The Role Of Filamin A On The Resistance To EGFR-TKIs In Non-small Cell Lung Cancer

Objective: Lung cancer is the malignant tumor with the highestmorbidity and mortality in the world. Approximately80%of lung cancers arenon-small cell lung cancer （NSCLC）. Although the five-survival rate isreportedly80%after surgical removal at early stage, but the majority ofpatients are diagnosed with advanced cancer and lost the chance of surgery.Despite the significant progress has been made in chemotherapy strategy inthe past30years, however, the prognosis for patients with advanced NSCLCremains poor. So to develop novel and more effective treatments for advancedNSCLC is one of the most important topics in the field of oncology. In thepast decade, the use of epidermal growth factor receptor-tyrosin kinaseinhibitors （EGFR-TKIs） such as Gefitinib, Erlotinib have achieved significantclinical benefit for NSCLC patients harboring activating EGFR mutations.Unfortunately, it was also reported that～20%of patients with an EGFRmutation do not respond to gefitinib. Furthermore, the majority of patientsdevelop resistance to gefitinib within a few years. Therefore, EGFR mutationcan not be the only criteria for the selection of treatment of EGFR-TKIs inNSCLC. In addition to EGFR mutation, it is possible that there are othermolecular mechanisms involved in the efficacy of EGFR-TKIs.Filamin A（FLNa）, also called actin-binding protein280（ABP-280） orFilamin-1, which organizes filamentous actin into orthogonal networks andstress fibers through its N-terminal actin-binding domain. FLNa also bindsvarious transmembrane proteins and provides a scaffold for a wide range ofcytoplasmic and nuclear signaling proteins which involved in the process ofcell’s proliferation, adhesion, invasion, migration and cancer progression.In order to clarify whether FLNa expression influences the sensitivity ofEGFR-TKIs in NSCLC, the following experiments were performed:（1） Western blot were employed to detect the protein levels of FLNa in differenttypes of NSCLC cell lines. Growth inhibition of NSCLC lines by Gefitinibwere examined by MTT.（2）The recombinant plasmid which express FLNasiRNA were constructed and transfected into NSCLC cells which originallyhigh express FLNa. Stable cell line with knockdown of FLNa was established.Simultaneously, we transfected the plasmid contains FLNa gene into NSCLCcells which originally low express FLNa and established the stable cell linewith over-expression of FLNa.（3）The biological behavior includingproliferation, migration and invasion were observed by MTT, wound healingassay and transwell cell invasion assay in the stable cell lines with knockdownor over-expression of FLNa after exposure to Gefitinib. The results of thisstudy will provide new target for overcoming resistance to EGFR-TKIs.Methods:1The protein levels of FLNa in different NSCLC cell linesHuman NSCLC GLC-82, H1975, A549and PC-9cells were cultured invitro, the cells were collected, then proteins were extracted from cells, and theprotein levels of FLNa were detected by Western blot.2Growth inhibition of NSCLC lines by GefitinibGLC-82, H1975, A549and PC-9cells were seeded in96-well plates inmedium containing10%FCS, respectively. We replaced the medium withRPMI1640containing different concentrations of Gefitinib next day,incubated cells for72h, and then MTT reagent were added, and they weresubsequently incubated for4hours. Absorbance at490nm was measuredusing a microplate reader. Growth inhibition rate and the the drugconcentration producing50%cell growth inhibition （IC50） were calculated.3Construction and identification of FLNa siRNA recombinant plasmidAccording to coding region sequence of human FLNa gene fromGeneBank （No. NM₀01456.3）, we designed specific sequence of smallinterfering RNA （siRNA） for FLNa and synthesised the shRNA templateoligonucleotides. After annealing, the shRNA template oligonucleotides wereinserted into the linearized shRNA expression vector to construct FLNa siRNA recombinant plasmid. The plasmid clones for the presence of shRNAinserts were screened by PCR, and comfirmed by DNA sequencing.4Screening of stable transfected cell linesThe expression plasmid of FLNa siRNA and plasmid containing FLNagene vector were transfected into H1975and PC-9cells, respectively. Aftertransfection for48h, puromycin and hygromycin were used to screentransfected cells for three weeks. The levels of FLNa mRNA and protein instably transfected cells were examined by real-time quantitative RT-PCR andWestern blot.5Growth inhibition of stably transfected cell lines by GefitinibCells with persistently knockdown of FLNa （H1975/FLNa siRNA） oroverexpress of FLNa （PC-9/FLNa） and their Control group （H1975/Control）or （PC-9/pREP4） were seeded in96-well plates, respectively. We replaced themedium with RPMI1640medium containing different concentrations ofGefitinib next day, incubated cells for72h, and then3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） reagent were added, and theywere subsequently incubated for4hours. Absorbance at450nm wasmeasured using a microplate reader. Growth inhibition rate and the IC50ofGefitinib were calculated.6The migration ability of cells were examined by wound healing assayOnce confluent, stably transfected cell lines H1975and PC-9werescratched with10μl pipette tips, the floating cells were washed away with PBS,then treated with Gefitinib. Images of the same field were taken every6h untilclosure of