Expression And Molecular Mechanism Of Oct4 In The Resistance Of Non-small Cell Lung Cancer To Gefitinib

BackgroundPrimary lung cancer(hereinafter referred to as lung cancer)ranks first in both incidence and mortality among all malignant tumors in China.Currently the newly confirmed lung cancer patients are about half a million per year,and will reach to 1 million in 2025.In recent 30 years,with the mortality rose 465%,lung cancer severely threatens human health.Non-small cell lung cancer(NSCLC),as the major pathological type of lung cancer,accounts for about 85%-90%in all cases.Compared with the small cell lung cancer,the NSCLC grows slower and migrate later,but there are only 15%-18%confirmed cases can be removed by surgery with a very high rate of recurrence and metastasis after the operation.As the remission rate of two-drug cisplatin-based chemotherapy is only around 30%,the effect of drug chemotherapy in advanced NSCLC patients’ survival is limited.Therefore NSCLC patients face a poor prognosis.Molecular typing methods have greatly improved our understanding of the pathophysiology of NSCLC,NSCLC has ushered in an era of precision medicine,of which the epidermal growth factor receptor(EGFR)has been the most well-known and well-applied targeting gene.Among NSCLC patients,the EGFR mutation mainly takes place in the intracellular coding domain(exon 18-21),including the deletion mutation of exon 19(delE746-A750)and the point mutation of exon 21(L858R),both of which accout for more than 90%of all the mutation of EGFR and are related to the sensitivity to epithelial growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI).Thus these mutations are called sensitizing mutations,which are found among 50%of Asian NSCLC patients.The EGFR-TKI propell the NSCLC therapy from standard two drug platinum-based chemotherapy into molecularly targeted therapy,as the first generation of EGFR-TKI,such as gefitinib and erlotinib have become the first-line treatment for patients who were positive for the EGFR sensitizing mutations.Such large,randomized clinical trials have proven that patients bearing EGFR sensitizing mutations have shown favourable clinical outcomes with EGFR-TKI treatment,whereas most patients will develop drug resistance to gefitinib after 8-16 months,and thus researches have been focused on the mechanism of EGFR-TKI resistance.The molecular mechanism of EGFR-TKI resistance in NSCLC includes T790M mutation in EGFR20 exon,MET gene amplification,epithelial-mesenchymal transition(EMT),transition to small cell lung cancer,and bypass activation of EGFR pathway,among which the T790M mutation in EGFR20 exon is the most common one.Another mechanism of lung cancer drug resistance is to obtain cancer stem cells(CSC)phenotype,which is mediated by the expression of pluripotent transcription factors(such as Oct4,Sox2,and Nanog).CSC is a small group of cancer cells that possess characteristics associated with normal stem cells,specifically the capacity of self-renewal,differentiation into multiple cancer cell lineages,and extensive proliferation.CSC is considered as the source of tumor cloning,a group of cells that drives the tumor to grow and develop.It is the root of the genesis,development,invasion and metastasis,drugs resistance and recurrence of the tumor.Many researches have proven the existence of CSC with high tumorigenicity,pluripotency,self-renewability,and high drug resistance in lung cancer.Octamer-binding transcription factor 4(Oct4)is one of the most important transcription factors,which plays critical role in maintaining pluripotency,as well as the marker for the embryonic stem cells.Oct4 is over expressed in many solid tumors and involved in the procedure of genesis,development,recurrence,and metastasis.Patients with Oct4 overexpression are associated with a faster growth,easier metastasis,and poorer prognosis.By using human non-small cell lung adenocarcinoma cell line and primary tumor cells generated by patients,it is reported that CSC expresses many pluripotent stem cell genes,especially Oct4,despite the different phenotypes.Relating to the multiple drugs resistance(MDR)phenotypes of CSC,Oct4 may play an important role in the development of chemotherapy resistance of malignant tumors,so Oct4 becomes a critical target in cancer therapy,especially to those tumors with chemotherapy resistance.However,studies on the role of Oct4 in drug-resistant cancers are limited.Induction of mutant EGFR in transgenic CCSP-rtTA/TetO-EGFR(L858R/T790M)mice correlated with increased Oct4 and Sox2 expression in the lung tissue of EGFR-TKI resistant tumors.However,the molecular