PAX3

Gene Summary

Gene:PAX3; paired box 3
Aliases: WS1, WS3, CDHS, HUP2
Location:2q35
Summary:This gene is a member of the paired box (PAX) family of transcription factors. Members of the PAX family typically contain a paired box domain and a paired-type homeodomain. These genes play critical roles during fetal development. Mutations in paired box gene 3 are associated with Waardenburg syndrome, craniofacial-deafness-hand syndrome, and alveolar rhabdomyosarcoma. The translocation t(2;13)(q35;q14), which represents a fusion between PAX3 and the forkhead gene, is a frequent finding in alveolar rhabdomyosarcoma. Alternative splicing results in transcripts encoding isoforms with different C-termini. [provided by RefSeq, Jul 2008]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:paired box protein Pax-3
HPRD
Source:NCBIAccessed: 17 August, 2015

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 17 August 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 17 August, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Entity Topic PubMed Papers
-PAX3 and Alveolar Rhabdomyosarcoma View Publications143
Soft Tissue SarcomaPAX3 and Soft Tissue Cancers View Publications36
MelanomaPAX3 and Melanoma View Publications31
Urinary System CancersPAX3 and Urinary System Cancers View Publications15
Rhabdomyosarcomat(2;13)(q35;q14) in Rhabdomyosarcoma
Alveolar rhabdomyosarcoma, a malignant tumour of skeletal muscle usually found in children and young adults, is characterised by a chromosomal translocation of the PAX3-FKHR genes: t(2;13)(q35;q14).

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: PAX3 (cancer-related)

Lagutina IV, Valentine V, Picchione F, et al.
Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in mouse myoblasts using CRISPR-Cas9 nuclease.
PLoS Genet. 2015; 11(2):e1004951 [PubMed] Free Access to Full Article Related Publications
Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.

Zhang L, Xia L, Zhao L, et al.
Activation of PAX3-MET pathways due to miR-206 loss promotes gastric cancer metastasis.
Carcinogenesis. 2015; 36(3):390-9 [PubMed] Related Publications
MicroRNAs (miRNAs) are thought to have an important role in tumor metastasis by regulating diverse cellular pathways. Here, we describe the function and regulation network of miR-206 in gastric cancer (GC) metastasis. MiR-206 expression was downregulated in GC cells especially in high metastatic potential cells and was also significantly decreased in metastatic lesions compared with their corresponding primary tumor samples. Both gain- and loss-of-function studies confirmed that miR-206 significantly suppressed GC cell invasion and metastasis both in vitro and in vivo. Mechanistically, paired box gene 3 (PAX3) was identified as a functional target of miR-206 in GC cells. MiR-206 inhibited GC metastasis by negatively regulating expression of PAX3. In addition, PAX3 expression was markedly higher in GC tissues than in adjacent non-cancerous tissues. GC patients with positive PAX3 expression had shorter overall survival times. Transwell assays and in vivo metastasis assays demonstrated that overexpression of PAX3 significantly promoted the invasiveness and pulmonary metastasis of GC cells. On the other hand, downregulation of PAX3 markedly reduced cell metastatic potential. Mechanistic investigations indicated that prometastasis function of PAX3 was mediated by upregulating downstream target MET. Moreover, we found that levels of PAX3 and MET were positively correlated in matched human GC specimens, and their coexpression was associated with poor prognoses. In conclusion, our results reveal that miR-206-PAX3-MET signaling is critical to GC metastasis. Targeting the pathway described here may open new therapeutic prospects to restrict the metastatic potential of GC.

Thalhammer V, Lopez-Garcia LA, Herrero-Martin D, et al.
PLK1 phosphorylates PAX3-FOXO1, the inhibition of which triggers regression of alveolar Rhabdomyosarcoma.
Cancer Res. 2015; 75(1):98-110 [PubMed] Related Publications
Pediatric tumors harbor very low numbers of somatic mutations and therefore offer few targets to improve therapeutic management with targeted drugs. In particular, outcomes remain dismal for patients with metastatic alveolar rhabdomyosarcoma (aRMS), where the chimeric transcription factor PAX3/7-FOXO1 has been implicated but problematic to target. In this report, we addressed this challenge by developing a two-armed screen for druggable upstream regulatory kinases in the PAX3/7-FOXO1 pathway. Screening libraries of kinome siRNA and small molecules, we defined PLK1 as an upstream-acting regulator. Mechanistically, PLK1 interacted with and phosphorylated PAX3-FOXO1 at the novel site S503, leading to protein stabilization. Notably, PLK1 inhibition led to elevated ubiquitination and rapid proteasomal degradation of the PAX3-FOXO1 chimeric oncoprotein. On this basis, we embarked on a preclinical validation of PLK1 as a target in a xenograft mouse model of aRMS, where the PLK1 inhibitor BI 2536 reduced PAX3-FOXO1-mediated gene expression and elicited tumor regression. Clinically, analysis of human aRMS tumor biopsies documented high PLK1 expression to offer prognostic significance for both event-free survival and overall survival. Taken together, these preclinical studies validate the PLK1-PAX3-FOXO1 axis as a rational target to treat aRMS.

