ABSTRACT
Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. Patients with relapsed disease have a poor prognosis despite intense treatment. In the present study, we aimed to identify chemoresistance gene expression signatures in vincristine resistant neuroblastoma cells. We found that vincristine-resistant neuroblastoma cells formed larger clones and survived under reduced serum conditions as compared with non-resistant parental cells. To identify the possible mechanisms underlying vincristine resistance in neuroblastoma cells, we investigated the expression profiles of genes known to be involved in cancer drug resistance. This specific gene expression patterns could predict the behavior of a tumor in response to chemotherapy and for predicting the prognosis of high-risk neuroblastoma patients. Our signature could help chemoresistant neuroblastoma patients in avoiding useless and harmful chemotherapy cycles.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Drug Resistance, Neoplasm/genetics , Neoplasm Proteins/biosynthesis , Neuroblastoma/genetics , Transcriptome , Vincristine/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Cycle/drug effects , Cell Line, Tumor , Cisplatin/pharmacology , Clone Cells , Etoposide/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Neoplasm Proteins/genetics , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Nocodazole/pharmacology , Prognosis , Vincristine/therapeutic useABSTRACT
Neuroblastoma is a childhood solid tumour originating from undifferentiated neural progenitor cells of the sympathetic nervous system. Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. In the present study, we aimed to identify novel drugs that can inhibit the growth and survival of chemoresistant neuroblastoma. High-throughput screening identified a small molecule, epi-enprioline that was able to induce apoptosis of vincristine-resistant neuroblastoma cells via the mitochondrial apoptotic pathway. Epi-enprioline reduced tumour growth in multiple preclinical models, including an orthotopic neuroblastoma patient-derived xenograft model in vivo. In summary, our data suggest that epi-enprioline can be considered as a lead compound for the treatment of vincristine-resistant neuroblastoma uncovering a novel strategy, which can be further explored as a treatment for drug-resistant neuroblastoma.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Neuroblastoma/drug therapy , Pyridines/pharmacology , Animals , Antineoplastic Agents/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Doxorubicin/pharmacology , Drug Screening Assays, Antitumor/methods , Female , High-Throughput Screening Assays/methods , Humans , Mice , Mice, Nude , Small Molecule Libraries/pharmacology , Vincristine/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.
Subject(s)
Nanoparticles/chemistry , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Animals , Chromatography, Liquid , Liposomes/chemistry , Mass Spectrometry , Mice , Particle SizeABSTRACT
BACKGROUND: Conditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation. RESULTS: To investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons. CONCLUSIONS: The finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.
Subject(s)
Gene Knock-In Techniques/methods , Genetic Techniques , Transgenes , Animals , Doxycycline , Green Fluorescent Proteins/genetics , Mice , Mice, Transgenic , TetracyclineABSTRACT
The T-box transcription factors TBX2 and TBX3 are overexpressed in many human cancers raising the need for a thorough understanding of the cellular function of these proteins. In Drosophila, there is one corresponding ortholog, Optomotor-blind (Omb). Currently, only two missense mutations are known for the two human proteins. Making use of the developmental defects caused by inactivation of omb, we have isolated and molecularly characterized four new omb mutations, three of them are missense mutations of amino acids fully conserved in all Tbx proteins. We interpret the functional defects in the framework of the known structure of the human TBX3 protein and provide evidence for loss of Omb DNA-binding activity in all three newly identified missense mutations.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Nerve Tissue Proteins/metabolism , T-Box Domain Proteins/metabolism , Amino Acid Sequence , Animals , Brachyura/genetics , Conserved Sequence , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Humans , Molecular Sequence Data , Mutation, Missense , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Protein Structure, Tertiary , T-Box Domain Proteins/chemistry , T-Box Domain Proteins/geneticsABSTRACT
