Breast Cancer Research and Treatment

, Volume 138, Issue 1, pp 47–57 | Cite as

Loss of E-cadherin is not a necessity for epithelial to mesenchymal transition in human breast cancer

  • Antoinette Hollestelle
  • Justine K. Peeters
  • Marcel Smid
  • Mieke Timmermans
  • Leon C. Verhoog
  • Pieter J. Westenend
  • Anouk A. J. Heine
  • Alan Chan
  • Anieta M. Sieuwerts
  • Erik A. C. Wiemer
  • Jan G. M. Klijn
  • Peter J. van der Spek
  • John A. Foekens
  • Mieke Schutte
  • Michael A. den Bakker
  • John W. M. Martens
Preclinical Study


Epithelial to mesenchymal transition (EMT) is typically defined by the acquisition of a spindle cell morphology in combination with loss of E-cadherin and upregulation of mesenchymal markers. However, by studying E-cadherin inactivation in 38 human breast cancer cell lines, we noted that not all cell lines that had undergone EMT had concomitantly lost E-cadherin expression. We further investigated this discrepancy functionally and in clinical breast cancer specimens. Interestingly, reconstitution of wild-type E-cadherin cDNA in a E-cadherin negative cell line that had undergone EMT (MDA-MB-231) did not revert the spindle morphology back to an epithelial morphology. Neither were changes observed in the expression of several markers known to be involved in the EMT process. Similarly, upregulation of E-cadherin via global DNA demethylation in eleven cell lines that had undergone EMT did not induce a change in cell morphology, nor did it alter the expression of EMT markers in these cells. Next, we extracted genes differentially expressed between cell lines that had undergone EMT versus cell lines that had not undergone EMT. Caveolin-1 was identified to be an excellent marker for EMT, irrespective of E-cadherin status (specificity and sensitivity of 100 %). Consistent with our observations in the breast cancer cell lines, expression of Caveolin-1 identified a subset of basal breast cancers, particularly of metaplastic pathology, and only 50 % of these lacked E-cadherin expression. The discrepancy between E-cadherin loss and EMT was thus reproduced in clinical samples. Together, these results indicate that in human breast cancer loss of E-cadherin is not causal for EMT and even not a necessity.


E-cadherin EMT Spindle cell morphology Basal subtype Metaplastic breast cancer Caveolin-1 



We appreciate the assistance of members of the pathology immunohistochemistry labs. We thank Prof. Wolter Oosterhuis for insightful discussions. This work was supported by the Dutch Cancer Society, Erasmus MC Mrace and Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10549_2013_2415_MOESM1_ESM.doc (30 kb)
Supplementary material 1 (DOC 29 kb)
10549_2013_2415_MOESM2_ESM.doc (34 kb)
Supplementary material 2 (DOC 34 kb)
10549_2013_2415_MOESM3_ESM.doc (75 kb)
Supplementary material 3 (DOC 75 kb)
10549_2013_2415_MOESM4_ESM.xls (58 kb)
Supplementary material 4 (XLS 57 kb)
10549_2013_2415_MOESM5_ESM.xls (260 kb)
Supplementary material 5 (XLS 260 kb)
10549_2013_2415_MOESM6_ESM.xlsx (16 kb)
Supplementary material 6 (XLSX 15 kb)
10549_2013_2415_MOESM7_ESM.doc (36 kb)
Supplementary material 7 (DOC 36 kb)


