The Effect Of Caveolin-1on The Sensitivity To TKIs In Non-small Cell Lung Cancer With EGFR Mutations

Objective: Lung cancer is a malignant carcinoma with most morbidity andmortality worldwide.1.2million new cases has been diagnosis annually.Among them, non-small cell lung cancer (NSCLC) accounts for80%to85%.Most patients were in advanced stage at the time of diagnosis. Platinum-baseddoublet chemotherapy is a conventional therapy for NSCLC patients withadvanced stage, but the overall survival and the quality of life in patients werenot improved ideally. In recent years, with the development of molecularbiology, molecule-targeted drugs which demonstrated superior efficacy andlower toxicity were widely used in the treatment of advanced NSCLC.Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor(RTK), belongs to ErbB (HER) family. ErbB family comprise of fourreceptors: ErbB1(EGFR), ErbB2(HER2), ErbB3(HER3) and ErbB4(HER4).EGFR is overexpressed in40%to80%of NSCLC. EGFR ligand bindingresults in the dimerization of the receptor and the subsequent recruitment ofdownstream molecules that mediate cell proliferation, differentiation andsurvival. EGFR-tyrosine kinase inhibitors (EGFR-TKIs) surpress thephosphorylation and tyrosine kinase activity of EGFR by competitive bindingto the intracellular adenosine triphosphate (ATP)-binding domain, thereafter,inhibit the tumor growth. Gefitinib is the most commonly used EGFR-TKI inadvanced NSCLC. But in the applications of gefitinib in clinical, most ofpatients developed acquired resistance within a year. EGFR T790M mutationand MET amplification are the two main mechanisms of acquired resistance togefitinib. But there are still other resistance mechanism has not yet beenidentified.Caveolin-1(Cav-1) is a2124kDa integral membrane protein and aprincipal structural component of caveolae. Cav-1was consist of178amino acid residues, it has a unique hairpin conformation where both N-terminal andC-terminal are exposed to the cytoplasm. Residues82to101of theN-terminal (scaffolding domain) was the main functional region, thisscaffolding domain can bind EGFR, and modulate the activity of EGFRtyrosine kinase. Therefore, we speculated that Cav-1may affect the sensitivityto Gefitinib in NSCLC cells with EGFR mutations.In order to verify this hypothesis, the EGFR-mutant cell line, PC-9, wasused to perform the following experiments in vitro:(1) Establishment ofstably transfected cell lines with overexpression of Cav-1.(2) Examining theeffects of gefitinib on the biological behavior of transfected cell lines,including proliferation, migration and invasion by MTT, wound healing assayand Transwell invasion assay.(3)Western blot was used to detect the effects ofgefitinib on the inhibition of EGFR phosphorylation and EGF-induceddownstream EGFR signaling. The aim of this study is to investigate the effectof Cav-1on the sensitivity to TKIs in NSCLC with EGFR mutations and itsmechanisms. It will provide a new target to solve the problem of EGFR-TKIsresistance.Methods:1Establishment of stably transfected cell linesPC-9cells were stably transfected with plasmids expressing human fullCav-1gene or empty plasmid and selected with G418, resulting in PC/Cav-1and PC-9/pcDNA3.1cell lines. The expression efficiency of Cav-1wasmeasuted by real-time quantitative (RT-PCR) and Western blot.2Proliferation assayPC-9/Cav-1and PC-9/pcDNA3.1cells were seeded in96-well plates,respectively. Cells were treated with different concentrations of gefitinib (0,0.04,0.08,0.16,0.32,0.64,1.28,2.56μmol/L) and continue to culture for72h,MTT assay was used to measure the OD values and to calculate the growthinhibition rate and the half maximal inhibitory concentration (IC50) ofgefitinib.3Migration assay Wound healing assay was used to examine the migration abilities ofstably transfected cell lines affected by gefitinib. PC-9/Cav-1andPC-9/pcDNA3.1cells were seeded in96-well plates, respectively. Cells werescratched with10l pipette tips and washed with PBS, then treated with orwithout gefitinib. Images of cells at the same field were taken every6h untilthe closure 