Oncogene (2014), 1–9 © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

REVIEW

PAX genes in childhood oncogenesis: developmental biology gone awry? P Mahajan1, PJ Leavey1 and RL Galindo1,2,3 Childhood solid tumors often arise from embryonal-like cells, which are distinct from the epithelial cancers observed in adults, and etiologically can be considered as ‘developmental patterning gone awry’. Paired-box (PAX) genes encode a family of evolutionarily conserved transcription factors that are important regulators of cell lineage specification, migration and tissue patterning. PAX loss-of-function mutations are well known to cause potent developmental phenotypes in animal models and underlie genetic disease in humans, whereas dysregulation and/or genetic modification of PAX genes have been shown to function as critical triggers for human tumorigenesis. Consequently, exploring PAX-related pathobiology generates insights into both normal developmental biology and key molecular mechanisms that underlie pediatric cancer, which are the topics of this review. Oncogene advance online publication, 21 July 2014; doi:10.1038/onc.2014.209

INTRODUCTION The developmental mechanisms necessary to generate a fully patterned, complex organism from a nascent embryo are precise. Undifferentiated primordia undergo a vast array of cell lineage specification, migration and patterning, and differentiate into an ensemble of interdependent connective, muscle, nervous and epithelial tissues. Dysregulation of these precise developmental programs cause various diseases/disorders, including—and relevant to this review—childhood cancer. Interestingly, the pathobiology of childhood cancer is different from adult neoplasia. Solid tumors in adults are most often epithelial in origin (for example, prostate and breast carcinoma), whereas pediatric solid tumors are typically comprised of histologically primitive blastemal/embryonal-type cells (for example, neuroblastoma, nephroblastoma, medulloblastoma and rhabdomyosarcoma (RMS)). Developmental dysregulation of precursor cell maturation and terminal differentiation, or ‘development gone awry,’ represents a seminal feature of childhood cancer. In this review, we discuss in particular the misregulation of Paired-Box (PAX) gene-mediated developmental programs as important underpinnings of childhood cancer. The evolutionarily conserved PAX transcription factors are master regulators of histogenesis and organogenesis. Spontaneous and targeted murine Pax gene mutations disrupt many aspects of tissue/organ patterning, as well as interfere with the maintenance of adult stem cells necessary for tissue-specific repair/regeneration. Here we profile how PAX genes normally direct precursor cell differentiation, whereas dysregulation and/or misexpression of PAX orthologs serve as critical participants in a broad spectrum of childhood malignancies (Table 1). Of note, we refer interested readers to Li et al.,1 Lang et al.2 and Robson et al.,3 for more detailed reviews of PAX

developmental mechanisms and PAX genes in medical (adult) oncology.

STRUCTURAL MOTIFS DEFINE THE PAX FAMILY SUBGROUPS The mammalian PAX family of transcription factors is comprised of nine members that function as ‘master regulators’ of organogenesis4 (Figure 1). The structural motif that defines this unique family of molecules is the evolutionarily conserved paired domain (PD). The PD, 128 amino acids in length, is a DNA-binding motif that recognizes highly related DNA sequences (TCACGC/G; minor variability can be seen for each PAX ortholog).5–7 Seven of the nine PAX molecules, PAX2 through -8, additionally possess a second DNA-binding domain, a homeodomain (HD) motif (consensus binding sequence TAAT–ATTA),7,8 which adds functional complexity to these PAX orthologs, as the PD and HD can operate cooperatively (consensus sequence AATTA–GTCACGC)7,9 or independently of one another. Unlike the PD, the HD varies structurally among the HD-containing PAX proteins, and is used to further organize the PAX orthologs into subfamilies (described in more detail below). Finally, except for PAX4 and -6, PAX molecules contain a conserved octapeptide motif (OP) positioned between the PD and HD. This eight-amino-acid element has been shown to function as a transcriptional inhibitory motif evolutionarily related to the Drosophila-engrailed family eh1 repression domain and Goosecoid protein, which behave as transcriptional repressors.10 Thus, PAX molecules, depending on context and cofactors, can function either as transcriptional activators or repressors, underscoring even more so their experimentally problematic functional complexity. Below, we discuss in more detail aspects of PAX-mediated development and pathobiology that pertain specifically to PAXrelated pediatric malignancies, organized into sections corresponding to the four PAX family subgroups.

