Inhibition of erbB1/2 by small molecule tyrosine kinase inhibitors, but not trastuzumab, affects metabolic pathways: Implications for cardiac toxicity.

PR-1

Background: Therapies targeting ErbB2 represent an attractive strategy in breast cancer. Trastuzumab, an anti-ErbB2 monoclonal antibody, is an approved treatment for patients with ErbB2-overexpressing breast cancers. Lapatinib is a potent, reversible inhibitor of ErbB2 and ErbB1 tyrosine kinases (TKI) and is currently in Phase III clinical trials in breast and other carcinomas. The principal adverse event attributable to trastuzumab is cardiac toxicity. This study was conducted to elucidate mechanisms that affect metabolic pathways by a TKI and an antibody directed to ErbB2 and their effects on breast cancer cells, primary human adipocytes and cardiomyocytes. Synergistic effects with cytotoxic agents were also tested.Materials and methods: Western blotting was used for p-Akt, p-Erk1/2, p-AMPKα and p-eEF2 (Cell Signaling, Beverly, MA); ERRα, ERRγ (R & D Systems Minneapolis, MN); PGC-1 (Chemicon International, Temecula, CA); MCAD (Cayman Chemicals, Ann Arbor, MI) and Actin (Sigma, St. Louis, MO). Lipid staining: cells were fixed in NBF and stained with Oil Red O (Sigma). Cells: AU565, BT474 breast cancer cells and primary cardiomyocytes were grown in RPMI supplemented with 15% BFS and treated with BAPTA/AM (Calbiochem): 5-30 μM; GW-2974 (Sigma): 1-25 μM; trastuzumab (Genentech): 5-50 mg/ml; Heregulin (LabVision, Fremont, CA): 5-100 ng/ml; doxorubicin: 50 ng/ml; TNFα: 100 ng/ml.Results: Our results show that treatment with GW-2974/lapatinib on ErbB1/2 inhibits fatty acid metabolic pathways through activation of adenosine monophosphate kinase (AMPK), a key regulator in mitochondrial energy producing pathways, in human cardiac cells, and breast cancer cells. The changes include phosphorylation of AMPK, eEF2, upregulation of ERRα and PGC-1, activators of fatty acid oxidation, in cardiomyocytes and breast cancer cells, downregulation of fatty acid synthase (FAS), and changes in ion and calcium channels. In addition to fatty acid oxidation, depletion of amino acids occurred. The metabolic changes were reversed by calcium chelation. Trastuzumab failed to activate AMPK, but downregulated survival pathways. There were dramatic differences in the cytotoxic synergistic effect between GW-2974 and trastuzumab.Conclusions: Our results show that treatment using TKI to ErbB1/ErbB2 pathways in breast cancer cells, and human cardiomyocytes results in activation of AMPK. AMPK regulates cellular energy homeostasis. Activation of AMPK after stress is associated with protection of cells against ischemia and nutrient depletion and cytotoxic agents, helping to preserve the levels of cellular ATP and cellular survival. Thus, activation of AMPK provide protection against cellular damage by ErbB targeted therapy alone or when given in combination with chemotherapy. The failure of trastuzumab to activate AMPK points to the source of its cardiac toxicity and synergy with cytotoxic agents.