TY - JOUR
T1 - L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness
AU - Machado, Raquel A.C.
AU - Stojevski, Dunja
AU - De Landtsheer, Sébastien
AU - Lucarelli, Philippe
AU - Baron, Alexandre
AU - Sauter, Thomas
AU - Schaffner-Reckinger, Elisabeth
N1 - Funding Information:
The authors wish to thank Dr. Christelle Bahlawane, Sonia Frasquilho, Tobias Okello, and Dr. Yervand Karapetyan (Integrated Biobank of Luxembourg) for coordination, technical assistance and expert evaluation of immunohistochemistry experiments. We also thank Christina Laurini and Marie Catillon for technical assistance with immunoblot analysis and plasmid construction as well as Dr. Andreas Girod for expert assistance with confocal microscopy analysis (DLSM, University of Luxembourg). The authors are grateful to Professor Dan Liu (Baylor College of Medicine, Houston, TX, USA) for providing SGK3 plasmids.
Funding Information:
This work was supported by a Grant from the Fondation Cancer Luxembourg (FC/2016/04).
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/2/22
Y1 - 2021/2/22
N2 - 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. [MediaObject not available: see fulltext.]
AB - 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. [MediaObject not available: see fulltext.]
KW - Actin-bundling
KW - ERK/MAPK pathway
KW - Extracellular matrix degradation
KW - Invadopodia
KW - Invasion
KW - L-plastin
KW - Metastasis
KW - PI3K pathway
KW - RSK
KW - SGK
UR - http://www.scopus.com/inward/record.url?scp=85101226943&partnerID=8YFLogxK
UR - https://www.ncbi.nlm.nih.gov/pubmed/33618712
U2 - 10.1186/s12964-021-00710-5
DO - 10.1186/s12964-021-00710-5
M3 - Article
C2 - 33618712
AN - SCOPUS:85101226943
SN - 1478-811X
VL - 19
SP - 22
JO - Cell Communication and Signaling
JF - Cell Communication and Signaling
IS - 1
M1 - 22
ER -