Skip to main content
Download PDF

Wnt signaling aberrant activation drives ameloblastoma invasion and recurrence: bioinformatics and in vitro insights

BMC Oral Health volume 24, Article number: 1421 (2024) Cite this article

Abstract

Objective

This study aims to explore the regulatory mechanisms of Wnt signaling in the invasion and recurrence of ameloblastoma (AM) to provide a new theoretical basis for its treatment.

Methods

Bulk RNA sequencing was employed to analyze samples from AM patients, and identify differentially expressed genes. Subsequently, bioinformatics methods such as Weighted Gene Co-Expression Network Analysis (WGCNA), DESeq2, and KEGG enrichment analysis were utilized to construct gene co-expression networks and identify pathways associated with invasion and recurrence. Furthermore, in vitro experiments, including Cell Counting Kit-8 (CCK-8), Wound healing assays, Western blotting, and qPCR were conducted to validate the effects of Wnt signaling on AM biological functions and the expression of related genes and proteins.

Results

Bioinformatics analysis revealed significant activation of the Wnt signaling pathway during AM invasion and recurrence, and differential gene analysis identified specific gene expression patterns associated with the Wnt signaling pathway. In vitro experiments further demonstrated that the standard Wnt/β-catenin pathway activator, Laduviglusib significantly activated Wnt signaling, leading to a marked increase in the mRNA and protein expression levels of TCF7, β-catenin, WNT2B, and LEF1, thereby enhancing the proliferation and migration capabilities of AM cells.

Conclusion

This study reveals the critical role of aberrant Wnt signaling activation in AM proliferation and migration, identifying it as a key driver of AM invasion and recurrence. The findings provide new insights into the mechanisms underlying AM invasion and recurrence, laying the foundation for developing novel therapeutic strategies.

Peer Review reports

Introduction

As the most common odontogenic benign tumor in the oral and maxillofacial region, Ameloblastoma (AM) can cause functional impairments, such as difficulties in chewing, swallowing, and breathing, due to tooth displacement, associated jawbone destruction and fractures [1, 2]. It exhibits complex biological behavior, characterized by significant invasiveness, a high recurrence rate, and a rare potential for malignant transformation [3, 4]. The recurrence of AM not only complicates treatment but also severely impacts the patient’s quality of life and prognosis. Therefore, elucidating the mechanisms of AM invasion and recurrence is crucial for optimizing treatment strategies and patient management. The Wnt/β-catenin signaling pathway plays a critical regulatory role in cell proliferation, differentiation, and various biological processes, including tissue development, homeostasis maintenance, and cell fate determination. Under normal physiological conditions, this pathway is tightly regulated. However, its aberrant activation is closely associated with tumorigenesis and progression in various cancers [5,6,7]. The role of Wnt/β-catenin signaling in AM remains controversial. Research by Kim et al. [8] suggests that Wnt activation inhibits AM tumor stem cells, whereas Li et al. [9] argue that Wnt/β-catenin activation helps maintain AM stemness and tumorigenicity. Additionally, some studies report that alterations in the Wnt signaling pathway are related to the malignant transformation process in cases where AM has transformed into ameloblastic carcinoma [10].These findings imply that the Wnt signaling pathway may play a significant role in AM invasion and recurrence; however, its specific mechanisms remain unclear. This study aims to elucidate the potential mechanisms underlying AM invasion and recurrence by employing Bulk RNA sequencing to identify specific gene expression patterns and key molecules associated with invasive and recurrent AM. Subsequently, in vitro experiments will be conducted to validate the effects of Wnt signaling on AM cells, aiming to uncover key regulatory mechanisms of AM invasion and recurrence and provide a scientific basis for developing new AM therapeutic strategies.

Materials and methods

Human biosample collection

The study samples were collected from ameloblastoma (AM) patients undergoing maxillofacial surgery at the Affiliated Stomatological Hospital of Kunming Medical University. Fresh AM tumor tissues and adjacent normal tissues were obtained during surgery. The diagnosis of AM was confirmed by histopathological examination. None of the patients had received radiotherapy or chemotherapy prior to surgery. A total of 7 normal tissue samples and 18 AM tissue samples were collected for Bulk RNA sequencing. Additionally, fresh tissues from 7 AM patients were used for primary cell culture. Recurrence was defined as patients undergoing surgery for a non-first-time occurrence. The study was approved by the Medical Ethics Committee (Approval No. KYKQ2024MEC0058), and all patients provided informed consent.

