PAX3 is located on chromosome 2q35. Full-length PAX3 consists of 10 exons encoding a 510 amino acid residue protein. Human PAX3 protein is 98% identical to the mouse orthologue . Studies in Xenopus show that both Pax3 and Zic are independently required for neural crest (NC) differentiation. The cooperative functions of Pax3 and Zic determine NC cells fate . PAX3 expression is necessary for proliferation and migration of NC cells and muscle cell precursors in the dorsal dermomyotome. It is also involved in developmental pathways that lead to melanocytes and neurons originating from the NC, and mature skeletal myocytes from the dorsal dermomyotome. Accordingly, PAX3 is implicated in the pathogenesis of tumours associated with these tissues, including RMS, melanoma and neuroblastoma. The following section will focus on the functions of PAX3 and the roles of its isoforms in myogenesis, melanogenesis, neurogenesis and related oncogenesis.
PAX3 in myogenesis and RMS
Skeletal muscles are formed from the paraxial mesoderm surrounding the neural tube. Cells of the dermomyotome exhibit early, restricted patterns in expression of Pax3 and Pax7, and then develop into skeletal muscles of the trunk and limbs. Pax3 is involved in induction and migration of myoblast precursors, and in the expression of the muscle-specific transcription factors, MyoD, Myf-5 and myogenin (Fig. 1A). However, Pax3 is down-regulated when muscle tissue begins to differentiate and the muscle-specific transcription factors are activated [32, 33]. Ectopic expression of Pax3 prevents the myogenic differentiation of myoblasts into myotubes, which might involve the cooperation of Msx1 and Notch genes [34–37]. In chicken embryos, the expression of Msx1 overlaps with Pax3 in migrating limb muscle precursors and Msx1 antagonizes the myogenic activity of Pax3 . Msx2, another Msx homeobox gene family member, was found to be an immediate downstream effector of Pax3. Pax3 represses Msx2 expression in the development of the murine cardiac neural crest . Pax3 potentiates the migration of hypaxial muscle precursors by directly modulating the expression of c-Met tyrosine kinase receptor [40, 41]. Interestingly, in muscle tumours, which often harbour an activated PAX3, c-MET is up-regulated . Therefore, both muscle development and tumourigenesis involve regulation of the MET pathway by PAX3. In P19 carcinoma cells, Wnt3 up-regulates Pax3 expression, which in turn, activates Six1, Eya2 and Dach2. This is followed by the down-regulation of Pax3 and activation of MyoD and myogenin expression [43, 44]. Thus evidence supports a role for Pax3 as a controller of a cascade of transcriptional events that are necessary and sufficient for skeletal myogenesis. This role deserves greater study from both scientific and clinical viewpoints. This hypothesis is also supported by observations that PAX3/Pax3 mutations are associated with limb muscle hypoplasia in Waardenburg syndrome patients and Splotch phenotype mice, respectively [33, 45]. PAX3 and PAX7 have similar structures and patterns of expression. Despite this, a lack of PAX3 expression during embryogenesis may not be compensated for by PAX7 or other genes [46, 47]. On the other hand, the distinct roles of Pax3 and Pax7 in regenerative myogenesis of adult mammals suggest that Pax3 may not compensate for Pax7 in postnatal muscle development . However, the function of Pax7 in specification of postnatal myogenic satellite cells is still controversial [49, 50]. The interaction between Pax3 and Pax7 needs further clarification.
Figure 1. (A) PAX3 and PAX7 are involved in myogenesis during embryonic development. The ectopic expression of PAX3 prevents terminal myogenic differentiation, possibly by regulating Msx1 and Notch signalling. (B) Schematic representation of the chromosomal translocations involving PAX3/PAX7 and FKHR, which are known to result in alveolar rhabdomyosarcoma.
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RMS is the most frequent soft tissue tumour in children under 15 years old. It develops as a consequence of disruption to the regulation of the growth and differentiation of myogenic precursor cells. In contrast to normal myogenic cells, RMS tumour cells remain in cell cycle and usually fail to differentiate completely into muscle cells.
