Hormone-dependent tumor regression in vivo by an inducible transcriptional repressor directed at the PAX3-FKHR oncogene.
In alveolar rhabdomyosarcomas (ARMSs), a specific chromosomal translocation creates a fusion transcription factor, PAX3-FKHR, that is oncogenic due to transcriptional activation. As a strategy for down-regulation of PAX3-FKHR target genes, we created conditional PAX3 repressors by fusing the PAX3 DNA-binding motifs to the hormone binding domain (HBD) of the estrogen receptor and to the KRAB repression domain. We validated proper expression, specific DNA binding, corepressor interaction, and nuclear localization for the KRAB-PAX3-HBD protein and showed it to be a 4-hydroxytamoxifen-dependent transcriptional repressor of transiently transfected and integrated PAX3 reporters in ARMS cells. We established ARMS cell lines that exhibited stable expression of the conditional PAX3 repressor proteins and used them to down-regulate the malignant growth under low serum or anchorage-independent conditions in a hormone-dependent manner. Terminal deoxynucleotidyl transferase-mediated nick end labeling assays revealed that hormonal activation of the PAX3 repressors induced extensive apoptosis that correlated with down-regulation of BCL-X(L) expression. SCID mice that were engrafted with the KRAB-PAX3-HBD ARMS cell lines and were implanted with 4-hydroxytamoxifen timed-release pellets exhibited suppression of tumor growth and an altered vascularity that was not observed in the control mice. These observations strongly suggest that we have directly repressed the PAX3 target genes that are deregulated by the PAX3-FKHR oncogene in ARMS.
Ayyanathan
K
K
The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Fredericks
W J
WJ
Berking
C
C
Herlyn
M
M
Balakrishnan
C
C
Gunther
E
E
Rauscher
F J
FJ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D019556:COS Cells; D002455:Cell Division / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D015536:Down-Regulation; D005260:Female; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D018390:Gene Targeting; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009857:Oncogenes / Q000235:genetics; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D011960:Receptors, Estrogen / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D018232:Rhabdomyosarcoma, Alveolar / Q000235:genetics* / Q000473:pathology; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; C494812:Bcl2l1 protein, mouse; D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; D019253:Proto-Oncogene Proteins c-bcl-2; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D051020:bcl-X Protein; D013629:Tamoxifen; C068686:Pax3 protein, mouse; C016601:afimoxifene; D008156:Luciferases