Neuroblastoma is the most common paediatric cancer type. Patients diagnosed with high-risk neuroblastoma have poor prognosis and occasionally tumours relapse. As a result, novel treatment strategies are needed for relapse and refractory neuroblastoma patients. Here, we found that high expression of Mps1 kinase (mitotic kinase Monopolar Spindle 1) was associated with relapse-free neuroblastoma patient outcomes and poor overall survival. Silencing and inhibition of Mps1 in neuroblastoma or PDX-derived cells promoted cell apoptosis via the caspase-dependent mitochondrial apoptotic pathway. The mechanism of cell death upon Mps1 inhibition was dependent on the polyploidization/aneuploidization of the cells before undergoing mitotic catastrophe. Furthermore, tumour growth retardation was confirmed in a xenograft mouse model after Mps1-inhibitor treatment. Altogether, these results suggest that Mps1 expression and inhibition can be considered as a novel prognostic marker as well as a therapeutic strategy for the treatment of high-risk neuroblastoma patients.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Mitosis , Neuroblastoma/enzymology , Neuroblastoma/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Biomarkers, Tumor/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Humans , Mice , Mitochondria/metabolism , Polyploidy , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Colorectal cancer is one of the most commonly diagnosed cancers and the third most common cause of cancer-related death. Metastasis is the leading reason for the resultant mortality of these patients. Accordingly, development and characterization of novel anti-cancer drugs limiting colorectal tumor cell dissemination and metastasis are needed. In this study, we found that the small molecule Reversine reduces the migration potential of human colon carcinoma cells in vitro. A coupled kinase assay with bio-informatics approach identified the c-Jun N-terminal kinase (JNK) cascade as the main pathway inhibited by Reversine. Knockdown experiments and pharmacological inhibition identified JNK1 but not JNK2, as a downstream effector target in cancer cell migration. Xenograft experiments confirm the effect of JNK inhibition in the metastatic potential of colon cancer cells. These results highlight the impact of individual JNK isoforms in cancer cell metastasis and propose Reversine as a novel anti-cancer molecule for treatment of colon cancer patients.
Subject(s)
Cell Movement/drug effects , Colonic Neoplasms/drug therapy , Mitogen-Activated Protein Kinase 8/antagonists & inhibitors , Molecular Targeted Therapy/methods , Morpholines/pharmacology , Purines/pharmacology , Xenograft Model Antitumor Assays/methods , Animals , Anthracenes/pharmacology , Cell Line , Cell Line, Tumor , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Humans , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mitogen-Activated Protein Kinase 8/metabolism , Tumor Burden/drug effectsABSTRACT
BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinating enzyme that is associated with multiprotein complexes that regulate key cellular pathways, including cell cycle, cellular differentiation, cell death, and the DNA damage response. In this study, we found that the reduced expression of BAP1 pro6motes the survival of neuroblastoma cells, and restoring the levels of BAP1 in these cells facilitated a delay in S and G2/M phase of the cell cycle, as well as cell apoptosis. The mechanism that BAP1 induces cell death is mediated via an interaction with 14-3-3 protein. The association between BAP1 and 14-3-3 protein releases the apoptotic inducer protein Bax from 14-3-3 and promotes cell death through the intrinsic apoptosis pathway. Xenograft studies confirmed that the expression of BAP1 reduces tumor growth and progression in vivo by lowering the levels of pro-survival factors such as Bcl-2, which in turn diminish the survival potential of the tumor cells. Patient data analyses confirmed the finding that the high-BAP1 mRNA expression correlates with a better clinical outcome. In summary, our study uncovers a new mechanism for BAP1 in the regulation of cell apoptosis in neuroblastoma cells.
