Abstract
Causal implication of S100A4 in inducing metastases was convincingly shown previously. However, the mechanisms that associate S100A4 with tumor progression are not well understood. S100A4 protein, as a typical member of the S100 family, exhibits dual, intracellular and extracellular, functions. This work is focused on the extracellular function of S100A4, in particular its involvement in tumor-stroma interplay in VMR (mouse adenocarcinoma cell line) tumor cells, which exhibit stroma-dependent metastatic phenotype. We demonstrated the reciprocal influence of tumor and stroma cells where tumor cells stimulate S100A4 secretion from fibroblasts in culture. In turn, extracellular S100A4 modifies the cytoskeleton and focal adhesions and triggers several other events in tumor cells. We found stabilization of the tumor suppressor protein p53 and modulation of its function. In particular, extracellular S100A4 down-regulates the pro-apoptotic bax and the angiogenesis inhibitor thrombospondin-1 genes. For the first time, we demonstrate here that the S100A4 protein added to the extracellular space strongly stimulates proteolytic activity of VMR cells. This activity most probably is associated with matrix metalloproteinases and, in particular, with matrix metalloproteinase-13. Finally, the application of the recombinant S100A4 protein confers stroma-independent metastatic phenotype on VMR tumor cells. In conclusion, our results indicate that metastasis-inducing S100A4 protein plays a pivotal role in the tumor-stroma environment. S100A4 released either by tumor or stroma cells triggers pro-metastatic cascades in tumor cells.
Highlights
Metastatic dissemination of cancer cells results in incurable disease and becomes one of the leading causes of cancer patient deaths
We demonstrated the important role of S100A4 in this interplay needed for the acquisition of the metastatic phenotype
S100A4 is a typical member of the S100 family of Ca2ϩbinding proteins with dual, intracellular and extracellular, functions
Summary
Cell Lines and Transfection—VMR and CSML-0 mouse adenocarcinoma cell lines derived from two independent spontaneous tumors in A/Sn mice [31] were used. Metastasis Assay and Tissue Processing—For experimental metastasis assay, 6-week-old A/Sn mice were given an intravenous lateral tail vein injection with 106 tumor cells suspended in 0.2 ml of PBS. 5– 6-week-old A/Sn mice were given an intravenous lateral tail vein injection with 106 tumor cells suspended in 0.2 ml of PBS along with 50 g of recombinant S100A4 or control Myo117 protein (recombinant 117 aa fragment of heavy chain of non-muscle myosin) into tail vein. Immunoprecipitation and Western Blot—0.5 ml of the CM were supplemented with a protease inhibitor mixture (1 mM dithiothreitol, 10 g/ml leupeptin, 2 g/ml aprotonin, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine) and 1% Nonidet P-40 and incubated with antiS100A4 polyclonal antibodies for 2 h in ice. The immunocomplexes were collected using a protein G-Sepharose suspension (50% v/v), separated on gradient SDS-PAGE (4 –20%), and transferred onto ImmobilonTM transfer membrane (Millipore). The same concentrated CM was used for Western blot analysis with anti-MMP-13 antibodies (Neomarkers) as described above
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