Breast Cancer Research and Treatment

, Volume 142, Issue 3, pp 537–548 | Cite as

Tamoxifen selects for breast cancer cells with mammosphere forming capacity and increased growth rate

  • Diego Raffo
  • Damian E. Berardi
  • Osvaldo Pontiggia
  • Laura Todaro
  • Elisa Bal de Kier Joffé
  • Marina SimianEmail author
Preclinical study


Using the M05 mouse mammary tumor model and the MCF-7 cell line, we investigated the effect of tamoxifen treatment on the fraction of breast cancer cells with self-renewing capacity both in vitro and in vivo. We found that pretreatment with 4-OH-tamoxifen leads to an increase in cells with the ability of forming mammospheres that express lower levels of ER-α and increased expression of transcription factors associated with pluripotency. Moreover, exposure on plastic to 4-OH-tamoxifen by itself leads to an upregulation of these transcription factors. M05 tumors grown in mice treated with tamoxifen have a higher percentage of cells with self-renewing capacity and this proportion is conserved when tumors are passaged to nontreated mice. Furthermore, interruption of tamoxifen leads to increased tumor growth compared to tumors grown in mice that were never exposed to the antiestrogen. In addition, these tumors are characterized by a higher number of CD24lCD29h cells compared to tumors grown in nontreated mice. Treatment in vitro with 4-OH-tamoxifen for 5 days leads to a long lasting increase in the proportion of cells with self-renewing capacity even after 1 month of growth in the absence of the antiestrogen. Finally, we compared the mammosphere forming capacity of hormone dependent and independent passages of the M05 tumor and found that hormone independence is associated to an increase in cells with self-renewing capacity. Our results support previous findings that suggest that endocrine treatment selects for cells with stem cell properties.


Breast cancer Tamoxifen resistance Stem cells Self-renewing capacity Estrogen receptor 



This work is supported by Grants from MINCYT (PICT 2008-0325) and Florencio Fiorini Foundation to MS, Conicet PIP 112 20110100557 grant to LT, and PICT 2010-01296 (MINCYT) and M00243 (UBACyT) to EBKJ.

Conflict of interests

The authors declare they have no competing interests.

Supplementary material

10549_2013_2760_MOESM1_ESM.eps (54 mb)
Fig. S1 Quantification of the changes in levels of transcription factors associated to stemness. a Densitometry of RT-PCR results shown in Fig. 2a corresponding to MCF-7 cells grown on plastic and treated with 4-OH-tamoxifen (2D-4-OH-TAM) or grown as mammospheres. Values are expressed as arbitrary units (A.U.) relative to the levels of GADPH. They are normalized against the bands corresponding to the cells grown on 2D in the absence of 4-OH-Tamoxifen (2D). b The same analysis was carried out for the results obtained with LM05-E cells. (EPS 55300 kb)


