| Objective:Lung cancer remains the leading cause of cancer-related mortality in the world. According to the online database GLOBOCAN 2012, an estimated 14.1million new cancer cases and 8.2 million cancer-related deaths occurred in2012. More than half of all cancers(56.8%) and cancer deaths(64.9%) in2012 occurred in less developed regions of the world. Non-small-cell lung cancer(NSCLC) account for 85% of all lung cancers. Although the treatments have been improved in the past 20 years, the prognosis for patients with advanced NSCLC remains poor. The recommended first-line therapy of a platinum-based doublet for advanced NSCLC has yielded response rate of only 20% and median overall survival(OS) of 8 ~ 10 months. Addition of bevacizumab to a platinum-based doublet chemotherapy increases the median OS to over 12 months slightly. The second-line chemotherapeutic agents of docetaxel and pemetrexed have yielded response rates of only 8%~9% with progression-free survival of less than 3~5 months. Gefitinib and erlotinib are reversible small molecule ATP analogues originally designed to inhibit the tyrosine kinase(TK) activity of wild-type epidermal growth factor receptor(EGFR). Paez and Lynch’s research indicated that tumor cells with EGFR activating mutations, such as exon 19 deletions and exon 21 point mutations,are sensitive to the epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKIs).Then EGFR mutation becomes an important indicator to chose lung cancer patients or predict the effectiveness of EGFR TKIs. These EGFR activating mutations were observed in approximately 10 ~ 15% of NSCLC patients in Western population and 40% of NSCLC patients in Asian population respectively.Unfortunately, although patients with EGFR activating mutations initiallyshowed good responses to first generation EGFR-TKIs, most patients eventually developed disease progression after about 9~14 months. Preclinical modeling and analysis of tumor tissue obtained from patients after the development of disease progression has led to the identification of a number of mechanisms that mediate EGFR TKI resistance. Such genetic and other signaling aberrations that drive resistance mechanisms include HER2 amplification, MET amplification, PIK3 CA mutation, BRAF mutation, NF1 loss and potentially FGFR signaling. In addition, resistant tumors have also been reported to show histologic changes such as small cell lung cancer(SCLC) transformation or epithelial mesenchymal transition(EMT). However,it is now well established that acquisition of a second mutation in EGFR,resulting in substitution of threonine at the “gatekeeper” amino acid 790 to methionine(T790M) is the most common resistance mechanism and is detected in tumor cells from more than 50% of patients after disease progression. The T790 M mutation is believed to render the receptor refractory to inhibition by these reversible EGFR TKIs through exerting effects on both steric hindrance and increased ATP affinity.Filamin-A(FLNA), also known as human actin-binding protein 280(ABP-280) or Filamin-1, is encoded by the X-linked gene FLNA. Human filamin-A is a homodimer with large subunits of 280 KD, forming a V-shaped structure. At the N-terminal of the monomer, there is an actin-binding domain(ABD), followed by 24 tandem repeats of ~96 amino acids in length. Between repeats 15 and 16, there is a hinge domain, and repeat 24 is separated from repeat 23 by a second hinge domain. The last 65 amino acids of repeat 24 mediates the dimerization of filamin-A subunits. Filamin-A binds and cross-links cortical actin filaments into a dynamic three-dimensional structure through its N-terminal actin-binding domain. In addition to filamentous actin,Filamin-A interacts with more than 60 functionally diverse cellular proteins,including transmembrane receptors, signaling molecules, and DNA damage repair proteins. These diverse interactions suggest that Filamin-A is a key component of a versatile signaling. It plays an important role in the process oftumor occurrence and development, especially in the metastatic tumor and the sensitivity of DNA damage, can be used for the treatment of tumor and prognosis evaluation.In order to test whether FLNa expression influences the sensitivity to EGFR-TKIs in NSCLC, this study will be carried out from the cellular and molecular level:(1) To screen out lung cancer cell lines which express high level of FLNa,Western blot were applied to detect the protein levels of FLNa in NSCLC cell lines,such as PC-9 and H1975.(2) Building up p SIF1-FLNa sh RNA plasmids and establishing stable cell line with knockdown of FLNa though cell transfection technique.(3) MTS, wound healing assay and transwell cell invasion assay are used to observe the biological behavior change, such as proliferation, migration and invasion, in the stable cell lines after exposure to gefitinib.