8842

PROM1

AC133|CD133|CORD12|MCDR2|PROML1|RP41|STGD4;prominin 1;PROM1;prominin 1

antigen AC133|hProminin|hematopoietic stem cell antigen|prominin-1|prominin-like 1|prominin-like protein 1

4p15.32

protein-coding

pediatric brain tumors are significant causes of morbidity and mortality. It hasbeen hypothesized that they derive from self-renewing multipotent neural stem cells. Here, we tested whether different pediatric brain tumors, including medulloblastomas and gliomas, contain cells with properties similar to neural stem cells. We find that tumor-derived progenitors form neurospheres that can bepassaged at clonal density and are able to self-renew. Under conditions promoting differentiation, individual cells are multipotent, giving rise to both neurons and glia, in proportions that reflect the tumor of origin. Unlike normal neural stem cells, however, tumor-derived progenitors have an unusual capacity to proliferate and sometimes differentiate into abnormal cells with multiple differentiation markers. Gene expression analysis reveals that both whole tumorsand tumor-derived neurospheres express many genes characteristic of neural and other stem cells, including CD133, Sox2, musashi-1, bmi-1, maternal embryonic leucine zipper kinase, and phosphoserine phosphatase, with variation from tumor to tumor. After grafting to neonatal rat brains, tumor-derived neurosphere cellsmigrate, produce neurons and glia, and continue to proliferate for more than 4 weeks. The results show that pediatric brain tumors contain neural stem-like cells with altered characteristics that may contribute to tumorigenesis. This finding may have important implications for treatment by means of specific targeting of stem-like cells within brain tumors.

BACKGROUND: Recently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and thedrug resistance property of CD133 positive cancer stem cells, however, are stillunknown. RESULTS: In this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol) and etoposide (VP16) compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors. CONCLUSION: Our study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumor's resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in tumors to improve the survival of brain tumor patients.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for up to 10% of ALL childhood malignancies. Cellular heterogeneity is a hallmark of this embryonal cancer, as distinct neural crest lineages can be found within the same tumor sample. The aim of our study was to investigate the presence of asubpopulation of immature cells with features of cancer-like stem cells in 10 neuroblastoma cell lines. RT-PCR and flow cytometry were performed in order to analyze different kinds of 'stemness genes' such as: NESTIN (NES), CD133, SOX-2,BMI1, c-KIT, MELK1, MUSASHI-1 (MSI1), FAS, CD44 and VIMENTIN (VIM). In addition,glial and neuronal markers such as NCAM1, GFAP and B-TUBULIN III (TUBB3) were analyzed. Epigenetic changes within the CD133 (Prominin-1) gene promoter were also analyzed. Neuroblastoma cell lines showed a particular pattern of expression, suggesting the presence of an immature cancer stem cell-like subpopulation. The CD133 protein, commonly used to enrich putative cancer propagating stem cell-like populations in different kinds of solid tumors, presented a half-methylated DNA state in 7 of the 12 neuroblastoma cell lines analyzed. An increase in RNA and protein levels of CD133 was achieved following demethylation by assays using 5-aza-2'-deoxycytidine (5-Aza-dC). Since cancer stem cells are believed to be responsible for tumor metastasis, escape from anticancer therapies and disease relapse, their therapeutic targeting and analysis is crucial in neuroblastoma. Moreover, the regulation of CD133 by epigenetic changes may provide an innovative mechanism of CD133 expression as its regulation still remains unclear.

The surface marker PROM1 is considered one of the most important markers of tumor-initiating cells, and its expression is believed to be an adverse prognostic factor in gliomas and in other malignancies. To date, to our knowledge, no specific studies of its expression in medulloblastoma series have been performed. The aims of our study were to evaluate the expression profile ofthe PROM1 gene in medulloblastoma and to assess its possible role as a prognostic factor. The PROM1 gene expression was evaluated by quantitative- polymerase chain reaction on 45 medulloblastoma samples by using specific dye-labeled probe systems. A significantly higher expression of PROM1 was found both in patients with poorer prognosis (P= .007) and in those with metastasis (P= .03). Kaplan-Meier analysis showed that both overall survival (OS) and progression-free survival (PFS) were shorter in patients with higher PROM1 mRNA levels than in patients with lower expression, even when the desmoplastic cases were excluded (P= .0004 and P= .002, for OS and PFS for ALL cases, respectively; P= .002 and P= .008 for OS and PFS for nondesmoplastic cases, respectively). Cox regression model demonstrated that PROM1 expression is an independent prognostic factor (hazard ratio, 4.56; P= .008). The result was validated on an independent cohortof 42 cases by microarray-based analysis (P= .019). This work suggests that highmRNA levels of PROM1 are associated with poor outcome in pediatric medulloblastoma. Furthermore, high PROM1 expression levels seem to increase the likelihood of metastases. Such results need to be confirmed in larger prospective series to possibly incorporate PROM1 gene expression into risk classification systems to be used in the clinical setting.

