Association of MDM2 Overexpression in Ameloblastomas with MDM2 Amplification and BRAFV600E Expression.

Ameloblastoma is a rare tumor but represents the most common odontogenic neoplasm. It is localized in the jaws and, although it is a benign, slow-growing tumor, it has an aggressive local behavior and high recurrence rate. Therefore, alternative treatment options or complementary to surgery have been evaluated, with the most promising one among them being a targeted therapy with the v-Raf murine sarcoma viral oncogene homologue B (BRAF), as in ameloblastoma the activating mutation V600E in BRAF is common. Studies in other tumors have shown that the synchronous inhibition of BRAF and human murine double minute 2 homologue (MDM2 or HDM2) protein is more effective than BRAF monotherapy, particularly in the presence of wild type p53 (WTp53). To investigate the MDM2 protein expression and gene amplification in ameloblastoma, in association with BRAFV600E and p53 expression. Forty-four cases of ameloblastoma fixed in 10% buffered formalin and embedded in paraffin were examined for MDM2 overexpression and BRAFV600E and p53 expression by immunohistochemistry, and for MDM2 ploidy with fluorescence in situ hybridization. Sixteen of forty-four (36.36%) cases of ameloblastoma showed MDM2 overexpression. Seven of sixteen MDM2-positive ameloblastomas (43.75%) were BRAFV600E positive and fifteen of sixteen MDM2-positive ameloblastomas (93.75%) were p53 negative. All MDM2 overexpressing tumors did not show copy number alterations for MDM2. Overexpression of MDM2 in ameloblastomas is not associated with MDM2 amplification, but most probably with MAPK activation and WTp53 expression. Further verification of those findings could form the basis for the use of MDM2 expression as a marker of MAPK activation in ameloblastomas and the trial of dual BRAF/MDM2 inhibition in the management of MDM2-overexpressing/BRAFV600E-positive/WTp53 ameloblastomas.

Prognostic value of the expression and localization of cell proliferation and apoptosis markers in unicystic ameloblastomas.

The aim of this study was to verify whether the expression of cell proliferation and apoptosis markers in different types of unicystic ameloblastoma (UA) is associated with the location of neoplastic cells. Immunohistochemical study with a sample of 32 cases of UA, 11 cases of conventional ameloblastoma (CAM) and ten dental follicles (DF) cases was performed. Cell proliferation was assessed using Ki-67 status, and apoptosis by caspase-3 expression. Mural UA (MUA) showed a higher immunostaining of Ki-67 (p < 0.05) and a lower immunostaining of Caspase-3 (p < 0.05) compared with luminal and intraluminal subtypes of UA and CAM. The neoplastic cells of the MUA's cystic capsule showed a higher expression of Ki-67 protein (p < 0.0001) and a lower expression of Caspase-3 (p < 0.0001) compared with the lumen. DF showed lower Ki-67 and Caspase-3 immunostaining (p < 0.05) than neoplasms. The higher immunoexpression of Ki-67 and the lower immunoexpression of Caspase-3 in MUA, in the parenchyma cells within the cystic capsule, suggest an association between the biological behaviour and location of neoplastic cells in a tumour.

Arl4c is involved in tooth germ development through osteoblastic/ameloblastic differentiation.

Murine tooth germ development proceeds in continuous sequential steps with reciprocal interactions between the odontogenic epithelium and the adjacent mesenchyme, and several growth factor signaling pathways and their activation are required for tooth germ development. The expression of ADP-ribosylation factor (Arf)-like 4c (Arl4c) has been shown to induce cell proliferation, and is thereby involved in epithelial morphogenesis and tumorigenesis. In contrast, the other functions of Arl4c (in addition to cellular growth) are largely unknown. Although we recently demonstrated the involvement of the upregulated expression of Arl4c in the proliferation of ameloblastomas, which have the same origin as odontogenic epithelium, its effect on tooth germ development remains unclear. In the present study, single-cell RNA sequencing (scRNA-seq) analysis revealed that the expression of Arl4c, among 17 members of the Arf-family, was specifically detected in odontogenic epithelial cells, such as those of the stratum intermedium, stellate reticulum and outer enamel epithelium, of postnatal day 1 (P1) mouse molars. scRNA-seq analysis also demonstrated the higher expression of Arl4c in non-ameloblast and inner enamel epithelium, which include immature cells, of P7 mouse incisors. In the mouse tooth germ rudiment culture, treatment with SecinH3 (an inhibitor of the ARNO/Arf6 pathway) reduced the size, width and cusp height of the tooth germ and the thickness of the eosinophilic layer, which would involve the synthesis of dentin and enamel matrix organization. In addition, loss-of-function experiments using siRNAs and shRNA revealed that the expression of Arl4c was involved in cell proliferation and osteoblastic cytodifferentiation in odontogenic epithelial cells. Finally, RNA-seq analysis with a gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis showed that osteoblastic differentiation-related gene sets and/or GO terms were downregulated in shArl4c-expressing odontogenic epithelial cells. These results suggest that the Arl4c-ARNO/Arf6 pathway axis contributes to tooth germ development through osteoblastic/ameloblastic differentiation.

