| BackgroundNasopharyngeal carcinoma(NPC)is a type of nasopharyngeal malignant tumor,which has an obvious regional specificity and tends to occur in southeast of Asia and southern of China.In recent years,with the understanding of the pathogenesis of nasopharyngeal carcinoma and the progress of treatment technology,especially the extensive application of radiotherapy technology and the optimization of chemotherapy strategies.Currently,recurrence and metastasis remain the biggest difficulties in clinical treatment of nasopharyngeal carcinoma,and are also the main causes of death in patients with nasopharyngeal carcinoma.Thus,it is crucial to clarify the mechanism of nasopharyngeal carcinoma metastasis.Tumor metastasis is an important factor affecting the morbidity and mortality of tumor patients,and the tumor-specific death caused by it accounts for about 90%of the total tumor mortality.Tumor metastasis is a complex dynamic process,in which tumor angiogenesis,as the morphological basis of malignant tumor growth and metastasis,plays a key role in the process of tumor metastasis.Vasculogenic mimicry(VM)is a novel tumor blood supply model that differs from classical endothelium-dependent angiogenesis.In 2016,a paper published in the journal Science pointed out that VM,as a supplement to traditional endothelial-dependent angiogenesis,could provide blood and nutrient supply for tumor growth.Therefore,scholars began to gradually pay attention to the key role of VM in the process of tumor development.The duct structure of VM was composed of highly aggressive tumor cells with PAS positive and CD31 negative.Its unique structure makes it easier for tumor cells to come into direct contact with flowing blood,greatly increasing the risk of distant metastasis.Recently,VM has been confirmed that present in various malignant tumors such as breast cancer,ovarian cancer,gastric cancer and liver cancer,and is closely related to the growth and metastasis of tumors.And in nasopharyngeal carcinoma,is only two.The study on the VM with sun yat-sen university is the most representative,their research confirmed that the EB virus can be induced by hypoxia inducing factor(HIF-alpha)to promote the formation of VM in nasopharyngeal carcinoma(NPC),but the VM role in nasopharyngeal carcinoma metastasis,and the specific regulatory mechanism has not been reported.Our previous studies have showed that miR-124 can prevent NPC metastasis by targeting FOXQ1,but the specific molecular mechanism remains unclear.FOXQ1,as an important member of the FOX family,has been proven to be involved in the occurrence,development,invasion and metastasis of a variety of tumors,and has a strong correlation with epithelial mesenchymal transformation(EMT).For example,in liver cancer,miR-4319 can inhibit cell proliferation,accelerate cell apoptosis,and inhibit epithelial-mesenchymal transformation(EMT)by targeting FOXQ1.In colon cancer,tumor-associated macrophages(TAM)mediate the occurrence of EMT by regulating the JAK2/STAT3/miR-506-3p/FOXQ1 signaling axis.Therefore,as an important regulator of EMT process in many tumors,FOXQ1 may be correlated with VM in nasopharyngeal carcinoma.Meanwhile,miR-124 has been shown to inhibit the formation of VM in cervical cancer,prostate cancer,oral cancer and other tumors.Based on the above research status of miR-124,FOXQ1 and VM,we have reasons to make the following scientific hypothesis:miR-124/FOXQ1 may inhibit the metastasis of nasopharyngeal carcinoma by regulating the formation of VM.In this study,we detected angiogenic mimicry in 114 cases of nasopharyngeal carcinoma by immunohistochemistry,and further analyzed the correlation between VM and FOXQ1 and clinical data of nasopharyngeal carcinoma.At the same time,the specific mech anism of miR-124/FOXQ1/EGFR regulating VM formation and nasopharyngeal carcinoma metastasis was further explored in vitro cell experiments and in vivo animal experiments.This study is helpful to further explore the specific mechanism of NPC metastasis.providing therapeutic targets for actively seeking effective,safe and reliable new treatment approaches,and providing theoretical support for the application of anti-VM therapy in the clinical treatment of nasopharyngeal carcinoma.Materials and MethodsThe expression of VM in NPC was detected by immunohistochemistry,and the correlation between VM and Foxq1,EGFR and NPC clinical data was further analyzed;In vitro.3D culture,qRT-PCR,Western blot and immunofluorescence were