Gene Fusions Involving PAX and FOX Family Members in Alveolar Rhabdomyosarcoma
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Oncogene (2001) 20, 5736 ± 5746 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma Frederic G Barr*,1 1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 36th Street and Hamilton Walk, Philadelphia, Pennsylvania, PA 19104-6082, USA The chromosomal translocations t(2;13)(q35;q14) and Clinical, pathologic and cytogenetic features of alveolar t(1;13)(p36;q14) are characteristic of alveolar rhabdo- rhabdomyosarcoma myosarcoma, a pediatric soft tissue cancer related to the striated muscle lineage. These translocations rearrange Alveolar rhabdomyosarcoma (ARMS) is one subtype PAX3 and PAX7, members of the paired box transcrip- of rhabdomyosarcoma (RMS), a family of pediatric tion factor family, and juxtapose these genes with soft tissue tumors that are related to the skeletal FKHR, a member of the fork head transcription factor muscle lineage (Wexler and Helman, 1997). ARMS was family. This juxtaposition generates PAX3 ± FKHR and initially distinguished from the other major RMS PAX7 ± FKHR chimeric genes that are expressed as subtype, embryonal rhabdomyosarcoma (ERMS), by chimeric transcripts that encode chimeric proteins. The histologic criteria. As the criteria were re®ned, the fusion proteins, which contain the PAX3/PAX7 DNA subtypes were found to be associated with distinct binding domain and the FKHR transcriptional activation clinical behaviors (Tsokos et al., 1992). ARMS presents domain, activate transcription from PAX-binding sites mainly in adolescents and young adults, often in the with higher potency than the corresponding wild-type vicinity of skeletal muscle in the extremities and trunk, PAX proteins. This increased function results from the and is associated with an unfavorable prognosis. This insensitivity of the FKHR activation domain to inhibitory unfavorable prognosis is related to the propensity for eects of N-terminal PAX3/PAX7 domains. In addition early and wide dissemination, often involving bone to altered function, the fusion products are expressed in marrow, and to poor response to chemotherapy. In ARMS tumors at higher levels than the corresponding contrast, ERMS mainly presents in children less than wild-type PAX products due to two distinct mechanisms. 10 years old in regions without abundant skeletal The PAX7 ± FKHR fusion is overexpressed as a result of muscle such as the head and neck, genitourinary tract, in vivo ampli®cation while the PAX3 ± FKHR fusion is and retroperitoneum, and has a more favorable overexpressed due to a copy number-independent prognosis. increase in transcriptional rate. Finally, though FKHR Diagnosis of ARMS is often complicated by scant subcellular localization is regulated by an AKT- evidence of striated muscle dierentiation and rela- dependent pathway, the fusion proteins are resistant to tively subtle histologic criteria for distinguishing these signals and show exclusively nuclear localization. ERMS and ARMS (Tsokos, 1994). The problem is Therefore, these translocations alter biological activity at compounded by the fact that other pediatric solid the levels of protein function, gene expression, and tumors including neuroblastoma, Ewing's sarcoma and subcellular localization with the cumulative outcome lymphoma can present as collections of poorly postulated to be aberrant regulation of PAX3/PAX7 dierentiated cells. Subtle evidence of myogenic target genes. This aberrant gene expression program is dierentiation can be detected with immunohistochem- then hypothesized to contribute to tumorigenic behavior ical reagents speci®c for muscle proteins (such as by impacting on the control of growth, apoptosis, MyoD, desmin, or muscle-speci®c actin) and electron dierentiation and motility. Oncogene (2001) 20, microscopic analysis of myo®laments. However, there 5736 ± 5746. is no well-established immunohistochemical or ultra- structural marker that distinguishes ARMS and Keywords: rhabdomyosarcoma; transcription factor; ERMS. translocation; paired box; fork head Chromosomal studies identi®ed nonrandom translo- cations that distinguish the majority of ARMS tumors from ERMS and other pediatric solid tumors. The most prevalent ®nding in ARMS is a translocation, t(2;13)(q35-37;q14), which was detected in 70% of ARMS cases (Douglass et al., 1987; Turc-Carel et al., 1986; Wang-Wuu et al., 1988) (Figure 1). In addition, a variant translocation, t(1;13)(p36;q14), was identi®ed in *Correspondence: FG Barr; E-mail: [email protected] a smaller subset of ARMS cases (Biegel et al., 1991; Alveolar rhabdomyosarcoma gene fusions FG Barr 5737 Douglass et al., 1991). The 2;13 and 1;13 translocations have not been detected in any other tumor type and appear to be speci®c and sensitive markers of ARMS. Mapping and cloning of loci involved in 2;13 and 1;13 translocations Based on physical mapping studies, PAX3 was localized to the vicinity of the