PLAUR Confers Resistance to Gefitinib Through EGFR/P-AKT/Survivin Signaling Pathway

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.

uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

“…Moreover, as reported by Liu et al, GLS2 negatively regulates the PI3K/AKT signaling pathway [32]. It has been widely known that, when uPAR is expressed at low levels, pAkt is downregulated [33] while pERK is strongly upregulated; uPAR loss, in fact, inhibits the PI3K/Akt pathway and forces the cells to use ERK as an alternative pathway to grow and survive [34]. Here, we verified this mechanism reporting that after uPAR loss, the phosphorylation of Akt was inhibited and, to compensate for this effect to keep the cell proliferating and surviving, the phosphorylation levels of ERK were enhanced.…”

High-throughput single-EV liquid biopsy: Rapid, simultaneous, and multiplexed detection of nucleic acids, proteins, and their combinations

“…In previous studies, we have demonstrated the feasibility of measuring miRNA or mRNA in EVs ( 13 , 14 ). In this study, we developed an HNCIB system and show that the system is capable of highly efficient capture of EVs and simultaneous detection of proteins and RNA in a single EV.…”

Glycerol kinase 5 confers gefitinib resistance through SREBP1/SCD1 signaling pathway

“…1a, b). To further understand the mechanism of gefitinib resistance, we generated gefitinib-resistant PC9R cells by exposing gefitinib-sensitive PC9 cells to increasing concentrations of gefitinib [39]. We found that in the gefitinib-resistant PC9R cells, GK5 was significant upregulated compared to that in gefitinib-sensitive PC9 by quantitative reverse transcription qRT-PCR and Western blotting analysis (Fig.…”

“…The human lung adenocarcinoma cell line PC9 and NCI-H1975 (H1975, intrinsically harbor EGFR L858R/T790 M) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). To induce gefitinib resistance, PC9 cells were exposed to increasing concentrations of gefitinib [39]. The gefitinib-sensitive PC9 and -resistance PC9R and H1975 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/streptomycin.…”