Hettmer S, Li Z, Billin AN, et al.
Rhabdomyosarcoma: current challenges and their implications for developing therapies.
Cold Spring Harb Perspect Med. 2014; 4(11):a025650 [PubMed] Related Publications
Rhabdomyosarcoma (RMS) represents a rare, heterogeneous group of mesodermal malignancies with skeletal muscle differentiation. One major subgroup of RMS tumors (so-called "fusion-positive" tumors) carries exclusive chromosomal translocations that join the DNA-binding domain of the PAX3 or PAX7 gene to the transactivation domain of the FOXO1 (previously known as FKHR) gene. Fusion-negative RMS represents a heterogeneous spectrum of tumors with frequent RAS pathway activation. Overtly metastatic disease at diagnosis is more frequently found in individuals with fusion-positive than in those with fusion-negative tumors. RMS is the most common pediatric soft-tissue sarcoma, and approximately 60% of all children and adolescents diagnosed with RMS are cured by currently available multimodal therapies. However, a curative outcome is achieved in <30% of high-risk individuals with RMS, including all those diagnosed as adults, those diagnosed with fusion-positive tumors during childhood (including metastatic and nonmetastatic tumors), and those diagnosed with metastatic disease during childhood (including fusion-positive and fusion-negative tumors). This white paper outlines current challenges in RMS research and their implications for developing more effective therapies. Urgent clinical problems include local control, systemic disease, need for improved risk stratification, and characterization of differences in disease course in children and adults. Biological challenges include definition of the cellular functions of PAX-FOXO1 fusion proteins, clarification of disease heterogeneity, elucidation of the cellular origins of RMS, delineation of the tumor microenvironment, and identification of means for rational selection and testing of new combination therapies. To streamline future therapeutic developments, it will be critical to improve access to fresh tumor tissue for research purposes, consider alternative trial designs to optimize early clinical testing of candidate drugs, coalesce advocacy efforts to garner public and industry support, and facilitate collaborative efforts between academia and industry.

Almazán-Moga A, Roma J, Molist C, et al.
Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry.
Cytometry A. 2014; 85(12):1020-9 [PubMed] Related Publications
Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Circulating tumor cells in peripheral blood or disseminated to bone marrow, a concept commonly referred to as minimal residual disease (MRD), are thought to be key to the prediction of metastasis and treatment efficacy. To date, two MRD markers, MYOD and MYOGENIN, have been tested; however, MRD detection continues to be challenging mainly owing to the closeness of the detection limit and the discordance of both markers in some samples. Therefore, the addition of a third marker could be useful for more accurate MRD assessment. The PAX3 gene is expressed during embryo development in all myogenic precursor cells in the dermomyotome. As RMS cells are thought to originate from these muscle precursor cells, they are expected to be positive for PAX3. In this study, PAX3 expression was characterized in cancer cell lines and tumors, showing wide expression in RMS. Detection sensitivities by quantitative polymerase chain reaction (qPCR) of the previously proposed markers, MYOD and MYOGENIN, were similar to that of PAX3, thereby indicating the feasibility of its detection. Interestingly, the flow cytometry experiments supported the usefulness of this technique in the quantification of MRD in RMS using PAX3 as a marker. These results indicate that flow cytometry, albeit in some cases slightly less sensitive, can be considered a good approach for MRD assessment in RMS and more consistent than qPCR, especially owing to its greater specificity. Furthermore, fluorescence-activated cell sorting permits the recovery of cells, thereby providing material for further characterization of circulating or disseminated cancer cells.

Choi H, Jin SH, Han MH, et al.
Human melanocytes form a PAX3-expressing melanocyte cluster on Matrigel by the cell migration process.
J Dermatol Sci. 2014; 76(1):60-6 [PubMed] Related Publications
BACKGROUND: The interactions between human epidermal melanocytes and their cellular microenvironment are important in the regulation of human melanocyte functions or in their malignant transformation into melanoma. Although the basement membrane extracellular matrix (BM-ECM) is one of major melanocyte microenvironments, the effects of BM-ECM on the human melanocyte functions are not fully explained at a molecular level.
OBJECTIVE: This study was aimed to characterize the molecular and cellular interactions between normal human melanocytes (NHMs) and BM-ECM.
METHODS: We investigated cell culture models of normal human melanocytes or melanoma cells on three-dimensional (3D) Matrigel to understand the roles of the basement membrane microenvironment in human melanocyte functions. Melanogenesis and melanobast biomarker expression in both primary human melanocytes and melanoma cells on 3D Matrigel were evaluated.
RESULTS: We found that NHMs migrated and formed reversible paired box 3 (PAX3) expressing cell clusters on three-dimensional (3D) Matrigel. The melanogenesis was significantly decreased in the PAX3 expressing cell cluster. The expression profile of PAX3, SOX10, and MITF in the melanocyte cluster on 3D Matrigel was similar to that of melanoblasts. Interestingly, PAX3 and SOX10 showed an inverse expression profile in NHMs, whereas the inverse expression pattern of PAX3 and SOX10 was disrupted in melanoma MNT1 and WM266-4 cells.
CONCLUSION: The human melanocyte culture on 3D Matrigel provides an alternative model system to study functions of human melanoblasts. In addition, this system will contribute to the elucidation of PAX3-related tumorigenic mechanisms to understand human melanoma.

