ErbB-2 inhibition activates Notch-1 and sensitizes breast cancer cells to a gamma-secretase inhibitor.
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, 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.
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ABSTRACT: Trastuzumab-based therapy improves survival for women with human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer. We conducted a multicenter phase II study to evaluate the efficacy and safety of trastuzumab combined with vinorelbine, and to assess cardiac surveillance algorithms and tumor markers as prognostic tools. Patients with HER2-positive (immunohistochemistry [IHC] 3+-positive or fluorescence in situ hybridization [FISH]-positive) metastatic breast cancer received first-line chemotherapy with trastuzumab and vinorelbine to determine response rate. Eligibility criteria were measurable disease and baseline ejection fraction >or= 50%. Serial testing for HER2 extracellular domain (ECD) was performed. Fifty-four women from 17 participating centers were entered onto the study. The overall response rate was 68% (95% confidence interval, 54% to 80%). Response rates were not affected by method of HER2 status determination (FISH v IHC) or by prior adjuvant chemotherapy. Median time to treatment failure was 5.6 months; 38% of patients were progression free after 1 year. Concurrent therapy was quite feasible with maintained dose-intensity. Patients received both chemotherapy and trastuzumab on 90% of scheduled treatment dates. Two patients experienced cardiotoxicity in excess of grade 1; one patient experienced symptomatic heart failure. A surveillance algorithm of screening left ventricular ejection fraction (LVEF) at 16 weeks successfully identified women at risk for experiencing cardiotoxicity. Other acute and chronic side effects were tolerable. Lack of decline in HER2 ECD during cycle 1 predicted tumor progression. Trastuzumab and vinorelbine constitute effective and well-tolerated first-line treatment for HER2-positive metastatic breast cancer. Patients with normal LVEF can be observed with surveillance of LVEF at 16 weeks to identify those at risk for cardiotoxicity.Journal of Clinical Oncology 08/2003; 21(15):2889-95. · 18.04 Impact Factor
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ABSTRACT: Notch receptors regulate cell fate determination, stem cell self-renewal, proliferation and apoptosis. We previously reported that elevated mRNA expression of the Notch ligand JAG1 identifies breast cancer patients with a poor prognosis. Here we show through immunohistochemical analysis of the same breast cancer cases (N=127) that patients with tumors expressing high levels of JAG1 protein had a worse outcome than those with tumors expressing low levels (10-year survival 26 vs 48%, and median survival 63 vs 108 months, respectively; P=0.03). We also describe the novel application of the Allred score to quantify JAG1 mRNA and protein expression levels. Using the Allred score, patients with tumors expressing high levels of JAG1 mRNA had a worse outcome than those with tumors expressing low levels (10-year survival 16 vs 47%, and median survival 43 months vs 100 months, respectively; P<0.001). Interestingly, when tumors were classified as either high or low for JAG1 mRNA or protein expression, there was only 65% agreement (=0.08) between the two methods of expression analysis. When JAG1 mRNA and protein data were combined, patients with tumors expressing low levels of both had a 10-year survival of 53% and median survival of 131 months. In comparison, patients with tumors expressing either high levels of JAG1 protein, mRNA or both had reduced 10-year survival and median survival (31%, 19%, 11% and 77, 43, 23 months respectively; P<0.0001). There was marginal evidence of an interaction effect (P=0.055), which indicated that the prognostic value of JAG1 protein was limited to the JAG1 mRNA-low subgroup. These data show that the Allred score can be used to rapidly quantify JAG1 mRNA and protein levels in breast cancer to identify patients who have a significant survival disadvantage and who may benefit from therapies (such as -secretase inhibitors) that target signaling through the Notch pathway.Keywords: breast cancer, gene expression, NOTCH1, JAG1, prognosis, survivalModern Pathology 05/2007; 20(6):685-693. · 5.25 Impact Factor
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ABSTRACT: Overexpression of the HER2 protein occurs in 25% to 30% of human breast cancers and leads to a particularly aggressive form of the disease. Efficacy and safety of recombinant humanized anti-HER2 monoclonal antibody as a single agent was evaluated in women with HER2-overexpressing metastatic breast cancer that had progressed after chemotherapy for metastatic disease. Two hundred twenty-two women, with HER2-overexpressing metastatic breast cancer that had progressed after one or two chemotherapy regimens, were enrolled. Patients received a loading dose of 4 mg/kg intravenously, followed by a 2-mg/kg maintenance dose at weekly intervals. Study patients had advanced metastatic disease and had received extensive prior therapy. A blinded, independent response evaluation committee identified eight complete and 26 partial responses, for an objective response rate of 15% in the intent-to-treat population (95% confidence interval, 11% to 21%). The median duration of response was 9.1 months; the median duration of survival was 13 months. The most common adverse events, which occurred in approximately 40% of patients, were infusion-associated fever and/or chills that usually occurred only during the first infusion, and were of mild to moderate severity. These symptoms were treated successfully with acetaminophen and/or diphenhydramine. The most clinically significant adverse event was cardiac dysfunction, which occurred in 4.7% of patients. Only 1% of patients discontinued the study because of treatment-related adverse events. Recombinant humanized anti-HER2 monoclonal antibody, administered as a single agent, produces durable objective responses and is well tolerated by women with HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. Side effects that are commonly observed with chemotherapy, such as alopecia, mucositis, and neutropenia, are rarely seen.Journal of Clinical Oncology 10/1999; 17(9):2639-48. · 18.04 Impact Factor