Bulk RNA sequencing

Fresh AM samples were rapidly frozen in liquid nitrogen for 1 h and stored at -80 °C. RNA integrity and purity were assessed using agarose gel electrophoresis, a NanoPhotometer, and an Agilent 2100 Bioanalyzer. The RNA libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit. After initial quantification with Qubit 2.0 and dilution to 1.5 ng/µL, the insert size was determined using an Agilent 2100, and precise quantification was performed by qRT-PCR. The qualified libraries were pooled and sequenced on an Illumina platform, generating 150 bp paired-end reads.

Bulk RNA sequencing data processing

The raw sequencing data were filtered to compute the Q20, Q30, and GC content of the clean data. HISAT2 v2.0.5 was used to index the reference genome and align the paired-end clean reads. Gene read counts were calculated using featureCounts 1.5.0-p3, and gene expression levels were assessed using the FPKM method.

WGCNA analysis based on bulk RNA sequencing data

The R package WGCNA v1.72-5 was used to analyze 25 samples, incorporating phenotypic information (“primary,” “recurrent,” and “normal”) to construct a gene co-expression network. An appropriate soft threshold was determined for hierarchical clustering. Significant gene modules were identified, followed by GOBP and KEGG pathway enrichment analysis.

DESeq2 analysis and pathway enrichment

Representative primary and recurrent AM RNA-seq data underwent batch effect correction using the DESeq2 package. We first prepared a metadata table containing sample information, including batch information for each sample. Batch was included as a factor in the design matrix of DESeq2 to identify and correct for batch effects. Following batch effect correction, DESeq2 was then used to identify differentially expressed genes (fold change > 2, P < 0.05). Heatmaps and volcano plots were generated to visualize the results. To highlight key pathways involved in gene expression, GOBP and KEGG enrichment analyses were conducted using ClusterProfiler v4.10.1.

Primary culture, purification, and drug intervention of AM tumor cells

Fresh AM tumor tissues were washed extensively with saline, preserved in tissue preservation solution, and transferred to a biosafety cabinet. After washing with PBS, the tissues were minced and digested with tissue digestive enzymes for 40 min. The digested tissues were then seeded into 24-well plates and cultured in high-glucose DMEM medium containing 10% fetal bovine serum (EVA, Hong Kong) and 1% penicillin-streptomycin (Hyclone, USA) at 37 °C with 5% CO₂. When epithelial cells reached 40% confluence, fibroblasts were removed by a 2-minute digestion with digestive enzymes (Biogenous, China). When AM epithelial cells reached 50% confluence, they were treated with Laduviglusib (MedChemExpress, USA), a standard activator of the Wnt/β-catenin pathway, for 24 h at concentrations of 1µM, 10µM, and 20µM. Experiments were performed in triplicate.

Cell proliferation

Cells in the logarithmic growth phase were treated with Laduviglusib for 24 h before the culture medium was removed. A mixture of 10µL CCK-8 reagent (GLPBIO, USA) and 90µL DMEM was added to each well, and the cells were incubated at 37 °C with 5% CO₂ for 2 h. Absorbance at 450 nm was measured using a microplate reader (Thermo, USA) to assess cell proliferation. Experiments were performed in triplicate.

Wound healing assay

Cells in the logarithmic growth phase were uniformly seeded to cover the bottoms of the wells. A vertical scratch was made using a 200µL pipette tip, and detached cells were removed with PBS. Images were captured using an inverted microscope. The scratched wells were divided into control (CON), 1µM, 10µM, and 20µM groups, with respective concentrations of Laduviglusib added. The plates were incubated at 37 °C with 5% CO₂ for 24 h, and images of the scratch were taken at 0, 12, and 24 h using an inverted microscope. Experiments were performed in triplicate. The scratch area was analyzed using ImageJ 154 software to determine wound healing rates at 12 h (WHR12) and 24 h (WHR24).