used to verify the miR-124/Foxq1/EGFR signal axis in regulating VM formation.In vivo,nude mouse subcutaneous tumor model and nude mouse tail vein injection lung metastasis model were used to verify that Foxq1 promotes VM formation and NPC metastasis by regulating EGFR.The dual luciferase experiment and the CHIP were used to verify that Foxq1 directly binds to the EGFR promoter region.Results1.Vasculogenic mimicry has a strong correlation with Foxq1 and NPC clinical stage,and is closely related to poor prognosis of patients with nasopharyngeal carcinoma.We detected the expression of Foxq1 in 114 samples of nasopharyngeal carcinoma(7,24,38,and 45 cases of grade Ⅰ,Ⅱ,Ⅲ,and Ⅳ)and 40 samples in control group of nasopharyngitis tissues.The statistical results showed that Foxq1 was significantly stronger in nasopharyngeal carcinoma than control group(P<0.001).While the VM in NPC tissues was tested by PAS/CD31 double staining,and different degrees of vasculogenic mimicry were found in nasopharyngeal carcinoma tissues,among which 78 cases(68%)were VM positive and 36 cases(32%)were VM negative.The statistical results showed that Foxq1(P<0.001),the NPC clinical stage(TNM stage)(P=0.013),and the N stage(P=0.004)were significantly different between VM positive and VM negative NPC patients,but there were no statistically significant differences in T stage(P=0.127),age(P=0.662),or gender(P=0.577).Then,kaplan-Meier survival analysis curve showed that the prognosis of VM-positive NPC patients was significantly worse than VM-negative patients(P=0.046),suggesting that VM was an independent adverse prognostic factor for NPC patients.2.In vitro,Foxq1 was positively correlated with VM formation in nasopharyngeal carcinoma cell lines.In vitro cell experiments,we found that nasopharyngeal carcinoma cell lines,including 5-8F,6-10B,CNE1,CNE2 and C666-1,could form different degrees of vascular-like structures during 3D culture.While the VM formation capacity of poorly differentiated nasopharyngeal carcinoma cell lines(5-8F,CNE2 and C666-1)was significantly higher than that of highly differentiated nasopharyngeal carcinoma cell lines(6-10B and CNE1).These results showed that VM formation ability of NPC cell line was significantly positively correlated with its malignant degree.Then we constructed 5-8F and CNE1 cell lines that stably over-expressed or interfered Foxq1,and verified them by QRT-PCR and Western blot.Subsequently,in vitro 3D culture,we found that VM formation in 5-8F and CNE1 cells that interfered Foxq1 was significantly inhibited,while over-expressed Foxq1 significantly promoted VM formation in 5-8F and CNE1 cells.These results suggested that Foxq1 could promote VM formation in nasopharyngeal carcinoma cell lines.3.Foxq1 can accelerate VM by promoting the expression of EGFR signaling pathway and VM-related genes,while Erlotinib can block Foxq1 induced VM formation.Although we have preliminary confirmed that Foxq1 is positively correlated with VM,the exact mechanism by which Foxq1 promotes VM is unclear.In order to further explore its specific mechanisms,we used bioinformatics databases to predict the downstream target genes of miR-124/Foxq1.Then qRT-PCR was carried out to verify these predicted genes in 5-8F that over-expressed Mir-124 or interfered Foxq1.The result showed that the expressions of genes such as EGFR,TEAD1,SIX4 and so on were all dysregulated.We then selected EGFR from these genes as the downstream target gene,and subsequently verified the expression of EGFR in 5-8F and CNE1 cells that interfered or overexpressed Foxq1 by QRT-PCR and Western blot.We found that up-regulating Foxq1 could promote the expression of EGFR,while interference with Foxq1 significantly inhibited the expression of EGFR.To verify the role of EGFR in Foxq1-induced VM formation,we treated nasopharyngeal carcinoma cells that overexpressed Foxq1 with Erlotinib(EGFR inhibitors)for 24 h,then detected the VM formation ability of them through 3D cell culture in vitro.Obviously,the VM formation ability of nasopharyngeal carcinoma cells that treated with Erlotinib was significantly suppressed.This result suggested that Erlotinib could block Foxq1 on promoting the formation of VM.In summary,Foxq1 can promote VM formation in nasopharyngeal carcinoma cells by regulating EGFR expression.Furthermore,we detected the expression of EGFR in 114 nasopharyngeal carcinoma samples by immunohistochemistry,and conducted statistical analysis.Meanwhile,Pearson correlation