Project description:A functional genetic screen to identify genes causing tamoxifen resistance in an estrogen-dependent human breast cancer cell model was performed. By insertion of defective retrovirus into the genome, individual genetic changes were introduced at random in ZR-75-1 cells. Subsequently, infected cells were selected for their ability to proliferate while being exposed to tamoxifen, and from these cultures stable cell lines were established. The retrovirus insertion sites were mapped enabling the identification of several candidate breast cancer anti-estrogen resistance (BCAR) genes. By cDNA transfection of estrogen-dependent cells resulting in their transformation into a tamoxifen-resistant phenotype, proof was provided for the causative role of BCAR genes.A functional genetic screen to identify genes causing tamoxifen resistance in an estrogen-dependent human breast cancer cell model was performed. By insertion of defective retrovirus into the genome, individual genetic changes were introduced at random in ZR-75-1 cells. Subsequently, infected cells were selected for their ability to proliferate while being exposed to tamoxifen, and from these cultures stable cell lines were established. The retrovirus insertion sites were mapped enabling the identification of several candidate breast cancer anti-estrogen resistance (BCAR) genes identified. By cDNA transfection of estrogen-dependent cells resulting in their transformation into a tamoxifen-resistant phenotype, proof was provided for the causative role of BCAR genes (Van Agthoven et al, Breast Cancer Res Treat DOI:10.1007/s10549-008-9969-5, 2008). To elucidate the mechanisms how these individual BCAR genes induce cell proliferation in growth-arrested ZR-75-1 cells, we assessed the gene expression patterns between the different groups of cell lines. Keywords: Expression profiling, reference design RNA was isolated from two independent cultures of 69 tamoxifen-resistant cell lines. Detailed information regarding these cell lines with respect to the viral integration sites, was described previously (Van Agthoven et al, Breast Cancer Res Treat DOI:10.1007/s10549-008-9969-5, 2008). Each sample was hybridized in duplicate/triplicate and once in a dye-swap. The mixture of cell lines used as a reference was detailed previously (Jansen et al, J Clin Oncol 23, 732, 2005).

Project description:Resistance to endocrine therapy agents has presented a clinical obstacle in the treatment of hormone-dependent breast cancer. Our laboratory has initiated a study of microRNA regulation of signaling pathways that may result in breast cancer progression on aromatase inhibitors (AI). Microarray analysis of microRNA expression identified 115 significantly regulated microRNAs, of which 49 microRNAs were believed to be hormone-responsive. Within the AI-resistant cells, microRNAs were differentially expressed between the steroidal and non-steroidal AI-resistant lines. Also, a group of microRNAs were inversely expressed in the AI-resistant lines versus LTEDaro and tamoxifen-resistant. We focused our work on hsa-miR-128a which was hormone-responsive and up-regulated in the letrozole-resistant cell lines. Human miR-128a was shown to negatively target TGFBRI protein expression by binding to the 3’UTR region of the gene. Loss of TGFBRI resulted in compromised sensitivity to the growth inhibitory effects of TGFB in the letrozole-resistant lines. Inhibition of endogenous miR-128a resulted in re-sensitization of the letrozole-resistant lines to TGFB growth inhibitory effects. This data suggests that the hormone-responsive miR-128a can modulate TGFB signaling and survival of the letrozole-resistant cell lines. To our knowledge, this is the first study to address the role of microRNA regulation as well as TGFB signaling in AI-resistant breast cancer cell lines. We believe that in addition to estrogen-modulation of gene expression, hormone-regulated microRNAs may provide an additional level of post-transcriptional regulation of signaling pathways critically involved in breast cancer progression and AI-resistance. To look at microRNA expression profiles of breast cancer cell lines derived from MCF-7 cells that are resistant to endocrine therapy agents. MCF-7 cells that overexpress aromatase (MCF-7aro) were cultured long-term in the presence of endocrine therapy agents until cells acquired resistance. Three different aromatase inhibitors (letrozole, anastrozole or exemestane) were used, as well as the ER antagonist tamoxifen, or the hormone-free long-term estrogen deprived cells (LTED). Three replicates of the control cells (MCF-7aro) and all resistant cells were used for microarray experiments. Total of 23 samples were analyzed by microarray.

