Anticancer therapy by anthracyclines often leads to the development of multidrug resistance (MDR), with subsequent treatment failure. Thiosemicarbazones have been previously suggested as suitable anthracycline partners due to their ability to overcome drug resistance through dual Pgp-dependent cytotoxicity-inducing effects. Here, we focused on combining anthracyclines (doxorubicin, daunorubicin, and mitoxantrone) and two thiosemicarbazones (DpC and Dp44mT) for treating cell types derived from the most frequent pediatric solid tumors. Our results showed synergistic effects for all combinations of treatments in all tested cell types. Nevertheless, further experiments revealed that this synergism was independent of Pgp expression but rather resulted from impaired DNA repair control leading to cell death via mitotic catastrophe. The downregulation of checkpoint kinase 1 (CHEK1) expression by thiosemicarbazones and the ability of both types of agents to induce double-strand breaks in DNA may explain the Pgp-independent synergism between anthracyclines and thiosemicarbazones. Moreover, the concomitant application of these agents was found to be the most efficient approach, achieving the strongest synergistic effect with lower concentrations of these drugs. Overall, our study identified a new mechanism that offers an avenue for combining thiosemicarbazones with anthracyclines to treat tumors regardless the Pgp status.

• Keywords
• anthracenedione, anthracyclines, checkpoint kinase 1, combined anticancer treatment, double strand breaks in DNA, pediatric solid tumors, thiosemicarbazones,
• MeSH
• Anthracyclines * pharmacology   MeSH
• Checkpoint Kinase 1 metabolism   MeSH
• Child MeSH
• Doxorubicin metabolism   pharmacology   MeSH
• Topoisomerase II Inhibitors MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism   MeSH
• DNA Damage MeSH
• Antibiotics, Antineoplastic MeSH
• Thiosemicarbazones * pharmacology   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anthracyclines * MeSH
• Checkpoint Kinase 1 MeSH
• CHEK1 protein, human MeSH Browser
• Doxorubicin MeSH
• Topoisomerase II Inhibitors MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 MeSH
• Antibiotics, Antineoplastic MeSH
• Thiosemicarbazones * MeSH

NANOG is a transcription factor involved in the regulation of pluripotency and stemness. The functional paralog of NANOG, NANOGP8, differs from NANOG in only three amino acids and exhibits similar reprogramming activity. Given the transcriptional regulatory role played by NANOG, the nuclear localization of NANOG/NANOGP8 has primarily been considered to date. In this study, we investigated the intriguing extranuclear localization of NANOG and demonstrated that a substantial pool of NANOG/NANOGP8 is localized at the centrosome. Using double immunofluorescence, the colocalization of NANOG protein with pericentrin was identified by two independent anti-NANOG antibodies among 11 tumor and non-tumor cell lines. The validity of these observations was confirmed by transient expression of GFP-tagged NANOG, which also colocalized with pericentrin. Mass spectrometry of the anti-NANOG immunoprecipitated samples verified the antibody specificity and revealed the expression of both NANOG and NANOGP8, which was further confirmed by real-time PCR. Using cell fractionation, we show that a considerable amount of NANOG protein is present in the cytoplasm of RD and NTERA-2 cells. Importantly, cytoplasmic NANOG was unevenly distributed at the centrosome pair during the cell cycle and colocalized with the distal region of the mother centriole, and its presence was markedly associated with centriole maturation. Along with the finding that the centrosomal localization of NANOG/NANOGP8 was detected in various tumor and non-tumor cell types, these results provide the first evidence suggesting a common centrosome-specific role of NANOG.

• Keywords
• NANOG, NANOGP8, centrosome, human, localization, mother centriole,
• MeSH
• Centrioles immunology   MeSH
• Centrosome immunology   MeSH
• Humans MeSH
• Nanog Homeobox Protein immunology   MeSH
• Cell Proliferation MeSH
• Transfection MeSH
• Transcription Factors MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NANOG protein, human MeSH Browser
• Nanog Homeobox Protein MeSH
• Transcription Factors 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