  1. 1.
    Gumbiner BM (2005) Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev Mol Cell Biol 6:622–634PubMedCrossRefGoogle Scholar
  2. 2.
    van Roy F, Berx G (2008) The cell-cell adhesion molecule E-cadherin. Cell Mol Life Sci 65(23):3756–3788Google Scholar
  3. 3.
    Perez-Moreno M, Fuchs E (2006) Catenins: keeping cells from getting their signals crossed. Dev Cell 11:601–612PubMedCrossRefGoogle Scholar
  4. 4.
    Scott JA, Yap AS (2006) Cinderella no longer: alpha-catenin steps out of cadherin’s shadow. J Cell Sci 119:4599–4605PubMedCrossRefGoogle Scholar
  5. 5.
    Berx G, Becker KF, Hofler H, van Roy F (1998) Mutations of the human E-cadherin (CDH1) gene. Hum Mutat 12:226–237PubMedCrossRefGoogle Scholar
  6. 6.
    Lei H, Sjoberg-Margolin S, Salahshor S, Werelius B, Jandakova E, Hemminki K, Lindblom A, Vorechovsky I (2002) CDH1 mutations are present in both ductal and lobular breast cancer, but promoter allelic variants show no detectable breast cancer risk. Int J Cancer 98:199–204PubMedCrossRefGoogle Scholar
  7. 7.
    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 10:437–449PubMedCrossRefGoogle Scholar
  8. 8.
    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 55:5195–5199PubMedGoogle Scholar
  9. 9.
    Peinado H, Olmeda D, Cano A (2007) Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7:415–428PubMedCrossRefGoogle Scholar
  10. 10.
    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 10:593–601PubMedCrossRefGoogle Scholar
  11. 11.
    Bracken CP, Gregory PA, Kolesnikoff N, Bert AG, Wang J, Shannon MF, Goodall GJ (2008) A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res 68:7846–7854PubMedCrossRefGoogle Scholar
  12. 12.
    Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, Spaderna S, Brabletz T (2008) A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO Rep 9:582–589PubMedCrossRefGoogle Scholar
  13. 13.
    Sarrio D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J (2008) Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res 68:989–997PubMedCrossRefGoogle Scholar
  14. 14.
    Lien HC, Hsiao YH, Lin YS, Yao YT, Juan HF, Kuo WH, Hung MC, Chang KJ, Hsieh FJ (2007) Molecular signatures of metaplastic carcinoma of the breast by large-scale transcriptional profiling: identification of genes potentially related to epithelial-mesenchymal transition. Oncogene 26:7859–7871PubMedCrossRefGoogle Scholar
  15. 15.
    Hollestelle A, Elstrodt F, Nagel JH, Kallemeijn WW, Schutte M (2007) Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines. Mol Cancer Res 5:195–201PubMedCrossRefGoogle Scholar
  16. 16.
    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 122:125–133PubMedCrossRefGoogle Scholar
  17. 17.
    Sieuwerts AM, Meijer-van Gelder ME, Timmermans M, Trapman AM, Garcia RR, Arnold M, Goedheer AJ, Portengen H, Klijn JG, Foekens JA (2005) How ADAM-9 and ADAM-11 differentially from estrogen receptor predict response to tamoxifen treatment in patients with recurrent breast cancer: a retrospective study. Clin Cancer Res 11:7311–7321PubMedCrossRefGoogle Scholar
  18. 18.
    Wasielewski M, Elstrodt F, Klijn JG, Berns EM, Schutte M (2006) Thirteen new p53 gene mutants identified among 41 human breast cancer cell lines. Breast Cancer Res Treat 99:97–101PubMedCrossRefGoogle Scholar
  19. 19.
    Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116–5121PubMedCrossRefGoogle Scholar
  20. 20.
    Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102:15545–15550PubMedCrossRefGoogle Scholar
  21. 21.
