International Journal of Molecular Sciences

Review Notch Signaling Affects Oral Neoplasm Cell Differentiation and Acquisition of Tumor-Specific Characteristics

Keisuke Nakano 1,*, Kiyofumi Takabatake 1, Hotaka Kawai 1, Saori Yoshida 1, Hatsuhiko Maeda 2, Toshiyuki Kawakami 3 and Hitoshi Nagatsuka 1 1 Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; [email protected] (K.T.); [email protected] (H.K.); [email protected] (S.Y.); [email protected] (H.N.) 2 Department of Oral Pathology, School of Dentistry, Aichi Gakuin University, Nagoya 464-8650, Japan; [email protected] 3 Hard Tissue Pathology Unit, Matsumoto Dental University Graduate School of Oral Medicine, Shiojiri 399-0781, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-086-235-6651



Received: 19 March 2019; Accepted: 21 April 2019; Published: 23 April 2019


Abstract: Histopathological findings of oral neoplasm cell differentiation and metaplasia suggest that tumor cells induce their own dedifferentiation and re-differentiation and may lead to the formation of tumor-specific histological features. Notch signaling is involved in the maintenance of tissue stem cell nature and regulation of differentiation and is responsible for the cytological regulation of cell fate, morphogenesis, and/or development. In our previous study, immunohistochemistry was used to examine Notch expression using cases of odontogenic tumors and pleomorphic adenoma as oral neoplasms. According to our results, Notch signaling was specifically associated with tumor cell differentiation and metaplastic cells of developmental tissues. Notch signaling was involved in the differentiation of the ductal epithelial cells of salivary gland tumors and ameloblast-like cells of odontogenic tumors. However, Notch signaling was also involved in squamous metaplasia, irrespective of the type of developmental tissue. In odontogenic tumors, Notch signaling was involved in epithelial–mesenchymal interactions and may be related to tumor development and tumorigenesis. This signaling may also be associated with the malignant transformation of ameloblastomas. Overall, Notch signaling appears to play a major role in the formation of the characteristic cellular composition and histological features of oral neoplasms, and this involvement has been reviewed here.

Keywords: odontogenic tumor; pleomorphic adenoma; notch signaling; cell differentiation; immunohistochemistry; epithelial-mesenchymal interaction; malignant transformation

1. Introduction Tooth germ is composed of epithelial and mesenchymal tissues, and epithelial–mesenchymal interactions are an essential process for tooth development. This complex process is associated not only with the development of teeth but also with the development of odontogenic neoplasms. Odontogenic tumors, like other tumors, are histologically classified into malignant and benign tumors. Malignant tumors include odontogenic carcinoma, odontogenic carcinosarcoma, and odontogenic sarcoma. Benign tumors include benign epithelial odontogenic tumors, benign mixed epithelial and mesenchymal odontogenic tumors, and benign mesenchymal odontogenic tumors. Ameloblastoma is one of the most often encountered benign odontogenic epithelial tumors. It is clinically characterized as a benign but locally invasive tumor with a high recurrence risk. Histologically,

Int. J. Mol. Sci. 2019, 20, 1973; doi:10.3390/ijms20081973 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 1973 2 of 14 it is characterized by a tumor nest similar to the enamel organ that constitutes tooth germ [1]. Ameloblastic fibroma is categorized as a benign mixed epithelial and mesenchymal odontogenic tumor composed of an ameloblastoma-like epithelial tissue and dental papilla-like mesenchyme, in which no dental hard tissue is present [2]. A calcifying odontogenic cyst is a developmental odontogenic cyst that was previously classified as an odontogenic mixed tumor. It is characterized by an epithelial lining of the cyst wall, which gives it a resemblance to the histological structure of the enamel organ that includes ghost cells and calcified foci within the odontogenic epithelial region. Dentin-like hard tissue and dysplastic dentin may be observed in the ectomesenchymal stromal tissue [3]. Odontogenic myxoma is a less common mesenchymal odontogenic tumor. Histopathologically, this tumor is characterized by a spindle or stellate cells proliferating in an abundant myxoid extracellular matrix with or without small masses of odontogenic epithelium [4]. Ameloblastic carcinoma is a rare malignant odontogenic tumor and is the malignant counterpart of ameloblastoma. This neoplasm combines a histological similarity to ameloblastoma with a strong cytological atypism [5]. Salivary gland tumors are common in oral neoplasms. Pleomorphic adenomas are especially prevalent in the neoplasm and arise from the salivary gland. They are classified as a benign epithelial salivary gland tumor [6]. Histopathologically,pleomorphic adenomas are characterized by an admixture of polygonal epithelial and neoplastic myoepithelial cells in a variable background stroma. Typically, a transition from the epithelial to the mesenchymal component creates characteristic myxomatous tissues around the glandular structure [6,7]. However, the neoplastic myoepithelial cells in tumor nests undergo further differentiation into plasmacytoid cells, squamous epithelioid cells, and chondrocytes, among other cell types. The characteristics of these oral neoplasms are shown in Table1.

Table 1. Clinical and histological features in different type of oral neoplasms.

Clinical Features Histological Features Odontogenic tumor/cyst Epithelial tumor nests are similar to the Locally invasive tumor. Clinically benign enamel organ. Tumor cells arranged at Ameloblastoma but has a high risk of recurrence. periphery of tumor nests are columnar or cuboidal. Tumor composed of ameloblastoma like Usually grows slowly and painlessly, Ameloblastic fibroma epithelial tissue and dental papilla causing swelling of the jawbone. like mesenchyme. Usually grows slowly and painlessly, causing swelling of the jawbone. Spindle or stellate tumor cells proliferating Odonotogenic myxoma Clinically benign but has a high risk in an abundant myxoid extracellular matrix. of recurrence. Ameloblastoma-like lining epithelium Usually grows slowly and painlessly, including ghost cells and calcified foci. Calcifying odontogenic cyst causing swelling of the jawbone. Dentin-like hard tissue and dysplastic dentin may be observed. Rare odontogenic carcinoma. Most cases Histological similarity of ameloblastoma Ameloblastic carcinoa arise in posterior segments of the jaws. with strong cytological atypism. Salivary gland tumour Admixture of polygonal epithelial and Tumor shows slowly and painless Pleomorphic adenoma neoplastic myoepithelial cells in a variable growing. Most cases arise in the parotid. background stroma.

Notch is an evolutionarily-conserved pathway that plays a decisive role in cell fate determination in various tissue types during embryonic development, as well as postnatally [8]. Notch activity affects a wide range of developmental processes, such as differentiation, proliferation, apoptosis, neurogenesis, somite formation, hematopoiesis, angiogenesis, growth/differentiation of keratinocytes, and craniofacial development, including tooth development [9]. Under neoplastic conditions, such factors are also Int. J. Mol. Sci. 2019, 20, x 3 of 14

Notch activity affects a wide range of developmental processes, such as differentiation, proliferation, Int.apoptosis, J. Mol. Sci. neurogenesis,2019, 20, 1973 somite formation, hematopoiesis, angiogenesis, growth/differentiation3 of of 14 keratinocytes, and craniofacial development, including tooth development [9]. Under neoplastic relatedconditions, to cell such diff erentiationfactors are [10, 11also]. Therefore, related immunohistochemistryto cell differentiation analysis [10,11]. was performedTherefore, basedimmunohistochemistry on the use of cell dianalysisfferentiation was performed markers reported based on in previousthe use of papers. cell differentiationMuraki et al. [markers12] and Takaminereported etin al.previous [13] performed papers. immunohistochemistry Muraki et al. [12] analysis and ofTakamine ameloblastomas et al. and[13]pleomorphic performed adenomasimmunohistochemistry to determine analysis the role of of ameloblastomas Notch in cell diff anderentiation. pleomorphic The adenomas results suggested to determine that Notch the role is involvedof Notch inin cellcell didifferentiation.fferentiation in The various results tumors, suggested partly that leading Notch to a is histologically involved in similarcell differentiation differentiation. in Thus,various in thistumors, review, partly we focusedleading onto thea histologically involvement ofsimilar Notch differentiation. in the cell differentiation Thus, in ofthis representative review, we odontogenicfocused on the tumors involvement and pleomorphic of Notch in adenomasthe cell diff usingerentiation the literature of representative previously odontogenic published tumors by our researchand pleomorphic group. adenomas using the literature previously published by our research group.

