Hormone-dependent tumor regression in vivo by an inducible transcriptionalrepressor directed at the PAX3-FKHR oncogene

In alveolar rhabdomyosarcomas (ARMSs), a specific chromosomal translocation cr catus a fusion transcription factor, PAX3-FKHR, that is oncogenic due t o transcriptional activation, ils a strategy for downregulation of PAX3-FKH R target genes, we created conditional PAX3 repressors by fusing the PAX3 D NA-binding motifs to the hormone binding domain (HBD) of the estrogen recep tor and to the KRAB repression domain. We validated proper expression, spec ific DNA binding, corepressor interaction, and nuclear localization for the KRAB-PAX3-HBD protein and showed it to he a 4-hydroxytamoxifen-dependent t ranscriptional repressor of transiently transfected and integrated PAX3 rep orters 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 con ditions In a hormone-dependent manner. Terminal deoxynucleotidyl transferas e-mediated nick end labeling assays revealed that hormonal activation of th e PAX3 repressors induced extensive apoptosis that correlated with down-reg ulation 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-r elease pellets exhibited suppression of tumor growth and an altered vascula rity that was not observed in the control mice. These observations strongly suggest that we have directly repressed the PAX3 target genes that are der egulated by the PAX3-FKHR oncogene in ARMS.