The survival rate for patients with high-risk neuroblastomas remains poor despite new improvements in available therapeutic modalities. A detailed understanding of the mechanisms underlying clinical responses to multimodal treatment is one of the important aspects that may provide precision in the prediction of a patient's clinical outcome. Our study was designed as a detailed comparative analysis of five selected proteins (DDX39A, HMGA1, HOXC9, NF1, and PBX1) in one cohort of patients using the same methodical approaches. These proteins were already reported separately as related to the resistance or sensitivity to retinoids and as useful prognostic markers of survival probability. In the cohort of 19 patients suffering from high-risk neuroblastomas, we analyzed initial immunohistochemistry samples obtained by diagnostic biopsy and post-induction samples taken after the end of induction therapy. The expression of DDX39A, HMGA1, HOXC9, and NF1 showed varied patterns with almost no differences between responders and non-responders. Nevertheless, we found very interesting results for PBX1: non-responders had significantly higher expression levels of this protein in the initial tumor samples when compared with responders; this expression pattern changed inversely in the post-induction samples, and this change was also statistically significant. Moreover, our results from survival analyses reveal the prognostic value of PBX1, NF1, and HOXC9 expression in neuroblastoma tissue. In addition to the prognostic importance of PBX1, NF1, and HOXC9 proteins, our results demonstrated that PBX1 could be used for the prediction of the clinical response to induction chemotherapy in patients suffering from high-risk neuroblastoma.

• Keywords
• HOXC9, NF1, PBX1, immunohistochemistry, neuroblastoma, predictive markers, prognostic markers,
• Publication type
• Journal Article MeSH

The three most frequent pediatric sarcomas, i.e., Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma, were examined in this study: three cell lines derived from three primary tumor samples were analyzed from each of these tumor types. Detailed comparative analysis of the expression of three putative cancer stem cell markers related to sarcomas-ABCG2, CD133, and nestin-was performed on both primary tumor tissues and corresponding cell lines. The obtained results showed that the frequency of ABCG2-positive and CD133-positive cells was predominantly increased in the respective cell lines but that the high levels of nestin expression were reduced in both osteosarcomas and rhabdomyosarcomas under in vitro conditions. These findings suggest the selection advantage of cells expressing ABCG2 or CD133, but the functional tests in NOD/SCID gamma mice did not confirm the tumorigenic potential of cells harboring this phenotype. Subsequent analysis of the expression of common stem cell markers revealed an evident relationship between the expression of the transcription factor Sox2 and the tumorigenicity of the cell lines in immunodeficient mice: the Sox2 levels were highest in the two cell lines that were demonstrated as tumorigenic. Furthermore, Sox2-positive cells were found in the respective primary tumors and all xenograft tumors showed apparent accumulation of these cells. All of these findings support our conclusion that regardless of the expression of ABCG2, CD133 and nestin, only cells displaying increased Sox2 expression are directly involved in tumor initiation and growth; therefore, these cells fit the definition of the cancer stem cell phenotype.

• Keywords
• Cancer stem cells, Markers, Pediatric sarcomas, Sox2, Tumorigenicity,
• MeSH
• ATP Binding Cassette Transporter, Subfamily G, Member 2 metabolism   MeSH
• AC133 Antigen metabolism   MeSH
• Child MeSH
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Mice, Inbred NOD MeSH
• Mice, SCID MeSH
• Mice MeSH
• Cell Transformation, Neoplastic metabolism   pathology   MeSH
• Biomarkers, Tumor metabolism   MeSH
• Cell Line, Tumor MeSH
• Neoplastic Stem Cells metabolism   pathology   MeSH
• Neoplasm Proteins metabolism   MeSH
• Nestin metabolism   MeSH
• Osteosarcoma metabolism   pathology   MeSH
• Child, Preschool MeSH
• Rhabdomyosarcoma metabolism   pathology   MeSH
• Sarcoma metabolism   pathology   MeSH
• SOXB1 Transcription Factors metabolism   MeSH
• Animals MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Mice MeSH
• Child, Preschool MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
• ABCG2 protein, human MeSH Browser
• AC133 Antigen MeSH
• Biomarkers, Tumor MeSH
• Neoplasm Proteins MeSH
• Nestin MeSH
• PROM1 protein, human MeSH Browser
• SOX2 protein, human MeSH Browser
• SOXB1 Transcription Factors MeSH