2. Histopathology of Ameloblastoma Ameloblastoma is one of the most frequently en encounteredcountered tumors arising from the odontogenic epithelium. It is characterized as a locally invasive tumor that is clinically benign but has a high risk of recurrence. Although the prognosis is good, a long-termlong-term follow-up survey is indispensable as the disease has been known to recur 10 years after the firstfirst treatment [[1].1]. Histologically, ameloblastoma shows a follicular or plexiform pattern within a fibrousfibrous stromal connective tissue [[1].1]. There are four basic histopathological variations: variations: (1) (1) conventional, conventional, (2) (2) unicystic, unicystic, (3) (3) extraosseous/peripheral, extraosseous/peripheral, and and (4) (4)metastasizing. metastasizing. There There are aretwo two major major conventional conventional histological histological patterns: patterns: follicular follicular and and plexiform. plexiform. The follicular type ameloblastoma is the most common histological type, which is composed of island-like odontogenic epithelial tumor nests resemblingresembling thethe enamelenamel organorgan ofof toothtooth germ.germ. Epithelial tumor nests are surrounded by fibrousfibrous interstitialinterstitial stromalstromal tissue (Figure1 1a).a). InIn thethe epithelialepithelial tumortumor nest of thethe follicularfollicular type type ameloblastoma, ameloblastoma, tumor tumor cells cells arranged arranged at at the the periphery periphery of theseof these tumor tumor nests nests are columnar,are columnar, cuboidal, cuboidal, or hyperchromatic. or hyperchromatic. Marginal Marginal cells are cells arranged are arranged in a palisade in a fashionpalisade to fashion surround to thesurround tumor the nest. tumor The nest. peripheral The peripheral layered cells layered resemble cells resemble the inner the enamel inner epithelium.enamel epithelium. The cells The located cells inlocated the center in the of center the tumor of the nest tumor show nest a stellate show a reticulum-like stellate reticulum-like structure. stru Thesecture. structures These structures exhibit a similarityexhibit a similarity to the enamel to the organ enamel of tooth organ germ of andtooth result germ from and the result differentiation from the differentiation of tumor cells intoof tumor tooth germcells into constituent tooth germ cells. constituent The central cells. region The central of the tumorregion nestof the often tumor undergoes nest often squamous undergoes metaplasia, squamous keratinization,metaplasia, keratinization, and cystic changes.and cystic The changes. tumor stromaThe tumor consists stroma of fibrousconsists connective of fibrous tissue.connective It is generallytissue. It is rich generally in collagenous rich in collagenous fibers and has fibers a low and cell has density. a low cell density. The plexiformplexiform type type ameloblastoma ameloblastoma is the is second-mostthe second-most common common histological histological type and type is composed and is ofcomposed cord-like of and cord-like anastomosing and anastomosing strands of odontogenicstrands of odontogenic epithelial tumorepithelial nests. tumor The nests. tumor The nests tumor join withnests eachjoin with other each to form other a network-liketo form a network-like structure (Figurestructure1b). (Figure In the 1b). tumor In nestthe tumor of the nest plexiform of the typeplexiform ameloblastoma, type ameloblastoma, the cuboidal the and cuboidal/or columnar and/or cells columnar are arranged cells are in thearranged peripheral in the region peripheral of the anastomosingregion of the anastomosing nests. The appearance nests. The of stellate appearance reticulum of stellate cells are reticulum ambiguous cells in theare centerambiguous of the tumorin the nests.center Theof the tumor tumor stroma nests. consists The tumor of loose stroma fibrous consis connectivets of loose tissue. fibrous It generallyconnective shows tissue. an It edematous generally andshows cyst-like an edematous stromal degeneration.and cyst-like stromal degeneration.

Figure 1. Histopathological features of ameloblastoma. The growth patterns of these tumors are classified as (a) follicular type and (b) plexiform type. HE staining, magnification 40. × Int. J. Mol. Sci. 2019, 20, 1973 4 of 14 Int. J. Mol. Sci. 2019, 20, x 4 of 14

