review article rhabdomyosarcoma: advances in molecular and

Review ArticleRhabdomyosarcoma Advances in Molecular andCellular Biology

Xin Sun123 Wei Guo3 Jacson K Shen12 Henry J Mankin12

Francis J Hornicek12 and Zhenfeng Duan12

1Department of Orthopaedic Surgery Massachusetts General Hospital 100 Blossom Street Boston MA 02114 USA2Sarcoma Biology Laboratory Center for Sarcoma and Connective Tissue Oncology Massachusetts General Hospital100 Blossom Street Boston MA 02114 USA3Department of Orthopaedic Oncology Peking University Peoplersquos Hospital 11 Xizhimen South Street Xicheng DistrictBeijing 100044 China

Correspondence should be addressed to Zhenfeng Duan zduanmghharvardedu

Received 4 May 2015 Accepted 16 August 2015

Academic Editor Enrique de Alava

Copyright copy 2015 Xin Sun et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood and adolescence The two major histologicalsubtypes of RMS are alveolar RMS driven by the fusion protein PAX3-FKHR or PAX7-FKHR and embryonic RMS which isusually genetically heterogeneous The prognosis of RMS has improved in the past several decades due to multidisciplinary careHowever in recent years the treatment of patients with metastatic or refractory RMS has reached a plateau Thus to improve thesurvival rate of RMS patients and their overall well-being further understanding of the molecular and cellular biology of RMS andidentification of novel therapeutic targets are imperative In this review we describe the most recent discoveries in the molecularand cellular biology of RMS including alterations in oncogenic pathways miRNA (miR) in vivomodels stem cells and importantsignal transduction cascades implicated in the development and progression of RMS Furthermore we discuss novel potentialtargeted therapies that may improve the current treatment of RMS

1 Introduction

Rhabdomyosarcoma (RMS) is the most prevalent soft tissuetumor in children and adolescents accounting for 5 of allpediatric tumors [1 2] It is estimated that 350 new cases ofRMS are diagnosed each year in patients under 20 years ofage in the United States [2] In contrast RMS is extremelyrare in adults There is a slight male predominance (14 timesmore common in males than in females) but there are nosignificant differences in the incidence rates among races ordifferent ethnic groups [3] As RMS is derived from primitivemesenchymal stem cells directed towards myogenesis it canarise in a variety of anatomic sites throughout the body [4]RMS can occur either as a primary malignancy or as a com-ponent of a heterogeneous malignancy such as a malignantteratomatous tumor [5] Additionally a small percentage ofcases are associated with known genetic disorders such as

neurofibromatosis type 1 and the Li-Fraumeni familial cancersyndrome [6]

The World Health Organization (WHO) recently revisedthe classification of RMS subtypes as alveolar rhabdomyo-sarcoma (ARMS) embryonal rhabdomyosarcoma (ERMS)pleomorphic rhabdomyosarcoma (PRMS) and sclerosingspindle cell rhabdomyosarcoma (SRMS) in 2013 [7] ARMS isa high-grademalignancy occurringmostly in adolescents andyoung adultsThemost common site for ARMS is in the deeptissue of extremities ERMS represents approximately 70of all childhood RMS usually afflicting infants or childrenunder 10 years of age ERMS often affects the head and neckregions especially the orbit PRMS usually occurs in adultmales in the deep tissue of extremities but may occur at anysite In adult patients the pleomorphic variant is associatedwith the worst prognosis [8]

Hindawi Publishing CorporationSarcomaVolume 2015 Article ID 232010 14 pageshttpdxdoiorg1011552015232010

2 Sarcoma

Table 1 Alterations of chromosome in RMS by CGH in recent 5 years

Materials Gain Amplification Loss Deletion Reference

25 RMS samples 2p 12q 6p 9q 10q 1p2q 6q 8q 15q 18q 3p 11p 6p Li et al 2009 [16]

13 RMS cell lines

1p213ndash132 1q126q26ndash27 7q213ndash3111q41 2p243 8q241220q132 20q1332

2p243 (MYCN)8p1123ndash1121

(FGFR1) 12q133(CDK4) 19q12 20q

3p142ndash1224q27ndash3236p251ndash243

9p243ndash241 13q143

Missiaglia et al 2009[17]

57 ARMSsamples 12q15 2p24 12q13ndashq14 Barr et al 2009 [18]

128 primaryRMS samples 7 8 11 20

2p241 (MYCN)8p112ndashp111 (FGFR1)12q133ndash141 (CDK4)MDM2 (12q143ndashq15)

Williamson et al2010 [12]

26 frozenprimary ERMSsamples

8 2 11 12 13 19 20

2p21 2q35 2q1422q361 5q352ndashq353

11p112 11q24212q133

6 9 10 14 15 16 181p3623 1q321 3p1424q351ndash352 9p21317q112 22q1331

Paulson et al 2011[13]

39 RMS samples 12q133 12q133ndashq14112q141 17q251

2q1312 12q13312q133ndashq141

9p12ndashp11210q1121ndashq112214q3233 16p112

22q111

1p211 2q141 5q1329p12 9q12 Liu et al 2014 [11 15]

RMS cellsderived fromrefractory RMS

NACA HSD17B6SDR9C7 RDH16GPR182 ZBTB39TAC3 MYO1A

NAB2 STAT6 LRP1

Park et al 2014 [14]

20 RMS samplesof Chinesepatients

12q2431 17q2511q211 7q112312q133ndashq141

9p133 12q133ndashq14112q15 16p1311

5q132 15q11214q3233 (IGHG

IGHM)

1p3633 1p131 2q1115q132 8p231 9p243

16p112Liu et al 2014 [11 15]

Since the 1970s the Intergroup RhabdomyosarcomaStudy Group (IRSG) has conducted a series of clinical trialscomparing risk-base and has established a series of treatmentguidelines [9] Currently multidisciplinary managementincluding chemotherapy and surgery with or without radia-tion has become the standard treatment for RMS The 5-yearsurvival rate of RMS has increased from 25 in 1970 up to60 since 2000 [1 10]However there has been little improve-ment in the oncological outcome of patients with RMS inrecent years Drug resistance andmetastatic disease representthe two most common phenomena for therapy failure Somerandomized chemotherapy trials have failed to improve out-come despite the introduction of newer or more intensivetherapies Thus there is an urgent need for alternative moreeffective treatment strategies Recent molecular and geneticanalysis of these tumors has produced substantial newinsights into molecular cell biology molecular cytogeneticsand tumorigenesis of RMS leading to a better understandingof RMS development at the molecular level These advancesmay ultimately lead to better clinical understanding and topotentially developing more potent targeted therapies Thepurpose of this review is to summarize these most recentfindings in RMS

2 Novel Discoveries of ChromosomalAlterations in RMS

Malignant transformation occurs cytogenetically due to theaccumulation of somatic mutations by the acquisition oftumor-specific chromosomal translocations

21 Gains and Losses of Chromosomes in RMS ComparativeGenomic Hybridization (CGH) analysis has revealed that allRMS have specific gains and losses [11ndash18] (Table 1) ERMSfrequently exhibits gains or losses of specific whole chromo-somes whereas ARMS is characterized by the presence ofregions of genomic amplification [11 19] The focal regionsand genes most of which have frequent gains and ampli-fications include 12q133ndashq141 and 8p112ndash112 and CDK4MYCN GLI MDM2 FGFR1 and FGFR4 respectively Fre-quently the genes differentially expressed in subtypes ofRMS particularly when they are from chromosomal regionsshow a high level of gains in cell lines Previous CGH studieshave shown that ARMS tumors tend to have fewer copynumber variants than ERMS tumors [12 20] For examplefrequent gains were detected in TYROBP HCST LRFN3and ALKBH6 (19q1312) in ERMS but not in ARMS [15]