Abraham J, Nuñez-Álvarez Y, Hettmer S, et al.
Lineage of origin in rhabdomyosarcoma informs pharmacological response.
Genes Dev. 2014; 28(14):1578-91 [PubMed] Free Access to Full Article Related Publications
Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

Bergantin E, Quarta C, Nanni C, et al.
Sulforaphane induces apoptosis in rhabdomyosarcoma and restores TRAIL-sensitivity in the aggressive alveolar subtype leading to tumor elimination in mice.
Cancer Biol Ther. 2014; 15(9):1219-25 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Rhadbomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is subdivided in the embryonal (ERMS) and alveolar (ARMS) subtypes, the latter being associated with the worst prognosis. We report that sulforaphane (SFN), a broccoli-derived anticancer isothiocyanate, causes dose- and time-dependent growth inhibition and apoptosis in both ERMS and ARMS cells. In ARMS, SFN induced the modulation of expression of crucial genes and proteins: mRNA and protein levels of PAX3-FKHR, MYCN, and MET decreased, while those of p21 and TRAIL-receptor DR5 (but not DR4) increased. Since DR5 expression increased specifically in ARMS, we treated ARMS cells with TRAIL, SFN, or their combination. While ARMS cells (RH30 and RH4) proved to be TRAIL-resistant, SFN restored their sensitivity to TRAIL-induced cell-growth inhibition, leading to a stronger effect in combination with TRAIL. ARMS cells transfected with siDR5 showed that SFN-induced DR5 acts as a key regulator, being directly related to the TRAIL-induced cell-growth inhibition. The in vivo anti-tumor activity of SFN and TRAIL was evaluated in a xenograft murine model of ARMS through microPET. The results showed that the systemic treatment (3 wk) of mice with SFN or TRAIL as single agents only delayed tumor evolution, while the combined treatment of SFN and TRAIL led to tumor elimination. These findings indicate that SFN triggers the apoptotic pathway in both alveolar and embryonal rhabdomyosarcomas and that combined treatment with SFN and TRAIL might be a promising therapy for the aggressive alveolar subtype.

Wang X, Bledsoe KL, Graham RP, et al.
Recurrent PAX3-MAML3 fusion in biphenotypic sinonasal sarcoma.
Nat Genet. 2014; 46(7):666-8 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Biphenotypic sinonasal sarcoma (SNS) is a newly described tumor of the nasal and paranasal areas. Here we report a recurrent chromosomal translocation in SNS, t(2;4)(q35;q31.1), resulting in a PAX3-MAML3 fusion protein that is a potent transcriptional activator of PAX3 response elements. The SNS phenotype is characterized by aberrant expression of genes involved in neuroectodermal and myogenic differentiation, closely simulating the developmental roles of PAX3.

de Souza RR, Oliveira ID, del Giúdice Paniago M, et al.
Investigation of IGF2, Hedgehog and fusion gene expression profiles in pediatric sarcomas.
Growth Horm IGF Res. 2014; 24(4):130-6 [PubMed] Related Publications
UNLABELLED: The childhood sarcomas are malignant tumors with high mortality rates. They are divided into two genetic categories: a category without distinct pattern karyotypic changes and the other category showing unique translocations that originate gene rearrangements. This category includes rhabdomyosarcoma (RMS), Ewing's sarcoma (ES) and synovial sarcoma (SS). Diverse studies have related development genes, such as; IGF2, IHH, PTCH1 and GLI1 and sarcomatogenesis.
OBJECTIVE: To characterize the RMS, ES and SS rearrangements, we quantify the expression of IGF2 IHH, PTCH1 and GLI1 genes and correlate molecular data with clinical parameters of patients.
DESIGN: We analyzed 29 RMS, 10 SS and 60 ES tumor samples by RT-PCR (polymerase chain reaction-reverse transcription) and qPCR (quantitative PCR).
RESULTS: Among the samples of ARMS, 50% had rearrangements of PAX3/7-FOXO1, 60% of ES samples were EWS-FLI1 positive and 90% of SS samples were positive for SS18-SSX1/2. In relation to the control reference samples (QPCR Human Reference Total RNA-Stratagene, Human Skeletal Muscle Total RNA-Ambion, Universal RNA Human Normal Tissues-Ambion), RMS samples showed a high IGF2 gene expression (p<0.0001). Moreover, ES samples showed a low IGF2 gene expression (p<0.0001) and high IHH (p<0.0001), PTCH1 (p=0.0173) and GLI1 (p=0.0113) gene expressions.
CONCLUSIONS: The molecular characterization of IGF and Hedgehog pathway in these pediatric sarcomas may collaborate to enable a better understanding of the biological behavior of these neoplasms.

