Cancer Cell Biology and Drug Discovery Group, Department of Life Sciences and Medicine, Faculty of Science, Technology and Medicine, University of Luxembourg, CAMPUS Belval, BT1, 7, avenue des Hauts-Fourneaux, 4362, Esch-sur-Alzette, Luxembourg.
Systems Biology Group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
Cell Commun Signal. 2021 Feb 22;19(1):22. doi: 10.1186/s12964-021-00710-5.
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.
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.
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.
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. Video abstract.
转移是癌症发病率和死亡率的主要原因，约占癌症死亡人数的 90%。肌动蛋白束蛋白 L-塑蛋白已被提议作为转移标记物，其残基丝氨酸 5 的磷酸化已知会增加其肌动蛋白束集活性。我们最近表明，ERK/MAPK 信号通路的激活导致 L-塑蛋白 Ser5 磷酸化，并且下游激酶 RSK1 和 RSK2 能够直接磷酸化 Ser5。在这里，我们研究了 PI3K 途径在 L-塑蛋白 Ser5 磷酸化中的参与以及该磷酸化事件在乳腺癌细胞中的功能影响。
为了解 L-塑蛋白 Ser5 磷酸化的上游信号转导网络，我们基于免疫印迹分析数据进行了计算建模，然后通过抑制/过表达研究和体外激酶测定进行了实验验证。为了评估 L-塑蛋白表达/Ser5 磷酸化在乳腺癌细胞中的功能影响，我们要么在最初表达内源性 L-塑蛋白的细胞系中沉默 L-塑蛋白，要么在缺乏内源性 L-塑蛋白的细胞系中表达 L-塑蛋白野生型和磷酸变体。建立的细胞系用于细胞生物学实验和共聚焦显微镜分析。
我们的建模方法表明，除了 ERK/MAPK 途径外，还取决于细胞环境，PI3K 途径通过其下游激酶 SGK3 促进 L-塑蛋白 Ser5 磷酸化。Transwell 侵袭/迁移实验的结果表明，BT-20 或 HCC38 细胞中 L-塑蛋白的 shRNA 介导敲低显着降低了细胞侵袭，而磷酸模拟 L-塑蛋白 Ser5Glu 变体的稳定表达导致 BT-549 和 MDA-MB-231 细胞的迁移和侵袭增加。最后，共聚焦图像分析结合酶谱实验和明胶降解实验提供了证据，表明 L-塑蛋白 Ser5 磷酸化促进 L-塑蛋白募集到侵袭伪足、MMP-9 活性和伴随的细胞外基质降解。
总之，我们的结果表明 L-塑蛋白 Ser5 磷酸化增加了乳腺癌细胞的侵袭性。作为 ERK/MAPK 和 PI3K/SGK 途径的下游分子，L-塑蛋白在此被提议作为一种潜在的治疗靶点，旨在阻断两条途径失调信号转导的结果，从而损害癌细胞的侵袭和转移形成。
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