Embryonal RMS (ERMS) and alveolar RMS (ARMS) are two major subtypes [51, 52]. A number of molecular genetic lesions are implicated in the development of RMS. The amplification of genes, such as PAX3/7-FKHR, MYCN, MDM2 and CDK4, is a characteristic feature of ARMS. Specific chromosomal gains, including chromosomes 2, 8,12 and 13, are associated with ERMS. In addition, the disruption of some genes, for example IGF2, P16, TP53 and of the HGF/c-MET signalling pathway has been implicated in the progression of RMS [42, 53–55]. Unlike normal muscle, a CpG island within PAX3 is hypermethylated in the majority of ERMS but not in most ARMS. This CpG methylation is inversely correlated with PAX3 expression .
Chromosomal translocations are characteristic of ARMS: with t(2;13)(q35;q14) and t(1:13)(q36;q14) occurring in about 75% and 25% of sufferers, respectively (Fig. 1B). The translocations lead to production of two fusion proteins: PAX3-FKHR and PAX7-FKHR. A microarray study of RMS identified a novel variant translocation t(2;2)(q35;q23), which generates a fusion protein composed of PAX3 and the nuclear receptor co-activator, NCOA1 . The PAX3-NCOA1 protein is a transcriptional activator with similar transactivation properties to PAX3-FKHR. PAX3-FKHR shows 10–100 times the transactivating and transforming capacity of wild-type PAX3. The Fas death domain-associated protein (Daxx) represses the transcriptional activity of Pax3 by approximately 80% but Pax3-FKHR is unresponsive to this repressive effect . Daxx-mediated repression of Pax3 is inhibited by the nuclear body associated protein PML . PAX3-FKHR induces the expression of a large set of genes involved in myogenesis, such as MyoD1, myogenin and Six1 Other downstream targets are BCL-XL, FKHR, TGF-α, PDGF-a receptor, insulin-like growth factor (IGF)-1 receptor, as well as genes that are not normally targets of wild-type PAX3 [51, 59]. A comparison of the gene expression profiles using microarrays revealed an overexpression of putative PAX3-FKHR target genes, such as DCX, CNR1, in PAX3-FKHR positive ARMS, relative to that in PAX3-FKHR negative ERMS . The role(s) of PAX3/7-FKHR in promoting the different molecular pathogeneses of ARMS and ERMS remains to be tested and would surely prove to be a fruitful topic of study.
PAX3-FKHR can induce cellular transformation and prevent apoptosis [61, 62]. Furthermore, it shows oncogenic effects, predominantly at relatively low levels although suppression of growth occurs at higher levels . The two DNA-binding domains of PAX3-FKHR, that is PD and the HD, are functionally separate influencing the control of growth suppression and transformation, respectively. In a landmark study, the mouse myoblast C2C12 cell line was transfected singly with cDNA for Pax3, PAX3-FKHR, IGF-II or cotransfected with IGF-II plus Pax3 or with IGF-II plus PAX3-FKHR genes. All transfectants showed altered morphologies, a lack of differentiation and higher proliferation rates in vitro. Moreover, the subcutaneous injection of C2C12 transfectants into nude mice produced tumours.
Tumours derived from IGF-II and PAX3-FKHR cotransfected cells were composed of undifferentiated cells showing most angio-genesis, least apoptosis and invaded normal muscle tissues. Schaaf et al. found that IGF-II is expressed at higher levels in RMS than in normal muscles , suggesting that PAX3 or PAX3-FKHR interact with IGF-II to play a critical role in RMS development, which is summarized in Fig. 2. Recently, the ability of PAX3 and PAX3-FKHR to promote RMS cell survival by regulating the expression of PTEN or TFAP2B was demonstrated [65, 66]. The involvement of PAX3 and PAX3-FKHR in RMS tumourigenesis is likely to be by at least partially altering the MET, PTEN or AP2 signalling pathways.
Figure 2. A diagrammatic representation summarizing how PAX3 and the chimeric protein, PAX3-FKHR could promote the development of rhabdomyosarcoma (ERMS, embryonal rhabdomyosarcoma and ARMS, alveolar rhabdomyosarcoma).
