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Alveolar rhabdomyosarcoma – The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis

Alveolar rhabdomyosarcoma – The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis Rhabdomyosarcoma is a soft tissue sarcoma arising from cells of a mesenchymal or skeletal muscle lineage. Alveolar rhabdomyosarcoma (ARMS) is more aggressive than the more common embryonal (ERMS) subtype. ARMS is more prone to metastasis and carries a poorer prognosis. In contrast to ERMS, the majority of ARMS tumors carry one of several characteristic chromosomal translocations, such as t(2;13)(q35;q14), which results in the expression of a PAX3-FOXO1 fusion transcription factor. In this review we discuss the genes that cooperate with PAX3-FOXO1, as well as the target genes of the fusion transcription factor that contribute to various aspects of ARMS tumorigenesis. The characterization of these pathways will lead to a better understanding of ARMS tumorigenesis and will allow the design of novel targeted therapies that will lead to better treatment for this aggressive pediatric tumor. Keywords: Alveolar rhabdomyosarcoma, PAX3-FOXO1, PAX7-FOXO1, FGFR4, CNR1, IRIZIO, N-MYC, IGF2, MET, CXCR4, p53, MDM2, P-Cadherin, TFAP2B, miR17-92 Introduction retroperitoneum [7,8]. The alveolar subtype (ARMS) con- According to the American Cancer Society, rhabdomyo- stitutes approximately another 20% of RMS cases [10] and sarcoma (RMS) comprises about three percent of child- occurs predominantly in adolescents. Histologically, hood cancers, with about 350 new cases occurring ARMS tumors typically contain alveoli structures similar annually in the US [1], and it affects slightly more males in appearance to those seen in the lung [7], though solid- than females [2]. RMS is a small, round, blue cell tumor variant ARMS does occur [11]. Primary ARMS tumors usually arising in skeletal muscle tissue, and it is thought typically arise in the extremities and trunk [7-9,12], and to originate from mesenchymal cells likely committed to they are more aggressive than their ERMS counterparts. the skeletal muscle lineage. Consistent with a myogenic ARMS is associated with a poorer prognosis, with a 5-year origin, RMS tumors express skeletal muscle markers such failure-free survival of 65% [8]. as skeletal muscle actin and myosin, desmin, myoglobin, A characteristic of the ARMS subtype is the occurrence Z-band protein, MYOD and often myogenin [3-7]. RMS of recurrent chromosomal translocations. The most com- consists of two major histological subtypes, embryonal mon of these is t(2;13)(q35;q14), which results in the and alveolar RMS. The embryonal subtype (ERMS) is expression of an oncogenic fusion protein. This fusion thought to be histologically reminiscent of embryonic protein consists of the paired and homeodomains of the developing skeletal muscle [7]. ERMS is the most preva- PAX3 transcription factor with the potent transcriptional lent of the subtypes, accounting for about 60% of RMS activation domain of FOXO1 (FKHR), a member of the cases [2]. It occurs mainly in children younger than 10 forkhead (FOX) family of transcription factors [13-15]. years and is usually associated with a favorable prognosis, The PAX3 homeodomain is required to recapitulate with a failure-free survival rate of 81% [8,9]. Tumors usu- PAX3-FOXO1-induced tumorigenesis, though the paired ally localize to the head and neck (including the extraocu- domain may play a minor role [16,17]. The PAX3-FOXO1 lar muscles of the eye), the genitourinary tract and the fusion protein can be detected in about 55% of ARMS cases [18]. A similar translocation of t(1;13)(p36;q14) fuses the PAX7 DNA-binding domains, the closest homolog of * Correspondence: gerard.grosveld@stjude.org PAX3, to FOXO1 [19]. This translocation occurs in a fur- Department of Genetics, St Jude Children’s Research Hospital, Memphis, TN 38105, USA ther 22% of ARMS cases [18]. Recently, further similar Full list of author information is available at the end of the article © 2012 Marshall and Grosveld; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 2 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 translocations have been found in individual ARMS cases: PAX3-FOXO1 transcriptional activity [37]. There is also t(2;X)(q35;q13), which results in PAX3-AFX fusion [20], evidence that MyoD transcriptional activity is abrogated in and t(2;2)(q35;p23) and t(2;8)(q35;q13), which generate a ERMS tumors [40]. fusion protein of PAX3-NCOA1 and PAX3-NCOA2, The remaining ARMS tumors are classed as fusion- respectively [21,22]. These “cryptic” rare fusion variants negative ARMS. However, fusion-negative ARMS are in- are thought to be present in up to another 10% of ARMS distinguishable on the levels of gene expression and in tumors [7]. During normal development, PAX3 expression clinical outcome from ERMS tumors, leading some to occurs in the neural tube and dermomyotome [23], and it argue that translocation status should be the defining is required for the normal migration of skeletal muscle factor of ARMS [41-44]. Within the ARMS subtype, precursors to the limb bud [24]. PAX7 expression is a prognosis can vary by disease stage at diagnosis as well marker of satellite cells in adult skeletal muscle [25] and is as translocation status. For example, patients presenting required for normal self-renewal [26]. Unlike skeletal with metastatic disease have an estimated 4-year overall muscle-specific PAX3 and PAX7, FOXO1A, AFX, survival rate of 75% for PAX7-FOXO1, while those with NCOA1 and NCOA2 are widely expressed and mediate the PAX3-FOXO1 translocation have only 8% estimated gene transcription downstream of cell signaling pathways 4-year overall survival [18]. Indeed, there is evidence [27-31]. All of the ARMS fusion proteins consist of the that the PAX3-FOXO1 is a more potent oncogene than PAX3/7 DNA-binding domains fused to the transcrip- PAX7-FOXO1. Barr et al. [45] found that only 1/24 tional activation domains of more potent transcription fac- PAX3-FOXO1-positive ARMS tumors had amplification tors (see Figure 1) [14,15,22]. Genome-wide transcription of the PAX3-FOXO1 gene, while PAX7-FOXO1 was factor-binding studies have not yet been performed to de- amplified in 10/11 PAX7-FOXO1 ARMS, implying that termine whether wild-type PAX3 and PAX7-binding sites genomic amplification of PAX7-FOXO1 is required for differ from these PAX3/7 fusion transcription factor- tumorigenesis, while a single copy of PAX3-FOXO1 is binding sites. sufficient. However the gene expression profiles of Expression of these ARMS fusion transcription factors is PAX3-FOXO1- and PAX7-FOXO1-expressing tumors thought to abrogate normal skeletal muscle differentiation, have not, to the author’s knowledge, been specifically allowing aberrant cell division and tumor development. compared to identify the gene set responsible for this PAX3 expression can inhibit myogenic differentiation of difference in prognosis between ARMS tumors with cultured myoblasts [35]. Although PAX3 protein is rapidly these two fusion genes. degraded during early myogenic differentiation, PAX3- FOXO1 has a significantly longer half-life than wild-type Review PAX3 [36]. PAX3/7-FOXO1 is capable of suppressing PAX3-FOXO1 is the most common fusion gene in MyoD expression and activity [37,38]. PAX7-FOXO1 ex- ARMS. This fusion transcription factor is thought to pression induces NFκB signaling, which inhibits myogen- drive the gene expression that causes the worse progno- esis via activation of cyclin D1/CDK4 complexes. These sis in ARMS tumors. Many studies have sought to iden- complexes sequester MyoD, which would normally drive tify the differences in gene expression between ERMS cell cycle withdrawal and myogenic differentiation [39]. In and ARMS, as well as the genes aberrantly regulated by addition transcriptionally inactive MyoD can enhance PAX3-FOXO1. In this review, we summarize these gene Figure 1 Gene translocation in alveolar rhabdomyosarcoma. Scale diagram showing the parent proteins and the resulting fusion proteins arising from chromosomal translocations occurring in ARMS. Green or yellow indicates the protein fusion sites [14,19-22]. Homologous domains are indicated in like colors. DNA-binding domains are indicated as: paired domain (PD), homeodomain (HD), fork head DNA-binding domain (FH) and basic helix-loop-helix domain (bHLH). Regions of the proteins known to act as transcriptional activation domains are indicated (TAD). Other domains include the octapeptide domain (O), PAS domains (PAS A/B), LXXLL motifs (L1-L7) and glutamine-rich region (Q-rich). Maps were derived from the following references: [14,19-22,32-34]. Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 3 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 expression changes specific to PAX3-FOXO1 expression reported in 0 to 22% of cases [12,56,57]. RMS cell lines and/or ARMS. Moreover, we consolidate these data into show a significantly higher rate of p53 abnormalities a list of genes that may well represent the means by with 60%, indicating establishment of these cell lines which ARMS tumors obtain a more aggressive pheno- through xenograft and cell culture increases the propor- type than ERMS. tion of cell lines with p53 alterations [56]. Looking at p53 and MDM2 expression levels, both are low in ARMS and ERMS. Metastatic ERMS tumors show sig- Cooperating mutations in ARMS tumors nificantly higher p53 protein expression, indicating that It is likely that PAX3/7-FOXO1 translocation is one of p53 gene alterations are a late event in rhabdomyosarco- the earliest events in ARMS tumorigenesis as it occurs magenesis. Again, p53 status did not show any correl- in the majority of ARMS cases, more often than any ation to prognosis [58]. other genetic lesion characterized in the disease. How- Chromosome 2 has been shown to be amplified at ever, PAX3-FOXO1 expression in normal cells is not 2p24 in 32 to 60% of ARMS tumors [49,51,59,60]. This sufficient to induce transformation, and other genetic region is known to contain the proto-oncogene N-MYC. alterations are required [46-48]. Genomic amplification Two independent studies have shown that a gain in the is common in ARMS tumors. The three most common genomic copy number of the N-MYC gene is associated amplifications seen in ARMS involve regions of chromo- with an unfavorable disease outcome [59,61]. In somes 2, 12 and 13 [49]. addition, N-MYC is more highly expressed in ARMS The region of chromosome 12 amplification spans cells lines than ERMS lines, despite the fact that it was 12q13-15 and is reported in 28% to 56% of ARMS found to only be genomically amplified in one of the five tumors [49-52]. This 12q13-15 region includes genes lines, indicating more than one mechanism of N-MYC such as C/EBP-homolog and transcription factor CHOP/ overexpression in ARMS. However, in this study no clear DDIT3/GADD153, sarcoma-amplified sequence and relationship in N-MYC expression was seen with regard transmembrane 4 superfamily member SAS/TSPAN31, to primary tumor samples [62]. alpha 