Dataset Information

A Drosophila model of the rhabdomyosarcoma initiator PAX7-FKHR.

ABSTRACT: Alveolar rhabdomyosarcoma (ARMS) is an aggressive myogenic-type tumor and a gain-of-function disease, caused by misexpression of the PAX3-FKHR or PAX7-FKHR fusion oncoprotein from structurally rearranged chromosomes. PAX3-FKHR misexpressed in terminally differentiating mouse myofibers can cause rhabdomyosarcoma at a low frequency, suggesting that skeletal muscle is an ARMS tissue of origin. Because patterned muscle is widely viewed as irreversibly syncytial, questions persist, however, regarding this potential pathogenetic mechanism for ARMS tumor initiation. To further explore this issue, we generated transgenic Drosophila lines that conditionally express human PAX-FKHR. Here we show that PAX7-FKHR causes nucleated cells to form and separate from syncytial myofibers, which then spread to nonmuscular tissue compartments, including the central nervous system, and that wild-type PAX3 demonstrates similar potential. We further show that Ras, which is known to interfere with the differentiation of myogenic cells, genetically interacts with PAX7-FKHR: constitutively activated Ras enhances PAX7-FKHR phenotypes, whereas loss-of-function ras alleles dominantly suppress PAX7-FKHR activity, including rescue of lethality. These results show that PAX-FKHR can drive the generation of discrete nucleated cells from differentiated myofibers in vivo, argue for syncytial muscle as an ARMS tissue of origin, and demonstrate that Drosophila provides a powerful system to screen for genetic modifiers of PAX-FKHR.

SUBMITTER: Galindo RL

PROVIDER: S-EPMC1569182 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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A Drosophila model of the rhabdomyosarcoma initiator PAX7-FKHR.

Galindo Rene L RL Allport Jay A JA Olson Eric N EN

Proceedings of the National Academy of Sciences of the United States of America 20060828 36

Alveolar rhabdomyosarcoma (ARMS) is an aggressive myogenic-type tumor and a gain-of-function disease, caused by misexpression of the PAX3-FKHR or PAX7-FKHR fusion oncoprotein from structurally rearranged chromosomes. PAX3-FKHR misexpressed in terminally differentiating mouse myofibers can cause rhabdomyosarcoma at a low frequency, suggesting that skeletal muscle is an ARMS tissue of origin. Because patterned muscle is widely viewed as irreversibly syncytial, questions persist, however, regarding ...[more]

PMID: 16938866

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Project description:BackgroundAlveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein's transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR-mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS.MethodsTo identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells' motility. We used microarray analysis to identify genes whose expression level decreased when PAX3-FKHR was downregulated. We used mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays to determine whether PAX3-FKHR binds to the promoter region of the target gene. We used siRNA and pharmacologic inhibitor to downregulate the target gene of PAX3-FKHR and investigated the effect of such downregulation on cell motility.ResultsWe found that when PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1A decreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR-mediated cell migration and metastasis.ConclusionsTaken together, we have identified CPT1A as a novel transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may represent a novel therapeutic target for the treatment of ARMS.

Project description:Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS can be parsed based on clinical outcome into two subtypes, fusion-positive RMS (FP-RMS) or fusion-negative RMS (FN-RMS) based on the presence or absence of either PAX3-FOXO1 or PAX7-FOXO1 gene fusions. In both RMS subtypes, tumor cells show histology and a gene expression pattern resembling that of developmentally arrested skeletal muscle. Differentiation therapy is an attractive approach to embryonal tumors of childhood including RMS; however, agents to drive RMS differentiation have not entered the clinic and their mechanisms remain unclear. MicroRNA-206 (miR-206) expression increases through normal muscle development and has decreased levels in RMS compared with normal skeletal muscle. Increasing miR-206 expression drives differentiation of RMS, but the target genes responsible for the relief of the development arrest are largely unknown. Using a combinatorial approach with gene and proteomic profiling coupled with genetic rescue, we identified key miR-206 targets responsible for the FN-RMS differentiation blockade, PAX7, PAX3, NOTCH3, and CCND2. Specifically, we determined that PAX7 downregulation is necessary for miR-206-induced cell cycle exit and myogenic differentiation in FN-RMS but not in FP-RMS. Gene knockdown of targets necessary for miR-206-induced differentiation alone or in combination was not sufficient to phenocopy the differentiation phenotype from miR-206, thus illustrating that miR-206 replacement offers the ability to modulate a complex network of genes responsible for the developmental arrest in FN-RMS. Genetic deletion of miR-206 in a mouse model of FN-RMS accelerated and exacerbated tumor development, indicating that both in vitro and in vivo miR-206 acts as a tumor suppressor in FN-RMS at least partially through downregulation of PAX7. Collectively, our results illustrate that miR-206 relieves the differentiation arrest in FN-RMS and suggests that miR-206 replacement could be a potential therapeutic differentiation strategy.

Dataset Information

A Drosophila model of the rhabdomyosarcoma initiator PAX7-FKHR.

Publications

A Drosophila model of the rhabdomyosarcoma initiator PAX7-FKHR.

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