3. Notch SignalingFigure 1. Histopathological in Ameloblastoma features of ameloblastoma. The growth patterns of these tumors are classified as (a) follicular type and (b) plexiform type. HE staining, magnification ×40. Notch plays an important role in cell fate determination in various tumors and also in normal tissue3. development.Notch Signaling Inin theAmeloblastoma craniofacial area, the development of the mandibular condyles is an importantNotch feature; plays as an such, important we examined role in cell Notch-related fate determination factors in various in this regiontumors [and14]. also Under in normal neoplastic conditions,tissue development. Notch-related In factorsthe craniofacial are also area, involved the development in cell differentiation of the mandibular [10,11]. Therefore,condyles is wean also examinedimportant odontogenic feature; as neoplasms, such, we examined such as benignNotch-rela ameloblastomated factors in this and region ameloblastic [14]. Under carcinoma neoplastic and its subtypesconditions, [12,15 ,Notch-related16], squamous factors odontogenic are also involved tumor, odontogenicin cell differentiation myxoma, [10,11 ameloblastic]. Therefore, fibromawe also [17], calcifyingexamined odontogenic odontogenic cyst neoplasms, [18], and calcifying such as benign epithelial ameloblastoma odontogenic and tumors. ameloblastic In the carcinoma above-mentioned and studies,its subtypes we histologically [12,15,16], examinedsquamous theodontogenic expression tumor, of Notch odontogenic and related myxoma factors, ameloblastic and showed fibroma that it is involved[17], incalcifying the development odontogenic of odontogeniccyst [18], and tumorscalcifying and epithelial the diff erentiationodontogenic of tumors. tumor cellsIn the as above- a potential role ofmentioned Notch signaling. studies, we Our histologically findings suggest examined that the Notch1 expression plays aof fundamental Notch and related role in factors the cytological and showed that it is involved in the development of odontogenic tumors and the differentiation of tumor differentiation of epithelial and mesenchymal components and the acquisition of tumor-specific cells as a potential role of Notch signaling. Our findings suggest that Notch1 plays a fundamental charactersrole in inthe these cytological odontogenic differentiation tumors of [epitheli12,15,16al ,and18], mesenchymal but not in odontogenic components myxomaand the acquisition [17]. In tooth development,of tumor-specific the expression characters of Notch1in these mRNAodontogenic was detected tumors [12,15,16,18], in the odontogenic but not epithelium in odontogenic of mouse molarmyxoma teeth, and [17]. was In foundtooth todevelopment, be transient the in theexpre innerssion enamel of Notch1 epithelium mRNA atwas the detected early bell in stage the and not inodontogenic the differentiated epithelium ameloblast of mouse cells molar [9]. teeth, Notch1 and and was its found ligand to Jagged1be transient mRNA in the expression inner enamel has been detectedepithelium in human at the tooth early germ bell stage at the and early not stage in the of differentiated dental crown ameloblast calcification. cells [9]. Thus, Notch1 Notch and signaling its has beenligand shown Jagged1 to mRNA be involved expression in tooth has been development detected in andhuman odontoblast tooth germdi atff theerentiation. early stage Inof dental a previous study,crown ameloblastoma calcification. was Thus, found Notch to signaling express Notch1has been and shown its ligandto be involved Jagged1 in in tooth polyhedral development tumor and cells in the centralodontoblast region differentiation. of the tumor In nest. a previous The state study, of ameloblastoma the Notch expression was found suggested to express that Notch1 the and tumor its nest ligand Jagged1 in polyhedral tumor cells in the central region of the tumor nest. The state of the Notch of ameloblastoma is similar to an early state of the enamel organ in tooth germ development. In a expression suggested that the tumor nest of ameloblastoma is similar to an early state of the enamel seriesorgan of odontogenic in tooth germ tumor development. studies, In we a series found of that odontogenic Notch was tumor expressed studies, inwe the found squamous that Notch metaplasia was of theexpressed odontogenic in the epithelium. squamous metaplasia Since Kumamoto of the odontogenic and Ohki epithelium. showed that Since the Kumamoto keratinizing and cellsOhki were Notchshowed negative, that wethe believekeratinizing the dicellsfference were liesNotch in nega the celltive, stages. we believe In the the squamous difference celllies diin fftheerentiation cell stage,stages. the keratinizing In the squamous cells cell showed differentiation the final stage, stage the of keratinizing differentiation. cells showed On the the contrary, final stage squamous of cellsdifferentiation. were found to On be the at contrary, the pre-final squamous stage. cells In were addition, found to our be at study the pre-final detected stage. Notch In addition, localization patternsour study in tumor detected cells. Notch The expressionlocalization ofpatterns Notch1 in intumor tumor cells. tissue The expression was localized of Notch1 to distinct in tumor parts of differentiation.tissue was Inlocalized the periphery to distinct of theparts tumor of differenti nest ofation. the ameloblastoma, In the periphery Notch of the expression tumor nest is of high the in the ameloblastoma, Notch expression is high in the columnar or ameloblast-like cells and low in the columnar or ameloblast-like cells and low in the cuboidal or basaloid cells. In the central region of the cuboidal or basaloid cells. In the central region of the ameloblastoma cell nests, Notch expression and ameloblastoma cell nests, Notch expression and localization changes according to cell differentiation; localization changes according to cell differentiation; however, Notch expression is low here however,compared Notch to expressionthat in the marginal is low hereregions compared [12] (Figure to that2). In in particular, the marginal in ameloblastoma, regions [12] Notch (Figure 2). In particular,signaling in is ameloblastoma,considered to play Notch a major signaling role in isthe considered differentiation to play of epithelial a major role tumor in the cells. di ffOnerentiation the of epithelialcontrary, tumor the expression cells. On of the Notch contrary, is scarce the in expressionthe stromal ofconnective Notch is tissue scarce of inthe the tumor, stromal suggesting connective tissuethat of theit does tumor, not play suggesting a positive that role it in does tumor not stroma play aformation. positive role in tumor stroma formation.

Figure 2. Immunohistochemical features of Notch1 in ameloblastoma. (a) Notch-positive reactions are observed in the cytoplasm of peripheral columnar cells and central squamous metaplastic cells. Magnification 100. (b) Notch-positive reactions in the budding and peripheral layer of the epithelial × nests. Magnification 100. Anti-Notch-1 intracellular domain antibody, Abcam, ab83232, 1:100. × Int. J. Mol. Sci. 2019, 20, x 5 of 14

Figure 2. Immunohistochemical features of Notch1 in ameloblastoma. (a) Notch-positive reactions are observed in the cytoplasm of peripheral columnar cells and central squamous metaplastic cells. Magnification ×100. (b) Notch-positive reactions in the budding and peripheral layer of the epithelial nests. Magnification ×100. Anti-Notch-1 intracellular domain antibody, Abcam, ab83232, 1:100. Int. J. Mol. Sci. 2019, 20, 1973 5 of 14 4. Histopathology of Ameloblastic Fibroma and Odontogenic Myxoma 4. HistopathologyAmeloblastic fibroma of Ameloblastic is a benign Fibroma odontogenic and Odontogenic tumor composed Myxoma of odontogenic epithelial tissue and mesenchymalAmeloblastic fibromatissue. It is is a benignusually odontogenicslow-growing, tumor and composedthe posterior of odontogenic area of the mandible epithelial tissueis the andmost mesenchymal common location. tissue. It can It is cause usually the slow-growing, expansion of the and jaw the bone posterior but rarely areaof reaches the mandible a remarkable is the mostsize. Histologically, common location. the epithelial It can cause component the expansion shows of a thepattern jaw boneof elongated but rarely strands reaches of athe remarkable epithelial size.tumor Histologically, nest and the enamel the epithelial organ-like component tumor nest. shows The a patternelongated of elongatedstrand tumor strands nest ofis thecomposed epithelial of tumortwo tight nest and and parallel-running the enamel organ-like layers of tumor columnar nest. and/or The elongated cuboidal strand cells. tumorThe thickening nest is composed at the ends of twoof strand tight andtumor parallel-running nests resemble layers the tooth of columnar bud. The and cells/or cuboidalsurrounding cells. the The enamel thickening organ-like at the endstumor of strandnest are tumor columnar. nests resemble The stellate the tooth reticulum bud. Theis loca cellsted surrounding in the central the enamel region organ-likeof the tumor tumor nest. nest The are columnar.mesenchymal The tumor stellate component reticulum is cell-rich located inand the myxoid central and region resembles of the tumor the dental nest. papilla The mesenchymal of the tooth tumorgerm. In component the mesenchymal is cell-rich region, and myxoidspindle andor stellate resembles-shaped the dentalcells proliferate papilla of in the a myxoid tooth germ. or mucoid In the mesenchymalextracellular matrix. region, These spindle are or frequently stellate-shaped associated cells with proliferate an embedded in a myxoid tooth or [2] mucoid (Figure extracellular 3a). matrix.Odontogenic These are frequentlymyxoma is associated the third-most with anfrequent embedded odontogenic tooth [2] tumor. (Figure It3 a).is classified as a benign mesenchymalOdontogenic odontogenic myxoma tu ismor the third-most and is most frequent likely to odontogenic occur in the tumor. molar It region is classified of the as mandible. a benign mesenchymalClinically, it usually odontogenic grows tumorslowly and and is painlessly, most likely and to occurcauses in swelling the molar of regionthe jawbone. of the mandible.A recent Clinically,retrospective it usuallycase study grows reported slowly that and odontogeni painlessly,c andmyxoma causes occurring swelling in of th thee maxillofacial jawbone. A region recent retrospectivetends to have case a studysignificant reported recurrence that odontogenic rate [4]. myxomaRadiographically, occurring inlesions the maxillofacial appear as regionunilocular, tends tomultilocular, have a significant and scalloped recurrence radioluc rateencies, [4]. Radiographically, sometimes with lesions root resorption. appear as Histologically, unilocular, multilocular, the tumor andtissue scalloped consists radiolucencies, of randomly sometimesdistributedwith stellate root or resorption. spindle-shaped Histologically, cells. Tumor the tumor cells tissue produce consists an ofabundant randomly alcianophilic distributed stellate extracellular or spindle-shaped matrix. Th cells.ese Tumormyxoid cells ground produce ansubstances abundant alcianophilicare rich in extracellularmucopolysaccharide matrix. Theseand hyaluronic myxoid ground acid and substances contain are small rich amounts in mucopolysaccharide of fine collagen and fibers hyaluronic (Figure acid3b). and contain small amounts of fine collagen fibers (Figure3b). The tumortumor cell cell component component of odontogenic of odontogenic myxoma my isxoma simply is derivedsimply fromderived odontogenic from odontogenic ectodermal mesenchyme.ectodermal mesenchyme. The epithelial–mesenchymal The epithelial–mesenchymal interactions essential interactions to tooth essential development to tooth are development not involved inare the not development involved in the of odontogenicdevelopment myxoma. of odontogeni On thec myxoma. contrary, On during the contrary tumorigenesis,, during the tumorigenesis, ameloblastic fibromathe ameloblastic is a tumor fibroma that isis considereda tumor that to is haveconsidered an epithelial–mesenchymal to have an epithelial–mesenchymal interaction between interaction the epithelialbetween the component epithelial andcomponent the mesenchymal and the mesenchy componentmal ofcomponent the tumor. of The the ditumor.fferentiation The differentiation stage of the ameloblasticstage of the ameloblastic fibroma tumor fibroma is clearly tumor diff iserent clearly from different the diff fromerentiation the differentiation stage of the tumor,stage of which the tumor, lacks epithelial–mesenchymalwhich lacks epithelial–mesenchymal interactions. interactions.

