EGFR Signaling Pathway Promotes EMT Process To Affect Vasculogenic Mimicry In Nasopharyngeal Carcinoma

Background:Nasopharyngeal carcinoma(NPC)is a malignant nasopharyngeal carcinoma with a high incidence in Southeast Asia.Guangdong Province is an area with a high incidence of morbidity in my country.Nasopharyngeal carcinoma is a highly sensitive tumor to radiation and chemotherapy.Early and locally advanced nasopharyngeal carcinoma patients are mainly treated with radiotherapy alone or combined with radiotherapy and chemotherapy.In recent years,EGFR-targeted therapy has received more and more attention.However,the mechanism of molecularly targeted therapy remains unclear.Epidermal growth factor receptor(EGFR),a member of the ErbB family,is a 170kda transmembrane receptor that involves three domains,including an extracellular domain,a transmembrane domain,and a cellular domain.internal domain.The extracellular region can recognize and bind the corresponding ligand,and the intracellular part has tyrosine kinase activity.Once activated,EGFR forms homo-or heterodimers with other ErbB family members,and then phosphorylates tyrosine kinases,which in turn activate downstream signaling pathways,such as JAK-STAT and PI3K-Akt.These signals ultimately lead to tumor development and progression.Therefore,epidermal growth factor receptor(EGFR)is a valuable tumor therapeutic target.Accumulating evidence suggests that EGFR overexpression is associated with poor prognosis in nearly 85%of NPC patients.Treatments for EGFR include monoclonal antibodies,tyrosine kinase inhibitors,phosphatidylinositol-3-kinase(PI3K)inhibitors,and antisense gene therapy.At present,EGFR-targeted drugs(nimotuzumab/cetuximab)can competitively bind EGFR and block downstream signal transduction pathway mediated by EGFR,thus inhibiting tumor cell proliferation,inducing differentiation,promoting apoptosis,inhibiting tumor angiogenesis and enhancing the efficacy of radiotherapy and chemotherapy.However,the exact mechanism of molecular targeted therapy remains unclear.In the previous study of this subject,we found that Foxq1 can significantly promote the formation of angiogenic mimicry(VM),tumor growth and metastasis,and can be effectively inhibited by EGFR inhibitors(nimotuzumab or erlotinib).Angiogenic mimicry is a novel blood supply system first proposed by Maniotis et al in 1999 in aggressive human melanoma.It is a vascular-like structure formed by a highly aggressive tumor through self-deformation and remodeling of the extracellular matrix to satisfy its own blood supply.It is thought to be involved in the progression and metastasis of malignant tumors.However,what mediates the biological behavior of EGFR to promote vascular mimicry in nasopharyngeal carcinoma is a new question.We noticed that there are many common regulators between epithelial-mesenchymal transition(EMT)and angiogenic mimicry,such as hypoxia-inducible factor(HIF-1?),vascular endothelial growth factor(VEGF),matrix metalloproteinases(MMPS),etc..EMT is a process in which epithelial cells lose cell polarity,lose tight intercellular junctions and adhesion junctions,acquire the ability to infiltrate and migrate,and become mesenchymal cells with morphology and characteristics under the action of certain factors.Cadherin,Catenin,and Vimentin are considered markers of EMT.Cadherin is a cell surface molecule involved in the Ca2+-dependent adhesion mechanism.Among them,E-cadherin is one of the widely studied cadherin family members and is also a potent tumor suppressor.E-cadherin plays an important role in maintaining epithelial phenotype and regulating tissue homeostasis by regulating various signaling pathways.On the contrary,N-cadherin is an indicator of persistent EMT,and its expression is associated with the occurrence and development of various cancers.The cytoplasmic tail of Cadherin binds to ?-catenin,which in turn binds to ?-catenin to form the Cadherin-Catenin adhesion complex.When E-cadherin expression was downregulated,?-catenin no longer interacted with E-cadherin,and as E-cadherin levels decreased,?-catenin was either degraded or accumulated in the nucleus and involved in transcription.Vimentin is a major component of the intermediate filament(IF)protein family,which is ubiquitously expressed in normal mesenchymal cells,and its overexpression in cancer is closely related to tumor growth,invasion and poor prognosis.EMT has been found to be involved in the formation of angiogenic mimicry in some tumors,such as liver cancer,gastric cancer,and melanoma.However,no studies have clarified the relationship between angiogenic mimicry and EMT in nasopharyngeal carcinoma.Our study aimed to demonstrate whether EMT could act as a bridge to mediate the Foxq1/EGFR pathway to promote vascular mimicry in NPC.Materials and Methods:We detected the expressions of PAS,CD31,Vimentin and E-cadherin in 60 NPC patients by immunohistochemical staining,and analyzed the correlation between EMT and vasculogenic mimicry in the clinical data of NPC.In the 3D cell culture model,after adding Nimotuzumab,the number of vasculogenic mimicry was observed,and RT-PCR and Western Blot were used to detect the number of vasculogenic mimicry vessels and the expression of EMT indicators.Finally,we performed in vivo experiments in animals.The subcutaneous tumorigenesis model of nude mice was verified by immunohistochemical experiments to verify the relationship between EMT indexes and vasculogenic mimicry.Results1.In nasopharyngeal carcinoma tissue,VM was significantly correlated with EMT and clinical stage of nasopharyngeal carcinoma.Vasculogenic mimicry and EMT-related factors(E-cadherin and Vimentin)in 60 nasopharyngeal carcinoma tissues were quantified by PAS/CD31 double-fill staining and immunohistochemical staining.The statistical results showed that with the progression of NPC,Vasculogenic mimicry blood vessels(PAS positive,CD31 negative)increased,corresponding to the decrease of EMT inhibitor(E-cadherin)and the increase of EMT promoter(Vimentin).Immunohistochemical scoring was performed on 60 nasopharyngeal carcinoma tissues.The scores were then grouped and statistically analyzed by total stage,T stage,and N stage.We found that in the PAS+/CD31-score,the score of intermediate-advanced nasopharyngeal carcinoma tissues(?