Expression profiling tumor cells from MYCN-driven neuroblastoma upon BRD4 or AURKA inhibition
ABSTRACT: Amplification of MYCN is the most prominent genetic marker of high-stage neuroblastoma, a childhood tumor originating from the neural crest. We generated a cell line (mNB-A1) from tumors developed in transgenic mouse and treated these cells with DMSO (n=6), the BRD4-inhibitor JQ1 (n=3) or the AURKA-inhibitor MLN8237 (n=3) for 24 h. The expression profiles of vehicle (DMSO)-treated mNB-A1 cells were compared to JQ1- or MLN8237-treated mNB-A1 cells.
Project description:The goal of this experiment was to understand the changes in gene expression in the human basal-like breast cancer cell line HCC1143 following treatment with the MEK inhibitor Trametinib (T), PI3K/mTOR inhibitor BEZ235 (B), the BET inhibition JQ1 (JQ), Trametinib + JQ1 (TJ), or BEZ235 + JQ1(BJ), compared to a DMSO control (D). Samples were treated for 72hr and run in triplicate. Overall design: The human basal-like breast cancer cell line HCC1143 was treated for 72hr with 1uM Trametinib (T), 1uM BEZ235, 1uM JQ1 (JQ), 1uM Trametinib + 1uM JQ1 (TJ), 1uM BEZ235 + 1uM JQ1 (BJ), or a 0.05% DMSO control. Total RNA was isolated using a QIAGEN total RNA RNeasy kit, libraries were generated with a Truseq kit, and samples were run on the Nextseq500, data processing is described below.
Project description:Estrogen signaling pathway is critical for breast cancer development and has remained the major adjuvant therapeutic target for this disease. Tamoxifen has been used in clinic for many years to treat ER-positive breast cancer. However a great many (30%) suffer relapse due to drug resistance. In this study, the bromodomain inhibitor JQ1 was found to down-regulate ERalpha gene expression and have anti-tumor effect in cultured tamoxifen-resisant breast cancer cells. We used microarrays to detail the global programme of gene expression in tamoxifen-resistant MCF7 cells treated with the bromodomain inhibitor JQ1. Tamoxifen-resistant breast cancer MCF7 cells were treated with DMSO (vehicle) or JQ1 (0.2 uM) for 24 hours before total RNA was purified for microarray. Each sample was triplicated.
Project description:Bromodomain and extra terminal domain (BET) inhibition reduces occupancy of BET-family proteins at promoter and enhancer sites resulting in changes in the transcription of specific genes. We used microarray profiling to investigate the transcriptional changes induced by BET inhibitor JQ1 treatment in DV90 cells to identify the underlying changes of gene regulation that lead to JQ1 sensitivity. DV90 cells (JQ1 sensitive non-small cell lung cancer cell line) were treated with 135 nM (IC50) or 785 nM (IC90) of JQ1 for 4h and 24h. DMSO treated controls served as reference and at least four replicates per condition were collected. RNA was extracted and hybridized to Affymetrix HuGene-2.1ST microarrays to identify treatment induced transcriptional changes.
Project description:Bromodomain and extra terminal domain (BET) proteins are important epigenetic regulators facilitating the transcription of genes in chromatin areas linked to acetylated histones. JQ1, a BET protein inhibitor, has antiproliferative activity against many cancers, mainly through inhibition of c-MYC and upregulation of p21. In this research, we investigated the use of JQ1 for human osteosarcoma (OS) treatment. JQ1 significantly inhibited the proliferation and survival of OS cells inducing G1 cell cycle arrest, premature senescence, but little effect on apoptosis. Interestingly, c-MYC protein levels in JQ1-treated cells remained unchanged, whereas the upregulation of p21 protein was still observable. Although effective in vitro, JQ1 alone failed to reduce the size of the MNNG/HOS xenografts in immunocompromised mice. To overcome the resistance of OS cells to JQ1 treatment, we combined JQ1 with rapamycin, an mTOR inhibitor. JQ1 and rapamycin synergistically inhibited the growth and survival of OS cells in vitro and in vivo. We also identified that RUNX2 is a direct target of BRD4 inhibition by JQ1 in OS cells. Chromatin immunoprecipitation (ChIP) showed that enrichment of BRD4 protein around RUNX2 transcription start sites diminished with JQ1 treatment in MNNG/HOS cells. Overexpression of RUNX2 protected JQ1-sensitive OS cells from the effect of JQ1, and siRNA-mediated inhibition of RUNX2 sensitized the same cells to JQ1. In conclusion, our findings suggest that JQ1, in combination with rapamycin, is an effective chemotherapeutic option for OS treatment. We also show that inhibition of RUNX2 expression by JQ1 partly explains antiproliferative activity of JQ1 in OS cells. MNNG/HOS cells treated with 7.5mM JQ1, 12.5nM Rapamycin or both were used for RNA extraction and hybridization on Affymetrix microarrays. We compared these microarray samples with the corresponding control (treated with DMSO).
