Osteopontin Contributes To Acquired Resistance In Lung Adenocarcinoma Cells Through The Integrin/FAK Signalling Pathway

Objective:Lung cancer is the most prevalent malignant tumor worldwide,fortunately with the gradual popularized of targeted therapy in clinical practice,about 60%patients with non-small cell lung cancer harbouring EGFR sensitive mutations have obtained better quality of life and better therapeutic effects from the application of targeted therapy.However it was found that patients who were relieved after the targeted therapy usually developed acquired resistance in about one year,showing that the primary lesion progressed again or metastases lesion appeared.At present,it is found that the mechanisms of acquired resistance in lung cancer mainly include:EGFR secondary mutations,mutations of genes downstream to EGFR,bypass activation and histological transformation.Previous studies have suggested that osteopontin(OPN)which was a secreted glycated phosphoprotein,plays a role in promoting tumor progression or metastasis in a variety of cancers.In this study,we found that focal adhesion kinase(FAK)protein exhibited abnormal activation in lung adenocarcinoma cells resistant to EGFR-TKI.It was known that the activation of FAK is closely related to integrin,and OPN can transmit cellular signals through integrin.Therefore,this study aims to elucidate the possible mechanisms of OPN-mediated FAK bypass activation leading to acquired drug-resistance in lung adenocarcinoma cells,by in vitro experiments and clinical specimen detection,and to explore therapeutic measures for this mechanism.Methods:1.Western Blot was performed to detect the activity of proteins downstream to EGFR in PC-9 and PC-9GR cells in the effect of EGFR-TKI,proteome profiler array analysis and Western Blot were used to compare the activity of various proteins in lung adenocarcinoma cell line PC-9 and its resistance line PC-9GR(PC-9 gefitinib-resistant),and the downstream protein activity was observed after knocking down FAK expression in PC-9GR cells by transfection of si-RNA to explore whether there is alternative activation of FAK in resistant lung adenocarcinoma cells.2.Inhibiting the activity of EGFR and FAK in PC-9GR cells by combining the application of EGFR-TKI and FAK inhibitor,or combining EGFR-TKI and si-RNA of FAK,detecting cell resistance of cells through colony-forming unit assay,apoptosis analyze and cell viability experiments.Western Blot was used to detect the protein activity.Aiming at exploring whether the FAK bypass activation partially mediates acquired resistance in drug-resistant lung adenocarcinoma cells.3.Proteome profiler array analysis and Western Blot were used to compare the expression of soluble receptors and their ligands between PC-9 and PC-9GR,and to observe the expression of integrin av and β3 and the activity of downstream proteins with the application of EGFR-TKI in PC-9 cells.Aiming at exploring whether integrin αvβ3 in lung adenocarcinoma cells caused FAK activation.4.Transfection of si-RNA was used to knock do wn integrin av and β3 in PC-9GR cells.Cell viability experiment and Western Blot were performed to explore whether integrin av and β3 mediate acquired resistance in drug-resistant lung adenocarcinoma cells.5.Seven lung adenocarcinoma patients developed resistance to first-generation EGFR-TKIs who were hospitalized between February 2018 and February 2019 were selected,collecting their pathological sections of tissue biopsies which undertaken before and after drug-resistance emerging.Integrin av expression levels of lung cancer tissues were detected by immunohistochemical experiment,and the expression quantitative score was carried out.The difference of expression level was calculated by t test.6.qRT-PCR,Western Blot and ELISA assays were performed to compare the mRNA,protein expression and secretion levels of osteopontin in PC-9 cells and PC-9GR cells,and detect the mRNA,protein expression and secretion levels of osteopontin in PC-9 cells under the application of EGFR-TKI,which were to explore whether osteopontin is elevated in drug-resistant lung adenocarcinoma cells.7.Recombinant human osteopontin(rOPN)was added to the PC-9 