Identification of ferroptosis-related proteins in ameloblastoma based on proteomics analysis.

PURPOSE: We used proteomic sequencing and experimental verification to identify the potential ferroptosis-related proteins in ameloblastoma. METHODS: Samples of ameloblastoma (n = 14) and normal gingival tissues (n = 5) were collected for proteomic sequencing to identify differentially expressed proteins (DEPs) in ameloblastoma. Ferroptosis-related genes were downloaded from FerrDb V2, which were then compared with DEPs to obtain ferroptosis-related DEPs (FR-DEPs). A functional enrichment analysis was performed, and a protein-protein interaction network was built. The hub proteins were screened using the Cytoscape software, and potential drugs targeting them were retrieved from the DrugBank database. A hub protein was selected for immunohistochemical validation, and its expression was assessed in ameloblastomas, odontogenic keratocysts, dentigerous cysts, and normal gingival tissues. The primary ameloblastoma cells were cultured to explore the effect of the protein on the migratory properties of the tumour cells. RESULTS: A total of 58 FR-DEPs were screened, and six hub proteins were identified: mTOR, NFE2L2, PRKCA, STAT3, EGFR, and CDH1. Immunohistochemical analysis showed that mTOR expression was upregulated in ameloblastomas compared with that in odontogenic keratocysts, dentigerous cysts, and normal gingival tissues. p-mTOR was highly expressed in ameloblastomas, with a positivity rate of 83.3%. In addition, rapamycin, an inhibitor of mTOR, can inhibit the migratory capacity of primary cultured ameloblastoma cells. CONCLUSION: Our results revealed the ferroptosis-related proteins in ameloblastomas and their underlying biological processes. Additionally, mTOR was overexpressed and was found to be associated with the aggressiveness of ameloblastomas, which may be a potential target for future treatments.

Expression of stem cell markers SALL4, LIN28A, and KLF4 in ameloblastoma.

BACKGROUND: Ameloblastoma (AME) is a benign odontogenic tumour of epithelial origin characterised by slow but aggressive growth, infiltration, and recurrence; it is capable of reaching large dimensions and invading adjacent structures. Stem cell research has proven to be significant in the sphere of tumour biology through these cells' possible involvement in the aetiopathogenesis of this tumour. METHODS: Immunohistochemistry was performed on AME, dentigerous cyst (DC), and dental follicle (DF) samples, and indirect immunofluorescence was performed on the AME-hTERT cell line to determine the expression of SALL4, LIN28A, and KLF4. RESULTS: Expression of proteins related to cellular pluripotency was higher in AME cells than in DC and DF cells. The analysis revealed that the proteins in question were mainly expressed in the parenchyma of AME tissue samples and were detected in the nuclei of AME-hTERT cells. CONCLUSIONS: Stem cells may be related to the origin and progression of AME.

Differential expression of PEA3 in odontogenic cysts and tumors.