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PAX3 in melanogenesis and melanoma
Melanocytes are dendritic pigment-producing cells that originate from non-pigmented precursors of the NC melanoblasts. Several transcription factors, including PAX3 and microphthalmia-associ-ated transcription factor (MITF), are involved in this transformation. Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas MITF facilitates melanoblast survival within and immediately after, migration from the dorsal neural tube . The expression of Pax3 is probably necessary, but not sufficient, for maintaining melanocytes in their differentiated state . PAX3 promotes melanocyte lineage commitment while simultaneously preventing their differentiation (Fig. 3) . Furthermore, PAX3 alone or in synergy with SOX10, activates MITF. Failures in this regulation arising from PAX3 mutations cause the auditory-pigmentary symptoms in Waardenburg syndrome 1 patients .
Figure 3. The common origin of melanocytes and nerve cells from the neural crest and the development and progression of normal melanocytes to metastatic melanoma. The roles of PAX3 in neurogenesis, melanogenesis and melanoma development are proposed.
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Melanocytes can develop into cutaneous and ocular melanoma. Pigmented ocular tumours can also develop from proliferating cells of the retinal pigment epithelium . The incidence and mortality of cutaneous melanoma have increased at annual rates of 2–3% worldwide over the last 30 years, with the greatest increases seen in elderly men . Melanomas can clinically progress through four subtypes: benign naevi to dysplastic naevi then radial and vertical growth phase melanoma and finally metastatic melanoma (Fig. 3) . Dysplastic naevus has been suggested as the precursor of cutaneous melanoma.
The transition of melanocytes into clinically characterized melanoma is associated with changes in the function of numerous genes. Melanoma susceptibility genes are CDKN2A and CDK4, those for growth factors, such as bFGF, PDGF and EGF, and for proteins in signalling pathways, especially MAPK, STAT and Nodal[75–78]. Several transcription factors, such as PAX3, MITF, SOX10, c-MYC, PTEN, RAS and c-RET, also play roles in the pathogenesis of melanoma . PAX3 is a key transcription factor in regulating the expression of a variety of melanocytic genes [69, 80].
PAX3 is expressed in primary melanomas and melanoma cell lines but not in the surrounding normal tissues or skin sections. Transfection of melanoma cells with antisense PAX3 oligonucleotides triggers cell death by inducing apoptosis [81, 82]. The down-regulation of Pax3 in interleukin-6 receptor/interleukin-6 induced melanoma cells (B16F10.9) is linked to arrested growth and transdifferentiation to a glial cell phenotype. Pax3 reduction also induces a loss of melanogenesis, followed by a sharp decrease in MITF mRNA and gene promoter activity . Recently, PAX3 was identified as a regulator of the melanoma susceptibility and progression genes SCF, TGF-β, MUC18, RhoC and TIMP3 using microarray analyses [84, 85]. In quiescent cells, the retinoblastoma tumour suppressor protein, pRB, in its unphos-phorylated state, interacts with the transcription factor E2F and inhibits the transcription of E2F-responsive genes, which are essential for cell cycle progression. PAX3 interacts strongly with pRB .
PAX3 in neurogenesis and neuroblastoma
Pax3 is expressed in the developing nervous system during early neurogenesis (Fig. 3) . The induction of Pax3 in P19 embryonal carcinoma stem cells is closely linked to subsequent neu-ronal differentiation . Koblar etal. found that Pax3 regulates the generation of sensory neurons from precursors that originate from the NC . They demonstrated that Pax3 mRNA was initially expressed in all NC cells but was later restricted to neurons. The addition of FGF2 to the cultures significantly increased Pax3 mRNA expression in NC cells and resulted in increased neurogenesis.
Neuroblastoma is the commonest extracranial solid tumour in childhood. It is a disease of the sympathicoadrenal lineage of the neural crest, and therefore primary tumours may originate in all sites of peripheral sympathetic ganglia and paraganglia [90, 91]. Many genetic abnormalities have been implicated in neuroblastomas, including amplification of the N-MYC oncogene . Overexpression of transfected N-MYC induces cellular transformation. For example, transgenic mice overexpressing N-MYC in NC-derived tissues frequently develop neuroblastomas. However, a reduced expression of N-MYCIn cultured human neuroblastoma cell lines decreases proliferation and induces differentiation . Abnormally elevated expression of PAX3 has also been found in some neuroblastoma cell lines and tumours . Deletion and mutagenesis experiments have shown that Pax3 contains the inverted E box sequence CGCGTG (or CACGCG) in the 5' promoter region, which responds to regulation by N-Myc and c-Myc. Mouse N-Myc and c-Myc directly activate the Pax3 promoter and the ectopic expression of N-Myc and c-Myc increases Pax3 expression . Whether PAX3 initiates pathogenesis of neuroblastoma or N-MYC induces neuroblastoma by modulating PAX3 expression, is a question worthy of further investigation.
