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.

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.

Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.

The specific targeting of signal transduction by low-molecular-weight inhibitors or monoclonal antibodies represents a very promising personalized treatment strategy in pediatric oncology. In this study, we present the successful and clinically relevant use of commercially available phospho-protein arrays for analyses of the phosphorylation profiles of a broad spectrum of receptor tyrosine kinases and their downstream signaling proteins in tumor tissue samples. Although these arrays were made for research purposes on human biological samples, they have already been used by several authors to profile various tumor types. Our study performed a systematic analysis of the advantages and pitfalls of the use of this method for personalized clinical medicine. In certain clinical cases and their series, we demonstrated the important aspects of data processing and evaluation, the use of phospho-protein arrays for single sample and serial sample analyses, and the validation of obtained results by immunohistochemistry, as well as the possibilities of this method for the hierarchical clustering of pediatric solid tumors. Our results clearly show that phospho-protein arrays are apparently useful for the clinical consideration of druggable molecular targets within a specific tumor. Thus, their potential validation for diagnostic purposes may substantially improve the personalized approach in the treatment of relapsed or refractory solid tumors.

Mitochondria are central for cancer responses to therapy-induced stress signals. Refractory tumors often show attenuated sensitivity to apoptotic signaling, yet clinically relevant molecular actors to target mitochondria-mediated resistance remain elusive. Here, we show that MYC-driven neuroblastoma cells rely on intact mitochondrial ribosome (mitoribosome) processivity and undergo cell death following pharmacological inhibition of mitochondrial translation, regardless of their multidrug/mitochondrial resistance and stem-like phenotypes. Mechanistically, inhibiting mitoribosomes induced the mitochondrial stress-activated integrated stress response (ISR), leading to downregulation of c-MYC/N-MYC proteins prior to neuroblastoma cell death, which could be both rescued by the ISR inhibitor ISRIB. The ISR blocks global protein synthesis and shifted the c-MYC/N-MYC turnover toward proteasomal degradation. Comparing models of various neuroectodermal tumors and normal fibroblasts revealed overexpression of MYC proteins phosphorylated at the degradation-promoting site T58 as a factor that predetermines vulnerability of MYC-driven neuroblastoma to mitoribosome inhibition. Reducing N-MYC levels in a neuroblastoma model with tunable MYCN expression mitigated cell death induction upon inhibition of mitochondrial translation and functionally validated the propensity of neuroblastoma cells for MYC-dependent cell death in response to the mitochondrial ISR. Notably, neuroblastoma cells failed to develop significant resistance to the mitoribosomal inhibitor doxycycline over a long-term repeated (pulsed) selection. Collectively, we identify mitochondrial translation machinery as a novel synthetic lethality target for multidrug-resistant MYC-driven tumors.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Its dismal prognosis is often attributed to the presence of cancer stem cells (CSCs) that have been identified in PDAC using various markers. However, the co-expression of all of these markers has not yet been evaluated. Furthermore, studies that compare the expression levels of CSC markers in PDAC tumor samples and in cell lines derived directly from those tumors are lacking. Here, we analyzed the expression of putative CSC markers-CD24, CD44, epithelial cell adhesion molecule (EpCAM), CD133, and nestin-by immunofluorescence, flow cytometry and quantitative PCR in 3 PDAC-derived cell lines and by immunohistochemistry in 3 corresponding tumor samples. We showed high expression of the examined CSC markers among all of the cell lines and tumor samples, with the exception of CD24 and CD44, which were enriched under in vitro conditions compared with tumor tissues. The proportions of cells positive for the remaining markers were comparable to those detected in the corresponding tumors. Co-expression analysis using flow cytometry revealed that CD24+/CD44+/EpCAM+/CD133+ cells represented a significant population of the cells (range, 43 to 72%) among the cell lines. The highest proportion of CD24+/CD44+/EpCAM+/CD133+ cells was detected in the cell line derived from the tumor of a patient with the shortest survival. Using gene expression profiling, we further identified the specific pro-tumorigenic expression profile of this cell line compared with the profiles of the other two cell lines. Together, CD24+/CD44+/EpCAM+/CD133+ cells are present in PDAC cell lines derived from primary tumors, and their increased proportion corresponds with a pro-tumorigenic gene expression profile.