2-macroglobulin receptor A2MR/LRP1, Sonic Another chromosomal region frequently amplified in hedgehog (SHH) pathway effector and zinc finger tran- ARMS is 13q31-32, showing amplification in between 14 scription factor GLI1, cyclin-dependent kinase cell cycle and 19% of ARMS tumors [49,51]. Presence of this amp- regulator CDK4 and p53 pathway modulator MDM2. In lification is significantly associated with poorer failure- most cases, gene amplification accompanies an increase free survival in ARMS [63]. The minimum overlapping in gene expression [50,53]. region of amplification at this region was originally Though GLI1 is amplified genetically, the expression defined as only containing two genes: GPC5 and of this gene is not always associated with its genetic C13ORF25. The C12ORF25 gene encodes the micro- amplification. When GLI1 is overexpressed in RMS, it RNA cluster miR-17-92 (MIR17HG) in an intron [64]. has been associated with an undifferentiated subtype ra- GPC5 overexpression can increase cell proliferation ther than ERMS or ARMS, indicating that GLI1 may through the modulation of the growth factor activity of play a role in tumors that show primitive histopatho- FGF2, HGF and WNT1a [64]. However, more thorough logical features [54]. Thus, GLI1 overexpression cannot mapping of the genetic amplification showed that the be well associated with the ARMS pathology. entire GPC5 locus was only amplified in 12.5% of 13q31 MDM2 is perhaps the best candidate oncogene in this re- amplified ARMS tumors, while the minimally amplified gion because of its inhibitory effect on p53 function [55]. region contains only the peptidylprolyl isomerase However, MDM2 is not always included in this 12q13-15 pseudogene (LOC390419) and MIR17HG. This amplifi- amplification. RH30, an ARMS cell line, lacks amplification cation is particularly prevalent in PAX7-FOXO1-positive ARMS tumors. The miR-17-92 cluster of micro-RNAs of MDM2 but shows amplification and overexpression of SAS, CHOP, GLI1 and A2MR [53]. In addition, the fre- has been shown to play a role in a variety of cancer types quency of MDM2 gene amplification specifically may be as (for review, see [65]). In PAX7-FOXO1, but not PAX3- FOXO1 expressing ARMS, overexpression of miR-17, low as 10% in ARMS tumors [56]. One study found only 2 of 34 ARMS samplestobehighlyimmunoreactivefor -19a, -19b, 20a and 92a is specifically associated with an MDM2 [12]. Moreover, MDM2 expression shows no asso- increased rate of 2-year treatment failure. This indicates a possible pro-tumorigenic interaction between PAX7- ciation with patient prognosis or other clinicopathologic parameters [12]. Thus, it may be amplification of one of the FOXO1 and miR-17-92 locus overexpression [63]. other genes at this chromosome 12 locus that is the import- Rhabdomyosarcoma can also be associated with a loss of heterozygosity (LOH) or loss of imprinting (LOI) at ant cooperating mutation with PAX3-FOXO1. Other alterations in the p53 pathway have been found 11p15.5 [66,67]. This region contains several imprinted in ARMS. In ARMS tumor samples mutated p53 was genes such as IGF2, which is maternally imprinted Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 4 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 (paternal allele is expressed), and H19 and p57/Kip2, [73,81] and in ARMS versus non ARMS tumor cell lines which are paternally imprinted (maternal allele is [78]. A tamoxifen (4-OHT)-inducible PAX3-FOXO1-ER expressed) [68-70]. IGF2 expression appears to be spe- construct induced upregulation of both N-MYC mRNA cifically upregulated by changes in imprinting or LOH at and protein in the transduced ERMS cell line, RD, and this locus in RMS. ERMS tumors are associated predom- this was not sensitive to cycloheximide treatment, indi- inantly with a LOH at the IGF2 locus, though there is cating that N-MYC is a direct transcriptional target of some discrepancy in the proportion of ERMS tumors PAX3-FOXO1. However the PAX3-FOXO1 regulatory showing this change: 23% according to Anderson et al. region for N-MYC did not appear to be contained within [66] and 72% according to Visser et al. [67]. Conversely, −1871 to +1058 of the N-MYC gene. Consistent with a IGF2 is upregulated by LOI in 46% of fusion-positive transforming role for both PAX3-FOXO1 and N-MYC, ARMS tumors, while imprinting of H19 is conserved in the two genes synergized in soft agar colony-forming 93% [66]. This indicates that an increase in IGF2 expres- assays [81]. In addition, knockdown of N-MYC expres- sion in RMS is important for tumorigenesis, though the sion identified a positive feedback loop between N-MYC mechanism of this upregulation, either LOH or LOI, and PAX3-FOXO1 [93]. varies by subtype. IGF2 was shown to be specifically overexpressed in A screen for PAX3-FOXO1-interacting proteins using ARMS compared to Ewing’s sarcoma cell lines [75], ARF−/− primary mouse myoblasts expressing PAX3- which is perhaps not surprising given that LOI of the FOXO1 and an RH30 cDNA expression library identi- IGF2 is seen in almost half of ARMS tumors [66]. How- fied a gene that could induce tumor formation where ever, Khan et al. [5] have shown that, in NIH3T3 cells, ARF−/− myoblasts expressing PAX3-FOXO1 alone did PAX3-FOXO1 expression can induce the upregulation not. The RH30 gene expression library expressed a trun- of IGF2 mRNA. Interestingly, H19 expression was also cated fragment of this novel gene dubbed IRIZIO, and found to be upregulated in response to PAX3-FOXO1 expression of either this truncated form or the full- expression in these cells. Khan et al. [5] did not investi- length wild-type IRIZIO were protumorigenic in this gate whether PAX3-FOXO1 can regulate imprinting of model [71]. Due to the nature of the screen, and given this locus, and the mechanism of PAX3-FOXO1 regula- that abrogation of the p53 and pRb pathways are tion of IGF2 mRNA expression remains unknown. required for PAX3-FOXO1-driven cell transformation Two factors shown to be upregulated in PAX3- [46,47], this screen was expected to identify proteins that FOXO1-expressing cells, MET [73,81,90,91] and CXCR4 could abrogate the pRb pathway [71]. The mechanism of [85,86], are thought to play a role in the increased pro- the interaction between IRIZIO and pRb, however, has pensity for metastasis seen with ARMS. yet to be identified. MET is the receptor for hepatocyte growth factor/scatter factor (HGF/SF) [94]. HGF-MET signaling has been shown PAX3-FOXO1 target genes to play an important role in both normal skeletal muscle Many gene expression studies have been performed by development and regeneration, and it is involved in regu- various groups to try to identify genes that are either lating myogenic cell migration, survival, proliferation and downstream of PAX3-FOXO1 gene expression in vari- differentiation [95,96]. MET appears to be a downstream ous cell types or are indicative of ARMS tumor gene target of PAX3. Splotch mice, which express a mutant expression profiles (see Table 1). Only a small propor- PAX3 and fail to form limb muscles because of an inhib- tion of these studies have gone on to further investigate ition of myogenic precursor cell migration, show a the mechanism of PAX3-FOXO1 regulation of these decreased expression of MET [24,97]. MET has also been genes and/or what role these genes may play in ARMS found in five independent studies to be downstream of the tumorigenesis. PAX3-FOXO1 fusion protein in ARMS [73,81,90-92]. However, in the case of ARMS, it appears that HGF may Two of these genes have already been mentioned as cooperating mutations seen in ARMS tumors, N-MYC play a role in chemoattraction of tumor cells to the bone and IGF2. The N-MYC locus is known to be amplified marrow, which is a common site of metastasis in ARMS cases [98,99]. ARMS cell line CW9019 shows chemotaxis in a proportion of ARMS tumors, and the IGF2 locus is known to show LOI in ARMS tumors (see cooperating toward bone marrow-derived-fibroblast-conditioned media mutations in ARMS tumors). However, these studies in vitro, and this migration is inhibited by the MET- blocking agent, K-252a. Moreover, both RH30 and also indicate that PAX3-FOXO1 may regulate the gene expression from these loci. CW9019 ARMS lines home to the bone marrow in lethally N-MYC expression has been shown to be upregulated irradiated mice, while the ERMS cell lines RD, SMS-CTR, and RH18 do not [99]. Conversely, siRNA against PAX3- in four independent studies using PAX3-FOXO1 target- ing siRNA in the ARMS cell line, RH4 [72], PAX3- FOXO1 prevents migration of cells in wound-healing FOXO1 overexpression in the RD (ERMS) cell line assays of RH30 cells in response to HGF. In addition to a Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 5 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Table 1 PAX3-FOXO1 target genes in ARMS Gene Description Regulation References PF target ABAT 4-Aminobutyrate aminotransferase; Up [72-74] Yes ACTC Actin, alpha, cardiac muscle 1 Up [5,75] Yes ADAM10 A disintegrin and metalloproteinase domain 10 Up [73][72] Yes ADRA2A Alpha-2 adrenergic receptor subtype C10 Up [72,74,76] Yes ADRA2C Alpha-2 adrenergic receptor subtype C4 Up [22,74,76] Yes ALK ALK tyrosine kinase receptor, anaplastic lymphoma kinase Ki-1 Up [22,72,74,76,77] Yes ANK2 Ankyrin 2 Up [73,74,76] Yes ASS Argininosuccinate synthase Up [73,74,78]? ASTN2 Astrotactin 2 Up [72,76] Yes BIN1 Bridging integrator 1 Down [72,79] Yes BMP5 Bone morphogenic protein 5 Up [72-74,76] Yes C10ORF6 Family with sequence similarity 178, member A Up [75,76] Yes CCND1 Cyclin D1 Down [75][79]? CD9 CD9 molecule/Tetraspanin-29 Up [73,76] Yes CDH3 Cadherin 3, type 1, P-cadherin (placental) Up [72,76,80] Yes CHD7 Chromodomain helicase DNA-binding protein 7 Up [76,81] Yes CKM Creatine kinase M chain, muscle Up [5,79] Yes CNR1 Cannabinoid receptor 1 Up [22,72,74,82-84] Yes COL18A1 Collagen type 18 α1Up[73,74]? CXCR4 C-X-C chemokine receptor type 4 Up [75,76,85-87] Yes CXCR7 C-X-C chemokine receptor type 7 Down [76,87] Yes DCX Neuronal migration protein doublecortin Up [73,74,81]? DES Desmin Down [72,79] Yes DKFZP762M127 Unknown Up [73,74,81] Yes DUSP4 Dual specificity phosphatase 4 Down [73,81]? DZIP3 DAZ interacting protein 3, zinc finger Up [72,74,81] Yes ELA1 Elastase-1 Up [22,72,74] Yes ENC1 Ectodermal-neural cortex 1 (with BTB-like domain) Up [75,76] Yes ENO3 Enolase 3 (beta, muscle) Up [74,79]? EPHA4 Ephrin type-A receptor 4 Up/down [76,81,82] Yes FGFR2 Fibroblast growth factor receptor 2 Up [72,76] Yes FGFR4 Fibroblast growth factor receptor 4 Up [22,73,76,88,89] Yes FLNB Filamin B, beta Down [76,82] Yes FNBP1 Formin-binding protein 1 Down [22,75,76]? FOXF1 Forkhead box protein F1 Up [22,72,74,76,81] Yes FOXO1 Forkhead box O1 Up [75,76] Yes GADD45A Growth arrest and DNA-damage-inducible protein GADD45 alpha Up [73,76,81] Yes GRAF GTPase regulator associated with FAK Up [22,72] Yes GTF3C1 General transcription factor 3C Up [72,81] Yes H19 Imprinted maternally expressed gene, untranslated mRNA Up [5,79] Yes HDAC5 Histone deacetylase 5 Up [73,76] Yes HUMMLC2B Myosin regulatory