cancer stem cells (CSCs) have been identified in a number of solid tumors, but not yet in rhabdomyosarcoma (RMS), the most frequently occurring soft tissue tumor in childhood. Hence, the aim of this study was to identify and characterize a CSC population in RMS using a functional approach. We found that embryonal rhabdomyosarcoma (eRMS) cell lines can form rhabdomyosarcoma spheres (short rhabdospheres) in stem cell medium containing defined growth factors over several passages. Using an orthotopic xenograft model, we demonstrate that a 100 fold less sphere cells result in faster tumor growth compared to the adherent population suggesting that CSCs were enriched in the sphere population. Furthermore, stem cell genes such as oct4, nanog, c-myc, pax3 and sox2 are significantly upregulated in rhabdospheres which can be differentiated into multiple lineages such as adipocytes, myocytes and neuronal cells. Surprisingly,gene expression profiles indicate that rhabdospheres show more similarities withneuronal than with hematopoietic or mesenchymal stem cells. Analysis of these profiles identified the known CSC marker CD133 as one of the genes upregulated in rhabdospheres, both on RNA and protein levels. CD133(+) sorted cells were subsequently shown to be more tumorigenic and more resistant to commonly used chemotherapeutics. Using a tissue microarray (TMA) of eRMS patients, we found that high expression of CD133 correlates with poor overall survival. Hence, CD133 could be a prognostic marker for eRMS. These experiments indicate that a CD133(+) CSC population can be enriched from eRMS which might help to develop novel targeted therapies against this pediatric tumor.

The AC133 epitope has been used as a marker for both normal and cancer stem cells from multiple tissue lineages. To identify transcription factors that regulate CD133 expression, we conducted parallel large-scale RNA interference screens in Caco-2 cancer cells that endogenously express CD133 and in engineered HEK293 cells that express CD133 from a heterologous promoter. The transcription factor AF4 was identified following a comparative analysis between the two screens. We then showed that AF4 is a promoter of CD133 transcription in multiple cancer cell lines. Knockdown of AF4 resulted in a dramatic reduction in CD133 transcript levels. Importantly, a subset of pediatric acute lymphoblastic leukemias (ALL) harbor a fusion oncogene results from a chromosomal translocation that juxtaposes the mixed-lineage leukemia (MLL) gene and the AF4 gene. An investigation of the functional role of CD133 in the MLL-AF4-dependent ALL cells revealed that CD133 was required for leukemia cell survival. Together, our findings show AF4-dependent regulation of CD133 expression, which is required for the growth of ALL cells. CD133 may therefore represent a therapeutic target in a subset of cancers.

Neuroblastoma (NB) is the most common and lethal extracranial solid tumor of childhood. Despite aggressive therapy, more than half of the children with advanced NB will die of uncontrolled metastatic disease. After chemotherapy, tumor-initiating cells (TICs) could persist, cause relapses and metastasis. The aim of this study is to demonstrate the tumor-initiating properties of CD133highNB cells and to identify new specific genetic abnormalities. Isolation of the CD133high cell population from NB cell lines was followed by neurosphere formation, soft agar assays, and orthotopic injections in NOD/SCID/IL2Rgammac-null mice. A differential genotyping analysis was performed with Affymetrix SNP 6.0 arrays on CD133low and CD133high populations and the frequency of the abnormalities of 36 NB tumors was determined. Our results show that CD133high NB cells possess tumor-initiating properties, as CD133high cells formed significantly more neurospheres and produced significantly more colonies in soft agar than CD133low. Injection of 500 CD133high cells was sufficient to generate primary tumors and frequent metastases in mice. Differential genotypinganalysis demonstrated two common regions with gains (16p13.3 and 19p13.3) including the gene EFNA2 in the CD133high population, and two with loss of heterozygosity (16q12.1 and 21q21.3) in the CD133low population. The gain of EFNA2 correlated with increased expression of the corresponding protein. These abnormalities were found in NB samples and some were significantly correlated with CD133 expression. Our results show that CD133high NB cells have TICs properties and present different genotyping characteristics compared to CD133lowcells. Our findings reveal insights into new therapeutic targets in NB TICs.#CI- Copyright (c) 2012 Wiley Periodicals, Inc.

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor.Major research efforts have focused on characterizing and targeting putative brain tumor stem or propagating cell populations from the tumor mass. However, less is known about the relationship between these cells and highly invasive MB cells that evade current therapies. Here, we dissected MB cellular heterogeneityand directly compared invasion and self-renewal. Analysis of higher versus lowerself-renewing tumor spheres and stationary versus migrating adherent MB cells revealed differential expression of the cell surface markers CD271 [p75 neurotrophin receptor (p75NTR)] and CD133. Cell sorting demonstrated that CD271 selects for subpopulations with a higher capacity for self-renewal, whereas CD133 selects for cells exhibiting increased invasion in vitro. CD271 expression is higher in human fetal cerebellum and primary samples of the Shh MB molecular variant and lower in the more aggressive, invasive group 3 and 4 subgroups. Global gene expression analysis of higher versus lower self-renewing MB tumor spheres revealed down-regulation of a cell movement transcription program in thehigher self-renewing state and a novel potential role for axon guidance signaling in MB-propagating cells. We have identified a cell surface signature based on CD133/CD271 expression that selects for MB cells with a higher self-renewal potential or invasive capacity in vitro. Our study underscores a previously unappreciated role for CD271 in selecting for MB cell phenotypes and suggests that successful treatment of pediatric brain tumors requires concomitant targeting of a spectrum of transitioning self-renewing and highly infiltrative cell subpopulations.