RNA-seq of coding and non-coding RNA from miR-522 pulldown in human MDA-MB-468 breast cancer cells
Ontology highlight
ABSTRACT: Identifying miRNA-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical seed base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic (this dataset). Within these targets, mostly non-canonical MREs were identified by sequencing RNase-resistant fragments.
Project description:Identifying miRNA-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical seed base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic. Within these targets, mostly non-canonical MREs were identified by sequencing RNase-resistant fragments (this dataset).
Project description:A number of studies have indicated that thyroid hormone receptor β1 (TRβ1) functions as a tumor suppressor. TRs mediate transcriptional responses through a highly conserved DNA-binding domain (DBD). A novel rat TRβ isoform (rTRβΔ) was previously identified, in which a novel exon, N (108 bp), is located between exons 3 and 4 within the DBD; this exon represents the only difference between rTRβΔ and rTRβ1. In vitro, rTRβΔ exhibits a stronger tumor-suppressive capacity than rTRβ1, and further analysis revealed a high level of conservation between the rat and human DBD sequences. In the present study, an artificially modified human TRβ1 (m-hTRβ1) was constructed via the introduction of the 108-bp sequence into the corresponding position of the wild-type human TRβ1 (wt-hTRβ1) DBD. An electrophoretic mobility shift assay and transfection experiments confirmed that m-hTRβ1 is functional. Overexpression of m-hTRβ1 inhibits the proliferation of MDA-MB-468 cells in the presence of triiodothyronine by promoting apoptosis, which may be associated with the upregulation of Caspase-3 and Bak gene expression and the activation of the Caspase-3 protein. In addition, the pro-apoptotic effect of m-hTRβ1 was stronger, compared with wt-hTRβ1. These results indicated that m-hTRβ1 may act as a tumor suppressor in MDA-MB-468 cells. These data provided a novel insight into gene therapy for breast cancer.
Project description:Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process of effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. Results: Colony formation and sulforhodamine B (IC50 < 1nM) assays, and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI), of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset, RMI was significantly associated with tumor size or lymph node status. High (>75) percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis, RMI (P = 0.029), tumor size (P = 0.015) and lymph node status (P = 0.01) were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41). In Wang dataset, RMI predicted time to disease relapse (P = 0.09). Conclusions: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment. Mol Cancer. 2009 Sep 24;8(1):75. Experiment Overall Design: Rapamycin treatment of MDA-MB-468 breast cancer cell line: Experiment Overall Design: MDA-MB-468 cell line was treated by DMSO (vehicle) and 100 nM rapamycin for 24 hours. We sought to identify differentially expressed genes. Experiment Overall Design: Rapamycin treatment of breast tumor xenografts: Experiment Overall Design: MDA-MB-468 cells were inoculated in the mammary fat pad of female nude mice. When resulting tumor volumes had reached 75-150 mm3, the mice were divided in four groups. Groups 1 and 2 received a single injection of DMSO (vehicle) or rapamycin (15 mg/kg) intraperitoneally and sacrificied 24 h later (1-day groups). Groups 3 and 4 received weekly injections of DMSO or rapamycin for 3 weeks and sacrificied 24 h after the last injection (22-day groups).
Project description:Exposure to cigarette smoke (CS) has been associated with an increased risk of fatal breast cancers and recurrence, along with chemoresistance and chemotherapy impairment. This strengthens the interest in chemopreventive agents to be exploited both in healthy and oncological subjects to prevent or repair CS damage. In the present study, we evaluated the chemopreventive properties of the natural sesquiterpene β-caryophyllene towards the damage induced by cigarette smoke condensate (CSC) in triple negative breast cancer MDA-MB-468 cells. Particularly, we assessed the ability of the sesquiterpene to interfere with the mechanisms exploited by CSC to promote cell survival and chemoresistance, including genomic instability, cell cycle progress, autophagy/apoptosis, cell migration and related pathways. β-Caryophyllene was found to be able to increase the CSC-induced death of MDA-MB-468 cells, likely triggering oxidative stress, cell cycle arrest and apoptosis; moreover, it hindered cell recovery, autophagy activation and cell migration; at last, a marked inhibition of the signal transducer and activator of transcription 3 (STAT3) activation was highlighted: this could represent a key mechanism of the chemoprevention by β-caryophyllene. Although further studies are required to confirm the in vivo efficacy of β-caryophyllene, the present results suggest a novel strategy to reduce the harmful effect of smoke in cancer patients and to improve the survival expectations in breast cancer women.
Project description:BackgroundIncreased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies.MethodsThe basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique.ResultsLow and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies.ConclusionProgressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.
Project description:Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process of effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. Results: Colony formation and sulforhodamine B (IC50 < 1nM) assays, and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI), of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset, RMI was significantly associated with tumor size or lymph node status. High (>75) percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis, RMI (P = 0.029), tumor size (P = 0.015) and lymph node status (P = 0.01) were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41). In Wang dataset, RMI predicted time to disease relapse (P = 0.09). Conclusions: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment. Mol Cancer. 2009 Sep 24;8(1):75.
