The E3 Ubiquitin Ligase NEDD4 Mediates Cell Migration Signaling Of EGFR In Lung Cancer Cells

Objective:This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling,and determine how NEDD4 promotes the EGFR-dependent lung cancer cell migration.The results in this study may help to develop an effective targeted therapy for lung cancer.Methods: 1.Lentiviral vector-loaded NEDD4 sh RNA was used to deplete endogenous NEDD4 in lung cancer A549 and H1650 cells,and the effects of knockdown were examined by western blotting.Recoveringthe expression of NEDD4 by re-introducing NEDD4 c DNA in the knockdown cell line.2.Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound healing and transwell assays.3.Lung cancer A549 or H358 cells were stimulated with EGFR for 0-4 h.Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation.Co-expression of NEDD4 with EGFR was determined by immunohistochemical(IHC)staining in 63 lung adenocarcinoma tissue samples and quantitied and analyzed the NEDD4 and EGFR positive rate respectively,and then co-expression rate was obtained.4.Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulationwere examined using the GST-UBA pulldown assay or immunoblotting.Co-expression of NEDD4 with PTEN in 63 lung adenocarcinoma tissue sampleswas determined by IHC staining.5.NEDD4 or its ligasedead mutant NEDD4(NEDD4-LD,NEDD4-C867A)was ectopically expressed in A549 cells using a lentiviral expression system.The LAMP2-positive vesicles atthe cell edges were observed and quantified in the NEDD4 or NEDD4-LD overexpressedcells upon EGF stimulation for 30 min.The secreted lysosomal cathepsin B in the culturing medium of the vector control and sh NEDD4 cells were examined using ELISA assay.6.A549 cells were treated with the specific cathepsin B inhibitor CA-074 Me and the role of cathepsin B in lung cancer cell migration,either EGF-dependent or EGF-independent,was determined by the wound healing and transwell assays.Results: 1.Knockdown of NEDD4 significantly reduced both the basal and the EGF-stimulated migration of lung cancer A549 cells.Furthermore,re-expression of the sh RNA-resistant NEDD4 in the knockdown cells recovered cell migration capacity.Consistent with the results in A549 cells,knockdown of NEDD4 in H1650 cells impaired the cell migration.2.EGFR was phosphorylated and degraded upon EGF stimulation.NEDD4 was co-immunoprecipitated with the activated EGFR.3.The immunofluorescent staining showed that NEDD4 was co-localized with the internalized EGFR upon EGF stimulation,while there was no co-localization without EGF stimulation.Both NEDD4 and EGFR were overexpressed in 41 out of 63 lung adenocarcinoma tumor tissue samples,with a 65% expression rate.Furthermore,NEDD4 and EGFR were always co-expressedin lung adenocarcinoma tumor tissue.4.NEDD4 did not down-regulate PTEN expression by poly-ubiquitinating PTEN;and the expression level of PTEN was not reversely correlated with that of NEDD4 in lung adenocarcinoma samples.5.Treatment with chloroquine,a lysosomal inhibitor,diminished the EGF-dependent migration in A549 cells.6.The LAMP2-positive vesicles appeared at the cell edges in the NEDD4-overexpressed cells upon EGF stimulation for 30 min,while no LAMP2-positive vesicle structure was observedat the cell edgesin the NEDD4-LD overexpressed cells.7.In the A549 vector control cells,EGF dramatically stimulated cathepsin B secretion.In the NEDD4 knockdown cells,the basal level of the secreted cathepsin B was dropped about 50%, and the EGF-stimulated secretion of cathepsin B was eliminated.8.Treatment with 10 mM CA-074 Me,a specific inhibitor of cathepsin B, significantlyinhibitedboth the basal and the EGF-stimulated lung cancer cell migration.Conclusions: 1.Knockdown of NEDD4 significantly reduces the basal and EGF-stimulated cell migration in non-small cell lung carcinoma(NSCLC)cells.2.NEDD4 mediates lung cancer cell migration by interaction with the EGFR signaling complex,independent on the PTEN/PI3K/AKT signaling pathway.3.NEDD4 ligase activity promotes lysosomal secretion.4.Cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration.