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E-cadherin promotor methylation and mutation are inversely related to motility capacity of breast cancer cells

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Abstract

Inactivation of the tumor suppressor E-cadherin is an important event during breast tumorigenesis, as its decreased expression is linked to aggressiveness and metastasis. However, the relationship between the different modes of E-cadherin inactivation (mutation versus promotor hypermethylation) and breast cancer cell behavior is incompletely understood. The high correlation between E-cadherin inactivation status and cell morphology in vitro suggests different biological roles for the two inactivation modes during breast tumorigenesis. Because E-cadherin has been linked to cell invasion and metastasis, and cell motility is a crucial prerequisite to form metastases, we here compared the cell motility capacities of breast cancer cell lines with known E-cadherin status. Using barrier migration assays and time-lapse microscopy, we analyzed the migratory capacity of nine well-characterized human breast cancer cell lines (MDA-MB-231, MCF-7, T47D, BT549, MPE600, CAMA-1, SUM159PT, SUM52PE, and SK-BR-3). This subset was chosen based on E-cadherin gene status (wild-type, mutated, and promotor hypermethylated): three cell lines of each group. In addition, cell proliferation assays were performed for all conditions, to dissect migratory from proliferative effects. In this study, we demonstrate an overt association between the mode of E-cadherin inactivation and cell migration. Promotor hypermethylated E-cadherin cell lines showed a higher migration capacity, while cell lines with mutated E-cadherin were less motile compared to wild-type E-cadherin cell lines. Migration induction by fibronectin and basic fibroblast growth factor did not alter the cell motility association differences. Cell proliferation assays showed that the associations found were not caused by proliferation differences. Inhibition and overexpression of E-cadherin as well as DNA demethylation confirmed the relationship between E-cadherin and breast cancer cell motility. Our results demonstrate an association between the mode of E-cadherin inactivation and migration of breast cancer cells, which justifies more detailed research on the role of E-cadherin inactivation in cell migration and metastasis.

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Abbreviations

bFGF:

Basic fibroblast growth factor

ECM:

Extracellular matrix

ED:

Effective distance

EMT:

Epithelial–mesenchymal transition

FN:

Fibronectin

HDGC:

Hereditary diffuse-type gastric cancer

MFD:

Mean front displacement

SRB:

Sulphorhodamine B

TD:

Total distance

5-Aza:

5-Azacytidine

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Acknowledgments

We thank the Erasmus Medical Instrumentation Service (EMI) for technical assistance with development of materials, and we thank Olga Ilina for E-cadherin siRNA and antibodies. This work was supported by a pilot-study grant from the Mrace Translational Research Fund of the Erasmus University MC (RvH).

Conflict of interest

The authors declare that they have no conflict of interest.

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Correspondence to Remco van Horssen or Timo L. M. ten Hagen.

Electronic supplementary material

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10549_2012_2261_MOESM1_ESM.doc

Table S1 Migration parameters for MCF-7-NT, MCF-7-siEcad, MDA-MB-231, MDA-MB-231-Ecad and 5-azacytidine treated MDA-MB-231 cells. (DOC 24 kb)

10549_2012_2261_MOESM2_ESM.tif

Fig. S1 Setup of the barrier migration assay. a Cell culture chambers containing cover glasses for optimal microscopy are coated with FN. b A migration insert (the barrier) is placed in the middle of the chamber generating a two-compartment system. Cells are seeded around this barrier, in the outer compartment. c Upon removal of the barrier cells are able to move into the clean cell free area towards the centre of the chamber. d Cells are imaged by live cell time lapse microscopy for 24 hr. (TIFF 1040 kb)

10549_2012_2261_MOESM3_ESM.tif

Fig. S2 Immunofluorescence staining for beta-catenin localization. One cell line per group was selected and grown under the four conditions as tested for cell motility (none, FN-coated, bFGF-treated and both FN/bFGF). Beta-catenin was mainly present at cell-cell contacts in MCF-7 cells, cytosolic in MDA-MB-231 cells and absent in SK-BR-3 cells. Coating and/or treatment did not make a difference. Nuclei are stained with DAPI. Bar: 10 μm. (TIFF 3857 kb)

Movie 1 MCF-7 epithelial breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 1a, upper panels. (MPG 4485 kb)

Movie 2 SUM52PE epithelial breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 1a, middle panels. (MPG 4544 kb)

Movie 3 T47D epithelial breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 1a, lower panels. (MPG 5151 kb)

Movie 4 MDA-MB-231 spindle-shaped breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 2a, upper panels. (MPG 5456 kb)

Movie 5 SUM159PT spindle-shaped breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 2a, middle panels. (MPG 5613 kb)

Movie 6 BT549 spindle-shaped breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 2a, lower panels. (MPG 5641 kb)

Movie 7 SK-BR-3 rounded breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 3a, upper panels. (MPG 5135 kb)

Movie 8 CAMA-1 rounded breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 3a, middle panels. (MPG 4394 kb)

Movie 9 MPE600 rounded breast cancer cells migrating for 24 h without (left panels) and with (right panels) FN-coating, treated with (lower panels) and without bFGF (upper panels). Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 3a, lower panels. (MPG 5124 kb)

10549_2012_2261_MOESM13_ESM.mpg

Movie 10 MCF-7-NT (left panel) and MCF-7-siEcad cells migrating for 24 hr without coating. Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 4b. (MPG 4528 kb)

10549_2012_2261_MOESM14_ESM.mpg

Movie 11 MDA-MB-231 (untreated, left panel), MDA-MB-231-Ecad (middle panel) and 5-azacytidine treated MDA-MB-231 cells (right panel) migrating for 24 hr without coating. Time-lapse phase-contrast microscopy was performed taking an image every 12 min. Display rate is 10 frames/s. Movie corresponds with Figure 4d. (MPG 7122 kb)

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van Horssen, R., Hollestelle, A., Rens, J.A.P. et al. E-cadherin promotor methylation and mutation are inversely related to motility capacity of breast cancer cells. Breast Cancer Res Treat 136, 365–377 (2012). https://doi.org/10.1007/s10549-012-2261-8

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