METTL3 suppresses anlotinib sensitivity by regulating m6A modification of FGFR3 in oral squamous cell carcinoma.

BACKGROUND: N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA, but there were few studies on its role in cancer drug sensitivity and resistance. Anlotinib has been proved to have effective antitumor effects in oral squamous cell carcinoma (OSCC) in our previous study. Here, we sought to investigate the treatment target of anlotinib and the function and mechanisms of m6A modification in regulating anlotinib effect in OSCC. METHODS: Anlotinib treatment in a dose-dependent manner, western blotting, qRT-PCR and cell lost-of-function assays were used to study the treatment target of anlotinib in OSCC. RNA m6A dot blot assays, the m6A MeRIP-seq and MeRIP-qPCR, RNA and protein stability assays were used to explore the m6A modification of the treatment target of anlotinib. Cell lost-of-function assays after METTL3 depletion were conducted to investigate the effect of m6A modification level on the therapeutic effect of anlotinib in OSCC. Patient-derived tumor xenograft (PDX) models and immunohistochemistry staining were performed to study the relationship of METTL3 and antitumor sensitivity of anlotinib in vivo. RESULTS: Anlotinib targeted FGFR3 in the treatment of OSCC and inhibited tumor cell proliferation and promoted apoptosis by inactivating the FGFR3/AKT/mTOR signaling pathway. METTL3 was identified to target and modify FGFR3 m6A methylation and then decrease the stability of mRNA. METTL3 expression level was related to the anlotinib sensitivity in OSCC cells in vitro and METTL3 knockdown promoted anlotinib sensitivity of OSCC cells by inhibiting the FGFR3 expression. PDX models samples furthermore showed that METTL3 and FGFR3 levels were tightly correlated with the anlotinib efficacy in OSCC. CONCLUSIONS: In summary, our work revealed that FGFR3 was served as the treatment target of anlotinib and METTL3-mediated FGFR3 m6A modification played a critical function in the anlotinib sensitivity in OSCC.

SOD2 confers anlotinib resistance via regulation of mitochondrial damage in OSCC.

OBJECTIVE: Previous studies had revealed that anlotinib had outstanding anti-tumor efficacy on oral squamous cell carcinoma. However, the underlying mechanism is still unclear. MATERIALS AND METHODS: Anlotinib resistant OSCC cells were established and analyzed by RNA-sequencing. The correlations between SOD2 expression and anlotinib resistance were investigated in OSCC cells and PDX models. Functional assays were performed to verify the SOD2 expression and anlotinib resistance in OSCC cells. RESULTS: Anlotinib resistant genes were enriched in the biological processes of mitochondrion organization and the gene pathway of reactive oxygen species. SOD2 expression level was positively correlated with the resistance of anlotinib in OSCC cells and PDX models. Higher SOD2 expression of OSCC cells was more resistant to anlotinib. Anlotinib induced ROS generation, apoptosis and mitochondrial damage in OSCC cells, which can be enhanced by SOD2 knockdown and decreased by SOD2 overexpression. Mitochondrial damage was identified as swelling and cristae disappearance morphology under TEM, decreased mitochondrial membrane potential and lower MFN2 expression. CONCLUSIONS: SOD2 may be capable of protecting mitochondria by downregulating ROS generation, which contributes to the resistance of anlotinib in OSCC cells. SOD2 can be utilized as a potential therapeutic target to improve the anti-cancer efficacy of anlotinib in OSCC.

Arecoline induces epithelial-mesenchymal transformation and promotes metastasis of oral cancer by SAA1 expression.

