Cyclophilin B supports the survuval of glioblastoma multiforme cells
ABSTRACT: We have found that cyclophilin B (CypB) expression is important for malignant glioblastoma multiforme (GBM) cell proliferation. To identify molecular mechanisms that could explain CypB-dependent survival in human GBM cells, a microarray analysis was performed using RNA prepared from U251MG GBM cells transduced with lentiviral CypB shRNA. These data revealed that about 130 genes were more than 2-fold affected by CypB depletion. Significant alterations in the expression of genes related to cell death, cell proliferation and cell migration were found in the shCypB cells. U251 glioblastoma cells transduced with lentivirus expressing non-target control shRNA (shCON) were compared to cells transduced with lentivirus expressing shRNA sequence against cyclophilin B (shCypB). Cells transduced with the lentiviral shRNA (shCON VS shCypB) for 5 days before total RNA extraction. Three biological replicates of cells treated with each shRNA (shCON or shCypB) were profiled.
Project description:We have found that cyclophilin B (CypB) expression is important for malignant glioblastoma multiforme (GBM) cell proliferation. To identify molecular mechanisms that could explain CypB-dependent survival in human GBM cells, a microarray analysis was performed using RNA prepared from U251MG GBM cells transduced with lentiviral CypB shRNA. These data revealed that about 130 genes were more than 2-fold affected by CypB depletion. Significant alterations in the expression of genes related to cell death, cell proliferation and cell migration were found in the shCypB cells. Overall design: U251 glioblastoma cells transduced with lentivirus expressing non-target control shRNA (shCON) were compared to cells transduced with lentivirus expressing shRNA sequence against cyclophilin B (shCypB). Cells transduced with the lentiviral shRNA (shCON VS shCypB) for 5 days before total RNA extraction. Three biological replicates of cells treated with each shRNA (shCON or shCypB) were profiled.
Project description:Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Recent studies including our own identified that the mitotic kinase, maternal embryonic leucine-zipper kinase (MELK), is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we provide evidence that the kinase activity of MELK is essential for the action of MELK in GSCs and vital for GBM growth. We utilized in silico structure-based analysis for protein-compound interaction to predict that a recently identified small molecule, Compound 1 (C1), binds to the kinase-active site of MELK protein and eliminates MELK kinase activity in nanomolar concentrations. When treated with C1, GSCs undergo mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed using MELK shRNA-mediated knockdown. C1 treatment strongly induces tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuates growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors. Microarray-based expression analysis of glioma stem cells treated with MELK-signaling inhibitors
Project description:To determine the biological effects of MPS1 inhibition (both by siRNA and Drug (NMSP715)) on signaling pathways in GBM cells (U251 &U87), we profiled the modulation of phosphorylated and non-phosphorylated proteins using RPPA Relative protein levels for each sample were determined by interpolation of each dilution curves from the standard curve antibody slide. All the data points were normalized for protein loading and transformed to a linear value. Linear values were transformed to Log2 value and then median‐centered for hierarchical cluster analysis.
Project description:We over-expressed an epigenetic regulator in a glioblastoma (GBM) primary culture from an adult patient. These GBM cells have cancer stem cell phenotypes, as they have self-renewal properties and tumor initiation potential when transplanted in immunocompromised mice. An epigenetic regulator (ER) was over-expressed in the GBM primary culture G514NS. EGFP was expressed from the same vector backbone as a control. N = 3 biological replicates for each of EGFP- and ER-overexpressing cells. Please note that complete data output (with 74,342 data rows) from Partek analysis contains several identifiers which are not represented in the GPL17586, and therefore is linked as Series supplementary file.
Project description:To identify differentially expressed genes in HT GCB-DLBCL cell lines transduced with non-silencing control shRNA or DNMT1 shRNA. Overall design: HT cells were transduced with lentivirus vectors expressing shRNA non-silencing control or shRNA targeting DNMT1 (shDNMT1) in duplicate for each shRNA.
Project description:p53-repressed transcripts have recently been shown to play important roles in various biological processes, such as stem cell differentiation and cancer. We identified a transcript named Apela that is repressed by p53 and highly expressed in mouse ES cells. To see which transcripts are affected by Apela knockdown, we performed gene expression microarray using Affymetrix Gene ST 1.0 array. Two short hairpin RNAs (shRNAs) targeting Apela were used to decrease the RNA levels of Apela to about 20% of the control (a shRNA target luciferase, shLuc). We designed two lentivirus-based shRNAs against Apela and used shRNA against luciferase as a control. Lentiviruses were made and used to transduced mouse ES cells. For each shRNA, three repeats were done.
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.
Project description:Glioblastoma (GBM) is the most aggressive form of astrocytoma and is difficult to diagnose at early stage. In this study, our goal is to find and validate biomarkers for GBM through serum proteome expression profiling, which would assist in the early diagnosis, and management of GBMs. In the discovery phase, we performed shotgun proteomics of control and GBM pooled serum samples using untargeted 4-plex iTRAQ (isobaric Tags for Relative and Absolute Quantitation) mass spectrometry to identify candidate biomarkers with differential abundance The two candidates, S100A8 and S100A9 were prioritized for verification based on their maximal abundance in GBM serum in our discovery phase.
Project description:To determine the biological mechanisms underlying the oncogenic properties of YAP in ccRCC the human ccRCC cell line MZ1774 was transduced with lentivirus containing a shRNA-cassette targeting YAP-mRNA. Expression profiles of MZ1774 YAP knockdown cells were compared to mock-transduced control cells. Total RNA from MZ1774 cells stably expressing shRNA directed against YAP compared to mock-transduced MZ1174 cells
Project description:IRE1a is a critical modulator of the unfolded protein response. Its RNAse activity generates the mature transcript for the XBP1 transcription factor and also degrades other ER associated mRNAs in a process termed Regulated IRE1a Dependent mRNA Decay or RIDD. To determine if IRE1a is critical in the response to oncogenic Ras we used ShRNA to knockdown Ire1a or Xbp1 in primary mouse epidermal keratinocytes transduced with a v-HRAS retrovirus. Overall design: Primary mouse keratinocytes were transduced with a v-Hras retrovirus and either a control ShRNA lentivirus or ShRNA against either Ire1a or Xbp1. RNA isolated from each group at 4 days post lentiviral infection (n=3 biological replicates per group) was used for microarrays. Primary mouse keratinocytes transduced with control ShRNA lentivirus served as the reference set for gene expression analysis (n=3 biological replicates per group).