Skip to main content

Advertisement

SpringerLink
Log in

EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

HER2 is gene amplified or over-expressed in 20–25% of breast cancers resulting in elevated HER2 activation. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, despite prolonged survival, treated breast cancer patients develop resistance. Resistance to trastuzumab occurs upon inactivation of HER2 regulatory proteins or upon up-regulation of alternative receptors. In particular, elevated levels of EGFR, present in estrogen receptor (ER) positive, trastuzumab-resistant BT-474 xenografts caused, a trastuzumab-resistant phenotype (Ritter et al. Clin Cancer Res 13:4909–4919, 2007). However, the role of EGFR in acquired trastuzumab resistance in ER negative cell models is not well defined. In this study, SKBR3 cell line clones expressing EGFR were generated to examine the role of EGFR over-expression on trastuzumab sensitivity in an, ER-negative breast carcinoma cell line. A stable clone, SKBR3/EGFR (clone 4) expressing moderate levels of EGFR remained sensitive to trastuzumab, whereas a stable clone, SKBR3/EGFR (clone 5) expressing high levels of EGFR, became resistant to trastuzumab. Depletion of EGFR by EGFR small-interfering RNAs in the SKBR3/EGFR (clone 5) reversed trastuzumab resistance. However, the SKBR3/EGFR (clone 5) cell line remained sensitive to lapatinib, an EGFR/HER2 inhibitor. Biochemical analysis using co-immunoprecipitation and proximity-based quantitative VeraTag assays demonstrated that high levels of EGFR phosphorylation, EGFR/EGFR homo-dimerization, and EGFR/HER2 hetero-dimerization were present in the trastuzumab-resistant cells. We conclude that EGFR over-expression can mediate trastuzumab resistance in both ER positive and ER negative cells and hypothesize that a threshold level of EGFR, in the absence of autocrine ligand production, is required to induce the resistant phenotype.

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.

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

Similar content being viewed by others

References

  1. Cho HS, Masonn K, Ramyar KX et al (2003) Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature 421:756–760

    Article  CAS  PubMed  Google Scholar 

  2. Yarden Y, Sliwkowski MX (2001) Untangling the ERBB signalling network. Nat Mol Cell Biol 2:127–137

    Article  CAS  Google Scholar 

  3. Slamon DJ, Clark GM, Wong SG et al (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177–182

    Article  CAS  PubMed  Google Scholar 

  4. Slamon DJ, Leyland-Jones B, Shak S et al (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpress HER2. N Engl J Med 344:783–792

    Article  CAS  PubMed  Google Scholar 

  5. Romond EH, Perez EA, Bryant J et al (2005) Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 353:1673–1684

    Article  CAS  PubMed  Google Scholar 

  6. Nagata Y, Lan KH, Zhou X et al (2004) PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6:117–127

    Article  CAS  PubMed  Google Scholar 

  7. Ginestier C, Adelaide J, Gonclaves A, et al (2007) ERBB2 phosphorylation and trastuzumab sensitivity of breast cancer cell lines. Oncogene 26:7163–7169

    Google Scholar 

  8. Shattuck DL, Miller JK, Carraway KL et al (2008) Met receptor contributes to trastuzumab resistance of Her-2 overexpressing breast cancer cells. Cancer Res 68:1471–1477

    Article  CAS  PubMed  Google Scholar 

  9. Nahta R, Yuan LX, Zhang B et al (2005) Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells. Cancer Res 65:1118–1128

    Article  Google Scholar 

  10. Lu Y, Zi X, Zhao Y et al (2001) Insulin-like growth factor-I receptor signaling and resistance to Trastuzuman (Herceptin). J Natl Cancer Inst 93:1852–1857

    Article  CAS  PubMed  Google Scholar 

  11. Ritter CA, Marianela PT, Rinehart C et al (2007) Human breast cancer cells selected for resistance in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network. Clin Cancer Res 13:4909–4919

    Article  CAS  PubMed  Google Scholar 

  12. Prat A, Baselga J (2008) The role of hormonal therapy in the management of hormonal-receptor-positive breast cancer with co-expression of HER2. Nat Clin Pract Oncol 5:531–542

    Article  CAS  PubMed  Google Scholar 

  13. Mass RD, Vogel C, Murphy M et al (2001) Relationship of estrogen receptor (ER) status to clinical benefit in clinical trials of Herceptin. Eur J Cancer 37(suppl 6):S190

    Article  Google Scholar 

  14. Brufsky A, Lembersky B, Schiffman K et al (2005) Hormone receptor status does not affect the clinical benefit of trastuzumab therapy for patients with metastatic breast cancer. Clin Breast Cancer 6:247–252