Arecoline, the main alkaloid of areca nut, is well known for its role in inducing submucosal fibrosis and oral squamous cell carcinoma (OSCC), however the mechanism remains unclear. The aim of this study was to establish an arecoline-induced epithelial-mesenchymal transformation (EMT) model of OSCC cells and to investigate the underlying mechanisms. CAL33 and UM2 cells were induced with arecoline to establish an EMT cell model and perform RNA-sequence screening. Luminex multiplex cytokine assays, western blot, and RT-qPCR were used to investigate the EMT mechanism. Arecoline at a concentration of 160 µg/ml was used to induce EMT in OSCC cells, which was confirmed using morphological analysis, transwell assays, and EMT marker detection. RNA-sequence screening and Luminex multiplex cytokine assays showed that many inflammatory cytokines (such as serum amyloid A1 [SAA1], interleukin [IL]-6, IL-36G, chemokine [CCL]2, and CCL20) were significantly altered during arecoline-induced EMT. Of these cytokines, SAA1 was the most highly upregulated. SAA1 overexpression induced EMT and promoted the migration and invasion of CAL33 cells, while SAA1 knockdown attenuated arecoline-induced EMT. Moreover, arecoline enhanced cervical lymph node metastasis in an orthotopic xenograft model of the tongue established using BALB/c nude mice. Our findings revealed that arecoline induced EMT and enhanced the metastatic capability of OSCC by the regulation of inflammatory cytokine secretion, especially that of SAA1. Our study provides a basis for understanding the mechanism of OSCC metastasis and suggests possible therapeutic targets to prevent the occurrence and development of OSCC associated with areca nut chewing.

METTL3 Promotes Tumorigenesis and Metastasis through BMI1 m6A Methylation in Oral Squamous Cell Carcinoma.

RNA modification plays an essential function in regulating gene expression and diverse biological processes. RNA modification enzyme methyltransferase-like 3 (METTL3) affects tumor progression by regulating the N6-methyladenosine (m6A) modification in the mRNAs of critical oncogenes or tumor suppressors, but its effect in oral squamous cell carcinoma (OSCC) remains unknown. In this study, we revealed that METTL3 was consistently upregulated in two OSCC cohorts, and high METTL3 expression was associated with poor prognosis. Functionally, cell proliferation, self-renewal, migration, and invasion ability in vitro and tumor growth and metastasis in vivo were decreased after METTL3 knockdown in OSCC cells. In contrast, the opposite results were obtained after METTL3 overexpression. In addition, the results obtained with the Mettl3 genetically modified mouse model validated the essential role of Mettl3 in chemical-induced oral carcinogenesis. In mechanism, methylated RNA immunoprecipitation sequencing (MeRIP-seq), MeRIP-quantitative real-time PCR, and luciferase reporter and mutagenesis assays identified that METTL3 mediates the m6A modification in the 3' UTR of BMI1 mRNA. METTL3 promotes BMI1 translation in OSCC under the cooperation with m6A reader IGF2BP1. Our findings revealed that METTL3 promotes OSCC proliferation and metastasis through BMI1 m6A methylation, suggesting that the METTL3-m6A-BMI1 axis may serve as a prognostic biomarker or therapeutic target in patients with OSCC.

MCTS1 promotes invasion and metastasis of oral cancer by modifying the EMT process.

BACKGROUND: The oncogene, malignant T-cell-amplified sequence 1 (MCTS1), has been found to be highly expressed in a variety of cancer cell lines. It has been shown to be involved in cell cycle progression and to confer a growth advantage for lymphomas and breast cancer. Nevertheless, the role of MCTS1 in contributing to the development of oral cancer remains elusive. METHODS: We analyzed the gene expression profiles of MCTS1 in normal oral keratinocytes and cancerous cells. Cellular proliferation, invasion, and migration experiments were performed to detect the effect of MCTS1 on the biological evolution of oral cancer. The in vitro results were verified by the in vivo lymphatic metastasis test. The underlying mechanism of MCTS1 in promoting oral cancer invasion and metastasis correlated with the epithelial-mesenchymal transition (EMT) process as revealed by western blotting. RESULTS: The results showed that MCTS1 was aberrantly expressed in oral cancer cells. MCTS1 overexpression significantly promoted tumor cell growth, proliferation, migration, and invasion. MCTS1-mediated lymphatic metastasis was verified in vivo using an intraplantar tumor model. Biomarkers associated with EMT progression were positively or negatively regulated upon knockdown or overexpression of MCTS1, respectively. CONCLUSIONS: Higher MCTS1 expression in oral cancer may be connected with an unfavorable prognosis due to involvement of MCTS1. MCTS1 potentiates the growth and proliferation of oral cancer cells and subsequent metastasis by regulating cell cycle and modifying the EMT process. KEYWORDS: Oral cancer; oncogene; malignant T-cell-amplified sequence 1 (MCTS1); metastasis; invasion.

