Role of Tensin3 during plasticity of cell adhesion
Tensin3 在细胞粘附可塑性中的作用
基本信息
- 批准号:411726325
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:德国
- 项目类别:Research Grants
- 财政年份:2018
- 资助国家:德国
- 起止时间:2017-12-31 至 2021-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
Semi-stable changes of cell-matrix adhesion - hereafter referred to as adhesion plasticity - are hallmarks of (patho-)physiological cell behaviour. This plasticity includes switches in the mode of motility and adhesion of tumor cells, i.e. the change from mesenchymal to amoeboid migration, or between low and high matrix adhesion during colonisation. To gain insights into the underlying mechanisms, we have recently generated a MDA-MB 468 human breast cancer subline by simple selection for adhesion-deficient cells. This stable isogenic subline line is anoikis-resistent and exhibits impaired contraction of 3D-collagen matrices, indicating defects in ECM contact formation and force transmission. To analyse the underlying changes in gene expression, unbiased transcriptome analysis was performed. We found a profound downregulation of Tensin3 (Tns3) in the adhesion-deficient cell line, whilst Tns1, Tns2, Vinculin and Talin were unaffected. Moreover, we showed that loss of Tns3 is causative for the cellular phenotype. In this grant we want to analyse in detail the role of Tns3 for tumor cell adhesion, migration, and tumorigenesis. Tensins are thought to act as linkers between integrins and the actin cytoskeleton. Thus we will re-express Tns3 and various deletion mutants in the adhesion-deficient subline and overexpress Tns3-GFP and dominant negative constructs in the parental cells. In the resulting cell lines, cell adhesion, cell spreading, collagen contraction, and migration will be measured. The observed influence of Tns3 expression on Integrins α6, β1 and β4 will be mechanistically and functionally analysed. We will determine the focal adhesion dynamics of the obtained cell lines by confocal life imaging and costaining with known markers such as Vinculin and F-actin. We expect that Tns3, and certain protein domains within, is specifically required for adhesion maturation and force transmission, but less for initial protrusion formation. To functionally validate our findings, we will further assess the role of Tns3 in 3D mammary acini formation by MCF-10a cells. This model showed in preliminary work a particular sensitivity to changes in cell adhesion components and mechanotransduction. Thus we expect pre-malignant lesion formation and luminal filling by elevated Tns3 expression. In xenograft tumorigenesis assays in nude mice, we will finally analyse the role of Tns3 for colonisation by HCT-8 carcinoma cells in vivo. In this model, tumor formation critically requires coinjection of tumor-promoting fibroblasts and the integrin-ECM interactions of HCT-8 cells, making it a suitable system to assess Tns3 functions. We expect to better understand the specific molecular roles of Tns3 and to delineate these to the mechanism of cell-matrix adhesion assembly, migration and tumor cell colonisation.
细胞-基质粘附的半稳定变化-下文称为粘附可塑性-是(病理)生理细胞行为的标志。这种可塑性包括肿瘤细胞的运动和粘附模式的转换,即从间充质迁移到变形虫迁移的变化,或在定殖期间在低和高基质粘附之间的变化。为了深入了解潜在的机制,我们最近通过简单选择粘附缺陷细胞产生了MDA-MB 468人乳腺癌亚系。这种稳定的等基因亚系是抗失巢凋亡的,并且表现出3D-胶原基质的收缩受损,表明ECM接触形成和力传递的缺陷。为了分析基因表达的潜在变化,进行了无偏转录组分析。我们发现在粘附缺陷细胞系中Tensin 3(Tns 3)的显著下调,而Tns 1、Tns 2、tagulin和Talin不受影响。此外,我们发现,Tns 3的损失是细胞表型的原因。在这项研究中,我们希望详细分析Tns 3在肿瘤细胞粘附、迁移和肿瘤发生中的作用。张力蛋白被认为是整合素和肌动蛋白细胞骨架之间的接头。因此,我们将在粘附缺陷亚系中重新表达Tns 3和各种缺失突变体,并在亲本细胞中过表达Tns 3-GFP和显性阴性构建体。在所得细胞系中,将测量细胞粘附、细胞铺展、胶原蛋白收缩和迁移。将从机制和功能上分析观察到的Tns 3表达对整合素α6、β1和β4的影响。我们将通过共聚焦生命成像和与已知标记物如纤维蛋白和F-肌动蛋白共染色来确定所获得的细胞系的粘着斑动力学。我们预计Tns 3及其内的某些蛋白结构域是粘附成熟和力传递所特别需要的,但对于初始突起形成则较少。为了在功能上验证我们的发现,我们将进一步评估Tns 3在MCF-10a细胞形成3D乳腺腺泡中的作用。该模型在初步工作中显示出对细胞粘附组分和机械转导的变化特别敏感。因此,我们预期癌前病变的形成和管腔填充的升高Tns 3的表达。在裸鼠异种移植瘤发生试验中,我们将最终分析Tns 3在体内HCT-8癌细胞定植中的作用。在该模型中,肿瘤形成关键需要共注射肿瘤促进成纤维细胞和HCT-8细胞的整合素-ECM相互作用,使其成为评估Tns 3功能的合适系统。我们期望更好地了解特定的分子作用的Tns 3和描绘这些细胞-基质粘附组装,迁移和肿瘤细胞定植的机制。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Professor Dr. Guido Posern其他文献
Professor Dr. Guido Posern的其他文献
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{{ truncateString('Professor Dr. Guido Posern', 18)}}的其他基金
The role of actin nucleators and nucleation promoting factors in nuclear gene expression via MRTF
肌动蛋白成核剂和成核促进因子在 MRTF 核基因表达中的作用
- 批准号:
170443560 - 财政年份:2010
- 资助金额:
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Priority Programmes
Molekularer Mechanismus des G-Actin-Schalters bei der Regulation der Transkription
G-肌动蛋白开关转录调控的分子机制
- 批准号:
5455741 - 财政年份:2005
- 资助金额:
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Research Grants
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Actin-MAL信号转导下游靶基因:差异调节转录物在增殖和凋亡中的作用
- 批准号:
20559762 - 财政年份:2005
- 资助金额:
-- - 项目类别:
Research Grants