Subject(s)
14-3-3 Proteins/metabolism , Apoptosis , Cell Cycle , Gene Expression Regulation, Neoplastic , Neuroblastoma/metabolism , Tumor Suppressor Proteins/biosynthesis , Ubiquitin Thiolesterase/biosynthesis , 14-3-3 Proteins/genetics , Animals , Cell Line, Tumor , Female , Humans , Mice , Mice, Nude , Neuroblastoma/genetics , Neuroblastoma/pathology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Tumors are phenotypically heterogeneous and include subpopulations of cancer cells with stemlike properties. The natural product salinomycin, a K+-selective ionophore, was recently found to exert selectivity against such cancer stem cells. This selective effect is thought to be due to inhibition of the Wnt signaling pathway, but the mechanistic basis remains unclear. Here, we develop a functionally competent fluorescent conjugate of salinomycin to investigate the molecular mechanism of this compound. By subcellular imaging, we demonstrate a rapid cellular uptake of the conjugate and accumulation in the endoplasmic reticulum (ER). This localization is connected to induction of Ca2+ release from the ER into the cytosol. Depletion of Ca2+ from the ER induces the unfolded protein response as shown by global mRNA analysis and Western blot analysis of proteins in the pathway. In particular, salinomycin-induced ER Ca2+ depletion up-regulates C/EBP homologous protein (CHOP), which inhibits Wnt signaling by down-regulating ß-catenin. The increased cytosolic Ca2+ also activates protein kinase C, which has been shown to inhibit Wnt signaling. These results reveal that salinomycin acts in the ER membrane of breast cancer cells to cause enhanced Ca2+ release into the cytosol, presumably by mediating a counter-flux of K+ ions. The clarified mechanistic picture highlights the importance of ion fluxes in the ER as an entry to inducing phenotypic effects and should facilitate rational development of cancer treatments.
ABSTRACT
AIM: We wanted to assess the potency of a trifunctional nanoparticle (NP) that targeted and activated CD8+ dendritic cells (DC) and delivered an antigen to induce antitumor responses. MATERIALS & METHODS: The DC targeting and activating properties of ferrous NPs conjugated with immunostimulatory CpG-oligonucleotides, anti-DEC205 antibody and ovalbumin (OVA) as a model antigen to induce antigen-specific T-cell responses and antitumor responses were analyzed. RESULTS: OVA-loaded NP conjugated with immunostimulatory CpG-oligonucleotides and anti-DEC205 antibody efficiently targeted and activated CD8+ DC in vivo, and induced strong OVA-specific T-cell activation. Vaccination of B16/OVA tumor-burdened mice with this NP formulation resulted in tumor growth arrest. CONCLUSION: CD8+ DC-targeting trifunctional nanocarriers bear significant potential for antitumor immunotherapy.
Subject(s)
CD8 Antigens/metabolism , Dendritic Cells/immunology , Magnetite Nanoparticles/chemistry , Melanoma, Experimental/therapy , Oligonucleotides/immunology , Ovalbumin/immunology , Animals , Antibodies/chemistry , Antibodies/immunology , Antigens, CD/immunology , Cell Proliferation , CpG Islands , Dendritic Cells/metabolism , Dextrans/chemistry , Fluorescent Dyes/chemistry , Humans , Immunotherapy , Lectins, C-Type/immunology , Lymphocyte Activation , Magnetite Nanoparticles/therapeutic use , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice, Inbred C57BL , Minor Histocompatibility Antigens/immunology , Oligonucleotides/chemistry , Receptors, Cell Surface/immunology , Surface Properties , Tumor Burden , VaccinationABSTRACT
Reporter gene activity in enhancer trap lines is often implicitly assumed to mirror quite faithfully the endogenous expression of the "trapped" gene, even though there are numerous examples of enhancer trap infidelity. optomotor-blind (omb) is a 160 kb gene in which 16 independent P-element enhancer trap insertions of three different types have been mapped in a range of more than 60 kb. We have determined the expression pattern of these elements in wing, eye-antennal and leg imaginal discs as well as in the pupal tergites. We noted that one pGawB insertion (omb (P4) ) selectively failed to report parts of the omb pattern even though the missing pattern elements were apparent in all other 15 lines. We ruled out that omb (P4) was defective in the Gal4 promoter region or had inactivated genomic enhancers in the integration process. We propose that the Gal4 reporter gene in pGawB may be sensitive to orientation or promoter proximity effects.
Subject(s)
Drosophila Proteins/genetics , Drosophila/genetics , Nerve Tissue Proteins/genetics , T-Box Domain Proteins/genetics , Animals , Arthropod Antennae/metabolism , Chromosome Mapping , Drosophila/metabolism , Drosophila Proteins/metabolism , Enhancer Elements, Genetic , Extremities , Eye/metabolism , Genes, Reporter , Imaginal Discs/metabolism , Mutagenesis, Insertional/methods , Nerve Tissue Proteins/metabolism , Promoter Regions, Genetic , Pupa/metabolism , T-Box Domain Proteins/metabolism , Wings, Animal/metabolismABSTRACT
The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.