BACKGROUND: PEA3 transcription factor has been identified as a downstream target of the MAPK and PI3K pathways, and PEA3 overexpression has been observed in a variety of tumor types. We aimed to evaluate PEA3 expression in odontogenic cysts and tumors and compare the expression among odontogenic lesions. In addition, the correlations between PEA3 expression and clinicopathological characteristics of conventional ameloblastoma and unicystic ameloblastoma were investigated. METHODS: This study was performed on 165 samples of odontogenic cysts and tumors including 20 dentigerous cysts, 20 odontogenic keratocysts, 16 adenomatoid odontogenic tumors, 5 ameloblastic fibromas, 45 unicystic ameloblastomas, and 59 conventional ameloblastomas. The sections were immunohistochemically stained with mouse monoclonal anti-PEA3 antibody and PEA3 expression was evaluated as the immunoreactive score. RESULTS: PEA3 expression was absent in all dentigerous cysts (DCs) and odontogenic keratocysts, while all adenomatoid odontogenic tumors showed either no (75%) or low (25%) expression of PEA3. Most of the ameloblastic fibromas (60%) displayed no PEA3 expression. A high expression of PEA3 was observed in a substantial number of unicystic ameloblastomas (48.9%) and conventional ameloblastomas (49.2%) in our study. PEA3 expression in DCs, odontogenic keratocysts and adenomatoid odontogenic tumors were significantly different from that in conventional ameloblastomas and that in unicystic ameloblastomas (p < 0.05). The expression of PEA3 was significantly different in the age groups of unicystic ameloblastomas and histological subtypes of conventional ameloblastomas (p < 0.05). CONCLUSION: PEA3 overexpression is predominant in unicystic ameloblastomas and conventional ameloblastomas compared to other odontogenic lesions, indicating a pivotal role of PEA3 as a downstream effector of MAPK pathway in these two odontogenic lesions.

Connexin 43 expression in tooth germ and benign odontogenic tumors.

OBJECTIVE: The aim of this study was to investigate and compare the immunohistochemical expression of connexin 43 (Cx43) in tooth germs (TGs), ameloblastic fibromas (AFs), ameloblastic fibro-odontomas (AFOs), and conventional ameloblastomas (AMs). STUDY DESIGN: Nine TGs, 12 AFs, 12 AFOs, and 27 AMs were evaluated for Cx43 expression by immunohistochemistry. RESULTS: Most of the TGs expressed Cx43 in the mesenchyme (77.6%) and in the late stages of odontogenesis. Cx43 was more highly expressed (P < .05) in the mesenchymal layer of all groups than in the epithelial layer except for the AFOs. When comparing the expression of Cx43 in the different layers of the analyzed groups, statistically significant differences were observed between AFO vs AM (*P = .0158) in the epithelial layer and between AF vs AFO (P** = .0046) in the mesenchymal layer. CONCLUSIONS: The results obtained in this study showed that Cx43 is a protein with important expression in the mesenchymal layer of the embryonic and odontogenic tissues studied. It could be speculated that Cx43 participates in mineralization events based on the relationship of the expression of this protein between the epithelial and mesenchymal layers of odontogenic tissues.

IL-6 Plays a Critical Role in Stromal Fibroblast RANKL Induction and Consequent Osteoclastogenesis in Ameloblastoma Progression.

Ameloblastoma (AB) is the most common benign, epithelial odontogenic tumor that occurs in the jawbone. AB is a slow-growing, benign epithelial tumor but shows locally invasive growth, with bone resorption or recurrence if not adequately resected. From these points of view, understanding the mechanism of AB-induced bone resorption is necessary for better clinical therapy and improving patients' quality of life. In bone resorption, osteoclasts play critical roles, and RANKL is a pivotal regulator of osteoclastogenesis. However, the source of RANKL-expressing cells in the AB tumor microenvironment is controversial, and the mechanism of osteoclastogenesis in AB progression is not fully understood. In this study, we investigated the distribution of the RNA expression of RANKL in AB specimens. We found that PDGFRα- and S100A4-positive stromal fibroblasts expressed RANKL in the AB tumor microenvironment. Moreover, we analyzed the mechanisms of osteoclastogenesis in the AB tumor microenvironment using the human AB cell line AM-1 and a human primary periodontal ligament fibroblast cells. The results of histopathologic and in vitro studies clarified that the interaction between AB cells and stromal fibroblasts upregulated IL-6 expression and that AB cells induced RANKL expression in stromal fibroblasts and consequent osteoclastogenesis in AB progression.

Immunoexpression of stem cell markers SOX-2, NANOG AND OCT4 in ameloblastoma.

