A novel miRNA fingerprint inversely associated with breast cancer progression in SKBR3 cells challenged with all-trans retinoic acid and lapatinib
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ABSTRACT: In SKBR3 cells, simultaneous targeting of RARM-kM-1 with all-trans retinoic acid (ATRA) and HER2 with lapatinib results in synergistic anti-tumor responses. SKBR3 cells were treated with vehicle (DMSO), lapatinib (100 nM), ATRA (100 nM) or lapatinib+ATRA for 36 hours and miRNA expression profile was determined by one-color Agilent microarray experiments.
Project description:SKBR3 cells, which bear both an HER2 and a RARA gene amplification, were treated for 12 or 48 hours with 100 nM retinoic acid, 100 nM lapatinib or the combination.The two drugs synergize and induce massive apoptosis. The aim is to find the molecular mechanism(s) of this synergism. Gene expression profiling was performed using Agilent two-color 4X44K arrays. Original processed data are available in the archive: http://www.ebi.ac.uk/arrayexpress/files/E-MEXP-3192/E-MEXP-3192.additional.zip
Project description:Ablation of ERRalpha significantly delays ERBB2-induced mammary tumorigenesis and ERRalpha regulates genes of the ERBB2 amplicon. To further investigate the relationship between ERRalpha activity and RTK signaling, we mapped ERRalpha binding sites in SKBr3 cells upon EGF treatment or heregulin treatment. Inhibition of ERBB2 signaling using the RTK inhibitor lapatinib impacts on ERRalpha stability, while cells resistant to lapatinib treatment exhibit restored ERRalpha expression. We therefore mapped ERRalpha binding sites in parental (sensitive) cells (pSKBr3) as well as in lapatinib-resistant cells (LRSKBr3). ChIP-Seq analysis of ERRalpha binding profile in SKBr3 or BT-474 breast cancer cells.
Project description:Ablation of ERRalpha significantly delays ERBB2-induced mammary tumorigenesis and ERRalpha regulates genes of the ERBB2 amplicon. To further investigate the relationship between ERRalpha activity and RTK signaling, we depleted ERRalpha in SKBr3 cells upon serum starvation, EGF treatment or heregulin treatment. Inhibition of ERBB2 signaling using the RTK inhibitor lapatinib impacts on ERRalpha stability. Since we have shown that the development of lapatinib-resistance restaures the expression of ERRalpha in breast cancer cells, we performed depletion of ERRalpha in SKBr3 cells that have developped resistance to lapatinib treatment in order to identify a potential reprogramming of ERRa transcriptional activity associated to lapatinib resistance, For the study of growth factor effec on ERRalpha activity, total RNA was obtained from human SKBr3 breast cancer cells cultured in DMEM deprived of FBS (starved) for 24 hours and treated with PBS, EGF (100uM) or Heregulin (100uM) for an additional 24h. Cells were transfected with siRNA against ERRalpha or with siControl for 60 hours prior to harvesting. For the effect of ERRalpha in lapatinib resistance cells, parental SKBr3 cells (pSKBr3) and Lapatinib-resistant cells (LRSKBr3, maintained in 2uM lapatinib) were transfected with siControl (siC) or siERRalpha for 60 hours prior to harvesting and RNA extraction
Project description:These studies are aimed at understanding gene expression chnages in a Her2 positive breast cancer cell line that has developed acquired resistance to lapatinib. Samples include SKBR3 parental and resistant (SKBR3-R) under basal conditions and in response to 0.1 and 1uM lapatinib treatment after 24 hours.
Project description:The contribution of aberrant DNA methylation and the downstream effects in tumorogenesis through silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations can be reversed, we investigated the effects of the epigenetic therapy in breast cancer cell lines. We used microarrays to investigate the global microRNA expression profile after demethylation treatment with 5-aza-2’-deoxycytidine (DAC) in breast cancer cell lines and identified distinct classes of early and late systematic stable or transient effects of the treatment. Three selected breast cell lines including MDA-MB231, SKBR3, BT549, HS578T, MCF7 and HB2 (a breast epithelial cell line as control) were subject for miRNA isolation before treatment, after treatment with DAC and at five point follow-ups (1st, 3rd, 5th, 7th and 10th passages) at “drug holiday” condition and hybridized on Affymetrix microarrays.
Project description:Ablation of ERRalpha significantly delays ERBB2-induced mammary tumorigenesis and ERRalpha regulates genes of the ERBB2 amplicon. To further investigate the relationship between ERRalpha activity and RTK signaling, we depleted ERRalpha in SKBr3 cells upon serum starvation, EGF treatment or heregulin treatment. Inhibition of ERBB2 signaling using the RTK inhibitor lapatinib impacts on ERRalpha stability. Since we have shown that the development of lapatinib-resistance restaures the expression of ERRalpha in breast cancer cells, we performed depletion of ERRalpha in SKBr3 cells that have developped resistance to lapatinib treatment in order to identify a potential reprogramming of ERRa transcriptional activity associated to lapatinib resistance,
Project description:We carried out a genome-wide investigation of the primary transcriptional targets of 1α,25(OH)2D3 in breast epithelial cancer cells using RNA-Seq technology. We identified early transcriptional targets of 1α,25(OH)2D3 involved in adhesion, growth regulation, angiogenesis, actin cytoskeleton regulation, hexose transport, inflammation and immunomodulation, apoptosis, endocytosis and signaling. Furthermore, we found several transcription factors to be regulated by 1α,25(OH)2D3 that subsequently amplify and diversify the transcriptional output driven by 1α,25(OH)2D3 leading finally to a growth arrest of the cells. Moreover, we could show that 1α,25(OH)2D3 elevates the trimethylation of histone H3 lysine 4 at several target gene promoters. Our present transcriptomic analysis of differential expression after 1α,25(OH)2D3 treatment provides a resource of primary 1α,25(OH)2D3 targets that might drive the antiproliferative action in breast cancer epithelial cells. ChIP-Seq for trimethylated histone H3K4 with SKBr3 cells treated for 2h with 100nM 1α,25(OH)2D3 or vehicle as control.
Project description:This study was designed to investigate the Metformin mode of action in different subtypes of breast cancer using cell and molecular, and systems biology techniques. To that end, several concentrations of Metformin have been used. Besides, five different breast cancer cell lines representing the five breast cancer phenotypes have been employed in this study. These cell lines were BT-474, MCF-7, MDA-MB-231, MDA-MB-468, and SkBr3 as representative for (Luminal B, Luminal A, Claudin-low, Basal-like, and HER2) subtypes respectively. Interestingly, Metformin treatment significantly reduced cancer cell viability and proliferation while inducing cell apoptosis and enhanced cell necrosis of the Basal-like (MDA-MB-468), although, the less sensitive subtype is HER2 (SkBr3).
Project description:SKBR3 breast cancer cell extracts were digested with trypsin (or LysC) on short time scales (7min, 15min, 30min, 1h, and 18h), and the results were compared to overnight digestion protocols.