The Underlying Mechanism Study Of Erlotinib Resistance In Oral Tongue Squamous Cell Cancer

Background and purpose:Oral tongue squamous cell carcinoma(OTSCC)is a common malignant tumor,the incidence of disease has been increasing in young adults in recent years,which is a serious threat to human health.Squamous cell carcinoma(SCC)is one of the most common types of tongue cancer with poor prognosis.At present,the treatment of tongue squamous cell carcinoma is still mainly surgical resection of primary lesions and neck lymph node removal,and then combined with chemotherapy before or after surgery.Although the methods for the treatment of OTSCC had made great progress recently,chemotherapy is still an important adjuvant therapeutic choice in clinic for the critical roles in inhibiting the proliferation,invasion and metastasis of cancer cells.Recently,targeting epidermal growth factor receptor(EGFR)has been applied to clinical practice and improved the therapeutic effect of OTSCC.EGFR,a receptor tyrosine kinase,is an important member of ERBB receptor famil,It is expressed at high levels in many human cancers,including oral cancer,lung cancer,breast cancer,but at relative low levels in normal tissue cells.When binding with ligand,EGFR occurs homologous and heterodimerization,activating its downstream signaling pathways including PI3K-AKT and MEK/ERK pathways,promoting cell proliferation,angiogenesis and inhibiting cell apoptosis.The commonly used EGFR inhibitors can be classified as two categories:(1)Antibody such as cetuximab and nimotuzumab and(2)small chemical agents such as erlotinib,gefitinib and icotinib.Antibody drugs,especially Cetuximab,are widely used in the treatment of oral cancer,while small molecule EGFR tyrosine kinase inhibitor(EGFR-TKI)such as erlotinib and gefitinib are seldom used.However,clinical trials revealed that the efficacy of EGFR inhibitors in OTSCC cells was very poor and limited the clinicalapplication.According to the mechanism by which drug resistance occurred,the resistance of cancer cells to chemical agents can be defined as acquired resistance and primary resistance.Acquired resistance is a process in which tumor cells gradually transformed from sensitivity to resistance in the course of drug treatment.At present,acquired resistance is one of the most important reasons for the resistance to chemical agents in human cancers.The reasons for acquired resistance of tumor to EGFR-TKI could be attributed to EGFR mutation(mainly T790M mutation)and overactivation of c-MET and autophagy.It has been well known that c-Met,a receptor tyrosine kinase(RTK),binds to hepatocyte growth factor(HGF)and caused the homo-and heterodimerization of c-MET,which leads to the activation of c-MET through autophosphorylation and activates its downstream signal pathways such as PI3K-AKT,MEK/ERK.Over activation of the c-MET signaling pathway could activate PI3K-AKT,MEK/ERK signaling pathways through bypass activation,leading to the resistance of tumor cells to EGFR inhibitors.Autophagy is an evolutionarily conserved lysosomal degradation pathway that maintains cell survival under stress conditions such as hypoxia,nutrient deprivation and drug treatment and plays important roles in maintaining the homeostasis of cells and tissues.According to the mechanism of autophagy initiation,autophagy can be divided into canonical autophagy and non-canonical autophagy.The initiation of canonical autophagy is dependent on the activation of ULK complex due to nutrient deficiency and growth factor starvation.However,the initiation of non-canonicalautophagy can bypass the ULK complex and Beclinl-hVPS34 complex and activate autophagy directly by activating downstream autophagy related genes.At present autophagy has been considered as one of the most important reasons of tumor cell therapy resistance.People have been trying to treat malignant tumor by inhibiting autophagy.Chloroquine(CQ),hydroxychloroquine(HCQ)has been used in the clinical treatment of cancer.A large number of experiments and clinical evidence have shown that CQ and HCQ can increase the sensitivity of tumor