ABSTRACT
In Ewing's sarcoma family of tumours (ESFT), the clinically most adverse prognostic parameters are the presence of tumour metastasis at time of diagnosis and poor response to neoadjuvant chemotherapy. To identify genes differentially regulated between metastatic and localised tumours, we analysed 27 ESFT specimens using Affymetrix microarrays. Functional annotation of differentially regulated genes revealed 29 over-represented pathways including PDGF, TP53, NOTCH, and WNT1-signalling. Regression of primary tumours (n=20) induced by polychemotherapy was found to be correlated with the expression of genes involved in angiogenesis, apoptosis, ubiquitin proteasome pathway, and PI3 kinase and p53 pathways. These findings could be confirmed by in vitro cytotoxicity assays. A set of 46 marker genes correctly classifies these 20 tumours as responding versus non-responding. We conclude that expression signatures of initial tumour biopsies can help to identify ESFT patients at high risk to develop tumour metastasis or to suffer from a therapy refractory cancer.
Subject(s)
Bone Neoplasms/genetics , Gene Expression Profiling/methods , Microarray Analysis/methods , Sarcoma, Ewing/genetics , Adolescent , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bone Neoplasms/drug therapy , Cell Communication/genetics , Cell Line, Tumor , Child , Child, Preschool , Drug Resistance, Neoplasm/genetics , Female , Genes, p53 , Humans , Male , Mutation, Missense/genetics , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/secondaryABSTRACT
Clear cell sarcoma of soft tissue (CCSST) represents a highly malignant tumor of the musculoskeletal system that is characterized by the chromosomal translocation t(12;22)(q13;q12) of the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1). In a former microarray expression study, we identified ERBB3, a member of the epidermal growth factor receptor (EGFR) family, as a promising new diagnostic marker in the differential diagnosis of CCSST. Here we show that, besides ErbB3, all CCSST cell lines (n = 8) also express the ErbB2 receptor or the ErbB4 receptor, representing an adequate coreceptor of ErbB3. The phosphorylation status of ErbB3 revealed these receptor pairs to be either constitutively activated in CCSST cells with high neuregulin-1 (NRG1) expression (n = 4) or activatable by exogenic NRG1 in cells showing low amounts of NRG1 mRNA (n = 4). Exogenous NRG1 stimulated the growth of a subset of CCSST cells but did not affect the kinetics of another subset. This difference was not strictly dependent on endogenous NRG1 expression; however, the growth-inhibiting effect of the pan-ErbB tyrosine kinase inhibitor CI-1033 or PD158780 clearly correlated with NRG1 expression indicating an autocrine growth stimulation loop which may constitute an interesting target of new therapeutic strategies in this tumor entity.
Subject(s)
Gene Expression Regulation, Neoplastic , Neuregulin-1/metabolism , Receptor, ErbB-3/metabolism , Sarcoma, Clear Cell/metabolism , Signal Transduction , Soft Tissue Neoplasms/metabolism , Alleles , Cell Line, Tumor , Humans , Kinetics , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Oligonucleotides/chemistry , Phosphorylation , Pyrimidines/pharmacologyABSTRACT
Clear cell sarcoma of soft tissue (CCSST), also known as malignant melanoma of soft parts, represents a rare lesion of the musculoskeletal system usually affecting adolescents and young adults. CCSST is typified by a chromosomal t(12;22)(q13;q12) translocation resulting in a fusion between the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1), of which the activity in nontransformed cells is regulated by cyclic AMP. Our aim was to identify critical differentially expressed genes in CCSST tumor cells in comparison with other solid tumors affecting children and young adults to better understand signaling pathways regulating specific features of the development and progression of this tumor entity. We applied Affymetrix Human Genome U95Av2 oligonucleotide microarrays representing approximately 12,000 genes to generate the expression profiles of the CCSST cell lines GG-62, DTC-1, KAO, MST2, MST3, and Su-CC-S1 in comparison with 8 neuroblastoma, 7 Ewing tumor, and 6 osteosarcoma cell lines. Subsequent hierarchical clustering of microarray data clearly separated all four of the tumor types from each other and identified differentially expressed transcripts, which are characteristically up-regulated in CCSST. Statistical analysis revealed a group of 331 probe sets, representing approximately 300 significant (P < 0.001) differentially regulated genes, which clearly discriminated between the CCSST and other tumor samples. Besides genes that were already known to be highly expressed in CCSST, like S100A11 (S100 protein) or MITF (microphthalmia-associated transcription factor), this group shows an obvious portion of genes that are involved in cyclic AMP response or regulation, in pigmentation processes, or in neuronal structure and signaling. Comparison with other expression profile analyses on neuroectodermal childhood tumors confirms the high robustness of this strategy to characterize tumor entities based on their gene expression. We found the avian erythroblastic leukemia viral oncogene homologue 3 (ERBB3) to be one of the most dramatically up-regulated genes in CCSST. Quantitative real-time PCR and Northern blot analysis verified the mRNA abundance and confirmed the absence of the inhibitory transcript variant of this gene. The protein product of the member of the epidermal growth factor receptor family ERBB3 could be shown to be highly present in all of the CCSST cell lines investigated, as well as in 18 of 20 primary tumor biopsies. In conclusion, our data demonstrate new aspects of the phenotype and the biological behavior of CCSST and reveal ERBB3 to be a useful diagnostic marker.
Subject(s)
Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 22/genetics , Genes, erbB/genetics , Sarcoma, Clear Cell/genetics , Soft Tissue Neoplasms/genetics , Translocation, Genetic , Blotting, Northern , Cell Line, Tumor , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genetic Markers/genetics , Humans , Male , Middle Aged , Neuroblastoma/genetics , Polymerase Chain Reaction/methods , RNA-Binding Protein EWS/genetics , Receptor, ErbB-3/biosynthesis , Receptor, ErbB-3/genetics , Sarcoma, Clear Cell/metabolism , Sarcoma, Ewing/genetics , Soft Tissue Neoplasms/metabolism , Up-RegulationABSTRACT
The first series of synthetic 1-aza-9-oxafluorenes with cytostatic activities in the micromolar range was evaluated as cyclin-dependent kinase (CDK1) inhibitors. Activity was found to be selective in comparison to the inhibition of other kinases within the CDK family. Compounds were shown to inhibit the membrane-efflux pump P-glycoprotein responsible for multidrug resistance in cancer cells. First structure-activity relationships are discussed.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Antineoplastic Agents/chemical synthesis , Aza Compounds/chemical synthesis , CDC2 Protein Kinase/antagonists & inhibitors , Fluorenes/chemical synthesis , Intercalating Agents/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Aza Compounds/chemistry , Aza Compounds/pharmacology , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Fluorenes/chemistry , Fluorenes/pharmacology , Intercalating Agents/chemistry , Intercalating Agents/pharmacology , Mice , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
A first series of novel 1-aza-9-oxafluorenes has been prepared from 3-carbonyl substituted 1,4-dihydropyridines and p-benzoquinone as small-sized cytostatics. Biological evaluation has been carried out in various cancer cell-lines. First structure-activity relationships proved the 4-phenyl substituent to be more favorable than the 4-methyl substituent. Cytostatic properties are discussed.
