An engineered PAX3-KRAB transcriptional repressor inhibits the malignant phenotype of alveolar rhabdomyosarcoma cells harboring the endogenous PAX3-FKHR oncogene.
The t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma tumors (ARMS) creates an oncogenic transcriptional activator by fusion of PAX3 DNA binding motifs to a COOH-terminal activation domain derived from the FKHR gene. The dominant oncogenic potential of the PAX3-FKHR fusion protein is dependent on the FKHR activation domain. We have fused the KRAB repression module to the PAX3 DNA binding domain as a strategy to suppress the activity of the PAX3-FKHR oncogene. The PAX3-KRAB protein bound PAX3 target DNA sequences and repressed PAX3-dependent reporter plasmids. Stable expression of the PAX3-KRAB protein in ARMS cell lines resulted in loss of the ability of the cells to grow in low-serum or soft agar and to form tumors in SCID mice. Stable expression of a PAX3-KRAB mutant, which lacks repression function, or a KRAB protein alone, lacking a PAX3 DNA binding domain, failed to suppress the ARMS malignant phenotype. These data suggest that the PAX3-KRAB repressor functions as a DNA-binding-dependent suppressor of the transformed phenotype of ARMS cells, probably via competition with the endogenous PAX3-FKHR oncogene and repression of target genes required for ARMS tumorigenesis. The engineered repressor approach that directs a transcriptional repression domain to target genes deregulated by the PAX3-FKHR oncogene may be a useful strategy to identify the target genes critical for ARMS tumorigenesis.
Fredericks
W J
WJ
The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Ayyanathan
K
K
Herlyn
M
M
Friedman
J R
JR
Rauscher
F J
FJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015197:Carcinogenicity Tests; D002455:Cell Division / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D018232:Rhabdomyosarcoma, Alveolar / Q000235:genetics* / Q000473:pathology*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D000076183:Tripartite Motif-Containing Protein 28; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C109484:ZNF10 protein, human; C068686:Pax3 protein, mouse; C101116:TRIM28 protein, human; C103190:Trim28 protein, mouse; D000076183:Tripartite Motif-Containing Protein 28