The Expression of Selected Wnt Pathway Members (FZD6, AXIN2 and β-Catenin) in Canine Oral Squamous Cell Carcinoma and Acanthomatous Ameloblastoma

Simple Summary The expression patterns of selected Wnt pathway molecules were analyzed in two of the most common canine oral neoplasia—canine oral squamous cell carcinoma (COSCC) and canine acanthomatous ameloblastoma (CAA). We found an overlap of areas with high expression of FZD6 and SOX2 in COSCC, while cytokeratin expression was low in these areas, indicating the low differentiation status of these cells. In CAA, FZD6-positive areas expressed cytokeratin and exhibited features of squamous metaplasia. Moreover, the expression of β-catenin and AXIN2 was higher in both CAA and COSCC than in the healthy canine oral epithelium. This work uncovered the distinct expression patterns of Wnt molecules in both lesions, indicating the involvement of this pathway in the pathology of canine oral cancers, which presents opportunities for their usage for the prediction of cell behavior or in the development of new therapeutic approaches. Abstract The Wnt signaling pathway is well known to be involved in many types of human cancer; however, in veterinary medicine, the investigation of individual Wnt members’ expression, and their role in or association with oral tumor pathogenesis, is still underevaluated. We aim to determine the expression pattern of Frizzled-6 (FZD6) as one of the Wnt receptors in two of the most common canine oral neoplastic lesions—canine oral squamous cell carcinoma (COSCC) and canine acanthomatous ameloblastoma (CAA). While COSCC is a malignant tumor with aggressive biological behavior and a tendency to metastasize, CAA is a benign tumor with high local invasiveness. In CAA, the expression of FZD6 was mostly located in the center of the epithelial tumorous tissue, and cells exhibiting features of squamous metaplasia were strongly positive. In well-differentiated COSCC, FZD6 was expressed in the tumorous epithelium as well as the tumorous stroma. There was a negative correlation between cytokeratin expression and FZD6 expression in COSCC, where the central parts of the epithelial tumorous tissue were often FZD6-negative. The non-differentiated COSCC with low expression of cytokeratin exhibited a diffuse FZD6 signal. The invasive front with areas of tumor budding exhibited high FZD6 expression with a loss of cytokeratin expression. Moreover, the expression of β-catenin and AXIN2 was increased in comparison to gingiva. In conclusion, our study revealed significant differences in the expression patterns and the levels of FZD6 between COSCC and CAA, indicating the differential engagement of the Wnt pathway in these tumors.


Introduction
With the rapid developments in the field of veterinary oncology, there is an increasing need for a deeper understanding of the molecular regulations of animal cancer develop-

Canine Tissues
In total, 30 tumors were immunohistochemically evaluated, out of which 15 were COSCC and 15 CAA. Normal canine gingiva was used as a control tissue (total number: 5). Samples were acquired from the archive of the Department of Pathological Morphology and Parasitology of the Veterinary and Pharmaceutical University Brno (Czech Republic) in the form of paraffin tissue blocks. All samples were obtained from dogs of private owners.

Immunohistochemical Analysis and Immunofluorescent Detection
Tissue sections of 5 µm thickness were prepared and stained with Hematoxylin-Eosin (HE). The alternative sections were used for immunohistochemical and immunofluorescent analysis. The following primary antibodies were applied for the detection of specific protein expression by immunohistochemistry (Table 1) or immunofluorescence ( Table 2): β-Catenin PY 489, FZD6, pan-cytokeratin, Sex determining region Y box 2 (SOX2), Ki-67. In the case of immunohistochemistry, 3,3 -Diaminobenzidine (DAB) was used for visualization of the signal and Hematoxylin was used to counterstain the nuclei. For visualization of nuclei in the case of immunofluorescence, DRAQ5™ Fluorescent Probe Solution was used (Thermo Scientific™, Shanghai, China). Sections were photographed under bright-field illumination with a Leica compound microscope DMLB2 (Leica, Wetzlar, Germany) or SP8 Resonant Scanning Confocal microscope (Leica, Wetzlar, Germany) in the case of immunofluorescence.