Western blot analysis

AM cells from different treatment groups were washed with PBS, lysed with cell lysis buffer, and centrifuged at 12,000 rpm for 25 min at 4 °C. The supernatant was collected, and protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology, China). After adding 5× loading buffer (Epizyme Biotech, China), the proteins were denatured by heating at 100 °C for 5 min. SDS-PAGE separation and stacking gels were prepared, and electrophoresis was performed at 80 V. After electrophoresis, the gels were cut, activated PVDF membranes were prepared, and proteins were transferred at a constant current of 300 mA for 90 min. The primary antibodies were anti-TCF7 (1:1000, Cell Signaling Technology, 2203), anti-β-catenin (1:5000, Proteintech, 66379-1-Ig), anti-WNT2B (1:1000, Affinity, DF12538), and anti-LEF1 (1:800, ZENBIO, 3382664), incubated at 4 °C for 12 h. Secondary antibodies, goat anti-rabbit (1:5000, Proteintech, SA00001-2) and goat anti-mouse (1:6000, Proteintech, SA00001-1), were incubated at room temperature for 90 min. The PVDF membranes were then soaked in a developing solution and imaged using a gel imaging system. Experiments were performed in triplicate. The grayscale values were analyzed with ImageJ 154 software.

RT-qPCR

AM cells from different treatment groups were washed with PBS, and RNA was extracted using an RNA extraction kit (AGBIO, China). The concentration and purity of total RNA were measured using a Quawell Q3000 spectrophotometer. The extracted RNA was reverse transcribed into cDNA using the Evo M-MLV RT PreMix kit (AGBIO, China). Real-time PCR was performed using the SYBR Green Pro Taq HS Premix qPCR kit II (with ROX) (AGBIO, China). Experiments were performed in triplicate. The primer sequences for RT-qPCR are shown in Table 1.

Table 1 The primer sequences
Full size table

Statistical methods

Statistical analysis was performed using SPSS 22.0 software. Data were presented as mean ± standard deviation (x̅±s). ANOVA was used for comparisons among multiple groups. Specifically, we performed Levene’s test for homogeneity of variance and the Shapiro-Wilk test for normality. Following ANOVA, LSD and Tamhane’s post-hoc tests were applied to identify specific group differences, and P < 0.05 was considered statistically significant.

Results

WGCNA identifies wnt signaling as a key factor in AM invasion and recurrence

In the constructed RNA-seq gene co-expression network for ameloblastoma (AM) and normal tissues, a soft threshold of 16 was determined using a scale-free topology fitting index to optimize network connectivity (Fig. 1A, B). Hierarchical clustering analysis identified 56 gene expression modules, with the MEblue module significantly associated with invasive and recurrent AM (correlation = 0.88, P < 1e-200, Fig. 1C, D). This module contained 2,348 key genes. Further enrichment analysis showed that these module genes were significantly enriched in the Wnt and Hippo signaling pathways. KEGG analysis indicated that these genes were involved in pattern specification processes, Wnt signaling, and its regulation, suggesting a critical role for Wnt signaling in AM invasion and recurrence (Fig. 1E, F).

Fig. 1
figure 1

Gene co-expression analysis conducted using WGCNA on samples from normal gingiva and ameloblastoma. A: Heatmap showing sample correlations. B: Plot of scale-free topology fitting index illustrating different connectivity levels corresponding to various soft thresholds. C: Dendrogram of hierarchical clustering for co-expressed genes; each leaf represents a gene, with major branches representing different modules, color-coded accordingly. D: Plot showing the correlation between modules and phenotypes, where rows represent modules and columns represent different phenotypes. The cell values indicate correlation coefficients between modules and phenotypes, with significance indicated by P-values in parentheses. E: Scatter plot depicting module membership (MM) and gene significance (GS) from the MEblue module in the recurrence cohort. F: Enrichment analysis of GOBP pathways for the top 600 differentially expressed genes. G: Enrichment analysis of KEGG pathways for the top 600 differentially expressed genes. WGCNA stands for weighted gene co-expression network analysis, while GS and MM denote gene significance and module membership, respectively

Full size image

Differential gene analysis between primary and recurrent AM RNA-seq samples

A total of 583 differentially expressed genes were identified between primary and recurrent AM samples, with 479 significantly upregulated and 104 downregulated. Notably, IRS4, ZBBX, NWD2, and FREM2 were significantly upregulated in recurrent samples, while GPR12 was downregulated (Fig. 2A, B). These upregulated genes exhibited high expression in the recurrent group but low expression in the primary group. KEGG pathway enrichment analysis revealed that these genes were involved in critical biological processes, including the Wnt signaling pathway (Fig. 2C). Further Gene Set Enrichment Analysis (GSEA) confirmed the significant activation of the canonical Wnt signaling pathway and cell-cell signaling by Wnt in recurrent AM, emphasizing the regulatory role of Wnt signaling in AM invasion and recurrence (Fig. 2D, E, F and G).