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%.4Invasion assayThe invasion assays were carried out using Transwell chamber with10mm diameter and8m pore size polycarbonate membrane coated withMatrigel. After starved with serum-free1640medium for4h, PC-9/Cav-1andPC-9/pcDNA3.1cells were seeded into upper chamber with serum-free1640medium with or without gefitinib, respectively. Lower chamber were addedinto10%serum1640medium. After incubated for24h, the invaded cells werefixed with95%ice-cold ethanol and then stained with hematoxylin and eosin(HE).5Western blottingPC-9/Cav-1cells and PC-9/pcDNA3.1cells were seeded into35mmdishes. After the treatment of gefitinib (0.015μmol/L) for0and8h, totalproteins were extracted from cells. Western blot was used to examine thelevels of Cav-1, p-EGFR, EGFR, p-ERK, and ERK, p-Akt, and Akt.-actinwas used as a loading control.Results:1The overexpression efficiency of Cav-1in stably transfected cell lines1.1Cav-1mRNA levels of stably transfected cell linesThe relative quantitive values of Cav-1mRNA in PC-9/Cav-1cells（4.07±0.08） were significantly higher than PC-9/pcDNA3.1cells(1.04±0.09)(P<0.01).1.2Cav-1protein levels of stably transfected cell linesThe relative levels of Cav-1protein in PC-9/Cav-1cells (2.51±0.08) were significantly higher than in PC-9/pcDNA3.1group cells (0.19±0.01)(P<0.01).2The sensitivity of gefitinib in stably transfected cell lines2.1The IC50value of gefitinib in stably transfected cell linesThe cell growth inhibition rates of PC-9/Cav-1cells at differentconcentrations (0,0.04,0.08,0.16,0.32,0.64,1.28,2.56μmol/L) of gefitinibwere markedly lower than those of PC-9/pcDNA3.1cells (P<0.05,respectively). The IC50value of PC-9/Cav-1cells (0.04±0.01) weresignificantly higher than that of PC-9/pcDNA3.1cells (0.015±0.01)(P<0.01).2.2Migration abilities of stably transfected cell lines treated with orwithout gefitinibMigration rates of PC-9/Cav-1cells without gefitinib for6h,12h and18h(20.10±0.01%，26.64±0.01%，50.00±0.00%) were significantly higher thanthat of PC-9/pcDNA3.1(15.25±0.00%，20.44±0.02%，22.47±0.01%)(P<0.01,respectively). Migration rates of PC-9/Cav-1cells with gefitinib(0.015μmol/L) exposure for6h,12h and18h (14.89±0.02%，22.44±0.01%，25.08±0.02%) were significantly higher than that in PC-9/pcDNA3.1cells(5.61±0.01%，9.90±0.02%，12.10±0.00%)(P<0.01, respectively).2.3Invasion abilities of stably transfected cell lines treated with orwithout gefitinibThe average invasion cell numbers of PC-9/Cav-1cells without gefitinib(93.00±11.68) were significantly more than that in PC-9/pcDNA3.1cells(66.40±6.02)(P<0.01). The average invasion cell numbers of PC-9/Cav-1group with gefitinib (0.015μmol/L) exposure (56.20±4.87) were significantlymore than that in PC-9/pcDNA3.1group (23.20±3.19)(P<0.01).3Levels of EGFR phosphorylation and the downstream signal moleculesin stably transfected cell lines treated with gefitinibThe levels of different proteins in stably transfected cells were detectedby Western blot. After treating with gefitinib for0μmol/L and0.015μmol/L,the levels of p-EGFR in PC-9/Cav-1group（1.22±0.02，1.03±0.01）weresignificantly higher as compared with PC-9/pcDNA3.1group (0.74±0.04, 0.51±0.02)(P<0.01, respectively). The levels of p-ERK in PC-9/Cav-1group(0.42±0.00,0.18±0.00) were significantly higher as compared withPC-9/pcDNA3.1group (0.22±0.01,0.04±0.01)(P<0.01, respectively). Thelevels of p-Akt in PC-9/Cav-1group (1.88±0.30,0.56±0.04) weresignificantly higher as compared with PC-9/pcDNA3.1group (1.27±0.17,0.28±0.04)(P<0.01, respectively).Conclusion:1Increasing the expression of Cav-1can promote the proliferation,migration and invasion abilities in PC-9cells; Cav-1may affect biologicalbehaviour of PC-9cells through regulating EGFR activation (phosphorylation)and its downstream MAPK/ERK and PI3K/Akt signal pathways.2Increasing the expression of Cav-1can reduce the inhibitory effects ofgefitinib on the proliferation, migration and invasion potential in PC-9cellsvia decreasing the inhibition of gefitinib on the EGFR activation(phosphorylation) and its downstream MAPK/ERK and PI3K/Akt signalpathways.