RESUMO
Germline mutations in the tumour suppressor BRCA2 predispose to breast, ovarian and a number of other human cancers. Brca2-deficient mouse models are used for preclinical studies but the pattern of genomic alterations in these tumours has not yet been described in detail. We have performed whole-exome DNA sequencing analysis of mouse mammary tumours from Blg-Cre Brca2(f/f) Trp53(f/f) animals, a model of BRCA2-deficient human cancer. We also used the sequencing data to estimate DNA copy number alterations in these tumours and identified a recurrent copy number gain in Met, which has been found amplified in other mouse mammary cancer models. Through a comparative genomic analysis, we identified several mouse Blg-Cre Brca2(f/f) Trp53(f/f) mammary tumour somatic mutations in genes that are also mutated in human cancer, but few of these genes have been found frequently mutated in human breast cancer. A more detailed analysis of these somatic mutations revealed a set of genes that are mutated in human BRCA2 mutant breast and ovarian tumours and that are also mutated in mouse Brca2-null, Trp53-null mammary tumours. Finally, a DNA deletion surrounded by microhomology signature found in human BRCA1/2-deficient cancers was not common in the genome of these mouse tumours. Although a useful model, there are some differences in the genomic landscape of tumours arising in Blg-Cre Brca2(f/f) Trp53(f/f) mice compared to human BRCA-mutated breast cancers. Therefore, this needs to be taken into account in the use of this model.
Assuntos
Genes BRCA2/fisiologia , Neoplasias Mamárias Experimentais/genética , Proteína Supressora de Tumor p53/deficiência , Animais , Antígenos CD/genética , Neoplasias da Mama/genética , Proteínas Cromossômicas não Histona/genética , Variações do Número de Cópias de DNA/genética , DNA de Neoplasias/genética , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Mutação em Linhagem Germinativa/genética , Humanos , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Neoplasias Ovarianas/genética , Proteínas Serina-Treonina Quinases/genética , Receptores Imunológicos/genética , Análise de Sequência de DNA , Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
We describe a screen for cellular response to drugs that makes use of haploid embryonic stem cells. We generated ten libraries of mutants with piggyBac gene trap transposon integrations, totalling approximately 100,000 mutant clones. Random barcode sequences were inserted into the transposon vector to allow the number of cells bearing each insertion to be measured by amplifying and sequencing the barcodes. These barcodes were associated with their integration sites by inverse PCR. We exposed these libraries to commonly used cancer drugs and profiled changes in barcode abundance by Ion Torrent sequencing in order to identify mutations that conferred sensitivity. Drugs tested included conventional chemotherapeutics as well as targeted inhibitors of topoisomerases, poly(ADP-ribose) polymerase (PARP), Hsp90 and WEE1.