Induction of apoptosis in rhabdomyosarcoma cells through down-regulation of PAX proteins (original) (raw)
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transcriptional activator than PAX3. rhabdomyosarcomas is a more potent t(2;13) translocation in alveolar The PAX3-FKHR fusion protein created by the http://mcb.asm.org/content/15/3/1522 Updated information and services can be found at: These include: CONTENT ALERTS more» cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on February 21, 2014 by guest Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH 2 -terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family.
The hybrid PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma transforms fibroblasts in culture
Proceedings of the National Academy of Sciences, 1996
Pediatric alveolar rhabdomyosarcoma is characterized by a chromosomal translocation that fuses parts of the PAX3 and FKHIR genes. PAX3 codes for a transcriptional regulator that controls developmental programs, and FKHR codes for a forkhead-winged helix protein, also a likely transcription factor. The PAX3-FKHR fusion product retains the DNA binding domains of the PAX3 protein and the putative activator domain of the FKHR protein. The PAX3-FKHR protein has been shown to function as a transcriptional activator. Using the RCAS retroviral vector, we have introduced the PAX3-FKHR gene into chicken embryo fibroblasts. Expression of the PAX3-FKHR protein in these cells leads to transformation: the cells become enlarged, grow tightly packed and in multiple layers, and acquire the ability for anchorage-independent growth. This cellular transformation in vitro will facilitate studies on the mechanism of PAX3-FKHR-induced oncogenesis.
Proceedings of the National Academy of Sciences, 1996
The t(2;13) translocation of alveolar rhabdomyosarcoma results in tumor-specific expression of a chimeric transcription factor containing the N-terminal DNA-binding domain of PAX3 and the C-terminal transactivation domain of FKHR. Here we have tested the hypothesis that PAX3-FKHR gains function relative to PAX3 as a consequence of switching PAX3 and FKHR transactivation domains, which were previously shown to have similar potency but distinct structural motifs. In transient cotransfection assays with human expression constructs, we have demonstrated the increased ability of PAX3-FKHR to activate transcription of a reporter gene located downstream of multimerized e5, PRS-9, or CD19 DNA-binding sites in three cell lines. For example, PAX3-FKHR was 100-fold more potent than PAX3 as an activator binding to e5 sites in NIH 3T3 cells. To compare transactivation potency independent of PAX3-specific DNA binding, we tested GAL4 fusions of full-length PAX3 and PAX3-FKHR or their respective C-terminal transactivation domains on a reporter with GAL4 DNA-binding sites. In this context, full-length PAX3-FKHR was also much more potent than PAX3. Additionally, the activity of each full-length protein was decreased relative to its C-terminal domain, demonstrating that N-terminal sequences are inhibitory. By deletion analysis, we mapped a bipartite cis-acting inhibitory domain to the same subregions within the DNA-binding domains of both PAX3 and PAX3-FKHR. We have shown, however, that the structurally distinct transactivation domains of PAX3 and PAX3-FKHR differ 10- to 100-fold in their susceptibility to inhibition, thus elucidating a mechanism by which PAX3 gains enhanced function during oncogenesis.
Fusion of the Paired Box 3 (PAX3) and Myocardin (MYOCD) Genes in Pediatric Rhabdomyosarcoma
Cancer Genomics & Proteomics, 2021
Background/Aim: Fusions of the paired box 3 gene (PAX3 in 2q36) with different partners have been reported in rhabdomyosarcomas and biphenotypic sinonasal sarcomas. We herein report the myocardin (MYOCD on 17p12) gene as a novel PAX3-fusion partner in a pediatric tumor with adverse clinical outcome. Materials and Methods: A rhabdomyosarcoma found in a 10-year-old girl was studied using a range of genetic methodologies. Results: The karyotype of the tumor cells was 48,XX,add(2)(q11),+del(2)(q35),add(3)(q?25),-7,del(8)(p21),-15,add(17)(p11),+20,+der(?)t(?;15) (?;q15),+mar[8]/46,XX[2]. Fluorescence in situ hybridization detected PAX3 rearrangement whereas array comparative genomic hybridization revealed genomic imbalances affecting hundreds of genes, including MYCN, MYC, FOXO3, and the tumor suppressor gene TP53. A PAX3-MYOCD fusion transcript was found by RNA sequencing and confirmed by Sanger sequencing. Conclusion: The investigated rhabdomyosarcoma carried a novel PAX3-MYOCD fusion gene and extensive additional aberrations affecting the allelic balance of many genes, among them TP53 and members of MYC and FOXO families of transcription factors. Alveolar rhabdomyosarcomas are cytogenetically characterized by the specific