Abstract
Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ~50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14 bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.
Keywords:
EWS/FLI1; Ewing sarcoma; PI3K pathway; promoter analysis.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Antineoplastic Agents / pharmacology
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Binding Sites
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Bone Neoplasms / drug therapy
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Bone Neoplasms / enzymology*
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Bone Neoplasms / genetics
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Bone Neoplasms / pathology
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Cell Cycle Checkpoints
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Cell Line, Tumor
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Dose-Response Relationship, Drug
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Gene Expression Regulation, Neoplastic* / drug effects
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Humans
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Imidazoles / pharmacology
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Oncogene Proteins, Fusion / genetics
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Oncogene Proteins, Fusion / metabolism*
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Phosphatidylinositol 3-Kinase / genetics
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Phosphatidylinositol 3-Kinase / metabolism*
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Phosphoinositide-3 Kinase Inhibitors
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Promoter Regions, Genetic
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Protein Binding
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Protein Kinase Inhibitors / pharmacology
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Proto-Oncogene Protein c-fli-1 / genetics
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Proto-Oncogene Protein c-fli-1 / metabolism*
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Proto-Oncogene Proteins c-akt / metabolism*
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Quinolines / pharmacology
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RNA Interference
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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RNA-Binding Protein EWS / genetics
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RNA-Binding Protein EWS / metabolism*
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Sarcoma, Ewing / drug therapy
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Sarcoma, Ewing / enzymology*
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Sarcoma, Ewing / genetics
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Sarcoma, Ewing / pathology
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Signal Transduction* / drug effects
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Sp1 Transcription Factor / genetics
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Sp1 Transcription Factor / metabolism*
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Transcription, Genetic* / drug effects
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Transfection
Substances
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Antineoplastic Agents
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EWS-FLI fusion protein
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Imidazoles
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Oncogene Proteins, Fusion
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Phosphoinositide-3 Kinase Inhibitors
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Protein Kinase Inhibitors
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Proto-Oncogene Protein c-fli-1
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Quinolines
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RNA, Messenger
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RNA-Binding Protein EWS
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Sp1 Transcription Factor
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SP1 protein, human
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Phosphatidylinositol 3-Kinase
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Proto-Oncogene Proteins c-akt
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dactolisib