Although methotrexate (MTX) is the most well-known antifolate included in many standard therapeutic regimens, substantial toxicity limits its wider use, particularly in pediatric oncology. Our study focused on a detailed analysis of MTX effects in cell lines derived from two types of pediatric solid tumors: medulloblastoma and osteosarcoma. The main aim of this study was to analyze the effects of treatment with MTX at concentrations comparable to MTX plasma levels in patients treated with high-dose or low-dose MTX. The results showed that treatment with MTX significantly decreased proliferation activity, inhibited the cell cycle at S-phase and induced apoptosis in Daoy and Saos-2 reference cell lines, which were found to be MTX-sensitive. Furthermore, no difference in these effects was observed following treatment with various doses of MTX ranging from 1 to 40 µM. These findings suggest the possibility of achieving the same outcome with the application of low-dose MTX, an extremely important result, particularly for clinical practice. Another important aspect of treatment with high-dose MTX in clinical practice is the administration of leucovorin (LV) as an antidote to reduce MTX toxicity in normal cells. For this reason, the combined application of MTX and LV was also included in our experiments; however, this application of MTX together with LV did not elicit any detectable effect. The expression analysis of genes involved in the mechanisms of resistance to MTX was a final component of our study, and the results helped us to elucidate the mechanisms of the various responses to MTX among the cell lines included in our study.

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.

Tyrosine kinase inhibitors (TKIs) are frequently used in combined therapy to enhance treatment efficacy and overcome drug resistance. The present study analyzed the effects of three inhibitors, sunitinib, gefitinib, and lapatinib, combined with iron-chelating agents, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) or di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). Simultaneous administration of the drugs consistently resulted in synergistic and/or additive activities against the cell lines derived from the most frequent types of pediatric solid tumors. The results of a detailed analysis of cell signaling in the neuroblastoma cell lines revealed that TKIs inhibited the phosphorylation of the corresponding receptor tyrosine kinases, and thiosemicarbazones downregulated the expression of epidermal growth factor receptor, platelet-derived growth factor receptor, and insulin-like growth factor-1 receptor, leading to a strong induction of apoptosis. Marked upregulation of the metastasis suppressor N-myc downstream regulated gene-1 (NDRG1), which is known to be activated and upregulated by thiosemicarbazones in adult cancers, was also detected in thiosemicarbazone-treated neuroblastoma cells. Importantly, these effects were more pronounced in the cells treated with drug combinations, especially with the combinations of lapatinib with thiosemicarbazones. Therefore, these results provide a rationale for novel strategies combining iron-chelating agents with TKIs in therapy of pediatric solid tumors.

CD133 (also known as prominin-1) is a cell surface glycoprotein that is widely used for the identification of stem cells. Furthermore, its glycosylated epitope, AC133, has recently been discussed as a marker of cancer stem cells in various human malignancies. During our recent experiments on rhabdomyosarcomas (RMS), we unexpectedly identified an atypical nuclear localization of CD133 in a relatively stable subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of CD133 has not yet been proven or analyzed in detail in cancer cells. In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines. The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies. Although the role of CD133 in the cell nucleus remains unclear, these results clearly indicate that this atypical nuclear localization of CD133 in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines.

The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor-2 (FGF-2), which activates FGF receptors (FGFRs) and stimulates the mitogen-activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin-like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF-2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF-2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation-related genes and downregulation of stem cell genes. Thus, exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling. Consistent with this hypothesis, expression of endogenous FGF-2 decreased during hESC differentiation and FGF-2 knockdown-induced hESC differentiation. In addition, FGF-2 signaling via FGFR2 activated MAPK kinase/extracellular signal-regulated kinase and AKT kinases, protected hESC from stress-induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self-renewal, cell survival, and adhesion by exogenous and endogenous FGF-2 may synergize to maintain the undifferentiated growth of hESCs.

Combining low-dose chemotherapies is a strategy for designing less toxic and more potent childhood cancer treatments. We examined the effects of combining the novel thiosemicarbazones, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), or its analog, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), with the standard chemotherapies, celecoxib (CX), etoposide (ETO), or temozolomide (TMZ). These combinations were analyzed for synergism to inhibit proliferation of three pediatric tumor cell-types, namely osteosarcoma (Saos-2), medulloblastoma (Daoy) and neuroblastoma (SH-SY5Y). In terms of mechanistic dissection, this study discovered novel thiosemicarbazone targets not previously identified and which are important for considering possible drug combinations. In this case, DpC and Dp44mT caused: (1) up-regulation of a major protein target of CX, namely cyclooxygenase-2 (COX-2); (2) down-regulation of the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), which is known to affect TMZ resistance; (3) down-regulation of mismatch repair (MMR) proteins, MSH2 and MSH6, in Daoy and SH-SY5Y cells; and (4) down-regulation in all three cell-types of the MMR repair protein, MLH1, and also topoisomerase 2α (Topo2α), the latter of which is an ETO target. While thiosemicarbazones up-regulate the metastasis suppressor, NDRG1, in adult cancers, it is demonstrated herein for the first time that they induce NDRG1 in all three pediatric tumor cell-types, validating its role as a potential target. In fact, siRNA studies indicated that NDRG1 was responsible for MGMT down-regulation that may prevent TMZ resistance. Examining the effects of combining thiosemicarbazones with CX, ETO, or TMZ, the most promising synergism was obtained using CX. Of interest, a positive relationship was observed between NDRG1 expression of the cell-type and the synergistic activity observed in the combination of thiosemicarbazones and CX. These studies identify novel thiosemicarbazone targets relevant to childhood cancer combination chemotherapy.

