ABSTRACT
Bone and bone marrow are among the most frequent metastatic sites of cancer. The occurrence of bone metastasis is frequently associated with a dismal disease outcome. The prevention and therapy of bone metastases is a priority in the treatment of cancer patients. However, current therapeutic options for patients with bone metastatic disease are limited in efficacy and associated with increased morbidity. Therefore, most current therapies are mainly palliative in nature. A better understanding of the underlying molecular pathways of the bone metastatic process is warranted to develop novel, well-tolerated and more successful treatments for a significant improvement of patients' quality of life and disease outcome. In this review, we provide comparative mechanistic insights into the bone metastatic process of various solid tumors, including pediatric cancers. We also highlight current and innovative approaches to biologically targeted therapy and immunotherapy. In particular, we discuss the role of the bone marrow microenvironment in the attraction, homing, dormancy and outgrowth of metastatic tumor cells and the ensuing therapeutic implications. Multiple signaling pathways have been described to contribute to metastatic spread to the bone of specific cancer entities, with most knowledge derived from the study of breast and prostate cancer. However, it is likely that similar mechanisms are involved in different types of cancer, including multiple myeloma, primary bone sarcomas and neuroblastoma. The metastatic rate-limiting interaction of tumor cells with the various cellular and noncellular components of the bone-marrow niche provides attractive therapeutic targets, which are already partially exploited by novel promising immunotherapies.
Subject(s)
Bone Neoplasms/diagnosis , Bone Neoplasms/secondary , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bone Neoplasms/therapy , Epithelial-Mesenchymal Transition , Humans , Immunotherapy , Models, Biological , Tumor MicroenvironmentABSTRACT
Recent genome analyses have identified recurrent mutations in the cohesin complex in a wide range of human cancers. Here we demonstrate that the most frequently mutated subunit of the cohesin complex, STAG2, displays a strong synthetic lethal interaction with its paralog STAG1. Mechanistically, STAG1 loss abrogates sister chromatid cohesion in STAG2 mutated but not in wild-type cells leading to mitotic catastrophe, defective cell division and apoptosis. STAG1 inactivation inhibits the proliferation of STAG2 mutated but not wild-type bladder cancer and Ewing sarcoma cell lines. Restoration of STAG2 expression in a mutated bladder cancer model alleviates the dependency on STAG1. Thus, STAG1 and STAG2 support sister chromatid cohesion to redundantly ensure cell survival. STAG1 represents a vulnerability of cancer cells carrying mutations in the major emerging tumor suppressor STAG2 across different cancer contexts. Exploiting synthetic lethal interactions to target recurrent cohesin mutations in cancer, e.g. by inhibiting STAG1, holds the promise for the development of selective therapeutics.
Subject(s)
Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Synthetic Lethal Mutations , Cell Cycle Proteins , Cell Division , Cell Line, Tumor , Cell Survival , HumansABSTRACT
Ewing sarcoma (EwS) is the second most common bone cancer in children and adolescents with a high metastatic potential. EwS development is driven by a specific chromosomal translocation resulting in the generation of a chimeric EWS-ETS transcription factor, most frequently EWS-FLI1.Nicotinamide adenine dinucleotide (NAD) is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. This study describes targeting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD synthesis, by FK866 in EwS cells. Here we report that blocking NAMPT leads to exhaustive NAD depletion in EwS cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shRNA revealed that EWS-FLI1 depletion significantly reduces the sensitivity of EwS cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 EwS cell lines of different genotypes with 5 Non-EwS cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive EwS cells, with IC50 values mostly below 1nM.Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of EwS cells and suggest NAMPT inhibition as a potential new treatment approach for Ewing sarcoma.
Subject(s)
Acrylamides/pharmacology , Cytokines/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Oncogene Proteins, Fusion/metabolism , Piperidines/pharmacology , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS/metabolism , Bone Neoplasms/drug therapy , Bone Neoplasms/enzymology , Bone Neoplasms/metabolism , Cell Line, Tumor , Cytokines/metabolism , Drug Resistance, Neoplasm , HeLa Cells , Humans , NAD/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/enzymology , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathologyABSTRACT
A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.