PAX3 splicing and tumours
The PAX3 isoforms are designated as PAX3a, PAX3b, PAX3c, PAX3d, PAX3e, PAX3g and PAX3h (Fig. 4) [30, 96, 97]. PAX3a and PAX3b are encoded by exons 1–4 and lack the HD and the carboxy terminal TD. PAX3c,ί/and e contain 8, 9 and 10 exons, respectively, and their isoforms possess intact HD and TD. Both PAX3c and PAX3d are evolutionary conserved in human beings and mice. Intron 8 is retained in PAX3c transcript and translation proceeds from exon 8 for five codons into intron 8 before reaching a stop codon. Intron 8 is spliced in PAX3d, and translation proceeds from exon 8 to exon 9. The predicted amino acid residues of PAX3c and PAX3d are identical except at the extreme carboxy termini. In vitro DNA-binding and transactivation studies suggested that PAX3d is functionally similar to PAX3c [30, 46]. PAX3g and PAX3h are truncated isoforms of PAX3d and PAX3e, respectively , both lack part of the TD encoded by exon 8.
Figure 4. PAX3 isoforms. The diagram illustrates the structure of PAX3 isoforms a-h DNA-binding domains: PD, paired domain; HD, homeodomain and TD, transactivation domain (not to scale). Lower vertical arrows and numbers indicate the nucleotide position of exon boundaries and the additional exons 9 and 10 are also indicated.
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Alternative transcripts of PAX3 have been identified in a variety of tissues, including human adult skeletal muscle and mouse embryos. Pax3g, also named Pax3Δ8, occurs in primary mouse myoblasts . A transient transfection assay demonstrated that Pax3g was transcriptionally inactive, although its presence effectively inhibited the activity of Pax3d, presumably by competing with it for Pax3-binding sites. A further alternative splicing occurs in Pax3 at the intron 2/exon 3 junction and results in the inclusion or exclusion of a glutamine (Q) residue (Pax3/Q− and Pax3/Q∼ forms, respectively) in the linker between the amino and carboxy terminal paired box domains . The Pax3/Q∼ form has stronger binding affinity and higher transcriptional activity than the Pax3/Q+ but both Q+/“ forms are co-expressed in equal abundance during multiple developmental stages in mice. A functional study of PAX3 isoforms in mouse melanocytes in vitro demonstrated they differ in biological functions . PAX3c, d and h promote melanocyte proliferation, migration, transformation and survival. Other isoforms have negative or no discernable effects on melanocytes.
PAX3 splicing variants show distinct expression profiles in different tumours. Compared to PAX3a, a high proportion of neuroblastomas and RMS express PAX3b, although the levels of both are low. Truncated isoforms of PAX6 act in a dominant negative manner when co-expressed with wild-type PAX6. Similar results have been found with PAX4 and PAX5. Thus PAX3a and PAX3d might interact in a similar way with other PAX3 isoforms to regulate their functions.
PAX3d is present in Ewing's sarcoma, melanoma and ERMS cell lines but is generally absent in neuroblastoma . PAX3c and PAX3d are preferentially expressed in melanocytes, melanoma cell lines and melanoma tissues but only faintly in testes, muscle, brain and brain tumours and are absent in the other normal tissues and cancer cell lines . PAX36 is the main isoform present in ARMS cell lines tested although PAX3c is expressed strongly in half of them (our unpublished data). With regard to ERMS cell lines, PAX3c and PAX3dare expressed at low-to-moderate levels .
Thus the published literature suggests that PAX3 isoforms may have significant roles in the development and progression of tumours of NC origin, specifically, PAX3c and PAX3d in melanomas and PAX3g and PAX3h in neuroblastomas [46,94, 97, 100, 102]. PAX3a and PAX3b may function by regulating the transactivation properties of the other isoforms, as indeed, do the truncated isoforms of PAX4, PAX5 and PAX6. The precise roles of these PAX3 spliced variants in normal developmental processes and in oncogenesis remain to be elucidated, and surely warrants an extended series of investigations.