1 Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA; 2Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA and 3Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA. Correspondence: Dr Galindo, Department of Pathology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9072, USA. E-mail: [email protected] Received 3 April 2014; revised 10 June 2014; accepted 11 June 2014

PAX genes in childhood cancers P Mahajan et al

2 Table 1. PAX genes

PAX genes in normal development and childhood cancers Subgroup Normal development

PAX3 PAX7

III

PAX2 PAX5 PAX8

II

PAX4 PAX6 PAX1 PAX9

IV I

Pediatric cancer

Skeletal muscle Rhabdomyosarcoma development and regeneration Kidney Wilms tumor Renal cell carcinoma Thyroid Thyroid carcinoma B cell B-cell acute lymphoblastic leukemia Acute myeloblastic leukemia B-cell lymphomas T-lymphoblastic leukemia Medulloblastoma Neuroblastoma Pancreas Retinoblastoma? Eye Musculoskeletal Pediatric bone-related malignancies?

PAX-MEDIATED DEVELOPMENT, PATHOBIOLOGY AND ONCOGENICITY PAX3 and PAX7 are canonical proto-oncogenes PAX3 and PAX7, members of subgroup III, are structurally the most complex PAX orthologs, being the only two family members containing a PD, a three-helix-length HD and an OP motif. In addition to being master regulators of neural tube, neural crest and somatic muscle development, PAX3 and -7 are also the clearest examples of how PAX can function as proto-oncogenes and drive human tumorigenesis, as clearly illustrated in the context of the skeletal muscle-lineage soft tissue sarcoma, rhabdomyosarcoma (RMS). PAX3 and PAX7 in skeletal muscle development and regeneration. Mutation of the Pax3 locus was identified as the molecular basis for the spontaneous-occurring semidominant lethal murine splotch mutation.11 Splotch homozygous mice were initially characterized for phenotypes related to neural tube- and neural crest-derived tissues. Further analysis showed that the splotch mouse also served as a model for skeletal muscle patterning (myogenesis). Developmental studies uncovered that Pax3 is expressed in the somites and mesenchymal limb buds,12 and that splotch mice, although exhibiting a mild reduction in body wall muscle, showed a striking failure to develop normal limb musculature.13 The observation that Pax3 mutation blocks the onset of myogenesis in a subset of somitic cells otherwise fated to become myoblasts (no expressions of the myogenic transcription factors Myogenin and MyoD, for example, were detected in these precursors) clearly pointed to Pax3 as a critical upstream myogenesis determinant.14 The importance of PAX3 in development, including myogenesis, is also seen in humans: mutations in the PAX3 gene have been identified in patients with Waardenburg Syndrome, a rare autosomal dominant disorder characterized by deafness, pigmentation anomalies and disruptions in myogenesis.15 Similar to its ‘sibling’ Pax3, Pax7 is also expressed in embryonic muscle progenitor cells, most notably those arising in the central dermomyotome.16 Pax7 homozygous mutant embryos, however, demonstrate no obvious muscle patterning defects, as mice with targeted null mutations for Pax7 express myogenesis markers (for example, MyoD, Myf5) in a normal spatiotemporal pattern.17 Pax7 loss-of-function muscle phenotypes, instead, are revealed postnatally, as Pax7 mutant mice die within few weeks after birth from failure to thrive due to severe muscular atrophy.17 Further probing of these animals revealed an absence of muscle-specific Oncogene (2014), 1 – 9

Figure 1. The mammalian family of PAX transcription factors. The PAX family of proteins is comprised of nine members, which are further classified into four distinct subgroups I–IV based on structural composition. All nine PAX proteins have a highly conserved PD. Group I includes PAX1 and 9; group II consists of PAX2, 5 and 8; group III includes PAX3 and 7; and PAX4 and 6 belong to group IV. PAX2, 3, 4, 5, 6, 7 and 8 contain a HD and all of the PAX proteins, with the exception of PAX4 and 6, contain an OP. TAD, transcription activation domain.

