ErbB-2 inhibition activates Notch-1 and sensitizes breast cancer cells to a gamma-secretase inhibitor.
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.
Osipo
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Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL 60153, USA. cosipo@lumc.edu
Patel
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Rizzo
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Clementz
A G
AG
Hao
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Golde
T E
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Miele
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D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors*; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016923:Cell Death / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D034741:RNA, Small Interfering / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000276:immunology; D051881:Receptor, Notch1 / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D000068878:Trastuzumab; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects
D053829:Amyloid Precursor Protein Secretases; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D051881:Receptor, Notch1; D000068878:Trastuzumab; C497761:NOTCH1 protein, human