Inhibition Of FoxO3a On The Tumorigenesis In NSCLC By Down-regulating EPS8 Directly

The treatment of epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs,such as gefitinib or erlotinib)can significantly prolongs the survival time of lung cancer patients with EGFR mutations.Gefitinib is the first EGFR-TKIs that be applied in clinical.In 2003,Gefitinib was approved by FDA as a third line therapy for NSCLC.In 2005,it was approved by China's food and drug administration to be used in treatment of NSCLC patients with locally advanced or metastatic NSCLC who have received chemotherapy before.However,some of patients are primary resistant to Gefitinib.Fox O3 a,a transcription factor of forked head family,can participate in many cellular processes,but its detailed mechanism in acquired EGFR-TKIs resistance and regulation of tumor function remains unclear.Growth factors can regulate a range of cell physiological processes,including proliferation,differentiation and metabolism.The intrinsic catalytic activity of growth factor receptor tyrosine kinases(RTK)is activited by binding its downstream ligands.And then the autophosphorylated RTK will phosphorylate the intracellular substrate and deliver the signal.Thus,identificating proteins that are tyrosyl phosphorylated by RTKs will be the key to elucidate RTKs-mediated signaling pathways.Here,we study on EGFR pathway substrate 8(EPS8)and explore its interaction with Fox O3 a.Furthermore,we discuss its biological functions and its influence on the drug resistance,proliferation and metastasis of NSCLC.Our research provide a new treatment strategy to solve the problem of Gefitinib resistance in clinical and provide experimental data for support.Objectives We systematically investigate the influnce of Fox O3 a and EPS8 in the Gefitinib resistance,proliferation and metastasis of NSCLC through the basic and clinical,in vivo and in vitro experiments by overexpressing and silencing target genes.Here we illustrate the role of Fox O3 a and EPS8 in the inhibition of primary EGFR-TKIs resistance,and discuss the vital function of EPS8 as new anticancer therapeutic target in clinical.Methods 1 The expression of Fox O3 a in NSCLC patients Detect the mutation type of EGFR in 75 NSCLC patients by fluorescence in situ hybridization(FISH)and measure the expression of Fox O3 a in same patients through immumohistochemical staining of their tissue microarray.2 Effects of Fox O3 a on drug resistance of PC9 cells In order to verify the effect of Fox O3 a on EGFR-TKIs resistance in lung adenocarcinoma PC9 cells in vitro,Fox O3 a overexpression and silencing was performed by liposome transfection.Then the sensitivity of PC9 cellline to Gefitinib were detected by MTT assay,plotting the suppression curve and calculating the IC50.3 Exploring the relationship between Fox O3 a and EPS8 in intro In order to study the regulatory mechanism of Fox O3 a,we detect the changes of EPS8,CD31,CD44,Cyclin D1 and Bcl-2,which were closely related to tumor resistance,cycle,apoptosis and metastasis,by Western Blot.Meanwhile,in order to verify the relationship between the Fox O3 a and EPS8,we measure the change of EPS8 m RNA content after overexpressing of Fox O3 a through RT-PCR.We detect the expression of Fox O3 a and EPS8 in PC9 cell and A549 cell by Western Blot.We measure the sensitivity of PC9 cellline to Gefitinib after overexpressing and silencing of EPS8 through MTT assay.5 Effects of Fox O3 a and EPS8 on migration and invasion of PC9 cells Before and after overexpressing and silencing of Fox O3 a and EPS8,the changes of migration and invasion of PC9 cells and A549 cell were detected by Transwell model.6 Effects of Fox O3 a and EPS8 on PC9 cell apoptosis and cell cycle Fox O3 a and EPS8 were overexpressed and silenced respectively by liposome transfection.Followed by Annexin V-7AAD / PE two fluorescent dyes double staining(cell apoptosis)or PI single staining(cell cycle).Flow cytometry was used to detect the changes of early and late apoptosis of PC9 cells before and after two gene changes at 488 nm.7 Effects of Fox O3 a and EPS8 on BALB / c Nude Mice Tumor Growth In order to study the effect of Fox O3 a and EPS8 on the development of tumor in vivo environment,Fox O3 a and EPS8 were over-expressed and silenced respectively.The cell concentration was adjusted to 2×107 / ml,after a week of adaptability,then injected into