analysis indicated that Foxq1 was positively correlated with EGFR in the nasopharyngeal carcinoma(P<0.001,r=0.5303).Further statistics of the expression of EGFR in VM positive and VM negative NPC.It showed that the expression of EGFR in VM positive NPC patients was significantly stronger than VM negative NPC patients,indicating that EGFR was also significantly correlated with VM(P<0.001).Finally,we detected the expression of EGFR signaling pathway and VM-related gene in 5-8F and CNE1 cells that overexpressed or interfered with Foxq1 by QRT-PCR,Western blot,and IF,including AKT,P-Akt,Bcl-2,MMP2,MMP9,and VE-cadherin.The results showed that the EGFR signaling pathway and VM-related genes were higher expressed in the 5-8F and CNE1 cells that overexpressed Foxq1,while the EGFR signaling pathway and VM-related genes were inhibited in the 5-8F and CNE1 cells that interfered Foxq1.These results showed that Foxq1 was positively related to EGFR signaling pathway and VM related gene expression in NPC cell lines.In summary,our results confirm that Foxq1 can promote VM formation by targeting THE EGFR signaling pathway and VM-related genes in vitro.4.Foxq1 could directly bind to the EGFR and regulate its transcription.In order to clarify the specific mechanism of Foxq1 and EGFR,we used bioinformatics database,such as UCSC and JASPAR databases,to analyze the binding sites between Foxq1 and EGFR.We found that Foxq1 had multiple predicted binding sites in the EGFR promoter region,and selected two of the most likely binding sites for double luciferase reporter assay verification.First,a full-length(FL)EGFR promoter(1KB)and three mutant EGFR promoters were cloned into luciferase reporter plasmid respectively.The mutated EGFR promoters included m1-site mutation(ATAGTTTAAA),M2-site mutation(AATTAAACATTA)and both mutated simultaneously.The luciferase reporter gene was transfected with Foxq1 or control plasmid into 293T cells and the fluorescence signal was detected 36h later.The results showed that the transcriptional activity of EGFR in Foxq1 group increased in different degrees than the control group,while the transcriptional activity of EGFR on the group that mutant M1 or M2 was significantly reduced,and the transcriptional activity of EGFR on the group that mutant Ml and M2 simultaneously was no longer significant.This suggests that FOXQ1 promotes transcription by acting on the EGFR promoter.Subsequently,we further demonstrated that Foxq1 directly bind to EGFR using the chromatin immunoprecipitation(CHIP)assay.The chromatin co-precipitated by anti-FoxQ1 or anti-IgG antibody was analyzed by QRT-PCR and agarose electrophoresis.We found that the EGFR promoter was significantly enriched in the anti-FoxQ1 group compared with the anti-IgG group.In summary,Foxq1 promotes EGFR transcription by directly binding to the M1 and M2 sites in the EGFR promoter.5.In vivo,Foxq1 could promote VM formation,NPC growth and metastasis.In vivo,we confirmed the correlation between Foxq1 and VM using a subcutaneous xenograft model in nude mice.Firstly,1×105 5-8F cells stably overexpressed Foxq1 or control 5-8F cells were subcutaneously injected into the armpits of nude mice.Tumor growth curves were drawn by testing tumor volume every 3 days.Two weeks later,the mice were euthanized,and the tumors were taken out.The results showed that the growth rate and weight of the xenografts in the overexpressing Foxq1 group were significantly increase.It could be seen that the xenografts of the overexpressing Foxq1 group were lobulated,while the xenografts of the control group were rounded and smooth.These results suggest that Foxq1 promotes the malignant growth of nasopharyngeal carcinoma in vivo.Immunohistochemical analysis of xenograft specimens showed that the expression of Foxq1,EGFR and VM in the overexpressed Foxq1 group were significantly increased compared with the control group.After total RNA and total proteins were extracted by grinding the xenografts,the expression of Foxq1,EGFR signaling pathways and VM-related genes in the xenografts were analyzed by QRT-PCR and Western blot.The statistical results indicated that the EGFR signaling pathway and VM-related genes in the xenografts of overexpressed Foxq1 group were also significantly increased compared with the control group.To verify the effect of Foxq1 on NPC metastasis,we injected overexpressing Foxq1 5-8F cells and control cells through the tail vein of nude mice to construct a lung metastasis model of nude mice.After 