Oncogene database

2308

FOXO1

forkhead box O1;FOXO1;forkhead box O1

forkhead box protein O1|forkhead box protein O1A|forkhead, Drosophila, homolog of, in rhabdomyosarcoma

13q14.1

protein-coding

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

The t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma tumors (ARMS) creates an oncogenic transcriptional activator by fusion of PAX3 DNA binding motifs to a COOH-terminal activation domain derived from the FKHR gene. The dominant oncogenic potential of the PAX3-FKHR fusion protein is dependent on the FKHR activation domain. We have fused the KRAB repression module to the PAX3 DNA binding domain as a strategy to suppress the activity of the PAX3-FKHR oncogene. The PAX3-KRAB protein bound PAX3 target DNA sequences and repressed PAX3-dependent reporter plasmids. Stable expression of the PAX3-KRAB protein in ARMS cell lines resulted in loss of the ability of the cells to grow in low-serum or soft agar and to form tumors in SCID mice. Stable expression of a PAX3-KRAB mutant, which lacks repression function, or a KRAB protein alone, lacking a PAX3 DNA binding domain, failed to suppress the ARMS malignant phenotype. These data suggest that the PAX3-KRAB repressor functions as a DNA-binding-dependent suppressor of the transformed phenotype of ARMS cells, probably via competition with the endogenous PAX3-FKHR oncogene and repression of target genes required for ARMS tumorigenesis. The engineered repressor approach that directs a transcriptional repression domain to target genes deregulated by the PAX3-FKHR oncogene may be a useful strategy to identify the target genes critical for ARMS tumorigenesis.

In alveolar rhabdomyosarcomas (ARMSs), a specific chromosomal translocation creates a fusion transcription factor, PAX3-FKHR, that is oncogenic due to transcriptional activation. As a strategy for down-regulation of PAX3-FKHR target genes, we created conditional PAX3 repressors by fusing the PAX3 DNA-binding motifs to the hormone binding domain (HBD) of the estrogen receptor and to the KRAB repression domain. We validated proper expression, specific DNA binding, corepressor interaction, and nuclear localization for the KRAB-PAX3-HBD protein and showed it to be a 4-hydroxytamoxifen-dependent transcriptional repressor of transiently transfected and integrated PAX3 reporters in ARMS cells. We established ARMS cell lines that exhibited stable expression of the conditional PAX3 repressor proteins and used them to down-regulate the malignant growth under low serum or anchorage-independent conditions in a hormone-dependent manner. Terminal deoxynucleotidyl transferase-mediated nick end labeling assays revealed that hormonal activation of the PAX3 repressors induced extensive apoptosis that correlated with down-regulation of BCL-X(L) expression. SCID mice that were engrafted with the KRAB-PAX3-HBD ARMS cell lines and were implanted with 4-hydroxytamoxifen timed-release pellets exhibited suppression of tumor growth and an altered vascularity that was not observed in the control mice. These observations strongly suggest that we have directly repressed the PAX3 target genes that are deregulated by the PAX3-FKHR oncogene in ARMS.

Chromosomal translocations that disrupt the molecular organization of transcription factors are typical of a variety of solid and hematopoietic cancers. Alveolar rhabdomyosarcoma (ARMS), a paediatric soft tissue malignant tumour, is characterized by the recurrent translocation t(2;13)(q35;q14) that fuses the 5 DNA binding domain-encoding sequences of the Pax3 gene with the 3 sequences of the FKHR gene. The insulin-like growth factor (IGF) system has an important role in muscle development as well as in the aetiology of paediatric sarcomas, including ARMS. In the present study the potential regulation of the IGF-I receptor (IGF-I-R) gene by PAX3-FKHR at the transcriptional level was investigated. PAX3-FKHR was able to transactivate the IGF-I-R promoter in sarcoma-derived cell lines, whereas PAX3 exhibited a reduced potency in comparison to the fusion protein. Furthermore, transfection of the chimera induced a significant increase in the endogenous levels of IGF-I-R protein, suggesting that the IGF-I-R gene is a physiologically-relevant molecular target for the PAX3-FKHR oncogene.

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.

tumors have evolved elaborate mechanisms for evading immune detection, such as production of immunoinhibitory cytokines and down-regulation of major histocompatibility complex (MHC) expression. We have studied PAX3-FKHR as an example of an oncogenic fusion protein associated with an aggressive metastatic cancer. We show that PAX3-FKHR alters expression of genes that are normally regulated by Janus kinase/signal transducer and activator of transcription (STAT) signaling pathways. This occurs as a result of a specific interaction between PAX3-FKHR and the STAT3 transcription factor, which results in a dramatic reduction in tumor MHC expression, and an alteration in local cytokine concentrations to inhibit surrounding inflammatory cells and immune detection. Collectively, these data show that an oncogenic transcription factor can promote tumor growth and tissue invasion while inhibiting local inflammatory and immune responses. This is the first time that an immunomodulatory role has been described for an oncogenic fusion protein.