cell culture medium,si-RNA was transfected in PC-9 cells to knock down OPN expression.Then immunofluorescence experiment,Western Blot were were undertaken to explore whether rOPN resulted in bypass activation of integrin/FAK pathway in lung adenocarcinoma cells.8.si-RNA were transfecting to knockdown osteopontin expression in PC-9GR cells,to explore whether osteopontin mediates acquired resistance in drug-resistant lung adenocarcinoma cells through cell viability experiment,ELISA assay,and Western Blot.Results:1.AKT and ERK were partially activated in PC-9GR cells under the fully effect of EGFR-TKI,and FAK is abnormally activated;FAK also activated in PC-9 cells under the action of EGFR-TKI;Knocking down of FAK expression in PC-9GR cells mediated decrease of p-AKT and p-ERK.It is suggested that FAK bypass activation during the emerging of acquired resistance in lung adenocarcinoma cells activate AKT and MAPK signaling pathways.2.Treating PC-9GR cells with combining FAK and EGFR inhibitors can significantly block the AKT pathway,inhibit FAK re-activation,reduce cell mitosis and increase cell apoptosis,which enhanced the effect of EGFR-TKI;Knocking down FAK expression in PC-9GR cells and meanwhile application of EGFR-TKI enhanced the effect of blocking AKT and MAPK pathways.It is suggested that FAK bypass activation partially mediates resistance in drug-resistant lung adenocarcinoma cells.3.The expression level of integrin av and β3 were higher in PC-9GR cells than in PC-9 cells;Increased integrin av and β3 were detected in PC-9 cells under the action of EGFR-TKI,and the FAK protein was activated simultaneously,also the activation of FAK protein possibly induced AKT and ERK reactivation;Application of EGFR-TKI in PC-9 cells that transfected with si-RNA of ITG-AV didn’t give rise to the activation of FAK.It is suggested that the activation of FAK protein in lung adenocarcinoma cells is mediated by integrin αvβ3 dimer.4.After Knocking down integrin av and β3 expression in PC-9GR cells,resistance to EGFR-TKI was weakened,activity of FAK,AKT,and ERK decreased.It is suggested that integrin av and β3 in drug-resistant lung adenocarcinoma cells can mediate drug resistance by activating downstream proteins.5.Five of seven(71.4%)drug-resistant patients had increased expression of integrin av,and there was a statistical difference(p=0.041)through quantitative analysis,suggesting that integrin expression up-regulation may occur after EGFR-TKI resistance in lung adenocarcinoma.6.Compared with PC-9 cells,PC-9GR cells have increased osteopontin(OPN)expression level and up-regulated secreted osteopontin(sOPN);PC-9 cells under the action of EGFR-TKI showed increased OPN expression level and up-regulated sOPN secretion.It is suggested that OPN secondary to lung adenocarcinoma cells increased after acquired drug resistance.7.PC-9 cells cultured with recombinant human osteopontin(rOPN)showed increased membranous distribution of integrin αv,elevated level of integrin av or β3 expression,and subsequently bypass activated FAK;PC-9 cells transfected with si-RNA of OPN no longer occurs FAK activation under the action of EGFR-TKI.It is suggested that secondary elevation of OPN in lung adenocarcinoma cells can activate FAK through integrin αvβ3.8.After knocking down OPN expression in PC-9GR cell,sOPN secretion decreased,resistance to EGFR-TKI attenuated,and activity of FAK,AKT,ERK declined.Suggesting that OPN in drug-resistant lung adenocarcinoma cells can partially mediate acquired resistance by activating downstream proteinsConclusions:OPN increased in lung adenocarcinoma cells during the therapy of EGFR-TKIs,and secreted more sOPN.The glycated phosphoprotein then activated integrin αvβ3 dimer and FAK downstream,thereby inducing the activation of AKT and MAPK pathways.The bypass activation suspended the tumor cell survival and partially mediates acquired resistance of lung adenocarcinoma cells.Decreasing the expression of OPN and integrin αv or β3,and inhibiting the activity of FAK protein in drug-resistant lung adenocarcinoma cells can reduce tumor cell resistance,which suggests a new treatment strategy for lung adenocarcinoma patients who resistant to EGFR-TKIs.