Breast Cancer Research and Treatment

, Volume 129, Issue 2, pp 623–628 | Cite as

Simultaneous measurement of ERα, HER2, and PhosphoERK1/2 in breast cancer cell lines by flow cytometry

  • Ulas Darda Bayraktar
  • Tae Kon Kim
  • Katherine Drews-Elger
  • Cara Benjamin
  • Dorraya El-Ashry
  • Eric Wieder
  • Krishna V. Komanduri
Brief Report


The activation of human epidermal growth factor receptor-2 (HER2) results in the activation of the mitogen-activated protein kinase (MAPK) cascade that may lead to the resistance to anti-estrogen therapy in estrogen receptor (ERα) expressing breast cancer by means of phosphorylation of ERα in the N-terminal region by phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and by means of decreasing ERα expression. Immunohistochemistry is the most widely used technique for the detection of ERα and HER2 in breast cancer specimens, however, is inadequate in its ability to assess the relationship between ERα, HER2, and MAPK cascade at the single cell level. To clear this major hurdle, we devised a novel flow cytometric method to quantify the expression of ERα, HER2, and the activation of MAPK cascade simultaneously in single cells. The method was validated by concurrent Western blotting in established cell lines: MDA-231 (ERα and HER2-negative), MCF-7 (ERα-positive, HER2-negative), MCF-7 cells overexpressing ERα after long-term incubation in estrogen-free medium, and HER2 transfected MCF7 cells. Using the flow cytometry method, we confirmed the previous finding that ERα expression is down-regulated upon epidermal growth factor mediated ERK1/2 phosphorylation in EGFR/MCF-7 cells. To our knowledge, this is the first such assay to incorporate simultaneous single cell measurement for all of these pathways, which may prove useful to determine the intratumoral heterogeneity in breast tumors or the receptor status in circulating tumor cells.


Estrogen receptor Human epidermal growth factor receptor Extracellular signal-regulated kinase Multicolor flow cytometry 


