AG311, a small molecule inhibitor of complex I and hypoxia-induced HIF-1α stabilization

Highlights

• AG311 competitively inhibits ubiquinone-binding to complex I and prevents electron transfer and mitochondrial respiration.

• Cellular response to hypoxia is reduced by AG311, including HIF-1α protein levels, target genes, and oxygen tension.

• Combination of AG311 and dichloroacetate augments cytotoxicity and tumor growth reduction.

Abstract

Cancer cells have a unique metabolic profile and mitochondria have been shown to play an important role in chemoresistance, tumor progression and metastases. This unique profile can be exploited by mitochondrial-targeted anticancer therapies. A small anticancer molecule, AG311, was previously shown to possess anticancer and antimetastatic activity in two cancer mouse models and to induce mitochondrial depolarization. This study defines the molecular effects of AG311 on the mitochondria to elucidate its observed efficacy. AG311 was found to competitively inhibit complex I activity at the ubiquinone-binding site. Complex I as a target for AG311 was further established by measuring oxygen consumption rate in tumor tissue isolated from AG311-treated mice. Cotreatment of cells and animals with AG311 and dichloroacetate, a pyruvate dehydrogenase kinase inhibitor that increases oxidative metabolism, resulted in synergistic cell kill and reduced tumor growth. The inhibition of mitochondrial oxygen consumption by AG311 was found to reduce HIF-1α stabilization by increasing oxygen tension in hypoxic conditions. Taken together, these results suggest that AG311 at least partially mediates its antitumor effect through inhibition of complex I, which could be exploited in its use as an anticancer agent.

Introduction

In addition to genetic and epigenetic heterogeneity, the concept of metabolic heterogeneity has increased recently. Considerable heterogeneity exists between different tumors, but also within the same tumor. This intratumoral heterogeneity is predominately mediated by genetic diversity, energy demand, and the proximity to vasculature, dictating glucose and oxygen availability. Until recently, cancer cells were thought to rely only on glycolysis for ATP production, even in the presence of sufficient oxygen supply [1], but metabolic heterogeneity demonstrates that there are multiple metabolic phenotypes, going beyond the upregulated glycolysis as observed by Warburg. One of these metabolic phenotypes is mitochondrial respiration, as cancer cells have been shown to have functional mitochondria capable of producing ATP [2], [3], [4].

Anticancer agents acting through mitochondrial inhibition have been described. For example, the anti-diabetic biguanides metformin and phenformin have been shown to exert their anticancer effect by inhibiting complex I, the first enzyme in the mitochondrial electron transport chain (ETC) [5], [6], [7], [8]. BAY-87-2243, another complex I inhibitor, has shown efficacy in a preclinical model of resistant melanoma [9]. The small molecule, IACS-10759, was specifically designed as a complex I inhibitor to target chemoresistant dormant tumors [9], [10]. Complex I, NADH-ubiquinone oxidoreductase, is a crucial player in mitochondrial respiration. It transfers electrons from NADH to reduce ubiquinone to ubiquinol resulting in proton translocation into the mitochondrial intermembrane space, which establishes an electrochemical gradient that is used for ATP synthesis. In order for this process to continue, molecular oxygen is required as the final electron acceptor. Regions of solid tumors often exhibit low oxygen tension (hypoxia), which has been shown to be involved in tumor development, chemo- and radioresistance, and metastasis in addition to tumor bioenergetics [11], [12]. These alterations in bioenergetic processes are mediated in part by increasing gene expression involved in glycolysis and by lowering the activity of the electron transport chain [13], [14], [15]. The major cellular adaptive response to hypoxia is tightly regulated by hypoxia-inducible transcription factor-1α, HIF-1α, which is stabilized in low oxygen tensions, thus an inhibition of complex I can prevent electron transfer and decrease oxygen consumption, which in turn could decrease HIF-1α stabilization [16], [17]. HIF-1α is capable of inducing a broad range of cellular responses including angiogenesis, resistance to apoptosis and tumor energetics/metabolism. Due to the importance of mitochondrial oxidative phosphorylation in hypoxia and in supporting tumor growth, progression and metastasis [18], [19], the development of mitochondrial inhibitors seems well justified.

Herein we investigate the molecular mechanism of a small molecule antitumor agent, AG311. We have previously shown that AG311 significantly reduced primary tumor growth and lung metastases in two breast cancer mouse models, by 81–85% [20]. Upon further investigation, a distinct metabolic mechanism for AG311 emerged. We reported that AG311 rapidly induced necrotic cell death, depolarized the mitochondrial membrane and severely reduced intracellular ATP levels [20]. In the current study, we further define the molecular and antitumor mechanisms and identify complex I of the electron transport chain (ETC) as a likely molecular target of AG311 in isolated cells and in tumors. We show that as a downstream consequence of complex I inhibition, AG311 reduced hypoxia-induced HIF-1α stabilization. Additionally, we show that AG311 synergizes with dichloroacetate (DCA) to increase cell death in cancer cells and increase tumor volume in a xenograft tumor mouse model.