chromosome translocations t(2;13)(q36;q14) and t(1;13)(p36;q14) (1-4). The t(2;13)(q36;q14) results in fusion of the paired box 3 (PAX3) gene from 2q36 with the forkhead box O1 gene (FOXO1, also known as FKHR) from 13q14 (5-7), whereas t(1;13)(p36;q14) fuses the paired box 7 (PAX7) gene from 1p36 with FOXO1 (8). The abovementioned chromosome aberrations and their corresponding fusion genes are found in 80% of alveolar rhabdomyosarcomas (9). In the remaining 20%, fusions of PAX3 with the genes FOXO4 (also known as AFX, in Xq13), nuclear receptor coactivator 1 (NCOA1, in 2p23), nuclear receptor coactivator 2 (NCOA2, in 8q13) or INO80 complex subunit D (INO80D, in 2q33) were found (9-12). Apart from alveolar rhabdomyosarcomas, PAX3-FOXO1, PAX3-NCOA1, and fusion of PAX3 with the mastermind-like transcriptional coactivator 3 gene (MAML3, from 4q31.1; recombination occurs through a 2q35;4q31-chromosomal translocation) were also detected in biphenotypic sinonasal sarcomas (13-16). Furthermore, a PAX3-NCOA2 fusion was reported in embryonal rhabdomyosarcoma (11, 17). In the present study, we report the finding in a pediatric rhabdomyosarcoma of a novel fusion of PAX3 with the myocardin (MYOCD) gene which maps to 17p12 and codes for a smooth and cardiac muscle-specific transcriptional coactivator of the serum response factor. Materials and Methods Ethics statement. The study was approved by the Regional Ethics Committee (Regional komité for medisinsk forskningsetikk Sør-Øst, Norge, http://helseforskning.etikkom.no). All patient information has been de-identified. Case description. The patient was a ten-year-old girl with an advanced stage of rhabdomyosarcoma. The tumor presented as a pelvic mass with spreading to pelvic and abdominal lymph nodes, several pelvic and abdominal viscera, and tumorous nodules within the abdominal cavity. Examination of a diagnostic biopsy showed a 723 This article is freely accessible online.
Identification of target genes of PAX3-FOXO1 in alveolar rhabdomyosarcoma
Oncology Reports, 2013
Background: The majority of alveolar rhabdomyosarcoma (ARMS) are distinguished through the paired box 3−forkhead box protein O1 (PAX3−FOXO1) fusion oncoprotein, being generated by a 2;13 chromosomal translocation. This fusion-positive ARMS is the most clinically difficult type of rhabdomyosarcoma. The present study characterized four genes [gremlin 1 (GREM1), deathassociated protein kinase-1 (DAPK1), myogenic differentiation-1 (MYOD1), and hairy/enhancer-of-split related with YRPW motif-1 (HEY1)] as targets of PAX3−FOXO1. Materials and Methods: The expression of the four genes, PAX3−FOXO1, and v-myc myelocytomatosis viral-related oncogene, neuroblastoma-derived (avian) (MYCN) was determined in various ARMS cell models and primary tumors. The roles of PAX3−FOXO1 and MYCN expression were examined. Results: Pulse-chase and cycloheximide experiments suggest that GREM1, DAPK1, and MYOD1 are directly regulated by PAX3−FOXO1. PAX3−FOXO1 appears to indirectly down-regulate HEY1 by up-regulating MYCN. Data reveal that the growthsuppressive activity of high PAX3−FOXO1 expression is closely-associated with up-regulation of the GREM1 and DAPK1 tumor-suppressor genes. Conclusion: This study characterized four downstream targets of PAX3−FOXO1 that contribute to the biological activities of growth suppression and myogenic differentiation. Rhabdomyosarcoma is the most prominent type of pediatric soft tissue sarcoma, being associated with the skeletal muscle lineage. Among pediatric rhabdomyosarcoma, there are two major subtypes based on their histological appearance: embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS) (1). ARMS has a higher frequency of metastases at the initial diagnosis than ERMS, commonly conferring a poorer prognosis than ERMS (2, 3). A common characteristic of ERMS is a loss of heterozygosity at 11p15, however ERMS has not been reported to exhibit a diagnostic genetic alteration. In contrast, chromosomal translocation is frequently observed for ARMS (4, 5). The translocation t(2;13)(q35;q14) generating the paired box 3−forkhead box protein O1 (PAX3−FOXO1) gene fusion was found to occur in 55% of ARMS cases, while the translocation t(1;13)(q36;q14) generating the paired box 7−forkhead box protein O1 (PAX7−FOXO1) gene fusion occurred in 22% of cases, and 23% of ARMS were fusionnegative (2). The 2;13 translocation present in ARMS is characterized by an overexpression of PAX3−FOXO1 relative to wild-type PAX3 (6). Although PAX3−FOXO1-positive ARMS is the most clinically intractable fusion subtype of pediatric rhabdomyosarcoma, it can be difficult to histologically classify rhabdomyosarcoma into ARMS and ERMS in some cases (7), and no specific drugs are available for treating a specific fusion or histological subtype (1, 2, 7, 8). Therefore, there is a strong