Breast Cancer Research and Treatment

, Volume 123, Issue 2, pp 387–396 | Cite as

Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells

  • Amelia D’Alessio
  • Antonella De Luca
  • Monica R. Maiello
  • Luana Lamura
  • Anna Maria Rachiglio
  • Maria Napolitano
  • Marianna Gallo
  • Nicola Normanno
Preclinical study
First Online:
Received:
Accepted:

Abstract

Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27kip1 and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27kip1, with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27kip1 mRNA and in the nuclear levels of the p27kip1 transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27kip1 mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27kip1 mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27kip1 expression.

Keywords

EGFR ErbB-2 Breast cancer Cell cycle 
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Notes

Acknowledgments

This study was partially supported, through research grants, by the nonprofit “Associazione Italiana per la Ricerca sul Cancro” (AIRC) and Ministero della Salute to N.N.

Supplementary material

10549_2009_649_MOESM1_ESM.pdf (18 kb)
Western blot analysis for phosphorylated EGFR and ErbB-2 in SK-Br-3 and BT-474 cells following treatment with gefitinib (2 μM SK-Br-3 and 1 μM BT-474) and/or trastuzumab (20 μg/ml) for 2 h. C control, untreated cells, G gefitinib, T trastuzumab, G + T gefitinib plus trastuzumab. (PDF 17 kb)
10549_2009_649_MOESM2_ESM.pdf (38 kb)
Effects of cycloheximide treatment (10 μM at 2, 4 and 8 h) on the levels of expression of p27kip1 in BT-474 cells. a Western blot analysis for p27kip1 expression in cells untreated or treated for 48 h with gefitinib and trastuzumab, alone or in combination. b Densitometric analysis of the blots shown in a. (PDF 37 kb)

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Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • Amelia D’Alessio
    • 1
  • Antonella De Luca
    • 1
  • Monica R. Maiello
    • 1
  • Luana Lamura
    • 1
  • Anna Maria Rachiglio
    • 2
  • Maria Napolitano
    • 3
  • Marianna Gallo
    • 1
  • Nicola Normanno
    • 1
  1. 1.Cell Biology and Biotherapy UnitINT-Fondazione PascaleNaplesItaly
  2. 2.Centro Ricerche Oncologiche Mercogliano – CROMMercoglianoItaly
  3. 3.Immunology UnitINT-Fondazione PascaleNaplesItaly

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