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Transcriptional effect of an off-target shRNA targeting nucleostemin in human glioblastoma-derived cancer stem cells

ABSTRACT: Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples. The significance of its expression was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). We found that an off-target nucleostemin RNAi (shRNA22) abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is severely compromised. Attempts were made to identify the primary target of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways. The use of this shRNA may offer a new therapeutic approach for this incurable type of brain tumors. The transcriptional profile of neurosphere cultures infected with lentiviral particles containing shRNA22 was compared with the profile of neurosphere cultures infected with lentiviral particles containing control shRNA. Experiments were done in triplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Gil-Ranedo J

PROVIDER: S-ECPF-GEOD-30448 | biostudies-other | 2011

REPOSITORIES: biostudies-other

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Transcriptional effect of an off-target shRNA targeting nucleostemin in human glioblastoma-derived cancer stem cells

Project description:Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples. The significance of its expression was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). We found that an off-target nucleostemin RNAi (shRNA22) abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is severely compromised. Attempts were made to identify the primary target of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways. The use of this shRNA may offer a new therapeutic approach for this incurable type of brain tumors. The transcriptional profile of neurosphere cultures infected with lentiviral particles containing shRNA22 was compared with the profile of neurosphere cultures infected with lentiviral particles containing control shRNA. Experiments were done in triplicate.

2011-10-20 | E-GEOD-30448 | biostudies-arrayexpress

Transcriptional effect of an off-target shRNA targeting nucleostemin in human glioblastoma-derived cancer stem cells

2011-10-21 | GSE30448 | GEO

shRNA profiling of human DZNep-treated glioblastoma multiforme cancer stem cells

Project description:Overexpression of the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) occurs in diverse malignancies, including prostate cancer, breast cancer, and glioblastoma multiforme (GBM) (1). Based on its ability to modulate transcription of key genes implicated in cell cycle control, DNA repair and cell differentiation, EZH2 is believed to play a crucial role in tissue-specific stem cell maintenance and tumor development. Here we show that targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep), or its specific down-regulation by shRNA, strongly impairs GBM cancer stem cell self-renewal in vitro and tumor-initiating capacity in vivo. Using genome-wide expression analysis of DZNep-treated GBM cancer stem cells, we found the expression of c-myc, recently reported to be essential for GBM cancer stem cells, to be strongly repressed upon EZH2 depletion. Specific shRNA-mediated down-regulation of EZH2 in combination with chromatin immunoprecipitation (ChIP) experiments revealed that c-myc is a direct target of EZH2 in GBM cancer stem cells. Taken together, our observations provide evidence that direct transcriptional regulation of c-myc by EZH2 may constitute a novel mechanism underlying GBM cancer stem cell maintenance and suggest that EZH2 may be a valuable new therapeutic target for GBM management. Experiment Overall Design: Three samples of cancer stem-cell enriched gliospheres from primary glioblastoma multiforme cell cultures were treated with DZNep. Untreated gliospheres from the same cultures were used as controls.

| S-ECPF-GEOD-18150 | biostudies-other

An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells.

Project description:Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133(+) neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples, and that its expression, conversely to what it has been described for ordinary stem cells, does not disappear when cells are differentiated. The significance of nucleostemin expression in CSCs was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). In doing so, we found an off-target nucleostemin RNAi (shRNA22) that abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is significantly delayed. Attempts were made to identify the primary target/s of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways or multiple targets. The use of this shRNA may contribute to develop new therapeutic approaches for this incurable type of brain tumor.

| S-EPMC3236221 | biostudies-literature

Lectins identify glycan biomarkers on glioblastoma-derived cancer stem cells.

Project description:Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Despite aggressive therapy with surgery, radiotherapy, and chemotherapy, nearly all patients succumb to disease within 2 years. Several studies have supported the presence of stem-like cells in brain tumor cultures that are CD133-positive, are capable of self-renewal, and give rise to all cell types found within the tumor, potentially perpetuating growth. CD133 is a widely accepted marker for glioma-derived cancer stem cells; however, its reliability has been questioned, creating a need for other identifiers of this biologically important subpopulation. We used a panel of 20 lectins to identify differences in glycan expression found in the glycocalyx of undifferentiated glioma-derived stem cells and differentiated cells that arise from them. Fluorescently labeled lectins that specifically recognize ?-N-acetylgalactosamine (GalNAc) and ?-N-acetylglucosamine (GlcNAc) differentially bound to the cell surface based on the state of cellular differentiation. GalNAc and GlcNAc were highly expressed on the surface of undifferentiated cells and showed markedly reduced expression over a 12-day duration of differentiation. Additionally, the GalNAc-recognizing lectin Dolichos biflorus agglutinin was capable of specifically selecting and sorting glioma-derived stem cell populations from an unsorted tumor stock and this subpopulation had proliferative properties similar to CD133(+) cells in vitro and also had tumor-forming capability in vivo. Our preliminary results on a single cerebellar GBM suggest that GalNAc and GlcNAc are novel biomarkers for identifying glioma-derived stem cells and can be used to isolate cancer stem cells from unsorted cell populations, thereby creating new cell lines for research or clinical testing.

| S-EPMC3425159 | biostudies-literature

CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells.