Fig. 2
figure 2

Differential gene expression analysis between primary and recurrent ameloblastoma samples conducted using DESeq2. A: A volcano plot visualized differential gene expression, highlighting genes with a fold change > 2 and P < 0.05 in green, indicating significant differences. B: Heatmap of gene expression patterns in primary and recurrent ameloblastoma samples, with genes grouped based on expression levels; red indicates high expression, and blue indicates low expression. C: Bubble plot showing KEGG pathway enrichment among differentially expressed genes. D: Expression profile of genes related to the Wnt signaling pathway in recurrent ameloblastoma. E, F, and G: GSEA for GOBP gene sets to further explore the involvement of these pathways

Full size image

Activation of wnt signaling increases TCF7, β-catenin, WNT2B, and LEF1 protein and mRNA levels in AM primary cells

Following a 24-hour treatment of AM primary cells with Laduviglusib, the expression levels of TCF7, β-catenin, WNT2B, and LEF1 proteins increased. Compared with the control group, TCF7 showed a significant difference at 10 µM (95% CI: 0.25 to 0.59, P = 0.00045), β-catenin showed a significant difference at 20 µM (95% CI: 0.26 to 0.95, P = 0.0038), WNT2B exhibited significant differences across all groups with the highest expression at 20 µM (95% CI: 0.41 to 0.78, P = 0.000064), and LEF1 showed significant differences across all groups with the highest expression at 10 µM (95% CI: 0.85 to 1.17, P = 0.0000) (Fig. 3A-E). Similarly, the mRNA expression levels also showed a dose-dependent increase with the drug concentration. Compared to the control group, TCF7 showed significant differences at 10 µM (95% CI: 1.63 to 2.90, P = 0.000035) and 20 µM (95% CI: 5.53 to 6.80, P = 1.6254E-8). CTNNB1 (95% CI: 5.57 to 7.50, P = 0.0000) and WNT2B (95% CI: 7.13 to 9.73, P = 3.957E-7) exhibited significant differences at 20 µM. LEF1 showed notable differences between groups, with a significant difference already observable at 1 µM (95% CI: 0.2 to 1.2, P = 0.013) (Fig. 3F-I).

Fig. 3
figure 3

Changes in Key Gene and Protein Expression in the Wnt Pathway Under Different Concentrations of Laduviglusib. A-E: Differences in the expression of TCF7, β-catenin, WNT2B, and LEF1 proteins. F-I: Differences in mRNA expression levels of TCF7, CTNNB1, WNT2B, and LEF1

Full size image

Wnt signaling activation promotes AM cell proliferation and migration

After treating AM primary cells with 1µM, 10µM, and 20µM of Laduviglusib for 24 h, cell proliferation significantly increased with the rise in drug concentration, showing a statistically significant difference at 1µM (95% CI: 0.19 to 0.33, P = 0.000029) compared to the control group (Fig. 4A). These results suggest that Laduviglusib significantly enhances AM cells proliferation. Additionally, 1µM Laduviglusib significantly promoted AM cells migration, with wound healing rates at 12 h (95% CI: 0.35 to 0.70, P = 0.000012) and 24 h (95% CI: 0.27 to 0.44, P = 0.000119) being higher than those in the other groups (Fig. 4B, C).

Fig. 4
figure 4

Effects of Different Concentrations of Laduviglusib on AM Cell Proliferation and Migration. A: Changes in AM cell proliferation under various concentrations of Laduviglusib. B: Differences in AM cell migration capabilities at 12 h and 24 h after Laduviglusib treatment. C: Wound healing status of AM cells at 12 h and 24 h following Laduviglusib intervention, with images magnified at 4× and scale bars at 200 μm

