5077

PAX3

CDHS|HUP2|WS1|WS3;paired box 3;PAX3;paired box 3

paired box homeotic gene 3|paired box protein Pax-3|paired domain gene 3|paired domain gene HuP2

2q35-q37|2q35

protein-coding

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 setsof resources, we investigated several candidate PAX3-FKHR target genes. First, we demonstrated in both short-term and long-term systems that PAX3-FKHR upregulatesexpression 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 tumorsrevealed 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 ARMStumorigenesis and progression.

Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, likely results from deregulation of the skeletal myogenesis program. Although associations between PAX3, PAX7, FOXO1A, and RMS tumorigenesis are well recognized, the entire spectrum of genetic factors underlying RMS development and progression is unclear. Using a combined approach of spectral karyotyping, array-based comparative genomic hybridization (CGH), and expression analysis, we examined 10primary RMS tumors, including embryonal, alveolar, and the rare adult pleomorphic variant, to explore the involvement of different genes and genetic pathways in RMS tumorigenesis. A complete karyotype established for each tumor revealed a high aneuploidy level, mostly tetraploidy, with double minutes and additional structural aberrations. Quantitative expression analysis detected the overexpression of the AURKA gene in ALL tumors tested, suggesting a role for this mitotic regulator in the aneuploidy and chromosomal instability observed in RMS.Array-based CGH analysis in primary RMS tumors detected copy number changes of genes involved in multiple genetic pathways, including transcription factors such as MYC-related gene from lung cancer and the cytoskeleton and cell adhesion-encoding genes laminin gamma-2 and p21-activated kinase-1. Our data suggest the involvement of genes encoding cell adhesion, cytoskeletal signaling,and transcriptional and cell cycle components in RMS tumorigenesis.

Wilms tumor (WT) is the most frequent renal neoplasm of childhood; a myogenic component is observed in 5% to 10% of tumors. We demonstrate for the first time that myogenic WTs are associated with expression of PAX3, a transcription factorknown to specify myoblast cell fate during muscle development. In a panel of 20 WTs, PAX3 was identified in 13 of 13 tumor samples with myogenic histopathology but was absent in 7 of 7 tumors lacking a myogenic component. Furthermore, we show that PAX3 is expressed in the metanephric mesenchyme and stromal compartment of developing mouse kidney. Modulation of endogenous PAX3 expression in human embryonic kidney (HEK293) cells influenced cell migration in in vitro assays. mutations of WT1 were consistently associated with PAX3 expression in WTs, and modulation of WT1 expression in HEK293 cells was inversely correlated with the level of endogenous PAX3 protein. We demonstrate abundant PAX3 and absence of PAX2 expression in a novel cell line (WitP3) isolated from the stromal portion of a WT bearing a homozygous deletion of the WT1 gene. We hypothesize that PAX3 sets stromal cell fate in developing kidney but is normally suppressed by WT1 during the mesenchyme-to-epithelium transition leading to nephrogenesis. Loss of WT1 permits aberrant PAX3 expression in a subset of WTs with myogenic phenotype.

The expression of a number of human paired box-containing (PAX) genes has been correlated with various types of tumors. Novel fusion genes encoding chimeric fusion proteins have been found in the pediatric malignant tumor alveolar rhabdomyosarcoma (RMS). They are generated by two chromosomal translocations t(2;13) and t(1;13) juxtaposing PAX3 or PAX7, respectively, with a forkhead domain gene FKHR. Here we describe that specific down-regulation of the t(2;13) translocation product in alveolar RMS cells by antisense oligonucleotides results in reduced cellular viability. Cells of embryonal RMS, the other major histiotype of this tumor, were found to express either wild type PAX3 or PAX7 at elevated levels when compared with primary human myoblasts. Treatment of corresponding embryonal RMS cells with antisense olignucleotides directed against the mRNA translational start site of either one of these two transcription factors similarly triggers cell death, which is most likely due to induction of apoptosis. Retroviral mediated ectopic expression of mouse Pax3 in a PAX7 expressing embryonal RMS cell line could partially rescue antisense induced apoptosis. These data suggest that the PAX3/FKHR fusion gene and wild-type PAX genes play a causative role in the formation of RMS and presumably other tumor types, possibly by suppressing the apoptotic program that would normally eliminate these cells.