Fusobacterium Nucleatum Promotes Cisplatin-resistance And Migration Of TSCC Via WNT/NFATc3 Pathway

Bacterial infection contributes to tumor development and malignant progression.Fusobacterium nucleatum(F.nucleatum)is a known gram-negative anaerobic periodontal pathogen,related to a variety of human diseases including periodontal disease,oral cancer,inflammatory bowel disease,and colorectal cancer.Oral squamous cell carcinoma(OSCC)usually occurs in cells of the epidermis or appendages and is the most common malignant tumor in the oral and maxillofacial region.Tongue squamous cell carcinoma(TSCC)is one of the most common types of OSCC.Cis-diaminodichloroplatinum(CDDP)is one of the most common chemotherapy drugs to treat OSCC by increasing P53 expression and inducing apoptosis.However,tumor recurrence is high in TSCC due to chemoresistance and metastasis.With the development of metagenomics,studies have found that the abundance of Fusobacterium nucleatum in patients with TSCC is not only significantly higher than that of healthy people but also increases gradually with the progress of the cancer stage.Objective: A pilot study suggested that F.nucleatum might activate the WNT signal pathway when co-cultured with TSCC cells Cal-27 and HSC-3.This study aimed to observe the biological characteristics of Cal-27 and HSC-3 influenced by F.nucleatum,especially cisplatin-resistance and migration,and to explore the mechanism related to the WNT signal pathway.Methods: Cal-27 and HSC-3 were treated with different concentrations of F.nucleatum.The cells were divided into experimental groups(MOI = 100,200,500,1000)and control group(MOI = 0).The CCK-8 analysis was used to evaluate the effect of F.nucleatum on cell proliferation and adhesion.We conducted a cisplatinresistant cell line Cal-27/CDDP and evaluated cell apoptosis by CCK-8 assay,flow cytometric detection of mitochondrial membrane potential,and caspase 3/7 activity assay under the stimulation of 10 ?M CDDP.Western blotting method was used to detect the levels of endogenous apoptosis-related proteins BAX/BCL-2 and P53.Balb/c Nude mice aged 5-6 weeks were induced by subcutaneous injection of Cal-27/CDDP to conduct mouse models of TSCC.CDDP was injected intraperitoneally,and the tumors of the experimental group were injected with F.nucleatum.The size and weight of tumors were compared between the experimental group and the control group.We observed the cell morphology under the microscope and detected the influence of cell migration ability by scratch experiment and Western blotting method.The expression levels of WNT pathway genes and Nuclear factors of activated T-cells(NFATs)were evaluated by Real-time PCR and Western blotting.A plasmid of p NFATLuc was utilized to conduct a luciferase reporter gene assay of total NFAT.To validate the signal pathway reversely,we repeated the above assays along with NFATc3 knockdown cell lines and NFAT inhibitor VIVIT.Results: In treated cells,the proliferation curves of Cal-27 and HSC-3 were consistent with the control groups.The numbers of adhesive cells were more in treated groups than in the control groups,especially the MOI = 500 groups.The survival rates were significantly higher in the treated groups.The expressions of apoptosis-related proteins like cleaved-caspase 3,BAX,BCL-2 and P53 were lower relatively examined by Western blotting.The size and weight of mice treated were bigger and heavier than the control.Cells in MOI = 500 groups showed flat polygonal,round,irregular or fusiform-shaped cell morphology under the microscope,arranged in a paving stonelike structure.The expression of E-cadherin was higher.The results of the scratch wound assay also showed that the migration area and distance were larger than the control.A plasmid of p NFAT-Luc was utilized to conduct a luciferase reporter gene assay of NFAT.The expression levels of WNT pathway proteins WNT5 A and NFATc3 were notably higher in treated cells detected by Real-time PCR and Western blotting.The migration indicators like migration area and E-cadherin expression of NFATc3knocked-down cell lines were reduced.With the inhibition effect of NFAT inhibitor VIVIT,the apoptotic rates and P53 expressions of F.nucleatum-treated groups in were up-regulated reversely.Conclusion:We concluded that F.nucleatum might promote cisplatin-resistance,migration and adhesion of TSCC cell lines,without influencing proliferation.F.nucleatum could activate the WNT5A/NFATc3 pathway to promote migration and cisplatin-resistance of Cal-27 and HSC-3.