De Salvo M, Raimondi L, Vella S, et al.
Hyper-activation of Notch3 amplifies the proliferative potential of rhabdomyosarcoma cells.
PLoS One. 2014; 9(5):e96238 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.

Rudzinski ER, Anderson JR, Lyden ER, et al.
Myogenin, AP2β, NOS-1, and HMGA2 are surrogate markers of fusion status in rhabdomyosarcoma: a report from the soft tissue sarcoma committee of the children's oncology group.
Am J Surg Pathol. 2014; 38(5):654-9 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Pediatric rhabdomyosarcoma (RMS) is traditionally classified on the basis of the histologic appearance into alveolar (ARMS) and embryonal (ERMS) subtypes. The majority of ARMS contain a PAX3-FOXO1 or PAX7-FOXO1 gene fusion, but about 20% do not. Intergroup Rhabdomyosarcoma Study stage-matched and group-matched ARMS typically behaves more aggressively than ERMS, but recent studies have shown that it is, in fact, the fusion status that drives the outcome for RMS. Gene expression microarray data indicate that several genes discriminate between fusion-positive and fusion-negative RMS with high specificity. Using tissue microarrays containing a series of both ARMS and ERMS, we identified a panel of 4 immunohistochemical markers-myogenin, AP2β, NOS-1, and HMGA2-which can be used as surrogate markers of fusion status in RMS. These antibodies provide an alternative to molecular methods for identification of fusion-positive RMS, particularly in cases in which there is scant or poor-quality material. In addition, these antibodies may be useful in fusion-negative ARMS as an indicator that a variant gene fusion may be present.

Lynn M, Shah N, Conroy J, et al.
A study of alveolar rhabdomyosarcoma copy number alterations by single nucleotide polymorphism analysis.
Appl Immunohistochem Mol Morphol. 2014; 22(3):213-21 [PubMed] Related Publications
Rhabdomyosarcoma, the most common pediatric soft tissue malignancy arises in 2 major histologic forms: embryonal and alveolar. Classically, the alveolar subtype is characterized by a chromosomal translocation t(2;13)(q35;q14) or t(1;13)(p36;q14) fusing the PAX3 or PAX7 gene, respectively, to the FOXO1 gene, although fusion-negative cases of alveolar rhabdomyosarcoma (ARMS) occur; these share considerably more with the genomic profiles and biological behavior of embryonal rhabdomyosarcoma than with fusion-positive ARMS. The current understanding of any additional genetic aberrations in fusion-positive ARMS is limited. In this study, we evaluated tumor-specific copy number alterations in a cohort of fusion-positive ARMSs using high-resolution technology. The results presented here include previously described changes as well as completely novel findings of copy number alterations in BCR and DICER. The study furthermore highlights associations between fusion type and genotype, as well as outcomes and genotype. Rearrangement of PAX7 is strongly associated with copy number alteration of Glypican 5 (GPC5) and moderately with amplification of IGF1R. There is a moderate association between death from/relapse of disease and, on the one hand, amplification of 12q13.3 (DDIT3; Gli1), and on the other hand, copy number alteration of Wnt6 or LRP1B. Gains of both LRP1B and Gli1 in turn are strongly associated with MycN amplification.

Liu C, Li D, Hu J, et al.
Chromosomal and genetic imbalances in Chinese patients with rhabdomyosarcoma detected by high-resolution array comparative genomic hybridization.
Int J Clin Exp Pathol. 2014; 7(2):690-8 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children. Although associations between ARMS tumorigenesis and PAX3, PAX7, and FKHR are well recognized, the complete genetic etiology underlying RMS pathogenesis and progression remains unclear. Chromosomal copy number variations (CNVs) and the involved genes may play important roles in the pathogenesis and progression of human malignancies. Using high-resolution array comparative genomic hybridization (aCGH), we examined 20 formalin-fixed, paraffin-embedded (FFPE) RMS tumors to explore the involvement of the relevant chromosomal regions with resident genes in RMS tumorigenesis. In RMS, frequent gains were identified on chromosome regions 12q13.3-q14.1, 12q24.31, 17q25.1, 1q21.1, and 7q11.23, whereas frequent losses were observed on chromosome regions 5q13.2, 14q32.33, and 15q11.2. Amplifications were observed on chromosome regions 9p13.3, 12q13.3-q14.1, 12q15, and 16p13.11, whereas deletions were detected on chromosome regions 1p36.33, 1p13.1, 2q11.1, 5q13.2, 8p23.1, 9p24.3, and 16p11.2. Frequent gains were detected in GLI1, GEFT, OS9, and CDK4 (12q13.3-q14.1), being 60% in embryonal rhabdomyosarcoma (ERMS) and 66.67% in alveolar rhabdomyosarcoma (ARMS), respectively. However, frequent losses were detected in IGHG1, IGHM, IGHG3, and IGHG4 (14q32.33), being 70% in ERMS and 55.56% in and ARMS, respectively. Frequent gains were detected in TYROBP, HCST, LRFN3, and ALKBH6 (19q13.12) in ERMS but not in ARMS. The frequency of TYROBP, HCST, LRFN3, and ALKBH6 gains is significantly different in ERMS versus ARMS (P=0.011). The results suggest that novel TYROBP, HCST, LRFN3, and ALKBH6 genes may play important roles in ERMS. The technique used is a feasible approach for array comparative genomic hybridization analysis in archival tumor samples.