Sarcoma 3

PAX3

Chromosome 2

PAX3-FKHR

t(213)(q37q14)

NCOA1

PAX7

Chromosome 1

PAX7-FKHR

t(113)(p36q14)

PAX3

Chromosome 2

FKHR

Chromosome 13

PAX3-NCOA1

t(22)(q35q23)

Paired domain Homeodomain

DNA binding domain Transactivation domain

PB

PB

PB

PB HD

HD

HD

HD TAD

TAD

TAD

TAD

TAD

TAD

HD

HD

PB

PB

100 amino acids

Fusion site

Fusion site

FD

DNA binding heterodimerization domain Nuclear receptor interaction domain

LXXLL HAT

HAT

bHLHPAS

Fusion site

(a)

(b)

(c)

Figure 1The chromosomal rearrangements in ARMS 80 of ARMS classified as translocation-positive ARMS carry characteristic chromo-somal translocations demonstrated as t(213)(q35q14) t(113)(p36q14) and (22)(q35p23) In (a) and (b) the translocations fuse the FKHR(a member of the forkheadHNF-3 transcription factor family) locus on chromosome 13 to either PAX3 on chromosome 2 or PAX7 onchromosome 1 In (c) the translocation generated a fusion protein composed of PAX3 and the nuclear receptor coactivator NCOA1 havingsimilar transactivation properties as PAX3FKHR

These studies have identified a number of genetic alterationsin RMSMany of these chromosomal changesmay be respon-sible for tumor progression and proliferation These geneticalterations may be potential treatment targets in RMS [11]

22 Chromosomal Translocations in ARMS Chromosomalanalyses have demonstrated two translocations associatedwith ARMS t(213)(q35q14) and t(113)(p36q14) [21] Initialstudies detected these two gene fusions in 80 of ARMS[22] These characteristic chromosomal translocations areadjacent to the 51015840 DNA-binding domains of PAX a memberof the paired box transcription factor family and the trans-activation domain at the 31015840 end of FKHR a member of theforkheadHNF-3 transcription factor family Approximately75 of these structural rearrangements translocate the PAX3gene at 2q35 to the FKHR gene at 13q14 as t(213)(q35q14)less frequently in the other 25 the t(13)(q36q14) transloca-tion fuses PAX7 to FKHR [23] Previous studies have demon-strated that PAX3FKHR fusion gene status significantlyimproves current risk stratification while the presence ofPAX7FKHR and rarer variant fusion gene products requirefurther investigation [24] The remaining 20 of ARMS isPAX gene fusion-negative (PFN) and forms a more hetero-geneous group which remains a challenge to detect due to

the lack of consistent chromosomal rearrangements PFNARMS has a similar clinical course to ERMS which suggeststhat the fusion gene status provides more accurate informa-tion about patient outcomes than did the histologic subtypeDespite the low overall burden of somatic mutations infusion-positive RMSmultiple genes were recurrently alteredincluding NRAS KRAS HRAS FGFR4 PIK3CA CTNNB1FBXW7 and BCOR [25]

Another novel translocation t(22)(q35p23) was identi-fied in ARMS biopsy samples by gene expression signatures[26] The chromosomal translocation generates a fusion pro-tein composed of PAX3 and the nuclear receptor coacti-vator NCOA1 which has similar transactivation propertiesas PAX3FKHR [26] These biologic effects contribute totumorigenesis bymodulatingmyogenic differentiation alter-ing growth and apoptotic pathways and stimulating motilityand other metastatic pathways (Figure 1)

23 Chromosomal Alterations in ERMS ERMS exhibits a lossof imprinting (LOI) leading to a twofold gene dosage effect[27] Most tumors have at least one 15-Mb region with loss ofheterozygosity (LOH) along chromosome 11 [27] The allelo-type of ERMS demonstrates a high frequency of LOH onchromosomes 11p 11q and 16q [28] ERMS tumorigenesis

4 Sarcoma

Table 2 The histological types of rhabdomyosarcoma

Histological type Predilection population Predilection site Risk category Genetic change

ERMS Infants or children under 10years old Head and neck region Intermediate LOH at chromosome 11p155

ARMS Adolescents and young adults Deep tissue of extremity High t(213)(q35q14) t(113)(p36q14)

PRMS Adult males Throughout the body HighJUN (1p31) MYC (8q24)CCND1 (11q13) INT2 (11q133)MDM2 (12q143ndashq15)

SRMSIn the first decade of life with asecond mode centered aroundthe fifth decade

The extremities headand neck Superior SRF-NCOA2 MYOD1 mutation

ERMS embryonal rhabdomyosarcomaARMS alveolar rhabdomyosarcomaPRMS pleomorphic rhabdomyosarcomaSRMS spindle cellsclerosing rhabdomyosarcoma

can result from the inactivation of the parental bias of chro-mosome 11p15 which is the most common rearrangementin ERMS [29] The proportion of ERMS with LOH alongchromosome 11 is considerably higher than in other subtypes[27] In histopathological analysis ERMS expresses lowPAX3levels and elevated PAX7 levels [30] Hosoi et al identified ahidden 2q35 breakpoint as a novel PAX3 rearrangement incomplex chromosomal translocations in ERMS [31]

24 Chromosomal Alterations in Other RMS There are lim-ited studies on the biological pathways involved in othersubtypes of RMS compared with the two major subtypesARMS andERMS Fluorescence In SituHybridization (FISH)reveals amplification of JUN (1p31) MYC (8q24) CCND1(11q13) INT2 (11q133) MDM2 (12q143ndashq15) and MALT(18q21) in these tumor cells contributing to the pathogenesisof PRMS [32] MYOD1 homozygous mutations are alsoreported as frequent recurrent and pathognomonic eventsin adult-type SRMS [33 34] (Table 2) Furthermore four outof five pediatric tumors showedMYOD1mutations in a study[34] Yoshida et al described a 8q13 locus (NCOA2) generearrangement in a small subset of SRMS occurring uniquelyin the infantilecongenital setting fused with key transcrip-tion factors involved in skeletal muscle differentiation suchas SRF and TEAD1 [35] They also identified NCOA2 as acandidate PAX3 partner geneThePAX3-NCOA2 fusion geneplays a dual role in the tumorigenesis of RMS promotion ofthe proliferation and inhibition of the myogenic differenti-ation of RMS cells [35] As PAX3-NCOA2-induced tumorsgrow more slowly SRMS that are PAX3-NCOA2 fusion-positive could be associated with a very favorable prognosis[36]

Not all RMSs occur as sporadic primary tumors Occa-sionally RMS inherits a mutant gene as part of an establishedfamilial syndrome For example Beckwith-Wiedemann Syn-drome (BWS) with RMS involves dysregulation or alterationof imprinted genes in the 11p155 chromosomal regionincluding IGF2 H19 and CDKN1C (p57KIP2) [37]

3 Cell of Origin in RMS

There have been a number of studies aimed at deciphering thecell origin for RMS [56ndash61]