Project description:Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-? production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

| S-EPMC4496728 | biostudies-literature

Isolation and characterization of cancer stem cells derived from human glioblastoma.

Project description:Cancer stem cell (CSC) is considered as a cause of cancer recurrence and metastasis. Simultaneously CSCs are responsible for the heterogeneous population in tumor tissues due to their differentiation potential. However, the characterizations of CSCs are still not enough and cancer stem cell lines widely available is desired to be established for the advancement of cancer research. In this study, we tried to isolate and characterize stem like cells from human glioblastoma cell line U-251MG cells. U-251MG P1 cells, which was previously condensed in the presence of hyaluronic acid as CD44 positive population were subjected to single cell isolation procedure. Although 5 clones were isolated, only one clone exhibited high expression of CD44, Nanog, OCT3/4 and SOX2, and named U-251MGSC1. The sphere forming ability of U-251MGSC1 cell was significantly higher than the parental U-251MG cells. Tumorigenicity of U-251MG-SC1 cells were higher than that of U-251MG cells. U-251MGSC1 cells exhibited higher expression of CD44, SOX2, Nestin and A2B5 than U-251MG cells in vitro and in vivo. The expression of GFAP and NF-M was enhanced when the cells were treated with the conditioned medium of U-251MG cells indicating the potential of differentiation. Sphere forming ability was more efficient than that of U-251MG cells and was enhanced in the presence of hyaluronic acid, which enhanced the cell growth as well. U-251MGSC1 cells exhibited rapid growth tumor in nude mice and efficient metastatic ability in transmembrane assay when compared with U-251MG cells. As the result, we concluded U-251MGSC1 cell was a glioblastoma CSC line derived from the parental U-251MG cells. U-251MGSC1 cells will be a good tool to develop effective therapeutic agents against CSCs and to elucidate the properties of glioma derived CSCs and the mechanism of tumor development in brain.

| S-EPMC7868757 | biostudies-literature

Potential Therapeutic Effects of the Neural Stem Cell-Targeting Antibody Nilo1 in Patient-Derived Glioblastoma Stem Cells.

Project description:Glioblastoma (GBM) is the most devastating and least treatable brain tumor with median survival <15 months and extremely high recurrence rates. Promising results of immune checkpoint blockade obtained from pre-clinical studies in mice did not translate to clinic, and new strategies are urgently needed, particularly those targeting GBM stem cells (GSCs) that are held responsible for drug resistance and tumor recurrence. Patient-derived GSC cultures are critical for finding effective brain tumor therapies. Here, we investigated the ability of the recently described monoclonal antibody Nilo1 to specifically recognize GSCs isolated from GBM surgical samples. We employed five patient-derived GSC cultures with different stemness marker expression and differentiation potential, able to recapitulate original tumors when xenotransplanted in vivo. To answer whether Nilo1 has any functional effects in patient-derived GSCs lines, we treated the cells with Nilo1 in vitro and analyzed cell proliferation, cell cycle, apoptosis, sphere formation, as well as the expression of stem vs. differentiation markers. All tested GSCs stained positively for Nilo1, and the ability of Nilo1 to recognize GSCs strongly relied on their stem-like phenotype. Our results showed that a subset of patient-derived GSCs were sensitive to Nilo1 treatment. In three GSC lines Nilo1 triggered differentiation accompanied by the induction of p21. Most strikingly, in one GSC line Nilo1 completely abrogated self-renewal and led to Bax-associated apoptosis. Our data suggest that Nilo1 targets a molecule functionally relevant for stemness maintenance and pinpoint Nilo1 as a novel antibody-based therapeutical strategy to be used either alone or in combination with cytotoxic drugs for GSC targeting. Further pre-clinical studies are needed to validate the effectiveness of GSC-specific Nilo1 targeting in vivo.

| S-EPMC7468525 | biostudies-literature

Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients.

Project description: Not available

| S-EPMC5310181 | biostudies-literature

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells.

Project description:Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

| S-EPMC4648292 | biostudies-literature

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