monopotential stem cells, termed satellite cells, which are critically required for skeletal muscle physiologic growth, maintenance and regeneration.17,18 Realix et al.19 further demonstrated that the loss of satellite cells is progressive in Pax7 mutants, pointing toward an antiapoptic role of Pax7 for which Pax3 is not compensatory. In addition, Pax7 deletion in primary murine myoblasts and satellite cells resulted in cell cycle arrest and early differentiation,20 highlighting that Pax7 is necessary for maintaining satellite cells in a stem cell-like state for ongoing muscle renewal. Of note, although numerous findings support the notion that Pax7 is indispensible for the proper establishment and function of perinatal satellite cells, Cre/loxP lineage tracing and tamoxifeninducible conditional gene inactivation interestingly reveal that loss of Pax7 in adult satellite cells does not compromise muscle regeneration. In these studies, Lepper et al.21 showed that adult satellite cells lacking Pax7 (as well as cells lacking both Pax7 and Pax3) proliferate and properly reoccupy the sublaminal niche during muscle repair from injury, and report that Pax7 is only required in satellite cells up to the juvenile period, at which point these progenitors make a transition into quiescence. The authors of these studies appropriately remind us that stem cell biology is not static, but is instead dynamic throughout an organism’s life cycle, and that developmental windows exist that are important to consider. © 2014 Macmillan Publishers Limited

PAX genes in childhood cancers P Mahajan et al

3 Translocation involving PAX3 and PAX7 drives RMS. Turning toward pediatric oncology, RMS is the most common pediatric soft tissue tumor, with an incidence rate of 4.5 per million US children.22,23 The estimated 5-year failure-free survival rate for lowrisk RMS patients is 90%.24 However, despite intensive therapies, the 3-year event-free survival for children with high-risk RMS is only 20%, with no significant improvement in the past 25 years.25 RMS is a malignancy comprised of skeletal muscle precursors that fail to exit the cell cycle and are blocked from differentiating into syncytial muscle. RMS is typically divided into two distinct histologic subgroups, each with differing clinical features: embryonal RMS, which is more common, and alveolar RMS (A-RMS), which is notoriously more aggressive. RMS cells are known to misexpress/overexpress PAX3 and/or PAX7, thus suggesting a potential pathogenetic role for PAX3/PAX7 in RMS.26–28 A critical breakthrough regarding PAX3’s potent myogenic influence on RMS occurred when a recurrent chromosomal translocation uniquely associating with A-RMS, t(2;13)(q35;q14), was molecularly characterized and found to fuse PAX3 on chromosome 2 with the FOXO1 forkhead transcription factor locus on chromosome 13 (Figure 2). The newly encoded PAX3– FOXO1 chimera, which fuses the PAX3 DNA-binding domains with the stronger FOXO1 transcriptional activation domain, is misexpressed from the endogenous PAX3 enhancer/promoter, which dysregulates myogenesis in vivo and drives RMS tumorigenesis. A similar finding occurred not long after, when a second (though less common) recurrent translocation, t(1;13)(p36;q14), was characterized and found to encode an equivalent PAX7–FOXO1 oncogenic fusion.29 Thus, A-RMS is a genetically distinct disease driven by the PAX–FOXO1 fusion oncoprotein, which is both unique to and diagnostic for ARMS.30 Clinically, expression of the PAX–FOXO1 fusion is now widely used as a diagnostic marker (identified by PCR/FISH technology), which harbors prognostic significance.30 PAX3–FOXO1, the more frequent chimera encountered, is associated with a worse clinical outcome among patients with metastatic disease,30 as PAX3– FOXO1-positive patients with metastatic disease have a significant increased risk for treatment failure. Fusion gene status has also been shown to be associated with distinct clinical phenotypes, where PAX7–FOXO1 tumors are more often localized and typically present in younger patients within the extremities.31 Of note, tumors that are classified as A-RMS based on histologic morphology but are otherwise PAX–FOXO1 fusion negative (~20–30% of all A-RMS cases) have been shown to behave similarly to E-RMS with regard to clinical outcome.32 As the PAX–FOXO1 fusions contain the native PAX3/7 DNAbinding domains, it is postulated that myogenic genes normally regulated by PAX3 and PAX7 underlie PAX–FOXO1 A-RMS pathogenesis. PAX3 has been shown to exert ongogenic potential by modulating the c-MET receptor in RMS cell lines with a t(2;13) translocation. In addition, genome-wide analysis of PAX3–FOXO1 RMS points toward ALK, FGFR4, MYCN and IGFIR as direct targets of the fusion gene.33 Interestingly, high levels of PAX7 expression are noted in RMS tumors lacking a PAX–FOXO1 gene fusion, suggesting that overexpression of PAX7 may facilitate fusionnegative RMS.26,27 Of note, molecular genetic studies have identified additional rare, novel fusion proteins in A-RMS, such as fusion of PAX3 to the nuclear receptor coactivator NCOA1, which appears to demonstrate similar transactivation properties as PAX3–FOXO1.34 Shern et al.35 have also reported a new PAX3NCOA1 fusion in RMS, as well as a PAX3 fusion involving the C terminus of INO80D. The influence that these rare fusions exert on the clinical behavior of these RMS variants at present remains unclear. As mentioned above, alterations of PAX genes in RMS presumably induce aberrant myogenesis, tumor growth and resistance to apoptosis. The observation that PAX3–FOXO1, in © 2014 Macmillan Publishers Limited