Qadir MA, Zhan SH, Kwok B, et al.
ChildSeq-RNA: A next-generation sequencing-based diagnostic assay to identify known fusion transcripts in childhood sarcomas.
J Mol Diagn. 2014; 16(3):361-70 [PubMed] Related Publications
Childhood sarcomas can be extremely difficult to accurately diagnose on the basis of morphological characteristics alone. Ancillary methods, such as RT-PCR or fluorescence in situ hybridization, to detect pathognomonic gene fusions can help to distinguish these tumors. Two major deficiencies of these assays are their inability to identify gene fusions at nucleotide resolution or to detect multiple gene fusions simultaneously. We developed a next-generation sequencing-based assay designated ChildSeq-RNA that uses the Ion Torrent platform to screen for EWSR1-FLI1 and EWSR1-ERG, PAX3-FOXO1 and PAX7-FOXO1, EWSR1-WT1, and ETV6-NTRK3 fusions of Ewing sarcoma (ES), alveolar rhabdomyosarcoma, desmoplastic small round cell tumor, and congenital fibrosarcoma, respectively. To rapidly analyze resulting data, we codeveloped a bioinformatics tool, termed ChildDecode, that operates on a scalable, cloud-computing platform. Total RNA from four ES cell lines plus 33 clinical samples representing ES, alveolar rhabdomyosarcoma, desmoplastic small round cell tumor, and congenital fibrosarcoma tumors was subjected to ChildSeq-RNA. This accurately identified corresponding gene fusions in each tumor type, with no examples of false positive fusion detection in this proof-of-concept study. Comparison with previous RT-PCR findings demonstrated high sensitivity (96.4%; 95% CI, 82.3%-99.4%) and specificity (100%; 95% CI, 56.6%-100%) of ChildSeq-RNA to detect gene fusions. Herein, we propose ChildSeq-RNA as a novel tool to detect gene fusions in childhood sarcomas at single-nucleotide resolution.

Kikuchi K, Hettmer S, Aslam MI, et al.
Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma.
PLoS Genet. 2014; 10(1):e1004107 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.

Shern JF, Chen L, Chmielecki J, et al.
Comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion-positive and fusion-negative tumors.
Cancer Discov. 2014; 4(2):216-31 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
UNLABELLED: Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Children's Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma.
SIGNIFICANCE: This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention.

Win KT, Lee MY, Tan TD, et al.
Nasopharyngeal alveolar rhabdomyosarcoma expressing CD56: a mimicker of extranodal natural killer/T-cell lymphoma.
Int J Clin Exp Pathol. 2014; 7(1):451-5 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Alveolar rhabdomyosarcoma (ARMS) is remarkably rare in adults older than 45 years. Histologically, the tumor is composed of blue round cells with frequent expression of CD56 in addition to myogenic markers. Recent studies of ARMS have shown two specific recurrent translocations: PAX3-FKHR [t(2;13)(q35;q14)] or PAX7-FKHR [t(1;13)(p36;q14)]. Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) occurs most frequently in the upper aerodigestive tract with a male preference in East Asia and Central and South Americas with neoplastic cells frequently expressing CD56. We report a 53-year-old Taiwanese man presenting with a nasopharyngeal mass, cervical lymphadenopathy, and multiple bone metastases. Histologically, the nasopharyngeal biopsy revealed diffuse sheets of small blue round tumor cells without obvious alveolar pattern, angioinvasion or tumor necrosis. An initial erroneous diagnosis of ENKTL was made due to CD56 expression using fresh tumor tissue with flow cytometric analysis and the patient was treated accordingly. Retrospective study showed that the tumor cells expressed CD56, desmin, and myogenin. Fluorescence in situ hybridization revealed that the tumor cells were positive for FKHR gene rearrangement, confirming the diagnosis of ARMS. Our case illustrates that a diagnosis of ENKTL based solely on CD56 expression can be misleading for a nasopharyngeal small blue round cell tumor. ARMS should be included as a differential diagnosis, and a correct diagnosis can be reached only after a high index of suspicion and a thorough histological examination with the aid of ancillary studies.