light chain 2, skeletal muscle isoform Down [72,73] Yes IGF2 Insulin-like growth factor II Up [5,75] Yes IGFBP3 IGF-binding protein 3 Down [73,81] Yes Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 6 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Table 1 PAX3-FOXO1 target genes in ARMS (Continued) IGFBP5 IGF-binding protein 5 Up [5] Yes IL4R Interleukin 4 receptor Up [72,73,75,76] Yes JAKMIP2 Janus kinase and microtubule-interacting protein 2 Up [72-74,76] Yes KCNN3 Small conductance calcium-activated potassium channel protein 3 Up [73,74,81] Yes KCNS3 Potassium voltage-gated channel subfamily S member 3 Up [73,74]? LRRFIP2 Leucine-rich repeat (in FLII) interacting protein 2 Up [72,74] Yes MARCH3 Membrane-associated RING finger protein 3 Up [73,81] Yes MCAM Melanoma cell adhesion molecule Up [73,81] Yes MEG3 Maternally expressed 3 Up [73-75]? MN1 Meningioma (disrupted in balanced translocation) 1 Up [73,76] Yes MET Hepatocyte growth factor receptor Up [73,76,81,90-92] Yes MTUS2 Microtubule associated tumor suppressor candidate 2 Up [72,76] Yes MYBPH Myosin-binding protein H Up/down [5,72,75] Yes MYCN N-MYC proto-oncogene protein Up [72,73,76,78,81] Yes MYH8 Myosin, heavy chain 8, skeletal muscle, perinatal Up/down [5,72] Yes MYL1 Myosin, light chain 1, alkali; skeletal, fast Up/down [5,79] Yes MYL4 Myosin, light chain 4, alkali; atrial, embryonic Up/down [5,72] Yes MYOD Myoblast determination protein 1 Up [5,73,76,81] Yes MYOG Myogenin (myogenic factor 4) Up [5,90] Yes NEBL Nebulette Up/down [72,73,76,81] Yes NELL1 NEL-like protein 1 Up [22,72-74,76] Yes NHLH1 Nescient helix loop helix 1 Up [22,76] Yes NPTX2 Neuronal pentraxin II Down [76,81] Yes NRCAM Neuronal cell adhesion molecule Up [72-74] Yes OLIG2 Oligodendrocyte transcription factor 2 Up [22,74]? PALMD Palmdelphin Down [72,76] Yes PBK PDZ-binding kinase Up [73,76] Yes PCDH7 Protocadherin 7 Up [76,82] Yes PDZRN3 PDZ domain containing ring finger 3 Up [74,76] Yes PGBD5 PiggyBac transposable element-derived protein 5 Up [22,72,74] Yes PHF17 PHD finger protein 17 Up [74,76] Yes PIPOX Pipecolic acid oxidase Up [22,72,74,76] Yes PKP1 Plakophilin 1 (ectodermal dysplasia/skin fragility syndrome) Up [72,76] Yes PLAG1 Pleiomorphic adenoma gene 1 protein Down [75,81] Yes PLK2 Polo-like kinase 2 Down [73,76] Yes PODXL Podocalyxin-like protein 1 Up [22,74,76] Yes POU4F1 Brain-specific homeobox/POU domain protein 3A Up [72,73,76] Yes PPARGC1A Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha Up [22,76] Yes PRKAR2B Protein kinase, cAMP-dependent, regulatory, type II, beta Up [73,81,82] Yes PRKCA Protein kinase C, alpha Up [73,76] Yes PSEN2 Presenilin 2 (Alzheimer’s disease 4) Up [73,74]? PTHLT Parathyroid hormone-like hormone Up [76,82] Yes QDPR Quinoid dihydropteridine reductase Up [74,81] Yes RASSF4 Ras association (RalGDS/AF-6) domain family 4 Up [72,74,76] Yes RRP22 Ras-like protein family member 10A, on chm 22 Up [73,74]? Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 7 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Table 1 PAX3-FOXO1 target genes in ARMS (Continued) RYR1 Skeletal muscle-type ryanodine receptor Up [5,74] Yes RYR3 Brain-type ryanodine receptor Up [74,76,81] Yes SLC24A3 Solute carrier family 24 (sodium/potassium/calcium exchanger), member 3 Up [74,76] Yes SIX1 SIX homeobox 1 Up [5,75] Yes SOX14 SRY (sex determining region Y)-box 14 Up [22,76] Yes STX11 Syntaxin 11 Up [76,82] Yes SULF1 Sulfatase 1 Up [73,76] Yes SVIL Supervillin Down [72,76] Yes TCF712 Transcription factor 7-like 2 (T-cell specific, HMG-box) Up [73,81] Yes TGFB1 Transforming growth factor, beta 1 Up [5,76] Yes TFAP2B Transcription factor AP-2 beta Up [22,72] Yes TIAF1 TGF-beta-1-induced antiapoptotic factor 1 Up [73,74]? TM4SF10 Transmembrane 4 superfamily member 10 Up [73,81] Yes TNFAIP3 Tumor necrosis factor, alpha-induced protein 3 Up [73,76] Yes TNNC2 Troponin C type 2 (fast) Up/down [5,72,73,79] Yes TNNI2 Troponin I type 2 (skeletal, fast) Up/down [5,72] Yes TNNT2 Troponin T type 2 (cardiac) Up [5,79] Yes TNNT3 Troponin T type 3 (skeletal, fast) Down [72,79] Yes TRAM2 Translocation-associated membrane protein 2 Up [73,76] Yes TSC22D2 TSC22 domain family, member 2 Up [74,76] Yes UBE2G2 Ubiquitin-conjugating enzyme E2G 2 (UBC7 homolog, yeast) Up [22,76] Yes WSCD1 WSC domain-containing protein 1 Up [22,74]? WVA5A Von Willebrand factor A domain containing 5A Up [72-74] Yes Summary of genes found to be differentially regulated in more than one reference in ARMS tumors and/or cell lines, or by PAX3-FOXO1 overexpression, in various cell types. Gene name and description are indicated and also the relative expression in ARMS or PAX3-FOXO1. Also indicated is whether the study indicates that these genes are downstream of PAX3-FOXO1 expression. Genes indicated in bold will be further discussed. migratory function for MET in ARMS, knockdown of reduces migration, and combined inhibition reveals syner- MET by shRNA in ERMS and ARMS inhibits cell prolifera- gism between these receptors [99]. SDF-1 can also induce tion and induces apoptosis. Moreover, shRNA-mediated proliferation of the ARMS cell line, RH30 [103]. Consist- knockdown of MET inhibits anchorage-independent ent with CXCR4 expression being downstream of PAX3- growth of ARMS and ERMS, and mutant MET-expressing FOXO1 transgene expression, CXCR4 expression in RMS MEFS prevent PAX3-FKHR transformation [91]. Consist- correlates with the ARMS histology, unfavorable primary ent with MET as a PAX3-FOXO1 target, high MET expres- site, advanced disease at diagnosis and bone marrow in- sion in RMS correlates with ARMS histology, advanced volvement [100]. disease at diagnosis and bone marrow involvement [100]. Other genes have been further confirmed as down- CXCR4 is normally expressed in satellite cells within stream genes of PAX3-FOXO1. Cannabinoid receptor 1 skeletal muscle and is used as a marker of mononucleated (CNR1) is specifically upregulated at both the mRNA cells capable of differentiating into myofibers [101]. and protein level in fusion-positive ARMS cells [74]. CXCR4 is a cell surface receptor; it binds and mediates CNR1 is normally highly expressed in brain [104] but is the signaling of stromal-derived factor-1 (SDF-1) and also expressed in skeletal muscle at levels detectable by induces cell chemotaxis [102]. SDF-1 can induce migra- RT-PCR [105]. CNR1 has been confirmed, using ChIP tion and chemotactic invasion in ARMS cell lines, but not analysis, as a direct target of both PAX3 and PAX3- ERMS cell lines [85], and this migration can be inhibited FOXO1 transcriptional activity [82]. Furthermore, the by SDF-1-neutralizing antibody or the CXCR4 inhibitor homeodomain of PAX3-FOXO1 appears to be the im- AMD3100 [103]. Moreover, expression of CXCR4 and portant domain for the regulation of CNR1 expression MET in ARMS lines appears to synergize to induce the [72]. CNR1 has been proposed to be a potential drug migration of cells toward bone marrow-derived fibroblast target in ARMS. Treatment with CNR1 agonists can in- conditioned media in vitro. Inhibition of each receptor duce apoptosis in some ARMS cell lines [83]. In addition, Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 8 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 CNR1 expression has been linked with an increase propen- the biology of ARMS. These include ARMS cell lines sity for PAX3-FOXO1 expressing mouse myoblast invasive- derived from human tumors in the laboratory of Dr. Peter ness and lung metastasis formation. Moreover, treatment Houghton: RH3, RH4, RH10, RH28, RH30 and RH41, all of with an inverse agonist to CNR1 can abrogate in vitro inva- which express the PAX3-FOXO1 fusion protein sion and in vivo lung metastasis formation [84]. Thus, [13,109,110] and have been widely used in the field. In CNR1 may represent a viable therapeutic target specific for addition, the NCI-supported Pediatric Preclinical Testing the increased metastatic capacity of PAX3-FOXO1 expres- Program (PPTP) uses RH10, RH28, RH30, RH30R, RH41 sing ARMS. and RH65 subcutaneous xenograft tumors to test drug effi- Transcription factor AP2β (TFAP2B) has been shown to cacy in a well-characterized preclinical model of many be a downstream target of PAX3-FOXO1 and appears to pediatric cancers [111-113]. require the paired domain of PAX3-FOXO1 to be induced. Other in vitro models that have been used involve the TFAP2B promoter expression is induced by PAX3, which introduction of the PAX3/7-FOXO1 fusion proteins into has been shown to bind to the TFAP2B promoter by ChIP both myogenic and non-myogenic cell lines including fibro- analysis. siRNA targeting TFAP2B introduced into PAX3- blast cell lines, ERMS cell lines, MEFs, mesenchymal stem FKHR-positive ARMS induces apoptosis, indicating that cells (MSC), normal or immortalized human or mouse TFAP2B can mediate cell survival in ARMS, downstream myogenic cells and even an osteosarcoma cell line of PAX3-FOXO1 [72]. [5,81,82,91,114-119]. Given that the cell of origin for ARMS FGFR4 has been identified as a direct transcriptional tar- has yet to be identified, perhaps this variation in model cell get of PAX3 and PAX3-FOXO1, which bind to a down- linesisprudent.However,itislikelythatARMSand ERMS stream enhancer region [76,106]. Accordingly, FGFR4 is tumors are derived from a mesenchymal cell likely of the significantly upregulated by PAX3-FOXO1 expression the myogenic lineage because of skeletal muscle lineage- [22,73,76,88,89,107]. However, upregulation of FGFR4 specific gene expression seen in these tumors [7]. downstream of PAX3-FOXO1 in primary myoblasts does Conversely, some studies have used endogenous not appear to act as an effector of PAX3-FOXO1-mediated PAX3-FOXO1 in ARMS cell lines to determine the tran- myoblast transformation given that wild-type FGFR4 upre- scriptional targets of this fusion protein within the gulation is not required for PAX3-FOXO1-induced prolif- ARMS tumor cell context. Both Kikuchi et al. [90] and eration, transformation, invasion or inhibition of myogenic Ebauer et al. [72] used siRNA specifically targeting differentiation [89]. However, knockdown of FGFR4 in PAX3-FOXO1 or both PAX3 and PAX3-FOXO1 RMS cell lines does show a reduction in cell proliferation sequences, respectively. Inhibition of PAX3-FOXO1 and an increase in apoptosis, suggesting that at later stages expression reduced cell proliferation and motility and of ARMS tumorigenesis FGFR4 overexpression may inter- allowed some myogenic differentiation [90]. In addition, act with other unknown genetic lesions within these cell comparative gene expression studies were performed lines to induce pro-survival and proliferation effects [107]. identifying over 100 PAX3-FOXO1 gene targets. Cao It is interesting to note however that kinase domain- et al. [76] performed ChIP sequencing studies using a activating mutations in FGFR4 have been identified in 7.5% PAX3-FOXO1-specific antibody and were able to iden- of RMS, including fusion-positive ARMS [108], and can tify 1,463 putative PAX3-FOXO1-binding sites in the contribute to myoblast growth advantage and transform- human