Inducible short-term and stable long-term cell culture systems reveal that the PAX3-FKHR fusion oncoprotein regulates CXCR4, PAX3, and PAX7 expression

• Published in: Laboratory investigation 2004-08, Vol.84 (8), p.1060-1070
• Main Authors: Tomescu, Oana, Xia, Shujuan J, Strezlecki, Donna, Bennicelli, Jeannette L, Ginsberg, Jill, Pawel, Bruce, Barr, Frederic G
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites - genetics; Biological and medical sciences; Biotechnology; Cell Division; Cell Line, Tumor; chromosomal translocation; DNA Primers - genetics; DNA, Neoplasm - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Forkhead Box Protein O1; Forkhead Transcription Factors; Fundamental and applied biological sciences. Psychology; gene expression; Gene Expression Regulation, Neoplastic; gene fusion; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Humans; Investigative techniques, diagnostic techniques (general aspects); Laboratory Medicine; Medical sciences; Medicine; Medicine & Public Health; Oncogene Proteins, Fusion - genetics; Oncogene Proteins, Fusion - metabolism; Paired Box Transcription Factors; Pathology; PAX3 Transcription Factor; PAX7 Transcription Factor; Receptors, CXCR4 - genetics; Receptors, CXCR4 - metabolism; Receptors, Estrogen - genetics; Receptors, Estrogen - metabolism; research-article; rhabdomyosarcoma; Rhabdomyosarcoma, Embryonal - genetics; Rhabdomyosarcoma, Embryonal - metabolism; Rhabdomyosarcoma, Embryonal - pathology; transcription factor; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 0023-6837; 1530-0307
• DOI: 10.1038/labinvest.3700125

In the pediatric cancer alveolar rhabdomyosarcoma (ARMS), the 2;13 chromosomal translocation juxtaposes the PAX3 and FKHR genes to generate a chimeric transcription factor. To explore molecular pathways altered by this oncoprotein, we generated an inducible form by fusing PAX3-FKHR to a modified estrogen receptor ligand-binding domain and expressed this construct in the RD embryonal rhabdomyosarcoma cell line. This inducible system permits short-term evaluation of downstream expression targets of PAX3-FKHR and complements a panel of stable long-term RD subclones constitutively expressing PAX3-FKHR. Using these two sets of resources, we investigated several candidate PAX3-FKHR target genes. First, we demonstrated in both short-term and long-term systems that PAX3-FKHR upregulates expression of the gene encoding the chemokine receptor CXCR4. In addition, we found that expression of wild-type PAX3 is upregulated, whereas expression of wild-type PAX7 is downregulated by PAX3-FKHR. In the presence of cycloheximide, CXCR4 and PAX3 are still inducible, supporting the hypothesis that these genes are direct transcriptional targets of PAX3-FKHR. Finally, studies of ARMS tumors revealed CXCR4, PAX3, and PAX7 expression levels consistent with our cell culture results. These findings of genes regulated by PAX3-FKHR will direct future biological and clinical investigation to important pathways contributing to ARMS tumorigenesis and progression.