Figure 3. Histopathological features of (a) ameloblasticameloblastic fibromafibroma andand ((bb)) odontogenicodontogenic myxoma.myxoma. HE staining, magnification 100. staining, magnification ××100. 5. Notch Signaling in Ameloblastic Fibroma and Odontogenic Myxoma

According to the literature [17], immunohistochemical analysis revealed that Notch expression is observed in both the epithelial and mesenchymal tumor tissue of ameloblastic fibroma. Within the epithelial tumor nests, a strong immunohistochemical Notch reaction is observed in the central region in comparison to the peripheral region. The immunohistochemical expression pattern of Notch in the epithelial component of the tumor is partially similar to that of ameloblastoma. However, Int. J. Mol. Sci. 2019, 20, x 6 of 14

5. Notch Signaling in Ameloblastic Fibroma and Odontogenic Myxoma According to the literature [17], immunohistochemical analysis revealed that Notch expression is observed in both the epithelial and mesenchymal tumor tissue of ameloblastic fibroma. Within the epithelial tumor nests, a strong immunohistochemical Notch reaction is observed in the central region Int.in comparison J. Mol. Sci. 2019 to, 20 the, 1973 peripheral region. The immunohistochemical expression pattern of Notch in6 ofthe 14 epithelial component of the tumor is partially similar to that of ameloblastoma. However, the pattern theof Notch pattern expression of Notch in expression the columnar in the cells columnar at the periphery cells at theof the periphery tumor nest of the is different. tumor nest Histologically is different. Histologicallycomparing the comparingfollicular type the of follicular ameloblastoma type of ameloblastomawith ameloblastic with fibroma, ameloblastic there are fibroma, a large there number are aof largeameloblast-like number of high ameloblast-like columnar cells high in columnar the epithelial cells intumor the epithelial nest of ameloblastic tumor nest offibroma. ameloblastic These fibroma.findings indicate These findings that the indicate differentiation that the distagefferentiation of peripheral stage high of peripheral columnar high cells columnar is more advanced cells is more in advancedameloblastic in ameloblasticfibroma than fibroma ameloblastoma. than ameloblastoma. In the follicular In the folliculartype of ameloblastoma, type of ameloblastoma, intense intenseNotch Notchexpression expression appears appears in the inperipheral the peripheral columnar columnar or cuboidal or cuboidal cells cellsof the of thetumor tumor nest. nest. Moreover, Moreover, in inameloblastic ameloblastic fibromas, fibromas, Notch Notch expression expression appears appears in in the the peripheral peripheral columnar columnar cells; cells; however, however, it it is weak. Immunohistochemically, Immunohistochemically, Notch Notch signaling signaling is is believed believed to to be involved in the differentiation differentiation of ameloblasts or columnar cells from the odontogenicodontogenic epithelium. In the mesenchymal component of ameloblastic fibroma, fibroma, a strong immunohistochemical positivity of Notch1 is observed in the tumor cells forming the the dental dental papilla-like papilla-like tissue tissue (Figur (Figuree 4a).4a). The The epithelial–mesenchymal epithelial–mesenchymal interaction interaction is an is animportant important event event of cell of proliferat cell proliferationion and for and differentiation for differentiation during during the development the development of various of various organs organsand tissues. and tissues.During Duringtooth developm tooth development,ent, epithelial–mesenchymal epithelial–mesenchymal interactions interactions are required, are required, and Notch and Notchexpression expression is observed is observed in both in both odontogenic odontogenic epithelial epithelial and and mesenchymal mesenchymal tissues, tissues, suggesting suggesting its involvement in cell didifferentiationfferentiation [[19].19]. Our immu immunohistochemicalnohistochemical examination revealed that Notch expression isis not not observed observed in thein the tumor tumor tissue tissue of odontogenic of odontogenic myxoma myxoma [17] (Figure [17] 4(Figureb). These 4b). findings These suggestfindings that suggest Notch that signaling Notch is signaling involved inis theinvolved epithelial–mesenchymal in the epithelial–mesenchymal interaction of tumorigenesis interaction of odontogenictumorigenesis tumors, of odontogenic and that the tumors, differentiation and that stage the ofdifferentiation the tumoral tissue stage in of odontogenic the tumoral myxoma tissue in is lessodontogenic advanced myxoma than that is inless ameloblastic advanced than fibroma. that in ameloblastic fibroma.

Figure 4.4. ImmunohistochemicalImmunohistochemical features features of Notch1 expression.expression. ( (aa)) Notch-positive Notch-positive reactions are observed in ameloblastic fibroma. Magnification 100. (b) Notch expression is not observed in the observed in ameloblastic fibroma. Magnification ×100.× (b) Notch expression is not observed in the tumor tissue of odontogenic myxoma. Magnification 100. Anti-Notch-1 intracellular domain antibody, tumor tissue of odontogenic myxoma. Magnificatio× n ×100. Anti-Notch-1 intracellular domain Abcam,antibody, ab83232, Abcam, 1:100. ab83232, 1:100. 6. Histopathology of Calcifying Odontogenic Cyst 6. Histopathology of Calcifying Odontogenic Cyst Calcifying odontogenic cyst is a rare odontogenic cyst, and forms part of the group of ghost cell Calcifying odontogenic cyst is a rare odontogenic cyst, and forms part of the group of ghost cell lesions of the jaw. Histologically, lesional tissues composed of a fibrous wall lined by ameloblastoma-like lesions of the jaw. Histologically, lesional tissues composed of a fibrous wall lined by ameloblastoma- epithelium are characterized by cuboidal cells, basal cells, preameloblast-like cells, and stellate like epithelium are characterized by cuboidal cells, basal cells, preameloblast-like cells, and stellate reticulum-like cells with scattered clusters of ghost cells and squamous metaplastic cells. Focal reticulum-like cells with scattered clusters of ghost cells and squamous metaplastic cells. Focal accumulations of ghost cells with calcifications may also be observed. The peripheral layered cells of accumulations of ghost cells with calcifications may also be observed. The peripheral layered cells of the lined epithelial resemble the inner enamel epithelium. The cells located in the central region of the the lined epithelial resemble the inner enamel epithelium. The cells located in the central region of lined epithelial show a stellate reticulum-like structure. A variable amount of dentinoid may appears the lined epithelial show a stellate reticulum-like structure. A variable amount of dentinoid may adjacent to the lining epithelium [3] (Figure5). Occasionally, odontoma, ameloblastic fibroma, and adenomatoid odontogenic tumors can be detected in the same area. Int. J. Mol. Sci. 2019, 20, x 7 of 14 appears adjacent to the lining epithelium [3] (Figure 5). Occasionally, odontoma, ameloblastic fibroma, and adenomatoid odontogenic tumors can be detected in the same area. Int. J. Mol. Sci. 2019, 20, 1973 7 of 14

