PURPOSE: Pediatric sarcomas are bone and soft tissue tumors that often exhibit high metastatic potential and refractory stem-like phenotypes, resulting in poor outcomes. Aggressive sarcomas frequently harbor a disrupted p53 pathway. However, whether pediatric sarcoma stemness is associated with abrogated p53 function and might be attenuated via p53 reactivation remains unclear. METHODS: We utilized a unique panel of pediatric sarcoma models and tumor tissue cohorts to investigate the correlation between the expression of stemness-related transcription factors, p53 pathway dysregulations, tumorigenicity in vivo, and clinicopathological features. TP53 mutation status was assessed by next-generation sequencing. Major findings were validated via shRNA-mediated silencing and functional assays. The p53 pathway-targeting drugs were used to explore the effects and selectivity of p53 reactivation against sarcoma cells with stem-like traits. RESULTS: We found that highly tumorigenic stem-like sarcoma cells exhibit dysregulated p53, making them vulnerable to drugs that restore wild-type p53 activity. Immunohistochemistry of mouse xenografts and human tumor tissues revealed that p53 dysregulations, together with enhanced expression of the stemness-related transcription factors SOX2 or KLF4, are crucial features in pediatric osteosarcoma, rhabdomyosarcoma, and Ewing's sarcoma development. p53 dysregulation appears to be an important step for sarcoma cells to acquire a fully stem-like phenotype, and p53-positive pediatric sarcomas exhibit a high frequency of early metastasis. Importantly, reactivating p53 signaling via MDM2/MDMX inhibition selectively induces apoptosis in aggressive, stem-like Ewing's sarcoma cells while sparing healthy fibroblasts. CONCLUSIONS: Our results indicate that restoring canonical p53 activity provides a promising strategy for developing improved therapies for pediatric sarcomas with unfavorable stem-like traits.

• Keywords
• Cancer stemness, Pediatric sarcomas, Prognosis, Targeted therapy, p53,
• MeSH
• Child MeSH
• Kruppel-Like Factor 4 * MeSH
• Humans MeSH
• Adolescent MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Neoplastic Stem Cells * metabolism   pathology   MeSH
• Tumor Suppressor Protein p53 * metabolism   genetics   MeSH
• Child, Preschool MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Sarcoma * genetics   pathology   metabolism   MeSH
• Signal Transduction MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Mice MeSH
• Child, Preschool MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• KLF4 protein, human MeSH Browser
• Klf4 protein, mouse MeSH Browser
• Kruppel-Like Factor 4 * MeSH
• Tumor Suppressor Protein p53 * MeSH

Prominin-1 (CD133) is a cholesterol-binding membrane glycoprotein selectively associated with highly curved and prominent membrane structures. It is widely recognized as an antigenic marker of stem cells and cancer stem cells and is frequently used to isolate them from biological and clinical samples. Recent progress in understanding various aspects of CD133 biology in different cell types has revealed the involvement of CD133 in the architecture and dynamics of plasma membrane protrusions, such as microvilli and cilia, including the release of extracellular vesicles, as well as in various signaling pathways, which may be regulated in part by posttranslational modifications of CD133 and its interactions with a variety of proteins and lipids. Hence, CD133 appears to be a master regulator of cell signaling as its engagement in PI3K/Akt, Src-FAK, Wnt/β-catenin, TGF-β/Smad and MAPK/ERK pathways may explain its broad action in many cellular processes, including cell proliferation, differentiation, and migration or intercellular communication. Here, we summarize early studies on CD133, as they are essential to grasp its novel features, and describe recent evidence demonstrating that this unique molecule is involved in membrane dynamics and molecular signaling that affects various facets of tissue homeostasis and cancer development. We hope this review will provide an informative resource for future efforts to elucidate the details of CD133's molecular function in health and disease.

• Keywords
• CD133, Cancer, Cancer stem cell, Cell signaling, Cilium, Exosome, Lipid raft, Microvillus, Prominin-1, Stem cell,
• MeSH
• AC133 Antigen metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Phosphatidylinositol 3-Kinases * metabolism   MeSH
• Neoplastic Stem Cells metabolism   MeSH
• Signal Transduction * MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• AC133 Antigen MeSH
• Phosphatidylinositol 3-Kinases * MeSH

The main objective of this study was to analyze changes in the antiproliferative effect of vitamin D3, in the form of calcitriol and calcidiol, via its combined application with all-trans retinoic acid (ATRA) in osteosarcoma cell lines. The response to treatment with calcitriol and calcidiol alone was specific for each cell line. Nevertheless, we observed an enhanced effect of combined treatment with ATRA and calcitriol in the majority of the cell lines. Although the levels of respective nuclear receptors did not correlate with the sensitivity of cells to these drugs, vitamin D receptor (VDR) upregulation induced by ATRA was found in cell lines that were the most sensitive to the combined treatment. In addition, all these cell lines showed high endogenous levels of retinoic acid receptor α (RARα). Our study confirmed that the combination of calcitriol and ATRA can achieve enhanced antiproliferative effects in human osteosarcoma cell lines in vitro. Moreover, we provide the first evidence that ATRA is able to upregulate VDR expression in human osteosarcoma cells. According to our results, the endogenous levels of RARα and VDR could be used as a predictor of possible synergy between ATRA and calcitriol in osteosarcoma cells.

• Keywords
• all-trans retinoic acid, calcidiol, calcitriol, osteosarcoma, retinoic acid receptor α, vitamin D receptor,
• MeSH
• Retinoic Acid Receptor alpha metabolism   MeSH
• Calcifediol administration & dosage   MeSH
• Calcitriol administration & dosage   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Osteosarcoma drug therapy   metabolism   MeSH
• Antineoplastic Agents administration & dosage   MeSH
• Antineoplastic Combined Chemotherapy Protocols MeSH
• Receptors, Calcitriol metabolism   MeSH
• Drug Screening Assays, Antitumor MeSH
• Tretinoin administration & dosage   MeSH
• Vitamins administration & dosage   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Retinoic Acid Receptor alpha MeSH
• Calcifediol MeSH
• Calcitriol MeSH
• Antineoplastic Agents MeSH
• Receptors, Calcitriol MeSH
• Tretinoin MeSH
• Vitamins 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

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.

• MeSH
• Adenocarcinoma diagnosis   metabolism   MeSH
• Epithelial Cell Adhesion Molecule metabolism   MeSH
• AC133 Antigen metabolism   MeSH
• CD24 Antigen metabolism   MeSH
• Hyaluronan Receptors metabolism   MeSH
• Stem Cells metabolism   MeSH
• Cells, Cultured MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Middle Aged MeSH
• Humans MeSH
• Biomarkers, Tumor MeSH
• Pancreatic Neoplasms diagnosis   metabolism   MeSH
• Nestin metabolism   MeSH
• Prognosis MeSH
• Flow Cytometry MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Aged MeSH
• Transcriptome MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Epithelial Cell Adhesion Molecule MeSH
• AC133 Antigen MeSH
• CD24 Antigen MeSH
• Hyaluronan Receptors MeSH
• EPCAM protein, human MeSH Browser
• Biomarkers, Tumor MeSH
• NES protein, human MeSH Browser
• Nestin MeSH
• PROM1 protein, human MeSH Browser