Skip to main content

Advertisement

SpringerLink
Log in

Characterization of the expression of the pro-metastatic MenaINV isoform during breast tumor progression

  • Research Paper
  • Published:
Clinical & Experimental Metastasis Aims and scope Submit manuscript

Abstract

Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the MenaINV isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed MenaINV within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a MenaINV isoform-specific monoclonal antibody and used it to examine MenaINV expression patterns in mouse mammary and human breast tumors. MenaINV expression increases during tumor progression and to examine the relationship between MenaINV expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while MenaINV robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-MenaINV-isoform specific antibody, and provide the first description of endogenous MenaINV protein expression in mouse and human tumors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

DCIS:

Ductal carcinoma in situ

EGF:

Epidermal growth factor

EMT:

Epithelial to mesenchymal transition

FFPE:

Formalin fixed paraffin embedded

GFP:

Green fluorescent protein

IP:

Immunoprecipitation

LN:

Lymph node

SMA:

Smooth muscle actin

TMEM:

Tumor microenvironment of metastasis

References

  1. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674

    Article  CAS  PubMed  Google Scholar 

  2. Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SFT, Csiszar K, Giaccia A, Weninger W, Yamauchi M, Gasser DL, Weaver VM (2009) Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139(5):891–906

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lu P, Weaver VM, Werb Z (2012) The extracellular matrix: a dynamic niche in cancer progression. J Cell Biol 196(4):395–406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Pouysségur J, Dayan F, Mazure NM (2006) Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441(7092):437–443

    Article  PubMed  Google Scholar 

  5. Joyce JA, Pollard JW (2009) Microenvironmental regulation of metastasis. Nat Rev Cancer 9(4):239–252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gertler F, Condeelis J (2011) Metastasis: tumor cells becoming MENAcing. Trends Cell Biol 21(2):81–90

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Roussos ET, Wang Y, Wyckoff JB, Sellers RS, Wang W, Li J, Pollard JW, Gertler FB, Condeelis JS (2010) Mena deficiency delays tumor progression and decreases metastasis in polyoma middle-T transgenic mouse mammary tumors. Breast Cancer Res 12(6):R101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Harney AS, Arwert EN, Entenberg D, Wang Y, Guo P, Qian B-Z, Oktay MH, Pollard JW, Jones JG, Condeelis JS (2015) Real-time imaging reveals local, transient vascular permeability, and tumor cell intravasation stimulated by TIE2hi macrophage-derived VEGFA. Cancer Discov

  9. Robinson BD, Sica GL, Liu Y-F, Rohan TE, Gertler FB, Condeelis JS, Jones JG (2009) Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination. Clin Cancer Res 15(7):2433–2441

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rohan TE, Xue X, Lin H-M, D’Alfonso TM, Ginter PS, Oktay MH, Robinson BD, Ginsberg M, Gertler FB, Glass AG, Sparano JA, Condeelis JS, Jones JG (2014) Tumor microenvironment of metastasis and risk of distant metastasis of breast cancer. J Natl Cancer Inst 106(8):1–11

    Article  Google Scholar 

  11. Goswami S, Philippar U, Sun D, Patsialou A, Avraham J, Wang W, Di Modugno F, Nistico P, Gertler FB, Condeelis JS (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):153–159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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 124(Pt 13):2120–2131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Agarwal S, Gertler FB, Balsamo M, Condeelis JS, Camp RL, Xue X, Lin J, Rohan TE, Rimm DL (2012) Quantitative assessment of invasive mena isoforms (Menacalc) as an independent prognostic marker in breast cancer. Breast Cancer Res 14(5):R124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Forse CL, Agarwal S, Pinnaduwage D, Gertler F, Condeelis JS, Lin J, Xue X, Johung K, Mulligan AM, Rohan TE, Bull SB, Andrulis IL (2015) Menacalc, a quantitative method of metastasis assessment, as a prognostic marker for axillary node-negative breast cancer. BMC Cancer 15:483

    Article  PubMed  PubMed Central  Google Scholar 

  15. Philippar U, Roussos ET, Oser M, Yamaguchi H, Kim H-D, Giampieri S, Wang Y, Goswami S, Wyckoff JB, Lauffenburger DA, Sahai E, Condeelis JS, Gertler FB (2008) A mena invasion isoform potentiates EGF-induced carcinoma cell invasion and metastasis. Dev Cell 15(6):813–828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pignatelli J, Goswami S, Jones JG, Rohan TE, Pieri E, Chen X, Adler E, Cox D, Maleki S, Bresnick A, Gertler FB, Condeelis JS, Oktay MH (2014) Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration. Sci Signal 7(353):ra112

    Article  PubMed  PubMed Central  Google Scholar 

  17. Roussos ET, Goswami S, Balsamo M, Wang Y, Stobezki R, Adler E, Robinson BD, Jones JG, Gertler FB, Condeelis JS, Oktay MH (2011) Mena invasive (Mena(INV)) and Mena11a isoforms play distinct roles in breast cancer cell cohesion and association with TMEM. Clin Exp Metastasis 28(6):515–527

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gertler FB, Niebuhr K, Reinhard M, Wehland J, Soriano P (1996) Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87(2):227–239

    Article  CAS  PubMed  Google Scholar 

  19. Lebrand C, Dent EW, Strasser GA, Lanier LM, Krause M, Svitkina TM, Borisy GG, Gertler FB (2004) Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron 42:37–49

    Article  CAS  PubMed  Google Scholar 

  20. Bear JEJE, Loureiro JJJJ, Libova II, Fässler RR, Wehland JJ, Gertler FBFB (2000) Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101(7):717–728

