Unknown,Transcriptomics,Genomics,Proteomics

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Data integration from two microarray platforms identifies genetic inactivation of RIC8A in a breast cancer cell line

ABSTRACT: Using array comparative genomic hybridization (aCGH), a large number of deleted genomic regions have been identified in human cancers. However, subsequent efforts to identify target genes selected for inactivation in these regions have often been challenging. We integrated here genome-wide copy number data with gene expression data and non-sense mediated mRNA decay rates in breast cancer cell lines to prioritize gene candidates that are likely to be tumour suppressor genes inactivated by bi-allelic genetic events. The candidates were sequenced to identify potential mutations. This integrated genomic approach led to the identification of RIC8A at 11p15 as a putative candidate target gene for the genomic deletion in the ZR-75-1 breast cancer cell line. We identified a truncating mutation in this cell line, leading to loss of expression and rapid decay of the transcript. We screened 127 breast cancers for RIC8A mutations, but did not find any pathogenic mutations. No promoter hypermethylation in these tumours was detected either. However, analysis of gene expression data from breast tumours identified a small group of aggressive tumours that displayed low levels of RIC8A transcripts. Real-time PCR analysis of 38 breast tumours showed a strong association between low RIC8A expression and the presence of TP53 mutations (P=0.006). We demonstrate a data integration strategy leading to the identification of RIC8A as a gene undergoing a classical double-hit genetic inactivation in a breast cancer cell line, as well as in vivo evidence of loss of RIC8A expression in a subgroup of aggressive TP53 mutant breast cancers. Gene expression data: Samples GSM388181-GSM388198. The experiment utilized six breast cancer cell lines: MDA-MB-468, MDA-MB-231, ZR-75-1, MCF7, BT-474 and T-47D, and three non-malignant cell lines: HMECs (non-malignant human mammary epithelial cells), IMR90 (normal lung fibroblasts) and WS1 (normal skin fibroblasts). All cell lines were obtained from American Type Culture Collection and grown in accordance with the distributor's instructions. Both malignant and non-malignant cell lines were treated with the translation inhibitor emetine dihydrochloride hydrate. For each cell line, parallel cell cultures were grown in 175 cm2 flasks until 70-80 % confluence. Half of the subconfluent cultures were treated with 100 Î¼g ml-1 of emetine dihydrochloride hydrate while the other half were left as untreated controls. Genome-wide copy number data: Samples GSM388211-GSM388216. The experiment utilized six breast cancer cell lines; MDA-MB-468, MDA-MB-231, ZR-75-1, MCF7, BT-474 and T-47D. All cell lines were obtained from American Type Culture Collection and grown in accordance with the distributor's instructions. All samples were hybridized once on 44k Agilent Human Genome CGH microarrays according to manufacturers instructions. Genomic DNA pooled from healthy female donors was used as a reference in all hybridizations. DNA from cell line samples were labeled with Cy5 and DNA from reference were labeled with Cy3.

ORGANISM(S): Homo sapiens

SUBMITTER: Olli Kallioniemi

PROVIDER: E-GEOD-15477 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Data integration from two microarray platforms identifies bi-allelic genetic inactivation of RIC8A in a breast cancer cell line.

Muggerud Aslaug Aamodt AA Edgren Henrik H Wolf Maija M Kleivi Kristine K Dejeux Emelyne E Tost Jörg J Sørlie Therese T Kallioniemi Olli O

BMC medical genomics 20090511

<h4>Background</h4>Using array comparative genomic hybridization (aCGH), a large number of deleted genomic regions have been identified in human cancers. However, subsequent efforts to identify target genes selected for inactivation in these regions have often been challenging.<h4>Methods</h4>We integrated here genome-wide copy number data with gene expression data and non-sense mediated mRNA decay rates in breast cancer cell lines to prioritize gene candidates that are likely to be tumour suppr ...[more]

PMID: 19432969

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Project description:Background: Research using in vitro canine mammary carcinoma cell lines and studies involving naturally occurring canine mammary tumours are not only fundamental models used to advance the understanding of cancer in veterinary patients but are also regarded as excellent translational models for human breast cancer. In human medicine, following disease classification and staging breast cancer is typically treated with multimodal therapies including radiotherapy. The response of a breast tumour to radiation depends not only on its innate radiosensitivity but also on tumour repopulation by cells that have developed radioresistance. Comparative canine and human studies investigating the molecular mechanisms of radioresistance may lead to the development of effective cancer treatments that benefit both species. Methods: Human (MDA-MB-231, MCF-7 and ZR-751) and canine (REM-134) radioresistant cell lines were established by exposing parental cells to increasing weekly doses of radiation. The development of radioresistance was evaluated through proliferation and colony formation assays. Phenotypic characterisation included migration and invasion assays and immunohistochemistry. Intrinsic differences between parental and radioresistant cells were investigated by whole-transcriptome gene expression analysis. Gene enrichment and pathway-focused analyses identified signalling networks differentially activated in radioresistant cells. Results: Colony formation assays identified a range of intrinsic radiosensitivities in the human and canine parental cell lines with the REM-134 cell line showing significantly greater radioresistance compared to the human cell lines. Colony formation and proliferation assays confirmed radioresistance in all developed radioresistant cell lines. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition. Gene analysis identified that following the acquisition of radioresistance in MCF-7 and ZR-751 cell lines the cell lines changed subtype classification from their parental luminal A to HER2-overexpressing (MCF-7 RR) and normal-like (ZR-751 RR) subtypes, indicating the extent of phenotypic changes and cellular plasticity involved in this process. MDA-MB-231 and MDA-MB-231 RR cell lines both classified as basal, whereas the REM-134 and REM-134 RR cell lines with enriched for HER2 overexpression. Whole-transcriptome gene expression analysis identified down-regulation of ER signalling genes and up-regulation of genes associated with PI3K, MAPK and WNT pathway activity in radioresistant cell lines derived from ER+ cells; this was confirmed by western blot, which showed increased p-AKT and p-ERK expression following radiation. Conclusions: To our knowledge, our study is the first to develop a canine radioresistant cell line and provide a comparative genetic and phenotypic analysis of the mechanisms of acquired radioresistance between canine and human cell lines. The cellular process that occur with the development of acquired radioresistance are similar between the human and canine cell lines; this suggests that not only is the canine model an appropriate model to study human disease but that treatment strategies used in human medicine may also be applicable to veterinary patients.

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Data integration from two microarray platforms identifies genetic inactivation of RIC8A in a breast cancer cell line

Publications

Data integration from two microarray platforms identifies bi-allelic genetic inactivation of RIC8A in a breast cancer cell line.

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