the six weeks BALB/c female nude mice on the right armpit subcutaneous,each injection of 0.1 ml,6 per group.From the 6th day of injection,the long diameter and short diameter of nude mice were measured with vernier caliper every three days.The calculation formula of nude mice was 1/2(short diameter2×long diameter).Nude mice were sacrificed after 21 days,the tumor was weighed and photographed.8 Clinical validation of Fox O3 a and EPS8 correlations We had explored the biological function of Fox O3 a and EPS8 as well as the possible interaction between them in vitro and animal experiments,respectively.Then we again through immunohistochemical staining of EPS8 protein in blank tissue 4 Effects of EPS8 on drug resistance of PC9 cells microarray of the same 75 patients with lung adenocarcinoma,to explore correlation between EPS8 and Fox O3 a expression and the difference between them in cancer and adjacent tissues.9 To explore the regulation of Fox O3 a to EPS8 in vitro.In order to futher explore the specific regulation mechanism of Fox O3 a to EPS8,we first searched the NCBI website for the 2000 bp length promoter region from EPS8 gene,and then predicted the possible binding sites of Fox O3 a in the EPS8 promoter region by http://jaspar.genereg.net/.The corresponding RT-PCR primers were then designed so that the amplified product contained the predicted binding sequence.Determine whether Fox O3 a plays a transcriptional regulation function by direct binding to the promoter region of EPS8 by chromatin immunoprecipitation method.Results 1 Fox O3 a high expression is associated with sensitivity to EGFR-TKIs The nuclear staining intensity of Fox O3 a was stronger than cytoplasmic staining in NSCLC patients.The high expression ratio of Fox O3 a in the EGFR-TKIs-resistant group(n=42)was 12%,while that was 36% in the EGFR-TKIs-sensitive group(n=33),the difference was statistically significant(P <0.001).ALK is the target of Crizotinib,an ATP-competitive tyrosine kinase inhibitor.There has no statistically significant in ALK-positive mutations and EGFR-TKIs resistance(P=0.138).2 Fox O3 a improves the sensitivity of PC9 cell to Gefitinib Compared with the control group(IC50=68.04±3.25 n M),Fox O3 a overexpression significantly enhanced the concentration-dependent growth inhibition of Gefitinib on PC9 cells(IC50=22.69±1.33 n M)(P<0.05).The concentration of Gefitinib on PC9 cells was significantly attenuated by si RNA silencing(IC50=286.14±11.03 n M)(P<0.01). 3 Fox O3 a regulates expression of EPS8 negatively After Fox O3 a overexpressing and silencing,we examined the changes of CD31,CD44,Cyclin D1,Bcl-2 and EPS8 protein contents which closely related to the development of tumor by Western blot.The results showed that Fox O3 a overexpression significantly inhibited the protein expression of EPS8(P<0.05);EPS8 was significantly increased after Fox O3 a silencing(P<0.01).Through transfecting Fox O3 a overexpressed plasmid,results of RT-PCR show that overexpression of Fox O3 a m RNA significantly inhibited the expression of EPS8 m RNA(P<0.05).4 EPS8 reduces the sensitivity of PC9 cell to Gefitinib Western blot analysis showed that the relative protein content of Fox O3 a was significantly lower in A549 cells than in PC9 cells(P<0.05),while compared with PC9 cell,there was higher protein expression of EPS8 in A549 cells(P<0.05).MTT assay showed that compared with Control group(IC50=68.04±3.25 n M),overexpression of EPS8 significantly inhibited the sensitivity of cells to Gefitinib(IC50=387.33±20.74 n M)(P<0.05);si RNA silencing of EPS8 significantly increased the sensitivity of cells to Gefitinib(IC50=15.93±2.60 n M)(P<0.05).5 Effects of overexpression and silence of Fox O3 a and EPS8 on migration and invasion of PC9 cell In Transwell migration experiment,compared to NC group(264±12 migratory cell),the number of migratory cells in EPS8 overexpression group increased to 391±23,while the number of migratory cells in Fox O3 a overexpression group decreased to 123±8(P<0.05).The number of migratory cells in si-EPS8 group reduced to 69,while the number of migratory cells in si-Fox O3 a group raised to 213(P<0.01).In Transwell invasion experiment,compared to NC group(111±9 migratory cell),the number of migratory cells in EPS8 overexpression group increased to 220±36,while the number of migratory cells in Fox O3 a overexpression group decreased to 79±4(P<0.05).The number of migratory cells in si-EPS8 group reduced to 57±6,while the number of migratory cells in si-Fox O3 a group raised to 152±14(P<0.01). 