High expression of the PAX3-FKHR oncoprotein is required to promote tumorigenesis of human myoblasts.

PAX3-FKHR is a fusion oncoprotein generated by the 2;13 chromosomal translocation in alveolar rhabdomyosarcoma (ARMS), a cancer associated with the skeletal muscle lineage. Previous studies determined that high-level PAX3-FKHR expression is a consistent feature in ARMS tumors. To investigate the relationship between expression and phenotype in human myogenic cells, PAX3-FKHR was introduced into immortalized human myoblasts to produce a low overall PAX3-FKHR expression level. Although PAX3-FKHR alone failed to exert transforming activity, a combination of PAX3-FKHR and MYCN induced transforming activity in cell culture assays. Furthermore, myoblasts expressing PAX3-FKHR with or without MYCN formed tumors in SCID mice. These tumors demonstrated invasive features and expressed myogenic markers, consistent with rhabdomyosarcoma. Comparisons of tumor and parental cells revealed that only a subset of parental cells developed into tumors and that tumor cells expressed high PAX3-FKHR levels compared with transduced parental cells. Subcloning of parental PAX3-FKHR/MYCN-transduced myoblasts identified rare high PAX3-FKHR-expressing subclones with high transforming and tumorigenic activity; however, most subclones expressed low PAX3-FKHR and showed neither transforming nor tumorigenic activity. Finally, RNA interference experiments in myoblast-derived tumor and ARMS cells revealed that high PAX3-FKHR expression plays a crucial role in regulating proliferation, transformation, and differentiation. These findings support the premise that high PAX3-FKHR-expressing cells are selected during tumorigenesis.

Identification of PAX3-FKHR-regulated genes differentially expressed between alveolar and embryonal rhabdomyosarcoma: focus on MYCN as a biologically relevant target.

Rhabdomyosarcoma is a family of myogenic soft tissue tumors subdivided into two main subtypes: alveolar (ARMS) and embryonal (ERMS). ARMS is characterized by a frequent 2;13 chromosomal translocation that creates a PAX3-FKHR fusion transcription factor. To identify downstream targets of PAX3-FKHR, we introduced an inducible form of PAX3-FKHR into human RD ERMS cells. Microarray analysis identified 39 genes (29 upregulated and 10 downregulated) that are modulated by PAX3-FKHR in RD cells and differentially expressed between ERMS and PAX3-FKHR-positive ARMS tumors. Functional annotation demonstrated that genes involved in regulation of transcription and development, particularly neurogenesis, are represented in this group. MYCN was one notable neural-related transcription factor-encoding gene identified in this set, and its regulation by PAX3-FKHR was further confirmed at the RNA and protein levels. The findings of cycloheximide inhibition and time-course studies are consistent with the hypothesis that the PAX3-FKHR protein acts directly on the MYCN gene at the transcriptional level. Functional studies established that MYCN cooperates with PAX3-FKHR to enhance oncogenic activity. In conclusion, we identified a selected set of biologically relevant genes modulated by PAX3-FKHR, and demonstrated that PAX3-FKHR contributes to the expression of MYCN and in turn MYCN collaborates with PAX3-FKHR in tumorigenesis.

Analysis of genetic events that modulate the oncogenic and growth suppressive activities of the PAX3-FKHR fusion oncoprotein.

Alveolar rhabdomyosarcoma (ARMS) is associated with chromosomal translocations that generate PAX3-FKHR and PAX7-FKHR fusion oncoproteins. Based on studies demonstrating that high PAX3-FKHR expression causes growth suppression, the hypothesis is proposed that, during ARMS tumorigenesis, the translocations cause low oncoprotein expression and are followed by collaborating events that block growth suppression pathways and permit upregulation of oncoprotein expression. To investigate oncogenic function at low expression levels, PAX3-FKHR was introduced into NIH3T3 cells in the pBabe retroviral vector. Compared to high expression systems, PAX3-FKHR expression from pBabe was lower and did not suppress growth, but showed transforming activity in the soft agar assay. As a possible collaborating event, PAX3-FKHR paired box mutations were previously shown in high expression systems to reverse growth suppressive effects. In the low expression system, the paired box mutation enhanced transformation in soft agar and focus formation assays. Although these mutations are candidate collaborating events, sequencing of paired box regions in ARMS tumors did not identify mutations. Finally, genes from known genetic alterations in ARMS were introduced, alone or combined, into NIH3T3 cells with high PAX3-FKHR expression and did not rescue growth suppression. In summary, these studies provide a model for an event in ARMS tumorigenesis that enhances PAX3-FKHR oncogenicity and abrogates growth suppression, but do not demonstrate a known event occurring in ARMS tumors that fulfills these criteria.

Chromosome translocations in sarcomas and the emergence of oncogenic transcription factors.

