1. ¹Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, USA
2. ²The Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, USA
3. ³Molecular and Cellular Biochemistry Program, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, USA
4. ⁴Molecular Biology Program, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, USA
5. ⁵Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
6. ⁶Department of Pharmacology and Molecular Therapeutics, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, USA

Correspondence: Dr C Osipo, The Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153, USA. E-mail: cosipo@lumc.edu

Received 18 July 2007; Revised 28 March 2008; Accepted 28 March 2008; Published online 12 May 2008.

Abstract

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, -secretase inhibitor (GSI) or Notch-1 small-interfering RNA (siRNA). Trastuzumab decreased Notch-1™ 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.