Neuroblastoma Differentiation: The Untapped Potential of Mitochondrial Uncouplers

Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of all pediatric cancer deaths (1). These cancers arise from precursor cells of the sympathetic nervous system and predominantly present as masses in the abdomen, although widespread disease is common. Treatment options for neuroblastoma are influenced by risk stratification which is based on clinical, biological, and histologic factors such as patient age, cancer spread, ploidy, segmental chromosome aberrations at 1p and 11q, and MYCN DNA copy number (amplified or not; ref. 2). MYCN amplification is associated with advanced disease and therapy resistance and subsequently confers poorer prognosis (1). In younger patients, neuroblastoma can spontaneously regress or differentiate into benign ganglioneuroma even without treatment. Standard treatments for neuroblastoma include chemotherapy, radiotherapy, and surgery. However, myeloablative chemotherapy, tandem autologous stem cell transplant and immunotherapies improve survival rates for high-risk patients (2). The retinoic acid isotretinoin (13-cis-retinoic acid) is also used in high-risk patients to target minimal residual disease (3). Retinoic acids are vitamin A metabolites that induce differentiation and subsequently halt proliferation of neuroblastoma cells and other cancer types (4). The molecular mechanisms by which retinoic acids mediate these affects are diverse, and include targeting retinoic acid receptors (transcription factors) and disrupting oncogenic signalling pathways such as Wnt/β-catenin and MYCN (4). Retinoic acids can also alter mitochondrial function (4), and some increase mitochondrial respiration and content in adipocytes (5). New research by Jiang and colleagues shows the mitochondrial uncoupler niclosamide ethanolamine (NEN) also induces neuroblastoma cell differentiation (6).

Mitochondrial uncoupling is a natural process that occurs via endogenous mechanisms e.g., uncoupling proteins, or by pharmacologic means using small molecules termed mitochondrial uncouplers. NEN is a small molecule protonophore that transports protons from the mitochondrial intermembrane space into the mitochondrial matrix (Fig. 1). In doing so, mitochondrial uncouplers allow protons to reenter the matrix without producing ATP and thereby, ‘uncouple’ nutrient oxidation from ATP production (Fig. 1). Because mitochondrial uncoupling forces cells to oxidize more nutrients to maintain the proton motive force and ATP production, this process can be exploited for the treatment of diseases associated with excess nutrient intake such as obesity. However, for many years researchers have also been investigating the anticancer effects of mitochondrial uncouplers in different cancer types (7).

In the study by Jiang and colleagues, NEN induced neurite formation, halted proliferation of neuroblastoma cells in vitro and inhibited the growth of orthotopic neuroblastoma tumors in vivo (6). They postulated that the anticancer effects of NEN were through its ability to reverse the Warburg effect and tip the balance of metabolites towards a state that favors reshaping of the cancer epigenome and cell differentiation. Specifically, NEN increased pyruvate/lactate, NAD⁺/NADH, and α-ketoglutarate (α-KG)/2-hydroxyglutarate (2-HG) ratios, which indicates increased flux through the citric acid cycle. Treatment of neuroblastoma cells with α-KG also stimulated neurite formation. Interestingly, inhibition of glutaminase (which converts glutamine to glutamate that is subsequently converted to α-KG) inhibited NEN-induced neurite outgrowth. These latter experiments supported the role of α-KG, and glutamine metabolism, in neuroblastoma cell differentiation. Importantly, the authors validated the mitochondrial uncoupling mechanism in neuroblastoma cell differentiation by repeating key experiments with the structurally unrelated mitochondrial uncoupler BAM15 (8). BAM15 also induced neurite formation and increased the ratios of NAD⁺/NADH and α-KG/2-HG. Furthermore, the growth inhibitory and metabolic phenotypes induced by NEN in vitro were confirmed in neuroblastoma tumors in vivo. In these studies, NEN was fed to orthotopic tumor-bearing mice and did not cause loss of body weight or signs of toxicity.

The effects of NEN on the epigenome of neuroblastoma cells were shown through analysis of DNA methylation. NEN treatment promoted hypomethylation of CpG islands in regions close to transcriptional start sites of gene networks regulating neuron development and differentiation. Under conditions of hypoxia, NEN also promoted DNA demethylation and decreased hypoxia-inducible factor 1 (HIF1) target genes, including lactate dehydrogenase A, which converts pyruvate to lactate. NEN treatment reduced the expression of the MYCN transcription factor, and gene set enrichment analyses demonstrated downregulation of its target genes. Other oncogenic factors impacted by NEN treatment included upregulation of the tumor suppressor p53 and inhibition of Wnt/β-catenin signaling; findings supported by other studies of NEN in different cancer types (7, 8). Finally, Jiang and colleagues linked the gene expression effects of NEN in neuroblastoma cells to prognostic signatures in patients with neuroblastoma. Specifically, genes upregulated by NEN were mostly enriched with gene sets associated with favorable prognosis while genes that were downregulated by NEN treatment were associated with unfavorable prognoses.

The current study by Jiang and colleagues did not examine the effects of NEN in neuroblastoma cells without MYCN amplification. Furthermore, they showed that some genes upregulated by NEN were enriched in the unfavorable prognosis gene set derived from neuroblastomas without MYCN amplification. It was unclear from their study whether NEN may be beneficial for cancers without MYCN amplification. However, a study by Huang and colleagues showed NEN induced anticancer effects in both MYCN-amplified and non-amplified neuroblastoma cells in vitro and xenografts in vivo (9). Interestingly, proteomic analyses in the Huang and colleagues study revealed glutaminase was downregulated by NEN in nonamplified cells (9), which poses the question of whether NEN would induce neurite outgrowth in neuroblastoma cells without MYCN amplification. Nevertheless, the study by Jiang and colleagues compliments the findings of Huang and colleagues in that they both highlight the potential of NEN for the treatment of neuroblastoma. Future studies could examine if NEN-induced epigenome changes and differentiation mechanisms relate to spontaneous regression characteristics observed in some children with advanced disease and if it could be used to promote this process.

In summary, the study by Jiang and colleagues has demonstrated a novel role for the mitochondrial uncouplers NEN and BAM15 in neuroblastoma cell differentiation. NEN is currently in Phase I/II clinical trials for pediatric patients with relapsed/refractory AML, hormone-resistant prostate cancer, and familial adenomatous polyposis. However, NEN is limited by its poor pharmacokinetic properties (7). Therefore, NEN derivatives and other uncouplers that have better pharmacokinetic properties, i.e., improved oral bioavailability, may be better suited pharmacotherapies for cancer. Overall, this study has shed light on the therapeutic potential of mitochondrial uncouplers for inducing cancer cell differentiation.

F.L. Byrne reports nonfinancial support from Life Biosciences outside the submitted work. No disclosures were reported by the other author.