Clinical & Experimental Metastasis

, Volume 28, Issue 6, pp 515–527 | Cite as

Mena invasive (MenaINV) and Mena11a isoforms play distinct roles in breast cancer cell cohesion and association with TMEM

  • Evanthia T. Roussos
  • Sumanta Goswami
  • Michele Balsamo
  • Yarong Wang
  • Robert Stobezki
  • Esther Adler
  • Brian D. Robinson
  • Joan G. Jones
  • Frank B. Gertler
  • John S. Condeelis
  • Maja H. Oktay
Research Paper


Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the Mena invasive (MenaINV) and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-MenaINV expressing tumor cells are significantly less cohesive and have discontinuous cell–cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more MenaINV mRNA than early non-metastatic ones. Furthermore, MenaINV expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that MenaINV is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that MenaINV expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk.


Breast cancer Metastasis Cell motility Intravasation TMEM 



Epidermal growth factor


Enhanced green fluorescent protein


Fluorescence activated cell sorting


Formalin-fixed paraffin-embedded


Fine needle aspiration






Intravital imaging


Quantitative real time polymerase chain reaction


Severe combined immunodeficiency


Tumor microenvironment for metastasis


Average primary tumor cells

Supplementary material

10585_2011_9388_MOESM1_ESM.docx (817 kb)
Supplementary material 1 (DOCX 816 kb)


