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The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells

BACKGROUND: EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. Howev...

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Autores principales: Shao, Genbao, Wang, Ranran, Sun, Aiqin, Wei, Jing, Peng, Ke, Dai, Qian, Yang, Wannian, Lin, Qiong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817799/
https://www.ncbi.nlm.nih.gov/pubmed/29455656
http://dx.doi.org/10.1186/s12943-018-0784-2
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author Shao, Genbao
Wang, Ranran
Sun, Aiqin
Wei, Jing
Peng, Ke
Dai, Qian
Yang, Wannian
Lin, Qiong
author_facet Shao, Genbao
Wang, Ranran
Sun, Aiqin
Wei, Jing
Peng, Ke
Dai, Qian
Yang, Wannian
Lin, Qiong
author_sort Shao, Genbao
collection PubMed
description BACKGROUND: EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling. METHODS: Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration. RESULTS: Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4 significantly reduced extracellular amount of cathepsin B induced by EGF. Consistent with the role of NEDD4, cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration. Our studies propose a novel mechanism underlying the EGFR-promoted lung cancer cell migration that is mediated by NEDD4 through regulation of cathepsin B secretion. CONCLUSION: NEDD4 mediates the EGFR lung cancer cell migration signaling through promoting lysosomal secretion of cathepsin B.
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spelling pubmed-58177992018-02-23 The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells Shao, Genbao Wang, Ranran Sun, Aiqin Wei, Jing Peng, Ke Dai, Qian Yang, Wannian Lin, Qiong Mol Cancer Research BACKGROUND: EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling. METHODS: Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration. RESULTS: Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4 significantly reduced extracellular amount of cathepsin B induced by EGF. Consistent with the role of NEDD4, cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration. Our studies propose a novel mechanism underlying the EGFR-promoted lung cancer cell migration that is mediated by NEDD4 through regulation of cathepsin B secretion. CONCLUSION: NEDD4 mediates the EGFR lung cancer cell migration signaling through promoting lysosomal secretion of cathepsin B. BioMed Central 2018-02-19 /pmc/articles/PMC5817799/ /pubmed/29455656 http://dx.doi.org/10.1186/s12943-018-0784-2 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Shao, Genbao
Wang, Ranran
Sun, Aiqin
Wei, Jing
Peng, Ke
Dai, Qian
Yang, Wannian
Lin, Qiong
The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells
title The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells
title_full The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells
title_fullStr The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells
title_full_unstemmed The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells
title_short The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells
title_sort e3 ubiquitin ligase nedd4 mediates cell migration signaling of egfr in lung cancer cells
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817799/
https://www.ncbi.nlm.nih.gov/pubmed/29455656
http://dx.doi.org/10.1186/s12943-018-0784-2
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