Project description:Efficacy and safety of anticancer drugs are traditionally studied using cancer cell lines and animal models. Recently, a potential anticancer agent, JQ1, an inhibitor of bromodomain and extra terminal (BET) protein, has been shown to promote apoptosis of cancerous cells by arresting them in G1 phase of the cell cycle. However, the effect of JQ1 on normal cells is poorly understood. In this study, we investigated the safety of JQ1 by using human umbilical cord mesenchymal stem cells (MSCs) as an in vitro model system. Our results indicated that JQ1 induced cell cycle arrest in G1 phase of MSCs, but did not promote apoptosis. Microarray analysis of MSCs treated with JQ1 indicated that it down-regulated genes involved not only in cell cycle regulation but also DNA replication, mitosis, and cell division. Although many studies have suggested the potential of JQ1 as an anticancer agent, our findings suggest that it caused a deleterious effect on normal cells and may not be safe for anticancer therapy. Human umbilical cord derived MSCs were cultured in vitro and treated with either 100 nM or 500 nM JQ1 for 24 hrs. Gene expression of treated cells was compared to untreated cultured cells.
Project description:Type II testicular germ cell cancers (GCC) are the most frequently diagnosed tumors in young men (20 - 40 years) and are classified as seminoma or non-seminoma. GCCs are commonly treated by orchiectomy and chemo- or radiotherapy. However, a subset of metastatic non-seminomas display only incomplete remission or relapse and require novel treatment options. Recent studies have shown effective application of the small-molecule inhibitor JQ1 in tumor therapy, which interferes with the function of bromodomain and extra-terminal (BET)-proteins. Here, we demonstrate that upon JQ1 doses ≥ 250 nM GCC cell lines and Sertoli cells display compromised survival and induction of cell cycle arrest. JQ1 treated GCC cell lines display upregulation of genes indicative for DNA damage and a cellular stress response. Additionally, downregulation of pluripotency factors and induction of mesodermal differentiation was detected. GCCs xenografted in vivo showed a reduction in tumor size, proliferation and angiogenesis when subjected to JQ1 treatment. The combination of JQ1 and the histone deacetylase inhibitor romidepsin further enhanced the apoptotic effect in vitro and in vivo. Thus, we propose that JQ1 alone, or in combination with romidepsin may serve as a novel therapeutic option for GCCs. Overall design: TCam-2 (seminoma), NCCIT (embryonal carcinoma), FS1 (Sertoli) and MPAF (fibroblast) cells were treated with 100 nM JQ1 or DMSO as solvent control. Samples were taken after 24h and 72h
Project description:We analyzed the transcriptional consequences of the BET bromodomain inhibitor JQ1 in the T-ALL cell line LOUCY by microarray analysis. Short-term exposure to a low dose of JQ1 (4h, 250nM) provided insights in the genes whose expression was immediately affected by BET bromodomain inhibition. Significantly downregulated genes upon short-term drug treatment included stem-cell associated genes and putative oncogenes such as BAALC, WT1, MN1, MEF2C, LMO1 and LMO2. Genes associated with the 500 highest ranked enhancer regions in LOUCY, were significantly enriched in genes downregulated after JQ1 treatment. Overall design: LOUCY cells were treated with 250nM (+)-JQ1 or DMSO for 4h. Three biological replicates of this treatment were performed.
Project description:We here use B-cell tumors as a model to address the mechanism of action of JQ1, a widely used BET inhibitor. Overall design: 250000 cells/mL of RAJI cells were cultured in a total volume of 40 mL. 24h after the plating DMSO or 100 nM of JQ1 were added to the culture for 24h. Total RNA from 107 RAJI was purified using TRIzol reagent (Invitrogen) according to manufacturer’s instructions, treated with TurboDNase (Ambion) and processed for oligonucleotide microarray profile through Affymetrix Human Gene 1.0 ST arrays platform.
Project description:The bromodomain inhibitor JQ1 and the histone deacetylase inhibitor panobinostat induce synergistic anticancer effects We analyzed whether JQ1 and panobinostat synergistically modulate gene expression Neuroblastoma SK-N-BE(2) cells were treated with vehicle control, 1 microM JQ1, 10 nM panobinostat, or combination of JQ1 and panobinostat for 6 hours, and subjected to differential gene expression studies with Affymetrix microarrays.