Abstract 1631: The ribonucleoside analogue, 8-amino-adenosine, inhibits AKT and MAPK signaling in breast cancer cells

Abstract

Abstract In contrast to DNA-directed, deoxyribose or arabinose containing nucleoside analogs that are currently established for cancer therapeutics, 8-amino-adenosine (8-NH2-Ado) possesses a ribose sugar. This distinct nucleoside analog is RNA-directed and is cytotoxic to a number of primary malignant hematological cells as well as cell lines. The analog's cytotoxicity is associated with a high accumulation of its metabolite, 8-NH2-Ado-triphosphate (8-NH2-ATP), reaching to levels as high as 8 mM with 10 μM 8-NH2-Ado treatment. This accumulation is coupled with a parallel decrease in the level of the endogenous ATP pool by as much as 90%. Unlike DNA-directed nucleoside analogs, in solid tumors the triphosphate of 8-substituted-Ado-analogs accumulates to high levels; thus warrants use of 8-NH2-Ado in these types of malignancies. We report that 10 μM 8-NH2-Ado rapidly induced apoptosis in the breast cancer cell lines, BT-474 and MCF-7 as cleavage of PARP was readily detected in 2 h and 4 h, respectively. To further understand the mechanisms of action of 8-NH2-Ado, we examined its effect on key growth and survival pathways using purified enzymes and cell line models. Because 8-NH2-ATP is an ATP analog, it is feasible that it may alter other processes requiring ATP such as protein kinase activity. To determine if 8-NH2-ATP can directly inhibit the activity of key kinases, we employed Invitrogen's SelectScreen Kinase Profiling Service. Members of the MAPK family were inhibited by 8-NH2-ATP at ratios of 8-NH2-ATP: ATP readily achievable in cells. Specifically, 8-NH2-ATP was a potent, selective inhibitor of the dual specificity mitogen-activated protein kinase (MEK) 1 and 2 were inhibited 100% even at 8-NH2-ATP: ATP ratios of 1.0. Additionally, MEK6 was also inhibited. MEK1 and 2 are kinases that feed the MAPK cascade into the Ras/Raf pathway, and they directly phosphorylate and activate ERK 1/2 which leads to the activation of various transcription factors and cell cycle regulatory proteins to induce growth and survival. MEK6 phosphorylates and activates p38 MAP kinase and includes a p38 isoform that binds ERK 1/2 and regulates ERK nuclear activity. The in vitro inhibition of MEK1, 2, and 6 by 8-NH2-ATP was consistent with the altered phosphorylation status of several important cell-signaling molecules in breast cancer cells. By Western blot analysis, 8-NH2-Ado treatment promoted a dramatic loss of ERK1/2 and AKT phosphorylation in both breast cancer cell lines within 6 h. MEK1, 2, 6 and AKT are attractive targets for cancer therapeutics as during tumor progression these kinases promote numerous processes such as inflammation, growth, survival, and therapeutic resistance. We conclude that 8-NH2-Ado by the accumulation of 8-NH2-ATP can directly inhibit MEK1, 2, and 6. This inhibition may contribute to the cytotoxic actions of 8-NH2-Ado in breast cancer cells and other malignancies dependent on the MEK/ERK pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1631.