cells to chemotherapeutic drugs by inhibiting autophagy.In this study,we first established erlotinib resistant tongue squamous cell carcinoma cell line by exposing TCA8113 cells to high concentrations of erlotinib for a long time.Then,we explored the role of c-Met in the development of acquired drug resistance to EGFR tyrosine kinase inhibitor in human tongue squamous carcinoma cells.Moreover,we also investigated the possible roles of autophagy in this process.Taken together,these works provide a new insight for the mechanism by which OTSCC developed acquired resistance to erlotinib.Methods:1.We screened and established erlotinib resistant cell line TCA-8113-ER by chronic exposing TCA-8113 cells to increase concentration of erlotinib.Cell survival analysis was used to detect the sensitivity of TCA-8113-ER cells to erlotinib.Transwell assay was used to detect the invasion and metastasis potential of drug resistant cells.Western blot analysis was used to detect the expression and phosphorylation of EGFR and c-MET.2.TCA-8113-ER cells were treated with c-MET kinase inhibitor SU11274 alone or in combination with erlotinib.MTT assay and colony formation assay were used to detect the viability of erlotinib-resistant TCA-8113-ER,Hochest33342 staining and flow cytometry analysis were used to detect the apoptosis of TCA-8113-ER cells.Western blot analysis was used to detect the expression of Caspase-3 and phosphorylation of AKT and ERK.Wound healing test and Transwell assay were used to detect the alterations of cell migration and invasion.3.RNA interference assay was used to silence the expression of c-MET in TCA-8113-ER cells,Western blot was used to detect the expression and phosphorylation of c-MET.Cell viability assay was used to detect the sensitivity of cells to erlotinib,Hochest33342 staining and flow cytometry analysis were used to detect the apoptosis of TCA-8113-ER1 cells.4.The xenograft tumor model was established by subcutaneous injection of TCA-8113-ER cells(107/200μl per animal)into BALB/C Nu mice.When tumor volume reached 100mm3,mice were treated with SU11274 alone or combined with erlotinib.After 14 days,mice were executed painlessly and the tumor weights were measured.5.TCA-8113 and CAL-27 cell lines were transiently transfected with LC3-EGFP.After 24 hours,cell was treated by erlotinib(10μM)for 48 hours and the distribution of LC3-EGFP was observed by inverted fluorescence microscope.The expression of LC3-Ⅱ/Ⅰ,mTOR,p-mTOR,AMPK and p-AMPK was examined by western blot.6.TCA-8113 and CAL-27 cells were treated with HCQ(10μM),3-MA(2.5mM),Rapa(1mM)alone or in combination with erlotinib for 48 hours.MTT assay was used to detect cell viability.Flow cytometry analysis was used to detect apoptosis.7.TCA-8113 and CAL-27 cells were co-transfected with siAtg5 or siBeclin 1 with LC3-EGFP.After 48 hours of transfection,cells were treated with erlotinib for 24 hours and the formation of autophagosomes was observed by inverted fluorescence microscope.Cell survival was detected by MTT assay and the expression of LC3-Ⅱ/Ⅰwas detected by western blot.Results:1.The phosphorylation level of c-MET was significantly increased,by contrast the phosphorylation level of EGFR was obviously decreased in TCA-8113-ER cells as compared with that in corresponding parental TCA-8113 cells.2.Treatment of TCA-8113-ER cells with SU11274 caused a significant growth inhibition in a short term,whereas failed to inhibit the growth of erlotinib resistant cells in a long term.Joint treatment of erlotinib resistant cells with SU11274(5μM)and erlotinib(5μM)significantly inhibited the growth of tumor cells in a both short and long term as compared with treatment with SU11274 alone;Flow cytometry analysis showed that the apoptotic rate was 10.27%in erlotinib alone and 11.8%in SU11274 alone.The joint treatment with SU11274 and erlotinib was 45.51%;Western blot analysis showed that treatment of erlotinib resistant cells with SU11274 in combination with erlotinib significantly promoted the cleavage of Caspase-3 and suppressed the phosphorylation of ERK and AKT;Wound healing assay and transwell assay