    Shipitsin M, Campbell LL, Argani P, Weremowicz S, Bloushtain-Qimron N, Yao J, Nikolskaya T, Serebryiskaya T, Beroukhim R, Hu M, Halushka MK, Sukumar S, Parker LM, Anderson KS, Harris LN, Garber JE, Richardson AL, Schnitt SJ, Nikolsky Y, Gelman RS, Polyak K (2007) Molecular definition of breast tumor heterogeneity. Cancer Cell 11:259–273PubMedCrossRefGoogle Scholar
  22. 22.
    Pothof J, Verkaik NS, van IW, Wiemer EA, Ta VT, van der Horst GT, Jaspers NG, van Gent DC, Hoeijmakers JH, Persengiev SP (2009) MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response. EMBO J 28:2090–2099PubMedCrossRefGoogle Scholar
  23. 23.
    Hollestelle A, Nagel JHA, Smid M, Lam S, Elstrodt F, Wasielewski M, Ng S, French PJ, Peeters JK, Rozendaal MJ, Riaz M, Koopman DG, ten Hagen TLM, de Leeuw HCGM, Zwarthoff EC, Teunisse A, van der Spek PJ, Klijn JGM, Dinjens WNM, Ethier SP, Clevers H, Jochemsen AG, den Bakker MA, Foekens JA, Martens JWM, Schutte M (2010) Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines. Breast Cancer Res Treat 121:53–64PubMedCrossRefGoogle Scholar
  24. 24.
    Mahler-Araujo B, Savage K, Parry S, Reis-Filho JS (2008) Reduction of E-cadherin expression is associated with non-lobular breast carcinomas of basal-like and triple negative phenotype. J Clin Pathol 61:615–620PubMedCrossRefGoogle Scholar
  25. 25.
    Lindley LE, Briegel KJ (2010) Molecular characterization of TGFbeta-induced epithelial-mesenchymal transition in normal finite lifespan human mammary epithelial cells. Biochem Biophys Res Commun 399:659–664PubMedCrossRefGoogle Scholar
  26. 26.
    Mani SA, Yang J, Brooks M, Schwaninger G, Zhou A, Miura N, Kutok JL, Hartwell K, Richardson AL, Weinberg RA (2007) Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci USA 104:10069–10074PubMedCrossRefGoogle Scholar
  27. 27.
    Lombaerts M, van Wezel T, Philippo K, Dierssen JW, Zimmerman RM, Oosting J, van Eijk R, Eilers PH, van de Water B, 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 94:661–671PubMedGoogle Scholar
  28. 28.
    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 25:629–642PubMedCrossRefGoogle Scholar
  29. 29.
    Sieuwerts AM, Kraan J, Bolt J, van der Spoel P, Elstrodt F, Schutte M, Martens JW, Gratama JW, Sleijfer S, Foekens JA (2009) Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells. J Natl Cancer Inst 101:61–66PubMedCrossRefGoogle Scholar
  30. 30.
    Moreno-Bueno G, Cubillo E, Sarrio D, Peinado H, Rodriguez-Pinilla SM, Villa S, Bolos V, Jorda M, Fabra A, Portillo F, Palacios J, Cano A (2006) Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for Snail, Slug, and E47 factors in epithelial-mesenchymal transition. Cancer Res 66:9543–9556PubMedCrossRefGoogle Scholar
  31. 31.
    Bloushtain-Qimron N, Yao J, Snyder EL, Shipitsin M, Campbell LL, Mani SA, Hu M, Chen H, Ustyansky V, Antosiewicz JE, Argani P, Halushka MK, Thomson JA, Pharoah P, Porgador A, Sukumar S, Parsons R, Richardson AL, Stampfer MR, Gelman RS, Nikolskaya T, Nikolsky Y, Polyak K (2008) Cell type-specific DNA methylation patterns in the human breast. Proc Natl Acad Sci USA 105:14076–14081PubMedCrossRefGoogle Scholar
  32. 32.
    Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz JI, He X, Perou CM (2010) Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 12:R68PubMedCrossRefGoogle Scholar
  33. 33.
    Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133:704–715PubMedCrossRefGoogle Scholar
  34. 34.
    Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, Speed T, Spellman PT, DeVries S, Lapuk A, Wang NJ, Kuo WL, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527PubMedCrossRefGoogle Scholar
  35. 35.
    Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98:10869–10874PubMedCrossRefGoogle Scholar
  36. 36.
    Pinilla SM, Honrado E, Hardisson D, Benitez J, Palacios J (2006) Caveolin-1 expression is associated with a basal-like phenotype in sporadic and hereditary breast cancer. Breast Cancer Res Treat 99:85–90PubMedCrossRefGoogle Scholar
  37. 37.
    Rodriguez-Pinilla SM, Sarrio D, Honrado E, Moreno-Bueno G, Hardisson D, Calero F, Benitez J, Palacios J (2007) Vimentin and laminin expression is associated with basal-like phenotype in both sporadic and BRCA1-associated breast carcinomas. J Clin Pathol 60:1006–1012PubMedCrossRefGoogle Scholar
  38. 38.
    Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA (2008) Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 68:3645–3654PubMedCrossRefGoogle Scholar
  39. 39.
    Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez-Boussard T, Livasy C, Cowan D, Dressler L, Akslen LA, Ragaz J, Gown AM, Gilks CB, van de Rijn M, Perou CM (2004) Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10:5367–5374PubMedCrossRefGoogle Scholar
  40. 40.
    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 216:141–150PubMedCrossRefGoogle Scholar
  41. 41.
    Weigelt B, Kreike B, Reis-Filho JS (2009) Metaplastic breast carcinomas are basal-like breast cancers: a genomic profiling analysis. Breast Cancer Res Treat 117:273–280Google Scholar
  42. 42.
    Reis-Filho JS, Milanezi F, Steele D, Savage K, Simpson PT, Nesland JM, Pereira EM, Lakhani SR, Schmitt FC (2006) Metaplastic breast carcinomas are basal-like tumours. Histopathology 49:10–21PubMedCrossRefGoogle Scholar
  43. 43.
    van Horssen R, Hollestelle A, Rens JA, Eggermont AM, Schutte M, Ten Hagen TL (2012) E-cadherin promotor methylation and mutation are inversely related to motility capacity of breast cancer cells. Breast Cancer Res Treat 136(2):365–377Google Scholar
  44. 44.
    Dumont N, Wilson MB, Crawford YG, Reynolds PA, Sigaroudinia M, Tlsty TD (2008) Sustained induction of epithelial to mesenchymal transition activates DNA methylation of genes silenced in basal-like breast cancers. Proc Natl Acad Sci USA 105:14867–14872PubMedCrossRefGoogle Scholar
  45. 45.
    You JS, Jones PA (2012) Cancer genetics and epigenetics: two sides of the same coin? Cancer Cell 22:9–20PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Antoinette Hollestelle
    • 1
    • 3
    • 8
  • Justine K. Peeters
    • 2
  • Marcel Smid
    • 1
    • 3
  • Mieke Timmermans
    • 1
    • 3
  • Leon C. Verhoog
    • 4
  • Pieter J. Westenend
    • 5
  • Anouk A. J. Heine
    • 1
    • 3
  • Alan Chan
    • 6
    • 7
  • Anieta M. Sieuwerts
    • 1
    • 3
  • Erik A. C. Wiemer
    • 1
  • Jan G. M. Klijn
    • 1
  • Peter J. van der Spek
    • 2
  • John A. Foekens
    • 1
    • 3
  • Mieke Schutte
    • 1
  • Michael A. den Bakker
    • 4
  • John W. M. Martens
    • 1
    • 3
  1. 1.Department of Medical Oncology, Josephine Nefkens InstituteErasmus University Medical Center-Daniel den Hoed Cancer CenterRotterdamThe Netherlands
  2. 2.Department of BioinformaticsErasmus University Medical CenterRotterdamThe Netherlands
  3. 3.Cancer Genomics CentreRotterdamThe Netherlands
  4. 4.Department of Pathology, Josephine Nefkens InstituteErasmus University Medical CenterRotterdamThe Netherlands
  5. 5.Laboratory for PathologyDordrechtThe Netherlands
  6. 6.PamGene International B.V.s-HertogenboschThe Netherlands
  7. 7.PercurosLeidenThe Netherlands
  8. 8.Department of Medical Oncology, Josephine Nefkens InstituteErasmus University Medical CenterRotterdamThe Netherlands

Personalised recommendations