Figure 2. PAX–FOXO1 fusion gene is both unique and diagnostic for ARMS. The PAX–FOXO1 translocation gene involves FOXO1 on chromosome 13 and PAX3 or PAX7 on chromosomes 2 and 1, respectively. This translocation retains the wild-type PAX DNAbinding site with the transactivation domain from the FOXO1 gene fused to the 3′ end. FD, forkhead domain.

comparison with wild-type PAX3, potently inhibits the differentiation of the C2C12 murine myoblasts’ cell line6 suggests that dysregulated PAX3/7 activity contributes to the A-RMS phenotype by interfering with terminal differentiation in the myogenic pathway, as well as the discrete process of myoblast cell–cell fusion.36,37 It is important to note, however, that even though PAX–FOXO1 expression is viewed as the driver mutation in precursor cells that newly acquire a PAX–FOXO1-encoding translocation, PAX–FOXO1 alone is apparently a weak driver of RMS tumorigenesis. For example, expression of PAX3–FOXO1 or PAX7–FOXO1 in cultured mesenchymal cells generates A-RMS-like tumors in mice only in the presence of additional oncogenesispromoting mutations, such as p53, RB or Ras pathway disruptions.38,39 In addition, mutations in p53 or INK/ARF are also known to significantly increase A-RMS tumorigenesis in vivo in genetically engineered PAX3–FOXO1 mouse models.40 Interestingly, genomic analysis of a comprehensive collection of human RMS tumors,41 however, showed that—in comparison with the E-RMS, for example—A-RMS has far fewer genetic alterations overall, with no lesions isolated in well-known genes (for example, p53) recurrently mutated in cancer. Thus, the influence that cooperative genetic mutations might impart on PAX–FOXO1mediated tumorigenesis in humans remains an intriguing, openended question. In summary, together these studies clearly identify PAX dysregulation and overexpression as RMS underpinnings, and that PAX3 and -7 function as canonical proto-oncogenes. PAX2, PAX5 and PAX8 in development and childhood cancer Similar to PAX3/7, PAX 2, PAX5 and PAX8, which belong to subgroup II, contain a PD, HD and OP motif, but are distinguished structurally by a truncated, single-helix HD. The developmental functions of PAX2, -5 and -8 are diverse and impinge on renal, thyroid, eye and lymphocyte development.42–46 Here we will focus on three aspects of PAX2/5/8 in developmental programming and childhood cancer: (1) PAX2 and -8 in renal patterning and neoplasia; (2) PAX8 in thyroid development and follicular carcinoma; and (3) PAX5 in B-cell lineage specification and leukemia/lymphoma. PAX2 and PAX8 in kidney development. PAX2 is critical in excretory system development—in particular, the urogenital tract. Pax2 is normally expressed throughout the developing branching uteric bud and Wolffian/Müllerian ducts, which persists into the nephrogenic mesenchyme and the early epithelia of patterning Oncogene (2014), 1 – 9