7. Notch Signaling in Calcifying Odontogenic Cyst 7. NotchKawakami Signaling et al. in reported Calcifying histological Odontogenic Notch Cyst expression on calcifying odontogenic cysts [20,21]. ImmunohistochemicalKawakami et al. reported analysis histologicalrevealed that Notch Notch expression expression on is calcifying observed odontogenic in the cytoplasm cysts of [20 both,21]. Immunohistochemicalepithelial and mesenchymal analysis cells. revealed In the thatenamel Notch organ-like expression epitheli is observedal component, in the cytoplasmNotch expression of both epithelialis similar and to mesenchymal that of ameloblastoma. cells. In the enamel Epithelial organ-like cells, epithelial including component, ghost Notch cells, expression express is similarimmunohistochemical to that of ameloblastoma. Notch-positi Epithelialvity (Figure cells, including5). Furthermore, ghost cells, the expressmesenchymal immunohistochemical spindle and/or Notch-positivityreticulum cells scattered (Figure 5in). the Furthermore, stromal connective the mesenchymal tissues express spindle Notch-positivity and /or reticulum (Figure cells scattered5). In the indentinoid the stromal formation connective area, tissues Notch express expression Notch-positivity is present (Figurein the 5cytoplasm). In the dentinoid of adjacent formation cells of area, the Notchdentinoid expression tissues is and present the in embedded the cytoplasm cells of adjacent(Figure cells5). According of the dentinoid to the tissues literature and the [20], embedded Notch cellsexpression (Figure is5). also According found toin the ghost literature cells. [ 20Notch], Notch localization expression has is alsobeen found previously in ghost found cells. Notchto be localizationcytoplasmic hasand/or been membranous, previously found and toNotch be cytoplasmic expression andis also/or membranous,observed in metaplastic and Notch expressionsquamous iscells. also On observed the contrary, in metaplastic the adja squamouscent epithelium cells. Onexpresses the contrary, weak theNotch adjacent positivity. epithelium These expresses findings weaksuggest Notch that positivity.probably Notch These signal findings activation suggest in that ghost probably cells exerts Notch a signallateral activationinhibitory ineffect ghost on cells the exertsadjacent a lateral epithelium, inhibitory blocking effect them on the from adjacent adopting epithelium, the same blocking cell fate. them In our from study, adopting Notch the expression same cell fate.was notIn our observed study, Notchin any expression of the cases was of not odonto observedgenic in myxoma any of the [17]. cases The of odontogenicloss of Notch myxoma expression [17]. Thesuggests loss of that Notch neoplastic expression cells suggests and tissues that neoplastic have no cytological cells and tissues differentiation. have no cytological On the contrary, differentiation. as in Onthe thecase contrary of calcifying, as in the odontogenic case of calcifying cysts, odontogenic strong Notch cysts, strongexpression Notch expressionhas been hasdetected been detected in the inodontogenic the odontogenic mixed mixedtumor, tumor,ameloblastic ameloblastic fibroma. fibroma. These Notch-related These Notch-related immuno immunohistochemicalhistochemical positive positivefeatures indicate features that indicate Notch that signaling Notch signalingis closely involved is closely in involved the epithelial–mesenchymal in the epithelial–mesenchymal interaction interactionand that cytodifferentiation and that cytodifferentiation occurs in calcifying occurs in odontogenic calcifying odontogenic cysts. cysts.

Figure 5. Microscopic features of (a) calcifying odontogenic cysts. HE staining, Magnification 100. Figure 5. Microscopic features of (a) calcifying odontogenic cysts. HE staining, Magnification ××100. (b) Notch1-positiveNotch1-positive reactions reactions are observed are observed in both ghost in cellsboth and ghost epithelial cells cells. and Immunohistochemical epithelial cells. staining, Magnification 100. (c) Notch1-positive reactions are observed in ectomesenchymal spindle Immunohistochemical staining,× Magnification ×100. (c) Notch1-positive reactions are observed in cells. Immunohistochemical staining, Magnification 100. (d) Notch1-positive reactions are observed ectomesenchymal spindle cells. Immunohistochemical× staining, Magnification ×100. (d) Notch1- adjacent to dentinoid tissues. Immunohistochemical staining, Magnification 100. Anti-Notch-1 × intracellular domain antibody, Abcam, ab83232, 1:100. Int. J. Mol. Sci. 2019, 20, x 8 of 14

positive reactions are observed adjacent to dentinoid tissues. Immunohistochemical staining, Magnification ×100. Anti-Notch-1 intracellular domain antibody, Abcam, ab83232, 1:100. Int. J. Mol. Sci. 2019, 20, 1973 8 of 14

8. Histopathology of Ameloblastic Carcinoma 8. HistopathologyAmeloblastic carcinoma of Ameloblastic is a rare Carcinoma primary epithelial odontogenic malignant neoplasm. This neoplasmAmeloblastic is characterized carcinoma by isa acombination rare primary of epithelialthe histological odontogenic features malignant of ameloblastoma neoplasm. and This a neoplasmstrong cytological is characterized atypism. by Ameloblastic a combination carcinoma of the histological can have features a follicular of ameloblastoma or plexiform andpattern a strong and cytologicalcan be formed atypism. of nests, Ameloblastic sheets, carcinomaor broad canribbo havens of a folliculartumor epithelium or plexiform [5]. pattern The histological and can be formedcharacteristics of nests, are sheets, a ortall broad columnar ribbons ofcellul tumorar epitheliummorphology [5]. Thewith histological pleomorphism, characteristics nuclear are ahyperchromatism, tall columnar cellular focal necrosis, morphology and withmitotic pleomorphism, activity (Figure nuclear 6). The hyperchromatism, peripheral cell layer focal of the necrosis, tumor andnest mitoticshows activitypalisading. (Figure Usually,6). The peripheralreverse nuclea cell layerr polarity of the tumor is focally nest shows present. palisading. The organized Usually, reversestratification nuclear of columnar polarity iscells, focally stratum present. intermed Theium, organized and stellate stratification reticulum of found columnar in ameloblastoma cells, stratum intermedium,are lost (Figure and 6). stellateThis histological reticulum tendency found in is ameloblastoma more marked arein higher-grade lost (Figure6 ).lesions. This histological The central tendencyregion of isepithelial more marked tumor in nests higher-grade or sheets lesions. may be The replaced central regionby an ofatypical epithelial basaloid tumor nestsepithelium or sheets or mayspindle be replacedepithelial by cells. an atypical The central basaloid region epithelium of epithelial or spindle tumor epithelial nests may cells. show The centralacanthomatous region of epithelialmetaplasia tumor and cystic nests degeneration. may show acanthomatous In addition, ameloblastic metaplasia andcarcinomas cystic degeneration. show a high proliferation In addition, ameloblasticindex compared carcinomas to ameloblastomas show a high [5]. proliferation index compared to ameloblastomas [5].