Project description:Breast cancer is a genetically and phenotypically complex disease. To understand the role of microRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer (BC) cell lines to discover miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations.Using a microarray carrying LNA™ modified oligonucleotide capture probes (Exiqon), expression levels of 725 human miRNAs were measured in 51 BC cell lines. MiRNA expression was explored by unsupervised cluster analysis and then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer. Unsupervised cluster analysis using the most variably expressed miRNAs divided the 51 BC cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of BC cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters of which half were related to the ER-status of the cell lines. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group of cell lines, 39 miRNAs were associated with ERBB2 overexpression and 24 miRNAs with E-cadherin gene mutations, which are frequent in this subtype of BC cell lines. In contrast, 31 different miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in BC cell lines that are not of luminal origin. The differential expression of 30 miRNAs were associated with p16INK4 status while only a few differentially expressed miRNAs were associated with BRCA1, or PIK3CA/PTEN, TP53 mutation status of the cell lines (P-value < 0.05). Twelve miRNAs were associated with DNA copy number variation of the respective locus. Luminal-basal and epithelial-mesenchymal associated miRNAs determine the overall subdivision of miRNA transcriptome of BC cell lines. Specific sets of miRNAs were associated with ERBB2 overexpression, p16INK4aor E-cadherin mutation or E-cadherin methylation status, which implies that these miRNAs may contribute to the driver role of the genetic aberrations. Additionally, miRNAs, which are located in a genomic region showing recurrent genetic aberrations, may themselves play a driver role in breast carcinogenesis or contribute to a driver gene in their vicinity. In short, our study provides detailed molecular miRNA portraits of BC cell lines, which can be exploited for functional studies of clinically important miRNAs. Affymetrix plus2PM arrays were hybridized according to the manufacturer's procedure using RNA extracted from 52 cultured breast cancer cell lines. Most cellines were analyzed in triplicate. Gene expression data in log2 scale arrays were calculated and associated with diverse characteristics. This submission represents the gene expression component of the study only

Project description:A functional genetic screen to identify genes causing tamoxifen resistance in an estrogen-dependent human breast cancer cell model was performed. By insertion of defective retrovirus into the genome, individual genetic changes were introduced at random in ZR-75-1 cells. Subsequently, infected cells were selected for their ability to proliferate while being exposed to tamoxifen, and from these cultures stable cell lines were established. The retrovirus insertion sites were mapped enabling the identification of several candidate breast cancer anti-estrogen resistance (BCAR) genes. By cDNA transfection of estrogen-dependent cells resulting in their transformation into a tamoxifen-resistant phenotype, proof was provided for the causative role of BCAR genes.A functional genetic screen to identify genes causing tamoxifen resistance in an estrogen-dependent human breast cancer cell model was performed. By insertion of defective retrovirus into the genome, individual genetic changes were introduced at random in ZR-75-1 cells. Subsequently, infected cells were selected for their ability to proliferate while being exposed to tamoxifen, and from these cultures stable cell lines were established. The retrovirus insertion sites were mapped enabling the identification of several candidate breast cancer anti-estrogen resistance (BCAR) genes identified. By cDNA transfection of estrogen-dependent cells resulting in their transformation into a tamoxifen-resistant phenotype, proof was provided for the causative role of BCAR genes (Van Agthoven et al, Breast Cancer Res Treat DOI:10.1007/s10549-008-9969-5, 2008). To elucidate the mechanisms how these individual BCAR genes induce cell proliferation in growth-arrested ZR-75-1 cells, we assessed the gene expression patterns between the different groups of cell lines. Keywords: Expression profiling, reference design RNA was isolated from two independent cultures of 69 tamoxifen-resistant cell lines. Detailed information regarding these cell lines with respect to the viral integration sites, was described previously (Van Agthoven et al, Breast Cancer Res Treat DOI:10.1007/s10549-008-9969-5, 2008). Each sample was hybridized in duplicate/triplicate and once in a dye-swap. The mixture of cell lines used as a reference was detailed previously (Jansen et al, J Clin Oncol 23, 732, 2005).

Dataset Information

Modulation of the leptin receptors expression in breast cancer cell lines exposed to leptin and tamoxifen.

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Modulation of the leptin receptors expression in breast cancer cell lines exposed to leptin and tamoxifen.

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