Despite constant advances in the field of pediatric oncology, the survival rate of high-risk neuroblastoma patients remains poor. The molecular and genetic features of neuroblastoma, such as MYCN amplification and stemness status, have established themselves not only as potent prognostic and predictive factors but also as intriguing targets for personalized therapy. Novel thiosemicarbazones target both total level and activity of a number of proteins involved in some of the most important signaling pathways in neuroblastoma. In this study, we found that di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) potently decreases N-MYC in MYCN-amplified and c-MYC in MYCN-nonamplified neuroblastoma cell lines. Furthermore, DpC succeeded in downregulating total EGFR and phosphorylation of its most prominent tyrosine residues through the involvement of NDRG1, a positive prognostic marker in neuroblastoma, which was markedly upregulated after thiosemicarbazone treatment. These findings could provide useful knowledge for the treatment of MYC-driven neuroblastomas that are unresponsive to conventional therapies.

• Keywords
• DpC, EGFR, MYC, NDRG1, lipid droplet, neuroblastoma, thiosemicarbazone,
• MeSH
• Gene Amplification drug effects   MeSH
• Models, Biological MeSH
• Iron Chelating Agents pharmacology   MeSH
• Down-Regulation drug effects   MeSH
• ErbB Receptors metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Stress, Physiological drug effects   MeSH
• Intracellular Signaling Peptides and Proteins metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Neuroblastoma metabolism   pathology   MeSH
• Cell Cycle Proteins metabolism   MeSH
• N-Myc Proto-Oncogene Protein metabolism   MeSH
• Proto-Oncogene Proteins c-akt metabolism   MeSH
• Pyridines pharmacology   MeSH
• Signal Transduction * MeSH
• Thiosemicarbazones pharmacology   MeSH
• Cell Shape drug effects   MeSH
• Gene Silencing drug effects   MeSH
• Up-Regulation drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Iron Chelating Agents MeSH
• di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone MeSH Browser
• ErbB Receptors MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• N-myc downstream-regulated gene 1 protein MeSH Browser
• Cell Cycle Proteins MeSH
• N-Myc Proto-Oncogene Protein MeSH
• Proto-Oncogene Proteins c-akt MeSH
• Pyridines MeSH
• Thiosemicarbazones MeSH

Serial xenotransplantation of sorted cancer cells in immunodeficient mice remains the most complex test of cancer stem cell (CSC) phenotype. However, we have demonstrated in various sarcomas that putative CSC surface markers fail to identify CSCs, thereby impeding the isolation of CSCs for subsequent analyses. Here, we utilized serial xenotransplantation of unsorted rhabdomyosarcoma cells in NOD/SCID gamma (NSG) mice as a proof-of-principle platform to investigate the molecular signature of CSCs. Indeed, serial xenotransplantation steadily enriched for rhabdomyosarcoma stem-like cells characterized by enhanced aldehyde dehydrogenase activity and increased colony and sphere formation capacity in vitro. Although the expression of core pluripotency factors (SOX2, OCT4, NANOG) and common CSC markers (CD133, ABCG2, nestin) was maintained over the passages in mice, gene expression profiling revealed gradual changes in several stemness regulators and genes linked with undifferentiated myogenic precursors, e.g., SOX4, PAX3, MIR145, and CDH15. Moreover, we identified the induction of a hybrid epithelial/mesenchymal gene expression signature that was associated with the increase in CSC number. In total, 60 genes related to epithelial or mesenchymal traits were significantly altered upon serial xenotransplantation. In silico survival analysis based on the identified potential stemness-associated genes demonstrated that serial xenotransplantation of unsorted rhabdomyosarcoma cells in NSG mice might be a useful tool for the unbiased enrichment of CSCs and the identification of novel CSC-specific targets. Using this approach, we provide evidence for a recently proposed link between the hybrid epithelial/mesenchymal phenotype and cancer stemness.

• Keywords
• cancer stem cells, epithelial/mesenchymal phenotype, in vivo tumorigenicity assay, rhabdomyosarcoma, serial xenotransplantation, stem-like state, stemness,
• Publication type
• Journal Article MeSH