Background: Ameloblastoma (AME) is characterized by a locally invasive growth pattern. In an attempt to justify the aggressiveness of neoplasms, the investigation of the role of stem cells has gained prominence. The SOX-2, NANOG and OCT4 proteins are important stem cell biomarkers. Methodology: To verify the expression of these proteins in tissue samples of AME, dentigerous cyst (DC) and dental follicle (DF), immunohistochemistry was performed and indirect immunofluorescence were performed on the human AME (AME-hTERT) cell line. Results: Revealed expression of SOX-2, NANOG and OCT4 in the tissue samples and AME-hTERT lineage. Greater immunostaining of the studied proteins was observed in AME compared to DC and DF (p < 0.001). Conclusions: The presence of biomarkers indicates a probable role of stem cells in the genesis and progression of AME.

[BRAF V600E expression in ameloblastomas, ameloblastic carcinomas and cysts].

PURPOSE: To investigate protein and genetic status of BRAF V600E in ameloblastomas, ameloblastic carcinomas and cysts, and to explore if recurrence and malignant transformation of ameloblastomas depends on BRAF status. METHODS: Twenty cysts, 25 primary ameloblastomas, 25 recurrent ameloblastomas and 8 ameloblastic carcinomas were analysed by immunohistochemistry, and 2 cysts, 5 primary ameloblatomas, 5 recurrent ameloblastomas, 3 atypical ameloblastomas and 6 ameloblastic carcinomas were analysed by quantitative real-time PCR(qPCR). SPSS 26.0 software package was used for data analysis. RESULTS: There was no correlation between BRAF V600E expression and recurrence. Cysts and carcinomas had lower frequencies of BRAF V600E mutations than ameloblastomas. There was a concordance between positive staining of BRAF V600E protein and BRAF V600E mutation. CONCLUSIONS: BRAF V600E mutation may be useful in the differential diagnosis of ameloblastomas with cysts and ameloblastic carcinomas. Immunohistochemistry may be a beneficial method to screen for BRAF V600E mutation in ameloblastoma.

Differential protein expression of osteoclastogenic factors in odontogenic cysts and tumors.

The osteolytic activity of odontogenic cysts and tumors is directly associated with their growth and aggressiveness. The influence of proteins expressed by epithelial and mesenchymal cells on this biological event differs between indolent cystic lesions, aggressive cystic lesions, and odontogenic tumors. The objective of this study was to compare the immunohistochemical expression of factors that stimulate (receptor activator of nuclear factor kappa-Β ligand - RANKL, cathepsin K - CatK and matrix metallopeptidase 8 - MMP-8) and inhibit (osteoprotegerin - OPG) osteoclastogenesis between dentigerous cyst (DC), glandular odontogenic cyst (GOC), odontogenic keratocyst (OKC), and ameloblastoma (AB). Paraffin-embedded sections of nine DCs, nine GOCs, 20 OKCs, 21 ABs, and four dental follicles (DFs) were subjected to immunohistochemistry. Immunoreactivity was analyzed semiquantitatively and quantitatively in epithelium and connective tissue, respectively. The proteins were immunoexpressed in epithelial and mesenchymal cells of all lesions studied. The expression of RANKL and CatK was higher in OKC, AB, and GOC (p<0.005). Higher expression of OPG was found in DF and DC compared to the other markers (p<0.005). MMP-8 expression was high in GOC and OKC. This study demonstrated the differential expression of factors that inhibit and stimulate bone resorption during the development of DC, GOC, OKC, and AB. Higher expression of RANKL and CatK was observed in more aggressive lesions. OPG appears to be one of the molecules responsible for the slower growth of DC.

Immunohistochemical study of the plasminogen activator system in benign epithelial odontogenic lesions.

