Jian Zhou1,2, Kwang Joo Kwak3, Zuoren Wu4, Dawei Yang1,2, Jing Li1,2, Meijia Chang1,2, Yuanlin Song1,2, Hengshan Zeng4, L James Lee3, Jie Hu1,2, Chunxue Bai1,2,5. 1. Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, China. 2. Shanghai Respiratory Research Institute, Shanghai, China. 3. The Ohio State University, Department of Chemical and Biomolecular Engineering, Columbus, Ohio, USA. 4. Hangzhou Dixiang Co. Ltd., Hangzhou, China. 5. State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.
Abstract
BACKGROUND/AIMS: Tyrosine kinase inhibitor gefitinib significantly improves the survival of patients with non-small-cell lung cancer (NSCLC) by inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase. However, patients eventually develop resistance to gefitinib through uncharacterized mechanisms. It is known that plasminogen activator urokinase receptor (PLAUR) plays an important role in cell proliferation, migration and apoptosis. However, the role of PLAUR, particularly exosomal PLAUR in gefitinib resistance in NSCLC has not been reported. The aim of this study is to determine the relationship between PLAUR and gefitinib resistance. METHODS: In this study, a tethered cationic lipoplex nanoparticle (TCLN) biochip containing molecular beacons was used as probes to detect PLAUR mRNA in plasma exosomes from patients with gefitinib-sensitive and -resistant NSCLC. In vitro, Real-time PCR was used to examine the expression of PLAUR mRNA and Western blot was applied to examine the expression of related proteins. The gene knockdown was achieved by Lentivirus based RNA silence technique. The cell counting kit-8 assay and EdU incorporation were used to examine cell proliferation. The flow cytometry was applied to determine cell apoptosis and cell cycle, while the mitochondrial membrane potential was measured by JC-1 dye assay. Signaling pathway affected by PLAUR knockdown was identified by cDNA Microarray. The effect of PLAUR knockdown on tumorigenesis was analyzed in vivo. RESULTS: We found that the exosomal PLAUR mRNA in the plasma of gefitinib-resistant NSCLC patients was significantly increased compared to that of gefitinib-sensitive NSCLC patients. The PLAUR mRNA and soluble PLAUR protein were also significantly increased in gefitinib-resistant human lung adenocarcinoma PC9R cells compared to gefitinib-sensitive PC9 cells. Silencing PLAUR in PC9R cells impaired mitochondrial membrane potential and increased cell apoptosis via EGFR/p-AKT/survivin signaling pathway. Furthermore, EGFR was upregulated in the geftinib-resistant PC9R cells, and knockdown of EGFR significantly increased cell apoptosis. CONCLUSIONS: Taken together, our results demonstrated that PLAUR induces geftinib-resistance through EGFR/p-AKT/survivin signaling pathway in gefitinib-resistant human lung adenocarcinoma cells. PLAUR could be a novel therapeutic target for gefitinib-resistant NSCLC patients.
BACKGROUND/AIMS: Tyrosine kinase inhibitor gefitinib significantly improves the survival of patients with non-small-cell lung cancer (NSCLC) by inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase. However, patients eventually develop resistance to gefitinib through uncharacterized mechanisms. It is known that plasminogen activator urokinase receptor (PLAUR) plays an important role in cell proliferation, migration and apoptosis. However, the role of PLAUR, particularly exosomal PLAUR in gefitinib resistance in NSCLC has not been reported. The aim of this study is to determine the relationship between PLAUR and gefitinib resistance. METHODS: In this study, a tethered cationic lipoplex nanoparticle (TCLN) biochip containing molecular beacons was used as probes to detect PLAUR mRNA in plasma exosomes from patients with gefitinib-sensitive and -resistant NSCLC. In vitro, Real-time PCR was used to examine the expression of PLAUR mRNA and Western blot was applied to examine the expression of related proteins. The gene knockdown was achieved by Lentivirus based RNA silence technique. The cell counting kit-8 assay and EdU incorporation were used to examine cell proliferation. The flow cytometry was applied to determine cell apoptosis and cell cycle, while the mitochondrial membrane potential was measured by JC-1 dye assay. Signaling pathway affected by PLAUR knockdown was identified by cDNA Microarray. The effect of PLAUR knockdown on tumorigenesis was analyzed in vivo. RESULTS: We found that the exosomal PLAUR mRNA in the plasma of gefitinib-resistant NSCLCpatients was significantly increased compared to that of gefitinib-sensitive NSCLCpatients. The PLAUR mRNA and soluble PLAUR protein were also significantly increased in gefitinib-resistant humanlung adenocarcinoma PC9R cells compared to gefitinib-sensitive PC9 cells. Silencing PLAUR in PC9R cells impaired mitochondrial membrane potential and increased cell apoptosis via EGFR/p-AKT/survivin signaling pathway. Furthermore, EGFR was upregulated in the geftinib-resistant PC9R cells, and knockdown of EGFR significantly increased cell apoptosis. CONCLUSIONS: Taken together, our results demonstrated that PLAUR induces geftinib-resistance through EGFR/p-AKT/survivin signaling pathway in gefitinib-resistant humanlung adenocarcinoma cells. PLAUR could be a novel therapeutic target for gefitinib-resistant NSCLCpatients.
Authors: Letizia Porcelli; Michele Guida; Simona Serratì; Amalia Azzariti; Simona De Summa; Roberta Di Fonte; Ivana De Risi; Marianna Garofoli; Mariapia Caputo; Antonio Negri; Sabino Strippoli Journal: J Immunother Cancer Date: 2021-05 Impact factor: 13.751