genome. Furthermore, PAX3-FOXO1-binding ation [89]. Thus, FGFR4-activating mutations likely repre- sites adjacent to MyoD, FGFR4 and IGF1R were verified sent cooperating mutations in RMS and upregulation of as transcriptionally regulated by PAX3-FOXO1. FGFR4 in fusion-positive ARMS would enhance this effect. The CDH3/P-cadherin gene has been identified as a dir- In vivo models of ARMS ect transcriptional target of PAX3/7-FOXO1 [72,76,80]. P- Many different transgenic and knock-in animal models cadherin expression in the C2C12 myoblast cell line inhi- have been attempted to recapitulate ARMS tumor for- bits myogenic differentiation and maintains a proliferative mation in vivo. Several of these models have attempted state through maintaining cyclin D1 expression. This in to constitutively express PAX3/7-FOXO1 fusion proteins turn results in transformation of C2C12 cells, allowing col- in the skeletal muscle lineage during development, only ony formation in soft agar. Additionally, P-cadherin expres- to result in developmental defects and not tumor forma- sion resulted in enhanced cell motility, as well as cadherin tion [120-123]. Transgenic mice expressing PAX3- switching, a hallmark of epithelial to mesenchymal transi- FOXO1 under the control of the PAX3 promoter and tion and metastatic progression [80]. enhancer regions resulted in expression of PAX3- FOXO1 in the dorsal neural tube and lateral dermomyo- In vitro models of ARMS tome. PAX3-FOXO1 expression in this context appeared Many different cell lines have been derived from human to interfere with normal PAX3 developmental functions ARMS tumors; these are regularly used to investigate including neural tube and neural crest abnormalities Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 9 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 similar to those seen in PAX3 mutant Splotch mice. The under control of myosin heavy-chain Gal4, also resulted majority of defects appeared to be in neural develop- in developmental defects in the fly, evidenced by disor- ment, though defects were seen in hind limb skeletal ganized myogenic patterning. Though nothing resem- muscle; however, no tumors developed [121,122]. bling tumor formation was seen, this group did note Lagutina et al. [120] developed a model where PAX3- dissemination and infiltration of non-native tissue by FOXO1 was knocked into the PAX3 locus. This knock-in PAX7-FOXO1 expressing mononucleated cells, indicat- locus expressed low amounts of PAX3-FOXO1, which in ing an increase in invasive capacity of these cells. heterozygous pups was sufficient to result in developmental Keller et al. [47] used a conditional PAX3-FOXO1 defects in the heart and diaphragm, leading to congestive knock-in into the PAX3 locus, and Myf6-driven Cre ex- heart failure and perinatal death, as well as malformations pression. This allowed, upon Cre recombination, expres- of some hypaxial muscles. However, neither chimeric adults sion of PAX3-FOXO1 driven by the PAX3 promoter and nor their newborn heterozygous pups developed malignan- 3’ FOXO1 genomic sequences that potentially contain cis- cies. It was hypothesized that PAX3-FOXO1 expression regulatory elements, a region absent from previous PAX3 from the PAX3 control sequences was insufficient to cause knock-in strategies. This was the first animal model that ARMS formation, and downstream regions of the FOXO1 successfully recapitulated the formation of ARMS, though locus may be required to induce sufficient PAX3-FOXO1 at the low frequency of approximately 0.4% (1/228) and expression to induce tumor development. with latency of over 1 year (383 days). However, this fre- A PAX7-FOXO1 model of ARMS was also attempted quency was greatly enhanced, and latency greatly reduced, P3Fa/P3Fa in Drosophila [123]. Expression of UAS-hPAX7-FOXO1, in homozygote PAX3 mice also lacking Trp53 or Figure 2 Review summary: Fusion gene regulated genes contributing to alveolar rhabdomyosarcoma. Rhabdomyosarcoma develops from an unknown cell of origin from the mesodermal lineage that may be skeletal muscle specified. This cell likely expresses both PAX3/7 and FOXO1 and may also express Myf6. A gene fusion event resulting in a PAX3/7 DNA-binding domain fused to a more potent transcriptional activation domain occurs. This fusion transcription factor is capable of inducing a group of PAX3-FOXO1-regulated genes that contribute to ARMS development in conjunction with other genetic lesions. Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 10 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Ink4a/Arf. Subsequently, ARMS tumors have devel- From these animal models it is apparent that the tim- oped in this conditional PAX3-FOXO1 knock-in ing of PAX3-FOXO1 expression is critical for ARMS model with a Pax7-CreER and M-Cre (Pax3 hypaxial development. Too early and widespread expression of muscle enhancer) also lacking functional Trp53 PAX3-FOXO1 expression can result in developmental [124]. Moreover, histologically diagnosed fusion- defects and no apparent tumor development [120-123], negative ARMS tumors have been found to develop whereas later expression of PAX3-FOXO1, via a Myf6- +/− −/− in conditional Ptch1 Trp53 mice when Cre is driven Cre recombinase, does cause disease, though at a expressed from Pax7-CreER. The latency and incidence of low frequency [47]. Perhaps inducible expression, driven ARMS tumor development in these different models have by various myogenic genes with carefully characterized yet to be compared. expression profiling, would result in an increased fre- Clearly the problems that have arisen during the devel- quency of disease and help to narrow down the exact opment of an animal model for ARMS indicate that the stage in which PAX3-FOXO1 expression drives ARMS timing and the cell lineage targeted for PAX3-FOXO1 tumorigenesis. Nevertheless, the cell of origin for expression are very important for the development of ARMS is yet to be identified, and animal models of ARMS tumor formation and for avoiding developmental ARMS will no doubt play an important role in its defects. In a review [125] following the publication of identification. the animal model [47], Keller et al. discuss the possibil- ities for the cell of origin for ARMS; because Keller Conclusion et al. achieved the formation of ARMS tumors in their To date, numerous factors (outlined in Figure 2) have mouse model using Myf6-Cre-driven conditional PAX3- been identified that contribute to ARMS tumor develop- FOXO1, and Myf6 is usually expressed in differentiating ment and its aggressive clinical phenotype. These consist skeletal muscle myotubes, they propose a potential de- of both PAX3/7-FOXO1 target genes, such as N-MYC, differentiation mechanism for ARMS development. IGF2, MET, CXCR4, CNR1, TFAP2B, FGFR4 and P-cad- However, the formation of a fusion gene such as PAX3- herin, and PAX3/7-FOXO1 cooperating factors, such as FOXO1 suggests that the cell of origin for ARMS should the abrogation of the p53 pathway, IGF2 deregulation, express both PAX3 and FOXO1 at the time that the N-MYC and miR17-92 amplification, and IRIZIO translocation occurs, given that open chromatin is likely expression. Future ARMS research will continue to dis- required for these two genomically distinct regions to cover the mechanisms by which ARMS tumorigenesis translocate. Anecdotal evidence for this includes that the occurs. This will involve the identification of more PAX3- genome translocations that occur in many different can- FOXO1 target and cooperating genes; more importantly, cer types occur between genes that are expressed in the the mechanisms by which these genes contribute to cell type of origin. A recent study by Osborne et al. tumorigenesis will be elucidated. It is critical that we [126] showed that the MYC and IGH genes, which are develop a mechanistic understanding of how these factors involved in a chromosomal translocation common in contribute and interact to perpetrate ARMS tumo- Burkitt lymphoma, are colocalized at the same transcrip- rigenesis. This will allow new opportunities to develop tion factories more often in activated B-cells, the origin- specifically targeted therapies for this aggressive pediatric ating cell of Burkitt lymphoma, than resting B cells. This disease. colocalization at the same transcription factory allows for close proximity of these gene loci in euchromatin, Abbreviations providing the circumstances where these genes would be ARMS: Alveolar rhabdomyosarcoma; bHLH: Basic helix loop helix domain; CDK: Cyclin-dependent kinase; CNR1: Cannabinoid receptor 1; in close association, facilitating the specific translocation ERMS: Embryonal rhabdomyosarcoma; FH: Forkhead DNA-binding domain; event. PAX3 is rapidly downregulated upon myoblast FKHR: Forkhead in rhabdomyosarcoma (now known as FOXO1); differentiation, so it would be unlikely that the PAX3 HD: Homeodomain DNA-binding domain; HGF/SF: Hepatocyte growth factor/scatter factor; LOH: Loss of heterozygosity; LOI: Loss of imprinting; loci would be expressed in a nascent myotube expressing MEF: Mouse embryonic fibroblast; miR: Micro RNA; MSC: Mesenchymal stem Myf6, making it difficult to understand how transloca- cells; PD: Paired box DNA-binding domain; PPTP: Pediatric Preclinical Testing tion could occur in nascent myotubes and therefore cast Program; RMS: Rhabdomyosarcoma; SDF-1: Stromal-derived factor-1; SHH: Sonic hedgehog; siRNA: Short interfering RNA; TFAP2B: Transcription some doubt on whether the dedifferentiation model is factor AP2 b. feasible. However, it is possible that Myf6 expression does rarely occur in a small subset of undifferentiated Competing interests myogenic cells in conjunction with PAX3. This could The authors have no competing interests to declare. allow for this model to produce ARMS tumors and ac- Authors’ contributions count for the low frequency at which these tumors are AM was responsible for the drafting of the manuscript. GG was responsible seen as well as the requirement for homozygous PAX3- for critical revision of the content and approved the final version of the FOXO1 knock-in alleles [47]. manuscript. All authors read and approved the final manuscript. Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 11 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Author details 18. Sorensen PH, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, Maurer HM, Bridge Department of Genetics, St Jude Children’s Research Hospital, Memphis, TN JA, Crist WM, Triche TJ, Barr FG: PAX3-FKHR and PAX7-FKHR gene fusions 38105, USA. 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Osborne CS, Chakalova L, Mitchell JA, Horton A, Wood AL, Bolland DJ, Corcoran AE, Fraser P: Myc dynamically and preferentially relocates to a transcription factory occupied by Igh. PLoS Biol 2007, 5:e192. doi:10.1186/2044-5040-2-25 Cite this article as: Marshall and Grosveld: Alveolar rhabdomyosarcoma – The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis. Skeletal Muscle 2012 2:25. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Alveolar rhabdomyosarcoma – The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis

Skeletal Muscle , Volume 2 (1) – Dec 3, 2012

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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Rhabdomyosarcoma is a soft tissue sarcoma arising from cells of a mesenchymal or skeletal muscle lineage. Alveolar rhabdomyosarcoma (ARMS) is more aggressive than the more common embryonal (ERMS) subtype. ARMS is more prone to metastasis and carries a poorer prognosis. In contrast to ERMS, the majority of ARMS tumors carry one of several characteristic chromosomal translocations, such as t(2;13)(q35;q14), which results in the expression of a PAX3-FOXO1 fusion transcription factor. In this review we discuss the genes that cooperate with PAX3-FOXO1, as well as the target genes of the fusion transcription factor that contribute to various aspects of ARMS tumorigenesis. The characterization of these pathways will lead to a better understanding of ARMS tumorigenesis and will allow the design of novel targeted therapies that will lead to better treatment for this aggressive pediatric tumor. Keywords: Alveolar rhabdomyosarcoma, PAX3-FOXO1, PAX7-FOXO1, FGFR4, CNR1, IRIZIO, N-MYC, IGF2, MET, CXCR4, p53, MDM2, P-Cadherin, TFAP2B, miR17-92 Introduction retroperitoneum [7,8]. The alveolar subtype (ARMS) con- According to the American Cancer Society, rhabdomyo- stitutes approximately another 20% of RMS cases [10] and sarcoma (RMS) comprises about three percent of child- occurs predominantly in adolescents. Histologically, hood cancers, with about 350 new cases occurring ARMS tumors typically contain alveoli structures similar annually in the US [1], and it affects slightly more males in appearance to those seen in the lung [7], though solid- than females [2]. RMS is a small, round, blue cell tumor variant ARMS does occur [11]. Primary ARMS tumors usually arising in skeletal muscle tissue, and it is thought typically arise in the extremities and trunk [7-9,12], and to originate from mesenchymal cells likely committed to they are more aggressive than their ERMS counterparts. the skeletal muscle lineage. Consistent with a myogenic ARMS is associated with a poorer prognosis, with a 5-year origin, RMS tumors express skeletal muscle markers such failure-free survival of 65% [8]. as skeletal muscle actin and myosin, desmin, myoglobin, A characteristic of the ARMS subtype is the occurrence Z-band protein, MYOD and often myogenin [3-7]. RMS of recurrent chromosomal translocations. The most com- consists of two major histological subtypes, embryonal mon of these is t(2;13)(q35;q14), which results in the and alveolar RMS. The embryonal subtype (ERMS) is expression of an oncogenic fusion protein. This fusion thought to be histologically reminiscent of embryonic protein consists of the paired and homeodomains of the developing skeletal muscle [7]. ERMS is the most preva- PAX3 transcription factor with the potent transcriptional lent of the subtypes, accounting for about 60% of RMS activation domain of FOXO1 (FKHR), a member of the cases [2]. It occurs mainly in children younger than 10 forkhead (FOX) family of transcription factors [13-15]. years and is usually associated with a favorable prognosis, The PAX3 homeodomain is required to recapitulate with a failure-free survival rate of 81% [8,9]. Tumors usu- PAX3-FOXO1-induced tumorigenesis, though the paired ally localize to the head and neck (including the extraocu- domain may play a minor role [16,17]. The PAX3-FOXO1 lar muscles of the eye), the genitourinary tract and the fusion protein can be detected in about 55% of ARMS cases [18]. A similar translocation of t(1;13)(p36;q14) fuses the PAX7 DNA-binding domains, the closest homolog of * Correspondence: gerard.grosveld@stjude.org PAX3, to FOXO1 [19]. This translocation occurs in a fur- Department of Genetics, St Jude Children’s Research Hospital, Memphis, TN 38105, USA ther 22% of ARMS cases [18]. Recently, further similar Full list of author information is available at the end of the article © 2012 Marshall and Grosveld; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 2 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 translocations have been found in individual ARMS cases: PAX3-FOXO1 transcriptional activity [37]. There is also t(2;X)(q35;q13), which results in PAX3-AFX fusion [20], evidence that MyoD transcriptional activity is abrogated in and t(2;2)(q35;p23) and t(2;8)(q35;q13), which generate a ERMS tumors [40]. fusion protein of PAX3-NCOA1 and PAX3-NCOA2, The remaining ARMS tumors are classed as fusion- respectively [21,22]. These “cryptic” rare fusion variants negative ARMS. However, fusion-negative ARMS are in- are thought to be present in up to another 10% of ARMS distinguishable on the levels of gene expression and in tumors [7]. During normal development, PAX3 expression clinical outcome from ERMS tumors, leading some to occurs in the neural tube and dermomyotome [23], and it argue that translocation status should be the defining is required for the normal migration of skeletal muscle factor of ARMS [41-44]. Within the ARMS subtype, precursors to the limb bud [24]. PAX7 expression is a prognosis can vary by disease stage at diagnosis as well marker of satellite cells in adult skeletal muscle [25] and is as translocation status. For example, patients presenting required for normal self-renewal [26]. Unlike skeletal with metastatic disease have an estimated 4-year overall muscle-specific PAX3 and PAX7, FOXO1A, AFX, survival rate of 75% for PAX7-FOXO1, while those with NCOA1 and NCOA2 are widely expressed and mediate the PAX3-FOXO1 translocation have only 8% estimated gene transcription downstream of cell signaling pathways 4-year overall survival [18]. Indeed, there is evidence [27-31]. All of the ARMS fusion proteins consist of the that the PAX3-FOXO1 is a more potent oncogene than PAX3/7 DNA-binding domains fused to the transcrip- PAX7-FOXO1. Barr et al. [45] found that only 1/24 tional activation domains of more potent transcription fac- PAX3-FOXO1-positive ARMS tumors had amplification tors (see Figure 1) [14,15,22]. Genome-wide transcription of the PAX3-FOXO1 gene, while PAX7-FOXO1 was factor-binding studies have not yet been performed to de- amplified in 10/11 PAX7-FOXO1 ARMS, implying that termine whether wild-type PAX3 and PAX7-binding sites genomic amplification of PAX7-FOXO1 is required for differ from these PAX3/7 fusion transcription factor- tumorigenesis, while a single copy of PAX3-FOXO1 is binding sites. sufficient. However the gene expression profiles of Expression of these ARMS fusion transcription factors is PAX3-FOXO1- and PAX7-FOXO1-expressing tumors thought to abrogate normal skeletal muscle differentiation, have not, to the author’s knowledge, been specifically allowing aberrant cell division and tumor development. compared to identify the gene set responsible for this PAX3 expression can inhibit myogenic differentiation of difference in prognosis between ARMS tumors with cultured myoblasts [35]. Although PAX3 protein is rapidly these two fusion genes. degraded during early myogenic differentiation, PAX3- FOXO1 has a significantly longer half-life than wild-type Review PAX3 [36]. PAX3/7-FOXO1 is capable of suppressing PAX3-FOXO1 is the most common fusion gene in MyoD expression and activity [37,38]. PAX7-FOXO1 ex- ARMS. This fusion transcription factor is thought to pression induces NFκB signaling, which inhibits myogen- drive the gene expression that causes the worse progno- esis via activation of cyclin D1/CDK4 complexes. These sis in ARMS tumors. Many studies have sought to iden- complexes sequester MyoD, which would normally drive tify the differences in gene expression between ERMS cell cycle withdrawal and myogenic differentiation [39]. In and ARMS, as well as the genes aberrantly regulated by addition transcriptionally inactive MyoD can enhance PAX3-FOXO1. In this review, we summarize these gene Figure 1 Gene translocation in alveolar rhabdomyosarcoma. Scale diagram showing the parent proteins and the resulting fusion proteins arising from chromosomal translocations occurring in ARMS. Green or yellow indicates the protein fusion sites [14,19-22]. Homologous domains are indicated in like colors. DNA-binding domains are indicated as: paired domain (PD), homeodomain (HD), fork head DNA-binding domain (FH) and basic helix-loop-helix domain (bHLH). Regions of the proteins known to act as transcriptional activation domains are indicated (TAD). Other domains include the octapeptide domain (O), PAS domains (PAS A/B), LXXLL motifs (L1-L7) and glutamine-rich region (Q-rich). Maps were derived from the following references: [14,19-22,32-34]. Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 3 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 expression changes specific to PAX3-FOXO1 expression reported in 0 to 22% of cases [12,56,57]. RMS cell lines and/or ARMS. Moreover, we consolidate these data into show a significantly higher rate of p53 abnormalities a list of genes that may well represent the means by with 60%, indicating establishment of these cell lines which ARMS tumors obtain a more aggressive pheno- through xenograft and cell culture increases the propor- type than ERMS. tion of cell lines with p53 alterations [56]. Looking at p53 and MDM2 expression levels, both are low in ARMS and ERMS. Metastatic ERMS tumors show sig- Cooperating mutations in ARMS tumors nificantly higher p53 protein expression, indicating that It is likely that PAX3/7-FOXO1 translocation is one of p53 gene alterations are a late event in rhabdomyosarco- the earliest events in ARMS tumorigenesis as it occurs magenesis. Again, p53 status did not show any correl- in the majority of ARMS cases, more often than any ation to prognosis [58]. other genetic lesion characterized in the disease. How- Chromosome 2 has been shown to be amplified at ever, PAX3-FOXO1 expression in normal cells is not 2p24 in 32 to 60% of ARMS tumors [49,51,59,60]. This sufficient to induce transformation, and other genetic region is known to contain the proto-oncogene N-MYC. alterations are required [46-48]. Genomic amplification Two independent studies have shown that a gain in the is common in ARMS tumors. The three most common genomic copy number of the N-MYC gene is associated amplifications seen in ARMS involve regions of chromo- with an unfavorable disease outcome [59,61]. In somes 2, 12 and 13 [49]. addition, N-MYC is more highly expressed in ARMS The region of chromosome 12 amplification spans cells lines than ERMS lines, despite the fact that it was 12q13-15 and is reported in 28% to 56% of ARMS found to only be genomically amplified in one of the five tumors [49-52]. This 12q13-15 region includes genes lines, indicating more than one mechanism of N-MYC such as C/EBP-homolog and transcription factor CHOP/ overexpression in ARMS. However, in this study no clear DDIT3/GADD153, sarcoma-amplified sequence and relationship in N-MYC expression was seen with regard transmembrane 4 superfamily member SAS/TSPAN31, to primary tumor samples [62]. alpha 2-macroglobulin receptor