The aim of this study was to analyze and compare the immunohistochemical expression of plasminogen activator system (PAS) proteins (uPA, uPAR, and PAI-1) in ameloblastomas (AMBs), odontogenic keratocysts (OKCs), and dental follicles (DFs) representing normal odontogenic tissue, as well as to investigate possible correlations between these proteins. Twenty AMBs, 20 OKCs, and 10 DFs were selected for immunohistochemical analysis. In each case, the immunoexpression of uPA, uPAR, and PAI-1 was evaluated semiquantitatively based on the percentage of positivity in odontogenic epithelial and connective tissue cells. The epithelial immunoexpression of uPA was significantly lower in AMBs when compared to OKCs (p = 0.001) and DFs (p = 0.029). Significantly higher epithelial immunostaining for uPAR was observed in AMBs when compared to OKCs (p < 0.001). There were no significant differences in the epithelial immunoexpression of PAI-1 between AMBs and OKCs (p = 1.000). The correlations found for the expression of the studied proteins were not statistically significant (p > 0.05). However, the epithelial and connective tissue expressions of uPAR have a strong positive and statistically significant correlation in AMBs. The present results suggest that uPA is involved in the pathogenesis of OKCs and that uPAR may participate in tumorigenesis in AMBs. The high percentage of PAI-1-positive cells suggests a possible role for this protein in the development of AMBs and OKCs. Furthermore, the studied proteins do not seem to act synergistically in AMBs, OKCs, and DFs.

EXPRESSION OF ALPHA-SMOOTH MUSCLE ACTIN IN ODONTOGENIC CYSTS AND TUMORS.

BACKGROUND: Odontogenic cysts and tumors exhibit different degrees of aggressiveness in their biological behavior. There has been evidence that the presence of myofibroblasts (MFs) at the invasion front promotes tumor invasion. Our study is based on the fact that MFs are important in the biological behavior of odontogenic cysts and tumors. AIM: To assess immunohistochemically expression of alpha-smooth muscle actin (α-SMA) of MFs in odontogenic cysts and tumors and correlate this expression to their biological behavior. MATERIALS AND METHODS: The archival tissues collected for 1.5 years were obtained from the Department of Oral Pathology & Microbiology, People's Dental Academy, Bhopal (India). A total of 40 cases consisting of 10 cases each of odontogenic keratocysts, radicular cysts, dentigerous cysts and ameloblastomas formed the study group. An immunohistochemical analysis of α-SMA expression and localization was carried out. RESULTS: Mean MF counts were the highest in odontogenic keratocysts which was followed by ameloblastomas, entigerous cysts and radicular cysts. Weak α-SMA-expression was found in 50% of cases, moderate in 22.5% of cases, and intense - in 10% cases. MFs were arranged in the spindle, focal, or network patterns in 35; 27.5 and 20% of cases, respectively. CONCLUSION: The analysis revealed that the MFs were distinctly heterogeneous in distribution and pattern of arrangement. This provided persuasive evidence that stroma of these lesions harbor MFs as reflected by α-SMA immunopositive cells.

Immunohistochemical analysis of SHH, SMO and GLI-1 proteins in epithelial odontogenic lesions.

The present study analyzed the expression of proteins involved in the sonic hedgehog signaling pathway (SHH, SMO, and GLI-1) in benign epithelial odontogenic lesions (odontogenic keratocyst - OKC, ameloblastoma - AB, and adenomatoid odontogenic tumor - AOT) in order to identify the role of these proteins in the pathogenesis of these lesions. The sample consisted of 20 OKCs, 20 ABs, and 10 AOTs. The Kruskal-Wallis, Mann-Whitney U, and Spearman's (r) tests were used for statistical analysis, with the level of significance set at 5% (p < 0.05). The membrane/cytoplasmic expression of SHH was significantly higher in AB compared to AOT (p = 0.022) and OKC (p = 0.02). No differences were found in the membrane/cytoplasmic expression of SMO between the lesions studied. Regarding GLI-1, significant differences were observed at the nuclear level for AB and OKC compared to AOT (p < 0.0001). In addition, significant positive correlations were found between cytoplasmic and nuclear GLI-1 in AB (r = 0.482; p = 0.031) and OKC (r = 0.865; p < 0.0001), and between membrane/cytoplasmic SMO and cytoplasmic GLI-1 in AOT (r = 0.667; p = 0.035) and OKC (r = 0.535; p = 0.015). The results of this study confirm the participation of the sonic hedgehog signaling pathway in the pathogenesis of the lesions studied. Overexpression of SHH in ABs and nuclear expression of GLI-1 in ABs and OKCs indicate that these proteins contribute to the more aggressive behavior of these two lesions when compared to AOT.