6 Effects of overexpression and silence of Fox O3 a and EPS8 on cell cycle and apoptosis of PC9 cell Detecting apoptosis by flow cytometry,we found that the rate of early and late apoptosis in Fox O3 a overexpression group and si-EPS8 group are increased to 12% and 22.6%,respectively;while that in si-Fox O3 a group and EPS8 overexpression group are decreased to 6.05% and 4.92%,respectively.Measuring cell cycle by flow cytometry,we found that the PI index in Fox O3 a overexpression group and si-EPS8 group are reduced to 24.76% and 37.56%,respectively;while that in si-Fox O3 a group and EPS8 overexpression group are enhanced to 56.4% and 57.71%,respectively.7 Effects of overexpression and silence of Fox O3 a and EPS8 on tumor growth in BALB/c nude mice Cells in which Fox O3 a and EPS8 were over-expressed and silenced respectively were injected into the six weeks BALB/c female nude mice,detect the volume of tumor at 21 st day.The results show that compared with Control group(380±25mm3),the tumor volume in si-Fox O3 a group and EPS8 overexpression group is significantly increased(541±62 mm3,P<0.05;826±82 mm3,P<0.01);the tumor volume in Fox O3 a overexpression group and si-EPS8 group is significantly decreased(216±18mm3,P<0.05;249±35mm3,P<0.01).Weigh tumors of each mice after stripping,we find that compared with Control group(0.28±0.04g),the tumor weight of si-Fox O3 a group and EPS8 overexpression group is significantly increased(0.37±0.06 g,P<0.01;0.58±0.11 g,P<0.01);the tumor weight of Fox O3 a overexpression group and si-EPS8 group is significantly decreased(0.10±0.02 g,P<0.01;0.18±0.03 g,P<0.01).8 Negative correlation of Fox O3 a and EPS8 in patients with NSCLC The results of immunohistochemistry show that EPS8 is mainly expressed in cytoplasm.There are 8 EPS8 highly expressed patients in EGFR TKIs-mutant group(n=33),which accounts for 24%;while there are 24 EPS8 highly expressed patients in EGFR TKIs-native group(n=42),which accounts for 57%(P<0.01).There are 27 EPS8 highly expressed patients in Fox O3 a low expression group(n=58),which accounts for 47%;while there are 5 EPS8 highly expressed patients in Fox O3 a high expression group(n=17),which accounts for 29%(P<0.01).According to the comparison of expression of Fox O3 a and EPS8 in carcinoma tissue and adjacent tissue of same patient,we found H-scores of Fox O3a(EPS8)in carcinoma tissue and adjacent tissue are 32 and 88,respectively;H-scores of EPS8 in carcinoma tissue and adjacent tissue are 104 and 69,respectively.The differences are statistically significant(P<0.0001).9 Fox O3 a directly regulates expression of EPS8 The fragment of the EPS8 promoter region was found to be the core region by double luciferase reporter gene.Then in the chromatin immunoprecipitation technique(Ch IP),we designed EPS8 amplification primers near the predicted binding sites in this core region.Ch IP found that the enrichment factor of Fox O3 a / Ig G in EPS8 primer group was 8.9 times,the difference was statistically significant.The results show that the transcription factor Fox O3 a directly binds to the CAGTTTAC sequence in the EPS8 core promoter region and plays a negative role in the transcriptional regulation of the latter.Conclusion 1 The expression of Fox O3 a in EGFR-mutant NSCLC patients is high,the expression of EPS8 in EGFR-mutant NSCLC patients is low,which is negatively correlated with the expression of Fox O3 a.2 The expression of Fox O3 a in cancerous tissues was significantly lower than that in adjacent tissues,and the EPS8 in cancer tissues was significantly higher than that in adjacent tissues.3 Fox O3 a enhances the sensitivity of lung adenocarcinoma PC9 cells to Gefitinib;EPS8 reduces the sensitivity of lung adenocarcinoma PC9 cells to Gefitinib.4 Fox O3 a could inhibit PC9 cell migration and invasion,promote cell apoptosis as well as cause cell cycle arrest.However,EPS8 functions were just the opposite.5 High expression of Fox O3 a could significantly inhibit the tumor development;High expression of EPS8 has a significant role in promoting the development of tumors.6 The transcription factor Fox O3 a exerts a negative regulation of the EPS8 gene by directly binding the 5'-GTAAATAT-3 'sequence in EPS8 promoter region.