Figure 6.6. Histopathological features of ameloblastic carcinoma. ( (aa)) Follicular Follicular growth growth patterns of ameloblastic carcinoma. HE staining, magnification 40. (b) Follicular tumor nests of ameloblastic ameloblastic carcinoma. HE staining, magnification ××40. (b) Follicular tumor nests of ameloblastic carcinoma. HE staining, magnification 100. carcinoma. HE staining, magnification ××100. 9. Notch Signaling in Ameloblastic Carcinoma 9. Notch Signaling in Ameloblastic Carcinoma According to our immunohistochemical research [15], strong Notch-positive reactions are detected According to our immunohistochemical research [15], strong Notch-positive reactions are in most neoplastic cells of ameloblastic carcinoma. On the contrary, no Notch-positive reaction is detected in most neoplastic cells of ameloblastic carcinoma. On the contrary, no Notch-positive found in the stroma. In the epithelial tumor nest, Notch expression tends to be strong in the peripheral reaction is found in the stroma. In the epithelial tumor nest, Notch expression tends to be strong in columnar or cuboidal cells, and moderate in the central polyhedral cells (Figure7). Contrarily, the the peripheral columnar or cuboidal cells, and moderate in the central polyhedral cells (Figure 7). central region of the epithelial tumor nests is replaced by atypical cells, such as basaloid epithelium, Contrarily, the central region of the epithelial tumor nests is replaced by atypical cells, such as or spindle epithelial cells that show strong Notch expression (Figure7). Immunohistochemically, basaloid epithelium, or spindle epithelial cells that show strong Notch expression (Figure 7). there are two patterns in the cellular localization of Notch in ameloblastic carcinoma. One is a strong Immunohistochemically, there are two patterns in the cellular localization of Notch in ameloblastic positive reaction in the cytoplasm of tumor cells (Figure7), and the other is a strong positive reaction carcinoma. One is a strong positive reaction in the cytoplasm of tumor cells (Figure 7), and the other in the nuclei of tumor cells (Figure7). In addition, the former has a higher cell proliferation activity is a strong positive reaction in the nuclei of tumor cells (Figure 7). In addition, the former has a higher and a larger number of mitotic figures, and the latter has a lower cell proliferation activity and a cell proliferation activity and a larger number of mitotic figures, and the latter has a lower cell smaller number of mitotic figures. Ameloblastoma, which is the benign counterpart of ameloblastic proliferation activity and a smaller number of mitotic figures. Ameloblastoma, which is the benign carcinoma, usually does not appear in mitotic figures. Moreover, a few Ki-67 positive cells are observed counterpart of ameloblastic carcinoma, usually does not appear in mitotic figures. Moreover, a few in the proliferating tumor nests. In contrast, in the case of ameloblastic carcinoma, an increased Ki-67 positive cells are observed in the proliferating tumor nests. In contrast, in the case of number of Ki-67 positive cells have been found in almost all epithelial tumor nests. In certain cases ameloblastic carcinoma, an increased number of Ki-67 positive cells have been found in almost all of osteosarcoma, without epithelial components, Notch-positive reactions have been found in the epithelial tumor nests. In certain cases of osteosarcoma, without epithelial components, Notch- neoplastic mesenchymal cells [15]. In our case study of osteosarcoma, the expression of Notch was positive reactions have been found in the neoplastic mesenchymal cells [15]. In our case study of found to be stronger in the well-differentiated regions compared to poorly differentiated regions [21]. These observations suggest that Notch signaling is apparent in cells when differentiation is in progress, and regulates cell differentiation and proliferation. Notch signaling is also associated with oncogenesis, Int. J. Mol. Sci. 2019, 20, x 9 of 14 osteosarcoma, the expression of Notch was found to be stronger in the well-differentiated regions compared to poorly differentiated regions [21]. These observations suggest that Notch signaling is Int. J. Mol. Sci. 2019, 20, 1973 9 of 14 apparent in cells when differentiation is in progress, and regulates cell differentiation and proliferation. Notch signaling is also associated with oncogenesis, whereby the impairment of its wherebytransmission the impairmentleads to malignant of its transmission transformation. leads The to disturbance malignant transformation.of the Notch signaling The disturbance pathway has of thebeen Notch detected signaling in several pathway cancers, has such been as detected breast incancer, several esophageal cancers, such cancer, as breastand lymphoblastic cancer, esophageal acute cancer,leukemia and (T-ALL) lymphoblastic [22–24]. acute Furthermore, leukemia (T-ALL)Notch has [22 –been24]. Furthermore,the focus of Notchstudies has on been the themetastasis, focus of studiesinvasion, on and the metastasis,malignant invasion,transforma andtion malignant of oral neoplasms, transformation such of as oral adenoid neoplasms, cystic suchcarcinoma as adenoid and cysticmalignant carcinoma ameloblastoma and malignant [15,25]. ameloblastoma Notch signaling [15,25 is]. believed Notch signaling to be a major is believed factor toin bethe a progression major factor inof themalignant progression neoplasms. of malignant neoplasms. In recent years, a cross-talk between the Wnt sign signalal and the the Notch Notch signal signal has has been been reported reported [26]. [26]. Wnt signalingsignaling isis anan essentialessential signal signal for for tooth tooth development, development, and and ameloblastoma ameloblastoma has has also also been been found found to expressto express Wnt Wnt and and its its related related factors factors [27 [27].]. In In the the process process of of Wnt Wnt signal signal transduction, transduction, the the existence existence of of a mechanisma mechanism that that stimulates stimulates Notch Notch signaling signaling has has become become clear.clear. ItIt hashas beenbeen reportedreported that the Jagged1 ligand is upregulated downstream of Wnt signaling,signaling, indicating that a cross-talkcross-talk between Wnt and Notch may be associated with tumor progression ofof ameloblastomaameloblastoma [[28].28].