Suppressed mitochondrial respiration via NOX5-mediated redox imbalance contributes to the antitumor activity of anlotinib in oral squamous cell carcinoma.

Anlotinib, a novel multitarget tyrosine kinase inhibitor, has shown promising results in the management of various carcinomas. This study aimed to investigate the antitumor activity of anlotinib in oral squamous cell carcinoma (OSCC) and the underlying molecular mechanism. A retrospective clinical study revealed that anlotinib improved the median progression-free survival (mPFS) and median overall survival (mOS) of patients with recurrent and metastatic (R/M) OSCC, respectively. Functional studies revealed that anlotinib markedly inhibited in vitro proliferation of OSCC cells and impeded in vivo tumor growth of OSCC patient-derived xenograft models. Mechanistically, RNA-sequencing identified that oxidative stress, oxidative phosphorylation and AKT/mTOR signaling were involved in anlotinib-treated OSCC cells. Anlotinib upregulated NADPH oxidase 5 (NOX5) expression, elevated reactive oxygen species (ROS) production, impaired mitochondrial respiration, and promoted apoptosis. Moreover, anlotinb also inhibited phospho-Akt (p-AKT) expression and elevated p-eIF2α expression in OSCC cells. NOX5 knockdown attenuated these inhibitory effects and cytotoxicity in anlotinib-treated OSCC cells. Collectively, we demonstrated that anlotinib monotherapy demonstrated favorable anticancer activity and manageable toxicities in patients with R/M OSCC. The antitumor activity of anlotinib in OSCC may be mainly involved in the suppression of mitochondrial respiration via NOX5-mediated redox imbalance and the AKT/eIF2α pathway.

Biomimetic titanium implant coated with extracellular matrix enhances and accelerates osteogenesis.

Aim: To evaluate the biological function of titanium implants coated with cell-derived mineralized extracellular matrix, which mimics a bony microenvironment. Materials & methods: A biomimetic titanium implant was fabricated primarily by modifying the titanium surface with TiO2 nanotubes or sand-blasted, acid-etched topography, then was coated with mineralized extracellular matrix constructed by culturing bone marrow mesenchymal stromal cells. The osteogenic ability of biomimetic titanium surface in vitro and in vivo were evaluated. Results:In vitro and in vivo studies revealed that the biomimetic titanium implant enhanced and accelerated osteogenesis of bone marrow stromal cells by increasing cell proliferation and calcium deposition. Conclusion: By combining surface topography modification with biological coating, the results provided a valuable method to produce biomimetic titanium implants with excellent osteogenic ability.

Anti-fungal drug itraconazole exerts anti-cancer effects in oral squamous cell carcinoma via suppressing Hedgehog pathway.

AIMS: To investigate the therapeutic potential of itraconazole in oral squamous cell carcinoma (OSCC) and its molecular mechanism. MATERIALS AND METHODS: The in vitro anti-cancer effects of itraconazole was determined by CCK-8 assay and colony formation assay. Transwell and wound healing assays were used to examine cell invasion and migration. The in vivo therapeutic efficacy of itraconazole was assessed by OSCC patient-derived xenograft (PDX) model. Western blot was performed to explore the anti-cancer mechanism. KEY FINDINGS: Itraconazole inhibited cell proliferation and colony formation of OSCC cells in a time and concentration dependent manner; induced cell cycle arrest and apoptosis, as well as inhibited cell invasion and migration. In the OSCC PDX model, itraconazole impeded tumor growth, reduced Ki-67 expression and induced apoptosis. Itraconazole downregulated the protein expression of Hedgehog pathway to inhibit proliferation and migration of oral squamous cell carcinoma cells, which can be revised by recombinant human sonic hedgehog protein (rSHH). SIGNIFICANCE: Itraconazole showed anti-cancer effects on OSCC via inhibiting the Hedgehog pathway.