(4) Western blot was used to detect the level of phosphorylation of EGFR, ERK and AKT signal molecule in the stable cell lines. The results of this Research will provide biomarkers for clinical choice and new target for overcoming resistance to EGFR-TKIs.Methods:1 Establishment of stably transfected cell linesPC-9 cells were stably transfected with control sh RNA(ctrl) or FLNa sh RNA(KD) plasmid and selected by puromycin, resulting in PC-9/FLNa(KD)and PC-9/ctrl cell lines. The knockdown efficiency of Flamin A was measuted by Western blot.2 Proliferation assayPC-9/FLNa(KD) and PC-9/ctrl cells were seeded in 96-well plates,respectively. Cells were treated with different concentrations of gefitinib(0,0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64μmol/L) and continue to culture for 48 h,MTS assay was used to measure the OD values and to calculate the growth inhibition rate and the half inhibitory concentration(IC50) of gefitinib.3 Migration assayWound healing assay was used to examine the migration abilities of stably transfected cell lines affected by gefitinib. PC-9/FLNa(KD) andPC-9/ctrl cells were seeded in 96-well plates, respectively. Cells were scratched with 10ml pipette tips and washed with PBS, then treated with or without gefitinib. Images of cells at the same field were taken every 6h until the closure of the scratch. The migration rate of cells were calculated by the following formula: Cell migration rate =(initial width of the scratch- current width) /2/ initial width of the scratch×100%).4 Invasion assayThe invasion assays were carried out using Transwell chamber with 10 mm diameter and 8 m m pore size polycarbonate membrane coated with Matrigel. After starved with serum-free 1640 medium for 4h, PC-9/FLNa(KD)and PC-9/ctrl cells were seeded into upper chamber with serum-free 1640 medium with or without gefitinib, respectively. Lower chamber were added into 10% serum 1640 medium. After incubated for 24 h, the invaded cells were fixed with 95% ice-cold ethanol and then stained with hematoxylin and eosin(HE).5 Western blottingPC-9/FLNa(KD) and PC-9/ctrl cells were seeded into 35 mm dishes.After the treatment of gefitinib(0.01μmol/L) for 0, 2, 4 and 8h, total proteins were extracted from cells. Western blot was used to examine the levels of Filamin A, p-EGFR, EGFR, p-ERK, and ERK. b-actin was used as a loading control.Results:1 Detecting the expression of Flamin A in stably transfected cell linesThe relative levels of FLNa protein in PC-9/FLNa(KD) cells(0.41±0.01) were significantly lower than in PC-9/ctrl group cells(0.77 ± 0.01)(P<0.01).2 The sensitivity to gefitinib in stably transfected cell lines2.1 The IC50 value of gefitinib in stably transfected cell linesThe cell growth inhibition rates of PC-9/FLNa(KD)cells at different concentrations(0, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64μmol/L) of gefitinib were markedly lower than those of PC-9/ctrl cells(P<0.05, respectively). Thebehaviour of PC-9 cells through regulating EGFR activation(phosphorylation)and its downstream MAPK/ERK signal pathways.2 Filamin A can increase the inhibitory effects of gefitinib on the proliferation, migration and invasion potential in PC-9 cells via increasing the inhibition of gefitinib on the EGFR activation(phosphorylation) and its downstream MAPK/ERK signal pathways.IC50 value of PC-9/FLNa(KD) cells(0.17±0.01) were significantly higher than that of PC-9/ctrl cells(0.08±0.00)(P<0.01).2.2 Migration abilities of stably transfected cell lines treated with or without gefitinibMigration rates of PC-9/FLNa(KD) cells without gefitinib for 6h, 12 h and 18h(17.45±0.13%, 33.13±0.36%, 50.00±0.00%) were significantly higher than that of PC-9/ctrl(9.75±0.14%, 19.63±0.20%, 35.62±0.37%)(P<0.01,respectively). Migration rates of PC-9/FLNa(KD) cells with gefitinib(0.08μmol/L) exposure for 6h, 12 h and 18h(11.00±0.25%, 27.64±0.47%,38.81±0.07%) were significantly higher than that in PC-9/ctrl cells(6.89±0.17,13.97±0.18%, 21.30±0.22%)(P<0.01, respectively).2.3 Invasion abilities of stably transfected cell lines treated with or without gefitinibThe average invasion cell numbers of PC-9/FLNa(KD) cells without gefitinib(147.00±0.58) were significantly more than that in PC-9/ctrl cells(82.00±1.15(P<0.01). The average invasion cell numbers of PC-9/FLNa( KD) group with gefitinib(0.08μmol/L) exposure(64.00±0.58) were significantly more than that in PC-9/ctrl group(7.00±1.15)(P<0.01).3 Levels of EGFR phosphorylation and the downstream signal molecules in stably transfected cell lines treated with gefitinibThe levels of different proteins in stably transfected cells were detected by Western blot. After the treatment of gefitinib(0.01μmol/L) for 0, 2, 4and8 h,, the levels of p-EGFR in PC-9/FLNa(KD) group(0.94±0.00, 0.71±0.00,0.82±0.00, 0.31±0.00)were significantly higher as compared with PC-9/ctrl group(0.23±0.00, 0.21±0.00, 0.19±0.00, 0.22±0.00)(P<0.01, respectively).The levels of p-ERK in PC-9/FLNa(KD) group(0.98±0.00, 0.36±0.00,0.35±0.00, 0.46±0.00) were significantly higher as compared with PC-9/ctrl group(0.74±0.00, 0.23±0.00, 0.06±0.00, 0.16±0.00)(P<0.01, respectively).Conclusion:1 Knockdown the expression of Filamin A can promote the proliferation,migration and invasion abilities in PC-9 cells. Flamin A may affect biological... |
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