31 Myogenic Differentiation in the Tumorigenesis Severalstudies have focused on elucidating the mechanisms govern-ing the impaired myogenic program in RMS [62 63] (Fig-ure 2) RMSoriginates as a consequence of regulatory disrup-tion of the growth and differentiation of myogenic precursorcells Progenitor cells reside in muscle and their activationresults in either proper myogenesis or aberrant signalingpathways leading to the development of RMS Based on theskeletal muscle lineage a complete transcriptome analysis ofRMS was performed to compare normal and fetal muscles[56] The high degree of similarity between fetal muscle andRMS expression profiles reflects the undifferentiated myo-genic nature of RMS The genes exclusively upregulated inRMS including FGFR4 NOTCH2 UBE2C UHRF1 andYWHAB genes contribute to the failure of RMS cells to com-plete normal skeletal muscle development and progress toan alternative fate [56] RMS cells represent an arrested statein the development of normal skeletal muscle with regionalsilencing of differentiation factors leading to the maturationdefect in RMS [57] The expression pattern of muscle-specific proteins regulating myogenic differentiation hasbeen extensively examined in RMS The family of myogenictranscription factors (MYOD MYF5 myogenin and MRF4)are considered to be responsible for the determination of stemcells intomyoblasts anddifferentiation intomyocytes [58 59]All these factors are activated during the onset or progressionof myogenesis and are subsequently silenced to reach a finalmuscular differentiation There are statistical differences inthe different subtypes of RMS tumors with MYOD or myo-genin staining patterns Amplification of MDM2 in an RMScell line interferes with MYOD activity and consequentlyinhibits overt muscle cell differentiation [61] Interleukin-4receptor (IL-4R) has also been proposed to be important for

Sarcoma 5

Table 3 Studies identifying stem cells in RMS

Markersubstrate Source Stem cell gene Functional characterization Reference

CD133 Orthotopicxenograft model

OCT4 NANOG c-MYCPAX3 and SOX2

Correlating with poor overallsurvival

Walter et al2011 [38]

PAX-FKHR Bone marrow ofC57BL6 mice

Myf5 MEF2 MyoD andmyogenin

Determining the molecularmyogenic and histologicphenotype of ARMS

Ren et al 2008[39]

V-ATPase RD cell line NANOG and OCT34

Driving mechanisms of areduced sensitivity to anticancerdrugs and activities related toinvasion and metastasis

Salerno et al2014 [40]

Survm-CRAsKYM-1 cell line(FGFR3-positive)

FGFR3Therapeutic effectiveness againstall cell populations and increasedeffectiveness against CSCs

Tanoue et al2014 [41]

Migration

specification

Injured muscle

Terminal differentiation

Fusion

PAX7

Myoblasts

Myoblast

MyoD

MyoD

Myf5

PAX3

Rb

Ezh2

IGF2

HDAC4YY1

YY1

ERK

AKT

RMS

Myogenin

P38 kinase

Myostatin

Progenitor

cell

N-RAS

Myofibers

FGFR4

UBE2C

UHRF1

NOTCH2

YWHAB

Satellite cells

RUNX1 MEF2C

JDP2 NFIC

Myocytes

Restoration of proliferation blockade of

differentiation

Proliferatio

n

Cell circle arrestEarlydifferentiation

TGF-1205731

Figure 2 Myogenic pathways in the tumorigenesis of RMS In aberrant neoplastic condition progenitor cells residing in muscle result inaberrant pathways which lead to malignant transformation and fail to differentiate proliferate uncontrollably and form RMS Sharp arrows(rarr) indicate upregulationactivation and blunt arrows (perp) indicate downregulationinhibition

the maturation of myotubes [64] An IL-4R blockade mighthelp therapeutically to modulate the expression of myogenictranscription factors (MYOD or myogenin) [57 64]

32 Potential Cancer Stem Cell in RMS Cancer Stem Cells(CSCs) may contribute to inherent refractory responses tocurrent therapies andmetastasis [65]Many CSCmodels playan important role in the development of RMS [38ndash41 66](Table 3)

A study explored a potential important role for mesen-chymal stem cells (MSCs) as the cell of origin of ARMS [67]Ren et al confirmed that PAX-FKHR fusion genes commitMSCs to a myogenic lineage by inhibiting terminal differen-tiation and contributing to ARMS formation [39] Anotherstudy showed that PAX-FKHR induces skeletal myogenesisin MSCs by transactivating MYOD and myogenin and trans-forms mesenchymal progenitor cells to the skeletal muscle lin-eage leading to malignant formation resembling ARMS [39]

6 Sarcoma

METCDKN2AB

PAX37-FKHR

MDM2

RAS

Raf

GLI1

PDK1p53Rb

Hh

Myo MEF2

p21

Ptch

FGFR4

IGF1R

p16INK4Ap14ARF

p19ARF

IGF2

FGF

Figure 3 Genetic analyses of RMS have pinpointed several common alterations including inactivation of a master regulator of p53 andRb pathways CDKN2AB and activation of FGFR4 RAS and Hedgehog (Hh) signaling The modifications of these pathways influenceoncogenesis and metastatic potential

As a human hematopoietic CSC marker Walter et alproposed that upregulated CD133 in ERMS can act as aprognostic marker and might help with the development ofnovel targeted therapies for ERMS [38]

4 Signaling Pathway Alterations in RMS

Recent studies have looked into signaling pathway alterationsand their downstream effects in RMSThese new findings notonly helped to improve the understanding of this malignancybut also offered novel potential therapeutic strategies forimproved treatment for patientswithRMS [68 69] (Figure 3)

41 RAS Signaling Pathway RASmutations commonlymain-tain the protein in its GTP bound state and therefore renderit constitutively active in RMS Zhang et al identified RASfamily members NRAS KRAS and HRAS in the RASpathway as themost commonlymutated genes in ERMS [70]the mutation status of these genes is significantly associatedwith the risk of ERMS development [70] A study foundthat a majority of ERMS demonstrated activation of the RASpathway exclusively either by homozygous deletion of NF1or by point mutations in one of the RAS family members asdescribed above [13] Despite remarkable genetic and molec-ular heterogeneity most (93 4144) RMS tumors hijackeda common receptor tyrosine kinaseRASPIK3CA geneticaxis [25] It could occur via two alternative mechanismsrearrangement of a PAX gene and accumulation of mutationsthat were downstream targets of the PAX fusion protein

Skeletal muscle cells have a robust antioxidant defensesystem to protect the DNA lipids and proteins from thedeleterious effects of excess Reactive Oxygen Species (ROS)

Cancer cells also have elevated ROS due to their increasedmetabolic activity oncogenic stimulation andmitochondrialdysfunction These findings implicate RAS mutations andoxidative stress as potential therapeutic targets for high-riskERMS

42 IGF Signaling Pathway There is clear preclinical datathat supports the involvement of the Insulin-like GrowthFactor (IGF) signaling pathway in RMS tumorigenesis andprogression [69] Zhu and Davie demonstrated that IGFpromotes the proliferation of RMS cells while blocking IGFsignaling interferes with cell growth in vivoThe IGF receptorIGF-1R is highly overexpressed both on the cell surface andin the nucleus of RMS cells furthermore RMS cell lines aresensitive to IGF-1R inhibitionThe IGF-2 locus shows a loss ofimprinting in both ERMS andARMS tumors and the expres-sion of PAX3-FKHR has been demonstrated to upregulateIGF-2 and activate IGF pathway in ARMS [69] Mariannaet al found that IGF-2 and tumor suppressors p19Arf andp21Cip1 are overexpressed and prominently upregulated inRMS mouse models [71]

43 TGF-120573 Signaling Pathway The regulatory role of Trans-forming Growth Factor-120573 (TGF-120573) in RMS has been recentlystudied [72] Wang et al demonstrated that the expression ofTGF-1205731 is significantly higher in RMS than in normal skeletalmuscle The inhibition of TGF-1205731 expression by shRNA-expressing vectors reverses themalignant behavior of RMSbyinhibiting cell growth and inducingmyogenic differentiationTGF-1205731 shRNA induces myogenin expression a regulator ofmyogenic differentiation genes Myogenin acts as a target fornegative regulation of myogenesis by TGF-1205731 signals with

Sarcoma 7

a differentiation regulatory cascade These results suggestthat the TGF-1205731 signaling pathway disrupts the differentia-tion of myogenic progenitors leading to the development ofRMS