Subject(s)
Bone Neoplasms/genetics , Microtubule Proteins/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/genetics , Bone Neoplasms/mortality , Bone Neoplasms/pathology , Cell Cycle/genetics , Cell Line, Tumor , Cell Survival/genetics , Doxycycline/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , High-Throughput Nucleotide Sequencing , Humans , Kaplan-Meier Estimate , Male , Microtubule Proteins/metabolism , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , RNA Interference , RNA-Binding Protein EWS/metabolism , Sarcoma, Ewing/mortality , Sarcoma, Ewing/pathology , WD40 RepeatsABSTRACT
The developmental receptor NOTCH plays an important role in various human cancers as a consequence of oncogenic mutations. Here we describe a novel mechanism of NOTCH-induced tumor suppression involving modulation of the deacetylase SIRT1, providing a rationale for the use of SIRT1 inhibitors to treat cancers where this mechanism is inactivated because of SIRT1 overexpression. In Ewing sarcoma cells, NOTCH signaling is abrogated by the driver oncogene EWS-FLI1. Restoration of NOTCH signaling caused growth arrest due to activation of the NOTCH effector HEY1, directly suppressing SIRT1 and thereby activating p53. This mechanism of tumor suppression was validated in Ewing sarcoma cells, B-cell tumors, and human keratinocytes where NOTCH dysregulation has been implicated pathogenically. Notably, the SIRT1/2 inhibitor Tenovin-6 killed Ewing sarcoma cells in vitro and prohibited tumor growth and spread in an established xenograft model in zebrafish. Using immunohistochemistry to analyze primary tissue specimens, we found that high SIRT1 expression was associated with Ewing sarcoma metastasis and poor prognosis. Our findings suggest a mechanistic rationale for the use of SIRT1 inhibitors being developed to treat metastatic disease in patients with Ewing sarcoma.
Subject(s)
Bone Neoplasms/drug therapy , Receptors, Notch/physiology , Sarcoma, Ewing/drug therapy , Sirtuin 1/physiology , Animals , Apoptosis , Basic Helix-Loop-Helix Transcription Factors/physiology , Bone Neoplasms/pathology , Cell Line, Tumor , Humans , Neoplasm Metastasis , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Protein c-fli-1/physiology , RNA-Binding Protein EWS/physiology , Repressor Proteins/physiology , Sarcoma, Ewing/pathology , Signal Transduction , Sirtuin 1/analysis , Sirtuin 1/antagonists & inhibitors , Tumor Suppressor Protein p53/physiology , ZebrafishABSTRACT
The European Network for Cancer Research in Children and Adolescents (ENCCA) provides an interaction platform for stakeholders in research and care of children with cancer. Among ENCCA objectives is the establishment of biology-based prioritization mechanisms for the selection of innovative targets, drugs, and prognostic markers for validation in clinical trials. Specifically for sarcomas, there is a burning need for novel treatment options, since current chemotherapeutic treatment protocols have met their limits. This is most obvious for metastatic Ewing sarcoma (ES), where long term survival rates are still below 20%. Despite significant progress in our understanding of ES biology, clinical translation of promising laboratory results has not yet taken place due to fragmentation of research and lack of an institutionalized discussion forum. To fill this gap, ENCCA assembled 30 European expert scientists and five North American opinion leaders in December 2011 to exchange thoughts and discuss the state of the art in ES research and latest results from the bench, and to propose biological studies and novel promising therapeutics for the upcoming European EWING2008 and EWING2012 clinical trials.
