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L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness

BACKGROUND: Metastasis is the predominant cause for cancer morbidity and mortality accounting for approximatively 90% of cancer deaths. The actin-bundling protein L-plastin has been proposed as a metastatic marker and phosphorylation on its residue Ser5 is known to increase its actin-bundling activi...

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Autores principales: Machado, Raquel A. C., Stojevski, Dunja, De Landtsheer, Sébastien, Lucarelli, Philippe, Baron, Alexandre, Sauter, Thomas, Schaffner-Reckinger, Elisabeth
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898450/
https://www.ncbi.nlm.nih.gov/pubmed/33618712
http://dx.doi.org/10.1186/s12964-021-00710-5
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author Machado, Raquel A. C.
Stojevski, Dunja
De Landtsheer, Sébastien
Lucarelli, Philippe
Baron, Alexandre
Sauter, Thomas
Schaffner-Reckinger, Elisabeth
author_facet Machado, Raquel A. C.
Stojevski, Dunja
De Landtsheer, Sébastien
Lucarelli, Philippe
Baron, Alexandre
Sauter, Thomas
Schaffner-Reckinger, Elisabeth
author_sort Machado, Raquel A. C.
collection PubMed
description BACKGROUND: Metastasis is the predominant cause for cancer morbidity and mortality accounting for approximatively 90% of cancer deaths. The actin-bundling protein L-plastin has been proposed as a metastatic marker and phosphorylation on its residue Ser5 is known to increase its actin-bundling activity. We recently showed that activation of the ERK/MAPK signalling pathway leads to L-plastin Ser5 phosphorylation and that the downstream kinases RSK1 and RSK2 are able to directly phosphorylate Ser5. Here we investigate the involvement of the PI3K pathway in L-plastin Ser5 phosphorylation and the functional effect of this phosphorylation event in breast cancer cells. METHODS: To unravel the signal transduction network upstream of L-plastin Ser5 phosphorylation, we performed computational modelling based on immunoblot analysis data, followed by experimental validation through inhibition/overexpression studies and in vitro kinase assays. To assess the functional impact of L-plastin expression/Ser5 phosphorylation in breast cancer cells, we either silenced L-plastin in cell lines initially expressing endogenous L-plastin or neoexpressed L-plastin wild type and phosphovariants in cell lines devoid of endogenous L-plastin. The established cell lines were used for cell biology experiments and confocal microscopy analysis. RESULTS: Our modelling approach revealed that, in addition to the ERK/MAPK pathway and depending on the cellular context, the PI3K pathway contributes to L-plastin Ser5 phosphorylation through its downstream kinase SGK3. The results of the transwell invasion/migration assays showed that shRNA-mediated knockdown of L-plastin in BT-20 or HCC38 cells significantly reduced cell invasion, whereas stable expression of the phosphomimetic L-plastin Ser5Glu variant led to increased migration and invasion of BT-549 and MDA-MB-231 cells. Finally, confocal image analysis combined with zymography experiments and gelatin degradation assays provided evidence that L-plastin Ser5 phosphorylation promotes L-plastin recruitment to invadopodia, MMP-9 activity and concomitant extracellular matrix degradation. CONCLUSION: Altogether, our results demonstrate that L-plastin Ser5 phosphorylation increases breast cancer cell invasiveness. Being a downstream molecule of both ERK/MAPK and PI3K/SGK pathways, L-plastin is proposed here as a potential target for therapeutic approaches that are aimed at blocking dysregulated signalling outcome of both pathways and, thus, at impairing cancer cell invasion and metastasis formation.
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spelling pubmed-78984502021-02-23 L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness Machado, Raquel A. C. Stojevski, Dunja De Landtsheer, Sébastien Lucarelli, Philippe Baron, Alexandre Sauter, Thomas Schaffner-Reckinger, Elisabeth Cell Commun Signal Research BACKGROUND: Metastasis is the predominant cause for cancer morbidity and mortality accounting for approximatively 90% of cancer deaths. The actin-bundling protein L-plastin has been proposed as a metastatic marker and phosphorylation on its residue Ser5 is known to increase its actin-bundling activity. We recently showed that activation of the ERK/MAPK signalling pathway leads to L-plastin Ser5 phosphorylation and that the downstream kinases RSK1 and RSK2 are able to directly phosphorylate Ser5. Here we investigate the involvement of the PI3K pathway in L-plastin Ser5 phosphorylation and the functional effect of this phosphorylation event in breast cancer cells. METHODS: To unravel the signal transduction network upstream of L-plastin Ser5 phosphorylation, we performed computational modelling based on immunoblot analysis data, followed by experimental validation through inhibition/overexpression studies and in vitro kinase assays. To assess the functional impact of L-plastin expression/Ser5 phosphorylation in breast cancer cells, we either silenced L-plastin in cell lines initially expressing endogenous L-plastin or neoexpressed L-plastin wild type and phosphovariants in cell lines devoid of endogenous L-plastin. The established cell lines were used for cell biology experiments and confocal microscopy analysis. RESULTS: Our modelling approach revealed that, in addition to the ERK/MAPK pathway and depending on the cellular context, the PI3K pathway contributes to L-plastin Ser5 phosphorylation through its downstream kinase SGK3. The results of the transwell invasion/migration assays showed that shRNA-mediated knockdown of L-plastin in BT-20 or HCC38 cells significantly reduced cell invasion, whereas stable expression of the phosphomimetic L-plastin Ser5Glu variant led to increased migration and invasion of BT-549 and MDA-MB-231 cells. Finally, confocal image analysis combined with zymography experiments and gelatin degradation assays provided evidence that L-plastin Ser5 phosphorylation promotes L-plastin recruitment to invadopodia, MMP-9 activity and concomitant extracellular matrix degradation. CONCLUSION: Altogether, our results demonstrate that L-plastin Ser5 phosphorylation increases breast cancer cell invasiveness. Being a downstream molecule of both ERK/MAPK and PI3K/SGK pathways, L-plastin is proposed here as a potential target for therapeutic approaches that are aimed at blocking dysregulated signalling outcome of both pathways and, thus, at impairing cancer cell invasion and metastasis formation. BioMed Central 2021-02-22 /pmc/articles/PMC7898450/ /pubmed/33618712 http://dx.doi.org/10.1186/s12964-021-00710-5 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data.
spellingShingle Research
Machado, Raquel A. C.
Stojevski, Dunja
De Landtsheer, Sébastien
Lucarelli, Philippe
Baron, Alexandre
Sauter, Thomas
Schaffner-Reckinger, Elisabeth
L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness
title L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness
title_full L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness
title_fullStr L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness
title_full_unstemmed L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness
title_short L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness
title_sort l-plastin ser5 phosphorylation is modulated by the pi3k/sgk pathway and promotes breast cancer cell invasiveness
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898450/
https://www.ncbi.nlm.nih.gov/pubmed/33618712
http://dx.doi.org/10.1186/s12964-021-00710-5
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