Zin A, Bertorelle R, Dall'Igna P, et al.
Epithelioid rhabdomyosarcoma: a clinicopathologic and molecular study.
Am J Surg Pathol. 2014; 38(2):273-8 [PubMed] Related Publications
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and is mostly represented by the embryonal (ERMS) and alveolar (ARMS) histotypes. Whereas ERMS shows variable genetic alterations including TP53, RB1, and RAS mutations, ARMS carries a gene fusion between PAX3 or PAX7 and FOXO1. Epithelioid RMS is a morphologic variant of RMS recently described in adults. Five cases of epithelioid RMS were identified after histologic review of 85 cases of ARMS enrolled in Italian therapeutic protocols. Immunostaining analyses (muscle-specific actin, desmin, myogenin, AP-2β, EMA, cytokeratins, INI-1) and reverse transcription polymerase chain reaction assays to detect MyoD1, myogenin, and PAX3/7-FOXO1 transcripts were performed. In 4 cases DNA sequencing of TP53 was performed; and RB1 allelic imbalance and homozygous deletion were analyzed by quantitative real-time polymerase chain reaction. Histologically, epithelioid RMS displayed sheets of large cells without rhabdomyoblastic differentiation or anaplasia in 3 and prominent rhabdoid cells in 2; necrosis was evident in 4, often with a geographic pattern. Immunostainings for INI, desmin, myogenin (scattered cells in 4, diffuse in 1) were positive in all; EMA and MNF116 were positive in 2; AP-2β was negative. PAX3/7-FOXO1 transcripts were absent. In all cases RB1 was wild type, and a TP53 mutation at R273H codon was found in 1. All patients are in complete remission, with a median follow-up of 6 years. Epithelioid RMS may occur in children and is probably related to ERMS, as suggested by lack of fusion transcripts, weak staining for myogenin, negative AP-2β, evidence of TP53 mutation (although only in 1 case), and a favorable clinical course.

Crose LE, Galindo KA, Kephart JG, et al.
Alveolar rhabdomyosarcoma-associated PAX3-FOXO1 promotes tumorigenesis via Hippo pathway suppression.
J Clin Invest. 2014; 124(1):285-96 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Alveolar rhabdomyosarcoma (aRMS) is an aggressive sarcoma of skeletal muscle characterized by expression of the paired box 3-forkhead box protein O1 (PAX3-FOXO1) fusion oncogene. Despite its discovery nearly two decades ago, the mechanisms by which PAX3-FOXO1 drives tumor development are not well characterized. Previously, we reported that PAX3-FOXO1 supports aRMS initiation by enabling bypass of cellular senescence checkpoints. We have now found that this bypass occurs in part through PAX3-FOXO1-mediated upregulation of RASSF4, a Ras-association domain family (RASSF) member. RASSF4 expression was upregulated in PAX3-FOXO1-positive aRMS cell lines and tumors. Enhanced RASSF4 expression promoted cell cycle progression, senescence evasion, and tumorigenesis through inhibition of the Hippo pathway tumor suppressor MST1. We also found that the downstream Hippo pathway target Yes-associated protein 1 (YAP), which is ordinarily restrained by Hippo signaling, was upregulated in RMS tumors. These data suggest that Hippo pathway dysfunction promotes RMS. This work provides evidence for Hippo pathway suppression in aRMS and demonstrates a progrowth role for RASSF4. Additionally, we identify a mechanism used by PAX3-FOXO1 to inhibit MST1 signaling and promote tumorigenesis in aRMS.

Kang Z, Yu Y, Zhu YJ, et al.
Downregulation of IGFBP2 is associated with resistance to IGF1R therapy in rhabdomyosarcoma.
Oncogene. 2014; 33(50):5697-705 [PubMed] Related Publications
Agents targeting the insulin-like growth factor-1 receptor (IGF1R) are in clinical development, but, despite some initial success of single agents in sarcoma, response rates are low with brief durations. Thus, it is important to identify markers predictive of response, to understand mechanisms of resistance, and to explore combination therapies. In this study, we found that, although associated with PAX3-FKHR translocation, increased IGF1R level is an independent prognostic marker for worse overall survival, particularly in patients with PAX3-FKHR-positive rhabdomyosarcoma (RMS). IGF1R antibody-resistant RMS cells were generated using an in vivo model. Expression analysis indicated that IGFBP2 is both the most affected gene in the insulin-like growth factor (IGF) signaling pathway and the most significantly downregulated gene in the resistant lines, indicating that there is a strong selection to repress IGFBP2 expression in tumor cells resistant to IGF1R antibody. IGFBP2 is inhibitory to IGF1R phosphorylation and its signaling. Similar to antibodies to IGF1/2 or IGF2, the addition of exogenous IGFBP2 potentiates the activity of IGF1R antibody against the RMS cells, and it reverses the resistance to IGF1R antibody. In contrast to IGF1R, lower expression of IGFBP2 is associated with poorer overall survival, consistent with its inhibitory activity found in this study. Finally, blocking downstream Protein kinase B (AKT) activation with Phosphatidylinositide 3-kinases (PI3K)- or mammalian target of rapamycin (mTOR)-specific inhibitors significantly sensitized the resistant cells to the IGF1R antibody. These findings show that constitutive IGFBP2 downregulation may represent a novel mechanism for acquired resistance to IGF1R therapeutic antibody in vivo and suggest various drug combinations to enhance antibody activity and to overcome resistance.