ABSTRACT
Notch can act as an oncogene or as a tumour suppressor and thus can either promote or inhibit tumour cell growth. To establish Notch status in Ewing's sarcoma family of tumours (ESFT), we investigated the Notch pathway by gene expression profiling meta-analysis or immunohistochemistry in samples obtained from 96 and 24 ESFT patients, respectively. We found that although Notch receptors were highly expressed, Notch did not appear to be active, as evidenced by the absence of Notch receptors in cell nuclei. In contrast, we show that Notch receptors known to be active in colon adenocarcinoma, hepatocarcinoma, and pancreatic carcinoma stain cell nuclei in these tumours. High expression of the Notch effector HES1 transcription factor, usually used as a surrogate marker for active Notch, was also restricted to outside of the nucleus in the majority of ESFT, and analysis of HES1 gene targets indicated HES1 to be transcriptionally inactive. Neither forced activation nor pharmacological or genetic blocking of Notch affected HES1 expression in ESFT cells, indicating HES1 expression to be uncoupled from the Notch pathway. Additional functional studies in ESFT cell lines confirmed Notch to be switched off. Finally, unlike experiments in which HES1 expression was modulated, experimental activation of Notch in ESFT cell lines via several means blocked cell proliferation and reduced their clonogenic potential in soft agar. These indicate that HES1 is uncoupled from Notch in ESFT, that EWS-FLI1-mediated inhibition of Notch contributes to ESFT aggressive cell growth, and support a role for Notch in ESFT tumour suppression, at least partly through the Notch effector HEY1.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Bone Neoplasms/metabolism , Homeodomain Proteins/metabolism , Receptors, Notch/metabolism , Sarcoma, Ewing/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Cell Nucleus/metabolism , Cell Proliferation , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Humans , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/pathology , Receptors, Notch/genetics , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathology , Signal Transduction/physiology , Transcription Factor HES-1 , Tumor Cells, CulturedABSTRACT
PURPOSE: We report a novel analytic method, named intercohort co-analysis or Ican, which aids in the discovery of genes with predictive value for the progression or outcome of diseases from small-size cohorts. We tested this premise in Ewing's sarcoma (ES), a highly metastatic cancer of bone and soft tissues that lacks validated molecular metastasis and prognostic indicators. EXPERIMENTAL DESIGN: To uncover genes significantly expressed in ES patient subsets, we first determined a nonarbitrary gene expression significance cutoff based on expression levels in validated expressing and nonexpressing tissues. We next searched for genes that were consistently significantly expressed in several ES cohort and cell line datasets. Significantly expressed genes were independently validated by quantitative reverse transcription-PCR in an additional ES cohort. RESULTS: Analysis of ES cohorts revealed marked intercohort gene expression variability. After filtering out the intercohort variability, CXCR4 and CXCR7 were found to be consistently associated with specific ES subsets. Pairwise analyses showed CXCR4 to correlate with ES metastases, and CXCR4 and CXCR7 to patient survival, but not with several other clinicopathological variables. CONCLUSION: Ican is a powerful novel method to identifying genes consistently associated with particular disease states in cancers for which large cohorts are not available, currently the case of most cancers. We report for the first time that high CXCR4 expression preferentially associates with metastatic ES, and that of CXCR7 with poor patient survival.
Subject(s)
Bone Neoplasms/diagnosis , Bone Neoplasms/genetics , Gene Expression Profiling/methods , Receptors, CXCR4/genetics , Receptors, CXCR/genetics , Sarcoma, Ewing/diagnosis , Sarcoma, Ewing/genetics , Bone Neoplasms/pathology , Cohort Studies , Humans , Phenotype , Prognosis , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Ewing/pathology , Tumor Cells, CulturedABSTRACT
Hypoxia is an important condition in the tumor cell microenvironment and approximately 1% to 1.5% of the genome is transcriptionally responsive to hypoxia with hypoxia-inducible factor-1 (HIF-1) as a major mediator of transcriptional activation. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. Because, by immunohistochemistry, HIF-1alpha expression was readily detectable in 18 of 28 primary Ewing's sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, we studied the effect of hypoxia on ESFT cell lines in vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is upregulated by hypoxia in a HIF-1alpha-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis-related genes, hypoxia stimulated the invasiveness and soft agar colony formation of ESFT cells in vitro. Our data represent the first transcriptome analysis of hypoxic ESFT cells and identify hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity with far-reaching consequences for the malignant properties of ESFT.