44 FGF Signaling Pathway Fibroblast Growth Factor (FGF)is crucial in embryonic development and functions to driveproliferation FGFs and their receptors (FGFRs) are essentialregulators in the processes of proliferation antiapoptosisdrug resistance and angiogenesis in RMS [69] RMS over-expresses the receptor tyrosine kinase FGFR4 which causesautophosphorylation and constitutive signaling in correlationwith poor differentiation and decreased survival [73] Recentwork has shown that FGF signaling can prevent ARMS cellsfrom apoptosis induced by targeting the IGF1-R-PI3K-mTORpathway [74]

45 ERK Signaling Pathway Previous studies have shownthat elevated myostatin expression deregulates ExtracellularRegulated Kinase (ERK) signaling and deficient activationof the p38 pathway contributes to the differentiation blockin RMS cells [62] The ERK pathway is frequently highlyactivated in RMS cells from embryonic derivation due to thepresence of activating RASmutations leading to reduced dif-ferentiation by blocking the p38 pathway [75]These findingsindicate that interventions that target themyostatinERKp38network could be an effective way to promote differentiationof RMS

46 Hippo Signaling Pathway Many upstream signal trans-duction proteins in response to cues from the cellular micro-environment regulate the core Hippo pathway [76] TheHippo pathway may limit tumorigenesis by inducing thecytosolic localization of the transcriptional cofactor Yes-Associated Protein 1 (YAP1) High YAP levels and activitiesincrease activated satellite cells and prevent differentiationby activating genes that inhibit differentiation [77] YAP1-TEAD1 was found to upregulate proproliferative and onco-genic genes and maintain the ERMS differentiation blockby interfering with MYOD1 and MEF2 prodifferentiationactivities [76] Inhibition of theHippo pathway is exhibited inRMS through downregulation of Hippo pathway tumor sup-pressors or upregulation of YAP [78] These data suggest thatHippo pathway dysfunction promotes RMS development

47 Notch Signaling Pathway and Wnt Signaling PathwayNotch pathway as an embryonic signaling pathway promotesmuscle stem cell maintenance by inhibiting myogenic earlydifferentiation and expanding pools of progenitor cells dur-ing both embryogenesis and postnatal muscle regenerationSimilar to Notch Wnt has various roles in embryonic fetaland neonatalmyogenesis during skeletalmyogenesis As bothpathways are regulators of myogenic lineage determinationand maturation RMS can arise from disordered regulationof these normal developmental pathways [79]

48 Caveolins in RMS Recent findings have shown that cav-eolins are expressed in a cell stage-dependent manner inRMS [80 81] Caveolins are scaffolding proteins that regulate

several targets and pathways (such as p53 IGF RASERK andTGF-120573myostatin signaling) associated with RMS develop-ment and therefore loss or gain of caveolin function is ableto generate multiple effects on the tumor behaviorThereforerecognizing their precise roles in RMS progression will becrucial to elaborate targeted therapies particularly as oneof the three different isoforms CAV1 could act as a potenttumor suppressor in ARMS Inhibition of CAV1 function cancontribute to aberrant cell proliferation leading to ARMSdevelopment [80]

49 DNA Repairing System Some studies indicate that RMSis characterized by germline mutations of DNA repair genes[82 83] DNA repair gene alterations in RMS occur sec-ondarily to malignant transformation The modificationsin DNA repair enzyme activity can result in resistance toadjuvant treatment in RMS tumor cells which limits theprognosis of RMS There are two pathways for repairingDNA breaks directly without affecting DNA structure andindirectly by DNA phosphodiester backbone cleavage [82](Figure 4) Direct repair includes repair during replicationand enzymatic repair Indirect repair is comprised of exci-sion repair systems including base excision repair (BER)nucleotide excision repair (NER) mismatch repair (MMR)and recombination repair (RR) [83] Defective DNA repairmechanisms result from point mutations or LOH in RMSand contribute to tumorigenesis MGMT and MMR proteinactivity and expression levels are used as predictor indices fortherapy outcome in RMS BER as the major DNA repair sys-tem against damage resulting from cellular metabolism canreverse the cytotoxic effects of alkylation agents used suchas antineoplastics subsequent tumor progression and deathimplicates an RMS mechanism of resistance to chemother-apeutic agents This may provide novel individualized ther-apeutics targeting downregulation of activated DNA repairenzymes or upregulation of DNA repair deficiencies [82]

5 MicroRNA (miRNA miR)Expression in RMS

Muscle-specific and ubiquitously expressed miRs appeardownregulated in RMS tumors and cell lines compared withthe normal counterparts These miRs often play crucial rolesas antioncogenes Inhibition of these miRs contributes toenhanced tumorigenesis through the modulation of diversemolecular pathways Upregulation of prooncogenic miRs hasbeen detected in RMS recently as well [84] (Table 4)

51 Antioncogenic miR in RMS Gain-of-function experimentshave demonstrated that reexpression of selected ldquotumor sup-pressorrdquo miRs impairs the tumorigenic behavior of RMS cells[84 85] As myo-miR family members miR-1 miR-133a andmiR-206 have been demonstrated to be downregulated inRMS and shown to have activity on mRNA expression bytargeting c-Met [42 43 86] Inhibition of thesemyo-miRNAsmay cause aberrant cell proliferation and migration in myo-genesis leading to RMS development especially for ERMSThere have been more miRs found as tumor suppressors inthis malignancy including miR-29 miR-450b-5p miR-203

8 Sarcoma

Table 4 MicroRNAs involved in myogenesis and RMS development

miRNA Expression Target Function Reference

miR-1 and miR-133a Downregulation MYH9Myogenic miRNA inhibit differentiationand promote proliferation in myogenesiscytostatic

Rao et al 2010 [42]

miR-206 Downregulation cMET Promote differentiation and proliferationin myogenesis

Yan et al 2009 [43]

miR-29 Downregulation HADC4 YY1 EZH2 Promote stabilization of RMS phenotype Marchesi et al 2014 [44]

miR-450-5p Downregulation ENOX PAX9 Promote differentiation and progression Sun et al 2014 [45]

miR-203 Downregulation JAK STAT Notch Inhibit differentiation and proliferation inmyogenesis tumor suppressor

Diao et al 2014 [46]

miR-214 Downregulation N-Ras Inhibit tumor cell growth and inducemyogenic differentiation and apoptosis

Huang et al 2014 [47]

miR-183 Upregulation EGR1 Promote migration and metastasis Sarver et al 2010 [48]

PMS2

MGMT

HR

Mismatch Recombination

NHEJ MMEJ

p53

Excision

BER NER

RMS

DNA repair

Direct repair Indirect repair

repair repairrepair

Figure 4The DNA repair systems in RMSThere are two pathways repairing the DNA lesions directly without affecting DNA structure andindirectly by DNA phosphodiester backbone cleavage The modifications in DNA repair enzymes expression or activity lead to resistanceto chemotherapy and radiation in RMS tumor cells MGMT O6-methylguanine-DNA methyltransferase BER base excision repair NERnucleotide excision repair HR homologous recombination NHEJ nonhomologous end-joining MMEJ microhomology-mediated end-joining

and miR-214 [44 46 47 84 87] They are all significantlydownregulated in RMS compared with normal skeletal mus-cles and function to inhibit tumor cell growth and inducemyogenic differentiation and apoptosis in RMS

52 Oncogenic miRNA in RMS The transcription factor EGR1is a tumor suppressor gene that is downregulated in RMS [48]miR-183 functions as an oncogene by targeting EGR1 and pro-moting tumor cell migration Either by direct anti-miR treat-ment or by indirect mechanisms that decrease transcriptmiR-183 targeted treatmentsmight provide a potential optionto RMS patients

