Mechanism Of EGFR Wt/v? Mediated Proliferation And Drug Resistance In Head And Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma(HNSCC),is one of the major tumors threatening human health,has become the world's sixth best cancer.HNSCC patients with 5-year survival rate of less than 50%,the world's fifth largest mortality rate.Malignant proliferation and chemotherapy resistance are important clinical manifestations of HNSCC.Therefore,to explore the molecular mechanism of HNSCC proliferation and chemotherapy resistance,to find a new effective therapeutic target,will become an important breakthrough in improving the treatment of head and neck squamous cell carcinoma.EGFR is a widely distributed cell surface receptor in mammals,plays an important role in the physiological processes of cell growth,proliferation and differentiation.The expression of EGFR in a variety of malignant tumor cells was higher than that in normal cells.In more than 90% of head and neck squamous cell carcinoma,EGFR expression increased.Based on this,the use of targeted drugs for EGFR is targeted for targeted therapy.However,when EFGR mutations,it will lead to clinical drug resistance.Many EGFR mutants have been found,the most common of which are EGFRv III type(the type III EGF deletion-mutants receptor).?-catenin as an important component of the Wnt signaling pathway,mainly involved in gene expression and mediated cell adhesion.Current studies have shown that ?-catenin gene mutations and overexpression,and tumor occurrence,development and prognosis are closely related.As a isoenzyme of pyruvate kinase,PKM2(Pyruvate kinase isozyme thpe M2)plays an important role not only in the glycolysis process but also in the activity of ?-catenin and the transcription of downstream genes.In this study,the function and molecular mechanism of EGFR wt / v III in tumor growth and chemotherapy resistance in head and neck squamous cell carcinoma were studied.The study is divided into three parts:In the first part,we extracted the protein from five head and neck squamous cell carcinoma cell lines,and the expression level of EGFR was detected by Western blot.Two cell lines of UM1 and SCC25 were selected and two types of wild type and v III mutant were established.Cell lines,CCK8 cell proliferation assay and clonal formation assay were used to detect cell proliferation ability.Cell resistance to cisplatin was measured by drug sensitivity test and flow cytometry.In the second part,EGFR wild-type cells were treated with EGF,and the level of PKM2 was detected by Western blot and immunofluorescence.EGFR wild-type and mutant cells were treated with EGF at the same time.The activity of PKM2 and the activity of ?-catenin were detected by Western blot.The expression and correlation of PKM2 and ?-catenin in head and neck squamous cell carcinoma were analyzed by analyzing the GEO database.In the third part,according to the literature,R399 / 400 locus was very important for PKM2 entry.Therefore,we established PKM2 overexpression and R399 / 400 A mutant stable clone.Western blot was used to detect PKM2 into ?-catenin pathway,The effect of downstream gene expression.The effect of PKM2 on cell proliferation and cisplatin resistance in squamous cell carcinoma of head and neck was detected by CCK8 proliferation assay,clone formation assay and flow cytometry.result:In the two cell lines of UM1,SCC25 wild type and v III mutant,we found that the proliferation of v III mutant cell lines was faster,with or without EGF stimulation.The plate clone formation ability and cisplatin resistance of v III mutant cell lines were also stronger than wild type cell lines.In the UM1,SCC25 wild-type and v III mutant cell lines,the use of ?-catenin inhibitor FH535 treatment found that the proliferation of both cell lines were inhibited,but EGFR v III mutant cell lines resistant to ?-Catenin inhibitor inhibitory effect.This suggests that EGFR v III mutant cell lines have higher beta-catenin activity in head and neck squamous cell carcinoma.Therefore,we believe that EGFR v III may mediate the growth of head and neck squamous cell carcinoma and cisplatin resistance by regulating the ?-catenin pathway.Through the analysis of HNSCC cell line and GEO database analysis,we found that PKM2 and ?-catenin were highly expressed in head and neck squamous cell carcinoma,and they were positively correlated.After EGF stimulation,EGFR was activated to mediate the entry of PKM2.At the same time,we found that EGFR v III could promote the entry of PKM2 and increase the phosphorylation level of ?-catenin compared with wild-type cell lines.It also proved that the activity of ?-catenin in EGFR v III mutant cell line was higher.After the mutation at R399 / 400,PKM2 nucleus was inhibited,leading to decreased activity of ?-catenin pathway and down-regulation of CCND1 gene in ?-catenin downstream gene.We found that PKM2 expression,the head and neck squamous cell carcinoma cell proliferation ability,clonal formation ability and cisplatin resistance significantly increased,and R399 / 400 site mutation,the function was no significant difference with the control group.in conclusion: 1.In head and neck squamous cell carcinoma,EGFR wt / v III mediates cell proliferation with cisplatin resistance.2.In the head and neck squamous cell carcinoma,the activity of ?-catenin pathway in EGFR wt / v III cells was increased.There was a positive correlation between PKM2 and ?-catenin in head and neck squamous cell carcinoma.EGFR wt / v III can promote PKM2 into the nucleus,activation of ?-catenin pathway.PKM2 R399 / 400 is very important for its entry,which affects the activity of ?-catenin pathway and the expression of downstream genes,and mediates the growth and drug resistance of head and neck squamous cell carcinoma.