Subject(s)
Biomarkers, Tumor/genetics , Bone Neoplasms/genetics , Gene Expression Regulation, Neoplastic/physiology , Hypoxia/metabolism , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , Sarcoma, Ewing/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Profiling , Gene Regulatory Networks , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunoenzyme Techniques , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Protein EWS , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Ewing/pathology , Transcriptional ActivationABSTRACT
BACKGROUND: EWS-FLI1 is a chimeric ETS transcription factor that is, due to a chromosomal rearrangement, specifically expressed in Ewing's sarcoma family tumors (ESFT) and is thought to initiate the development of the disease. Previous genomic profiling experiments have identified EWS-FLI1-regulated genes and genes that discriminate ESFT from other sarcomas, but so far a comprehensive analysis of EWS-FLI1-dependent molecular functions characterizing this aggressive cancer is lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a molecular function map of ESFT was constructed based on an integrative analysis of gene expression profiling experiments following EWS-FLI1 knockdown in a panel of five ESFT cell lines, and on gene expression data from the same platform of 59 primary ESFT. Out of 80 normal tissues tested, mesenchymal progenitor cells (MPC) were found to fit the hypothesis that EWS-FLI1 is the driving transcriptional force in ESFT best and were therefore used as the reference tissue for the construction of the molecular function map. The interrelations of molecular pathways were visualized by measuring the similarity among annotated gene functions by gene sharing. The molecular function map highlighted distinct clusters of activities for EWS-FLI1 regulated genes in ESFT and revealed a striking difference between EWS-FLI1 up- and down-regulated genes: EWS-FLI1 induced genes mainly belong to cell cycle regulation, proliferation, and response to DNA damage, while repressed genes were associated with differentiation and cell communication. CONCLUSIONS/SIGNIFICANCE: This study revealed that EWS-FLI1 combines by distinct molecular mechanisms two important functions of cellular transformation in one protein, growth promotion and differentiation blockage. By taking MPC as a reference tissue, a significant EWS-FLI1 signature was discovered in ESFT that only partially overlapped with previously published EWS-FLI1-dependent gene expression patterns, identifying a series of novel targets for the chimeric protein in ESFT. Our results may guide target selection for future ESFT specific therapies.
Subject(s)
Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Protein c-fli-1/physiology , Sarcoma, Ewing/genetics , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/genetics , DNA Damage/genetics , Gene Expression Profiling , Humans , Mesenchymal Stem Cells , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS , Sarcoma, Ewing/etiology , Sarcoma, Ewing/pathology , Transcription, GeneticABSTRACT
Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21(WAF1/CIP1) levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21(WAF1/CIP1) induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21(WAF1/CIP1). These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53.
Subject(s)
Genes, p53 , Oncogene Proteins, Fusion/physiology , Receptors, Notch/physiology , Sarcoma, Ewing/metabolism , Transcription Factors/physiology , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , Blotting, Western , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Primers , Fluorescent Antibody Technique , Gene Silencing , Humans , Oligonucleotide Array Sequence Analysis , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1 , RNA-Binding Protein EWS , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/geneticsABSTRACT
Caspase-14, a protease involved in skin barrier formation, is specifically expressed in epidermal keratinocytes (KCs). Here, we mapped three start sites of transcription of the human caspase-14 gene and analyzed the upstream chromosomal region for promoter activity. Reporter gene assays identified a core promoter region proximal to the first exon and a distal regulatory region which differentially suppressed promoter activity in KC and other cells. Sequence elements in the proximal promoter were bound by the transcription factors AP-1 (JunB, c-Jun, JunD, Fra-1 and Fra-2) and NFkappaB (p50 and RelB). Our data reveal the basic organization of the human caspase-14 promoter and suggest an important role of AP-1 and NFkappaB in the transcriptional control of caspase-14.
Subject(s)
Caspase 14/genetics , Epidermis/enzymology , Gene Expression Regulation , Keratinocytes/enzymology , NF-kappa B/metabolism , Transcription Factor AP-1/metabolism , Base Sequence , Binding Sites , Cells, Cultured , Cloning, Molecular , Electrophoretic Mobility Shift Assay , Epidermal Cells , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
The TEL/AML1 fusion gene results from the most frequent t(12;21)(p13;q22) translocation in childhood acute lymphoblastic leukemia (ALL). Its contribution to transformation is largely unknown, in particular with respect to survival and apoptosis. We therefore silenced TEL/AML1 expression in leukemic REH cells by RNA inhibition, which eventually led to programmed cell death. Microarray and 2D gel electrophoresis data demonstrated a differential regulation of heat-shock proteins (HSPs), among them HSP90, as well as of its client, survivin. Consistent with these findings, ectopic expression of TEL/AML1 in Ba/F3 cells increased protein levels of HSP90 and survivin and conferred resistance to apoptotic stimuli. Our data suggest that TEL/AML1 not only contributes to leukemogenesis by affecting an antiapoptotic network but also seems to be indispensable for maintaining the malignant phenotype. The functional relationship between TEL/AML1, HSP90, and survivin provides the rational for targeted therapy, be it the fusion gene or the latter 2 proteins.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Heat-Shock Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , RNA Interference , Base Sequence , Cell Line, Tumor , Cell Survival , Gene Expression Regulation, Neoplastic , Humans , Inhibitor of Apoptosis Proteins , SurvivinABSTRACT
Ewing's sarcoma family of tumors (ESFT) are a clinically and scientifically very demanding group of tumors in children and young adults with still unknown histogenesis. The rate-limiting oncogenic mutation in this disease has been identified as a chromosomal translocation, t(11;22)(q24;q12), that leads to the expression of a chimeric transcription factor, EWS-FLI1. We have studied the downstream pathway of EWS-FLI1 by a dual strategy including the isolation of direct target genes from ESFT chromatin and the monitoring of transcriptomic changes after silencing of EWS-FLI1 by RNA interference. This study has lead to the identification of several directly EWS-FLI1-regulated genes and the characterization of their genomic distribution. By comparing several ESFT cell lines, not only variation in overall gene expression patterns downstream of EWS-FLIl was observed, but also differential regulation of directly EWS-FLI1-bound genes. Interestingly, there was variation between members of the same functional gene families. Studies on CD99, another diagnostic hallmark of ESFT, in relation to EWS-FLI1 provided additional evidence for context dependence of fusion protein function. Together, our study represents a first approach to the separation of essential molecular consequences from noise generated by the EWS-FLI1 gene rearrangement in ESFT.