A2MR/LRP1, Sonic Another chromosomal region frequently amplified in hedgehog (SHH) pathway effector and zinc finger tran- ARMS is 13q31-32, showing amplification in between 14 scription factor GLI1, cyclin-dependent kinase cell cycle and 19% of ARMS tumors [49,51]. Presence of this amp- regulator CDK4 and p53 pathway modulator MDM2. In lification is significantly associated with poorer failure- most cases, gene amplification accompanies an increase free survival in ARMS [63]. The minimum overlapping in gene expression [50,53]. region of amplification at this region was originally Though GLI1 is amplified genetically, the expression defined as only containing two genes: GPC5 and of this gene is not always associated with its genetic C13ORF25. The C12ORF25 gene encodes the micro- amplification. When GLI1 is overexpressed in RMS, it RNA cluster miR-17-92 (MIR17HG) in an intron [64]. has been associated with an undifferentiated subtype ra- GPC5 overexpression can increase cell proliferation ther than ERMS or ARMS, indicating that GLI1 may through the modulation of the growth factor activity of play a role in tumors that show primitive histopatho- FGF2, HGF and WNT1a [64]. However, more thorough logical features [54]. Thus, GLI1 overexpression cannot mapping of the genetic amplification showed that the be well associated with the ARMS pathology. entire GPC5 locus was only amplified in 12.5% of 13q31 MDM2 is perhaps the best candidate oncogene in this re- amplified ARMS tumors, while the minimally amplified gion because of its inhibitory effect on p53 function [55]. region contains only the peptidylprolyl isomerase However, MDM2 is not always included in this 12q13-15 pseudogene (LOC390419) and MIR17HG. This amplifi- amplification. RH30, an ARMS cell line, lacks amplification cation is particularly prevalent in PAX7-FOXO1-positive ARMS tumors. The miR-17-92 cluster of micro-RNAs of MDM2 but shows amplification and overexpression of SAS, CHOP, GLI1 and A2MR [53]. In addition, the fre- has been shown to play a role in a variety of cancer types quency of MDM2 gene amplification specifically may be as (for review, see [65]). In PAX7-FOXO1, but not PAX3- FOXO1 expressing ARMS, overexpression of miR-17, low as 10% in ARMS tumors [56]. One study found only 2 of 34 ARMS samplestobehighlyimmunoreactivefor -19a, -19b, 20a and 92a is specifically associated with an MDM2 [12]. Moreover, MDM2 expression shows no asso- increased rate of 2-year treatment failure. This indicates a possible pro-tumorigenic interaction between PAX7- ciation with patient prognosis or other clinicopathologic parameters [12]. Thus, it may be amplification of one of the FOXO1 and miR-17-92 locus overexpression [63]. other genes at this chromosome 12 locus that is the import- Rhabdomyosarcoma can also be associated with a loss of heterozygosity (LOH) or loss of imprinting (LOI) at ant cooperating mutation with PAX3-FOXO1. Other alterations in the p53 pathway have been found 11p15.5 [66,67]. This region contains several imprinted in ARMS. In ARMS tumor samples mutated p53 was genes such as IGF2, which is maternally imprinted Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 4 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 (paternal allele is expressed), and H19 and p57/Kip2, [73,81] and in ARMS versus non ARMS tumor cell lines which are paternally imprinted (maternal allele is [78]. A tamoxifen (4-OHT)-inducible PAX3-FOXO1-ER expressed) [68-70]. IGF2 expression appears to be spe- construct induced upregulation of both N-MYC mRNA cifically upregulated by changes in imprinting or LOH at and protein in the transduced ERMS cell line, RD, and this locus in RMS. ERMS tumors are associated predom- this was not sensitive to cycloheximide treatment, indi- inantly with a LOH at the IGF2 locus, though there is cating that N-MYC is a direct transcriptional target of some discrepancy in the proportion of ERMS tumors PAX3-FOXO1. However the PAX3-FOXO1 regulatory showing this change: 23% according to Anderson et al. region for N-MYC did not appear to be contained within [66] and 72% according to Visser et al. [67]. Conversely, −1871 to +1058 of the N-MYC gene. Consistent with a IGF2 is upregulated by LOI in 46% of fusion-positive transforming role for both PAX3-FOXO1 and N-MYC, ARMS tumors, while imprinting of H19 is conserved in the two genes synergized in soft agar colony-forming 93% [66]. This indicates that an increase in IGF2 expres- assays [81]. In addition, knockdown of N-MYC expres- sion in RMS is important for tumorigenesis, though the sion identified a positive feedback loop between N-MYC mechanism of this upregulation, either LOH or LOI, and PAX3-FOXO1 [93]. varies by subtype. IGF2 was shown to be specifically overexpressed in A screen for PAX3-FOXO1-interacting proteins using ARMS compared to Ewing’s sarcoma cell lines [75], ARF−/− primary mouse myoblasts expressing PAX3- which is perhaps not surprising given that LOI of the FOXO1 and an RH30 cDNA expression library identi- IGF2 is seen in almost half of ARMS tumors [66]. How- fied a gene that could induce tumor formation where ever, Khan et al. [5] have shown that, in NIH3T3 cells, ARF−/− myoblasts expressing PAX3-FOXO1 alone did PAX3-FOXO1 expression can induce the upregulation not. The RH30 gene expression library expressed a trun- of IGF2 mRNA. Interestingly, H19 expression was also cated fragment of this novel gene dubbed IRIZIO, and found to be upregulated in response to PAX3-FOXO1 expression of either this truncated form or the full- expression in these cells. Khan et al. [5] did not investi- length wild-type IRIZIO were protumorigenic in this gate whether PAX3-FOXO1 can regulate imprinting of model [71]. Due to the nature of the screen, and given this locus, and the mechanism of PAX3-FOXO1 regula- that abrogation of the p53 and pRb pathways are tion of IGF2 mRNA expression remains unknown. required for PAX3-FOXO1-driven cell transformation Two factors shown to be upregulated in PAX3- [46,47], this screen was expected to identify proteins that FOXO1-expressing cells, MET [73,81,90,91] and CXCR4 could abrogate the pRb pathway [71]. The mechanism of [85,86], are thought to play a role in the increased pro- the interaction between IRIZIO and pRb, however, has pensity for metastasis seen with ARMS. yet to be identified. MET is the receptor for hepatocyte growth factor/scatter factor (HGF/SF) [94]. HGF-MET signaling has been shown PAX3-FOXO1 target genes to play an important role in both normal skeletal muscle Many gene expression studies have been performed by development and regeneration, and it is involved in regu- various groups to try to identify genes that are either lating myogenic cell migration, survival, proliferation and downstream of PAX3-FOXO1 gene expression in vari- differentiation [95,96]. MET appears to be a downstream ous cell types or are indicative of ARMS tumor gene target of PAX3. Splotch mice, which express a mutant expression profiles (see Table 1). Only a small propor- PAX3 and fail to form limb muscles because of an inhib- tion of these studies have gone on to further investigate ition of myogenic precursor cell migration, show a the mechanism of PAX3-FOXO1 regulation of these decreased expression of MET [24,97]. MET has also been genes and/or what role these genes may play in ARMS found in five independent studies to be downstream of the tumorigenesis. PAX3-FOXO1 fusion protein in ARMS [73,81,90-92]. However, in the case of ARMS, it appears that HGF may Two of these genes have already been mentioned as cooperating mutations seen in ARMS tumors, N-MYC play a role in chemoattraction of tumor cells to the bone and IGF2. The N-MYC locus is known to be amplified marrow, which is a common site of metastasis in ARMS cases [98,99]. ARMS cell line CW9019 shows chemotaxis in a proportion of ARMS tumors, and the IGF2 locus is known to show LOI in ARMS tumors (see cooperating toward bone marrow-derived-fibroblast-conditioned media mutations in ARMS tumors). However, these studies in vitro, and this migration is inhibited by the MET- blocking agent, K-252a. Moreover, both RH30 and also indicate that PAX3-FOXO1 may regulate the gene expression from these loci. CW9019 ARMS lines home to the bone marrow in lethally N-MYC expression has been shown to be upregulated irradiated mice, while the ERMS cell lines RD, SMS-CTR, and RH18 do not [99]. Conversely, siRNA against PAX3- in four independent studies using PAX3-FOXO1 target- ing siRNA in the ARMS cell line, RH4 [72], PAX3- FOXO1 prevents migration of cells in wound-healing FOXO1 overexpression in the RD (ERMS) cell line assays of RH30 cells in response to HGF. In addition to a Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 5 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Table 1 PAX3-FOXO1 target genes in ARMS Gene Description Regulation References PF target ABAT 4-Aminobutyrate aminotransferase; Up [72-74] Yes ACTC Actin, alpha, cardiac muscle 1 Up [5,75] Yes ADAM10 A disintegrin and metalloproteinase domain 10 Up [73][72] Yes ADRA2A Alpha-2 adrenergic receptor subtype C10 Up [72,74,76] Yes ADRA2C Alpha-2 adrenergic receptor subtype C4 Up [22,74,76] Yes ALK ALK tyrosine kinase receptor, anaplastic lymphoma kinase Ki-1 Up [22,72,74,76,77] Yes ANK2 Ankyrin 2 Up [73,74,76] Yes ASS Argininosuccinate synthase Up [73,74,78]? ASTN2 Astrotactin 2 Up [72,76] Yes BIN1 Bridging integrator 1 Down [72,79] Yes BMP5 Bone morphogenic protein 5 Up [72-74,76] Yes C10ORF6 Family with sequence similarity 178, member A Up [75,76] Yes CCND1 Cyclin D1 Down [75][79]? CD9 CD9 molecule/Tetraspanin-29 Up [73,76] Yes CDH3 Cadherin 3, type 1, P-cadherin (placental) Up [72,76,80] Yes CHD7 Chromodomain helicase DNA-binding protein 7 Up [76,81] Yes CKM Creatine kinase M chain, muscle Up [5,79] Yes CNR1 Cannabinoid receptor 1 Up [22,72,74,82-84] Yes COL18A1 Collagen type 18 α1Up[73,74]? CXCR4 C-X-C chemokine receptor type 4 Up [75,76,85-87] Yes CXCR7 C-X-C chemokine receptor type 7 Down [76,87] Yes DCX Neuronal migration protein doublecortin Up [73,74,81]? DES Desmin Down [72,79] Yes DKFZP762M127 Unknown Up [73,74,81] Yes DUSP4 Dual specificity phosphatase 4 Down [73,81]? DZIP3 DAZ interacting protein 3, zinc finger Up [72,74,81] Yes ELA1 Elastase-1 Up [22,72,74] Yes ENC1 Ectodermal-neural cortex 1 (with BTB-like domain) Up [75,76] Yes ENO3 Enolase 3 (beta, muscle) Up [74,79]? EPHA4 Ephrin type-A receptor 4 Up/down [76,81,82] Yes FGFR2 Fibroblast growth factor receptor 2 Up [72,76] Yes FGFR4 Fibroblast growth factor receptor 4 Up [22,73,76,88,89] Yes FLNB Filamin B, beta Down [76,82] Yes FNBP1 Formin-binding protein 1 Down [22,75,76]? FOXF1 Forkhead box protein F1 Up [22,72,74,76,81] Yes FOXO1 Forkhead box O1 Up [75,76] Yes GADD45A Growth arrest and DNA-damage-inducible protein GADD45 alpha Up [73,76,81] Yes GRAF GTPase regulator associated with FAK Up [22,72] Yes GTF3C1 General transcription factor 3C Up [72,81] Yes H19 Imprinted maternally expressed gene, untranslated mRNA Up [5,79] Yes HDAC5 Histone deacetylase 5 Up [73,76] Yes HUMMLC2B Myosin regulatory light chain 2, skeletal muscle