The basic strategy of current effective miR-based treat-ment studies is either efficient reexpression of miRs or restor-ing the function of miRs to inhibit the expression of certain

protein-coding genes and promote muscle differentiationMoreover miR expression profiling in tumors and possiblytheir detection in peripheral blood during treatment may beable to predict the response to chemotherapy or radiotherapyand be useful as a prognostic signature for the development oftreatment resistanceThe prognostic value of miR expressionlevels in RMS is a powerful tool in creating a better-tailoredstrategy for particular subsets of patients

53 Long Noncoding RNAs in RMS Long noncoding RNAs(LNCRNAs) are abundant in the mammalian transcriptomeand have been shown to play a role in RMS development [8889]TheH19 gene localized within a chromosomal region onhuman chromosome 11p15 encodes an imprinted untrans-lated RNA [90] H19 opposite tumor suppressor (HOTS)

Sarcoma 9

Table 5 In vivo animal models of RMS

Model TargetModel origin Reference

Inactivation Expression

DrosophilaPAX-FKHR Bipartite Gal4-UAS

expression systemGalindo et al2006 [49]

PAX7-FOXO1 rols Chromosomal deletionDf(3L)vin5

Avirneni-Vadlamudi et al2012 [50]

Zebrafishrag2 promoter c-Myc kRASG12D

Chen andLangenau 2011[51]

MAPKERK andAKTS6K1 PD98059 TPCK Tg(hsp70-HRASG12V) Le et al 2012

[52]

Mouse

Sufu N-myc Sfrp1 Ptch2and cyclin D1 Sufu+minus Lee et al 2007

[53]

Ink4aArf and Trp53 Pax3FKHR Pax3P3Fpwt Keller et al2004 [54]

Wnt120573-cateninsignaling pathway Wnt2 p53minusminusc-fosminusminus Singh et al 2010

[55]

a tumor growth inhibitor is encoded by an imprinted H19antisense transcript Overexpression of HOTS inhibits RMStumor cell growth Silencing HOTS by RNAi was shown toincrease in vitro colony formation as well as in vivo tumorgrowth of RMS [90]

6 In Vivo Models of RMS

Several in vivo models have recently been developed inDrosophila zebrafish and mice to further understand themolecular and cellular biology of RMS tumorigenesis [49ndash55 91ndash94] (Table 5)

61 Drosophila Models in RMS Despite the evolutionarydistance a strong conservation of genes pathways andregulatory molecular networks have been demonstratedbetween flies and humans Many human disease genes haverelated sequences in Drosophila In contrast to mammalianmodels the generation of Drosophila mutants is easy cheapand fast

For a better understanding of the pathogenic conse-quences of PAX-FKHR expression Drosophila was chosenbecause the animalrsquos transparent outer cuticle allows forsimple real-time monitoring of muscle abnormalities elicitedby PAX-FKHR including subtle or focal changes [49] Themutation in the myoblast fusion gene rolling pebbles (rols)was reported to dominantly suppress PAX-FKHR1-inducedlethality by using a Drosophila model of PAX-FKHR1-mediated transformation [50] PAX-FKHR1 signaling upreg-ulates the TANC1 gene and blocks myoblasts from terminaldifferentiation However downregulating the TANC1 genecould cause RMS cells to lose their neoplastic state undergofusion and form differentiation This novel finding uncov-ered a PAX-FKHR1-TANC1 neoplasia axis collaboratively ina Drosophila model and loss-gain-of-function studies inmammalian platforms [50]

62 Zebrafish Models in RMS In contrast to other mod-els the short tumor onset in zebrafish allows for rapididentification of essential genes in various processes suchas tumor growth self-renewal and maintenance There areother advantages for using zebrafish in the study of humandisease and development including the ease and low cost ofraising large numbers of fish and the highly conserved geneticand biochemical pathways between zebrafish and mammals[95]

As zebrafish RMS is highly similar to human ERMSby using fluorescent transgenic approaches it is more con-venient to understand histogenesis and different aspects oftumorigenesis in RMS A robust zebrafish model of RAS-induced RMS has been established sharing twomorphologicand immunophenotypic features resembling ERMS [51] Inone case both are associated with tissue-restricted geneexpression in RMS while in another both comprise a RAS-induced gene signature Cross-species microarray compar-isons confirm that conserved genetic tumor-specific andtissue-restricted pathways such as RAS and p53 pathwaysdrive RMS growth Tg(hsp70-HRASG12V) zebrafish embryoswere generated to evaluate gene expression that mimics RASpathway activation during tumorigenesis to defineRAS targetgenes [52] Another KRASG12D-induced zebrafish embryonalRMS was utilized to assess the therapeutic effects By theblockage of two major downstream signaling pathwaysMAPKERK and AKTS6K1 the model showed that inhibi-tion of translation initiation suppresses tumor cell prolifera-tion [52]

All the above zebrafish cancer models share similar cellu-lar features and molecular pathways with human RMS espe-cially ERMS and can demonstrate a response to therapiesin a similar manner with human RMS Thus a tremendousopportunity is available to implement these animal modelsinto different steps for novel targeted therapeutic develop-ment

10 Sarcoma

63 Mouse Models in RMS Mouse models established fromin vivo studies may contribute to the understanding of thegenetic basis in tumor development and progression andmayhelp to test the efficacies of novel antineoplastic agents

The importance of PAX3PAX7-FKHR fusion proteins inthe progression of ARMS tumorigenesis is evident from bothin vitro and in vivo studies Transgenic mouse models gener-ated with the PAX-FKHR fusion genemimic the formation ofhuman ARMS [67] A mouse model was developed with het-erozygous loss of Sufu a tumor suppressor inHedgehog (Hh)signaling combined with p53 loss driven ERMS with a lowpenetrance (9) [53] These studies indicate that the modelsconsistently formingARMSnot only need the introduction ofthe PAX-FKHR fusion gene but also need to be accompaniedby other genetic events such as p53 pathway disruption [54]

Compared with ARMS ERMS models are more complexto generate because of their lower tumor penetrance andlonger latency JW41 cells from the p53c-fos double mutantmice models resemble human ERMS cells morphologicallyand express similar characteristic markers [55] The overex-pression of the Wnt2 gene identified in JW41 cells confirmedin human RMS cell lines can allow for further analysis of theWnt signaling pathway Both of these results indicate that thedownregulation of theWnt pathway contributes to resistanceto apoptosis and the inhibition of myogenic differentiation inERMS

Using Ptch1 p53 andor Rb1 conditional mouse modelsand controlling prenatal or postnatal myogenic cell of originRubin et al demonstrated that the loss of p53 in maturingmyoblasts related to tumorigenesis of ERMS [96] They alsoindicated that Rb1 alteration with other oncogenic factorswas strongly associated with an undifferentiated phenotypeacting as a modifier In addition they highlighted a sub-set of p53-deficient ERMSs arising from Myf6-expressingmyoblasts which had the same latency with Pax7-expressingmurine satellite cells in the study

7 Potential Therapeutic Targets in RMS

Detection of genomic imbalances and identification of thecrucial effective genes can contribute to identifying novelpotential biomarkers and relevant targets for clinical therapyin RMS

Survivin-responsive conditionally replicating adenovi-ruses can regulate multiple factors (Survm-CRAs) [41]Although Survm-CRA can efficiently replicate and potentlyinduce cell death in RMS cells the cytotoxic effects are morepronounced in RSC-enriched or RSC-purified cells thanin RSC-exiguous or progeny-purified cells Injections ofSurvm-CRAs into tumor nodules generated by transplantingRSC-enriched cells induce significant death in RMS cells andregression of tumor nodulesThe unique therapeutic featuresof Survm-CRA include not only its therapeutic effectivenessagainst RMS but also its increasing effectiveness against CSCsuggesting that Survm-CRA may be a promising anticanceragent [41]

