The Role Of Notch1 And Its Cross-talk With Epidermal Growth Factor Receptor Signalling In Human Tongue Squamous Cell Carcinoma

Notch signalling plays a decisive role in mammalian cell fate determination including cell proliferation,differentiation and apoptosis.Perturbation of Notch signalling can manifest as tissue abnormalities ultimately leading to disease states such as cancer. Accumulating evidence demonstrated that Notch1,the main form of Notch receptors,is deregulated in various malignancies.It can either promote or suppress tumors depending on type and context of cells.It might be a potential therapeutic target in oncological applications.Up to now,there is no report on the tissue-specific expression of Notchl in tongue squamous cell carcinoma(TSCC),the most common oral cancer, and the potential role of Notchl in TSCC remains to be elucidated.Recently,increasing evidence indicated that the interplay between Notchl and epidermal growth factor receptor(EGFR) was involved in carcinogenesis of many cancers where the two pathways can be either antagonistic or synergistic,depending on type and context of cells.EGFR,as a member of erbB family of receptor tyrosine kinases(RTKs) has been extensively investigated.It is overexpressed in a large number of tumors including TSCC.Aberrant activation of EGFR signalling is frequently associated with transformation of primary cells,tumor progression,poor prognosis and decreased survival.EGFR signalling is considered a prime target for development of novel antineoplastic agents.Small molecule inhibitors of RTKs and monoclonal antibodies targeting EGFR signalling have been developed and clinically investigated for treatment of various cancers including head and neck cancers.However,the exact molecular mechanisms underlying sensitivity and resistance of tumor cells to EGFR inhibition remain to be elucidated.Hence,the identification of the potential interplay between EGFR and other signalling in cancers is of considerable significance.To the best of our knowledge,there is no report on the correlation between Notch1 and EGFR signalling in TSCC.This study was to investigate the tissue-specific expression and the role of Notch1 in TSCC,and to explore the potential cross-talk between Notch1 and EGFR signalling in TSCC.The study might provide significant clues to developing new and promising strategies of multitargeted molecular therapy for the treatment of oral cancers.PartⅠExpression of Notch1 and EGFR in human normal tongue mucosa, precancerous lesion and tongue squamous cell carcinomaObjective To investigate the expression of Notch1 and EGFR in human normal tongue mucosa,precancerous lesion and tongue squamous cell carcinoma(TSCC).Methods The expression of Notch1 and EGFR was detected in human normal tongue mucosa(n=15),tongue leukoplakia(LP)(n=59) and TSCC(n=65) by immunohistochemistry.Results(1) In normal tongue mucosa and LP,the positive staining of Notchl was mainly distributed in stratum corneum,partially in stratum granulosum and stratum spinosum,but not in stratum basale.With increase of the degree of epithelial dysplasia, the proportion of positive cells and expression levels of Notch1 gradually decreased(P < 0.05 ).The positive staining of EGFR was mainly distributed in stratum basale,rarely in stratum spinosum,but not in stratum granulosum and stratum corneum.With increase of the degree of epithelial dysplasia,the proportion of positive cells and expression levels of EGFR gradually increased(P < 0.05 ).(2) In TSCC,Notch1 expression was abrogated in peripheral cells of carcinomas except locations of squamous metaplasia of well-differentiated and moderately differentiated specimens where Notchl expression was elevated in stratifying cells.In poorly differentiated specimens,Notchl expression is negative in carcinomas.The higher the degree of differentiation of TSCC was,the higher the proportion of positive cells and expression levels of Notchl was(P<0.05). The higher the stage of TNM of TSCC was,the lower the proportion of positive cells and expression levels of Notchl was(P<0.05).EGFR expression was detected mainly in peripheral cells of carcinomas,but not in locations of squamous metaplasia.The higher the degree of differentiation of TSCC was,the lower the proportion of positive cells and expression levels of EGFR was(P<0.05).The higher the stage of TNM of TSCC was,the higher the proportion of positive cells and expression levels of EGFR was(P<0.05).Conclusions(1) The distribution of positive cells of Notchl was obviously different from that of EGFR in human normal tongue mucosa,precancerous lesion and tongue squamous cell carcinoma.(2) Notchl was down-regulated in the process of development and progression of tongue squamous cell carcinoma,while EGFR was up-regulated. PartⅡEffects of constitutive activation of Notch1 on cell growth and EGFR signalling in human tongue squamous cell carcinoma cells in vitroObjective To investigate effects of constitutive activation of Notchl in human tongue squamous cell carcinoma(TSCC) cells on cell growth and EGFR signalling in vitro.Methods Human TSCC cell line Tca8113 cells were transiently transfected with the eukaryotic expression plasmid pRAMIC-IRES2-EGFP encoding exogenous intracellular fragment of Notchl and control plasmid pIRES2-EGFP by Lipofectamine^TM2000,respectively.Untransfected parental Tca8113 cells served as control.The mRNA of Notchl and EGFR in Tca8113 cells was detected by reverse transcriptase polymerase chain reaction(RT-PCR).The protein levels of Notchl and its effector Hes1,EGFR and its downstream signaling molecule p-ERK,and p53 in Tca8113 cells were detected by Western Blot.The cell proliferation was evaluated by methyl thiazolyl tetrazolium(MTT) assay.The apoptosis was assessed by flow cytometry.The expression of Notch1 and EGFR protein in Tca8113 cells was detected by immunocytochemistry.Results After transfected with pRAMIC-IRES2-EGFP for 48h,when compared with untransfected parental Tca8113 cells,Notchl expression in Tca8113 cells significantly increased at mRNA(3.2-fold)(P<0.05) and protein level(6.1-fold)(P<0.05),and its effector Hes1 expression significantly increased at protein level(6.9-fold)(P<0.05), while EGFR expression significantly decreased at mRNA(12.7-fold)(P<0.05) and protein level(9.3-fold)(P<0.05),and its downstream signaling molecule p-ERK significantly decreased at protein level(16.6-fold)(P<0.05),and p53 significantly increased at protein level(9.7-fold)(P<0.05).The control plasmid pIRES2-EGFP had no effect.