PAX genes in childhood cancers P Mahajan et al

4 nephrons, with Pax2 expression downregulated as nephrogenesis concludes.43,47–49 Mutant mice further confirm the importance of Pax2 for proper nephrogenesis, as Pax2 heterozygotes demonstrate mild-to-moderate renal hypoplasia, whereas Pax2 homozygotes show renal agenesis, as well as the absence of additional genitourinary structures.48 Similar to these murine phenotypes, PAX2 heterozygous mutations in humans cause renal-coloboma syndrome, so named due to the presence of renal hypoplasia and optic nerve colobomas.46 Even though Pax8 is expressed in the developing renal vesicle and persists in conjunction with Pax2 during kidney development, Pax8 mutation itself does not disrupt nephrogenesis, suggesting a shared or compensatory role between Pax2 and -8.42,44,50,51 Compound Pax2/Pax8 mutant mouse models indeed demonstrate a cooperative Pax2/Pax8 relationship: mouse embryos heterozygous for Pax2 and Pax8 (Pax2+/ − /Pax8+/ − ) show enhanced growth impairment when compared with Pax2+/ − mice, with severe reduction in kidney size (~25–50% of normal size).51 These results illustrate the critical role of both PAX2 and PAX8 in renal cell specification, morphogenesis, growth and cell survival. Regulatory roles of PAX2 and PAX8 in cell survival and apoptosis. Consistent with the roles PAX2 and PAX8 provide in regulating the survival and propagation of nephrogenic-lineage precursors, PAX2/8 influence survival and proliferation in renal neoplasia. In renal cell carcinoma (RCC) cell lines, inhibition of PAX2 expression by RNA silencing results in growth inhibition.52 Similarly, silencing of PAX2 expression through RNA interference (RNAi) in additional genitourinary cancer cell lines (bladder, ovarian) induces apoptosis, despite the presence of cooperating p53 and/or HRAS mutations.53 In addition, PAX2 facilitates angiogenesis, tumor growth and proliferation, whereas downregulation of PAX2 expression in renal tumor-derived endothelial cells results in an increase in the PTEN tumor suppressor gene.54 In these same studies, normal human endothelial cells—when transfected with PAX2—induced a proangiogenic phenotype, further illustrating the pro-growth potential of PAX2. In invertebrate genetic models, two PAX2/5/8-related genes, egl-38 and pax-2, promote cell survival in Caenorhabditis elegans, thus showing evolutionary conservation of PAX2/5/8 anti-apoptotic activity.55 Finally, PAX8 has also been shown to promote tumor cell growth and cell cycling by transcriptionally regulating E2F1 and stabilizing the retinoblastoma protein (RB) protein.56 Aberrant PAX2 and PAX8 expressions associate with a variety of renal tumors, including Wilms tumor. PAX2 and PAX8 are implicated in both Wilms tumor and RCC pathobiology. Wilms tumor is the most common form of renal cancer in patients o 15 years of age, accounting for 95% of the childhood renal cancer diagnoses in this age group.57 The incidence rate is 8 cases per million children in the United States less than age 15 and ~ 500 new cases are diagnosed each year in the United States. It is most commonly diagnosed in children younger than 5 years of age and the 5-year survival rate is ~ 90%.58 In contrast, RCC rarely occurs in children, and occurrence is most frequently in the second decade of life, with the highest incidence among 15–19-year olds.59 Although Pax2 expression is necessary for nephrogenic precursor cell specification and survival, Pax2 must be downregulated for nephrogenesis to complete properly, as persistent transgenic expression of Pax2 results in nephric phenotypes similar to human nephrotic syndromes.60 Given that Wilms tumor (also referred to as nephroblastoma) is a blastemal neoplasm tumor, it is not surprizing that Pax2 expression persists in the epithelial blastema of these tumors. Further probing of Pax2 regulation in nephrogenesis revealed that downregulation of Pax2 expression coincided with the known tumor suppressor gene Wilms Tumor protein 1 (WT1), and that Pax2 expression is directly regulated by WT1,61 whereas additional studies have shown that Oncogene (2014), 1 – 9

