Activation of the AMPK regulated metabolic stress response by a small molecule HER2/EGFR tyrosine kinase inhibitor protects cardiac myocytes from apoptotic stimuli

Abstract

14000 Background: The HER2 receptor tyrosine kinase is a survival factor for human cardiomyocytes, providing a potential explanation for the increased incidence of cardiomyopathy associated with anti-HER2 monoclonal antibody trastuzumab. Here we show that GW2974, a HER2/EGFR TKI, but not trastuzumab activates AMP kinase (AMPK), initiating a metabolic stress response in human cardiomyocytes that protects against TNFa induced cell death. GW2974 stimulates calcium dependent fatty acid oxidation in vitro resulting in upregulation of ATP and in myocardium of GW2974 treated rodents. Methods: Western blot: p-Akt, p-Erk1/2, p-AMPKa and p- eEF2 (Cell Signaling); ERRa, ERR? (R&D Systems); PGC-1 (Chemicon); MCAD (Cayman Chemicals); Heregulin, NF?B, and Actin (Sigma). ATP was performed utilizing a bioluminescence assay kit H52 (Roche). Lipid staining was done with Oil Red O (Sigma). Cells: Au565 breast cancer and primary human cardiomyocytes (HMC) were grown in RPMI supplemented with 15% BFS treatments. BAPTA/AM and Compound C (Calbiochem); GW2974 and TNFa (Sigma); trastuzumab (Genentech). Results: Our results show that treatment with GW2974 activates catabolic pathways by activation of AMPK. AMPK is a key regulator in mitochondrial energy producing pathways in human cardiac cells. Phosphorylation of AMPK resulted in phosphorylation of eEF2, upregulation of ERRa and PGC-1 and upregulation of ATP levels. AMPK activation depends on Ca++ as the calcium chelation abolished it. Trastuzumab and Gleevec failed to activate AMPK survival pathways. In addition, GW2974 protected cardiac cells from TNFa killing, whereas treatment with TNFa and trastuzumab resulted in pervasive cellular death. Inhibition of AMPK by Compound C or siRNA resulted in cardiomyocyte killing. Conclusion: Activation of AMPK and its effects on metabolic pathways and energy production in HMC may explain the apparent reduced risk of cardiotoxicity associated with HER2 TKI compared with trastuzumab in treating patients with HER2 overexpressing breast cancers. Although activation of AMPK protected normal cardiac myocytes, this effect appears to be lethal to sensitive HER2 overexpressing breast cancer cells, which are “addicted” to glycolysis. No significant financial relationships to disclose.