+?)was higher than that of early-stage nasopharyngeal carcinoma tissues(stage ?+?)(p<0.001).At the same time,the E-cad score of mid-advanced nasopharyngeal carcinoma tissues was lower than that of early nasopharyngeal carcinoma tissues(p=0.005),and the VIM score of advanced nasopharyngeal carcinoma tissues was higher than that of early nasopharyngeal carcinoma tissues(p<0.001).The above results suggest that the later stage of tumor,the process of EMT and vasculogenic mimicry is promoted.At the same time,we found that the T stage and N stage of NPC had a similar trend.Taken together,EMT is associated with the formation of angiogenic mimicry in nasopharyngeal carcinoma tissues.The results suggest that EMT may be involved in the formation of vasculogenic mimicry.2.EGFR inhibitors inhibit the formation of VM in nasopharyngeal carcinoma cells.In order to demonstrate the effect of EGFR inhibitor(nimotuzumab)on the formation of vasculogenic mimicry in nasopharyngeal carcinoma cells,firstly,nimotuzumab was added to the culture medium of nasopharyngeal carcinoma cell lines 5-8F and CNE1,and mixed with 5-8F and CNE1 cells were cultured,and the morphological changes of the two groups were observed.We found that cell lines without nimotuzumab were more prone to linear cells in cell culture,but the number of such linear cells was significantly reduced with nimotuzumab.These cells were transferred to 3D medium and cultured for 24h.We observed that 5-8F and CNE1 cell lines to which Nimotuzumab was added were significantly less able to form angiogenic mimetic structures compared to cells not added to Nimotuzumab.These nasopharyngeal carcinoma cells were then extracted,and EGFR and its phosphorylation product(p-EGFR)were detected by Western blot.We found that the expression of EGFR did not change significantly after adding nimotuzumab,but the content of p-EGFR in the two cell lines decreased significantly(p<0.05).This suggests that nimotuzumab reduces the ability of NPC cells to form angiogenic mimicry by inhibiting EGFR phosphorylation.3.In vitro cell experiments,EMT was correlated with the ability of nasopharyngeal carcinoma cell line to form VM.To evaluate the relationship between EMT and angiogenic mimicry in in vitro cellular experiments.The changes of EMT-related factors E-cadherin,Vimentin,N-cadherin and ?-catenin before and after adding nimotuzumab were detected by qRT-PCR and Western blot.We found that the addition of nimotuzumab increased the expression of E-cadherin and decreased the expression of factors such as Vimentin.The EMT process is limited.We also designed a concentration gradient experiment,using qRT-PCR and Western blot to detect the EMT-related factor E-cadherin and vasculogenic mimicry-related factors in the medium of 5-8F and CNE1 cell lines in successively increasing concentrations of nimotuzumab Expression levels of Ve-cadherin.With the increase of the concentration of nimotuzumab in cell culture medium,the expression level of E-cadherin showed an upward trend,while that of Ve-cadherin showed a downward trend.In summary,in vitro,after treatment with an EGFR inhibitor(Nimotuzumab)in cell culture medium,the changes in cell morphology,vasculogenic mimetic ability,and EMT-related factors were observed,suggesting that EMT is related to the formation of vasculogenic mimicry.4.In vivo animal experiments,nimotuzumab can inhibit the EMT process and VM formation ability of nasopharyngeal carcinoma cell lines.In our previous study,to explore the relationship between angiogenic mimicry and Foxq1 and EGFR,we performed subcutaneous tumorigenesis experiments.We used BALB/c nude mice subcutaneously transplanted tumor model(n=6/group).BALB/c nude mice were injected subcutaneously with normal 5-8F cells and 5-8F cells stably overexpressing Foxq1.We then divided BALB/c nude mice subcutaneously injected with Foxq1-overexpressing 5-8F cells into two groups,and were treated with nimotuzumab and normal saline,respectively.We continued our experiments using previously preserved tumor tissue sections.In this study,we collected these tumor tissues for immunohistochemical staining and PAS/CD31 double staining.The staining results were scored by immunohistochemistry.We found that Nimotuzumab treated mouse tumor tissue was less prone to angiogenic mimicry than normal 5-8F cell tumor tissue.The expression of e-cadherin(P<0.05)was increased and that of Vimentin(P<0.05)was decreased,suggesting that the process of EMT promoting angiogenic mimicry was inhibited by Nimotuzumab.ConclusionsEMT may be the biological basis of vasculogenic mimicry.EGFR signaling pathway promotes vascular mimicry in nasopharyngeal carcinoma through EMT.