Full size image

Discussion

Wnt signaling plays a crucial role in stem cell biology, particularly in maintaining stem cell self-renewal across various tissues, such as embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, epidermal stem cells, and neural stem cells. Additionally, the Wnt signaling pathway is closely linked to cancer stem cell (CSC) characteristics within tumor cells. Studies have demonstrated that mutations in Wnt pathway components can promote cell proliferation, epithelial-mesenchymal transition (EMT), and metastasis, thereby driving cancer progression, metastasis, and recurrence [11,12,13]. Earlier studies have highlighted the differential expression of Wnt ligands in various AM subtypes, with classical and non-classical Wnt pathways being selectively activated or inhibited depending on the differentiation state of tumor cells [14]. This underscores the critical role of Wnt signaling in AM tumorigenesis. Recent research confirms that increased hydrostatic pressure activates Wnt/β-catenin pathway, and promotes AM cells migration and invasion thereby increasing expression of Wnt signalling downstream targets [15]. However, this evidence largely comes from in vitro studies. Moreover, mutations in the BRAF-V600E gene and its regulation of the MAPK/ERK pathway [16, 17], genes related to the Hedgehog pathway, and alterations in the tumor suppressor gene KMT2D [18] also play significant roles in the onset and progression of AM. However, the specific biological impacts of these changes on the pathogenesis of AM remain incompletely understood. Our study, through differential gene expression analysis, has identified a significant upregulation of genes associated with the Wnt signaling pathway in invasive and recurrent AM samples. Further gene set enrichment analysis (GSEA) confirmed the activation of the canonical Wnt signaling pathway in these samples, underscoring its central role in regulating AM invasion and recurrence. Besides, studies have shown that the post-transcriptional promotion of multiple Wnt pathway-related gene expressions by DDX56 is closely associated with early recurrence of lung squamous cell carcinoma [19]. In esophageal squamous cell carcinoma, Wnt/β-catenin signaling pathway activation stimulates cancer cell EMT, angiogenesis, and stemness, ultimately promoting tumor metastasis and recurrence [20]. These findings further underscore the importance of Wnt signaling activation in adverse prognostic phenotypes such as tumor metastasis and recurrence. Although our study has certain sample size limitations, the findings are consistent with previous related research. These findings align with previous research, suggesting that Wnt/β-catenin signaling activity is closely related to the maintenance of CSC properties in AM cells. Moreover, the activation of the Wnt pathway, along with other stemness-associated pathways, can enhance AM invasiveness and promote recurrence [8, 9, 21]. In conclusion, Wnt signaling activation plays a pivotal role in the invasive recurrence of AM.

Laduviglusib (CHIR99021) is a small organic molecule widely recognized as a standard activator of the Wnt/β-catenin pathway [22]. In this study, AM cells were treated with various concentrations of Laduviglusib. At concentrations of 1 µM and 10 µM, there was a higher expression of TCF7, β-catenin, WNT2B, and LEF1 compared to the control group, with corresponding increases in the mRNA levels of TCF7, CTNNB1, WNT2B, and LEF1. This indicates that Laduviglusib activates Wnt signaling at both the protein and RNA levels. Additionally, Wnt signaling activation significantly promoted AM cells proliferation and, at 1 µM and 10 µM concentrations, markedly enhanced AM cells migration. CTNNB1, which encodes β-catenin, is a crucial downstream effector of Wnt signaling. Previous studies have reported that hotspot mutations in CTNNB1 disrupt the activity of the β-catenin complex, leading to abnormal accumulation of β-catenin in the nucleus and cytoplasm. This accumulation triggers cells proliferation and metabolic dysregulation, contributing to tumorigenesis [23]. However, other studies did not observe β-catenin accumulation in the nuclei of AM cells, suggesting that the canonical Wnt pathway is not constitutively activated in these cells [21]. Nonetheless, upregulation of Wnt signaling-related proteins, such as Podoplanin and β-catenin, at the invasive front of recurrent and non-recurrent AMs indicates that Wnt signaling might promote cells migration and local invasion in recurrent AMs [24]. Despite these findings, the role of Wnt signaling in AM remains controversial. Some research suggests that the Wnt signaling activator valproic acid (VPA) leads to increased cell size and reduced proliferation in primary AM cells [8], which contradicts the observed promotion of adverse prognostic phenotypes in AM cells upon Wnt signaling activation in this study. Conversely, targeting the Wnt/β-catenin pathway’s negative regulator, CTNNBIP1, has been shown to enhance AM cell migration and invasion [25], while depletion of BRD4, leading to Wnt pathway inactivation, inhibits AM cell growth and tumorigenicity [21]. In conclusion, the activation or inhibition of the Wnt/β-catenin signaling pathway is crucial for tumorigenesis and progression in AM. Although there are conflicting findings regarding the specific mechanisms, our study demonstrates that Wnt signaling activation plays a key role in AM invasion and recurrence, providing new insights for future therapeutic strategies.

