Advertisement

Clinical & Experimental Metastasis

, Volume 33, Issue 3, pp 249–261 | Cite as

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

  • Madeleine J. Oudin
  • Shannon K. Hughes
  • Nazanin Rohani
  • Mira N. Moufarrej
  • Joan G. Jones
  • John S. Condeelis
  • Douglas A. Lauffenburger
  • Frank B. Gertler
Research Paper

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.

Keywords

Breast cancer Mena Hypoxia Stemness Microenvironment Metastasis 

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

Notes

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.

Supplementary material

10585_2015_9775_MOESM1_ESM.pdf (188 kb)
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)

References

  1. 1.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674CrossRefPubMedGoogle Scholar
  2. 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):891–906CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Lu P, Weaver VM, Werb Z (2012) The extracellular matrix: a dynamic niche in cancer progression. J Cell Biol 196(4):395–406CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Pouysségur J, Dayan F, Mazure NM (2006) Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441(7092):437–443CrossRefPubMedGoogle Scholar
  5. 5.
    Joyce JA, Pollard JW (2009) Microenvironmental regulation of metastasis. Nat Rev Cancer 9(4):239–252CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gertler F, Condeelis J (2011) Metastasis: tumor cells becoming MENAcing. Trends Cell Biol 21(2):81–90CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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):R101CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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 DiscovGoogle Scholar
  9. 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):2433–2441CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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):1–11CrossRefGoogle Scholar
  11. 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):153–159CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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):2120–2131CrossRefPubMedPubMedCentralGoogle Scholar
  13. 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):R124CrossRefPubMedPubMedCentralGoogle Scholar
  14. 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 15:483CrossRefPubMedPubMedCentralGoogle Scholar
  15. 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):813–828CrossRefPubMedPubMedCentralGoogle Scholar
  16. 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):ra112CrossRefPubMedPubMedCentralGoogle Scholar
  17. 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):515–527CrossRefPubMedPubMedCentralGoogle Scholar
  18. 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):227–239CrossRefPubMedGoogle Scholar
  19. 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 42:37–49CrossRefPubMedGoogle Scholar
  20. 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):717–728CrossRefPubMedGoogle Scholar
  21. 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):153–159CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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):2113–2126CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Valastyan S, Weinberg RA (2011) Tumor metastasis: molecular insights and evolving paradigms. Cell 147:275–292CrossRefPubMedPubMedCentralGoogle Scholar
  24. 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):61–74CrossRefPubMedPubMedCentralGoogle Scholar
  25. 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 12:165CrossRefPubMedPubMedCentralGoogle Scholar
  26. 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):307–313CrossRefPubMedGoogle Scholar
  27. 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):555–567CrossRefPubMedPubMedCentralGoogle Scholar
  28. 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):117ra8CrossRefPubMedPubMedCentralGoogle Scholar
  29. 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):515–527CrossRefPubMedPubMedCentralGoogle Scholar
  30. 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):e1002218CrossRefPubMedPubMedCentralGoogle Scholar
  31. 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):19280–19285CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Zardavas D, Baselga J, Piccart M (2013) Emerging targeted agents in metastatic breast cancer. Nat Rev Clin Oncol 10(4):191–210CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Madeleine J. Oudin
    • 1
  • Shannon K. Hughes
    • 1
    • 2
  • Nazanin Rohani
    • 1
  • Mira N. Moufarrej
    • 2
  • Joan G. Jones
    • 5
  • John S. Condeelis
    • 3
  • Douglas A. Lauffenburger
    • 1
    • 2
  • Frank B. Gertler
    • 1
    • 4
  1. 1.Koch Institute for Integrative Cancer ResearchMITCambridgeUSA
  2. 2.Department of Biological EngineeringMITCambridgeUSA
  3. 3.Department of Anatomy and Structural BiologyAlbert Einstein College of MedicineBronxUSA
  4. 4.Department of BiologyMITCambridgeUSA
  5. 5.Integrated Imaging ProgramAlbert Einstein College of MedicineBronxUSA

Personalised recommendations