β-actin was used as an internal control.MTT assay showed that the cell proliferation of Tca8113 cells transfected with pRAMIC-IRES2-EGFP was significantly inhibited as compared with controls(P<0.05).After transfected with pRAMIC-IRES2-EGFP for 48h,the rate of early apoptosis(%) of Tca8113 cells was significantly higher than that of Tca8113 cells transfected with pIRES2-EGFP and untransfected Tca8113 cells(P<0.05),and immunocytochemistry showed that the expression of Notch1 significantly up-regulated in Tca8113 cells transfected with pRAMIC-IRES2-EGFP,while EGFR expression significantly down-regulated,as compared with controls.Conclusions(1) Constitutive activation of Notch1 up-regulated Notch1 signaling molecules and down-regulated EGFR signaling molecules in TSCC cells in vitro.(2) Constitutive activation of Notch1 inhibited cell proliferation,induced apoptosis and up-regulated p53 protein in TSCC cells in vitro.PartⅢEffects of EGFR gene silencing and blocking EGFR signalling on cell growth and Notchl signalling in human tongue squamous cell carcinoma cells in vitroObjective To investigate effects of EGFR gene silencing and blocking EGFR signalling on cell growth and Notchl signalling in human tongue squamous cell carcinoma(TSCC) cells in vitro.Methods(1) Experiment 1:Human TSCC cell lines Tca8113 cells were transiently transfected with the eukaryotic expression plasmid shEGFR encoding the specific short hairpin RNA(shRNA) targeting EGFR and the control plasmid shNC encoding shRNA targeting unrelated sequence by Lipofectamine^TM2000,respectively.Untransfected parental Tca8113 cells served as control.(2) Experiment 2:Tca8113 cells were treated with AG1478,an inhibitor of receptor tyrosine kinases,at a total concentration of 5 or 10μM,respectively.(3) In Experiment 1 and 2,the mRNA of EGFR and Notchl in Tca8113 cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR).The protein levels of EGFR and its downstream signaling molecule p-ERK, Notch1 and its effector Hes1,and p53 in Tca8113 cells were detected by Western Blot. The expression of Notch1 and EGFR protein in Tca8113 cells was detected by immunocytochemistry.(4) In Experiment 1,the cell proliferation was evaluated by methyl thiazolyl tetrazolium(MTT) assay,and the apoptosis was assessed by flow cytometry,andResults(1) After transfection with shEGFR for 48h,when compared with untransfected parental Tca8113 cells,EGFR expression in Tca8113 cells significantly decreased at mRNA level(13.4-fold)(P<0.05) and protein level(10.0-fold)(P<0.05), and its downstream signaling molecule p-ERK significantly decreased at protein level (10.6-fold)(P<0.05),while Notchl expression in Tca8113 cells significantly increased at mRNA(2.2-fold)(P<0.05) and protein level(7.0-fold)(P<0.05),and its effector Hesl expression significantly increased at protein level(13.9-fold)(P<0.05),and p53 significantly increased at protein level(12.5-fold)(P<0.05).The control vector shNC had no effect.β-actin was used as an internal control.MTT assay showed that the cell proliferation of Tca8113 cells transfected with shEGFR was significantly inhibited as compared with controls(P<0.05).After transfected with shEGFR for 48h,the rate of early apoptosis(%) of Tca8113 cells was significantly higher than that of Tca8113 cells transfected with shNC and untransfected Tca8113 cells(P<0.05),and immunocytochemistry showed that the expression of EGFR protein significantly down-regulated,while Notchl expression significantly up-regulated in Tca8113 cells transfected with shEGFR,as compared with controls.(2) After treatment with AG1478 at a total concentration of 5 or 10μM in Tca8113 cells for 4h,when compared with untreated cells,the expression of mRNA and protein of EGFR had no significant difference,but its downstream signaling molecule p-ERK significantly decreased at protein level(2.0 or 3.5-fold)(P<0.05),while Notchl expression significantly increased at mRNA level(1.8 or 2.7-fold)(P<0.05) and protein level(1.3 or 2.9-fold)(P<0.01), and its effector Hesl expression significantly increased at protein level(4.4 or 14.8-fold) (P<0.05),and p53 significantly increased at protein level(1.8 or 13.2-fold)(P<0.05).β-actin was used as an internal control.Immunocytochemistry showed that the expression of EGFR protein had no obvious difference,while Notchl expression obviously up-regulated in Tca8113 cells treated with AG1478 at a total concentration of 5 or 10μM,as compared with controls.Conclusion(1) EGFR gene silencing mediated by shRNA down-regulated EGFR signaling molecules,up-regulated Notchl signaling molecules,inhibited cell proliferation,induced apoptosis and up-regulated p53 protein in TSCC cells in vitro.(2) Blocking EGFR signalling had no effect on the level of EGFR expression,but down-regulated its downstream signaling molecule p-ERK and up-regulated Notchl signaling molecules and p53 protein in TSCC cells in vitro.