in Wilms tumor sections where PAX2 is expressed, WT1 expression is absent.61 Interestingly, PAX2 and WT1 were subsequently shown to interact genetically and molecularly, as mice with heterozygous mutations in both genes harbor kidneys that are 50% smaller than wild type, whereas WT1 and PAX2 can function in a protein complex in vitro and in vivo.62 Thus, PAX2 and WT1 are critical components of nephrogenic pathobiology. PAX2 is also a specific and sensitive marker for tumors of renal origin, including RCC.63 Immunostaining in primary RCC tumors detects PAX2 expression in the malignant cells but not in the surrounding tissue.52 Furthermore, treatment of RCC cell lines with RNAi results in significant growth inhibition, supporting the role for PAX2 in proliferation.52 Thus, PAX2 function is postulated to facilitate RCC initiation and tumor maintenance. Similar to PAX2, PAX8 expression is observed in renal neoplasms.50,64 Analysis of normal human kidney and Wilms tumor showed a strong expression of PAX8 in Wilms tumors in comparison with the adult kidney.50 A study comparing PAX2 and PAX8 expressions in both primary renal tumors and metastatic tumors demonstrates that PAX2 and PAX8 may be useful diagnostic markers for both primary and metastatic tumors of the kidney.64 Regardless of histologic subtype, PAX8 immunostaining is observed in more cells with stronger intensity, and PAX8 expression is more frequent within metastatic RCC than PAX2. Therefore, PAX8 may be a more sensitive marker than PAX2 for both primary and metastatic tumors of renal histogenesis. These studies together suggest a role for PAX proteins as diagnostic and potentially important prognostic markers. PAX8 expressed in the thyroid and a role in cancer cell growth. In addition to the developing kidney, Pax8/PAX8 is also expressed in the developing and adult thyroid.44,50 Analysis of Pax8−/− mice illustrates the requirement of Pax8 for proper formation of the follicular cells of the thyroid gland:42 mice embryos with inactivated Pax8 revealed smaller thyroid glands with no detectable follicles. Interestingly, no other defects were observed in other Pax8-involved structures, likely given the redundancy of other Pax genes. These Pax8−/− mice demonstrated growth retardation and eventually demised, thus supporting a role for Pax8 in thyroid development and differentiation. PAX8 expression is observed in normal thyroids and differentiated thyroid cancers more frequently than undifferentiated thyroid cancers, demonstrating a potential role for PAX8 in differentiation.65 Furthermore, nuclear and cytoplasmic localization of PAX8 is seen in pediatric thyroid tissues, and cytoplasmic PAX8 is more frequently noted in thyroid cancers and is associated with more aggressive disease and increased risk of recurrence.66 Cytoplasmic PAX8 may lead to decreased transcription of thyroidspecific genes and therefore dysregulation of proper PAX8 function; however, the complex molecular mechanisms that underlie thyroid cancers are not clearly established and further work with larger sample sizes is needed to distinguish clinical significance. Thyroid carcinoma is overall the most common endocrine malignancy; however in pediatrics, thyroid cancer is rare with the highest incidence occurring in the 15–19 age group among children and adolescents.67 Of the pediatric thyroid carcinomas, papillary thyroid carcinoma is the most prevalent, representing 80% of differentiated thyroid carcinomas, whereas follicular thyroid carcinoma is uncommon.68,69 In children and adolescents, tumor recurrence has been shown to occur at higher rates in the presence of vascular invasion.68 Also noteworthy, PAX8 is involved in a recurrent chromosomal translocation observed in a subset of follicular thyroid cancers. The rearrangement, t(2;3)(q13;p25), involves PAX8 on chromosome 2 and the nuclear receptor, peroxisome proliferator-activated receptor gamma-1 (PPARγ) on chromosome 3. PAX8-PPARγ was initially proposed to be specific to follicular thyroid carcinomas, as it was not detected in follicular adenomas, papillary carcinomas or © 2014 Macmillan Publishers Limited

PAX genes in childhood cancers P Mahajan et al

5

Figure 3. PAX9 is misexpressed in bone/bone marrow-related malignancies. Shown are probes for PAX9 and PAX1 expressions in a battery of samples for Pre-B-cell ALL, T-cell ALL, embryonal rhabdomyosarcoma (E-RMS), A-RMS, Ewing sarcoma (EWS), and osteosarcoma (OS). Profiles are from cell lines (C), tumor xenografts (X) and primary human tumors (T). Also shown for comparison is a probe for MyoD, a gene typically expressed in E-RMS. Data are obtained from the Pediatric Tumor Affymetrix Database (Oncogenomics; http://home.ccr.cancer.gov/oncology/ oncogenomics/).