Project description:Triple-negative breast cancer (TNBC) is one of the most common malignant tumor types in females and its drug resistance is a major clinical issue. An increasing number of long non-coding RNAs (lncRNAs) have been reported as key regulators of drug resistance in TNBC. Plasmacytoma variant translocation 1 (PVT1) has been proved to promote the development of various cancer types. The present study suggested that PVT1 enhances the resistance of the TNBC cell line MDA-MB-231 to doxorubicin and uncovered the molecular mechanism. PVT1 function assays and its target gene analyses were performed. We revealed that PVT1 promoted the protein stability of nuclear factor erythroid 2 like 2 (Nrf2) by inhibiting the binding of kelch-like ECH-associated protein 1 (Keap1) to Nrf2, which is beneficial to the resistance of MDA-MB-231 cells to doxorubicin. These novel results enhance the current knowledge regarding the versatile roles of PVT1 and lay a foundation for future developments of clinical applications.
Project description:HOXB7 is often overexpressed in breast cancer cells and found to relate to poor prognosis. The search for the HOXB7 targets, as a transcription factor, has led to molecules involved in regulating cell proliferation, migration, invasion, and processes such as angiogenesis and therapy resistance. However, the specific targets affected by the deregulation of HOXB7 in breast cancer remain largely unknown in most molecular sub-types, such as triple-negative breast cancers (TNBC). To unveil the molecular basis behind these aggressive and often untreatable cancers, here we explored the contribution of HOXB7 deregulation for their aggressiveness. To this end, HOXB7 was silenced in TNBC Basal A cells MDA-MB-468, and the phenotype, gene/protein expression, and methylation profile of putative targets were analyzed. Lower migration and invasion rates were detected in HOXB7-silenced cells in comparison with the controls. In addition, these cells expressed more CDH1 and less DNMT3B, and the promoter methylation status of CDH1 diminished. Our data suggest that the HOXB7 transcription factor may act on TNBC Basal A cells by controlling CDH1 epigenetic regulation. This may occur indirectly through the up-regulation of DNMT3B, which then controls DNA methylation of the CDH1 promoter. Thus, future approaches interfering with HOXB7 regulation may be promising therapeutic strategies in TNBC treatment.
Project description:The lack of hormone receptors in triple negative breast cancer (TNBC) is associated with the inefficacy of anti-estrogen chemotherapies, leaving fewer options for patient treatment and higher mortality rates. Additionally, as with numerous types of inflammatory breast cancer, infiltration of tumor associated macrophages and other leukocyte sub-populations within the tumor inevitably lead to aggressive, chemo-resistant, metastatic and invasive types of cancer which escape immune surveillance. These processes are orchestrated by the release of potent cytokines, including TNF?, IL-6 and CCL2 from the stroma, tumor and immune cells within the tumor microenvironment. The present study evaluated apigenin modulating effects on the pro-inflammatory activating action of TNF? in TNBC MDA-MB-468 cells, derived from an African American woman. Initially, cell viability was determined to establish an optimal sub-lethal dose of TNF? and apigenin in MDA-MB-468 cells. Subsequently, various treatments effects were evaluated using whole transcriptomic analysis of mRNA and long intergenic non-coding RNA with Affymetrix HuGene-2.1-st human microarrays. Gene level differential expression analysis was conducted on 48,226 genes where TNF? caused significant upregulation of 53 transcripts and downregulation of 11 transcripts. The largest upward differential shift was for CCL2 [+61.86 fold change (FC); false discovery rate (FDR), P<0.0001]; which was down regulated by apigenin (to +10.71 FC vs. Control; FDR P-value <0.001), equivalent to an 83% reduction. Several TNF? deferentially upregulated transcripts were reduced by apigenin, including CXCL10, C3, PGLYRP4, IL22RA2, KMO, IL7R, ROS1, CFB, IKBKe, SLITRK6 (a checkpoint target) and MMP13. Confirmation of CCL2 experimentally induced transcript alterations was corroborated at the protein level by ELISA assays. The high level of CCL2 transcript in the cell line was comparable to that in our previous studies in MDA-MB-231 cells. The differential effects of TNF? were corroborated by ELISA, where the data revealed a >10-fold higher releasing rate of CCL2 in MDA-MB-468 cells compared with in MDA-MB-231 cells, both of which were attenuated by apigenin. The data obtained in the present study demonstrated a high level of CCL2 in MDA-MB-468 cells and a possible therapeutic role for apigenin in downregulating TNF?-mediated processes in these TNBC cells.
Project description:The present work describes the synthesis of a new triazole based ligand 3-(3,5-dimethyl-1H-pyrazol-1-yl)-1-methyl-1H-1,2,4-triazole (LM) and demonstration of its coordination diversity giving rise to a family of seven new coordination complexes, namely: [Ni(LM)3](ClO4)2·C2H6OS (5), [Co2(LM)6](ClO4)4·(C2H5)O (6), [Cd(LM)2Cl2] (7), [Cu(LM)2NO3]NO3 (8), [Fe(LM)3](BF4)2 (9), [Zn(LM)3](BF4)2 (10) and [Zn(LM)2NO3]NO3 (11), whose crystal structure was determined by single-crystal X-ray diffraction. Cytotoxic activity was evaluated against the MDA-MB-468 cancer cell line, which serves as a model for triple-negative breast cancer, and compared to the precursor molecule (L), as well as their coordination complexes (H3O){[NiL3](ClO4)3} (1), [CoL3](ClO4)2·2H2O (2), [CdL2Cl2] (3) and [CuL3](NO3)2 (4), for which the crystal structure was earlier determined. Notably, cadmium complexes 3 and 7 exhibit remarkable cytotoxicity and demonstrated a high selectivity index towards cancer cells when compared to peripheral blood mononuclear cells. Such activity highlights their potential function as anticancer agents.