Abstract
ECM1 overexpression is an independent predictor of poor prognosis in primary breast carcinomas, however the mechanisms by which ECM1 affects tumor progression have not been completely elucidated. ECM1 was silenced in the triple-negative breast cancer cell lines Hs578T and MDAMB231 using siRNA and the cells were evaluated for changes in morphology, migration, invasion and adhesion. Actin cytoskeleton alterations were evaluated by fluorescent staining and levels of activated Rho GTPases by pull down assays. ECM1 downregulation led to significantly diminished cell migration (p = 0.0005 for Hs578T and p = 0.02 for MDAMB231) and cell adhesion (p < 0.001 for Hs578T and p = 0.01 for MDAMB231). Cell invasion (matrigel) was reduced only in the Hs578T cells (p < 0.01). Silencing decreased the expression of the prometastatic molecules S100A4 and TGFβR2 in both cell lines and CD44 in Hs578T cells. ECM1–silenced cells also exhibited alterations in cell shape and showed bundles of F-actin across the cell (stress fibers) whereas NT-siRNA treated cells showed peripheral membrane ruffling. Downregulation of ECM1 was also associated with an increased F/G actin ratio, when compared to the cells transfected with NT siRNA (p < 0.001 for Hs578T and p < 0.00035 for MDAMB231) and a concomitant decline of activated Rho A in the Hs578T cells. Re-expression of S100A4 in ECM1-silenced cells rescued the phenotype in the Hs578T cells but not the MDAMB231 cells. We conclude that ECM1 is a key player in the metastatic process and regulates the actin cytoskeletal architecture of aggressive breast cancer cells at least in part via alterations in S100A4 and Rho A.
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Authors’ contributions
P. Gomez-Contreras: concept, design of experiments, data acquisition, analysis and interpretation of data, writing of manuscript. J. M. Ramiro-Díaz: design of experiments, data acquisition, analysis and interpretation of data, writing of manuscript and critical review. A. Sierra: data acquisition, writing of manuscript draft. C. Stipp: concept and design, writing of manuscript and critical review. F. E. Domann: interpretation of data, critical review of manuscript. R. J. Weigel: concept, interpretation of data, critical review of manuscript. G. Lal: concept, design, data analysis and interpretation, writing of manuscript and critical review.
Funding
This work was supported by NIH 1 K08 CA151658-01A1 (to GL) and the Department of Surgery at the University of Iowa. The funding sources have no role in the design of the study, data collection, analysis, interpretation of the data or writing of the manuscript.
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10585_2016_9827_MOESM1_ESM.tif
Supplementary Figure 1: (A) Wound scratch assay following Mitomycin C treatment of MDAMB231 cells. Images taken at 4X magnification. (B) A set of four pictures were analyzed using T-Scratch software. Percentage of healed wound area was normalized by setting the value at 0 h as 1. Statistically significant differences were again noted between siECM1 cells and NT cells (p = 0.01) and between NT cells and NT + Mitomycin groups (p = 0.03), indicated by *. There was a trend for ECM1 silenced + Mitomycin cells to migrate less than NT + mitomycin cells but this did not reach statistical significance (p = 0.2) (TIFF 815 kb)
10585_2016_9827_MOESM2_ESM.tif
Supplementary Figure 2: (A) Wound healing assay in MDAMB231 cells following S100A4 silencing compared to ECM1 silencing. (B) A set of four pictures were analyzed using T-Scratch software. Percentage of healed wound area was normalized by setting the value at 0 h as 1. Statistically significant reduction in the normalized healed wound area was found after ECM1 and S100A4 silencing compared to cells transfected with NT control (*p = 0.01 for both), but there was no difference in wound migration between ECM1 or S100A4 silenced cells (TIFF 1012 kb)
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Gómez-Contreras, P., Ramiro-Díaz, J.M., Sierra, A. et al. Extracellular matrix 1 (ECM1) regulates the actin cytoskeletal architecture of aggressive breast cancer cells in part via S100A4 and Rho-family GTPases. Clin Exp Metastasis 34, 37–49 (2017). https://doi.org/10.1007/s10585-016-9827-5
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DOI: https://doi.org/10.1007/s10585-016-9827-5