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SRP073103
Homo sapiens
42 Downloadable Samples
Illumina HiSeq 2500
Description
Treatment of cancer cells with anti-cancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor’s gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anti-cancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2''-deoxycytidine (Decitabine) has limited anti-tumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. Overall design: The transcriptomes, proteomes and HLA peptidomes of the U-87, T98G and LNT-229 GBM human cell lines were analyzed before and after treatment with Decitabine. Overall, the RNA-Seq transcriptome analyses resulted in the identification of above 26000 transcripts, the proteome analyses identified about 7500 proteins and the HLA class I peptidome analyses resulted in above 25000 identified HLA peptides. Two biological repetitions of the transcriptome, three of the proteome and three of the HLA peptidome were performed with each of the cell lines and treatment, resulting in highly reproducible datasets.
Publication Title
Human Leukocyte Antigen (HLA) Peptides Derived from Tumor Antigens Induced by Inhibition of DNA Methylation for Development of Drug-facilitated Immunotherapy.
Sample Metadata Fields
Specimen part, Cell line, Treatment, Subject
View Samples- Homo sapiens
- 12 Downloadable Samples
- IlluminaHiSeq2500
Description
Purpose: We identified KPC1 as the ubiquitin ligase that binds to the p105 precursor of NF-kB, ubiquitinates it and mediates its proteasomal processing to generate the p50 active subunit of the transcription factor. Using U87-MG human glioblastoma xenografts, we observed that overexpression of KPC1 results in strong inhibition of tumor growth mediated via excessive generation of p50.The goal of this RNASeq study was to analyze the profile of gene expression in xenografts overexpressing control (V0), KPC1 or p50 vectors, and to further understand how the altered gene expression patterns can explain the tumor suppressive effect we observed. Results:Transcript analysis of U87-MG xenografts overexpressing control (V0), KPC1 or p50 vector mapped to the human genome revealed: • A strong similarity between overexpression of p50 and KPC1 (correlation of 0.51, p-value<10-300 ) • A specific signature of NF-kB targets [21 of the consistently changed genes are known to be regulated by NF-kB (p-value<3.4×10-9 )] • A significant (p-value<1.4×10-18) increase in the expression of 40 tumor suppressor genes, with no significant change in other classes. • A significant down regulation of a cluster of genes including LIN28B, IL-6, HMAGA2 and VEGFA. This finding links well to an established regulatory axis involving LIN28B, Let-7 microRNA, and IL-6 in inflammation and cell transformation that is regulated by NF-kB. Overall design: Exponentially growing U87-MG cells were stably transfected with an empty vector (V0) or vectors coding for Myc-KPC1 or Flag-p50. Cells were dissociated with trypsin, washed with PBS, and brought to a concentration of 50×10^6 cells/ml. Cell suspension (5×10^6/0.1 ml) was inoculated subcutaneously at the right flank of 7-weeks old male Balb/C nude mice (n=7). Following 21 days, mRNA from U87-MG xenografts was isolated using an RNA purification kit, and analyzed using the Illumina HiSeq 2500 sequencer. The RNASeq analysis experiment was repeated twice independently. Run1 included a total of 7 samples. Samples 1-3 were isolated from V0 – control tumors (3 different tumors), samples 4-5 were isolated from KPC1-expressing tumors (2 different pools of 3 tumors each due to small tumor size), and samples 6-7 were isolated from p50-expressing tumors for (2 different pools of 2-3 tumors each, due to very small tumor size). Run2 included a total of 5 samples. Samples 8-10 were isolated from V0 (control) tumors (3 different tumors), samples 11-12 were isolated from KPC1 tumors (2 different pools of 3 tumors each due to small tumor size). Several repeated attempts to extract RNA from the p50-expressing tumors did not yield any results, as the tumors were miniscule.