Subject(s)
Bone Neoplasms/physiopathology , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/physiology , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/physiopathology , Bone Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Humans , Oncogene Proteins, Fusion/genetics , Sarcoma, Ewing/genetics , Translocation, GeneticABSTRACT
The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewing's sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH(2) terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4- and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies.
Subject(s)
Immunoglobulin Fragments/immunology , Oncogene Proteins, Fusion/immunology , Proto-Oncogene Protein c-fli-1/immunology , Sarcoma, Ewing/genetics , Sarcoma, Ewing/immunology , Amino Acid Sequence , Antibody Specificity , Carcinoma, Hepatocellular , Cell Line, Tumor , Epitopes/immunology , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/metabolism , Liver Neoplasms , Molecular Sequence Data , Neuroblastoma , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Protein Binding , Protein Folding , Proto-Oncogene Protein c-fli-1/chemistry , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS , Transcriptional Activation , TransfectionABSTRACT
Tumors of the Ewing's sarcoma family (ESFT), such as Ewing's sarcoma (EWS) and primitive neuroectodermal tumors (PNET), are highly aggressive malignancies predominantly affecting children and young adults. ESFT express chimeric transcription factors encoded by hybrid genes fusing the EWS gene with several ETS genes, most commonly FLI-1. EWS/FLI-1 proteins are responsible for the malignant phenotype of ESFT, but only few of their transcriptional targets are known. Using antisense and short hairpin RNA-mediated gene expression knockdown, array analyses, chromatin immunoprecipitation methods, and reexpression studies, we show that caveolin-1 (CAV1) is a new direct target of EWS/FLI-1 that is overexpressed in ESFT cell lines and tumor specimens and is necessary for ESFT tumorigenesis. CAV1 knockdown led to up-regulation of Snail and the concomitant loss of E-cadherin expression. Consistently, loss of CAV1 expression inhibited the anchorage-independent growth of EWS cells and markedly reduced the growth of EWS cell-derived tumors in nude mice xenografts, indicating that CAV1 promotes the malignant phenotype in EWS carcinogenesis. Reexpression of CAV1 or E-cadherin in CAV1 knockdown EWS cells rescued the oncogenic phenotype of the original EWS cells, showing that the CAV1/Snail/E-cadherin pathway plays a central role in the expression of the oncogenic transformation functions of EWS/FLI-1. Overall, these data identify CAV1 as a key determinant of the tumorigenicity of ESFT and imply that targeting CAV1 may allow the development of new molecular therapeutic strategies for ESFT patients.
