Mitochondrial uncoupler and retinoic acid synergistically induce differentiation and inhibit proliferation in neuroblastoma.

Neuroblastoma is a leading cause of death in childhood cancer cases. Unlike adult malignancies, which typically develop from aged cells through accumulated damage and mutagenesis, neuroblastoma originates from neural crest cells with disrupted differentiation. This distinct feature provides novel therapeutic opportunities beyond conventional cytotoxic methods. Previously, we reported that the mitochondrial uncoupler NEN (niclosamide ethanolamine) activated mitochondria respiration to reprogram the epigenome, promoting neuronal differentiation. In the current study, we further combine NEN with retinoic acid (RA) to promote neural differentiation both in vitro and in vivo. The treatment increased the expression of RA signaling and neuron differentiation-related genes, resulting in a global shift in the transcriptome towards a more favorable prognosis. Overall, these results suggest that the combination of a mitochondrial uncoupler and the differentiation agent RA is a promising therapeutic strategy for neuroblastoma.

Mitochondrial Uncoupling Induces Epigenome Remodeling and Promotes Differentiation in Neuroblastoma.

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 NAD+/NADH and pyruvate/lactate ratios and also the α-ketoglutarate/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 ß-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 ß-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. SIGNIFICANCE: Targeting cancer metabolism using the mitochondrial uncoupler niclosamide ethanolamine leads to methylome reprogramming and differentiation in neuroblastoma, providing a therapeutic opportunity to reverse the Warburg effect and suppress tumor growth. See related commentary by Byrne and Bell, p.167.

The Use of Fluorescence in situ Hybridization to Confirm PRKACA Gene Rearrangement in Fibrolamellar Hepatocellular Carcinoma: A Validation Study.

OBJECTIVE: The objectives of this study are to define the specificity of the DNAJB1-PRKACA fusion transcript for the fibrolamellar subtype of hepatocellular carcinoma (FL-HCC) by testing a targeted sampling of other hepatic neoplasms/proliferations and extrahepatic neoplasms seen in children and young adults and to develop a FISH assay using a commercially available PRKACA break apart probe for use in a CLIA-certified clinical laboratory. METHODS: Formalin fixed paraffin embedded tissue sections from 12 FL-HCC cases, 142 cases of other hepatic neoplasms/proliferations (conventional HCC, focal nodular hyperplasia (FNH), hepatocellular adenoma (HA) and hepatoblastoma (HB)) and extrahepatic neoplasms (neuroblastoma (NB), Wilms tumor (WT) and Gastrointestinal neuroendocrine tumor (GNET)) and 60 matched background normal control tissues underwent fluorescence in situ hybridization (FISH) testing using a break apart probe targeting the PRKACA gene locus on chromosome 19 using standard techniques. RESULTS: The PRKACA gene rearrangement was detected in 11/12 (92%) FL-HCC cases and 1/94 (1%) of conventional HCC cases. All other cases and background control tissues were negative for the PRKACA gene rearrangement. These findings establish a test sensitivity of 91.7% and specificity of 99.5%. CONCLUSION: This study shows that, using standard techniques, FISH testing with a commercially available break apart probe targeting the PRKACA gene can be used as a surrogate for the DNAJB1-PRKACA fusion commonly found in FL-HCC. Also, the PRKACA gene rearrangement is not expressed in other hepatic neo-plasms/proliferations or extrahepatic neoplasms seen in children and young adults. Finally, FISH testing can be used as a diagnostic tool to confirm the diagnosis of FL-HCC, in the appropriate clinical setting.