Project description:Background: Alveolar 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. Methods: To 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 microarrays 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 directly and specifically 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, Results: We found that When PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds directly and specifically to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel direct transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1Adecreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR-mediated cell migration and metastasis. Conclusions: Taken together, we have identified CPT1A as a novel direct 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:Background: Alveolar 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. Methods: To 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 microarrays 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 directly and specifically 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, Results: We found that When PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds directly and specifically to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel direct transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1Adecreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR-mediated cell migration and metastasis. Conclusions: Taken together, we have identified CPT1A as a novel direct 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. Identification of transcription targets of PAX3-FKHR in human alveolar rhabdomyosarcoma: By stably knocking down PAX3-FKHR in human alveolar rhabdomyosarcoma cell line Rh30 and comparing the gene expression profile of the knockdown clone (KD) to the parental Rh30, transcription targets of PAX3-FKHR have been identified. Gene expression in parental Rh30 cells was compared to that in either Rh30 cells stably expressing shRNA targeting PAX3-FKHR (KD) or control non-targeting shRNA (CON), in duplicate.

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Cell-to-cell variability in PAX3:FOXO1 expression determines tumorigenic potential in rhabdomyosarcoma

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