Rapamycin an inhibitor of mTOR can abrogate RMStumor growth in a xenograft mouse model [97] Thetumors in the rapamycin-treatedmice group show significant

reduction of proliferation and invasiveness and inductionof apoptosis The tumor growth inhibition is simultaneousassociated with the diminution of mTOR and Hh pathwayswhich are implicated in the pathogenesis of RMSThe resultsindicate that using rapamycin either alone or in combinationwith traditional chemotherapeutic drugs may represent apotential targeted agent for therapeutic intervention Ampli-fication and mutational activation of Fibroblast Growth Fac-tor Receptor 4 (FGFR4) in RMS cells promote tumor pro-gression [98] Ponatinib is the most potent FGFR4 inhibitorto block wild-type RMS cell growth mutate FGFR4 throughincreasing apoptosis and suppress the FGFR4 downstreamtarget STAT3 Ponatinib treatment slows tumor growth inRMS mouse models expressing mutated FGFR4 [98] Pona-tinib as an FGFR4 inhibitor shows potential to act as aneffective therapeutic agent for RMS tumorsThe expression ofCXC chemokine receptor 4 (CXCR4) CXCR7 and VascularEndothelial Growth Factor (VEGF) indicates poor prognosisin a variety of malignancies including RMS cell lines Mostsamples in a large series of clinical RMS cases show asso-ciation with high expression of these antagonists regardlessof their subtypes [99] However there are significant corre-lations with high expression of CXCR4 and VEGF in bothERMS and ARMS High VEGF expression is predictive ofadverse prognostic factors in RMS as the expression of theseangiogenesis factors was compared with clinicopathologicalparameters and prognosis Considering their overexpressionin RMS these chemokine receptors and VEGF could providepotential molecular therapeutic targets in RMS Some ARMScell lines have undergone apoptosis in response to antineo-plastic drugs such as bortezomib The proapoptotic BH3only family member NOXA acts as an upregulated proteindownstream PAX3-FKHR resulting in increased cell sensi-tivity to bortezomib Apoptosis in response to bortezomibcan be reversed by shRNA knockdown of Noxa PAX3-FKHR upregulation of Noxa creates a potential therapeuticinsight into inducing apoptosis in ARMS cellsThis apoptosispathway could represent a specific targeted therapy againstPAX3-FKHR-expressing ARMS cells [100]

Proton pump activation resulting from intracellular acid-ificationmay be an energetic mechanism to escape apoptosisV-ATPase is an ATP-driven proton pump acidifying theintracellular compartment and transports protons across theplasma membrane Esomeprazole as a V-ATPase inhibitor inthe PPI administration interferes with intensive ion trans-porter activity of RMS cells and induces remarkable cytotoxi-city RMS expresses a higher level ofV-ATPase comparedwithother sarcomasTherefore RMS should be very susceptible toesomeprazole treatment and V-ATPase could be consideredas a promising selective target for treatment [101]

8 Conclusion and Future Prospects

For RMS the current challenges include how to iden-tify promising novel therapeutics and integrate them intoexisting therapy For better understanding of congenitaland epigenetic modifications in the development of RMSthis review summarizes the recent genome-wide studies onmolecular and genetic alterations to decipher the underlying

Sarcoma 11

tumorigenesis mechanisms There is a link between geneticand epigenetic alterations responsible for a variety of tumorcell growth proliferation differentiation apoptosis andtherapy-resistance mechanisms The identification of theprognostic value of the PAX-FKHR fusion status in RMSis one of the most important shifts in the lineage and therisk assessment of RMS which exerts an oncogenic effectthrough multiple pathways and is incorporated into mostrelevant studies The misbalanced expression of a number ofmiRs involved in the regulation of myogenic differentiationalso implicates the tumorsrsquo escape suppressive mechanismsMoreover we described several in vivo models of RMSto explore underlying histogenesis and different aspects oftumorigenesis Recent work on CSCs has also contributedto understanding the cell origins refining molecular char-acterization and elucidating the underlying basis of refrac-tory responses to current adjuvant treatments of subsets oftumorsThese integrative approaches can provide opportuni-ties to identify diagnostic and prognostic biomarkers appliedto the individual targeted therapy and significantly improveRMS prognosis

Abbreviations

RMS RhabdomyosarcomaARMS Alveolar rhabdomyosarcomaERMS Embryonal rhabdomyosarcomaPRMS Pleomorphic rhabdomyosarcomaSRMS Sclerosingspindle cell

rhabdomyosarcomamiRNA miR MicroRNACSC Cancer stem cellMSC Mesenchymal stem cell

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported in part by grants from the Gat-tegno and Wechsler funds Dr Duan was supported in partthrough a grant from Sarcoma Foundation of America (SFA)a grant from National Cancer Institute (NCI)National Insti-tutes of Health (NIH) UO1 CA 151452 and a grant from anAcademic Enrichment Fund of MGH Orthopedic Surgery

References

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[2] D Egas-Bejar and W W Huh ldquoRhabdomyosarcoma in adoles-cent and young adult patients current perspectivesrdquo AdolescentHealth Medicine andTherapeutics vol 5 pp 115ndash125 2014

[3] J C Breneman E Lyden A S Pappo et al ldquoPrognosticfactors and clinical outcomes in children and adolescents withmetastatic rhabdomyosarcomamdasha report from the intergrouprhabdomyosarcoma study IVrdquo Journal of Clinical Oncology vol21 no 1 pp 78ndash84 2003

[4] V D Soleimani and M A Rudnicki ldquoNew insights into theorigin and the genetic basis of rhabdomyosarcomasrdquo CancerCell vol 19 no 2 pp 157ndash159 2011

[5] T Glass D D Cochrane S R Rassekh K Goddard and JHukin ldquoGrowing teratoma syndrome in intracranial non-ger-minomatous germ cell tumors (iNGGCTs) a risk for secondarymalignant transformationmdasha report of two casesrdquo Childrsquos Ner-vous System vol 30 no 5 pp 953ndash957 2014

[6] J Ji C Eng and K Hemminki ldquoFamilial risk for soft tissuetumors a nation-wide epidemiological study from SwedenrdquoJournal of Cancer Research and Clinical Oncology vol 134 no5 pp 617ndash624 2008

[7] C D M Fletcher J A Bridge P Hogendoorn and F MertensWHO Classification of Tumours of Soft Tissue and Bone vol 5IARC Press Paris France 4th edition 2013

[8] I Sultan I Qaddoumi S Yaser C Rodriguez-Galindo and AFerrari ldquoComparing adult and pediatric rhabdomyosarcoma inthe surveillance epidemiology and end results program 1973 to2005 an analysis of 2600 patientsrdquo Journal of Clinical Oncologyvol 27 no 20 pp 3391ndash3397 2009

[9] B Raney W Huh D Hawkins et al ldquoOutcome of patientswith localized orbital sarcoma who relapsed following treat-ment on Intergroup Rhabdomyosarcoma Study Group (IRSG)Protocols-III and -IV 1984ndash1997 a report from the ChildrenrsquosOncology Grouprdquo Pediatric Blood and Cancer vol 60 no 3 pp371ndash376 2013

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[13] V Paulson G Chandler D Rakheja et al ldquoHigh-resolutionarray CGH identifies common mechanisms that drive embry-onal rhabdomyosarcoma pathogenesisrdquo Genes Chromosomesand Cancer vol 50 no 6 pp 397ndash408 2011

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12 Sarcoma

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[43] D Yan X D Dong X Chen et al ldquoMicroRNA-1206 targets c-met and inhibits rhabdomyosarcomadevelopmentrdquoThe Journalof Biological Chemistry vol 284 no 43 pp 29596ndash29604 2009

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[46] Y Diao X Guo L Jiang et al ldquoMiR-203 a tumor suppressorfrequently down-regulated by promoter hypermethylation inrhabdomyosarcomardquo The Journal of Biological Chemistry vol289 no 1 pp 529ndash539 2014