Figure 7. Immunohistochemical features of Notch1 expression inin ameloblasticameloblastic carcinoma.carcinoma. (a) Notch expression tends to be strong in the peripheral columnarcolumnar cells. Positive reac reactionstions are mainly found in the cytoplasm. Magnification Magnification ×100.100. ( (bb)) Atypical Atypical cells, cells, such such as as basaloid basaloid epithelium epithelium or or spindle spindle epithelial × cells, show strong Notch expression. Positive reactionsreactions are mainly found in the nuclei.nuclei. MagnificationMagnification 100. Anti-Notch-1 intracellular domain antibody, Abcam, ab83232, 1:100. ××100. Anti-Notch-1 intracellular domain antibody, Abcam, ab83232, 1:100. 10. Histopathology of Pleomorphic Adenoma 10. Histopathology of Pleomorphic Adenoma In general, pleomorphic adenoma has a rounded shape and is covered with relatively thin fibrous In general, pleomorphic adenoma has a rounded shape and is covered with relatively thin connective tissues. Various types of cells, as well as a diverse range of differentiation, have been fibrous connective tissues. Various types of cells, as well as a diverse range of differentiation, have observed in this type of tumor. The tumor consists of small and/or large ductal-like/cystic spaces in the been observed in this type of tumor. The tumor consists of small and/or large ductal-like/cystic spaces tissue. The majority of the tumor consists of glandular structures of tumor nests surrounded by fibrous in the tissue. The majority of the tumor consists of glandular structures of tumor nests surrounded tissue. Histologically, the tumor is separated from normal salivary gland tissue. Neoplastic parenchyma by fibrous tissue. Histologically, the tumor is separated from normal salivary gland tissue. Neoplastic consists of duct-like and solid sheet-like structures composed mainly of tubular and myoepithelial parenchyma consists of duct-like and solid sheet-like structures composed mainly of tubular and cells. The inside of tumor nests is distinctively biphasic in appearance and consists of a sparse array of myoepithelial cells. The inside of tumor nests is distinctively biphasic in appearance and consists of relatively loose cells and epithelial cell nests. Numerous cells are grouped together to form solid cell a sparse array of relatively loose cells and epithelial cell nests. Numerous cells are grouped together nests. Inside the tumor cell nest, a tube-like structure is observed. Bilayers of basal cells and large cubic to form solid cell nests. Inside the tumor cell nest, a tube-like structure is observed. Bilayers of basal cells form a tube-like structure (Figure8). The mesenchymal area is a myxoma-like tissue and consists cells and large cubic cells form a tube-like structure (Figure 8). The mesenchymal area is a myxoma- of myoepithelial cells, spindle shaped cells, and round or oval-shaped cells. Most of the cells of the like tissue and consists of myoepithelial cells, spindle shaped cells, and round or oval-shaped cells. mesenchymal region are derived from myoepithelial cells and dissociate from the tumor parenchyma Most of the cells of the mesenchymal region are derived from myoepithelial cells and dissociate from to form myxoma-like tissues around the solid parenchymal region. In some areas, cartilage-like tissue the tumor parenchyma to form myxoma-like tissues around the solid parenchymal region. In some is characteristically observed (Figure8). In the myxomatous region, the myoepithelial cells sparsely areas, cartilage-like tissue is characteristically observed (Figure 8). In the myxomatous region, the form a cord-like structure, and the presence of mucus is confirmed. Myoepithelial cells differentiate myoepithelial cells sparsely form a cord-like structure, and the presence of mucus is confirmed. into various tissues. Cartilage-like tissue is found in many cases of polymorphous adenomas. It has Myoepithelial cells differentiate into various tissues. Cartilage-like tissue is found in many cases of also been shown that this specialized tissue also originates from myoepithelial cells. In cartilage-like tissues, the presence of a specific cartilage matrix and cartilage-like cells is observed. The neoplastic Int. J. Mol. Sci. 2019, 20, x 10 of 14 polymorphous adenomas. It has also been shown that this specialized tissue also originates from Int. J. Mol. Sci. 2019, 20, 1973 10 of 14 myoepithelial cells. In cartilage-like tissues, the presence of a specific cartilage matrix and cartilage- like cells is observed. The neoplastic epithelial cells dissociate from the mucus-like tissue via the secretionepithelial of cells their dissociate own substrate. from the Tumor mucus-like parenchyma tissuevia contributes the secretion to the of formation their own of substrate. a myxomatous Tumor parenchymaregion. Tumor contributes parenchymal to the formationcells coexist of awith, myxomatous and are region. able to Tumor penetrate, parenchymal the structures cells coexist of with,mesenchymal and are ablecells toand penetrate, cartilage-lik thee structures cells. Thus, of mesenchymalepithelial components cells and are cartilage-like dissociated cells.and mixed Thus, intoepithelial a mesenchymal-like components are tissue, dissociated resulting and in mixed a “mixed into a appearance.” mesenchymal-like In addition, tissue, resultingtumor cells in aundergo “mixed significantappearance.” squamous In addition, metaplasia. tumor cells undergo significant squamous metaplasia.

Figure 8. Histopathological featuresfeatures ofof pleomorphicpleomorphic adenoma adenoma (HE (HE staining). staining). (a ()a The) The tumor tumor consists consists of solid sheet-like tumor nests and myxomatous mesenchymal parts. Magnification 40. (b) Squamous of solid sheet-like tumor nests and myxomatous mesenchymal parts. Magnification× ×40. (b) metaplasia with keratin pearls located in the central region of the tumor nest. Magnification 100. Squamous metaplasia with keratin pearls located in the central region of the tumor× nest. 11. NotchMagnification Signaling ×100. in Pleomorphic Adenoma

11. NotchAccording Signaling to the in literature Pleomorphic [13], NotchAdenoma expression is found in the cytoplasm and nuclei of ductal epithelial cells and in neoplastic myoepithelial cells. Keratins are useful epithelial differentiation According to the literature [13], Notch expression is found in the cytoplasm and nuclei of ductal markers due to their expression being cell type-specific and differentiation-specific. Cytokeratin 7 epithelial cells and in neoplastic myoepithelial cells. Keratins are useful epithelial differentiation (CK7) is known to be expressed in ductal epithelial cells. In the pleomorphic adenoma, neoplastic markers due to their expression being cell type-specific and differentiation-specific. Cytokeratin 7 ductal epithelial cells expressing CK7 and Notch are also expressed in the nuclei of several ductal (CK7) is known to be expressed in ductal epithelial cells. In the pleomorphic adenoma, neoplastic epithelial cells. Double immunofluorescent staining revealed that Notch and CK7 are co-expressed in ductal epithelial cells expressing CK7 and Notch are also expressed in the nuclei of several ductal neoplastic ductal structures. Notch is expressed in the majority of the nucleus of neoplastic cells in the epithelial cells. Double immunofluorescent staining revealed that Notch and CK7 are co-expressed solid lesions of the tumor nest. Cytokeratin 13 (CK13) is normally expressed in squamous epithelial in neoplastic ductal structures. Notch is expressed in the majority of the nucleus of neoplastic cells in cells. In tumor cells that have formed squamous metaplasia, CK13 showed a strong expression in the the solid lesions of the tumor nest. Cytokeratin 13 (CK13) is normally expressed in squamous squamous cell layer. At the site of squamous metaplasia, histological differentiation from basal cells to epithelial cells. In tumor cells that have formed squamous metaplasia, CK13 showed a strong squamous epithelium was observed. Notch is strongly expressed in the cytoplasm and nucleus of basal expression in the squamous cell layer. At the site of squamous metaplasia, histological differentiation cells, and its expression is weakened as squamous epithelial cells differentiate. As the result of double from basal cells to squamous epithelium was observed. Notch is strongly expressed in the cytoplasm immunofluorescent staining of Notch and CK13, CK13 was found to be negative in basal cells at the and nucleus of basal cells, and its expression is weakened as squamous epithelial cells differentiate. periphery of the squamous metaplastic region but nuclear Notch was prominently positively expressed, As the result of double immunofluorescent staining of Notch and CK13, CK13 was found to be where CK13-positive squamous cell lost their nuclear positive expression of Notch (Figure9). negative in basal cells at the periphery of the squamous metaplastic region but nuclear Notch was Notch signaling is a major signaling pathway that controls cell fate and tissue growth. Notch is prominently positively expressed, where CK13-positive squamous cell lost their nuclear positive a single pass transmembrane receptor with domains outside and inside the cell membrane. When expression of Notch (Figure 9). Notch binds to its ligand, the Notch intracellular domain (NICD) is cleaved and moves into the nucleus. This conveys instructions to various morphogenesis and tissue differentiation processes during development. Notch plays an important role in intercellular signaling and is involved in the maintenance, differentiation, and neural function of stem cells in adults. Disturbances in the Notch signaling pathway are associated with oncogenesis, including esophageal cancer and breast cancer, among others [10,22]. Notch is also involved in the metastasis of malignant tumors, such as adenoid cystic carcinoma and malignant ameloblastoma [15,25], and is considered to be a major factor in the progression of malignant neoplasms. On the contrary, it has been suggested that Notch-Jagged1 is the primary mechanism for determining the fate of ghost cells, one being special keratinization [20]. Keratins are structural proteins that make up the cytoskeleton of keratinocytes, and Notch may be

Int. J. Mol. Sci. 2019, 20, 1973 11 of 14 involved in the expression of cytokeratin. The expression of CK7 and Notch in ductal epithelial cells suggests that Notch is involved in the neoplastic ductal cell differentiation of pleomorphic adenoma [29]. The expression of Notch was strong in basal cells, and was attenuated during their differentiation to squamous cells. This change in expression suggests that Notch is involved in the differentiation of basal cells into squamous cells [30,31]. In addition, the loss of expression of keratinocytes in the nucleus denotes the end of cell differentiation. In Okuda et al.’s study, the expression of Wnt was confirmed in small basal cells forming the ductal epithelium, with basal cells producing squamous epithelialization. This result is consistent with our current study, suggesting that Notch functions at sites where cell differentiation takes place [32,33]. Our data on the expression of Wnt in 30 cases of pleomorphic adenomas showed that Wnt is expressed in the ductal structure and squamous metaplasia of solid tumor nests. In these regions, the β-catenin pathway was activated, which strongly suggested that Wnt is involved in squamous metaplasia and differentiation of duct-like structures [32]. Notch signaling could, therefore, undergo cross-talk with Wnt signaling to play an important role in the cell diInt.ff J.erentiation Mol. Sci. 2019 of, 20 various, x tumors [34,35]. 11 of 14

