ErbB-2 inhibition activates Notch-1 and sensitizes breast cancer cells to a gamma-secretase inhibitor

Oncogene. 2008 Aug 28;27(37):5019-32. doi: 10.1038/onc.2008.149. Epub 2008 May 12.


ErbB-2 overexpression in breast tumors is associated with poor survival. Expression of Notch-1 and its ligand, Jagged-1, is associated with the poorest survival, including ErbB-2-positive tumors. Trastuzumab plus chemotherapy is the standard of care for ErbB-2-positive breast cancer. A proportion of tumors are initially resistant to trastuzumab and acquired resistance to trastuzumab occurs in metastatic breast cancer and is associated with poor prognosis. Thus, we investigated whether Notch-1 contributes to trastuzumab resistance. ErbB-2-positive cells have low Notch transcriptional activity compared to non-overexpressing cells. Trastuzumab or a dual epidermal growth factor receptor (EGFR)/ErbB-2 tyrosine kinase inhibitor (TKI) increased Notch activity by 2- to 6-fold in SKBr3, BT474 and MCF-7/HER2-18 cells. The increase in activity was abrogated by a Notch inhibitor, gamma-secretase inhibitor (GSI) or Notch-1 small-interfering RNA (siRNA). Trastuzumab decreased Notch-1trade mark precursor, increased amount and nuclear accumulation of active Notch-1(IC) and increased expression of targets, Hey1 and Deltex1 mRNAs, and Hes5, Hey1, Hes1 proteins. Importantly, trastuzumab-resistant BT474 cells treated with trastuzumab for 6 months expressed twofold higher Notch-1, twofold higher Hey1, ninefold higher Deltex1 mRNAs and threefold higher Notch-1 and Hes5 proteins, compared to trastuzumab-sensitive BT474 cells. The increase in Hey1 and Deltex1 mRNAs in resistant cells was abrogated by a Notch-1 siRNA. Cell proliferation was inhibited more effectively by trastuzumab or TKI plus a GSI than either agent alone. Decreased Notch-1 by siRNA increased efficacy of trastuzumab in BT474 sensitive cells and restored sensitivity in resistant cells. Trastuzumab plus a GSI increased apoptosis in sensitive cells by 20-30%. A GSI alone was sufficient to increase apoptosis in trastuzumab-resistant BT474 cells by 20%, which increased to 30% with trastuzumab. Notch-1 siRNA alone decreased cell growth by 30% in sensitive and more than 50% in resistant BT474 cells. Furthermore, growth of both trastuzumab sensitive and resistant cells was completely inhibited by combining trastuzumab plus Notch-1 siRNA. More importantly, Notch-1 siRNA or a GSI resensitized trastuzumab-resistant BT474 cells to trastuzumab. These results demonstrate that ErbB-2 overexpression suppresses Notch-1 activity, which can be reversed by trastuzumab or TKI. These results suggest that Notch-1 might play a novel role in resistance to trastuzumab, which could be prevented or reversed by inhibiting Notch-1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid Precursor Protein Secretases / antagonists & inhibitors*
  • Antibodies, Monoclonal / administration & dosage
  • Antibodies, Monoclonal / pharmacology*
  • Antibodies, Monoclonal, Humanized
  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use*
  • Breast Neoplasms / drug therapy*
  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Cell Death / drug effects
  • Drug Evaluation, Preclinical
  • Drug Resistance, Neoplasm / drug effects
  • Drug Resistance, Neoplasm / genetics
  • Enzyme Inhibitors / administration & dosage
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • RNA, Small Interfering / pharmacology
  • Receptor, ErbB-2 / antagonists & inhibitors*
  • Receptor, ErbB-2 / immunology
  • Receptor, Notch1 / antagonists & inhibitors
  • Receptor, Notch1 / metabolism*
  • Receptor, Notch1 / physiology
  • Trastuzumab
  • Tumor Cells, Cultured
  • Up-Regulation / drug effects


  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Enzyme Inhibitors
  • NOTCH1 protein, human
  • RNA, Small Interfering
  • Receptor, Notch1
  • Receptor, ErbB-2
  • Amyloid Precursor Protein Secretases
  • Trastuzumab