Vav1 is an hematopoietic-specific Rho guanine nucleotide exchange factor coupling tyrosine kinase receptors and Rac GTPases, and has been implicated in transformation of fibroblasts and pancreas. To determine the biologic effect and oncogenic potential of Vav1 in hematopoietic lineages, we stably express oncogenic mutant of Vav1 in primary bone marrow cells using retrovirus-mediated gene transfer. Contrary to the growth stimulatory effects observed in fibroblasts, oncogenic Vav1 inhibits hematopoietic stem cell/progenitor engraftment in vivo and progenitor cell expansion in vitro via inducing apoptosis. The oncogenic Vav1-induced apoptosis is associated with reduced expression of Bcl-2 and Bcl-xL proteins and effectively suppressed by transgenic overexpression of Bcl-2, suggesting Vav1-mediated signaling via Bcl-2 in apoptosis. Also, oncogenic Vav1 stimulates sustained activation of Rac GTPases and the biologic effects of oncogenic Vav1 are Rac-dependent. Further, when expressed in the p53-deficient cells, which express elevated Bcl-2 and Bcl-xL and are resistant to the apoptosis, oncogenic Vav1 enhances both proliferation and self-renewal of hematopoietic progenitor cells. These results demonstrate clear phenotypic differences between wild-type and p53(-/-) hematopoietic cells expressing oncogenic Vav1, and suggest oncogenic potential of Vav1-mediated pathways in primary hematopoietic cell when they collaborate with additional genetic hits that affect the p53 pathway.

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.

PAX3-FKHR, the product of a rearrangement of PAX3 with FKHR is the pathogenetic marker for alveolar rhabdomyosarcoma, an aggressive form of childhood cancer. In this work we show that PAX3-FKHR, which is a stronger transcriptional activator relative to PAX3, can lead to two apparently irreconcilable outcomes: transformation and terminal myogenic differentiation. Fibroblasts (10T1/2, NIH3T3, and a newly established murine line named Plus ) transduced by PAX3-FKHR acquire transformed features such as anchorage independence and loss of contact inhibition and concomitantly undergo various degrees of myogenic conversion depending on the host cells, including, in the case of the Plus line, terminal differentiation into contractile myotubes. This work highlights the potential of PAX3-FKHR to functionally operate as a deregulated Pangene and may have implications with regard to the identity of the precursor cell giving rise to alveolar rhabdomyosarcoma.

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.

Patients with translocation-positive alveolar rhabdomyosarcoma (ARMS), an aggressive childhood tumor primarily characterized by the PAX3-FOXO1 oncogenic fusion protein, have a poor prognosis because of lack of therapies that specifically target ARMS tumors. This fact highlights the need for novel pharmaceutical interventions. Posttranslational modifications such as phosphorylation are becoming attractive biological targets for the development of such interventions. Along these lines, we demonstrated that PAX3-FOXO1 is phosphorylated at three specific sites and that its pattern of phosphorylation is altered relative to wild-type Pax3 throughout early myogenesis and in ARMS tumor cells. However, little work has been performed examining the effect of directly inhibiting phosphorylation at these sites on ARMS development. To address this gap in knowledge, we used small molecule inhibitors or mutational analysis to specifically inhibit phosphorylation of PAX3-FOXO1 to investigate how altering phosphorylation of the oncogenic fusion protein affects ARMS phenotypes. We found that inhibiting the phosphorylation of PAX3-FOXO1 at Ser201 significantly reduced migration, invasion and proliferation in two independent ARMS tumor cell lines. Further, we found that inhibition of phosphorylation at Ser205 also decreased proliferation and anchorage-independent growth. Consistent with these in vitro results, we demonstrate for the first time that PAX3-FOXO1 is phosphorylated at Ser201 and Ser205 in a primary tumor sample and in tumor cells actively invading the surrounding normal tissue. This report is the first to demonstrate that the direct inhibition of PAX3-FOXO1 phosphorylation reduces ARMS tumor phenotypes in vitro and that these phosphorylation events are present in primary human ARMS tumors and invading tumor cells. These results identify phosphorylation of PAX3-FOXO1, especially at Ser201, as a novel biological target that can be explored as a promising avenue for ARMS therapies.