;46:668–76.