Mitochondrial Uncoupling Induces Epigenome Remodeling and Promotes Differentiation in Neuroblastoma

Abstract

The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the nicotinamide adenine dinucleotide (NAD)+/NADH and pyruvate/lactate ratios and also the α-ketoglutarate (α-KG)/2- hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and β-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and β-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma.

The magic bullet: Niclosamide

“…NEN treatment increases the intracellular pyruvate/lactate ratio, the a-KG/2-HG ratio, and total intracellular a-KG levels, leading to a reversal of the Warburg effect and the induction of cellular differentiation (Figure 2A). Consistent with these observations, NEN treatment induces promoter CpG island demethylation and epigenetic landscape remodeling (Figure 2B) (8). In neuroblastoma cells, many genes activated by NEN treatment are involved in neurogenesis, nervous system development and neuron differentiation.…”

“…The NEN-upregulated genes are enriched in the favorable prognosis gene signatures, while the NEN-downregulated genes are more enriched in unfavorable prognosis gene signatures. Consistent to the prognosis gene signatures changes, NEN treatment not only reduced the tumor growth but also prolonged the survival for tumor bearing mice (8). In vivo, NEN treatment also effectively increased the NAD+/NADH ratio and reduced lactate and 2-HG levels in xenograft tumors (8).…”

“…Consistent to the prognosis gene signatures changes, NEN treatment not only reduced the tumor growth but also prolonged the survival for tumor bearing mice (8). In vivo, NEN treatment also effectively increased the NAD+/NADH ratio and reduced lactate and 2-HG levels in xenograft tumors (8).…”

“…As described before, niclosamide dissipates the mitochondrial proton gradient requisite for ATP synthesis, leading to the reduction of intracellular ATP and an increased AMP/ATP ratio, culminating in the activation of AMP-activated protein kinase (AMPK) (8,15,18). Additionally, niclosamide may activate AMPK through a mechanism independent of the increased AMP/ATP ratio, namely through the AMPK b2 subunit (68).…”

“…Accumulating evidence indicates that niclosamide is a pleiotropic compound that targets multiple biological processes and signal pathways. Because niclosamide shuttles electrons across the mitochondrial inner membrane to activate the ETC, niclosamide reprograms intracellular metabolism ( 8 ), which can impact cellular epigenetic regulation at the transcriptional, translational, and post-translational levels ( 9 , 10 ). Furthermore, the ability of niclosamide to modify the global epigenetic landscape through metabolic reprogramming ( 8 ) may explain its ability to simultaneously inhibit oncogenic signaling pathways and activate tumor suppressor signaling pathways.…”