Yoshida H, Miyachi M, Sakamoto K, et al.
PAX3-NCOA2 fusion gene has a dual role in promoting the proliferation and inhibiting the myogenic differentiation of rhabdomyosarcoma cells.
Oncogene. 2014; 33(49):5601-8 [PubMed] Related Publications
We analyzed a complex chromosomal translocation in a case of embryonal rhabdomyosarcoma (RMS) and showed that it generates the fusion gene PAX3 (paired box 3)-NCOA2 (nuclear receptor coactivator 2). To understand the role of this translocation in RMS tumorigenesis, we established two types of stable mouse myoblast C2C12 cell lines expressing PAX3-NCOA2 and PAX3-FOXO1A (forkhead box O1A), respectively. Compared with control cells, PAX3-NCOA2 cells grew faster, were more motile, were less anchorage dependent, progressed more quickly through the G1/S phase of cell cycle and showed greater transcriptional activation of the PAX3 consensus-binding site. However, PAX3-NCOA2 cells proliferated more slowly and differentiated more weakly than did PAX3-FOXO1A cells. Both PAX3-NCOA2 cells and PAX3-FOXO1A cells formed tumors in nude mice, although the PAX3-NCOA2-induced tumors grew more slowly. Our results may explain why NCOA2 rearrangement is mainly found in embryonal rhabdomyosarcoma, which has a better prognosis than alveolar rhabdomyosarcoma, which expresses the PAX3-FOXO1A fusion gene. These results indicate that the PAX3-NCOA2 fusion gene has a dual role in the tumorigenesis of RMS: promotion of the proliferation and inhibition of the myogenic differentiation of RMS cells.

Ciarapica R, De Salvo M, Carcarino E, et al.
The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).
Oncogene. 2014; 33(32):4173-84 [PubMed] Related Publications
The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Fang WH, Wang Q, Li HM, et al.
PAX3 in neuroblastoma: oncogenic potential, chemosensitivity and signalling pathways.
J Cell Mol Med. 2014; 18(1):38-48 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Transcription factor PAX3/Pax3 contributes to diverse cell lineages during embryonic development and is important in tumourigenesis. We found that PAX3 is re-expressed in neuroblastoma and malignant neuroblastic (N-type) neuroblastoma cells had significantly higher PAX3 protein expression than their benign substrate-adherent (S-type) counterparts. Knock-down of PAX3 expression by siRNA transfection resulted in persistent cell growth inhibition in both types of neuroblastoma cell, owing to G1 cell cycle arrest and progressive apoptosis. Inhibition of PAX3 expression significantly decreased the attachment of S-type SH-EP1 cells to extra-cellular matrix proteins, fibronectin, laminin and collagen IV. Migration and invasion of both neuroblastoma cell types were markedly reduced after PAX3 down-regulation. PAX3 knock-down significantly augmented the cytotoxic effect of chemotherapeutic agents, etoposide, vincristine and cisplatin, commonly used to treat neuroblastoma. Microarray analyses revealed that particularly signalling pathways involving cell cycle, apoptosis, cell adhesion, cytoskeletal remodelling and development were altered by PAX3 down-regulation. Changes in PAX3 downstream genes identified by microarray analyses were validated in 47 genes by quantitative PCR. These novel findings lead us to propose that PAX3 might contribute to oncogenic characteristics of neuroblastoma cells by regulating a variety of crucial signalling pathways.

Bonvini P, Zin A, Alaggio R, et al.
High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma.
Br J Cancer. 2013; 109(12):3084-91 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
BACKGROUND: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome.
METHODS: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT-PCR, western blotting, IHC, and copy number analysis.
RESULTS: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (P<0.0001), and also correlated with larger tumour size (P<0.05) and advanced clinical stage (P<0.01), independently of fusion gene status. High ALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included.
CONCLUSION: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour.

Parham DM, Barr FG
Classification of rhabdomyosarcoma and its molecular basis.
Adv Anat Pathol. 2013; 20(6):387-97 [PubMed] Related Publications
Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, has traditionally been classified into embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS) for pediatric oncology practice. This review outlines the historical development of classification of childhood RMS and the challenges that have been associated with it, particularly problems with the diagnosis of "solid variant" ARMS and its distinction from ERMS. In addition to differences in clinical presentation and outcome, a number of genetic features underpin separation of ERMS from ARMS. Genetic differences associated with RMS subclassification include the presence of reciprocal translocations and their associated fusions in ARMS, amplification of genes in ARMS and its fusion subsets, chromosomal losses and gains that mostly occur in ERMS, and allelic losses and mutations usually associated with ERMS. Chimeric proteins encoded in most ARMS from the fusion of PAX3 or PAX7 with FOXO1 are expressed, result in a distinct pattern of downstream protein expression, and appear to be the proximate cause of the bad outcome associated with this subtype. A sizeable minority of ARMS lacks these fusions and shares the clinical and biological features of ERMS. A battery of immunohistochemical tests may prove useful in separating ERMS from ARMS and fusion-positive ARMS from fusion-negative ARMS. Because of limitation of predicting outcome solely based on histologic classification, treatment protocols will begin to utilize fusion testing for stratification of affected patients into low-risk, intermediate-risk, and high-risk groups.

