International Journal of Oncology. 2016 Mar;48(3):1258-70.
Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone
Yoshimoto S, Morita H, Matsubara R, Mitsuyasu T, Imai Y, Kajioka S, Yoneda M, Ito Y, Hirofuji T, Nakamura S, Hirata M.
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka, Japan
Special Patient Oral Care Unit, Kyushu University Hospital, Fukuoka, Japan
Department of General Dentistry, Fukuoka Dental College, Fukuoka, Japan
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, Fukuoka, Japan
Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Department of Physiology, School of Medicine, Kurume University, Kurume, Japan
Ameloblastoma is the most common benign odontogenic tumor in Japan. It is believed that it expands in the jaw bone through peritumoral activation of osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) released from the ameloblastoma, as in bone metastases of cancer cells. However, the clinical features of ameloblastoma, including its growth rate and patterns of invasion, are quite different from those of bone metastasis of cancer cells, suggesting that different underlying mechanisms are involved. Therefore, in the present study, we examined the possible mechanisms underlying the invasive expansion of ameloblastoma in the jaw bone. Expression levels of RANKL assessed by Western blotting were markedly lower in ameloblastoma (AM-1) cells than in highly metastatic oral squamous cell carcinoma (HSC-3) cells. Experiments coculturing mouse macrophages (RAW264.7) with AM-1 demonstrated low osteoclastogenic activity, as assessed by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell formation, probably because of low release of RANKL, whereas cocultures of RAW264.7 with HSC-3 cells exhibited very high osteoclastogenic activity. Thus, RANKL release from AM-1 appeared to be too low to generate osteoclasts. However, AM-1 cultured directly on calcium phosphate-coated plates formed resorption pits, and this was inhibited by application of bafilomycin A1. Furthermore, vacuolar-type H+-ATPase (V-ATPase) and H+/Cl− exchange transporter 7 (CLC-7) were detected on the surface of AM-1 cells by plasma membrane biotinylation and immunofluorescence analysis. Immunohistochemical analysis of clinical samples of ameloblastoma also showed plasma membrane-localized V-ATPase and CLC-7 in the epithelium of plexiform, follicular, and basal cell types. The demineralization activity of AM-1 was only 1.7 % of osteoclasts demineralization activity, and the growth rate was 20 % of human normal skin keratinocytes and HSC-3 cells. These results suggest that the slow expansion of several typical types of ameloblastomas in jaw bone is attributable to its slow growth and low demineralization ability.
Worldwide, ameloblastoma is a common odontogenic tumor and is characterized by slow but steady invasion into the maxillary and mandibular bones. A histopathological classification of ameloblastoma by the World Health Organization in 2005 defined four types: solid/multicystic, extraosseous/peripheral, desmoplastic, and unicystic. Solid/multicystic ameloblastoma is further divided into follicular and plexiform types, including the basal cell type (1,2).
Ameloblastoma is not a malignant lesion, however, treatment of any type of ameloblastoma is limited to surgical treatments such as enucleation and resection, although recurrence with significant morbidity is common after enucleation, particularly in young people (1-4). Accordingly, resection remains the best way to remove ameloblastoma, although this method is not without its drawbacks. Therefore, a better understanding of the pathophysiology of ameloblastoma is necessary, because there is a high demand for drugs capable of acting as selective inhibitors of ameloblastoma.
Expansion of solid/multicystic ameloblastoma in bone is thought to occur as a result of accelerated bone resorption activities by peritumoral osteoclasts. The activation of osteoclasts is triggered by the binding of receptor activator of nuclear factor kappa-B ligand (RANKL), which is released from vicinal ameloblastoma cells, to bind receptor activator of nuclear factor kappa-B (RANK) on the plasma membrane of osteoclasts, in a manner similar to that seen in bone-invasive cancers, particularly oral squamous cell carcinoma (SCC) (5-8). Furthermore, several matrix metalloproteinases (MMP; MMP-1, MMP-2, and MMP-9) released from ameloblastoma cells are also involved in progression of invasive lesions similar to that seen in oral invasive SCC (9-15).
However, ameloblastoma exhibits clinical features that differ from those of oral SCC, including its bone invasion patterns, rate of spread, and clinical symptoms. For example, solid/multicystic ameloblastoma and bone invasion of SCC show clear differences on X-ray transmission images (1,16,17). The border of a solid/multicystic ameloblastoma of the jaw bone is well defined, smooth, and scalloped, and the stroma exhibits a characteristic soap bubble or honeycomb appearance, often accompanied by knife-edge-like dental root resorption (Figure 1A). In contrast, the borders of other bone invasive cancers are poorly defined, often showing marked bone resorption similar to that seen in severe periodontitis, and are characterized by floating teeth without root resorption (Figure 1B). These differences are likely caused by the extremely slow spread of ameloblastoma relative to that of bone-invading cancer cells derived from oral tissues, breast, lung, and other organs, which tend to spread more rapidly (6).
We hypothesized that the expansion mechanism(s) of ameloblastoma in jaw bone differs from those of invasive cancer cells. Here, we compared the expression levels and release of RANKL in ameloblastoma and invasive oral SCC cell lines (AM-1 and HSC-3, respectively), to determine its effect on osteoclast differentiation (18). We also examined the possibility that ameloblastoma could directly resorb bone or dentine minerals. We found that ameloblastoma cells expressed lower amounts of RANKL than oral SCC cells but resorbed bone mineral materials by activation of vacuolar-type H+-ATPase (V-ATPase) and H+/Cl− exchange transporter 7 (CLC-7) on their plasma membranes (Figure 2).
Importance of the study:
Our results suggest that surface V-ATPase and CLC-7 on plasma membrane of ameloblastoma play important role for demineralization, and they would be new molecular targets as a novel internal treatment of ameloblastoma.
We thank Dr. Fumie Tanaka and Prof. Tetsuro Ikebe, Department of Oral Surgery, Fukuoka Dental College, for supplying panoramic X-ray pictures of ameloblastoma and gingival SCC. Written informed consent had been conducted in each patient for publication.
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