t(2;13) breakpoint on chromosome 2 (Barr et al., 1993). Analysis of somatic cell hybrids derived from ARMS cells indicated that PAX3 is split such that the 5' PAX3 region is located on the derivative chromosome 13 [der(13)] and the 3' PAX3 region is on the der(2) (Figure 2). In conjunction Figure 3 Comparison of wild-type and fusion products asso- with Southern blot ®ndings of PAX3 structural ciated with the 2;13 and 1;13 translocations. The paired box, alterations in ARMS cell lines, these studies established octapeptide, homeobox and fork head domain are indicated as open boxes, and transcriptional domains (DNA binding domain, that PAX3 is the chromosome 2 locus rearranged by DBD; transcriptional activiation domain, TAD; transcriptional the t(2;13). Northern analysis with a 5' PAX3 probe inhibitory domain, TID) are shown as solid bars. The sites demonstrated a novel 7.2 kb transcript unique to cell phosphorylated by Akt are indicated by stars, and the alternative lines with the t(2;13). Clones containing 5' PAX3 splice in the paired box is shown by an arrowhead. The vertical sequences fused to a novel sequence from chromosomal dash line indicates the translocation fusion point region 13q14 were then isolated from cDNA libraries constructed from ARMS cell lines (Galili et al., 1993; Shapiro et al., 1993) (Figure 3). The corresponding acid protein. A BLAST database search showed wild-type chromosome 13 gene generates a 6.5 kb homology to the fork head transcription factor family, widely expressed transcript that encodes a 655 amino and thus this chromosome 13 gene was named FKHR (fork head in rhabdomyosarcoma) (Galili et al., 1993); this gene was also termed ALV (Shapiro et al., 1993), and was recently renamed FOXO1 (Kaestner et al., 2000). These ®ndings indicate that the t(2;13) results in a chimeric transcript composed of 5' PAX3 sequences fused to 3' FKHR sequences. Analysis of the PAX3 ± FKHR cDNA revealed that the 5' PAX3 and 3' FKHR coding sequences are fused in-frame to encode an 836 amino acid fusion protein (Galili et al., 1993; Shapiro et al., 1993) (Figure 3). The consistency of this chimeric transcript was con®rmed by reverse transcriptase (RT) ± PCR experiments, and sequence analysis of these RT ± PCR products revealed an invariant fusion point. The reciprocal translocation Figure 1 Diagram of t(2;13)(q35;q14) and t(1;13)(p36;q14) product, the der(2) chimeric transcript consisting of 5' chromosomal translocations FKHR and 3' PAX3 exons, was not detected in ARMS lines by Northern blot analysis (Barr et al., 1993), but low level expression was detected by RT ± PCR analysis in a subset of ARMS cell lines and specimens (Galili et al., 1993). The ®ndings of higher, more consistent expression of the 5' PAX3-3' FKHR fusion indicate that the der(13) encodes the product involved in the pathogenesis of ARMS. In ARMS cases with the 1;13 translocation, attention focused on PAX7, another member of the paired box- containing transcription factor family located in chromosomal region 1p36 (Davis et al., 1994). RT ± PCR and Southern blot studies demonstrated that PAX7 is rearranged and fused to FKHR in tumors containing this translocation. This fusion results in a Figure 2 Generation of chimeric genes by the t(2;13)(q35;q14) chimeric transcript consisting of 5' PAX7 and 3' translocation in ARMS. The exons of the wild-type and fusion genes are shown as boxes above each map and the translocation FKHR regions (Figure 3), which is nearly identical in breakpoint distributions are shown as line segments below the structure and organization to the 5' PAX3 ± 3' FKHR map of the wild-type genes transcript formed by the 2;13 translocation. Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5738 Paired box family of transcription factors disrupted Pax7 gene demonstrate abnormalities in neural crest-derived structures and musculature (Man- PAX3 and PAX7 encode members of the paired box or souri et al., 1996; Seale et al., 2000). These studies thus PAX transcription factor family that is characterized provide functional evidence of an important role for by a conserved paired box DNA binding domain ®rst Pax3 and Pax7 in myogenic development. identi®ed in Drosophila segmentation genes (Tremblay and Gruss, 1994). There are nine human members of this family. Several family members also contain a Fork head family of transcription factors complete or truncated version of a homeobox DNA binding domain and a short conserved octapeptide The fork head domain was ®rst identi®ed as a 100 motif distal to the paired box. PAX3 and PAX7 amino acid region of sequence similarity between the constitute a subfamily within the PAX family and Drosophila fork head and the rat HNF-3a proteins, and encode very similar proteins containing an N-terminal was subsequently identi®ed in a large family of genes in DNA binding domain consisting of a paired box, species ranging from yeast to human (Kaufmann and octapeptide and complete homeodomain, and a pro- Knochel, 1996).