Project description:To study the effect of constitutive REST expression on DAOY, UW228 and UW426 cells, we generated low and high-REST (LR/HR) isogenic pairs of the three human medulloblastoma (MB) cell lines and performed RNA-Seq analysis. We also analyzed the expression profiles of D283 cells (Group3/4 MB cell line) with DAOY, UW228 and UW26 (SHH MB cell line ) to show the subgroup specific expression profiles.
Project description:Characterizing the impact of pharmacological and shRNA-mediated silencing of EAG2 in medulloblastoma. A medulloblastoma (MB) cell line was untreated, mock(DMSO)-treated and treated to inhibit EAG2 (both pharmacologically and via shRNA).
Project description:Astrocytes are important cells within the medulloblastoma microenvironment. Therefore, we assessed how astrocyte secreted factors may alter Daoy (human MB cell line; ATCC HTB-186) cell gene expression.
Project description:About 85% of WNT subgroup medulloblastomas exhibit the loss of one copy of chromosome 6. Loss of function mutations were identified in the ARID1B gene located on chromosome 6 in WNT subgroup tumors suggesting tumor suppressor role for ARID1B in WNT medulloblastomas. The expression of ARID1B was downregulated in the medulloblastoma cell line Daoy using two independent shRNA sequences in a doxycycline-inducible lentiviral vector tet-pLKO-puro and the effect of its downregulation on the gene expression profile was studied by the RNA-seq analysis
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.).
Project description:DDX3X is an ATP-dependent RNA helicase. Missense mutations in DDX3X gene are known to occur in WNT, SHH subgroup medulloblastomas. The role of DDX3X in medulloblastoma biology was studied by downregulating its expression in a SHH subgroup Daoy medulloblastoma cell line. DDX3X knockdown resulted in considerable reduction in proliferation, clonogenic potential and anchorage-independent growth of the medulloblastoma cells. Transcriptome analysis was performed to delineate the molecular mechanism underlying reduction in the malignant potential of the medulloblastoma cells upon DDX3X knockdown. Exogenous expression of three DDX3X missense mutants in the DDX3X knockdown cells could restore the malignant potential of the medulloblastoma cells.
Project description:The Hedgehog (Hh) signaling pathway regulates normal development and cell proliferation, whereas its aberrant activation causes tumor formation. Hh-induced tumors can arise from different tissues and can be indolent or highly aggressive, such as basal cell carcinoma (BCC) of the skin and neural progenitor-derived medulloblastoma (MB), respectively. Little is known about cell-intrinsic factors that control the development of such diverse Hh-dependent tumors. Transcription factor Zfx is required for the self-renewal of several stem cell types, whereas its role in malignant transformation remains controversial. We found that the deletion of Zfx prevented BCC formation and significantly delayed MB development caused by Hh activation in vivo. In contrast, Zfx was dispensable for the development of Hh-independent brain tumor glioblastoma. We used genome-wide expression and chromatin binding analysis in a human MB cell line to identify direct, evolutionarily conserved targets of Zfx. These targets included the Hh signal transducer Smoothened (Smo), suggesting that Zfx may directly control Hh pathway activation in tumors. Two additional targets of Zfx, Dis3L and Ube2j1, were also required for the growth of MB cells in vitro. These results identify a common cell-intrinsic regulator of diverse Hh-induced tumors, and suggest Zfx and Zfx-controlled genes as possible therapeutic targets in these malignancies.
Project description:The Hedgehog (Hh) signaling pathway regulates normal development and cell proliferation, whereas its aberrant activation causes tumor formation. Hh-induced tumors can arise from different tissues and can be indolent or highly aggressive, such as basal cell carcinoma (BCC) of the skin and neural progenitor-derived medulloblastoma (MB), respectively. Little is known about cell-intrinsic factors that control the development of such diverse Hh-dependent tumors. Transcription factor Zfx is required for the self-renewal of several stem cell types, whereas its role in malignant transformation remains controversial. We found that the deletion of Zfx prevented BCC formation and significantly delayed MB development caused by Hh activation in vivo. In contrast, Zfx was dispensable for the development of Hh-independent brain tumor glioblastoma. We used genome-wide expression and chromatin binding analysis in a human MB cell line to identify direct, evolutionarily conserved targets of Zfx. These targets included the Hh signal transducer Smoothened (Smo), suggesting that Zfx may directly control Hh pathway activation in tumors. Two additional targets of Zfx, Dis3L and Ube2j1, were also required for the growth of MB cells in vitro. These results identify a common cell-intrinsic regulator of diverse Hh-induced tumors, and suggest Zfx and Zfx-controlled genes as possible therapeutic targets in these malignancies.
Project description:The goal of this study was to profile the total proteome and transcriptome of the established medulloblastoma cell lines, Daoy and UW228, using label-free nano-LC-MS/MS-based quantitative proteomics and high-throughput RNA sequencing (RNA-Seq), coupled with pathway analysis to identify differentially expressed genes, proteins and signaling pathways with potential as prognostic markers. A total of 14250 and 12870 transcripts were detected for Daoy and UW228, respectively. Proteomic profiling identified 2630 and 1235 proteins in Daoy and UW228, representing 18% and 10% of detected transcripts, respectively. Interestingly, Daoy proteome included >50% unique proteins, while almost 90% of proteins expressed by UW228 were commonly expressed in Daoy. Differential expression of a number of adhesion, cytoskeletal and signaling molecules were observed between the two cell lines. Upregulation of a number of proteins and enrichment of key signaling pathways, including WNT, Sonic hedgehog (SHH) and integrin signaling pathways, were uniquely observed in MB cell lines, in particular in Daoy.