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Sarcoma 13

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[71] S C Marianna L Ianzano and G Nicoletti ldquoTumor sup-pressor genes promote rhabdomyosarcoma progression in p53heterozygousrdquo Oncotarget vol 5 no 1 pp 108ndash119 2013

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14 Sarcoma

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[88] S-Y Ng G K Bogu B Soh and L W Stanton ldquoThe longnoncoding RNA RMST interacts with SOX2 to regulate neu-rogenesisrdquoMolecular Cell vol 51 no 3 pp 349ndash359 2013

[89] C A Lynch B Tycko T H Bestor and C P Walsh ldquoReac-tivation of a silenced H19 gene in human rhabdomyosarcomaby demethylation of DNA but not by histone hyperacetylationrdquoMolecular Cancer vol 1 article 2 2002

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[91] C E Albacker N Y Storer E M Langdon et al ldquoThe his-tone methyltransferase SUV39H1 suppresses embryonal rhab-domyosarcoma formation in zebrafishrdquo PLoS ONE vol 8 no 5Article ID e64969 2013

[92] N Y Storer R M White A Uong et al ldquoZebrafish rhab-domyosarcoma reflects the developmental stage of oncogeneexpression during myogenesisrdquo Development vol 140 no 14pp 3040ndash3050 2013

[93] M E Hatley W Tang M R Garcia et al ldquoA mouse modelof rhabdomyosarcoma originating from the adipocyte lineagerdquoCancer Cell vol 22 no 4 pp 536ndash546 2012

[94] V Hosur A Kavirayani J Riefler et al ldquoDystrophin and dysfer-lin doublemutantmice a novelmodel for rhabdomyosarcomardquoCancer Genetics vol 205 no 5 pp 232ndash241 2012

[95] W Goessling T E North and L I Zon ldquoNew waves of discov-ery modeling cancer in zebrafishrdquo Journal of Clinical Oncologyvol 25 no 17 pp 2473ndash2479 2007

[96] B P Rubin K Nishijo H-I H Chen et al ldquoEvidence for anunanticipated relationship between undifferentiated pleomor-phic sarcoma and embryonal rhabdomyosarcomardquo Cancer Cellvol 19 no 2 pp 177ndash191 2011

[97] S Z Kaylani J Xu R K Srivastava L Kopelovich J G Presseyand M Athar ldquoRapamycin targeting mTOR and hedgehog sig-naling pathways blocks human rhabdomyosarcoma growth inxenograft murine modelrdquo Biochemical and Biophysical ResearchCommunications vol 435 no 4 pp 557ndash561 2013

[98] S Q Li A T Cheuk J F Shern et al ldquoTargeting wild-type andmutationally activated FGFR4 in rhabdomyosarcoma with theinhibitor ponatinib (AP24534)rdquoPLoSONE vol 8 no 10 ArticleID e76551 2013

[99] K Miyoshi K Kohashi F Fushimi et al ldquoClose correlationbetween CXCR4 and VEGF expression and frequent CXCR7expression in rhabdomyosarcomardquo Human Pathology vol 45no 9 pp 1900ndash1909 2014

[100] ADMarshall F Picchione R I KGeltink andGCGrosveldldquoPAX3-FOXO1 induces up-regulation of Noxa sensitizing alve-olar rhabdomyosarcoma cells to apoptosisrdquo Neoplasia vol 15no 7 pp 738ndash748 2013

[101] F Perut S Avnet C Fotia et al ldquoV-ATPase as an effective thera-peutic target for sarcomasrdquo Experimental Cell Research vol 320no 1 pp 21ndash32 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Sarcoma

Table 1 Alterations of chromosome in RMS by CGH in recent 5 years

Materials Gain Amplification Loss Deletion Reference

25 RMS samples 2p 12q 6p 9q 10q 1p2q 6q 8q 15q 18q 3p 11p 6p Li et al 2009 [16]

13 RMS cell lines

1p213ndash132 1q126q26ndash27 7q213ndash3111q41 2p243 8q241220q132 20q1332

2p243 (MYCN)8p1123ndash1121

(FGFR1) 12q133(CDK4) 19q12 20q

3p142ndash1224q27ndash3236p251ndash243

9p243ndash241 13q143

Missiaglia et al 2009[17]

57 ARMSsamples 12q15 2p24 12q13ndashq14 Barr et al 2009 [18]

128 primaryRMS samples 7 8 11 20

2p241 (MYCN)8p112ndashp111 (FGFR1)12q133ndash141 (CDK4)MDM2 (12q143ndashq15)

Williamson et al2010 [12]

26 frozenprimary ERMSsamples

8 2 11 12 13 19 20

2p21 2q35 2q1422q361 5q352ndashq353

11p112 11q24212q133

6 9 10 14 15 16 181p3623 1q321 3p1424q351ndash352 9p21317q112 22q1331

Paulson et al 2011[13]

39 RMS samples 12q133 12q133ndashq14112q141 17q251

2q1312 12q13312q133ndashq141

9p12ndashp11210q1121ndashq112214q3233 16p112

22q111

1p211 2q141 5q1329p12 9q12 Liu et al 2014 [11 15]

RMS cellsderived fromrefractory RMS

NACA HSD17B6SDR9C7 RDH16GPR182 ZBTB39TAC3 MYO1A

NAB2 STAT6 LRP1

Park et al 2014 [14]

20 RMS samplesof Chinesepatients

12q2431 17q2511q211 7q112312q133ndashq141

9p133 12q133ndashq14112q15 16p1311

5q132 15q11214q3233 (IGHG

IGHM)

1p3633 1p131 2q1115q132 8p231 9p243

16p112Liu et al 2014 [11 15]

Since the 1970s the Intergroup RhabdomyosarcomaStudy Group (IRSG) has conducted a series of clinical trialscomparing risk-base and has established a series of treatmentguidelines [9] Currently multidisciplinary managementincluding chemotherapy and surgery with or without radia-tion has become the standard treatment for RMS The 5-yearsurvival rate of RMS has increased from 25 in 1970 up to60 since 2000 [1 10]However there has been little improve-ment in the oncological outcome of patients with RMS inrecent years Drug resistance andmetastatic disease representthe two most common phenomena for therapy failure Somerandomized chemotherapy trials have failed to improve out-come despite the introduction of newer or more intensivetherapies Thus there is an urgent need for alternative moreeffective treatment strategies Recent molecular and geneticanalysis of these tumors has produced substantial newinsights into molecular cell biology molecular cytogeneticsand tumorigenesis of RMS leading to a better understandingof RMS development at the molecular level These advancesmay ultimately lead to better clinical understanding and topotentially developing more potent targeted therapies Thepurpose of this review is to summarize these most recentfindings in RMS

2 Novel Discoveries of ChromosomalAlterations in RMS

Malignant transformation occurs cytogenetically due to theaccumulation of somatic mutations by the acquisition oftumor-specific chromosomal translocations

21 Gains and Losses of Chromosomes in RMS ComparativeGenomic Hybridization (CGH) analysis has revealed that allRMS have specific gains and losses [11ndash18] (Table 1) ERMSfrequently exhibits gains or losses of specific whole chromo-somes whereas ARMS is characterized by the presence ofregions of genomic amplification [11 19] The focal regionsand genes most of which have frequent gains and ampli-fications include 12q133ndashq141 and 8p112ndash112 and CDK4MYCN GLI MDM2 FGFR1 and FGFR4 respectively Fre-quently the genes differentially expressed in subtypes ofRMS particularly when they are from chromosomal regionsshow a high level of gains in cell lines Previous CGH studieshave shown that ARMS tumors tend to have fewer copynumber variants than ERMS tumors [12 20] For examplefrequent gains were detected in TYROBP HCST LRFN3and ALKBH6 (19q1312) in ERMS but not in ARMS [15]

