Methylation profiling of neuro-ectodermal cell line with and without demethylating treatment.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE25225 | GEO | 2012/05/01
SECONDARY ACCESSION(S): PRJNA134465
REPOSITORIES: GEO
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Project description:Abstract: Epigenetic alterations are a fundamental aspect of cancer cells, and epigenetic drugs are currently used in clinical practice for hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and show epigenetic defects of apoptotic pathways, which makes the introduction of epigenetic drugs in this patient category logical. However, the young age of these patients is accompanied by ongoing developmental processes which are regulated epigenetic mechanisms, and prompted us to study molecular effects of nanomolar dosage epigenetic drugs in neuro-ectodermal tumor cell lines. Combination treatment of 5-aza-2`-deoxicytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages resulted in wide-spread demethylating effects in 17 NBL and 5 PNET cell lines in vitro. This widespread demethylation had large effects on gene-expression profiles. In NBL cell lines, almost every cellular pathway (193/200) investigated demonstrated altered expression upon treatment, and resulted in upregulation of known epigenetically regulated genes such as X-chromosomal, tissue-specific, and a few imprinted genes. Integration analysis of CpG island methylation array data and whole genome gene expression data identified 30 genes potentially upregulated by gene promoter demethylation. Homeobox genes frequently showed demethylation in both short term (72 hours) and long term cultures (3 months) of NBL lines. Continuous treatment with epigenetic drugs resulted in low rates of proliferation. The low rate of proliferation that might explain limited consecutive demethylation upon prolonged exposure. In conclusion, genome-wide methylation and gene expression changes are induced DAC and TSA treatment at nanomolar dosages. These effects affected more than 97% of cellular pathways investigated. Further studies towards the effects of epigenetic drug combinations are advised before being applied in clinical trials for pediatric patients.
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Project description:To study differentially expressed genes in neuro-ectodermal cell lines MYCN amplification (NMA) is the most important prognostic factor in neuroblastoma (NBL) patients, however 70% of advanced stage NBL are non-NMA and lack known driving oncogenic events. Gene expression profiles (HU133plus2.0 arrays, Affymetrix) of 17 NBL and 5 peripheral neuro-ectodermal cell lines were used to identify potential subgroups of NBL cell lines with a distinct gene signature. One group of non-NMA NBL cell lines was identified with a distinct gene expression profile and characterized by high expression of AXL. AXL is a tyrosine kinase receptor which plays a role in the metastatic process of cancer. We hypothesized that AXL contributes to the metastasizing potential of non-NMA NBL and tested if AXL silencing diminishes malignant properties of high AXL expressing cell lines. AXL was silenced in two non-NMA NBL cell lines by using a lentiviral shRNA construct that was able to transduce these cell lines with >90% infection efficiency. AXL mRNA expression level was efficiently knocked-down resulting in a severe decrease of migration of AXL positive cell lines GI-M-EN and SH-EP-2, and decreased invasion of GI-M-EN. Morphologically, AXL knockdown induced more rounded cells with a loss of contact. Intracellularly, we observed induction of stress fibers (immunofluorescence F-actin) in GI-M-EN. These changes in cytoskelet were associated with decreased migration. No effects were observed for cell proliferation, apoptosis or downstream pathways. In conclusion, AXL is identified as a possible mediator of NBL metastasis. Arrays were performed with 5 different PNET cell lines, which were used as controls for 17 NBL cell lines (GSE22771)
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Project description:To study differentially expressed genes in neuro-ectodermal cell lines MYCN amplification (NMA) is the most important prognostic factor in neuroblastoma (NBL) patients, however 70% of advanced stage NBL are non-NMA and lack known driving oncogenic events. Gene expression profiles (HU133plus2.0 arrays, Affymetrix) of 17 NBL and 5 peripheral neuro-ectodermal cell lines were used to identify potential subgroups of NBL cell lines with a distinct gene signature. One group of non-NMA NBL cell lines was identified with a distinct gene expression profile and characterized by high expression of AXL. AXL is a tyrosine kinase receptor which plays a role in the metastatic process of cancer. We hypothesized that AXL contributes to the metastasizing potential of non-NMA NBL and tested if AXL silencing diminishes malignant properties of high AXL expressing cell lines. AXL was silenced in two non-NMA NBL cell lines by using a lentiviral shRNA construct that was able to transduce these cell lines with >90% infection efficiency. AXL mRNA expression level was efficiently knocked-down resulting in a severe decrease of migration of AXL positive cell lines GI-M-EN and SH-EP-2, and decreased invasion of GI-M-EN. Morphologically, AXL knockdown induced more rounded cells with a loss of contact. Intracellularly, we observed induction of stress fibers (immunofluorescence F-actin) in GI-M-EN. These changes in cytoskelet were associated with decreased migration. No effects were observed for cell proliferation, apoptosis or downstream pathways. In conclusion, AXL is identified as a possible mediator of NBL metastasis.