  1. 1.
    Sengupta S, Jordan VC (2008) Selective estrogen modulators as an anticancer tool: mechanisms of efficiency and resistance. Adv Exp Med Biol 630:206–219PubMedCrossRefGoogle Scholar
  2. 2.
    Osborne CK, Schiff R (2011) Mechanisms of endocrine resistance in breast cancer. Annu Rev Med 62:233–247PubMedCrossRefGoogle Scholar
  3. 3.
    Davies C, Pan H, Godwin J, Gray R, Arriagada R, Raina V, Abraham M, Medeiros Alencar VH, Badran A, Bonfill X, Bradbury J, Clarke M, Collins R, Davis SR, Delmestri A, Forbes JF, Haddad P, Hou MF, Inbar M, Khaled H, Kielanowska J, Kwan WH, Mathew BS, Mittra I, Muller B, Nicolucci A, Peralta O, Pernas F, Petruzelka L, Pienkowski T, Radhika R, Rajan B, Rubach MT, Tort S, Urrutia G, Valentini M, Wang Y, Peto R, Adjuvant Tamoxifen: Longer Against Shorter Collaborative G (2013) Long-term effects of continuing adjuvant tamoxifen to 10 years versus stopping at 5 years after diagnosis of oestrogen receptor-positive breast cancer: ATLAS, a randomised trial. Lancet 381(9869):805–816. doi: 10.1016/S0140-6736(12)61963-1 Google Scholar
  4. 4.
    Ferrari P, Nicolini A, Carpi A (2013) Targeted therapies of metastatic breast cancer: relationships with cancer stem cells. Biomed Pharmacother 67(6):543–555. doi: 10.1016/j.biopha.2013.03.006 PubMedCrossRefGoogle Scholar
  5. 5.
    Eyler CE, Rich JN (2008) Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. J Clin Oncol 26(17):2839–2845PubMedCrossRefGoogle Scholar
  6. 6.
    Fillmore CM, Gupta PB, Rudnick JA, Caballero S, Keller PJ, Lander ES, Kuperwasser C (2010) Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling. Proc Natl Acad Sci USA 107(50):21737–21742. doi: 10.1073/pnas.1007863107 PubMedCrossRefGoogle Scholar
  7. 7.
    Simoes BM, Piva M, Iriondo O, Comaills V, Lopez-Ruiz JA, Zabalza I, Mieza JA, Acinas O, Vivanco MD (2011) Effects of estrogen on the proportion of stem cells in the breast. Breast Cancer Res Treat 129(1):23–35. doi: 10.1007/s10549-010-1169-4 PubMedCrossRefGoogle Scholar
  8. 8.
    Ao A, Morrison BJ, Wang H, Lopez JA, Reynolds BA, Lu J (2011) Response of estrogen receptor-positive breast cancer tumorspheres to antiestrogen treatments. PLoS One 6(4):e18810. doi: 10.1371/journal.pone.0018810 PubMedCrossRefGoogle Scholar
  9. 9.
    Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, Rimm DL, Wong H, Rodriguez A, Herschkowitz JI, Fan C, Zhang X, He X, Pavlick A, Gutierrez MC, Renshaw L, Larionov AA, Faratian D, Hilsenbeck SG, Perou CM, Lewis MT, Rosen JM, Chang JC (2009) Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA 106(33):13820–13825. doi: 10.1073/pnas.0905718106 PubMedCrossRefGoogle Scholar
  10. 10.
    Simian M, Manzur T, Rodriguez V, de Kier Joffe EB, Klein S (2009) A spontaneous estrogen dependent, tamoxifen sensitive mouse mammary tumor: a new model system to study hormone-responsiveness in immune competent mice. Breast Cancer Res Treat 113(1):1–8PubMedCrossRefGoogle Scholar
  11. 11.
    Pontiggia O, Rodriguez V, Fabris V, Raffo D, Bumaschny V, Fiszman G, de Kier Joffe EB, Simian M (2009) Establishment of an in vitro estrogen-dependent mouse mammary tumor model: a new tool to understand estrogen responsiveness and development of tamoxifen resistance in the context of stromal-epithelial interactions. Breast Cancer Res Treat 116(2):247–255PubMedCrossRefGoogle Scholar
  12. 12.
    Pontiggia O, Sampayo R, Raffo D, Motter A, Xu R, Bissell MJ, de Kier Joffe EB, Simian M (2012) The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through beta1 integrin. Breast Cancer Res Treat 133(2):459–471. doi: 10.1007/s10549-011-1766-x PubMedCrossRefGoogle Scholar
  13. 13.
    Dontu G, Wicha MS (2005) Survival of mammary stem cells in suspension culture: implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia 10(1):75–86PubMedCrossRefGoogle Scholar
  14. 14.
    Lengerke C, Fehm T, Kurth R, Neubauer H, Scheble V, Muller F, Schneider F, Petersen K, Wallwiener D, Kanz L, Fend F, Perner S, Bareiss PM, Staebler A (2011) Expression of the embryonic stem cell marker SOX2 in early-stage breast carcinoma. BMC Cancer 11:42. doi: 10.1186/1471-2407-11-42 PubMedCrossRefGoogle Scholar
  15. 15.
    Zhang L, Luo YB, Bou G, Kong QR, Huan YJ, Zhu J, Wang JY, Li H, Wang F, Shi YQ, Wei YC, Liu ZH (2011) Overexpression Nanog activates pluripotent genes in porcine fetal fibroblasts and nuclear transfer embryos. Anat Rec (Hoboken) 294(11):1809–1817. doi: 10.1002/ar.21457 CrossRefGoogle Scholar
  16. 16.
    Institute of Laboratory Animal Resources CoLSNRC (1996) Guide for the care and use of laboratory animals national. Academy Press, Washington, DCGoogle Scholar
  17. 17.
    Rios-Doria J, Day KC, Kuefer R, Rashid MG, Chinnaiyan AM, Rubin MA, Day ML (2003) The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells. J Biol Chem 278(2):1372–1379PubMedCrossRefGoogle Scholar
  18. 18.
    Simian M, Molinolo A, Lanari C (2006) Involvement of matrix metalloproteinase activity in hormone-induced mammary tumor regression. Am J Pathol 168(1):270–279. doi: 10.2353/ajpath.2006.050012 PubMedCrossRefGoogle Scholar
  19. 19.
    Sleeman KE, Kendrick H, Robertson D, Isacke CM, Ashworth A, Smalley MJ (2007) Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland. J Cell Biol 176(1):19–26PubMedCrossRefGoogle Scholar
  20. 20.
    Taddei I, Deugnier MA, Faraldo MM, Petit V, Bouvard D, Medina D, Fassler R, Thiery JP, Glukhova MA (2008) [beta]1 Integrin deletion from the basal compartment of the mammary epithelium affects stem cells. Nat Cell Biol 10(6):716–722PubMedCrossRefGoogle Scholar
  21. 21.
    Martin LA, Farmer I, Johnston SR, Ali S, Marshall C, Dowsett M (2003) Enhanced estrogen receptor (ER) alpha, ERBB2, and MAPK signal transduction pathways operate during the adaptation of MCF-7 cells to long term estrogen deprivation. J Biol Chem 278(33):30458–30468. doi: 10.1074/jbc.M305226200 PubMedCrossRefGoogle Scholar
  22. 22.
    Polyak K, Weinberg RA (2009) Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 9(4):265–273. doi: 10.1038/nrc2620 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Diego Raffo
    • 1
  • Damian E. Berardi
    • 1
  • Osvaldo Pontiggia
    • 1
  • Laura Todaro
    • 1
  • Elisa Bal de Kier Joffé
    • 1
  • Marina Simian
    • 1
    Email author
  1. 1.Research Area, Área InvestigaciónInstituto de Oncología “Angel H. Roffo”Buenos AiresArgentina

Personalised recommendations