Sarcoma 3

PAX3

Chromosome 2

PAX3-FKHR

t(213)(q37q14)

NCOA1

PAX7

Chromosome 1

PAX7-FKHR

t(113)(p36q14)

PAX3

Chromosome 2

FKHR

Chromosome 13

PAX3-NCOA1

t(22)(q35q23)



PB

PB

PB

PB HD

HD

HD

HD TAD

TAD

TAD

TAD

TAD

TAD

HD

HD

PB

PB

100 amino acids

Fusion site

Fusion site

FD


LXXLL HAT

HAT

bHLHPAS

Fusion site

(a)

(b)

(c)







4 Sarcoma















Sarcoma 5










Ren et al 2008[39]







Migration

specification

Injured muscle


Fusion

PAX7

Myoblasts

Myoblast

MyoD

MyoD

Myf5

PAX3

Rb

Ezh2

IGF2

HDAC4YY1

YY1

ERK

AKT

RMS

Myogenin

P38 kinase

Myostatin

Progenitor

cell

N-RAS

Myofibers

FGFR4

UBE2C

UHRF1

NOTCH2

YWHAB

Satellite cells

RUNX1 MEF2C

JDP2 NFIC

Myocytes


differentiation

Proliferatio

n


TGF-1205731





6 Sarcoma

METCDKN2AB

PAX37-FKHR

MDM2

RAS

Raf

GLI1

PDK1p53Rb

Hh

Myo MEF2

p21

Ptch

FGFR4

IGF1R

p16INK4Ap14ARF

p19ARF

IGF2

FGF










Sarcoma 7












8 Sarcoma




Rao et al 2010 [42]


Yan et al 2009 [43]








PMS2

MGMT

HR


NHEJ MMEJ

p53

Excision

BER NER

RMS

DNA repair


repair repairrepair







Sarcoma 9











[52]

Mouse


[53]



[55]









10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 3

PAX3

Chromosome 2

PAX3-FKHR

t(213)(q37q14)

NCOA1

PAX7

Chromosome 1

PAX7-FKHR

t(113)(p36q14)

PAX3

Chromosome 2

FKHR

Chromosome 13

PAX3-NCOA1

t(22)(q35q23)



PB

PB

PB

PB HD

HD

HD

HD TAD

TAD

TAD

TAD

TAD

TAD

HD

HD

PB

PB

100 amino acids

Fusion site

Fusion site

FD


LXXLL HAT

HAT

bHLHPAS

Fusion site

(a)

(b)

(c)







4 Sarcoma















Sarcoma 5










Ren et al 2008[39]







Migration

specification

Injured muscle


Fusion

PAX7

Myoblasts

Myoblast

MyoD

MyoD

Myf5

PAX3

Rb

Ezh2

IGF2

HDAC4YY1

YY1

ERK

AKT

RMS

Myogenin

P38 kinase

Myostatin

Progenitor

cell

N-RAS

Myofibers

FGFR4

UBE2C

UHRF1

NOTCH2

YWHAB

Satellite cells

RUNX1 MEF2C

JDP2 NFIC

Myocytes


differentiation

Proliferatio

n


TGF-1205731





6 Sarcoma

METCDKN2AB

PAX37-FKHR

MDM2

RAS

Raf

GLI1

PDK1p53Rb

Hh

Myo MEF2

p21

Ptch

FGFR4

IGF1R

p16INK4Ap14ARF

p19ARF

IGF2

FGF










Sarcoma 7












8 Sarcoma




Rao et al 2010 [42]


Yan et al 2009 [43]








PMS2

MGMT

HR


NHEJ MMEJ

p53

Excision

BER NER

RMS

DNA repair


repair repairrepair







Sarcoma 9











[52]

Mouse


[53]



[55]









10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Sarcoma















Sarcoma 5










Ren et al 2008[39]







Migration

specification

Injured muscle


Fusion

PAX7

Myoblasts

Myoblast

MyoD

MyoD

Myf5

PAX3

Rb

Ezh2

IGF2

HDAC4YY1

YY1

ERK

AKT

RMS

Myogenin

P38 kinase

Myostatin

Progenitor

cell

N-RAS

Myofibers

FGFR4

UBE2C

UHRF1

NOTCH2

YWHAB

Satellite cells

RUNX1 MEF2C

JDP2 NFIC

Myocytes


differentiation

Proliferatio

n


TGF-1205731





6 Sarcoma

METCDKN2AB

PAX37-FKHR

MDM2

RAS

Raf

GLI1

PDK1p53Rb

Hh

Myo MEF2

p21

Ptch

FGFR4

IGF1R

p16INK4Ap14ARF

p19ARF

IGF2

FGF










Sarcoma 7












8 Sarcoma




Rao et al 2010 [42]


Yan et al 2009 [43]








PMS2

MGMT

HR


NHEJ MMEJ

p53

Excision

BER NER

RMS

DNA repair


repair repairrepair







Sarcoma 9











[52]

Mouse


[53]



[55]









10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 5










Ren et al 2008[39]







Migration

specification

Injured muscle


Fusion

PAX7

Myoblasts

Myoblast

MyoD

MyoD

Myf5

PAX3

Rb

Ezh2

IGF2

HDAC4YY1

YY1

ERK

AKT

RMS

Myogenin

P38 kinase

Myostatin

Progenitor

cell

N-RAS

Myofibers

FGFR4

UBE2C

UHRF1

NOTCH2

YWHAB

Satellite cells

RUNX1 MEF2C

JDP2 NFIC

Myocytes


differentiation

Proliferatio

n


TGF-1205731





6 Sarcoma

METCDKN2AB

PAX37-FKHR

MDM2

RAS

Raf

GLI1

PDK1p53Rb

Hh

Myo MEF2

p21

Ptch

FGFR4

IGF1R

p16INK4Ap14ARF

p19ARF

IGF2

FGF










Sarcoma 7












8 Sarcoma




Rao et al 2010 [42]


Yan et al 2009 [43]








PMS2

MGMT

HR


NHEJ MMEJ

p53

Excision

BER NER

RMS

DNA repair


repair repairrepair







Sarcoma 9











[52]

Mouse


[53]



[55]









10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 Sarcoma

METCDKN2AB

PAX37-FKHR

MDM2

RAS

Raf

GLI1

PDK1p53Rb

Hh

Myo MEF2

p21

Ptch

FGFR4

IGF1R

p16INK4Ap14ARF

p19ARF

IGF2

FGF










Sarcoma 7












8 Sarcoma




Rao et al 2010 [42]


Yan et al 2009 [43]








PMS2

MGMT

HR


NHEJ MMEJ

p53

Excision

BER NER

RMS

DNA repair


repair repairrepair







Sarcoma 9











[52]

Mouse


[53]



[55]









10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 7












8 Sarcoma




Rao et al 2010 [42]


Yan et al 2009 [43]








PMS2

MGMT

HR


NHEJ MMEJ

p53

Excision

BER NER

RMS

DNA repair


repair repairrepair







Sarcoma 9











[52]

Mouse


[53]



[55]









10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















8 Sarcoma




Rao et al 2010 [42]


Yan et al 2009 [43]








PMS2

MGMT

HR


NHEJ MMEJ

p53

Excision

BER NER

RMS

DNA repair


repair repairrepair







Sarcoma 9











[52]

Mouse


[53]



[55]









10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 9











[52]

Mouse


[53]



[55]









10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















10 Sarcoma













Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 11


Abbreviations





Acknowledgments


References


















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















12 Sarcoma































Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 13

































14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















14 Sarcoma




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article rhabdomyosarcoma: advances in molecular and

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