Unknown,Transcriptomics,Genomics,Proteomics

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From mouse to humans: detecting preventing vaccination targets associated to breast cancer stem cells. Exon-level analysis

ABSTRACT: TuBo cell line (E) was compared to passage 1 (P1), 2 (P2) and 3 (P3) mammospheres to detect specific transcription isoforms associated to cancer stem cell. TuBo cell line (E) was compared to passage 1 (P1), 2 (P2) and 3 (P3) mammospheres. Exon-level transcription profiling was done using Affimetrix GeneChip Exon 1.0 ST. Exon-level analysis is more complex than gene-level analysis. The number of probesets to be investigated is at least 10 times larger in number than gene-level probesets. Thus results, upon statistical analysis, are massively contaminated by type I statistical errors, i.e. false positive [Della Beffa et al. 2008]. Furthermore, exon-level probesets are based only on four probes, which makes exon-level signal summarization more noisy than gene-level, where signal summarization is based on the overall probes encompassed by all exons of a gene. To moderate these issues we have expanded the number of experimental replications: six for TuBo and passage 1 mammosphere, four for passage 3 mammosphere and three for passage 2 mammosphere. Furthermore, after data summarization with RMA and normalization with sketch quantile method, alternative splicing detection between epithelial and mammospheres passages, was assessed using MiDAS, a two way anova developed by Affymetrix for the detection of alternative splicing events. We considered suitable for further investigations the splicing events in common between the sets of exons detected as spliced in each of the following comparisons: E vs P1, E vs P2 and E vs P3.

ORGANISM(S): Mus musculus

SUBMITTER: Raffaele Calogero

PROVIDER: E-GEOD-21475 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The efficacy of cancer treatments have improved constantly in the last decade. However, therapeutic resistance and the lack of curative treatments in metastatic disease, raises the question if conventional anticancer therapies target the right cells. Indeed, these treatments might miss cancer stem cells (CSCs), which might also represent a more chemoresistant and radioresistant subpopulation within cancer. In this view using vaccines in tertiary prevention of cancer, i.e. residual disease treatment, might be particularly effective if vaccine-elicited immune response is directed against CSC oncoantigens (OAs) M-bM-^@M-^T proteins required for the neoplastic process M-bM-^@M-^T the chance that the tumour will evade the vaccine should be reduced. An important task to devise effective CSC preventive vaccines is therefore the identification of CSC OAs. In this experiment we used a cell line (TuBo) derived by the mouse breast cancer model BALB-neuT and its CSC subpupolation, enriched by mean of mammosphere formation. Integrating data derived by gene and exon-level transcription profiling of the above experiments with public available normal and tumor datasets we identified two new breast cancer CSC OAs: TMPRSS4 and xCT. Both genes are linked to invasion, migration and metastasis. Furthermore, we observed that alternative splicing events discriminating TuBo cells, grown in adherent medium, with respect of TuBo mammospheres, produced growing in no-adherent medium, are very limited. We detect only one clear splcing event characterizing the beta-isoform of Rabgap1l gene. This SuperSeries is composed of the SubSeries listed below. The adherent TuBo epithelial cells were generated from a mammary carcinoma arising in a BALB/c mouse female transgenic for the activated rat ErbB2 oncogene (BALB-neuT) and were cultured in DMEM supplemented with 20% FCS. Single TuBo cells were plated in ultra low attachment flasks in serum-free DMEM-F12 medium supplemented with bFGF, EGF and insulin. Nonadherent spherical clusters of cells, named mammospheres, were collected by gentle centrifugation after 7 days and dissociated enzymatically and mechanically, using trypsin and pipetting, respectively.

Project description:Celiac disease (CD) is a chronic inflammation of the small intestine triggered by the ingestion of gluten in genetically predisposed individuals. Tissue transglutaminase (TG2) is a key factor in the pathogenesis of CD, because it catalyzes both the deamidation of specific glutamine residues and the formation of covalent Nε-(γ-glutamyl)-lysine isopeptide crosslinks resulting in TG2-gluten peptide complexes. These complexes are thought to activate B cells causing the secretion of anti-TG2 autoantibodies that serve as diagnostic markers for CD, although their pathogenic role remains unclear. To gain more insight into the molecular structures of TG2-gluten peptide complexes, we developed a reciprocal proteomic analysis strategy, which facilitates the comprehensive identification of isopeptides in a complex protein hydrolysate. The workflow consists of four steps: (1) performance of the model reaction between TG2 and gluten peptide(s) followed by tryptic hydrolysis, clean-up and untargeted nLC-MS/MS analysis (2), prediction of tryptic TG2-derived lysine peptides that serve as potential isopeptide modifications (3) the search for isopeptides by configuring the TG2-modifications in MaxQuant and search against the α-gliadin fasta file and (4) the reciprocal search for isopeptides by configuring the gluten peptide(s) as modification in MaxQuant and search against the TG2 fasta file. After verification by manual data curation and visualization, this strategy enabled the identification of 26 different isopeptides involving 19 TG2 lysine residues, only six of which were known previously. Experiments with different TG2-gluten peptide molar ratios revealed that K-425, K-590, K-600 and K-649 were the most preferred lysine residues involved in isopeptide crosslinking. Further, expanding the model system to three gluten peptides allowed the localization of the preferred glutamine crosslinking sites. The described strategy is generally applicable to any hydrolysate containing isopeptides and these new insights into the structure of TG2-gluten peptide complexes may help clarify the role of extracellular TG2 in CD autoimmunity and in other inflammatory diseases.

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From mouse to humans: detecting preventing vaccination targets associated to breast cancer stem cells. Exon-level analysis

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