  1. 1.
    Sahai E et al (2005) Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy. BMC Biotechnol 5:14PubMedCrossRefGoogle Scholar
  2. 2.
    Condeelis J, Singer RH, Segall JE (2005) The great escape: when cancer cells hijack the genes for chemotaxis and motility. Annu Rev Cell Dev Biol 21:695–718PubMedCrossRefGoogle Scholar
  3. 3.
    Wang W et al (2004) Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors. Cancer Res 64(23):8585–8594PubMedCrossRefGoogle Scholar
  4. 4.
    Heimann R et al (2000) Separating favorable from unfavorable prognostic markers in breast cancer: the role of E-cadherin. Cancer Res 60(2):298–304PubMedGoogle Scholar
  5. 5.
    Weigelt B, Peterse JL, van ‘t Veer LJ (2005) Breast cancer metastasis: markers and models. Nat Rev Cancer 5(8):591–602PubMedCrossRefGoogle Scholar
  6. 6.
    Di Modugno F et al (2007) Molecular cloning of hMena (ENAH) and its splice variant hMena+11a: epidermal growth factor increases their expression and stimulates hMena+11a phosphorylation in breast cancer cell lines. Cancer Res 67(6):2657–2665PubMedCrossRefGoogle Scholar
  7. 7.
    Gertler F, Condeelis J (2010) Metastasis: tumor cells becoming MENAcing. Trends Cell Biol 21(2):81–90PubMedCrossRefGoogle Scholar
  8. 8.
    Goswami S et al (2009) Identification of invasion specific splice variants of the cytoskeletal protein Mena present in mammary tumor cells during invasion in vivo. Clin Exp Metastasis 26(2):153–159PubMedCrossRefGoogle Scholar
  9. 9.
    Robinson BD et al (2009) Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination. Clin Cancer Res 15(7):2433–2441PubMedCrossRefGoogle Scholar
  10. 10.
    Di Modugno F et al (2006) The cytoskeleton regulatory protein hMena (ENAH) is overexpressed in human benign breast lesions with high risk of transformation and human epidermal growth factor receptor-2-positive/hormonal receptor-negative tumors. Clin Cancer Res 12(5):1470–1478PubMedCrossRefGoogle Scholar
  11. 11.
    Gurzu S et al (2008) The expression of cytoskeleton regulatory protein Mena in colorectal lesions. Rom J Morphol Embryol 49(3):345–349PubMedGoogle Scholar
  12. 12.
    Gurzu S et al (2009) The immunohistochemical aspects of protein Mena in cervical lesions. Rom J Morphol Embryol 50(2):213–216PubMedGoogle Scholar
  13. 13.
    Gertler FB et al (1996) Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87(2):227–239PubMedCrossRefGoogle Scholar
  14. 14.
    Bear JE et al (2000) Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101(7):717–728PubMedCrossRefGoogle Scholar
  15. 15.
    Drees F, Gertler FB (2008) Ena/VASP: proteins at the tip of the nervous system. Curr Opin Neurobiol 18(1):53–59PubMedCrossRefGoogle Scholar
  16. 16.
    Neel NF et al (2009) VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis. J Cell Sci 122(Pt 11):1882–1894PubMedCrossRefGoogle Scholar
  17. 17.
    Philippar U et al (2008) A Mena invasion isoform potentiates EGF-induced carcinoma cell invasion and metastasis. Dev Cell 15(6):813–828PubMedCrossRefGoogle Scholar
  18. 18.
    Urbanelli L et al (2006) Characterization of human Enah gene. Biochim Biophys Acta 1759(1–2):99–107PubMedGoogle Scholar
  19. 19.
    Welch DR, Neri A, Nicolson GL (1983) Comparison of ‘spontaneous’ and ‘experimental’ metastasis using rat 13762 mammary adenocarcinoma metastatic cell clones. Invasion Metastasis 3(2):65–80PubMedGoogle Scholar
  20. 20.
    Neri A et al (1982) Development and biologic properties of malignant cell sublines and clones of a spontaneously metastasizing rat mammary adenocarcinoma. J Natl Cancer Inst 68(3):507–517PubMedGoogle Scholar
  21. 21.
    Roussos ET, Balsamo M, Alford SK, Wyckoff JB, Gligorijevic B, Wang Y, Pozzuto M, Stobezki R, Goswami S, Segall JE, Lauffenburger DA, Bresnick AR, Gertler FB, Condeelis JS (2011) Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer. J Cell Sci. doi:10.1242/JCS086231 PubMedGoogle Scholar
  22. 22.
    Wyckoff J et al (2004) A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res 64(19):7022–7029PubMedCrossRefGoogle Scholar
  23. 23.
    Wang W et al (2002) Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling. Cancer Res 62(21):6278–6288PubMedGoogle Scholar
  24. 24.
    Wyckoff J et al (2010) High resolution multi-photon imaging of tumors in vivo. In: Goldman RD, Swedlow JR, Spector DL (eds) Live cell imaging; a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 441–462Google Scholar
  25. 25.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21(11):1369–1377PubMedCrossRefGoogle Scholar
  26. 26.
    Gupta PK, Baloch ZW (2002) Intraoperative and on-site cytopathology consultation: utilization, limitations, and value. Semin Diagn Pathol 19(4):227–236PubMedGoogle Scholar
  27. 27.
    Mouneimne G et al (2004) Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation. J Cell Biol 166(5):697–708PubMedCrossRefGoogle Scholar
  28. 28.
    Loureiro JJ et al (2002) Critical roles of phosphorylation and actin binding motifs, but not the central proline-rich region, for Ena/vasodilator-stimulated phosphoprotein (VASP) function during cell migration. Mol Biol Cell 13(7):2533–2546PubMedCrossRefGoogle Scholar
  29. 29.
    Lebrand C et al (2004) Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron 42(1):37–49PubMedCrossRefGoogle Scholar
  30. 30.
    Pino MS et al (2008) Human Mena+11a isoform serves as a marker of epithelial phenotype and sensitivity to epidermal growth factor receptor inhibition in human pancreatic cancer cell lines. Clin Cancer Res 14(15):4943–4950PubMedCrossRefGoogle Scholar
  31. 31.
    Lin EY et al (2003) Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol 163(5):2113–2126PubMedCrossRefGoogle Scholar
  32. 32.
    Symmans WF et al (2003) Total RNA yield and microarray gene expression profiles from fine-needle aspiration biopsy and core-needle biopsy samples of breast carcinoma. Cancer 97(12):2960–2971PubMedCrossRefGoogle Scholar
  33. 33.
    Di Modugno F et al (2004) Human Mena protein, a serex-defined antigen overexpressed in breast cancer eliciting both humoral and CD8+ T-cell immune response. Int J Cancer 109(6):909–918PubMedCrossRefGoogle Scholar
  34. 34.
    Giampieri S et al (2009) Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol 11(11):1287–1296PubMedCrossRefGoogle Scholar
  35. 35.
    Zeineldin R et al (2006) Mesenchymal transformation in epithelial ovarian tumor cells expressing epidermal growth factor receptor variant III. Mol Carcinog 45(11):851–860PubMedCrossRefGoogle Scholar
  36. 36.
    Guy CT, Cardiff RD, Muller WJ (1992) Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol 12(3):954–961PubMedGoogle Scholar
  37. 37.
    Lin EY et al (2001) Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med 193(6):727–740PubMedCrossRefGoogle Scholar
  38. 38.
    Wyckoff JB et al (2000) A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. Cancer Res 60(9):2504–2511PubMedGoogle Scholar
  39. 39.
    Wyckoff JB et al (2007) Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res 67(6):2649–2656PubMedCrossRefGoogle Scholar
  40. 40.
    Wang W et al (2005) Tumor cells caught in the act of invading: their strategy for enhanced cell motility. Trends Cell Biol 15(3):138–145PubMedCrossRefGoogle Scholar
  41. 41.
    Wang W et al (2007) Coordinated regulation of pathways for enhanced cell motility and chemotaxis is conserved in rat and mouse mammary tumors. Cancer Res 67(8):3505–3511PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Evanthia T. Roussos
    • 1
  • Sumanta Goswami
    • 1
    • 4
  • Michele Balsamo
    • 3
  • Yarong Wang
    • 1
  • Robert Stobezki
    • 4
  • Esther Adler
    • 7
  • Brian D. Robinson
    • 5
  • Joan G. Jones
    • 6
  • Frank B. Gertler
    • 3
  • John S. Condeelis
    • 1
    • 2
  • Maja H. Oktay
    • 7
  1. 1.Department of Anatomy and Structural BiologyAlbert Einstein College of MedicineBronxUSA
  2. 2.Gruss Lipper Biophotonics CenterAlbert Einstein College of MedicineBronxUSA
  3. 3.David H Koch Institute for Integrative Cancer ResearchMassachusetts Institute of TechnologyCambridgeUSA
  4. 4.Department of BiologyYeshiva UniversityNew YorkUSA
  5. 5.Department of PathologyJohns Hopkins HospitalBaltimoreUSA
  6. 6.Department of Pathology and Laboratory MedicineWeill Cornell Medical CollegeNew YorkUSA
  7. 7.Department of PathologyMontefiore Medical CenterBronxUSA

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