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Project description:Epigenetic alterations are a fundamental aspect of cancer cells, and epigenetic drugs are currently used in clinical practice for hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and show epigenetic defects of apoptotic pathways, which points to sensitivity towards epigenetic drugs in this patient group. The young age of these patients is accompanied by However, ongoing developmental processes regulated by epigenetic mechanisms may be deregulated by epigenetic drugs in this patient group that is characterized by young age. This prompted us to study molecular effects and side-effects of low dosage epigenetic drugs in neuro-ectodermal tumor cell lines of pediatric origin. Short term combination treatment of 5-aza-2`-deoxicytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages reduced proliferation, induced wide-spread demethylating effects in 17 NBL and 5 PNET cell lines, and was accompanied by large effects on gene-expression profiles. Approximately half of the genes that were significantly upregulated upon treatment demonstrated significant demethylating effects in their promoter regions. In NBL cell lines, almost every cellular pathway (193/200) investigated demonstrated altered expression upon treatment, and resulted in upregulation of known epigenetically regulated genes such as X-chromosomal, tissue-specific, and a limited number of imprinted genes, but also known tumor suppressor genes and oncogenes. In conclusion, genome-wide methylation and gene expression changes are induced DAC and TSA treatment at nanomolar dosages. This treatment affected more than 97% of cellular pathways investigated and further studies towards the effectiveness and side-effects of epigenetic drugs are desirable in pediatric tumors. Epigenetic alterations are a fundamental aspect of cancer cells, and epigenetic drugs are currently used in clinical practice for hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and show epigenetic defects of apoptotic pathways, which points to sensitivity towards epigenetic drugs in this patient group. The young age of these patients is accompanied by However, ongoing developmental processes regulated by epigenetic mechanisms may be deregulated by epigenetic drugs in this patient group that is characterized by young age. This prompted us to study molecular effects and side-effects of low dosage epigenetic drugs in neuro-ectodermal tumor cell lines of pediatric origin. Short term combination treatment of 5-aza-2`-deoxicytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages reduced proliferation, induced wide-spread demethylating effects in 17 NBL and 5 PNET cell lines, and was accompanied by large effects on gene-expression profiles. Approximately half of the genes that were significantly upregulated upon treatment demonstrated significant demethylating effects in their promoter regions. In NBL cell lines, almost every cellular pathway (193/200) investigated demonstrated altered expression upon treatment, and resulted in upregulation of known epigenetically regulated genes such as X-chromosomal, tissue-specific, and a limited number of imprinted genes, but also known tumor suppressor genes and oncogenes. In conclusion, genome-wide methylation and gene expression changes are induced DAC and TSA treatment at nanomolar dosages. This treatment affected more than 97% of cellular pathways investigated and further studies towards the effectiveness and side-effects of epigenetic drugs are desirable in pediatric tumors.
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