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.
Similar content being viewed by others
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
References
Condeelis J, Segall JE (2003) Intravital imaging of cell movement in tumours. Nat Rev Cancer 3(12):921–930
Yamaguchi H, Wyckoff J, Condeelis J (2005) Cell migration in tumors. Curr Opin Cell Biol 17(5):559–564
Wang W, Goswami S, Sahai E, Wyckoff JB, Segall JE, Condeelis JS (2005) Tumor cells caught in the act of invading: their strategy for enhanced cell motility. Trends Cell Biol 15(3):138–145
Friedl P, Wolf K (2010) Plasticity of cell migration: a multiscale tuning model. J Cell Biol 188(1):11–19. doi:10.1083/jcb.200909003
Kemler R (1993) From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet 9(9):317–321
Drees F, Pokutta S, Yamada S, Nelson WJ, Weis WI (2005) Alpha-catenin is a molecular switch that binds E-cadherin–beta-catenin and regulates actin-filament assembly. Cell 123(5):903–915
Yamada S, Pokutta S, Drees F, Weis WI, Nelson WJ (2005) Deconstructing the cadherin–catenin–actin complex. Cell 123(5):889–901
Frixen UH, Behrens J, Sachs M, Eberle G, Voss B, Warda A, Lochner D, Birchmeier W (1991) E-cadherin-mediated cell–cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol 113(1):173–185
Vleminckx K, Vakaet L Jr, Mareel M, Fiers W, van Roy F (1991) Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell 66(1):107–119
Siitonen SM, Kononen JT, Helin HJ, Rantala IS, Holli KA, Isola JJ (1996) Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer. Am J Clin Pathol 105(4):394–402
De Leeuw WJ, Berx G, Vos CB, Peterse JL, Van de Vijver MJ, Litvinov S, Van Roy F, Cornelisse CJ, Cleton-Jansen AM (1997) Simultaneous loss of E-cadherin and catenins in invasive lobular breast cancer and lobular carcinoma in situ. J Pathol 183(4):404–411. doi:10.1002/(sici)1096-9896(199712)183:4<404:aid-path1148>3.0.co;2-9
Guilford P, Hopkins J, Harraway J, McLeod M, McLeod N, Harawira P, Taite H, Scoular R, Miller A, Reeve AE (1998) E-cadherin germline mutations in familial gastric cancer. Nature 392(6674):402–405. doi:10.1038/32918
Gayther SA, Gorringe KL, Ramus SJ, Huntsman D, Roviello F, Grehan N, Machado JC, Pinto E, Seruca R, Halling K, MacLeod P, Powell SM, Jackson CE, Ponder BA, Caldas C (1998) Identification of germ-line E-cadherin mutations in gastric cancer families of European origin. Cancer Res 58(18):4086–4089
Berx G, Cleton-Jansen AM, Strumane K, de Leeuw WJ, Nollet F, van Roy F, Cornelisse C (1996) E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Oncogene 13(9):1919–1925
Berx G, Cleton-Jansen AM, Nollet F, de Leeuw WJ, van de Vijver M, Cornelisse C, van Roy F (1995) E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J 14(24):6107–6115
Becker KF, Atkinson MJ, Reich U, Becker I, Nekarda H, Siewert JR, Hofler H (1994) E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. Cancer Res 54(14):3845–3852
Becker KF, Atkinson MJ, Reich U, Huang HH, Nekarda H, Siewert JR, Hofler H (1993) Exon skipping in the E-cadherin gene transcript in metastatic human gastric carcinomas. Hum Mol Genet 2(6):803–804
Derksen PW, Liu X, Saridin F, van der Gulden H, Zevenhoven J, Evers B, van Beijnum JR, Griffioen AW, Vink J, Krimpenfort P, Peterse JL, Cardiff RD, Berns A, Jonkers J (2006) Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 10(5):437–449. doi:10.1016/j.ccr.2006.09.013
Hollestelle A, Elstrodt F, Timmermans M, Sieuwerts AM, Klijn JG, Foekens JA, den Bakker MA, Schutte M (2010) Four human breast cancer cell lines with biallelic inactivating alpha-catenin gene mutations. Breast Cancer Res Treat 122(1):125–133. doi:10.1007/s10549-009-0545-4
Graff JR, Herman JG, Lapidus RG, Chopra H, Xu R, Jarrard DF, Isaacs WB, Pitha PM, Davidson NE, Baylin SB (1995) E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res 55(22):5195–5199