Alteration of primary cilia morphology and associated signalling in ameloblastoma.

OBJECTIVES: Primary cilium is a cellular organelle with growing significance confirmed in tumour biology. Primary cilia have been associated with fine tuning of numerous cell signalling pathways and the role of this structure in cancer initiation and progression is recently at the forefront of attention. Here, we investigated possible alterations in the occurrence of primary cilia and changes of associated signalling in ameloblastoma, which represents the most common odontogenic tumour. METHODS: We performed immunohistochemistry to assess the number and morphology of primary cilia in ameloblastoma tissues. The gene expression of key SHH pathway members was analysed by qPCR. As a functional experiment, we treated a primary ameloblastoma cell line by a SHH pathway inhibitor Sonidegib (LDE225). RESULTS: We uncovered differences in primary cilia distribution and appearance in histological subtypes of ameloblastoma with the highest number of ciliated cells in plexiform and follicular subtypes. SHH protein was located close to primary cilia in ameloblastoma epithelial cells and the expression of molecules downstream of SHH signalling was upregulated. Moreover, the inhibition of SHH pathway by Sonidegib caused downregulation of SHH effector gene GLI1 and cell cycle regulator CCND1 in ameloblastoma primary cell line. The inhibition of SHH signalling also altered the expression of molecules involved in intraflagellar transport. CONCLUSIONS: In conclusion, our study uncovered alterations in number of ciliated cells and associated signalling in ameloblastoma, which indicate SHH inhibitors as potential therapeutic target to treat this disease.

Immunoexpression of BRAF, EGFR and CD10 in ameloblastoma.

The ameloblastoma is the most challenging odontogenic neoplasm to treat because of its locallyinvasive behaviour, severe clinical implication, risk of malignant transformation and high recurrence rate. Recent evidence suggests that BRAF, EGFR and CD10 have a role in the local invasiveness of ameloblastoma. However, the spatial distribution characteristics of these pro-invasive factors and their association with clinical parameters in this neoplasm are largely unexplored. We sought to address these issues in ameloblastoma subtypes and to determine their biological relevance. Nineteen unicystic (UA) and 20 conventional ameloblastoma (SMA) were subjected to immunohistochemical staining for BRAF, EGFR and CD10, and semiquantitative analysis was performed. All ameloblastoma cases (n=39/39; 100%) exhibited a BRAF+/EGFR+/CD10+immunoprofile. Their expression rates were significantly higher in SMA than UA (P<0.05). BRAF, essential for the progression and proliferation of ameloblastoma, was detected mainly in the cytoplasm of stellate reticulum-like>stromal>preameloblast- like cells (P<0.05). EGFR, a potent oncogenic protein, showed predominantly nuclear localisation. CD10, an apoptosis-inhibitory factor, was strongly expressed in the membrane of stellate reticulum-like cells. Taken together, present results suggest that the spatial distribution patterns of BRAF, EGFR and CD10 parallel the specific behaviours of SMA and UA. Their cellular and intracellular protein localisations have important targeted therapy implications.

A retrospective cohort study on predictors associated with skull base invasion of maxillary ameloblastomas.

PURPOSE: To identify factors associated with skull base involvement (SBI) of maxillary ameloblastomas (MA). METHODS: This retrospective cohort study was composed of MA patients treated during a 7-year period. Demographic, radiographic, and nine immunohistopathologic predictor variables were included. The outcome variable was presence of SBI (yes/no). Descriptive, bi- and multivariate statistics were computed, and P ≤ .05 in multivariate analyses was considered statistically significant. RESULTS: The sample comprised 23 subjects (34.8% females; 21.7% with SBI) with a mean age of 50.3 ± 18.2 years. Candidate predictors of an SBI in MAs were 1) male gender, 2) a low Karnofsky Performance Status score (KPS), 3) multilocular radiolucency, 4) ill-defined margins, 5) cortical perforation, 6) inclusion of an unerupted tooth, 7) moderate to strong reactivity to p53, Ki-67, CD10, astrocyte elevated gene-1 (AEG-1) protein, carbonic anhydrase IX (CA IX), calretinin (calbindin2; CALB2), and BRAF-V600E, and 8) negative to low immunopositivity to α-smooth muscle actin (α-SMA) and syndecan-1 (CD138). However, multivariate analyses confirmed the significant associations of SBI with negative/low syndecan-1 reactivity (P = .003; adjusted odds ratio [ORadj.], 4.04; 95% confidence interval [95% CI], -.89 to -.48; Pearson's Correlation Coefficient [r] = -.74) and with KPS (P = .003; ORadj., 4.04; 95% CI, -.78 to -.17; r = -.54) only. CONCLUSIONS: Our findings suggest an aggressive approach to MAs with negative to low syndecan-1 immunopositivity and/or in multi-morbid patients (who may have difficulty in access to health care). Otherwise, health care inequalities due to low KPS scores should be minimized or eliminated.

