Elevating Sox2 levels deleteriously affects the growth of glioblastoma and medulloblastoma cells.
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Angie Rizzinio
PROVIDER: E-GEOD-36947 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2012-02-20 | E-GEOD-34801 | biostudies-arrayexpress
Project description:Induction of the transcription factor Sox2 from a doxycycline-inducible promoter in iSox2-DAOY medulloblastoma cells. Affymetrix microarrays were used to characterize the gene expression profile of iSox2-DAOY cells in the absence and presence of doxycycline.
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Project description:In this study we investigated alterations in the plasma membrane (PM)-associated proteome of DAOY medulloblastoma cells. We used a surface biotinylation approach to label PM-associated proteins. We enriched for biotinylated proteins by streptavidin-pull-down and determined the composition by mass-spectrometry. We compared CRISPR/CAS9 control cells and CRISPR/CAS9 MAP4K4 (MAP4K4 depletion) cells under starvation and after 30 min stimulation with 20 ng/ml HGF. We performed two group analyses of all conditions.
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Project description:Bmi-1 and Mel-18 are close structural homologues that belong to the polycomb group (PcG) of transcriptional regulators of homeotic gene expression in development. They are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. A number of clinical and experimental observations have also implicated Bmi-1 in tumorigenesis and stem cell maintenance. Bmi-1 overexpression or amplification has been observed in a number of human malignancies, particularly in B-cell lymphomas, medulloblastomas and breast cancer. We report here that shRNA-mediated knock-down of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival and anchorage-independent growth, and suppression of xenograft tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increased clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone did not affect the growth of SK-OV-3 or U2OS cancer cell lines or normal human WI38 fibroblasts. Gene expression analysis of shRNA-expressing DAOY cells has demonstrated a significant overlap in the Bmi-1- and Mel-18-regulated genes and revealed novel gene targets under their control. Taken together, these results suggest that Bmi-1 and Mel-18 might have overlapping functions in human tumorigenesis. Experiment Overall Design: DAOY cells were transduced with the indicated lentiviral shRNA and stable cell lines were generated by selection in puromycin (1 µg/ml). 2 x 105 cells of each stable cell line were plated onto 60 mm dishes in triplicates and harvested 4 days later. Triplicate total RNA samples from shRNA-expressing and mock-transduced DAOY cells were isolated using affinity resin (Qiagen RNeasy Mini Kit, Qiagen AG). RNA Integrity and purity were assessed with the RNA 6000 Nano LabChip system on Bioanalyzer 2100 (Agilent Technologies). Gene array analysis was conducted at the Genomics Factory at Novartis PHARMA AG, Basel, Switzerland using Gene Chip Human Genome 133 2.0 Plus Expression Array (Affymetrix Inc.).
2007-10-31 | E-GEOD-7578 | biostudies-arrayexpress
Project description:Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor of many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling profoundly depend on interactions with other pathways such as epidermal growth factor receptor-mediated signaling which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. We demonstrate that the human medulloblastoma cell line Daoy possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic Hedgehog or Smoothened agonist induced expression of GLI1 protein and prevented processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed on the transcriptomic as well as proteomic level. Daoy cells responded to the co-treatment by downregulating GLI1, PTCH, and HHIP on the transcript level which was also seen when Amphiregulin (AREG) was used instead of EGF. The finding that EGFR signaling silences proteins acting as negative regulators of HH signaling is firstly described here as a novel crosstalk mechanism. Furthermore, combined EGFR/HH signaling maintains high GLI1 protein levels contrasting its downregulation on the transcript level. On the other hand, high level synergism was observed with respect to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile pointing to more wide-spread, yet context-dependent, interactions between HH/GLI and growth factor receptor signaling in human malignancies. To study interactions between HH- and EGF-induced signaling, time-resolved measurements were carried out over a period of 24 h at 14 different time points after stimulation by EGF with and without additional stimulation by SHH. Furthermore, as a control, cells without any stimulation by EGF and SHH (control) and cells in the presents of SHH were analyzed. Overall, three biological replicates of 60 different treatment/timepoints were analyzed yielding 180 different samples.
2014-07-31 | E-GEOD-46045 | biostudies-arrayexpress
Project description:Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is Ser/Thr protein kinase highly expressed in medulloblastoma (MB) patient samples which promotes cell migration and invasiveness downstream of growth factor receptors. Although MAP4K4 implication in the control of the membrane protrusions at the leading edge of cells during migration and invasion in MB is established, its downstream effectors governing and mediating this process in MB have not been identified. In this study we explored the proteomic neighborhood of MAP4K4 in MB cells and to identify candidate potentially affecting its activity and pro-migratory functions. Towards this aim, we expressed BioID fused versions of MAP4K4 (N- and C-terminal) in DAOY cells under presence of biotin. In the control setup, we expressed the BioID enzyme alone. After Streptavidin purification of biotinylated proteins, we performed mass spectrometry and quantified relative abundances compared to control conditions.
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Project description:Medulloblastoma is a highly aggressive pediatric brain tumor, in which expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However, the contribution of OCT4 transcript variants to tumor aggressiveness is still poorly understood. In this study, we found that transcripts encoding OCT4A, but not OCT4B or OCT4B1, were significantly correlated with LIN28A expression, which encodes another well-known pluripotency factor. LIN28A was found to specifically bind OCT4A transcripts and interact with poly(A) binding protein and RNA helicase A in polysomal fractions of medulloblastoma cells, favoring increased OCT4A protein levels in these cells. Medulloblastoma cells stably overexpressing OCT4A displayed significantly enhanced clonogenic activity, tumorsphere generation and invasion capability, as well as increased tumorigenicity. In an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic effects of OCT4A were found to be expression-level dependent and accompanied by distinct subchromosomal aberrations and differential expression of newly discovered, still poorly characterized, non-coding RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer. Total RNA of 2-3 clones of each cell line (Control and OCT4A overexpression) was extracted with the RNeasy Mini kit (Qiagen), following the manufacturerâs protocol. Gene expression profiling was carried out independently for each sample using Affymetrix GeneChip® Human Gene 2.0 ST whole-transcript arrays (Affymetrix, Santa Clara, CA, USA). The quality control and normalization of data were processed by Affymetrix® Expression Console Software (Affymetrix). Differentially expressed genes were identified with the One-Way ANOVA, with a p-value cutoff of 0.05, using Transcriptome Analysis Console v3.0 (Affymetrix).
2016-02-17 | E-GEOD-77947 | biostudies-arrayexpress
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2022-03-02 | E-MTAB-9823 | biostudies-arrayexpress
Project description:RNA sequencing of medulloblastoma cell lines DAOY and USP13-med; wild type and VAPB KO.
2023-06-28 | E-MTAB-11914 | biostudies-arrayexpress