    Article  CAS  PubMed  Google Scholar 

  21. Goswami S, Philippar U, Sun D, Patsialou A, Avraham J, Wang W, Di Modugno F, Nistico P, Gertler FB, Condeelis JS (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):153–159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, Pollard JW (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):2113–2126

    Article  PubMed  PubMed Central  Google Scholar 

  23. Valastyan S, Weinberg RA (2011) Tumor metastasis: molecular insights and evolving paradigms. Cell 147:275–292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Chaffer CL, Marjanovic ND, Lee T, Bell G, Kleer CG, Reinhardt F, D’Alessio AC, Young RA, Weinberg RA (2013) Poised chromatin at the ZEB1 promoter enables breast cancer cell plasticity and enhances tumorigenicity. Cell 154(1):61–74

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Brugnoli F, Grassilli S, Piazzi M, Palomba M, Nika E, Bavelloni A, Capitani S, Bertagnolo V (2013) In triple negative breast tumor cells, PLC-β2 promotes the conversion of CD133high to CD133low phenotype and reduces the CD133-related invasiveness. Mol Cancer 12:165

    Article  PubMed  PubMed Central  Google Scholar 

  26. Chen Y-C, Chen Y-W, Hsu H-S, Tseng L-M, Huang P-I, Lu K-H, Chen D-T, Tai L-K, Yung M-C, Chang S-C, Ku H-H, Chiou S-H, Lo W-L (2009) Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer. Biochem Biophys Res Commun 385(3):307–313

    Article  CAS  PubMed  Google Scholar 

  27. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1(5):555–567

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Murphy PA, Kim TN, Lu G, Bollen AW, Schaffer CB, Wang RA (2012) Notch4 normalization reduces blood vessel size in arteriovenous malformations. Sci Transl Med 4(117):117ra8

    Article  PubMed  PubMed Central  Google Scholar 

  29. Roussos ET, Goswami S, Balsamo M, Wang Y, Stobezki R, Adler E, Robinson BD, Jones JG, Gertler FB, Condeelis JS, Oktay MH (2011) Mena invasive (Mena(INV)) and Mena11a isoforms play distinct roles in breast cancer cell cohesion and association with TMEM. Clin Exp Metastasis 28(6):515–527

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shapiro IM, Cheng AW, Flytzanis NC, Balsamo M, Condeelis JS, Oktay MH, Burge CB, Gertler FB (2011) An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. PLoS Genet 7(8):e1002218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Di Modugno F, Iapicca P, Boudreau A, Mottolese M, Terrenato I, Perracchio L, Carstens RP, Santoni A, Bissell MJ, Nisticò P (2012) Splicing program of human MENA produces a previously undescribed isoform associated with invasive, mesenchymal-like breast tumors. Proc Natl Acad Sci USA 109(47):19280–19285

    Article  PubMed  PubMed Central  Google Scholar 

  32. Zardavas D, Baselga J, Piccart M (2013) Emerging targeted agents in metastatic breast cancer. Nat Rev Clin Oncol 10(4):191–210

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Department of Defense Breast Cancer Research Program Grants W81XWH-10-1-0040 to SKH and W81XWH-13-1-0031 to MJO, NIH Grant U54-CA112967 to FBG and DAL and GM58801 to FBG, funds from the Ludwig Center at MIT to FBG and NR, the KI NCI Core Grant P30-CA14051, and CA150344 to JC and CA100324 for JJ. JJ, JC and FG are compensated members if the scientific advisory board of MetaStat. No funding was provided by MetaStat for this work. We thank the Histology facilities in the KI Swanson Biotechnology Center for support. We thank Evanthia Roussos for gifts of tumor tissue.

Author contributions

MJO designed and performed experiments, data analysis and prepared the manuscript. SKH developed and validated the INV antibody, designed and performed experiments and completed data analysis. NR performed immunostaining experiments and image analysis. MNM developed MenaINV ELISA, performed experiments and completed data analysis. FBG and DAL guided overall experimental design. JJ and JC contributed human tissue sections and interpretation of them. All authors commented on the manuscript.

Author information

Author notes

    Authors and Affiliations

    1. Koch Institute for Integrative Cancer Research, MIT, 76-317, 77 Massachusetts Ave, Cambridge, MA, 02139, USA

      Madeleine J. Oudin, Shannon K. Hughes, Nazanin Rohani, Douglas A. Lauffenburger & Frank B. Gertler

    2. Department of Biological Engineering, MIT, Cambridge, MA, 02139, USA

      Shannon K. Hughes, Mira N. Moufarrej & Douglas A. Lauffenburger

    3. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA

      John S. Condeelis

    4. Department of Biology, MIT, Cambridge, MA, 02139, USA

      Frank B. Gertler

    5. Integrated Imaging Program, Albert Einstein College of Medicine, Bronx, NY, 10461, USA

      Joan G. Jones

    Authors
    1. Madeleine J. Oudin
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Shannon K. Hughes
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Nazanin Rohani
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Mira N. Moufarrej
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Joan G. Jones
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. John S. Condeelis
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Douglas A. Lauffenburger
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Frank B. Gertler
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Madeleine J. Oudin.

    Additional information

    Madeleine J. Oudin and Shannon K. Hughes have contributed equally to this work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    Figure S1

    Table with characteristics of human tumors. Table summarizing clinical features of human breast cancer tumors used for immunohistochemical analyses: tumor size, ER, PR or Her2 status, lymph node (LN) positivity, and site of metastasis (PDF 188 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Oudin, M.J., Hughes, S.K., Rohani, N. et al. Characterization of the expression of the pro-metastatic MenaINV isoform during breast tumor progression. Clin Exp Metastasis 33, 249–261 (2016). https://doi.org/10.1007/s10585-015-9775-5

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10585-015-9775-5

    Keywords

    Search

    Navigation