5 weeks,the lung tissues were collected for tissue fixation.Then embedded sections were performed,and lung metastatic nodules were statistically analyzed by HE staining.The results showed that the number of pulmonary metastatic nodules in the overexpressing FOXQ1 group was much more than the control group.In conclusion,Foxq1 could promote VM formation,nasopharyngeal cancer growth and metastasis in vivo.6.In vivo,Erotinib and Nimotuzumab could inhibit Foxq1-induced VM formation,nasopharyngeal cancer growth and metastasis.In vivo,1×105 overexpressing Foxq1 5-8F cells were injected subcutaneously or intravenously into mice,then treated with Erotinib(I.g,50mg/kg),Nimotuzumab(I.V,20mg/kg)or normal saline respectively.The results showed that the growth rate,volume and weight of xenografts in the erlotinib group and the Nimotuzumab group were lesser than the control group,and the histological analysis showed that the number of VM and lung metastatic nodules in the Erlotinib and Nimotuzumab group were significantly reduced.qRT-PCR and Western blot analysis of the gene expression in xenografts indicated that VM-related genes were significantly inhibited in the Erlotinib group and the Nimotuzumab group.Above results suggested that Erotinib and Nimotuzumab,as EGFR inhibitors,could effectively prevent Foxq1-induced VM formation,nasopharyngeal cancer growth and metastasis.7.Combination with anti-EGFR and anti-VEGF drugs resulted in improved antitumor efficacy.Above results have confirmed that anti-EGFR drugs can inhibit the growth and metastasis of NPC by inhibiting Foxq1-induced VM formation.At the same time,anti-VEGF drugs,which is used to prevent endothelium-dependent vessels(EDV),has been reported to have no inhibitory effect on VM,while it has been reported that VM is an key factor affecting the efficacy of anti-VEGF drugs.Therefore,we speculated that the combination of anti-EGFR and anti-VEGF drugs can simultaneously inhibit endothelium-dependent angiogenesis and VM,achieving better anti-tumor effect.We subcutaneously injected Foxq1 overexpressing 5-8F cells into BALB/C nude mice and treated them with Sunitinib,combination of Sunitinib and Erlotinib,or combination of Sunitinib and Nimotuzumab,respectively.The results showed that the growth rate,volume and weight of xenografts in the combination group were lower than those in sunitinib single drug group and control group.Through CD31-PAS dual histochemical staining,we found that Sunitinib had a strong inhibitory effect on EDV,but no significant effect on VM.While the combination of anti-EGFR and anti-VEGF drugs had obvious inhibitory effect on both EDV and VM.These results confirmed the synergistic effect of anti-VEGF and anti-EGFR,and provided a new idea and choice for the treatment of nasopharyngeal carcinoma.8.miR-124 could inhibit the expression of EGFR and VM formation induced by Foxq1.Our previous study has showed that miR-124 could inhibit NPC metastasis though targeting Foxq1.In this study,we further found that VM formation of overexpressing miR-124 5-8F cells was significantly inhibited through 3D cell culture in vitro.Subsequently,Foxq1 was further overexpressed in 5-8F cells that overexpressing Mir-124,and Foxq1 was found to reverse the inhibition of miR-124 on VM formation,which could also blocked by Erlotinib.In addition,through qRT-PCR,Western blot and IF,we found that overexpressed miR-124 could significantly reduce the expression of Foxq1,EGFR signaling pathway and VM-related genes.In summary,our results confirm that Mir-124 could target Foxq1 to inhibit the expression of EGFR signaling pathway and VM-related genes by,thereby inhibiting VM formation and nasopharyngeal carcinoma metastasis.Conclusions1.VM was positively correlated with Foxq1,EGFR and TNM stage in NPC,and was closely related to poor prognosis of NPC.2.In vitro,Foxq1 could promote VM formation through regulating EGFR signaling pathway and VM-related genes,while Erotinib could inhibit Foxq1-induced VM formation.3.In vivo,we demonstrated that Foxq1 could promote VM formation and NPC metastasis,which could be blocked by Erotinib or Nitutuzumab.4.Anti-EGFR and anti-VEGF drugs have a synergistic effect,which can simultaneously inhibit EDV and VM formation,thus achieving better anti-tumor effect.5.Foxq1 can directly bind to the EGFR and regulates EGFR transcription.6.miR-124/Foxq1/EGFR axis inhibits NPC metastasis through regulating VM formation. |
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