the scratch. The migration rate of cells were calculated by thefollowing formula: Cell migration rate=（initial width of the scratch-currentwidth）/2/initial width of the scratch×100%.7The invasion ability of cells were examined by transwell cell invasionassayThe invasion assays were carried out using Transwell chamber with10mm diameter and8m pore size polycarbonate membrane coated withMatrigel. Stably transfected cell lines H1975and PC-9cells were added into upper chamber of transwell contained serum-free medium with Gefitinib.After incubation for24h, the invaded cells were fixed with95%ice-coldethanol and then stained with hematoxylin and eosin （HE）. The invaded cellswere counted under the microscope. Five fields were counted for each assay.8The level of EGFR phosphorylation in H1975stable cell lineOnce confluent, the H1975stable cell line were incubated in the mediumcontaining Gefitinib for48hours, then total proteins were extracted from thecells. The levels of p-EGFR, EGFR, FLNa were detected by Western blot.β-actin was as a loading control.Results:1The protein levels of FLNa in different NSCLC cell linesThe results display that FLNa protein level of PC-9cell （0.79±0.02） issignificantly lower than that in GLC-82, H1975and A549cells （1.40±0.02,1.30±0.02,1.47±0.03）（P<0.01）.2Growth inhibition of NSCLC lines by GefitinibMTT assay shows that IC50values of GLC-82, H1975, A549cell（42.20±3.22，21.87±0.84，37.62±2.56） are distinctly higher than PC-9cell（0.05±0.01）（P<0.01）.3The correlation between the expression level of FLNa and sensitivity ofGefitinib in NSCLC cellsThe relevant analysis demonstrates a negative correlation between theexpression level of FLNa and sensitivity of Gefitinib in NSCLC cells. r=ï¹£0.95，r²=0.91, P<0.05.4Construction and identification of FLNa siRNA recombinant plasmidPCR results show that the FLNashRNA template oligonucleotides wereinserted into the shRNA expression vector correctly. DNA sequencingconfirms that the recombinant plasmid sequence is completely consistent withthe designed sequence.5The FLNa expression of stably transfected cell lines H1975and PC-9Real time quantitative RT-PCR detection shows that the level of FLNamRNA expression in H1975/FLNa siRNA cell group （0.44±0.01） is lower than in H1975/Control group （1.00±0.00）（P<0.01）. The level of FLNamRNA expression in PC-9/FLNa cell group （3.93±0.81） is higher than inPC-9/pREP4group（1.00±0.00）（P<0.01）.Western blot detection shows that the level of FLNa protein expressionin H1975/FLNa siRNA group cells （0.53±0.01） is lower than that inH1975/Control group cells （0.91±0.01）（P<0.05）. The level of FLNa proteinexpression in PC-9/FLNa group cells （0.67±0.01） is significantly higher thanin PC-9/pREP4group cells （0.40±0.01）（P<0.05）.6The sensitivity of Gefitinib in the stably transfected cell linesMTT test results showed that the cell growth inhibition rates of H1975/FLNa siRNA group cells at different concentrations of Gefitinib were clearlyhigher than those of H1975/Control group cells （P<0.05）. The IC50value ofH1975/FLNa siRNA group cells （13.44±0.99） is lower than that of H1975/Control group cells （20.43±2.47）（P<0.05）. The cell growth inhibition rates ofPC-9/FLNa group cells at different concentrations of Gefitinib were clearlylower than those of PC-9/pREP4group cells （P<0.05）. The IC50value ofPC-9/FLNa group cells （2.12±0.07） is clearly higher than that of PC-9/pREP4group cells （0.06±0.04）（P<0.01）.7Migration abilities of stably transfected cell linesThe wound healing assay showed that: migration rates of H1975/FLNasiRNA group cells in12h,24h and36h （5.02±0.61%，21.2±0.10%，7.07±0.10%, respectively） were significantly lower than that of H1975/Controlgroup cells （26.92±1.26%，41.81±1.50%，50.00±0.00%, respectively）（P<0.01）. Migration rates of PC-9/FLNa group cells in24h,48h and72h（13.06±0.69%，26.01±3.10%，50.00±0.00%, respectively） were significantlyhigher than that in PC-9/pREP4group cells （26.92±1.26%，41.81±1.50%，50.00±0.00%, respectively）（P<0.01）.8Invasion abilities of stably transfected cell linesTranswell cell invasion assay results: the numbers of cell permeating se-ptum in H1975/FLNa siRNA group cells （19.25±3.10） was significantly less than that in H1975/Control group cells （66.50±6.19）（P<0.01）. The numbersof cell permeating septum in PC-9/FLNa group cells （71.75±5.56） issignificantly more than that in PC-9/pREP4group cells （21.00±2.16）（P<0.01）.9The level of EGFR phosphorylation in H1975stable cell lineWestern blot detection shows that the level of p-EGFR expression inH1975/FLNa siRNA group cells is clearly less than that in H1975/Controlgroup cells.Conclusion:1The expression levels of FLNa are negatively correlated with thesensitivity of Gefitinib in NSCLC cells.2FLNa siRNA recombinant plasmid was successfully constructed andcan effectively inhibit the expression of FLNa mRNA and protein inNSCLC cells.3Knockdown of FLNa expression resulted in the enhancement insensitivity to Gefitinib in NSCLC cell lines; Increase of FLNa expression canreduce the sensitivity to Gefitinib in NSCLC cell lines.4Knockdown of FLNa expression can strengthen the inhibitory effect ofGefitinib for the proliferation, migration and invasion ability of NSCLC cell;Increase of FLNa expression can decrease the inhibitory effect of Gefitinib forthe proliferation, migration and invasion ability of NSCLC cell.5Knockdown of FLNa expression can enhance the inhibitory effect ofGefitinib on EGFR phosphorylation in NSCLC cell lines.