mutinodular hyperplasias.70 However, the presence of the PAX8PPARγ has since been noted in other thyroid neoplasms, including follicular adenomas.71–75 The fusion gene inhibits normal PPARγ function,70 thus highlighting the potential role of PPARγ as a tumor suppressor and possible therapeutic target. Functionally, PAX8-PPARγ expression increases cell cycle transit and decreases apoptosis,76 pointing toward an oncogenic potential for the fusion, whereas PAX8 immunostaining has been shown to be useful diagnostically.77–81 Further probing of how PAX8-PPARγ might underlie pediatric thyroid cancers remains to be determined, and the clinical/therapeutic significance of PAX8 and/or the PAX8-PPARγ fusion clarified. PAX5 in B-cell development, leukemias, lymphomas and other cancer types. Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and represents ~ 25% of childhood cancers. B-cell precursor ALL accounts for a majority of childhood leukemias and, in recent years, great improvements in ALL outcomes have been achieved, with a present-day 5-year survival rate of ~90%.82 Disruption of the genes involved in normal B-cell development, including PAX5, has been associated with leukemogenesis. PAX5, a B-cell-specific transcription factor, has a key role in B-cell lineage commitment83,84 and aberrant expression of PAX5 is associated with B-cell cancers, such as lymphoma and B-cell ALL.85–87 Pax5-deficient pro-B cells differentiate along myeloid and non-B-lymphoid lineages, whereas B-cell development is arrested,84 thus underscoring the critical importance of Pax5 in B-cell differentiation. A diverse array of PAX5 alterations, including PAX5 deletions and structural rearrangements, has been observed in both children and adults with acute B-cell ALL88 and recently germline PAX5 mutations have been linked with a susceptibility to pre B-cell ALL,89 implicating PAX5 in familial or hereditary ALL. PAX5 is a commonly targeted gene in ALL, as somatic mutations in PAX5 have been observed in ~ 30% of pediatric ALL cases.90 In ALL, PAX5 has been identified in various fusion genes.91–96 The incidence of PAX5 rearrangements in childhood B-cell precursor ALL is ~ 2.5%.97 In addition, intragenic PAX5 amplifications have been detected in pediatric B-cell precursor ALL patient samples, and although the importance of this amplification is unclear, PAX5 amplification may be a potential prognostic factor and preferentially associated with relapsed disease.98 PAX5 has also been associated with acute myeloblastic leukemia and PAX5 overexpression has been implicated in driving transcription of the B-cell-specific surface marker CD-19 in t(8;21) acute myeloblastic leukemia.99 PAX5 is additionally involved in a chromosomal rearrangement with the immunoglobulin heavy-chain locus on chromosome 14 [t(9;14)(p13;q32)], which results in the aberrant expression of PAX5 in variety of B-cell lymphomas,100,101 including aggressive B-cell non-Hodgkin’s lymphomas.102 Pax5 has also been shown to be a © 2014 Macmillan Publishers Limited

potent oncogene in the tumorgenesis of T-lymphoblastic lymphomas,103 suggesting that normal T-lymphoid development is interrupted with aberrant Pax5 expression. Loss-of-function Pax5 mutations in mice result in aggressive lymphomas, illustrating a role for Pax5 as a tumor suppressor gene.104 Although the prognostic implications of PAX5 alterations is unclear, PAX5 immunostaining has a potentially valuable diagnostic role in distinguishing B-cell lymphoid cancers from other lymphoid neoplasms.105 Of note, PAX5 expression is also observed in non-B-cell cancers, including nonhematopoietic solid tumors. PAX5 has been shown to be dysregulated in medulloblastoma106 and neuroblastoma.107 Medulloblastoma is the most common malignant pediatric brain tumor and neuroblastoma is the most common extracranial solid tumor in childhood. PAX5 is expressed in medulloblastoma, even though Pax5 transcripts are not detected in normal neonatal or adult cerebellar tissue, suggesting that PAX5 misexpression might be influential in medulloblastoma.106 Furthermore, downregulation of PAX5 in neuroblastoma cell lines decreases proliferation,107 highlighting the oncogenic potential of PAX5 in nonhematological malignancies. Interestingly, PAX5 has been shown to interact with the underphosphorylated form of the tumor suppressor RB108 and although this interaction warrants further investigation, PAX5 may interfere with normal RB function, thereby contributing to oncogenesis. PAX4 and PAX6 in the pancreas, eye and retinoblastoma PAX4 and PAX6, which contain the PD and full-length HD, but lack the OP motif, belong to subgroup IV. Pax4 and Pax6 are predominantly expressed in the pancreatic islet cells,109 whereas Pax6 is additionally critical in eye patterning.110–112 Pax4 specifically has a role in the differentiation of insulin-secreting β-cells in the pancreas113–116 and mutations in PAX4 have been associated with diabetes.117–119 In contrast, Pax6 is involved in the differentiation of the α-cells in the pancreas, which are responsible for synthesizing and secreting glucagon.120 Pax6/PAX6 is also a critical master regulator of eye development110–112 and has significant homology in Drosophila, mice and humans.121 Loss-of-function mutations in the evolutionary conserved Pax6 locus result in eye defects in mammals and flies, including the eyeless phenotype in Drosophila. Provocatively, ectopic compound eyes are induced within Drosophila wings, legs and antennae tissue by targeted misexpression of the Drosophila eyeless (ey) gene,122 as well as ectopic expression of murine Pax6, further illustrating PAX6’s role as a master regulator of eye patterning. Of note, an additional Drosophila Pax6 gene, twin of ey (toy), has also been identified and is required for the expression of ey to initiate eye development.123 Similar to the ey mutation, the small eye (Sey) mutation in mice, characterized by lack of eyes and nasal cavity, Oncogene (2014), 1 – 9