incentive to elucidate target genes of PAX3−FOXO1, which has the utility of being a therapeutic target and marker for the purpose of diagnosis and treatment of rhabdomyosarcoma. Several recent studies have utilized the gene expression profiles to classify rhabdomyosarcoma or identify target genes of only PAX3−FOXO1, or both PAX3−FOXO1 and PAX7−FOXO1, however, more research is needed to validate the biological function of the genes in ARMS development (9-15). Recently Ahn and co-authors identified 34 potential target genes and validated the four genes, gremlin-1 (GREM1), death-associated protein kinase-1 (DAPK1), myogenic differentiation (MYOD1), and hairy/enhancerof-split related with YRPW motif-1 (HEY1), as target genes of PAX3−FOXO1 in ARMS by analyzing gene 2029
Proceedings of the National Academy of Sciences, 2001
A unique chromosomal translocation involving the genes PAX3 and FKHR is characteristic of most human alveolar rhabdomyosarcomas. The resultant chimeric protein fuses the PAX3 DNA-binding domains to the transactivation domain of FKHR, suggesting that PAX3-FKHR exerts its role in alveolar rhabdomyosarcomas through dysregulation of PAX3-specific target genes. Here, we have produced transgenic mice in which PAX3-FKHR expression was driven by mouse Pax3 promoter͞enhancer sequences. Five independent lines expressed PAX3-FKHR in the dorsal neural tube and lateral dermomyotome. Each line exhibited phenotypes that correlated with PAX3-FKHR expression levels and predominantly involved pigmentary disturbances of the abdomen, hindpaws, and tail, with additional neurological related alterations. Phenotypic severity could be increased by reducing Pax3 levels through matings with Pax3-defective Splotch mice, and interference between PAX3 and PAX3-FKHR was apparent in transcription reporter assays. These data suggest that the tumor-associated PAX3-FKHR fusion protein interferes with normal Pax3 developmental functions as a prelude to transformation.
A novel PAX3 rearrangement in embryonal rhabdomyosarcoma
Cancer Genetics and Cytogenetics, 2009
Rhabdomyosarcoma is the most common soft tissue tumor seen in children and young adults, and it can be classified into 2 major histological subtypes, alveolar and embryonal. In the alveolar subtype, 2 recurrent chromosomal translocations, t(2;13)(q35;q14) and its variant t(1;13)(p36;q14), have been identified as the specific cytogenetic abnormalities. These translocations produce the PAX3-FOXO1 and PAX7-FOXO1 fusion genes, respectively. In the embryonal subtype, however, no recurrent chromosomal abnormalities have been identified. In this study, we analyzed the complex chromosomal translocation in one case with embryonal rhabdomyosarcoma by means of spectral karyotyping (SKY) and identified a novel translocation involving chromosome band 2q35, which is the locus of PAX3 gene. Furthermore, we identified the novel PAX3 rearrangement using fluorescence in situ hybridization (FISH) analysis. Additional identification of the partner gene may help disclose the molecular mechanism of the development of this embryonal subtype.
Genes & Development, 2004
To investigate the role of the translocation-associated gene Pax3:Fkhr in alveolar rhabdomyosarcomas, we generated a Cre-mediated conditional knock-in of Pax3:Fkhr into the mouse Pax3 locus. Exploring embryonic tumor cell origins, we replaced a Pax3 allele with Pax3:Fkhr throughout its expression domain, causing dominant-negative effects on Pax3 and paradoxical activation of the Pax3 target gene, c-Met. Ectopic neuroprogenitor cell proliferation also occurs. In contrast, activation later in embryogenesis in cells that express Pax7 results in viable animals with a postnatal growth defect and a moderately decreased Pax7+ muscle satellite cell pool, phenocopying Pax7 deficiency but remarkably not leading to tumors.
Alveolar rhabdomyosarcoma - The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis
Skeletal muscle, 2012
Rhabdomyosarcoma is a soft tissue sarcoma arising from cells of a mesenchymal or skeletal muscle lineage. Alveolar rhabdomyosarcoma (ARMS) is more aggressive than the more common embryonal (ERMS) subtype. ARMS is more prone to metastasis and carries a poorer prognosis. In contrast to ERMS, the majority of ARMS tumors carry one of several characteristic chromosomal translocations, such as t(2;13)(q35;q14), which results in the expression of a PAX3-FOXO1 fusion transcription factor. In this review we discuss the genes that cooperate with PAX3-FOXO1, as well as the target genes of the fusion transcription factor that contribute to various aspects of ARMS tumorigenesis. The characterization of these pathways will lead to a better understanding of ARMS tumorigenesis and will allow the design of novel targeted therapies that will lead to better treatment for this aggressive pediatric tumor.