mechanism of regulating CSC in gefitinib resistance remains unclear.The signal transducer and activator of transcription 3(STAT3)is a member of the highly conserved transcription factors family.It is involved not only in the regulation of cell cycle and apoptosis,but also in multiple procedures such as the tumor angiogenesis,tumor cell invasion/metastasis,and the immune escape.Meanwhile,it is important in maintaining the pluripotency of CSC by regulating the expression of the relative stem cell transcription factors.Survivin is an important member of the family of apoptotic proteins,and the survivin gene promoter contains STAT3 binding sites.The pathway of STAT3 is involved in lung cancer genesis;the activation of STAT3 is a mechanism of resistance to EGFR-TKI drug in NSCLC;there’s no research about whether Oct4 is involved in STAT3 signal pathway.The study of Oct4 expression as well as the role of Oct4 in the maintenance of gefitinib resistance could better explain the genesis and development of the lung cancer;a better understanding of the interactive mechanisms underlying Oct4 and EGFR-TKI resistance may help us to improve the medical efficiency and persistence of EGFR-TKI to the patients with EGFR mutations.ObjectivesTo study the expression of Oct4 mRNA and protein in the tumor tissue of NSCLC patients,and its correlation with the clinical pathological features of the patients;to establish the PC-9 and PC-9/GR cell lines with silencing Oct4 gene expression by using Oct4-siRNA transfection;to study the effect of Oct4 on gefitinib acquired resistance in PC-9/GR cell lines and its possible signal pathways.Methods(1)Expression and significance of Oct-4 in NSCLC86 samples of tumor tissues(as NSCLC group)and the corresponding adjacent normal tissues(as control group)from NSCLC patients were selected.The expression of Oct4 mRNA and protein in the tissue samples were detected by fluorescence quantitative PCR,immunohistochemistry and western blot respectively,and the correlations between the expression and clinical pathological features of the patients,including age,gender,smoking index,tumor size,differentiation degree,TNM stage,and pathologic type,were analyzed.(2)The role of Oct4 in the proliferation of NSCLC and its resistance to gefitinibHuman lung cancer cell line with EGFR exon 19 deletion mutation(PC-9)and gefitinib acquired resistance cell line(PC-9/GR)were selected as study objects;the expressions of Oct4 mRNA and protein of PC-9 cells and PC-9/GR cells were detected by fluorescence quantitative PCR and western blot respectively.Oct4-siRNA transfection was used to establish the PC-9 and PC-9/GR cell lines with specific silencing Oct4 gene expression,and 4 groups were designed as following:Con-siRNA PC-9 group(transfected with Con-siRNA),Oct4-siRNA PC-9 group(transfected with Oct4-siRNA),Con-siRNA PC-9/GR group(transfected with Con-siRNA),and Oct4-siRNA PC-9/GR group(transfected with Oct4-siRNA).Western blot was used to detect the expression of Oct4 protein in PC-9 cells and PC-9/GR cells after silencing Oct4 gene expression;MTT assay was used to detect the proliferative activities of PC-9 and PC-9/GR cells at 24h,48h,and 72h after the transfection.And then the PC-9 and PC-9/GR cells were both divided into 4 groups:Con-siRNA group(tranfected with Con-siRNA),Con-siRNA + gefitnib group(2.5 pM of gefitinib + transfected with Con-siRNA),Oct4-siRNA group(transfected with Oct4-siRNA),Oct4-siRNA+ gefitnib group(2.5 μM of gefitinib+ transfected with Oct4-siRNA).The apoptosis rate of each group was detected by the flow cytometry.(3)Mechanism of Oct4 in the resistance of NSCLC to gefitinibThe expression of p-STAT3 and survivin protein were detected by western blot in PC-9 cells and PC-9/GR cells;the expression of Oct4 and p-STAT3 protein in PC-9 cells and PC-9/GR cells were detected by immunofluorescence.Oct4-siRNA transfection was used to establish the PC-9 and PC-9/GR cell lines with specific silencing Oct4 gene expression,and 4 groups were designed as following:Con-siRNA PC-9 group(transfected with Con-siRNA),Oct4-siRNA PC-9 group(transfected with Oct4-siRNA),Con-siRNA PC-9/GR group(transfected with Con-siRNA),and Oct4-siRNA PC-9/GR group(transfected with Oct4-siRNA).Fluorescence quantitative PCR and western blot were used to detect the expression of Oct4,p-STAT3 and survivin mRNA and protein respectively in PC-9 cells and PC-9/GR cells after silencing Oct4 gene expression.Results(1)Expression and significance of Oct-4 in NSCLCThe expression of Oct4 mRNA in NSCLC group was significantly higher than that in control group by fluorescence quantitative PCR(t = 3.566,P =0.008).The