Publication Title
KPC1-mediated ubiquitination and proteasomal processing of NF-κB1 p105 to p50 restricts tumor growth.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 15 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
High anaplastic lymphoma kinase (ALK) protein levels may be correlated with an unfavorable prognosis in neuroblastoma (NBL) patients, regardless of ALK mutation status. We therefore examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated (MUT) and wild type (WT) NBL cell lines. Six of the nineteen NBL cell lines had a point mutation and four an amplification of the ALK gene. ALK amplified cell lines showed similar ALK levels and ALK inhibitor sensitivity as WT cell lines and were therefore co-analyzed. The ALK mRNA (p=0.043), ALK 220 kDa (p=0.009) and ALK 140 kDa (p=0.025) protein levels were higher in ALK mutant (n=6) than WT cell lines (n=13). ALK mRNA and protein levels significantly correlated with ERK1 and ERK2 protein levels, and also with PHOX2B mRNA levels, a neural differentiation marker which is mutated in NBL. Response to ALK inhibitor TAE684 was also significantly correlated with ALK levels. ALK mutant cell lines (n=4) demonstrated a higher sensitivity towards ALK inhibitor TAE684 (14.9 fold more sensitive, p=0.004) than eight WT cell lines. These results underline the importance of ALK mutations but also ALK levels for response to ALK inhibitors in NBL cell lines. Furthermore, the strong correlation of PHOX2B and ALK suggests that neural differentiation stage may be correlated with ALK levels in neuroblastoma. These data will enhance understanding of ALK inhibitor response in future patient trials.
Publication Title
Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated with ALK mutation status, ALK mRNA and protein levels.
Sample Metadata Fields
Sex, Specimen part
View Samples- Homo sapiens
- 42 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines.
Sample Metadata Fields
Sex, Specimen part, Disease, Cell line, Treatment
View Samples- Homo sapiens
- 42 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
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.
Publication Title
Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines.
Sample Metadata Fields
Sex, Specimen part, Cell line
View Samples- Mus musculus
- 111 Downloadable Samples
- NextSeq 500
Description
Purpose: To investigate alterations in subcutaneous white adipose gene expression induced by genetic AMPK activation in vivo, in mice fed a chow or a high-fat diet. Methods: Subcutaneous white adipose tissue mRNA profiles of wild-type transgenic (WT-Tg) mice and mice expressing a gain-of-function AMPK mutant gamma1 subunit (D316A-Tg) were generated by deep sequencing. Results: RNA sequencing revealed over 3000 differentially expressed genes between WT-Tg and D316A-Tg subcutaneous white adipose tissue (WATsc) from mice fed a high fat diet (HFD), of which many were classified as 'skeletal muscle-associated'. Interestingly, uncoupling protein 1 (UCP1), associated with 'beige' adipocyte formation in WATsc, was not differentially expressed. On a chow diet, many differentially expressed genes were also identified, with gene ontology analysis identifiying glycolysis, TCA cycle and brown fat differentiation as highly enriched; key features of brown adipocyte identity. HFD-associated skeletal-muscle associated gene expression was either not significantly altered, or significantly down-regulated on a chow diet, indicating a diet-induced gene signature in D316A-Tg WATsc. Conclusions: Our study revealed gene signatures indicative of brown adipocyte development on a chow diet, where no overt metabolic phenotype was observed in gain-of-function animals. When fed a HFD, WATsc from D316A-Tg mice displayed a muscle-like gene signature, expressing key components of creatine and calcium thermogenic cycles including Ckmt2 (creatine kinase, mitochondrial 2) Atp2a1 (SERCA1-sarco/endoplasmic reticulum ATPase 1) and ryr1 (ryanodine receptor 1). UCP1 expression was not altered between WT-Tg and D316A-Tg mice fed a HFD. Our findings suggest a novel role for AMPK in the regulation of white adipocyte identity and a potentially novel cell population that, when metabolically challenged, preferrentially utilise muscle-like thermogenic futile cycles independent of UCP1 to mediate whole organism energy expenditure. Overall design: Whole subcutaneous white adipose tissue mRNA profiles were generated from mice fed either chow or 45% high-fat diet.
Publication Title
AMPK activation protects against diet induced obesity through Ucp1-independent thermogenesis in subcutaneous white adipose tissue.
Sample Metadata Fields
Age, Specimen part, Cell line, Subject
View Samples