Expression of SOX2 and Ki-67 in COSCC and CAA of Dogs
COSCC and CAA exhibit distinct morphological features and the diagnosis is usually performed based on common histological staining. CAA usually grows invasively in the form of odontogenic epithelial trabeculae, which are surrounded by mesenchymal stroma (Figure 1A-D). In contrast, well-differentiated COSCC are characterized by an invasiveness and infiltrative "budding" of epithelial protrusions on the tumorous tissue periphery ( Figure 1E-H).  We performed analyses of the transcription factor SOX2's expression and determination of the proliferation activity in these tumors ( Figure 2). In the normal canine oral epithelium, the most SOX2-positive cells were located in the basal layer while the expression faded in the superficial direction through the epithelial layers. Compared to the state in the normal gingiva, where the SOX2 cells were located only sparsely on the basal layer of the oral epithelium, there was strong expression of SOX2 in both COSCC and CAA. In CAA, the expression was mainly distributed in the outer layer of the epithelial cells. There was strong positivity in the areas of COSCC "budding" and in the invasive front area.

Expression of FZD6 in CAA and COSCC
The expression of FZD6 was found in both analyzed tumors-CAA and COSCC-and the expression pattern of FZD6 varied significantly between these two lesions (Figures 3 and 4). The co-expression of FZD6 and cytokeratin was investigated to correlate FZD6 expression with the differentiation status of neoplastic cells.
In CAA, FZD6 expression was located in the membranes and Ki-67 protein was used to determine the areas of cell proliferation ( Figure 2). In normal gingiva, the Ki-67 cells were scattered only in the basal layer. In the case of CAA, the proliferating cells were distributed randomly in the middle areas of the tumorous epithelium; thus, the expression of SOX2 and Ki-67 was not colocalized in this tumor. In the case of well-differentiated COSCC, the proliferating cells were localized around the outer layer of the epithelial nests, resembling the expression stratification of the normal epithelium. In cases of a poorly differentiated COSCC, there was strong, diffuse Ki-67 expression.

Expression of FZD6 in CAA and COSCC
The expression of FZD6 was found in both analyzed tumors-CAA and COSCC-and the expression pattern of FZD6 varied significantly between these two lesions (Figures 3 and 4). The co-expression of FZD6 and cytokeratin was investigated to correlate FZD6 expression with the differentiation status of neoplastic cells.

Expression of β-Catenin and AXIN2 in CAA and COSCC
As we observed areas of increased expression of FZD6 in both types of tumors, we sought to uncover whether downstream molecules of Wnt signaling displayed upregulation in the analyzed areas. We selected two downstream targets-phosphorylated β-catenin (phospho PY489) and AXIN2. The expression of both markers was higher in both CAA and COSCC than in the healthy canine oral epithelium ( Figure 5E,F).
AXIN2 was expressed in numerous nuclei of both CAA and COCC ( Figure 5A,B). There was also a strong nuclear expression of phosphorylated β-catenin in CAA and COSCC ( Figure 5C,D). In CAA, less β-catenin-positive cells were located in the outer layer of the epithelial In CAA, FZD6 expression was located in the membranes and cytoplasm of epithelial cells (Figure 3). FZD6-positive cells were located in the center of epithelial tumorous tissue; however, a signal was located in the palisade-like outer layer of the tumorous tissue. Moreover, FZD6 expression was very strong in the areas of squamous metaplasia.
In the well-differentiated OSCC, FZD6-positive cells were located in the neoplastic epithelium and in the tumorous stroma ( Figure 4A). The poorly differentiated areas of OSCC, exhibiting low expression of cytokeratin, were diffusely positive for FZD6 (Figure 4). There was a distinct negative correlation between the intensity of cytokeratin and FZD6 expression in OSCC (Figure 4). Areas of tumor budding in the invasive front displayed high FZD6 expression with a loss of cytokeratin expression (Figure 4). In contrast, scattered cytoplasmic positivity of FZD6 was observed in the basal layer of the normal oral epithelium ( Figure 4C).