isoform Down [72,73] Yes IGF2 Insulin-like growth factor II Up [5,75] Yes IGFBP3 IGF-binding protein 3 Down [73,81] Yes Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 6 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Table 1 PAX3-FOXO1 target genes in ARMS (Continued) IGFBP5 IGF-binding protein 5 Up [5] Yes IL4R Interleukin 4 receptor Up [72,73,75,76] Yes JAKMIP2 Janus kinase and microtubule-interacting protein 2 Up [72-74,76] Yes KCNN3 Small conductance calcium-activated potassium channel protein 3 Up [73,74,81] Yes KCNS3 Potassium voltage-gated channel subfamily S member 3 Up [73,74]? LRRFIP2 Leucine-rich repeat (in FLII) interacting protein 2 Up [72,74] Yes MARCH3 Membrane-associated RING finger protein 3 Up [73,81] Yes MCAM Melanoma cell adhesion molecule Up [73,81] Yes MEG3 Maternally expressed 3 Up [73-75]? MN1 Meningioma (disrupted in balanced translocation) 1 Up [73,76] Yes MET Hepatocyte growth factor receptor Up [73,76,81,90-92] Yes MTUS2 Microtubule associated tumor suppressor candidate 2 Up [72,76] Yes MYBPH Myosin-binding protein H Up/down [5,72,75] Yes MYCN N-MYC proto-oncogene protein Up [72,73,76,78,81] Yes MYH8 Myosin, heavy chain 8, skeletal muscle, perinatal Up/down [5,72] Yes MYL1 Myosin, light chain 1, alkali; skeletal, fast Up/down [5,79] Yes MYL4 Myosin, light chain 4, alkali; atrial, embryonic Up/down [5,72] Yes MYOD Myoblast determination protein 1 Up [5,73,76,81] Yes MYOG Myogenin (myogenic factor 4) Up [5,90] Yes NEBL Nebulette Up/down [72,73,76,81] Yes NELL1 NEL-like protein 1 Up [22,72-74,76] Yes NHLH1 Nescient helix loop helix 1 Up [22,76] Yes NPTX2 Neuronal pentraxin II Down [76,81] Yes NRCAM Neuronal cell adhesion molecule Up [72-74] Yes OLIG2 Oligodendrocyte transcription factor 2 Up [22,74]? PALMD Palmdelphin Down [72,76] Yes PBK PDZ-binding kinase Up [73,76] Yes PCDH7 Protocadherin 7 Up [76,82] Yes PDZRN3 PDZ domain containing ring finger 3 Up [74,76] Yes PGBD5 PiggyBac transposable element-derived protein 5 Up [22,72,74] Yes PHF17 PHD finger protein 17 Up [74,76] Yes PIPOX Pipecolic acid oxidase Up [22,72,74,76] Yes PKP1 Plakophilin 1 (ectodermal dysplasia/skin fragility syndrome) Up [72,76] Yes PLAG1 Pleiomorphic adenoma gene 1 protein Down [75,81] Yes PLK2 Polo-like kinase 2 Down [73,76] Yes PODXL Podocalyxin-like protein 1 Up [22,74,76] Yes POU4F1 Brain-specific homeobox/POU domain protein 3A Up [72,73,76] Yes PPARGC1A Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha Up [22,76] Yes PRKAR2B Protein kinase, cAMP-dependent, regulatory, type II, beta Up [73,81,82] Yes PRKCA Protein kinase C, alpha Up [73,76] Yes PSEN2 Presenilin 2 (Alzheimer’s disease 4) Up [73,74]? PTHLT Parathyroid hormone-like hormone Up [76,82] Yes QDPR Quinoid dihydropteridine reductase Up [74,81] Yes RASSF4 Ras association (RalGDS/AF-6) domain family 4 Up [72,74,76] Yes RRP22 Ras-like protein family member 10A, on chm 22 Up [73,74]? Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 7 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Table 1 PAX3-FOXO1 target genes in ARMS (Continued) RYR1 Skeletal muscle-type ryanodine receptor Up [5,74] Yes RYR3 Brain-type ryanodine receptor Up [74,76,81] Yes SLC24A3 Solute carrier family 24 (sodium/potassium/calcium exchanger), member 3 Up [74,76] Yes SIX1 SIX homeobox 1 Up [5,75] Yes SOX14 SRY (sex determining region Y)-box 14 Up [22,76] Yes STX11 Syntaxin 11 Up [76,82] Yes SULF1 Sulfatase 1 Up [73,76] Yes SVIL Supervillin Down [72,76] Yes TCF712 Transcription factor 7-like 2 (T-cell specific, HMG-box) Up [73,81] Yes TGFB1 Transforming growth factor, beta 1 Up [5,76] Yes TFAP2B Transcription factor AP-2 beta Up [22,72] Yes TIAF1 TGF-beta-1-induced antiapoptotic factor 1 Up [73,74]? TM4SF10 Transmembrane 4 superfamily member 10 Up [73,81] Yes TNFAIP3 Tumor necrosis factor, alpha-induced protein 3 Up [73,76] Yes TNNC2 Troponin C type 2 (fast) Up/down [5,72,73,79] Yes TNNI2 Troponin I type 2 (skeletal, fast) Up/down [5,72] Yes TNNT2 Troponin T type 2 (cardiac) Up [5,79] Yes TNNT3 Troponin T type 3 (skeletal, fast) Down [72,79] Yes TRAM2 Translocation-associated membrane protein 2 Up [73,76] Yes TSC22D2 TSC22 domain family, member 2 Up [74,76] Yes UBE2G2 Ubiquitin-conjugating enzyme E2G 2 (UBC7 homolog, yeast) Up [22,76] Yes WSCD1 WSC domain-containing protein 1 Up [22,74]? WVA5A Von Willebrand factor A domain containing 5A Up [72-74] Yes Summary of genes found to be differentially regulated in more than one reference in ARMS tumors and/or cell lines, or by PAX3-FOXO1 overexpression, in various cell types. Gene name and description are indicated and also the relative expression in ARMS or PAX3-FOXO1. Also indicated is whether the study indicates that these genes are downstream of PAX3-FOXO1 expression. Genes indicated in bold will be further discussed. migratory function for MET in ARMS, knockdown of reduces migration, and combined inhibition reveals syner- MET by shRNA in ERMS and ARMS inhibits cell prolifera- gism between these receptors [99]. SDF-1 can also induce tion and induces apoptosis. Moreover, shRNA-mediated proliferation of the ARMS cell line, RH30 [103]. Consist- knockdown of MET inhibits anchorage-independent ent with CXCR4 expression being downstream of PAX3- growth of ARMS and ERMS, and mutant MET-expressing FOXO1 transgene expression, CXCR4 expression in RMS MEFS prevent PAX3-FKHR transformation [91]. Consist- correlates with the ARMS histology, unfavorable primary ent with MET as a PAX3-FOXO1 target, high MET expres- site, advanced disease at diagnosis and bone marrow in- sion in RMS correlates with ARMS histology, advanced volvement [100]. disease at diagnosis and bone marrow involvement [100]. Other genes have been further confirmed as down- CXCR4 is normally expressed in satellite cells within stream genes of PAX3-FOXO1. Cannabinoid receptor 1 skeletal muscle and is used as a marker of mononucleated (CNR1) is specifically upregulated at both the mRNA cells capable of differentiating into myofibers [101]. and protein level in fusion-positive ARMS cells [74]. CXCR4 is a cell surface receptor; it binds and mediates CNR1 is normally highly expressed in brain [104] but is the signaling of stromal-derived factor-1 (SDF-1) and also expressed in skeletal muscle at levels detectable by induces cell chemotaxis [102]. SDF-1 can induce migra- RT-PCR [105]. CNR1 has been confirmed, using ChIP tion and chemotactic invasion in ARMS cell lines, but not analysis, as a direct target of both PAX3 and PAX3- ERMS cell lines [85], and this migration can be inhibited FOXO1 transcriptional activity [82]. Furthermore, the by SDF-1-neutralizing antibody or the CXCR4 inhibitor homeodomain of PAX3-FOXO1 appears to be the im- AMD3100 [103]. Moreover, expression of CXCR4 and portant domain for the regulation of CNR1 expression MET in ARMS lines appears to synergize to induce the [72]. CNR1 has been proposed to be a potential drug migration of cells toward bone marrow-derived fibroblast target in ARMS. Treatment with CNR1 agonists can in- conditioned media in vitro. Inhibition of each receptor duce apoptosis in some ARMS cell lines [83]. In addition, Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 8 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 CNR1 expression has been linked with an increase propen- the biology of ARMS. These include ARMS cell lines sity for PAX3-FOXO1 expressing mouse myoblast invasive- derived from human tumors in the laboratory of Dr. Peter ness and lung metastasis formation. Moreover, treatment Houghton: RH3, RH4, RH10, RH28, RH30 and RH41, all of with an inverse agonist to CNR1 can abrogate in vitro inva- which express the PAX3-FOXO1 fusion protein sion and in vivo lung metastasis formation [84]. Thus, [13,109,110] and have been widely used in the field. In CNR1 may represent a viable therapeutic target specific for addition, the NCI-supported Pediatric Preclinical Testing the increased metastatic capacity of PAX3-FOXO1 expres- Program (PPTP) uses RH10, RH28, RH30, RH30R, RH41 sing ARMS. and RH65 subcutaneous xenograft tumors to test drug effi- Transcription factor AP2β (TFAP2B) has been shown to cacy in a well-characterized preclinical model of many be a downstream target of PAX3-FOXO1 and appears to pediatric cancers [111-113]. require the paired domain of PAX3-FOXO1 to be induced. Other in vitro models that have been used involve the TFAP2B promoter expression is induced by PAX3, which introduction of the PAX3/7-FOXO1 fusion proteins into has been shown to bind to the TFAP2B promoter by ChIP both myogenic and non-myogenic cell lines including fibro- analysis. siRNA targeting TFAP2B introduced into PAX3- blast cell lines, ERMS cell lines, MEFs, mesenchymal stem FKHR-positive ARMS induces apoptosis, indicating that cells (MSC), normal or immortalized human or mouse TFAP2B can mediate cell survival in ARMS, downstream myogenic cells and even an osteosarcoma cell line of PAX3-FOXO1 [72]. [5,81,82,91,114-119]. Given that the cell of origin for ARMS FGFR4 has been identified as a direct transcriptional tar- has yet to be identified, perhaps this variation in model cell get of PAX3 and PAX3-FOXO1, which bind to a down- linesisprudent.However,itislikelythatARMSand ERMS stream enhancer region [76,106]. Accordingly, FGFR4 is tumors are derived from a mesenchymal cell likely of the significantly upregulated by PAX3-FOXO1 expression the myogenic lineage because of skeletal muscle lineage- [22,73,76,88,89,107]. However, upregulation of FGFR4 specific gene expression seen in these tumors [7]. downstream of PAX3-FOXO1 in primary myoblasts does Conversely, some studies have used endogenous not appear to act as an effector of PAX3-FOXO1-mediated PAX3-FOXO1 in ARMS cell lines to determine the tran- myoblast transformation given that wild-type FGFR4 upre- scriptional targets of this fusion protein within the gulation is not required for PAX3-FOXO1-induced prolif- ARMS tumor cell context. Both Kikuchi et al. [90] and eration, transformation, invasion or inhibition of myogenic Ebauer et al. [72] used siRNA specifically targeting differentiation [89]. However, knockdown of FGFR4 in PAX3-FOXO1 or both PAX3 and PAX3-FOXO1 RMS cell lines does show a reduction in cell proliferation sequences, respectively. Inhibition of PAX3-FOXO1 and an increase in apoptosis, suggesting that at later stages expression reduced cell proliferation and motility and of ARMS tumorigenesis FGFR4 overexpression may inter- allowed some myogenic differentiation [90]. In addition, act with other unknown genetic lesions within these cell comparative gene expression studies were performed lines to induce pro-survival and proliferation effects [107]. identifying over 100 PAX3-FOXO1 gene targets. Cao It is interesting to note however that kinase domain- et al. [76] performed ChIP sequencing studies using a activating mutations in FGFR4 have been identified in 7.5% PAX3-FOXO1-specific antibody and were able to iden- of RMS, including fusion-positive ARMS [108], and can tify 1,463 putative PAX3-FOXO1-binding sites in the contribute to myoblast growth advantage and transform- human genome. Furthermore, PAX3-FOXO1-binding