In summary, a growing body of research has confirmed that dysfunction in the Wnt/β-catenin pathway significantly impacts the pathogenesis of various diseases, including solid tumors and sarcomas [5, 26, 27]. This study, using Bulk-RNA sequencing analysis of clinical AM samples combines with laboratory validation, reveals that the activation of Wnt signaling promotes AM invasion and recurrence, as well as enhances cell proliferation and migration. Recent studies have also shown that completely blocking Wnt signaling can significantly inhibit the growth of Wnt-dependent tumors [28]. In clinical trials, various Wnt signaling pathway inhibitors such as LGK974, Ipafricept and Porcupine inhibitors have been used to treat patients with poor prognosis cancers, including head and neck squamous cell carcinoma, ovarian cancer, and colorectal cancer [29]. Researchers subsequently noted that inhibiting the Wnt/β-catenin signaling could suppress the growth and metastasis of human osteosarcoma cells, suggesting that targeting Wnt may have potential therapeutic value for recurrent and metastatic osteosarcoma [30, 31].Therefore, we believe that targeting Wnt signaling for the treatment of invasive and recurrent AM holds promise, and we will further explore this approach in future research.

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/ Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA008531) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.

References

  1. Mohammed K, Aldelaimi A, Enezei H, Aldelaimi T. Tumors of Craniofacial Region in Iraq (Clinicopathological Study). J Res Med Dent Sci. 2021;9:66–71.

    Google Scholar 

  2. Aldelaimi A. A Clinical Analysis of Surgically Managed Mandibular Fractures: Epidemiology, Clinical Profile, Patterns, Treatments, and Outcomes. 2020;1–4.

  3. Liu W, Zheng C, Zhang X, Hu H. Recurrence and malignant risk of ameloblastoma: a demographic study of 1626 cases from east China. Oral Oncol. 2024;148:106651. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.oraloncology.2023.106651.

    Article  CAS  PubMed  Google Scholar 

  4. Xiong G, Xie N, Nie M, Ling R, Yun B, Xie J, Ren L, Huang Y, Wang W, Yi C, Zhang M, Xu X, Zhang C, Zou B, Zhang L, Liu X, Huang H, Chen D, Cao W, Wang C. Single-cell transcriptomics reveals cell atlas and identifies cycling tumor cells responsible for recurrence in ameloblastoma. Int J Oral Sci. 2024;16(1):21. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41368-024-00281-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Song P, Gao Z, Bao Y, Chen L, Huang Y, Liu Y, Dong Q, Wei X. Wnt/β-catenin signaling pathway in carcinogenesis and cancer therapy. J Hematol Oncol. 2024;17(1):46. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13045-024-01563-4.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Holzem M, Boutros M, Holstein TW. The origin and evolution of wnt signalling. Nat Rev Genet. 2024;25(7):500–12. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41576-024-00699-w.

    Article  CAS  PubMed  Google Scholar 

  7. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36(11):1461–73. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/onc.2016.304.

    Article  CAS  PubMed  Google Scholar 

  8. Kim HY, Li S, Lee DJ, Park JH, Muramatsu T, Harada H, Jung YS, Jung HS. Activation of wnt signalling reduces the population of cancer stem cells in ameloblastoma. Cell Prolif. 2021;54(7):e13073. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/cpr.13073.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Li S, Lee DJ, Kim HY, Kim JY, Jung YS, Jung HS. Unraveled roles of Cav1.2 in proliferation and stemness of ameloblastoma. Cell Biosci. 2022;12(1):145. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13578-022-00873-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hurník P, Putnová BM, Ševčíková T, Hrubá E, Putnová I, Škarda J, Havel M, Res O, Cvek J, Buchtová M, Štembírek J. Metastasising ameloblastoma or ameloblastic carcinoma? A case report with mutation analyses. BMC Oral Health. 2023;23(1):563. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12903-023-03259-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. MacLean MR, Walker OL, Arun RP, Fernando W, Marcato P. Informed by Cancer Stem cells of solid tumors: advances in treatments targeting tumor-promoting factors and pathways. Int J Mol Sci. 2024;25(7):4102. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms25074102.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yu M, Qin K, Fan J, Zhao G, Zhao P, Zeng W, Chen C, Wang A, Wang Y, Zhong J, Zhu Y, Wagstaff W, Haydon RC, Luu HH, Ho S, Lee MJ, Strelzow J, Reid RR, He TC. The evolving roles of wnt signaling in stem cell proliferation and differentiation, the development of human diseases, and therapeutic opportunities. Genes Dis. 2024;11(3):101026. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.gendis.2023.04.042.