Expression of β-Catenin and AXIN2 in CAA and COSCC
As we observed areas of increased expression of FZD6 in both types of tumors, we sought to uncover whether downstream molecules of Wnt signaling displayed upregulation in the analyzed areas. We selected two downstream targets-phosphorylated β-catenin (phospho PY489) and AXIN2. The expression of both markers was higher in both CAA and COSCC than in the healthy canine oral epithelium ( Figure 5E,F).
tumorous islets. While a strong nuclear expression of phosphorylated βcatenin was found in COSCC, the β-catenin signal was stronger in the larger and more differentiated cells in the middle of tumorous islets. In the physiological canine oral epithelium, the expression of phosphorylated β-catenin was very weak ( Figure 5F).  AXIN2 was expressed in numerous nuclei of both CAA and COCC ( Figure 5A,B). There was also a strong nuclear expression of phosphorylated β-catenin in CAA and COSCC ( Figure 5C,D). In CAA, less β-catenin-positive cells were located in the outer layer of the epithelial tumorous islets. While a strong nuclear expression of phosphorylated β-catenin was found in COSCC, the β-catenin signal was stronger in the larger and more differentiated cells in the middle of tumorous islets. In the physiological canine oral epithelium, the expression of phosphorylated β-catenin was very weak ( Figure 5F).

