Abstract
BackgroundMetastasis 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.MethodsTo 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.ResultsOur 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.ConclusionAltogether, 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.9rn5EeEuriEpmsCBgdyXncVideo abstract
Highlights
Metastasis is the predominant cause for cancer morbidity and mortality accounting for approxima‐ tively 90% of cancer deaths
We have shown that Extracellular signal regulated kinase (ERK)/Mitogen-activated protein kinase (MAPK) signalling leads to Ser5 phosphorylation of L-plastin and that this phosphorylation can be directly mediated by the ERK/MAPK pathway downstream kinases P90 ribosomal protein S6 kinase α-1 (RSK1) and P90 ribosomal protein S6 kinase α-3 (RSK2) [13]
Analysis of growth factor‐stimulated signalling in breast cancer cell lines A candidate network of the regulatory signalling pathways upstream of L-plastin was assembled by manually curating signalling pathways from literature (Fig. 1a)
Summary
Metastasis is the predominant cause for cancer morbidity and mortality accounting for approxima‐ tively 90% of cancer deaths. The ERK/MAPK and the PI3K pathways are two of the most commonly dysregulated signal transduction pathways in breast cancer [3] and harbour cancer driver genes that are associated with many cancer types [4]. Among other stimuli, both pathways are activated in response to growth factor binding to their respective receptors endowed with tyrosine kinase activity (RTKs) and their role in the control of cell proliferation, survival, differentiation, metabolism and invasion/migration has long been established [5]. There exists an extensive crosstalk between the two pathways and inhibition of one pathway may be compensated by activation of the other pathway, leading to attenuation of targeted therapeutic efficacy and contributing to drug resistance [6]
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