Inducible short-term and stable long-term cell culture systems reveal that the PAX3-FKHR fusion oncoprotein regulates CXCR4, PAX3, and PAX7 expression.
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.
Tomescu
Oana
O
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Xia
Shujuan J
SJ
Strezlecki
Donna
D
Bennicelli
Jeannette L
JL
Ginsberg
Jill
J
Pawel
Bruce
B
Barr
Frederic G
FG
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism*; D000072041:PAX3 Transcription Factor; D051763:PAX7 Transcription Factor; D051761:Paired Box Transcription Factors; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D018233:Rhabdomyosarcoma, Embryonal / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D018398:Homeodomain Proteins; D015514:Oncogene Proteins, Fusion; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051763:PAX7 Transcription Factor; C497321:PAX7 protein, human; D051761:Paired Box Transcription Factors; D019718:Receptors, CXCR4; D011960:Receptors, Estrogen; D014157:Transcription Factors; C068686:Pax3 protein, mouse