Figure 9. Immunofluorescent staining images of the formation of the ductal structure. (a) Green Figure 9. Immunofluorescent staining images of the formation of the ductal structure. (a) Green color color indicates the localization of CK7, and is found in ductal cells. (b) Red color indicates the indicates the localization of CK7, and is found in ductal cells. (b) Red color indicates the Notch1 Notch1 localization, and (c) merged image of CK7 and Notch1.; Immunofluorescent staining images localization, and (c) merged image of CK7 and Notch1.; Immunofluorescent staining images of of squamous metaplasia. (d) Green color indicates the CK13 localization, and is found in squamous squamous metaplasia. (d) Green color indicates the CK13 localization, and is found in squamous epithelial cells. (e) The nuclear localization of Notch1 is shown in red color in basal cells, and (f) merged epithelial cells. (e) The nuclear localization of Notch1 is shown in red color in basal cells, and (f) image of CK13 and Notch1. Magnification 100. Anti-Cytokeratin 7 antibody, abcam, [RCK105], merged image of CK13 and Notch1. Magnificatio× n ×100. Anti-Cytokeratin 7 antibody, abcam, ab9021, 1:100; anti-Cytokeratin 13 antibody, abcam, [AE8], ab16112, 1:100; anti-Notch-1 intracellular [RCK105], ab9021, 1:100; anti-Cytokeratin 13 antibody, abcam, [AE8], ab16112, 1:100; anti-Notch-1 domain antibody, Abcam, ab83232, 1:100. intracellular domain antibody, Abcam, ab83232, 1:100.

Notch signaling is a major signaling pathway that controls cell fate and tissue growth. Notch is a single pass transmembrane receptor with domains outside and inside the cell membrane. When Notch binds to its ligand, the Notch intracellular domain (NICD) is cleaved and moves into the nucleus. This conveys instructions to various morphogenesis and tissue differentiation processes during development. Notch plays an important role in intercellular signaling and is involved in the maintenance, differentiation, and neural function of stem cells in adults. Disturbances in the Notch signaling pathway are associated with oncogenesis, including esophageal cancer and breast cancer, among others [10,22]. Notch is also involved in the metastasis of malignant tumors, such as adenoid cystic carcinoma and malignant ameloblastoma [15,25], and is considered to be a major factor in the progression of malignant neoplasms. On the contrary, it has been suggested that Notch-Jagged1 is the primary mechanism for determining the fate of ghost cells, one being special keratinization [20]. Keratins are structural proteins that make up the cytoskeleton of keratinocytes, and Notch may be involved in the expression of cytokeratin. The expression of CK7 and Notch in ductal epithelial cells suggests that Notch is involved in the neoplastic ductal cell differentiation of pleomorphic adenoma [29]. The expression of Notch was strong in basal cells, and was attenuated during their differentiation to squamous cells. This change in expression suggests that Notch is involved in the differentiation of basal cells into squamous cells [30,31]. In addition, the loss of expression of keratinocytes in the nucleus denotes the end of cell differentiation. In Okuda et al.’s study, the expression of Wnt was confirmed in small basal cells forming the ductal epithelium, with basal cells

Int. J. Mol. Sci. 2019, 20, x 12 of 14 producing squamous epithelialization. This result is consistent with our current study, suggesting that Notch functions at sites where cell differentiation takes place [32,33]. Our data on the expression of Wnt in 30 cases of pleomorphic adenomas showed that Wnt is expressed in the ductal structure and squamous metaplasia of solid tumor nests. In these regions, the β-catenin pathway was activated, which strongly suggested that Wnt is involved in squamous metaplasia and differentiation of duct- like structures [32]. Notch signaling could, therefore, undergo cross-talk with Wnt signaling to play Int. J. Mol. Sci. 2019 20 an important role, in, 1973 the cell differentiation of various tumors [34,35]. 12 of 14

12. Conclusions Conclusions Ameloblastoma, ameloblastic ameloblastic fibroma, fibroma, calcifying odontoge odontogenicnic cyst, odontogenic myxoma, ameloblastic carcinoma, and and pleomorphic adenoma adenoma show show disease-specific disease-specific histological histological features. features. Although these these histological histological differences differences are are caused caused by a by variety a variety of factors, of factors, Notch Notchis considered is considered to play ato central play a centralrole. In role.odontogenic In odontogenic tumors tumorsand cysts, and Notch cysts, Notchsignaling signaling is believed is believed to be involved to be involved in the cytologicalin the cytological differentiation differentiation of ameloblast-like of ameloblast-like columnar columnar cells. In cells. pleomorphic In pleomorphic adenomas, adenomas, Notch signalingNotch signaling is believed is believedto be involved to be in involved the cytological in the differentiation cytological di offf erentiationneoplastic cells of neoplastic that form duct- cells likethat structures. form duct-like Moreover, structures. Notch Moreover,is involved Notchin the differentiation is involved in of the squamous differentiation metaplasia. of squamous In all of themetaplasia. tumors studied, In all of theNotch tumors was studied, found to Notch be strongly was found expressed to be strongly in cells expressed undergoing in cells differentiation, undergoing anddifferentiation, weakly expressed and weakly in cells expressed that had in cellsalready that differentiated. had already diff Furthermore,erentiated. Furthermore, the findings the discussed findings herediscussed suggest here that suggest Notch thatsignaling Notch induces signaling the induces differentiation the differentiation of odontogenic of odontogenic cells through cells epithelial- through mesenchymalepithelial-mesenchymal interactions interactions (Figure 10). (Figure Notch 10 sign). Notchaling signalingis also associated is also associated with oncogenesis with oncogenesis and the impairmentand the impairment of its signaling of its signaling may maybe associated be associated with with thethe malignant malignant transformation transformation of of ameloblastic ameloblastic carcinoma. In In the the pleomorphic adenoma, adenoma, the the Wnt Wnt ex expressionpression results coincided with the function of Notch at the sites of cell differentiation. differentiation. Regarding Regarding cell cell differentiation differentiation resulting resulting from from Wnt signaling, Notch signaling may undergo a cross-talk with Wnt signaling to play an important role in tumor cell differentiation.differentiation. This cross-talk between Wnt and Notch may be associ associatedated with the tumor tumor progression progression of ameloblastoma.

Figure 10. Notch signaling in cell differentiation of oral tumors. (a) Differentiation of ameloblast-like cell. Figure 10. Notch signaling in cell differentiation of oral tumors. (a) Differentiation of ameloblast-like (b) Differentiation of ductal epithelium. (c) Differentiation of squamous epithelium. (d) Notch signaling cell. (b) Differentiation of ductal epithelium. (c) Differentiation of squamous epithelium. (d) Notch is involved in epithelial-mesenchymal interactions of odontogenic tumors. N: Notch expression. signaling is involved in epithelial-mesenchymal interactions of odontogenic tumors. N: Notch expression. Funding: This study was funded by the Japan Society for Promotion of Science (JSPS) KAKENHI (Nos. 16K11441, 17K11862, 18K09789, 18K17224). Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. Sci. 2019, 20, 1973 13 of 14

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