    Article  CAS  PubMed  Google Scholar 

  13. Xiao M, Zhang X, Zhang D, Deng S, Zheng A, Du F, Shen J, Yue L, Yi T, Xiao Z, Zhao Y. Complex interaction and heterogeneity among cancer stem cells in head and neck squamous cell carcinoma revealed by single-cell sequencing. Front Immunol. 2022;13:1050951. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fimmu.2022.1050951.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Siar CH, Nagatsuka H, Han PP, Buery RR, Tsujigiwa H, Nakano K, Ng KH, Kawakami T. Differential expression of canonical and non-canonical wnt ligands in ameloblastoma. J Oral Pathol Med. 2012;41(4):332–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1600-0714.2011.01104.x.

    Article  PubMed  Google Scholar 

  15. Yang Z, Li K, Liang Q, Zheng G, Zhang S, Lao X, Liang Y, Liao G. Elevated hydrostatic pressure promotes ameloblastoma cell invasion through upregulation of MMP-2 and MMP-9 expression via Wnt/β-catenin signalling. J Oral Pathol Med. 2018;47(9):836–46. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/jop.12761.

    Article  CAS  PubMed  Google Scholar 

  16. Martins-de-Barros AV, Anjos RSD, Silva CCG, Silva E, Araújo F, Carvalho MV. Diagnostic accuracy of immunohistochemistry compared with molecular tests for detection of BRAF V600E mutation in ameloblastomas: systematic review and meta-analysis. J Oral Pathol Med. 2022;51(3):223–30. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/jop.13278.

    Article  CAS  PubMed  Google Scholar 

  17. Guimarães LM, Coura BP, Gomez RS, Gomes CC. The Molecular Pathology of Odontogenic tumors: expanding the spectrum of MAPK pathway driven tumors. Front Oral Health. 2021;2:740788. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/froh.2021.740788.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Marín-Márquez C, Kirby J, Hunter KD. Molecular pathogenesis of ameloblastoma. J Oral Pathol Med. 2024;53(5):277–93. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/jop.13538.

    Article  CAS  PubMed  Google Scholar 

  19. Wu Q, Luo X, Terp MG, Li Q, Li Y, Shen L, Chen Y, Jacobsen K, Bivona TG, Chen H, Zeng R, Ditzel HJ. DDX56 modulates post-transcriptional wnt signaling through miRNAs and is associated with early recurrence in squamous cell lung carcinoma. Mol Cancer. 2021;20(1):108. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12943-021-01403-w.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Li Q, Luo H, Dai FQ, Wang RT, Fan XQ, Luo YY, Deng MS, Wang Y, Long T, Guo W, Xu B, Xu CX, Jin H. SAMD9 promotes postoperative recurrence of esophageal squamous cell carcinoma by stimulating MYH9-Mediated GSK3β/β-Catenin signaling. Adv Sci (Weinh). 2023;10(11):e2203573. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/advs.202203573.

    Article  CAS  PubMed  Google Scholar 

  21. Xie J, Zhang J, Xiong G, Ouyang S, Yun B, Xu X, Wang W, Zhang M, Xie N, Chen D, Wang C. Targeting BRD4 attenuates the stemness and aggressiveness of ameloblastoma. Oral Dis. 2024;30(5):3212–24. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/odi.14762.

    Article  PubMed  Google Scholar 

  22. Bennett CN, Ross SE, Longo KA, Bajnok L, Hemati N, Johnson KW, Harrison SD, MacDougald OA. Regulation of wnt signaling during adipogenesis. J Biol Chem. 2002;277(34):30998–1004. https://doiorg.publicaciones.saludcastillayleon.es/10.1074/jbc.M204527200.

    Article  CAS  PubMed  Google Scholar 

  23. Kim S, Jeong S. Mutation hotspots in the β-Catenin gene: lessons from the Human Cancer Genome databases. Mol Cells. 2019;42(1):8–16. https://doiorg.publicaciones.saludcastillayleon.es/10.14348/molcells.2018.0436.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Siar CH, Ishak I, Ng KH, Podoplanin. E-cadherin, β-catenin, and CD44v6 in recurrent ameloblastoma: their distribution patterns and relevance. J Oral Pathol Med. 2015;44(1):51–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/jop.12203.

    Article  CAS  PubMed  Google Scholar 

  25. Liu S, Liu D, Liu J, Liu J, Zhong M. miR-29a-3p promotes migration and invasion in ameloblastoma via Wnt/β-catenin signaling by targeting catenin beta interacting protein 1. Head Neck. 2021;43(12):3911–21. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/hed.26888.