DNA methylation status of MutS genes in ameloblastoma.

OBJECTIVES: Ameloblastoma is an odontogenic epithelial tumour with a low expression of mismatch repair system components. We aimed to investigate the methylation status of the genes MSH2, MSH3 and MSH6 (MutS group) in conventional ameloblastomas. MATERIALS AND METHODS: The ameloblastoma and dental follicle samples (n = 10 each) were collected from 20 different patients. Each ameloblastoma sample was sectioned into two fragments: one was paraffin-embedded while the other one, likewise the dental follicle samples, was fixed in RNAlater and frozen at -196°C. All frozen samples were investigated for the MutS genes methylation levels, using the enzymatic restriction digestion and quantitative real-time PCR (qPCR) assay. The ameloblastoma paraffin-embedded samples were submitted to immunohistochemical reactions for MutS proteins detection and digitally quantification. Correlation analyses were performed between the immunohistochemical results and the respective gene methylation percentage. RESULTS: There are no significant differences between the MutS genes methylation levels in the ameloblastoma and the dental follicle. However, a strong negative correlation was found between MSH2 and MSH6 gene methylation status and their respective proteins expressions evaluated by immunohistochemistry. CONCLUSION: Our results show that the genes methylations is in part responsible for decreasing the expression of MSH2 and MSH6 genes in ameloblastoma.

αTAT1-induced tubulin acetylation promotes ameloblastoma migration and invasion.

Ameloblastoma (AB) is the most common benign epithelial odontogenic tumor occurring in the jawbone. AB is a slowly growing tumor but sometimes shows a locally invasive and an aggressive growth pattern with a marked bone resorption. In addition, the local recurrence and distant metastasis of AB also sometimes occurs, which resembles one of the typical malignant potentials. From these points of view, to understand better the mechanisms of AB cell migration or invasion is necessary for the better clinical therapy and improvements of the patients' quality of life. Microtubules in eukaryotic cells reveal the shape of hollow cylinders made up of polymerized alpha (α)- and beta (ß)-tubulin dimers and form the cytoskeleton together with microfilaments and intermediate filaments. Microtubules play important roles in cell migration by undergoing assembly and disassembly with post-translational modifications. Stability of microtubules caused by their acetylation is involved in cell migration. In this study, we investigated the expression and distribution of acetylated α-tubulin and alpha-tubulin N-acetyltransferase 1 (αTAT1), an enzyme which acetylates Lys-40 in α-tubulin, in AB specimens, and analyzed how tubulin was acetylated by αTAT1 activation in a human AB cell line, AM-1. Finally, we clarified that TGF-ß-activated kinase1 (TAK1) was phosphorylated by TGF-ß stimulation, then, induced tubulin acetylation via αTAT1 activation, which subsequently activated the migration and invasion of AB cells.

RAF1-MEK/ERK pathway-dependent ARL4C expression promotes ameloblastoma cell proliferation and osteoclast formation.

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogen-activated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C), induced by a combination of the EGF-MAPK pathway and Wnt/ß-catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS-MAPK pathway and Wnt/ß-catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non-tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C-RAF). Loss-of-function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1-MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1-ARL4C and BRAFV600E-MEK/ERK pathways promoted cell proliferation independently. ARL4C-depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co-cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1-MEK/ERK-ARL4C axis, which may function in cooperation with the BRAFV600E-MEK/ERK pathway, promotes ameloblastoma development. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.