Role of Tensin3 during plasticity of cell adhesion

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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.