Giant-cell tumor of bone (GCTB) is an intermediate type of primary bone tumor characterized by locally aggressive growth with metastatic potential. The aim of this study was to identify new druggable targets among the cell signaling molecules involved in GCTB tumorigenesis. Profiles of activated signaling proteins in fresh-frozen tumor samples and tumor-derived cell lines were determined using phosphoprotein arrays. Analysis of the obtained data revealed epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor beta (PDGFRβ) as potential targets, but only the PDGFR inhibitor sunitinib caused a considerable decrease in stromal cell viability in vitro. Furthermore, in the case of a 17-year-old patient suffering from GCTB, we showed that the addition of sunitinib to the standard treatment of GCTB with the monoclonal antibody denosumab resulted in the complete depletion of multinucleated giant cells and mononuclear stromal cells in the tumor tissue. To summarize, the obtained data showed that a specific receptor tyrosine kinase (RTK) signaling pattern is activated in GCTB cells and plays an important role in the regulation of cell proliferation. Thus, activated RTKs and their downstream signaling pathways represent useful targets for precision treatment with low-molecular-weight inhibitors or with other types of modern biological therapy.

Methotrexate is an important chemotherapeutic drug widely known as an inhibitor of dihydrofolate reductase (DHFR) which inhibits the reduction of folic acid. DHFR-mediated effects are apparently responsible for its primary antineoplastic action. However, other non-DHFR-mediated effects of methotrexate have been recently discovered, which might be very useful in the development of new strategies for the treatment of pediatric malignancies. The principal goal of this study was to analyze the possible impact of clinically achievable methotrexate levels on cell proliferation, mechanisms of epigenetic regulation (DNA methylation and histone acetylation), induced differentiation and the expression of differentiation-related genes in six osteosarcoma cell lines.

A detailed analysis of the expression of 440 cancer-related genes was performed after the combined treatment of medulloblastoma cells with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). The combinations of retinoids and celecoxib as a COX-2 inhibitor were reported to be effective in some regimens of metronomic therapy of relapsed solid tumors with poor prognosis. Our previous findings on neuroblastoma cells using expression profiling showed that LOX/COX inhibitors have the capability of enhancing the differentiating action of ATRA. Presented study focused on the continuation of our previous work to confirm the possibility of enhancing ATRA-induced cell differentiation in these cell lines via the application of LOX/COX inhibitors. This study provides more detailed information concerning the mechanisms of the enhancement of the ATRA-induced differentiation of medulloblastoma cells.

Infantile myofibromatosis represents one of the most common proliferative fibrous tumors of infancy and childhood. More effective treatment is needed for drug-resistant patients, and targeted therapy using specific protein kinase inhibitors could be a promising strategy. To date, several studies have confirmed a connection between the p.R561C mutation in gene encoding platelet-derived growth factor receptor beta (PDGFR-beta) and the development of infantile myofibromatosis. This study aimed to analyze the phosphorylation of important kinases in the NSTS-47 cell line derived from a tumor of a boy with infantile myofibromatosis who harbored the p.R561C mutation in PDGFR-beta. The second aim of this study was to investigate the effects of selected protein kinase inhibitors on cell signaling and the proliferative activity of NSTS-47 cells. We confirmed that this tumor cell line showed very high phosphorylation levels of PDGFR-beta, extracellular signal-regulated kinases (ERK) 1/2 and several other protein kinases. We also observed that PDGFR-beta phosphorylation in tumor cells is reduced by the receptor tyrosine kinase inhibitor sunitinib. In contrast, MAPK/ERK kinases (MEK) 1/2 and ERK1/2 kinases remained constitutively phosphorylated after treatment with sunitinib and other relevant protein kinase inhibitors. Our study showed that sunitinib is a very promising agent that affects the proliferation of tumor cells with a p.R561C mutation in PDGFR-beta.

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.