Genome-Wide Identification of PAX3-FKHR Binding Sites in Rhabdomyosarcoma Reveals Candidate Target Genes Important for Development and Cancer

Cao, Liang; Yu, Yunkai; Bilke, Sven; Walker, Robert L.; Mayeenuddin, Linnia H.; Azorsa, David O.; Yang, Fan; Pineda, Marbin; Helman, Lee J.; Meltzer, Paul S.

doi:10.1158/0008-5472.can-10-0582

Cited by 206 publications

(285 citation statements)

References 50 publications

(43 reference statements)

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“…To further confi rm the identity of the band, we performed Western blot analysis on the same set of samples with a PAX3-FOXO1-specifi c monoclonal antibody (PFM2). The specifi city of the antibody has been demonstrated before ( 9 ) and further confi rmed (Supplementary Fig. S4).…”

Section: Research Articlementioning

confidence: 54%

A Chimeric RNA Characteristic of Rhabdomyosarcoma in Normal Myogenesis Process

et al. 2013

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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 specifi c 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.

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Section: Research Articlementioning

confidence: 54%

A Chimeric RNA Characteristic of Rhabdomyosarcoma in Normal Myogenesis Process

et al. 2013

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“…Further, the regulatory sequences of the ALK gene were shown to contain a high-affinity binding site to the PAX3-FOXO1 protein. 38 Although a previous study 20 found no association between FOXO1 rearrangement and ALK immunoreactivity, this discordance may be owing to the difference in the number of cases studied, the ALK staining protocol used, and the molecular method employed to determine the gene rearrangement status.…”

Section: Discussionmentioning

confidence: 93%

Anaplastic lymphoma kinase status in rhabdomyosarcomas

Yoshida

Shibata²,

Wakai

et al. 2013

Modern Pathology

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Rhabdomyosarcoma is a rare soft tissue sarcoma that typically affects children, adolescents, and young adults. Despite treatment via a multidisciplinary approach, the prognosis of advance-stage rhabdomyosarcomas remains poor, and a new treatment strategy is needed. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is a potential target for specific inhibitors. In this study, we investigated 116 rhabdomyosarcomas using a polymer-based ALK immunostaining method and correlated the results with clinicopathological parameters. In addition, we examined ALK status using dual-color fluorescence in situ hybridization, PCR, and sequencing. In immunohistochemical analysis, ALK was detected in 2 (6%) of 33 embryonal rhabdomyosarcomas, 42 (69%) of 61 alveolar rhabdomyosarcomas, and 0 (0%) of 22 other subtypes, including pleomorphic, adult-spindle-cell/sclerosing, and epithelioid variants. Compared with ALK-negative alveolar rhabdomyosarcomas, ALK-positive ones are presented with metastatic spread more frequently and showed a greater extent of myogenin reactivity. Overall survival was not associated with ALK expression. FOXO1 rearrangement was significantly associated with ALK immunoreactivity. The median ALK copy number was greater in ALK-positive tumors than in ALK-negative tumors. Most (93%) cases tested showed no selective increase in the ALK gene dosage. ALK selective amplification and low-level selective gain were noted in one and three cases, respectively. Further, a high-polysomy pattern (Z4 ALK copies in Z40% of cells) was observed in seven cases. A significant increase in the ALK copy number was exclusive to the ALK-immunopositive cohort, but it was uncommon, accounting for only 30% of the 37 ALK-positive rhabdomyosarcomas. ALK gene rearrangement was not observed in either cohort, while an ALK somatic mutation (I1277T) was found in one ALK-negative embryonal case. Although it remains controversial whether ALK expression without gene rearrangement is therapeutically relevant, this comprehensive analysis may help future studies on the utility of ALK-targeted therapy for patients with rhabdomyosarcoma. Modern Pathology (2013) 26, 772-781; doi:10.1038/modpathol.2012 published online 11 January 2013 Keywords: anaplastic lymphoma kinase; fluorescence in situ hybridization; immunohistochemistry; rhabdomyosarcoma Rhabdomyosarcoma is a rare sarcoma with skeletal muscle differentiation that typically affects children, adolescents, and young adults. It is histologically classified into the embryonal, alveolar, and pleomorphic subtypes, 1-3 although other rare variants like sclerosing, adult-spindle-cell, and epithelioid rhabdomyosarcomas have also been proposed. [4][5][6][7] Each subtype is characterized by a distinct epidemiology, clinical behavior, and genetic background. 1-3 For example, alveolar rhabdomyosarcoma affects older patients compared with the embryonal subtype, is associated with poor survival, and in most cases, is characterized by a specific t(2;13) or t(1;13) translocation, which results i...

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“…50 However, there is no evidence for PAX3-FOXO1 directly regulating FBXO32 from analyses of ChIPseq data. 51 Recently, it has been shown that, in rat myoblasts in vitro, expression of FBXO32, which is induced in these cells by serum starvation, facilitates myogenesis through the repression of myocardin. 52 Intriguingly, both muscle atrophy and myogenesis involve apoptosis.…”

Section: Discussionmentioning

confidence: 99%

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx)

et al. 2013

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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.Oncogene ( Keywords: EZH2; FBXO32; histone methyltransferases; PAX3-FOXO1; rhabdomyosarcoma; Polycomb proteins INTRODUCTION Rhabdomyosarcomas (RMS) are heterogeneous highly malignant tumors, which account for 7-8% of all pediatric malignancies and over half of the soft-tissue sarcomas in children. RMS are classically subdivided in two major histotypes: embryonal (around 70-80%) and alveolar (around 20-30%), the latter often metastatic at diagnosis and showing a high risk of recurrence. 1 In 70% of cases, alveolar RMS is characterized by the chromosomal translocation t(2;13) or t(1;13), resulting in

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Genome-Wide Identification of PAX3-FKHR Binding Sites in Rhabdomyosarcoma Reveals Candidate Target Genes Important for Development and Cancer

Cited by 206 publications

References 50 publications

A Chimeric RNA Characteristic of Rhabdomyosarcoma in Normal Myogenesis Process

A Chimeric RNA Characteristic of Rhabdomyosarcoma in Normal Myogenesis Process

Anaplastic lymphoma kinase status in rhabdomyosarcomas

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx)

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