    Article  PubMed  Google Scholar 

  15. Sabnis G, Schayowitz A, Goloubeva O et al (2009) Trastuzumab reverses letrozole resistance and amplifies the sensitivity of breast cancer cells to estrogen. Cancer Res 69:1416–1428

    Article  CAS  PubMed  Google Scholar 

  16. Shi Y, Huang W, Tan Y et al (2009) A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue. Diagn Mol Pathol 18:11–21

    Article  PubMed  Google Scholar 

  17. Desmedt C, Sperinde J, Piette F et al (2009) Quantitation of HER2 expression or HER2: HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully selected for trastuzumab treatment primarily by FISH. Diagn Mol Pathol 18:22–29

    Article  CAS  PubMed  Google Scholar 

  18. Stommel JM, Kimmelman AC, Ying H et al (2007) Coactivation of receptor tyrosine kinase affects the response of tumor cells to targeted therapies. Science 318:287–290

    Article  CAS  PubMed  Google Scholar 

  19. Pegram MD, Lipton A, Hayes DF et al (1998) Phase II recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 22:2659–2671

    Google Scholar 

  20. Geyer CE, Forster J, Lindquist D et al (2006) Lapatinib plus capecitabine for HER-2 positive advanced breast cancer. N Engl J Med 355:2733–2743

    Article  CAS  PubMed  Google Scholar 

  21. Shepard FA, Periera JR, Ciuleanu T et al (2005) Erlotinib in previously treated non-small-lung cancer. N Engl J Med 353:123–132

    Article  Google Scholar 

  22. Moore MJ, Goldstein D, Hamm J et al (2007) Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada clinical trials group. J Clin Oncol 25:1960–1966

    Article  CAS  PubMed  Google Scholar 

  23. Juntilla TT, Akita RW, Parsons K, et al (2009) Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 15:429–440

    Google Scholar 

  24. Sliwkowski MX, Lofgren JA, Lewis GD et al (1999) Nonclinical studies addressing the mechanism of action of trastuzumab (herceptin). Sem Oncol 26:60–70

    CAS  Google Scholar 

  25. Kumar R, Shepard HM, Medelson J (1991) Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells. Mol Cell Biol 11:979–986

    CAS  PubMed  Google Scholar 

  26. Tao R, Maruyama I (2009) All EGF (ErbB) receptors have preformed homo- and heterodimeric structures in living cells. J Cell Biol 121:3207–3217

    Google Scholar 

  27. Yu X, Sharma KD, Takahasi T et al (2002) Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling. Mol Cell Biol 13:2547–2557

    Article  CAS  Google Scholar 

  28. Narayan M, Wilken JA, Harris LN et al (2009) Trastuzumab-induced HER reprogramming in “resistant” breast carcinoma cells. Cancer Res 69:2191–2194

    Article  CAS  PubMed  Google Scholar 

  29. Zhang D, Pal A, Bornmann WG et al (2008) Activity of lapatinib is independent of EGFR expression level in HER2-overexpressin breast cancer cells. Mol Cancer Ther 7:1846–1850

    Article  CAS  PubMed  Google Scholar 

  30. Rexer BN, Ghosh R, Arteaga CL (2009) Inhibition of PI3K and MEK: it is all about combinations and biomarkers. Clin Cancer Res 15:4518–4520

    Article  CAS  PubMed  Google Scholar 

  31. Scaltriti M, Verma C, Guzman M et al (2008) Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity. Oncogene 28:803–814

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The authors wish to thank John Winslow and Youssouf Badal for their input on the proximity-based assays. Lili Chen helped in FACS analysis. Yining Shi and Sailaja Pidaparthi helped in the initial development of VeraTag assays. Hasan Tahir helped in the synthesis of the VeraTag reporters. Jeff Sperinde helped in setting up a method to calculate analyte concentration by slope analysis.

Author information

Author notes

  1. Phets Nhonthachit

    Present address: Ross University School of Medicine, 630 US highway 1, North Brunswick, NJ, USA

Authors and Affiliations

  1. Oncology, Monogram Biosciences, 345 Oyster Point Blvd, South San Francisco, CA, USA

    Rajiv Dua, Jianhuan Zhang, Phets Nhonthachit, Elicia Penuel, Chris Petropoulos & Gordon Parry

Authors
  1. Rajiv Dua
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianhuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Phets Nhonthachit
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elicia Penuel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chris Petropoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gordon Parry
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rajiv Dua.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dua, R., Zhang, J., Nhonthachit, P. et al. EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance. Breast Cancer Res Treat 122, 685–697 (2010). https://doi.org/10.1007/s10549-009-0592-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10549-009-0592-x

Keywords

Search

Navigation