expression of Oct4 protein in NSCLC group was significantly higher than that in control group by western blot(t = 3.764,P = 0.007).The positive sites of Oct4 staining by immunohistochemistry were brown granules;compared with that in control group,the positive expression of Oct4 protein in NSCLC group was significantly higher(t = 8.033,P<0.001).The expression of Oct4 had no significantly difference by genders,ages,smoking index,and tumor size,(P>0.05);however,the expression of Oct4 was significantly higher among the patients with lung adenocarcinoma,poorly differentiated tumors,and TNM stage Ⅲ compared with the ones with big cell lung cancer/squamous-cell carcinoma,middle-highly differentiated tumors,and TNM stageⅠ-Ⅱ(P<0.05).(2)The role of Oct4 in the proliferation of NSCLC and its resistance to gefitinibCompared with those in PC-9 cells,the expression of Oct4 mRNA(t =4.21,P = 0.006)and Oct4 protein(t = 4.233,P = 0.005)were significantly increased in PC-9/GR cells.In PC-9 cells,the expression of Oct4 protein in Oct4-siRNA group was significantly lower than that in Con-siRNA group(t =3.985,P = 0.009).In PC-9/GR cells,the expression of Oct4 protein in Oct4-siRNA group was significantly lower than that in Con-siRNA group(t =4.117,P = 0.007).The proliferative activities of PC-9 and PC-9/GR cells in Oct4-siRNA groups were significantly lower than those in Con-siRNA groups at 24h,48h and 72 h after the transfection(P<0.05).Compared with the Con-siRNA group,the apoptosis rate of Con-siRNA + gefitnib group cells was significantly higher in PC-9 cells(X 2 = 8.644,P = 0.003);Compared with that of Oct4-siRNA group,the apoptosis rate of Oct4-siRNA + gefitnib group cells was significantly increased in PC-9 cells(X 2 = 9.019,P = 0.002).In the PC-9/GR cells,the apoptosis rate of Con-siRNA + gefitnib group cells was not statistically different compared with that of Con-siRNA group(X 2 = 1.413,P =0.872);compared with that of the Oct4-siRNA group,The apoptosis rate of Oct4-siRNA + gefitnib group cells was significantly increased(X 2 = 8.798,P =0.004).(3)Mechanism of OCT4 in the resistance of NSCLC to gefitinibCompared with that in PC-9 cells,the expression of p-STAT3 protein in PC-9/GR cells was significantly increased(t = 4.197,P-0.004).Compared with that in PC-9 cells,the expression of Survivin protein in PC-9/GR cells was significantly increased(t = 3.639,P = 0.008).The results of immunofluorescence showed that Oct4 and p-STAT3 protein were expressed in the nucleus of both PC-9 and PC-9/GR cells;and compared with those in PC-9 cells,the expressions of the two factors in PC-9/GR cells were significantly increased.In PC-9 cells,the expression of Oct4,p-STAT3 and survivin mRNA in Oct4-siRNA group was significantly lower than that in Con-siRNA group(t = 2.934,3.024,3.216,respectively,P =0.037,0.031,0.025,respectively);In PC-9/GR cells,the expression of Oct4,p-STAT3 and survivin mRNA in Oct4-siRNA group was significantly lower than that in Con-siRNA group(t = 3.108,2.014,2.572,respectively,P =0.009,0.028,0.017,respectively).In PC-9 cells,the expression of Oct4,p-STAT3 and survivin protein in Oct4-siRNA group was significantly lower than that in Con-siRNA group(t = 2.889,2.985,2.917,respectively,P = 0.018,0.007,0.013,respectively);In PC-9/GR cells,the expression of Oct4,p-STAT3 and survivin protein in Oct4-siRNA group was significantly lower than that in Con-siRNA group(t = 3.737,3.756,3.011,respectively,P = 0.002,0.002,0.007,respectively).Conclusions(1)The expression of Oct4 in NSCLC increased;Oct4 overexpression was associated with poorly differentiated NSCLC,high TNM stage and pathologic type,and Oct4 overexpression could be used as a predictor of poor prognosis of NSCLC.(2)Oct4 plays a crucial role in the maintenance of gefitinib-resistant NSCLC.Specific silencing Oct4 gene expression can reverse NSCLC acquired resistance to gefitinib and promote apoptosis.(3)The p-STAT3/survivin signaling pathway may be related to NSCLC acquired resistance to gefitinib.Down-regulation of Oct4 can reverse NSCLC acquired resistance to gefitinib,and its mechanism may be related to p-STAT3/survivin signaling pathway.InnovationIn this study,we concluded that OCT4 plays a crucial role in the maintenance of gefitinib resistance of lung adenocarcinoma cell PC-9/GR;Silence of OCT4 overexpression could reverse the gefitinib resistance of PC-9/GR cells;OCT4 might regulate lung cancer resistance to gefitinib through STAT3/survivin signal pathway,which is proposed for the first time.By further explore mechanisms which mediate gefitinib resistance,we should find new ways to targeted therapy for NSCLC cancer.