Subject(s)
Caveolin 1/physiology , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Protein c-fli-1/physiology , Sarcoma, Ewing/pathology , Animals , Cadherins/biosynthesis , Cadherins/genetics , Caveolin 1/antagonists & inhibitors , Caveolin 1/genetics , Caveolin 1/metabolism , Cell Adhesion/genetics , Cell Growth Processes/genetics , Cell Line, Tumor , Chromatin Immunoprecipitation , Down-Regulation , Gene Expression Regulation, Neoplastic , HT29 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , RNA, Antisense/genetics , RNA-Binding Protein EWS , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Snail Family Transcription Factors , Transcription Factors/biosynthesis , Transcription Factors/genetics , Up-RegulationABSTRACT
In all, 85% of Ewing's sarcoma family tumors (ESFT), a neoplasm of unknown histogenesis, express EWS-FLI1 transcription factor gene fusions. To characterize direct target genes avoiding artificial model systems, we cloned genomic DNA from ESFT chromatin precipitating with EWS-FLI1. We now present a comprehensive list of 99 putative transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction. Gene-derived chromatin fragments co-precipitating with EWS-FLI1 were nonrandomly distributed over the human genome and localized predominantly to the upstream region and the first two introns of the genes. At least 20% of putative direct EWS-FLI1 targets were neural genes. One-third of genes recovered showed a significant ESFT-specific expression pattern and were found to be altered upon RNAi-mediated knockdown of EWS-FLI1. Among them, MK-STYX, encoding a MAP kinase phosphatase-like protein, was consistently expressed in ESFT. EWS-FLI1 was found to drive MK-STYX expression by binding to a single ETS binding motif within the first gene intron. MK-STYX serves as precedence for successful recovery of direct EWS-FLI1 targets from the authentic ESFT cellular context, the most relevant system to study oncogenic mechanisms for the discovery of new therapeutic targets in this disease.
Subject(s)
Bone Neoplasms/genetics , Chromatin/genetics , DNA-Binding Proteins/genetics , Protein Tyrosine Phosphatases/pharmacology , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/genetics , Trans-Activators/genetics , DNA, Neoplasm/analysis , Gene Expression Profiling , Humans , Immunoprecipitation , Oligonucleotide Array Sequence Analysis , Protein Tyrosine Phosphatases/genetics , Proto-Oncogene Protein c-fli-1ABSTRACT
Azole antifungals are widely used to treat infections with dermatophyte fungi. Whereas it is well established that this class of drugs interferes with fungal ergosterol synthesis, little is known about its potential other biological effects. Here we report the isolation and structural organization of Microsporum canis metallothionein gene and demonstrate that fluconazole is able to downregulate the baseline as well as copper-induced expression of this gene. Since this effect occurred within 30 min after exposure of the fungus to fluconazole, it is unlikely that it is due to impaired ergosterol synthesis. Our additional demonstration that fluconazole enhances copper toxicity for M. canis suggests that inhibition of metallothionein expression by fluconazole is biologically relevant and may represent an important additional mode of the antifungal action of this drug. Therefore our data indicate that antifungal effects of azole derivatives might not only be due to interference with cell wall synthesis but may also affect other biological circuits within the fungal cells.
Subject(s)
Antifungal Agents/pharmacology , Copper/toxicity , Fluconazole/pharmacology , Gene Expression Regulation, Fungal/drug effects , Metallothionein/metabolism , Microsporum/drug effects , Amino Acid Sequence , Base Sequence , Cell Division/drug effects , Dose-Response Relationship, Drug , Down-Regulation , Drug Synergism , Metallothionein/genetics , Microsporum/genetics , Microsporum/metabolism , Molecular Sequence Data , RNA, Messenger/biosynthesis , Sequence AlignmentABSTRACT
Caspase-14 is the only member of the caspase family that shows a restricted tissue expression. It is mainly confined to epidermal keratinocytes and in contrast to other caspases, is not activated during apoptosis induced by ultraviolet irradiation or cytotoxic substances. As it is cleaved under conditions leading to terminal differentiation of keratinocytes we suggested that caspase-14 plays a part in the physiologic cell death of keratinocytes leading to skin barrier formation. Here we show that retinoic acid, at concentrations inhibiting terminal differentiation of keratinocytes, strongly suppressed caspase-14 mRNA and protein expression by keratinocytes in monolayer culture and in a three-dimensional in vitro model of differentiating human epidermis (skin equivalent). By contrast, the expression of the caspases 3 and 8, which are both activated during conventional apoptosis, was increased and unchanged, respectively, after retinoic acid treatment. In addition to inhibition of differentiation in skin equivalents, retinoic acid treatment led to keratinocyte apoptosis and activation of caspase-3, both of which were undetectable in differentiated control skin equivalents. As this occurred in the absence of detectable caspase-14, our data demonstrate that caspase-14 is dispensable for keratinocyte apoptosis. The fact that in contrast to caspase-3 and caspase-8, caspase-14, similarly to other keratinocyte differentiation-associated proteins, is downregulated by retinoids, strongly suggests that this caspase, but not caspase-3 and -8, plays a part in terminal keratinocyte differentiation and skin barrier formation.