ation [89]. Thus, FGFR4-activating mutations likely repre- sites adjacent to MyoD, FGFR4 and IGF1R were verified sent cooperating mutations in RMS and upregulation of as transcriptionally regulated by PAX3-FOXO1. FGFR4 in fusion-positive ARMS would enhance this effect. The CDH3/P-cadherin gene has been identified as a dir- In vivo models of ARMS ect transcriptional target of PAX3/7-FOXO1 [72,76,80]. P- Many different transgenic and knock-in animal models cadherin expression in the C2C12 myoblast cell line inhi- have been attempted to recapitulate ARMS tumor for- bits myogenic differentiation and maintains a proliferative mation in vivo. Several of these models have attempted state through maintaining cyclin D1 expression. This in to constitutively express PAX3/7-FOXO1 fusion proteins turn results in transformation of C2C12 cells, allowing col- in the skeletal muscle lineage during development, only ony formation in soft agar. Additionally, P-cadherin expres- to result in developmental defects and not tumor forma- sion resulted in enhanced cell motility, as well as cadherin tion [120-123]. Transgenic mice expressing PAX3- switching, a hallmark of epithelial to mesenchymal transi- FOXO1 under the control of the PAX3 promoter and tion and metastatic progression [80]. enhancer regions resulted in expression of PAX3- FOXO1 in the dorsal neural tube and lateral dermomyo- In vitro models of ARMS tome. PAX3-FOXO1 expression in this context appeared Many different cell lines have been derived from human to interfere with normal PAX3 developmental functions ARMS tumors; these are regularly used to investigate including neural tube and neural crest abnormalities Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 9 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 similar to those seen in PAX3 mutant Splotch mice. The under control of myosin heavy-chain Gal4, also resulted majority of defects appeared to be in neural develop- in developmental defects in the fly, evidenced by disor- ment, though defects were seen in hind limb skeletal ganized myogenic patterning. Though nothing resem- muscle; however, no tumors developed [121,122]. bling tumor formation was seen, this group did note Lagutina et al. [120] developed a model where PAX3- dissemination and infiltration of non-native tissue by FOXO1 was knocked into the PAX3 locus. This knock-in PAX7-FOXO1 expressing mononucleated cells, indicat- locus expressed low amounts of PAX3-FOXO1, which in ing an increase in invasive capacity of these cells. heterozygous pups was sufficient to result in developmental Keller et al. [47] used a conditional PAX3-FOXO1 defects in the heart and diaphragm, leading to congestive knock-in into the PAX3 locus, and Myf6-driven Cre ex- heart failure and perinatal death, as well as malformations pression. This allowed, upon Cre recombination, expres- of some hypaxial muscles. However, neither chimeric adults sion of PAX3-FOXO1 driven by the PAX3 promoter and nor their newborn heterozygous pups developed malignan- 3’ FOXO1 genomic sequences that potentially contain cis- cies. It was hypothesized that PAX3-FOXO1 expression regulatory elements, a region absent from previous PAX3 from the PAX3 control sequences was insufficient to cause knock-in strategies. This was the first animal model that ARMS formation, and downstream regions of the FOXO1 successfully recapitulated the formation of ARMS, though locus may be required to induce sufficient PAX3-FOXO1 at the low frequency of approximately 0.4% (1/228) and expression to induce tumor development. with latency of over 1 year (383 days). However, this fre- A PAX7-FOXO1 model of ARMS was also attempted quency was greatly enhanced, and latency greatly reduced, P3Fa/P3Fa in Drosophila [123]. Expression of UAS-hPAX7-FOXO1, in homozygote PAX3 mice also lacking Trp53 or Figure 2 Review summary: Fusion gene regulated genes contributing to alveolar rhabdomyosarcoma. Rhabdomyosarcoma develops from an unknown cell of origin from the mesodermal lineage that may be skeletal muscle specified. This cell likely expresses both PAX3/7 and FOXO1 and may also express Myf6. A gene fusion event resulting in a PAX3/7 DNA-binding domain fused to a more potent transcriptional activation domain occurs. This fusion transcription factor is capable of inducing a group of PAX3-FOXO1-regulated genes that contribute to ARMS development in conjunction with other genetic lesions. Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 10 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Ink4a/Arf. Subsequently, ARMS tumors have devel- From these animal models it is apparent that the tim- oped in this conditional PAX3-FOXO1 knock-in ing of PAX3-FOXO1 expression is critical for ARMS model with a Pax7-CreER and M-Cre (Pax3 hypaxial development. Too early and widespread expression of muscle enhancer) also lacking functional Trp53 PAX3-FOXO1 expression can result in developmental [124]. Moreover, histologically diagnosed fusion- defects and no apparent tumor development [120-123], negative ARMS tumors have been found to develop whereas later expression of PAX3-FOXO1, via a Myf6- +/− −/− in conditional Ptch1 Trp53 mice when Cre is driven Cre recombinase, does cause disease, though at a expressed from Pax7-CreER. The latency and incidence of low frequency [47]. Perhaps inducible expression, driven ARMS tumor development in these different models have by various myogenic genes with carefully characterized yet to be compared. expression profiling, would result in an increased fre- Clearly the problems that have arisen during the devel- quency of disease and help to narrow down the exact opment of an animal model for ARMS indicate that the stage in which PAX3-FOXO1 expression drives ARMS timing and the cell lineage targeted for PAX3-FOXO1 tumorigenesis. Nevertheless, the cell of origin for expression are very important for the development of ARMS is yet to be identified, and animal models of ARMS tumor formation and for avoiding developmental ARMS will no doubt play an important role in its defects. In a review [125] following the publication of identification. the animal model [47], Keller et al. discuss the possibil- ities for the cell of origin for ARMS; because Keller Conclusion et al. achieved the formation of ARMS tumors in their To date, numerous factors (outlined in Figure 2) have mouse model using Myf6-Cre-driven conditional PAX3- been identified that contribute to ARMS tumor develop- FOXO1, and Myf6 is usually expressed in differentiating ment and its aggressive clinical phenotype. These consist skeletal muscle myotubes, they propose a potential de- of both PAX3/7-FOXO1 target genes, such as N-MYC, differentiation mechanism for ARMS development. IGF2, MET, CXCR4, CNR1, TFAP2B, FGFR4 and P-cad- However, the formation of a fusion gene such as PAX3- herin, and PAX3/7-FOXO1 cooperating factors, such as FOXO1 suggests that the cell of origin for ARMS should the abrogation of the p53 pathway, IGF2 deregulation, express both PAX3 and FOXO1 at the time that the N-MYC and miR17-92 amplification, and IRIZIO translocation occurs, given that open chromatin is likely expression. Future ARMS research will continue to dis- required for these two genomically distinct regions to cover the mechanisms by which ARMS tumorigenesis translocate. Anecdotal evidence for this includes that the occurs. This will involve the identification of more PAX3- genome translocations that occur in many different can- FOXO1 target and cooperating genes; more importantly, cer types occur between genes that are expressed in the the mechanisms by which these genes contribute to cell type of origin. A recent study by Osborne et al. tumorigenesis will be elucidated. It is critical that we [126] showed that the MYC and IGH genes, which are develop a mechanistic understanding of how these factors involved in a chromosomal translocation common in contribute and interact to perpetrate ARMS tumo- Burkitt lymphoma, are colocalized at the same transcrip- rigenesis. This will allow new opportunities to develop tion factories more often in activated B-cells, the origin- specifically targeted therapies for this aggressive pediatric ating cell of Burkitt lymphoma, than resting B cells. This disease. colocalization at the same transcription factory allows for close proximity of these gene loci in euchromatin, Abbreviations providing the circumstances where these genes would be ARMS: Alveolar rhabdomyosarcoma; bHLH: Basic helix loop helix domain; CDK: Cyclin-dependent kinase; CNR1: Cannabinoid receptor 1; in close association, facilitating the specific translocation ERMS: Embryonal rhabdomyosarcoma; FH: Forkhead DNA-binding domain; event. PAX3 is rapidly downregulated upon myoblast FKHR: Forkhead in rhabdomyosarcoma (now known as FOXO1); differentiation, so it would be unlikely that the PAX3 HD: Homeodomain DNA-binding domain; HGF/SF: Hepatocyte growth factor/scatter factor; LOH: Loss of heterozygosity; LOI: Loss of imprinting; loci would be expressed in a nascent myotube expressing MEF: Mouse embryonic fibroblast; miR: Micro RNA; MSC: Mesenchymal stem Myf6, making it difficult to understand how transloca- cells; PD: Paired box DNA-binding domain; PPTP: Pediatric Preclinical Testing tion could occur in nascent myotubes and therefore cast Program; RMS: Rhabdomyosarcoma; SDF-1: Stromal-derived factor-1; SHH: Sonic hedgehog; siRNA: Short interfering RNA; TFAP2B: Transcription some doubt on whether the dedifferentiation model is factor AP2 b. feasible. However, it is possible that Myf6 expression does rarely occur in a small subset of undifferentiated Competing interests myogenic cells in conjunction with PAX3. This could The authors have no competing interests to declare. allow for this model to produce ARMS tumors and ac- Authors’ contributions count for the low frequency at which these tumors are AM was responsible for the drafting of the manuscript. GG was responsible seen as well as the requirement for homozygous PAX3- for critical revision of the content and approved the final version of the FOXO1 knock-in alleles [47]. manuscript. All authors read and approved the final manuscript. Marshall and Grosveld Skeletal Muscle 2012, 2:25 Page 11 of 14 http://www.skeletalmusclejournal.com/content/2/1/25 Author details 18. Sorensen PH, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, Maurer HM, Bridge Department of Genetics, St Jude Children’s Research Hospital, Memphis, TN JA, Crist WM, Triche TJ, Barr FG: PAX3-FKHR and PAX7-FKHR gene fusions 38105, USA. 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Osborne CS, Chakalova L, Mitchell JA, Horton A, Wood AL, Bolland DJ, Corcoran AE, Fraser P: Myc dynamically and preferentially relocates to a transcription factory occupied by Igh. PLoS Biol 2007, 5:e192. doi:10.1186/2044-5040-2-25 Cite this article as: Marshall and Grosveld: Alveolar rhabdomyosarcoma – The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis. Skeletal Muscle 2012 2:25. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Dec 3, 2012

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