Hurteau GJ, Carlson JA, Spivack SD, Brock GJ (2007) Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin. Cancer Res 67(17):7972–7976. doi:10.1158/0008-5472.can-07-1058
Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ (2008) The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 10(5):593–601. doi:10.1038/ncb1722
Savagner P (2001) Leaving the neighborhood: molecular mechanisms involved during epithelial–mesenchymal transition. BioEssays 23(10):912–923
Lombaerts M, van Wezel T, Philippo K, Dierssen JW, Zimmerman RM, Oosting J, van Eijk R, Eilers PH, van de WB, Cornelisse CJ, Cleton-Jansen AM (2006) E-cadherin transcriptional downregulation by promoter methylation but not mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines. Br J Cancer 94(5):661–671
Van Horssen R, Galjart N, Rens JA, Eggermont AM, ten Hagen TL (2006) Differential effects of matrix and growth factors on endothelial and fibroblast motility: application of a modified cell migration assay. J Cell Biochem 99(6):1536–1552
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82(13):1107–1112
Van Horssen R, ten Hagen TL (2011) Crossing barriers: the new dimension of 2D cell migration assays. J Cell Physiol 226(1):288–290. doi:10.1002/jcp.22330
Hollestelle A, Nagel JH, Smid M, Lam S, Elstrodt F, Wasielewski M, Ng SS, French PJ, Peeters JK, Rozendaal MJ, Riaz M, Koopman DG, Ten Hagen TL, de Leeuw BH, Zwarthoff EC, Teunisse A, van der Spek PJ, Klijn JG, Dinjens WN, Ethier SP, Clevers H, Jochemsen AG, den Bakker MA, Foekens JA, Martens JW, Schutte M (2010) Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines. Breast Cancer Res Treat 121(1):53–64. doi:10.1007/s10549-009-0460-8
Sisci D, Aquila S, Middea E, Gentile M, Maggiolini M, Mastroianni F, Montanaro D, Ando S (2004) Fibronectin and type IV collagen activate ERalpha AF-1 by c-Src pathway: effect on breast cancer cell motility. Oncogene 23(55):8920–8930
Friedl P, Alexander S (2011) Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147(5):992–1009
Ruoslahti E (1996) How cancer spreads. Sci Am 275(3):72–77
Steeg PS (2006) Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 12(8):895–904. doi:10.1038/nm1469
Sahai E (2007) Illuminating the metastatic process. Nat Rev Cancer 7(10):737–749. doi:10.1038/nrc2229
Handschuh G, Candidus S, Luber B, Reich U, Schott C, Oswald S, Becke H, Hutzler P, Birchmeier W, Hofler H, Becker KF (1999) Tumour-associated E-cadherin mutations alter cellular morphology, decrease cellular adhesion and increase cellular motility. Oncogene 18(30):4301–4312
Vos CB, Cleton-Jansen AM, Berx G, de Leeuw WJ, ter Haar NT, van Roy F, Cornelisse CJ, Peterse JL, van de Vijver MJ (1997) E-cadherin inactivation in lobular carcinoma in situ of the breast: an early event in tumorigenesis. Br J Cancer 76(9):1131–1133
Weigelt B, Horlings HM, Kreike B, Hayes MM, Hauptmann M, Wessels LF, de Jong D, Van de Vijver MJ, Van’t Veer LJ, Peterse JL (2008) Refinement of breast cancer classification by molecular characterization of histological special types. J Pathol 216(2):141–150. doi:10.1002/path.2407
Blick T, Widodo E, Hugo H, Waltham M, Lenburg ME, Neve RM, Thompson EW (2008) Epithelial mesenchymal transition traits in human breast cancer cell lines. Clin Exp Metastasis 25(6):629–642. doi:10.1007/s10585-008-9170-6
van de Wetering M, Barker N, Harkes IC, van der Heyden M, Dijk NJ, Hollestelle A, Klijn JG, Clevers H, Schutte M (2001) Mutant E-cadherin breast cancer cells do not display constitutive Wnt signaling. Cancer Res 61(1):278–284
Suyama K, Shapiro I, Guttman M, Hazan RB (2002) A signaling pathway leading to metastasis is controlled by N-cadherin and the FGF receptor. Cancer Cell 2(4):301–314
Hazan RB, Phillips GR, Qiao RF, Norton L, Aaronson SA (2000) Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol 148(4):779–790
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.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10549-012-2261-8