Conflict of interest



  1. 1.
    Lippman ME, Allegra JC, hompson EB et al (1978) The relation between estrogen receptors and response rate to cytotoxic chemotherapy in metastatic breast cancer. N Engl J Med 298:1223–1228PubMedCrossRefGoogle Scholar
  2. 2.
    Wolff AC, Hammond ME, Schwartz JN et al (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25:118–145PubMedCrossRefGoogle Scholar
  3. 3.
    Early Breast Cancer Trialists Collaborative Group (1998) Tamoxifen for early breast cancer: an overview of the randomized trials. Lancet 351:1451–1467CrossRefGoogle Scholar
  4. 4.
    Piccart-Gebhart MJ, Procter M, Leyland-Jones B et al (2005) Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353:1659–1672PubMedCrossRefGoogle Scholar
  5. 5.
    Vogel CL, Cobleigh MA, Tripathy D et al (2002) Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20(3):719–726PubMedCrossRefGoogle Scholar
  6. 6.
    Romond EH, Perez EZ, Bryant J et al (2005) Trastuzumab plus adjuvant chemotherapy for operable HER2 positive breast cancer. N Engl J Med 353:1673–1684PubMedCrossRefGoogle Scholar
  7. 7.
    Moeder CB, Giltnane JM, Harigopal M et al (2007) Quantitative justification of the change from 10% to 30% for human epidermal growth factor receptor 2 scoring in the American Society of Clinical Oncology/College of American Pathologists guidelines: tumor heterogeneity in breast cancer and its implications for tissue microarray based assessment of outcome. J Clin Oncol 25:5418–5425PubMedCrossRefGoogle Scholar
  8. 8.
    McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EWT, Chang F et al (2007) Roles of the raf/mek/erk pathway in cell growth, malignant transformation and drug resistance. Biochem Biophys Acta 1773(8):1263–1284. doi: 10.1016/j.bbamcr.2006.10.001 PubMedCrossRefGoogle Scholar
  9. 9.
    Ali S, Coombes RC (2002) Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer 2:101–112PubMedCrossRefGoogle Scholar
  10. 10.
    Oh AS, Lorant LA, Holloway JN, Kern FG, El-Ashry D (2001) Hyperactivation of MAPK induces loss of ERα expression in breast cancer cells. Mol Endocrinol 15(8):1344–1359PubMedCrossRefGoogle Scholar
  11. 11.
    Holloway JN, Murthy S, El-Ashry D (2004) A cytoplasmic substrate of MAPK is responsible for ERα down-regulation in breast cancer cells: role of NFκB. Mol Endocrinol 18:1396–1410PubMedCrossRefGoogle Scholar
  12. 12.
    Bayliss J, Hilger A, Vishnu P, Diehl K, El-Ashry D (2007) Reversal of the estrogen-negative phenotype and restoration of anti-estrogen response in breast cancer. Clin Cancer Res 13:7029–7036PubMedCrossRefGoogle Scholar
  13. 13.
    Dry JR, Pavey S, Pratilas CA, Harbron C, Runswick S, Hodgson D et al (2010) Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244). Cancer Res 70(6):2264–2273PubMedCrossRefGoogle Scholar
  14. 14.
    Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741PubMedCrossRefGoogle Scholar
  15. 15.
    Oyaizu T, Arita S, Hatano T, Tsubara A (1996) Immunohistochemical detection of estrogen and progesterone receptors performed with an antigen-retrieval technique on methacarn-fixed paraffin-embedded breast cancer tissues. J Surg Res 60:69–73PubMedCrossRefGoogle Scholar
  16. 16.
    Lostumbo A, Mehta D, Setty S, Nunez R (2006) Flow cytometry: a new approach for the molecular profiling of breast cancer. Exp Mol Pathol 80:46–53PubMedGoogle Scholar
  17. 17.
    Miller DL, El-Ashry D, Cheville AL, Liu Y, McLeskey SW, Kern FG (1994) Emergence of MCF-7 cells overexpressing a transfected epidermal growth factor receptor under estrogen-depleted conditions: evidence for a role of EGFR in breast cancer growth and progression. Cell Growth Differ 5:1263–1274PubMedGoogle Scholar
  18. 18.
    Liu Y, El-Ashry D, Chen D, Ding IYF, Kern FG (1995) MCF-7 breast cancer cells overexpressing transfected c-erbb2 have an in vitro growth advantage in estrogen-depleted conditions and reduced estrogen-dependence and tamoxifen-sensitivity in vivo. Breast Cancer Res Treat 34:97–117PubMedCrossRefGoogle Scholar
  19. 19.
    El-Ashry D, Miller DL, Kharbanda S, Lippman ME, Kern FG (1997) Constitutive raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis. Oncogene 15:423–435PubMedCrossRefGoogle Scholar
  20. 20.
    Jiménez E, Montiel M (2005) Activation of MAP kinase by muscarinic cholinergic receptors induces cell proliferation and protein synthesis in human breast cancer cells. J Cell Physiol 204(2):678–686PubMedCrossRefGoogle Scholar
  21. 21.
    Toi M, Sperinde J, Huang W et al (2010) Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer. BMC Cancer 10:56–65PubMedCrossRefGoogle Scholar
  22. 22.
    Colleoni M, Bagnardi V, Rotmensz N et al (2009) Increasing steroid hormone receptors expression defines breast cancer subtypes non responsive to preoperative chemotherapy. Breast Cancer Res Treat 116:359–369PubMedCrossRefGoogle Scholar
  23. 23.
    Tubbs RR, Hicks DG, Cook J et al (2007) Fluorescence in situ hybridization (FISH) as primary methodology for the assessment of HER2 Status in adenocarcinoma of the breast: a single institution experience. Diagn Mol Pathol 16(4):207–210PubMedCrossRefGoogle Scholar
  24. 24.
    Shin SJ, Hyjek E, Early E et al (2006) Intratumoral heterogeneity of HER-2/neu in invasive mammary carcinomas using fluorescence in situ hybridization and tissue microarray. Int J Surg Pathol 14:279–284PubMedCrossRefGoogle Scholar
  25. 25.
    Brunelli M, Manfrin E, Martignoni G et al (2009) Genotypic intratumoral heterogeneity in breast carcinoma with HER2/neu amplification evaluation according to ASCO/CAP criteria. Am J Clin Pathol 131:678–682PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Ulas Darda Bayraktar
    • 1
  • Tae Kon Kim
    • 1
  • Katherine Drews-Elger
    • 1
  • Cara Benjamin
    • 1
  • Dorraya El-Ashry
    • 1
  • Eric Wieder
    • 1
  • Krishna V. Komanduri
    • 1
  1. 1.Department of Medicine, Division of Hematology/OncologyUniversity of MiamiMiamiUSA

Personalised recommendations