showed that joint treatment with erlotinib and SU11274 inhibited the migration and invasion of the resistant cells to a greater extent as compared with treatment with SU11274 or erlotinib alone.3.Knockdown of c-MET significantly inhibited the growth of the resistant cells in a short and long term when treated with erlotinib,Short term survival analysis showed that the IC 50 value of erlotinib was about 1μM in c-MET knockdown cells,whereas it was more than 10 uM in control siRNA transfected cells;The long term survival analysis also showed that Knockdown of c-MET significantly inhibited the growth of the resistant cells treated with erlotinib.Flow cytometry analysis showed that treatment of c-MET knockdown cells with erlotinib significantly induced cell apoptosis(as compared with that in control siRNA transfected cells(40.22%vs 7.24%).4.Joint treatment with SU11274 and erlotinib significantly decreased the weight of tumor tissues than that in animals treated with erlotinib and SU11274 alone(combo vs control=0.37 vs 1.97;erlotinib vs control=1.42 vs 1.97;SU11274 vs control=1.38 vs1.97),the statistical analysis showed that the difference was significant(p<0.05).Western blot analysis showed that joint treatment with SU11274 and erlotinib significantly inhibited the phosphorylation of ERK and AKT as compared with that in the xenograft tumors.5.TCA-8113 and CAL-27 cells were transfected with LC3-EGFP to observe the punctated spots of LC3-Ⅱ,The results showed that the number of punctated LC3 more than 5 in erlotinib cells accounted for 60-70%of the total number of cells.while in control cells it only accounted for 30-40%of the total cells.These results suggested that erlotinib treatment promotes the formation of autophagosomes.Western blotting analysis showed significant increase in the LC3-Ⅰ to LC3-Ⅱ conversion(LC3-Ⅱ/Ⅰ ratio increased from 0.98 to 2.36 in CAL-27;1.12 to 2.28 in TCA-8113)and decreased expression of p62.6.TCA-8113 and CAL-27 cells were subjected to HCQ(10μM),3-MA(2.5mM)or Rapa(1mM)treatment in combination with erlotinib or alone.Cell viability analysis showed that incubation of HCQ significantly decreased cell viability upon erlotinib treatment as compared with control cells,while rapamycin treatment increased cell viability when treated with erlotinib.Flow cytometry showed that the apoptotic rate was 58.4%in HCQ and erlotinib combined treatment group and it was 43.5%in 3-MA and erlotinib combined treatment group,while it was 42.1%of erlotinib alone group.These data suggested that autophagy plays a protective role in erlotinib resistance.7.We detected the expression of autophagy related protein through western blot in erlotinib treated TCA-8113 and CAL-27cells.We found that erlotinib did not affect the expression of Beclinl in these two cell lines.However,the expression of ATG5,ATG3,ATG7 and LC3 were all increased after erlotinib treatment.We further transfected siRNA against ATG5 and Beclinl to investigate the role of ATG5 in the acquired resistance to erlotinib,Inverted fluorescence microscopy showed that the number of punctated LC3 spots in the cytosol in cells co-transfected siATG5 and LC3-EGFP were significantly decreased as compared with cells co-transfected control siRNA and LC3-EGFP,while did not affect LC3 activation in cells co-transfected with siBeclinl with LC3-GFP,Cell viability analysis showed that knockdown of ATG5 caused a marked increase in cell viability as compared with control siRNA.However,knockdown of Beclinl did not affect cell viability upon erlotinib treatment,These data suggested that it is ATG5 but not Beclinl plays essential role in the acquired resistance to erlotinib in OTSCC.Conclusions:1.c-MET overactivation plays critical role in the acquired resistance to erlotinib in oral tongue squamous by bypass activation of PI3K-AKT and MEK-ERK signaling pathway.2.Treatment of erlotinib induces autophagy in oral tongue squamous cell carcinoma in a BECLIN dependent manner by elevating the expression of ATG5 and autophagy mediated by ATG5 plays a protective role,maintains cell viability and confers the resistance to erlotinib.