Jothi M, Mal M, Keller C, Mal AK
Small molecule inhibition of PAX3-FOXO1 through AKT activation suppresses malignant phenotypes of alveolar rhabdomyosarcoma.
Mol Cancer Ther. 2013; 12(12):2663-74 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma.

Yuan H, Qin F, Movassagh M, et al.
A chimeric RNA characteristic of rhabdomyosarcoma in normal myogenesis process.
Cancer Discov. 2013; 3(12):1394-403 [PubMed] Related Publications
UNLABELLED: Gene fusions and their chimeric products are common features of neoplasia. Given that many cancers arise by the dysregulated recapitulation of processes in normal development, we hypothesized that comparable chimeric gene products may exist in normal cells. Here, we show that a chimeric RNA, PAX3-FOXO1, identical to that found in alveolar rhabdomyosarcoma, is transiently present in cells undergoing differentiation from pluripotent cells into skeletal muscle. Unlike cells of rhabdomyosarcoma, these cells do not seem to harbor the t(2;13) chromosomal translocation. Importantly, both PAX3-FOXO1 RNA and protein could be detected in the samples of normal fetal muscle. Overexpression of the chimera led to continuous expression of MYOD and MYOG-two myogenic markers that are overexpressed in rhabdomyosarcoma cells. Our results are consistent with a developmental role of a specific chimeric RNA generated in normal cells without the corresponding chromosomal rearrangement at the DNA level seen in neoplastic cells presumably of the same lineage.
SIGNIFICANCE: A chimeric fusion RNA, PAX3-FOXO1, associated with alveolar rhabdomyosarcoma, is also present in normal non-cancer cells and tissues. Its transient expression nature and the absence of t(2;13) chromosomal translocation are consistent with a posttranscriptional mechanism. When constantly expressed, PAX3-FOXO1 interfered with the muscle differentiation process, which presumably contributes to tumorigenesis.

Keller C, Guttridge DC
Mechanisms of impaired differentiation in rhabdomyosarcoma.
FEBS J. 2013; 280(17):4323-34 [PubMed] Related Publications
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, with presumed skeletal muscle origins, because of its myogenic phenotype. RMS is composed of two main subtypes, embryonal RMS (eRMS) and alveolar RMS (aRMS). Whereas eRMS histologically resembles embryonic skeletal muscle, the aRMS subtype is more aggressive and has a poorer prognosis. In addition, whereas the genetic profile of eRMS is not well established, aRMS is commonly associated with distinct chromosome translocations that fuse domains of the transcription factors Pax3 and Pax7 to the forkhead family member FOXO1A. Both eRMS and aRMS tumor cells express myogenic markers such as MyoD, but their ability to complete differentiation is impaired. How this impairment occurs is the subject of this review, which will focus on several themes, including signaling pathways that converge on Pax-forkhead gene targets, alterations in MyoD function, epigenetic modifications of myogenic promoters, and microRNAs whose expression patterns in RMS alter key regulatory circuits to help maintain tumor cells in an opportunistically less differentiated state.

Marshall AD, Picchione F, Geltink RI, Grosveld GC
PAX3-FOXO1 induces up-regulation of Noxa sensitizing alveolar rhabdomyosarcoma cells to apoptosis.
Neoplasia. 2013; 15(7):738-48 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Alveolar rhabdomyosarcoma (ARMS) has a much poorer prognosis than the more common embryonal subtype. Most ARMS tumors characteristically possess a specific genomic translocation between the genes of PAX3/7 and FOXO1 (FKHR), which forms fusion proteins possessing the DNA binding domains of PAX3/7 and the more transcriptionally potent transactivation domain of FOXO1. We have shown that the proapoptotic BH3-only family member Noxa is upregulated by the PAX3-FOXO1 fusion transcription factor in a p53-independent manner. The increased expression of Noxa renders PAX3-FOXO1-expressing cells more susceptible to apoptosis induced by a γ-secretase inhibitor (GSI1, Z-LLNle-CHO), the proteasome inhibitor bortezomib, and BH3 mimetic ABT-737. Apoptosis in response to bortezomib can be overcome by shRNA knockdown of Noxa. In vivo treatment with bortezomib reduced the growth of tumors derived from a PAX3-FOXO1-expressing primary myoblast tumor model and RH41 xenografts. We therefore demonstrate that PAX3-FOXO1 up-regulation of Noxa represents an unanticipated aspect of ARMS tumor biology that creates a therapeutic window to allow induction of apoptosis in ARMS cells.

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