A subset of sarcomas is characterised by recurrent chromosome translocations that generate novel fusion oncoproteins. One or both of the genes involved in these translocations often encode transcription factors, and the resulting fusion proteins have aberrant transcriptional function compared to their wild-type counterparts. These fusion transcription factors disrupt multiple biological pathways by altering expression of target genes, and thereby result in a variety of altered cellular properties that contribute to the tumourigenic process. However, experimental data indicate that the fusion gene alone is not sufficient for transformation in primary cells (EWS-FLI1) or tumourigenesis in the mouse (PAX3-FKHR, FUS-CHOP), suggesting that additional collaborating genetic alterations are required. In addition to improving our understanding of the etiology of these tumours, this accumulating knowledge of the oncogenic properties of these fusion proteins, their downstream targets, and cooperating genetic alterations will permit the development of a variety of novel approaches to improve the therapy of these cancers.

Co-expression of alternatively spliced forms of PAX3, PAX7, PAX3-FKHR and PAX7-FKHR with distinct DNA binding and transactivation properties in rhabdomyosarcoma.

PAX3 and PAX7 encode transcription factors implicated in the pathogenesis of rhabdomyosarcoma (RMS), including alveolar RMS in which chromosomal translocations generate PAX3-FKHR and PAX7-FKHR fusions. Previous studies of wild-type PAX3 and PAX7 identified alternative splicing events that modify the paired box and generate 2 isoforms of PAX3 (Q+ and Q-) and 4 isoforms of PAX7 (Q+GL+, Q+GL-, Q-GL+, Q-GL-). In our study, we investigated alternative splicing of the wild-type and fusion forms of PAX3 and PAX7 in alveolar and embryonal RMS and assessed the functional implications. For PAX3 and PAX3-FKHR, the Q+ and Q- isoforms were consistently co-expressed in RMS tumors with slightly higher levels of the Q+ isoform. For PAX7 and PAX7-FKHR, there was a consistent pattern of co-expression of the 4 isoforms in RMS tumors: Q+GL- > Q+GL+ >/= Q-GL- > Q-GL+. DNA binding analysis demonstrated that PAX3 and PAX3-FKHR Q- isoforms exhibit higher affinity than corresponding Q+ isoforms for class I sites and no difference for class II sites. For PAX7 and PAX7-FKHR, the relative affinity was Q-GL- > Q+GL- > Q-GL+ >/= Q+GL+ for class I sites and Q-GL-, Q+GL- > Q-GL+, Q+GL+ for class II sites. Finally, the transcriptional activities of the PAX3-FKHR and PAX7-FKHR isoforms on reporter plasmids varied over a 5-fold and 50-fold range, respectively, in accord with the differences in DNA binding activity. In conclusion, these studies reveal that PAX3, PAX7 and their fusions with FKHR are each expressed in RMS tumors as a consistent mixture of functionally distinct isoforms.

Analysis of the transforming and growth suppressive activities of the PAX3-FKHR oncoprotein.

The 2;13 chromosomal translocation occurs in most cases of the cancer alveolar rhabdomyosarcoma (ARMS), and juxtaposes the genes encoding the PAX3 and FKHR transcription factors. The resulting chimeric protein PAX3-FKHR is a potent transcriptional activator, and is hypothesized to function as a dominant acting oncogene. To investigate its biological function, PAX3-FKHR was transduced into three immortalized murine cell lines in either a constitutive or inducible manner. These cells only tolerate expression of low PAX3-FKHR levels, which is sufficient for transformation in NIH3T3 cells. In contrast, higher PAX3-FKHR levels, which are comparable to the endogenous level expressed in ARMS cells, result in growth suppression. To determine as to which PAX3 functional domains are needed for growth suppression and transformation, inactivating mutations were introduced into the paired box and homeodomain of PAX3-FKHR. In these experiments, the homeodomain is necessary for transformation, but not growth suppression; whereas the paired box is not required for transformation but mediates growth suppression. In summary, our findings demonstrate that the transforming and growth suppressive activities of PAX3-FKHR are dominant at different activity levels and are mediated by distinct functional domains. These findings are consistent with the hypothesis that distinct expression pathways are operative in these opposing phenotypic end points.

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.

Molecular pathogenesis of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a family of soft tissue tumors that are associated with the skeletal muscle lineage and generally occur in the pediatric population. Based on histopathologic features, two subtypes, embryonal (ERMS) and alveolar (ARMS), were identified and associated with distinct clinical characteristics and genetic alterations. ARMS is associated with 2;13 or 1;13 chromosomal translocations, which generate PAX3-FKHR and PAX7-FKHR fusion products, respectively. These translocations result in altered expression, function, and subcellular localization of the fusion products relative to the wild-type proteins, and ultimately contribute to oncogenic behavior by modifying growth, differentiation, and apoptosis pathways. In contrast to the specific translocations found in ARMS, most ERMS cases have allelic loss at chromosome 11p15.5. Chromosome fragment transfer studies demonstrated that this region represses tumor cell growth, suggesting the presence of tumor suppressor gene(s) in this region. In both ERMS and ARMS, there is evidence of collaborating alterations that affect common targets, such as the p53 and RB pathways. One mechanism for perturbing these pathways involves amplification of genes such as MDM2 and CDK4; these amplification events occur frequently in ARMS but only rarely in ERMS. Therefore, despite similarities in the downstream targets of these genetic alterations, the striking cytogenetic and molecular differences between ARMS and ERMS indicate distinct molecular etiologies in these two subtypes.