Discussion
The biological behavior of CAA and COSCC varies, especially considering the speed of growth and their local invasiveness [10]. Slow growth of CAA corresponds to the low numbers of Ki-67-positive cells found in this neoplasm, which is in contrast to COSCC, with high Ki-67 expression. The Ki-67 labeling index is considered to be a prognostic factor of many canine cancers [32][33][34][35], and, in COSCC, it was significantly associated with a lymph node metastasis [36]. Ki-67 expression might be also a negative prognostic marker for human patients with OSCC [37]. The distribution of the Ki-67-positive cells in CAA and COSCC has not been evaluated yet. In well-differentiated COSCC, the Ki-67-positive cells are located mostly in the areas of tumor budding and outer layers of the tumorous islets. This phenomenon corresponds to the invasive tumor front, where the Ki-67 expression was described to be high in human OSCC [38]. Similarly, in breast cancer tissue, the Ki-67-positive cells also tend to be located in the outer layers of the tumor nest rather than in the center [39]. There is no close counterpart in the human pathology to the CAA. CAA partially resembles human central and peripheral ameloblastoma, but there are some distinguishable features. There are four types of human ameloblastoma: conventional, peripheral and unicystic. By its macroscopic appearance, CAA resembles human peripheral ameloblastoma (HPA); however, HPA does not involve bone. Due to its bone invasion, CAA resembles human conventional ameloblastoma, of which the acanthomatous histological type is the most similar [40]. In this study, the Ki-67 proliferation rate was low, with positive cells encountered both to peripheral and central areas, similar to human ameloblastoma [41,42].
To illustrate the differences regarding the growth potential, we further performed analyses of the expression of transcription factor SOX2 and determination of cells with progenitor potential in CAA and COSCC, taking into consideration also expression in the normal gingiva as a reference structure. SOX2 expression has been suggested to act as a prognostic factor in various cancers in humans, and its increased expression has been associated with the malignancy and metastatic spread of tumors [43][44][45]. In oral lesions, SOX2 expression has been described in human odontogenic keratocysts and ameloblastoma [46]. In comparison to human ameloblastomas, where SOX2 expression is low, CAA exhibits higher expression of SOX2. Human odontogenic keratocysts are usually strongly SOX2-positive, resembling the expression in CAA. The SOX2-positive progenitor cells were mainly present in large numbers in the outer layer of the tumorous islets. This differential expression through individual layers was similar to the normal gingiva, where SOX2-positive cells were located in the basal layers. The high expression of SOX2 in the COSCC invasive front and tumor "budding" corresponds to its aggressive biological behavior, but the increased presence of SOX2-positive cells in outer layers of the CAA epithelium is surprising and further investigation of the fate of these cells is needed.
In the case of human oral cancer (OSCC), Wnt signaling components are often overexpressed [47,48]. In veterinary medicine, research on the Wnt pathway has, up to now, mostly concentrated on canine mammary tumors and canine cutaneous melanoma [49][50][51], while oral tumors have not been evaluated. Here, we selected for further analyses the Wnt receptor (FZD6) and also downstream molecules (AXIN2, β-catenin) to determine potential differences in Wnt pathway activity in CAA and COSCC.
In both analyzed lesions, high expression of FZD6 was observed. In the case of COSCC, the areas with strong FZD6 expression were also strongly SOX2-positive, indicating high numbers of progenitor cells in these areas. Similarly, alterations of several FZD receptors have been in many human cancers and FZD6 has been found to be increased in human liver, prostate, colorectal cancer and cutaneous SCC [28][29][30][31]. FZD6 is considered a potential cancer stem cell marker in human neuroblastoma and high expression is linked to a poor prognosis [52]. Ablation of FZD6 expression in human mammary cancer cell lines was found to inhibit cell invasion, lead to a more symmetrical growth pattern and inhibit the metastatic potential of tumorous cells [53]. Enforced expression of SOX2 leads to the inhibition of Wnt/β-catenin signaling activity [54]. In the veterinary literature, however, there is not yet evidence of FZD6 expression and its association with tumor progenitor and proliferation status, to the best of our knowledge. Considering the similar distribution of SOX2-and FZD6-positive cells in COSCC, the FZD6-positive tumors might demonstrate higher growth status and thus more aggressive biological behavior [53]. Additional experimental data are, however, needed to confirm the correlation between the level of FZD6 expression, SOX2 positivity and the prognosis of COSCC patients.
In COSCC, we noticed an association among the tumor differentiation of cell keratinization and FZD6 expression. Areas of weak cytokeratin expression displayed the highest FZD6 signal. The low cytokeratin expression in FZD6-positive areas of COSCC indicates that these areas correspond to the sites with low differentiation status. In CAA, squamous differentiation (metaplasia) is a fairly rare event [11]. In CAA with squamous metaplasia, we noticed exactly the opposite pattern to that in COSCC-the areas of strong cytokeratin expression exhibited high FZD6 expression. Abnormally increased expression of FZD6 in poorly differentiated cells of COSCC as well as well-differentiated areas of CAA suggest differences in the malfunctioning of the Wnt pathway in these tumors and variability in the cell response, where the alteration of Wnt signaling results in a completely different fate of neoplastic cells.
Next, we also wanted to analyze downstream components of the Wnt pathway in CAA and COSCC with the aim of uncovering whether FZD6 expression can be correlated with the alteration of downstream signaling and whether there are differences between these two oral lesions. We noticed overexpression of AXIN2 in both CAA as well as COSCC when compared to a normal gingiva. In human ameloblastoma, there was also strong immunostaining of AXIN2 in comparison to a normal oral mucosa [55], and high expression levels of AXIN2 in human SCC were associated with tumor size and recurrence [56]. Moreover, increased levels of AXIN2 were highly correlated with the malignant transformation of an oral leukoplakia in humans [57], indicating possibly similar associations in canine oral tumors.
β-catenin is a wide-ranging molecule in terms of its function. In the cell adhesion junctions, it acts as a link between α-catenin and cadherins [58,59]. The loss of β-catenin expression is therefore linked to the disruption of these cell contacts and it is associated with tumor spread-namely, metastasis initiation. Moreover, it is a well-studied prognostic factor used mainly in human SCC [60][61][62][63][64]. In COSCC, the loss of this adhesion molecule was also connected to the epithelial-mesenchymal transition in the invasive front of this tumor [21]. In terms of Wnt pathway activity and its involvement in the tumorigenesis of CAA and COSCC, the phosphorylated form of β-catenin expressed in the nucleus seems to be pivotal and there are no data in the literature about the expression of this form in canine cancers. During the activation of the Wnt pathway, β-catenin is phosphorylated at tyrosine 489 (PY489 β-catenin) and it is transferred to the nucleus, resulting in the presence of "nuclear" or "active" β-catenin, where it functions, among others, as an activator of oncogenic targets [65].
Activation of the canonical Wnt pathway seems to be dissimilar in these two canine oral neoplasia. In COSCC, the nuclear β-catenin expression was found to be strong and located focally mostly in the areas of well-differentiated cells and in the zones with low SOX2 expression. In contrast, the nuclear β-catenin expression was often lost in areas of SOX2-positive cells in the outer palisading layer of CAA. Because β-catenin is translocated to the nucleus in the "Wnt-on" state [66], we suggest that the well-differentiated cells in COSCC are those where Wnt signaling is activated, whereas in the less differentiated SOX2-positive (progenitor) cells, the Wnt signaling is disrupted, which might lead to the aggressive behavior of these cells.

Conclusions
Expression of FZD6, β-catenin and AXIN2 indicates the activation of the Wnt signaling pathway in both CAA and COSCC. Their expression pattern correlates with the progenitor marker SOX2 and the proliferation status of tumorous epithelial cells; however, the distribution of positive cells is distinct in these two neoplasms. Our results indicate the distinct involvement of Wnt signaling in these two oral pathogeneses in dogs.