    Article  PubMed  Google Scholar 

  26. Unno K, Chalmers ZR, Pamarthy S, Vatapalli R, Rodriguez Y, Lysy B, Mok H, Sagar V, Han H, Yoo YA, Ku SY, Beltran H, Zhao Y, Abdulkadir SA. Activated ALK cooperates with N-Myc via Wnt/β-Catenin signaling to induce neuroendocrine prostate cancer. Cancer Res. 2021;81(8):2157–70. https://doiorg.publicaciones.saludcastillayleon.es/10.1158/0008-5472.CAN-20-3351.

  27. Hawkins AG, Pedersen EA, Treichel S, Temprine K, Sperring C, Read JA, Magnuson B, Chugh R, Lawlor ER. Wnt/β-catenin-activated ewing sarcoma cells promote the angiogenic switch. JCI Insight. 2020;5(13). https://doiorg.publicaciones.saludcastillayleon.es/10.1172/jci.insight.135188.

  28. Liu Y, Qi X, Donnelly L, Elghobashi-Meinhardt N, Long T, Zhou RW, Sun Y, Wang B, Li X. Mechanisms and inhibition of Porcupine-mediated wnt acylation. Nature. 2022;607(7920):816–22. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41586-022-04952-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Krishnamurthy N, Kurzrock R. Targeting the Wnt/beta-catenin pathway in cancer: update on effectors and inhibitors. Cancer Treat Rev. 2018;62:50–60. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ctrv.2017.11.002.

    Article  CAS  PubMed  Google Scholar 

  30. Lu Q, Huang H, Wang X, Luo L, Xia H, Zhang L, Xu J, Huang Y, Luo X, Luo J. Echinatin inhibits the growth and metastasis of human osteosarcoma cells through Wnt/β-catenin and p38 signaling pathways. Pharmacol Res. 2023;191:106760. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.phrs.2023.106760.

    Article  CAS  PubMed  Google Scholar 

  31. Matsuoka K, Bakiri L, Wolff LI, Linder M, Mikels-Vigdal A, Patiño-García A, Lecanda F, Hartmann C, Sibilia M, Wagner EF. Wnt signaling and Loxl2 promote aggressive osteosarcoma. Cell Res. 2020;30(10):885–901. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41422-020-0370-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work is supported by the National Natural Science Foundation of China (81660187), the Medicine Leading Talent of Yunnan Province Health Care Committee (No.L-2018010) and Yunnan Province Xing Dian Talent (No.XDYC-MY-2022-0052).

Author information

Author notes

  1. Yemei Qian, Hongrong Zhang and Jingyi Li are co-first authors.

Authors and Affiliations

  1. Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Kunming Medical University, No. 1088 Mid Hai Yuan Road. Gaoxin District, Kunming, Yunnan, 650106, China

    Yemei Qian, Hongrong Zhang, Jingyi Li, Liangchong Huang, Yunfa Qin, Jian Zhang & Weihong Wang

  2. Yunnan Key Laboratory of Stomatology, Kunming, Yunnan, China

    Yemei Qian, Hongrong Zhang, Jingyi Li, Liangchong Huang, Yunfa Qin, Jian Zhang & Weihong Wang

Authors
  1. Yemei Qian
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Hongrong Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Jingyi Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Liangchong Huang
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Yunfa Qin
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Jian Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Weihong Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Yemei Qian, Hongrong Zhang and Jingyi Li completed the experiment and the initial draft of the paper. Liangchong Huang, Yunfa Qin and Jian Zhang participated in the experiment. Weihong Wang supervised the research. Yemei Qian , Hongrong Zhang and Jingyi Li are co-first authors for their equal contributions. All authors actively participated in the execution or analysis of the study, reviewed and approved the final manuscript, and agreed to the public release of the original data.

Corresponding author

Correspondence to Weihong Wang.

Ethics declarations

Ethics approval and consent to participate

The research protocol was conducted in accordance with the principles of the Helsinki Declaration and approved by the Ethics Committee of the Affiliated Stomatology Hospital of Kunming Medical University (Approval Number: KYKQ2024MEC0058), following Chinese national regulations. Informed consent was obtained from all participants involved in the study, including for the use of their information and photographs. No participants were enrolled without their explicit consent, and no consent waiver was required or granted by the Ethics Committee.

Consent for publication

Not applicable.

Clinical trial number

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qian, Y., Zhang, H., Li, J. et al. Wnt signaling aberrant activation drives ameloblastoma invasion and recurrence: bioinformatics and in vitro insights. BMC Oral Health 24, 1421 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12903-024-05003-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12903-024-05003-0

Keywords

BMC Oral Health

ISSN: 1472-6831

Contact us