ABSTRACT
BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS-FLI1 target genes still remain unknown and many have been identified in heterologous model systems. METHODS: We have developed a stable RNA interference model knocking down EWS-FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS-FLI1 and to identify genes that could contribute to tumourigenesis. RESULTS: EWS-FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS-FLI1 inhibition. We showed that TOPK is a new target gene of EWS-FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.
Subject(s)
Insulin-Like Growth Factor I/metabolism , Oncogene Proteins, Fusion/antagonists & inhibitors , Protein Serine-Threonine Kinases/biosynthesis , Receptor, IGF Type 1/metabolism , Sarcoma, Ewing/metabolism , Transcription Factors/antagonists & inhibitors , Animals , Apoptosis/physiology , Cell Line, Tumor , Cell Movement/physiology , Down-Regulation , Female , Humans , Insulin-Like Growth Factor I/antagonists & inhibitors , Insulin-Like Growth Factor I/biosynthesis , Insulin-Like Growth Factor I/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Mitogen-Activated Protein Kinase Kinases , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Protein c-fli-1 , RNA Interference , RNA-Binding Protein EWS , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/biosynthesis , Receptor, IGF Type 1/genetics , Sarcoma, Ewing/enzymology , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathology , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The congenital fibrosarcoma t(12;15)(p13;q25) rearrangement splices the ETV6 (TEL) gene on chromosome 12p13 in frame with the NTRK3 (TRKC) neurotrophin-3 receptor gene on chromosome 15q25. Resultant ETV6-NTRK3 fusion transcripts encode the helix - loop - helix (HLH) dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. We show here that ETV6-NTRK3 homodimerizes and is capable of forming heterodimers with wild-type ETV6. Moreover, ETV6-NTRK3 has PTK activity and is autophosphorylated on tyrosine residues. To determine if the fusion protein has transforming activity, NIH3T3 cells were infected with recombinant retroviral vectors carrying the full-length ETV6-NTRK3 cDNA. These cells exhibited a transformed phenotype, grew macroscopic colonies in soft agar, and formed tumors in severe combined immunodeficient (SCID) mice. We hypothesize that chimeric proteins mediate transformation by dysregulating NTRK3 signal transduction pathways via ligand-independent dimerization and PTK activation. To test this hypothesis, we expressed a series of ETV6-NTRK3 mutants in NIH3T3 cells and assessed their transformation activities. Deletion of the ETV6 HLH domain abolished dimer formation with either ETV6 or ETV6-NTRK3, and cells expressing this mutant protein were morphologically non-transformed and failed to grow in soft agar. An ATP-binding mutant failed to autophosphorylate and completely lacked transformation activity. Mutants of the three NTRK3 PTK activation-loop tyrosines had variable PTK activity but had limited to absent transformation activity. Of a series of signaling molecules well known to bind to wild-type NTRK3, only phospholipase-Cgamma (PLCgamma) associated with ETV6-NTRK3. However, a PTK active mutant unable to bind PLCgamma did not show defects in transformation activity. Our studies confirm that ETV6-NTRK3 is a transforming protein that requires both an intact dimerization domain and a functional PTK domain for transformation activity. Oncogene (2000) 19, 906 - 915.
Subject(s)
Adaptor Proteins, Signal Transducing , Cell Line, Transformed/enzymology , DNA-Binding Proteins/genetics , Receptor, trkC/genetics , Recombinant Fusion Proteins/genetics , Repressor Proteins , Transcription Factors/genetics , Translocation, Genetic , 3T3 Cells , Animals , Cell Line, Transformed/metabolism , GRB2 Adaptor Protein , Helix-Loop-Helix Motifs/genetics , Humans , Isoenzymes/metabolism , Mice , Mice, SCID , Molecular Sequence Data , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma , Protein Kinases/genetics , Protein Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Proto-Oncogene Proteins c-ets , Receptor, trkC/biosynthesis , Receptor, trkC/chemistry , Receptor, trkC/metabolism , Type C Phospholipases/metabolism , src Homology Domains/genetics , ETS Translocation Variant 6 ProteinABSTRACT
Biochips are collections of miniaturized test sites (microarrays) arranged on a solid substrate onto which a large number of biomolecules are attached with high density. Like a computer chip performing millions of mathematical operations in a few split seconds, a biochip allows for simultaneous analyses of thousands of biological reactions, such as decoding genes, in a few seconds. Biochip technologies can be applied to numerous fields including genomic, proteomic, and glycomic research, as well as pharmacology and toxicology. However, one of the most common applications is in the determination of gene expression in human cells and tissues. Global gene expression analysis has helped to identify important genes and signalling pathways in human malignant tumors. And there is hope that microarrays will make the step from "the (laboratory) bench to the bedside (of the patient)". Recent studies have indeed revealed that analysis of differential gene expression by microarrays may help to identify subtypes of malignant tumors, that allow a risk stratification of the patients. However, there are several issues that need to be addressed before microarrays may become a tool for routine diagnostics, such as problems with bioinformatic analysis, construction of disease or tissue specific microarrays with only limited numbers of genes of interest, standard operation procedures for tissue preparation to prevent RNA degradation, etc.. In this article, an overview over of the multifarious biochip applications and technologies, its limitations, challenges and future developments is provided.