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6 and human aniridia, in which the iris is absent, arises from mutations in the homologous Pax6/PAX6 gene.124–126 Consistent with PAX6’s intimate association with eye development, the hypothesis exists questioning whether PAX6 activity might be associated with pediatric ocular neoplasms—in particular, retinoblastoma. Retinoblastoma is the most common malignant ocular tumor in children and ~ 200 children are affected each year in the United States.127 The role of PAX6 in retinoblastoma has been investigated128,129 and PAX6 overexpression using recombinant lentiviral vectors induces retinoblastoma tumor cell proliferation with a reduction in G0/G1 arrest.129 Furthermore, silencing PAX6 with small interfering molecules inhibits proliferation and increases apoptosis in retinoblastoma cells, suggesting a role for PAX6 in retinoblastoma tumor cell survival.128 Investigation of a particular microRNA shown to be downregulated in retinoblastoma, MiR-365b-3b, demonstrated that expression of MiR-365b-3b promotes apoptosis by targeting PAX6 in retinoblastoma.130 Therefore, there is a potential role for PAX6 in the growth of retinoblastoma tumor cells. PAX1 and PAX9, a ‘benign’ subgroup? With regard to structure, subgroup I, comprised of PAX1 and PAX9, is the ‘minimalist’ subgroup, as these two PAX orthologs are the only members that contain no additional DNA-binding domains beyond the PD (Figure 1), and thus presumably activate (or repress via the OP motif) transcription from PD-specific DNA elements. On the basis of murine studies, Pax1 and Pax9 are best known for their critical roles in musculoskeletal development.131–133 Specifically, Pax1 is necessary for normal development of the vertebral column, sternum and scapula,132,134 whereas Pax9 homozygous loss-of-function mutations demonstrate craniofacial and hind-/forelimb abnormalities,133 showing slightly different expression profiles of Pax1 and -9. The importance of PAX1 and PAX9 in development is further revealed by their involvement in human disease. Consistent with Pax1 as integral for murine vertebral development, mutation of the PAX1 PD has been identified in spina bifida,135 while oligodontia has been associated with PAX9 mutations.136–139 These findings underscore the importance PAX1 and -9 for proper vertebral, craniofacial and tooth development.140 Keeping with the notion of subgroup 1 being the ‘minimalist’ subgroup, no definitive role for PAX1 or -9 has been uncovered with regard to human (or pediatric) tumorigenesis. We wish to introduce here, however, the observation that PAX9 is preferentially misexpressed in childhood tumors from the bone and bone marrow. For this review, we surveyed PAX1 and PAX9 expression levels in a large collection of pediatric cancer cell lines, xenograft tumors and primary tumors using the Pediatric Tumor Affymetrix Database (Oncogenomics; http://home.ccr.cancer.gov/oncology/ oncogenomics/).141 When compared with normal tissues and other pediatric malignancies, PAX9 showed a provocative and significant upregulation in three malignancies related to the bone or bone marrow: T-cell ALL, Ewing sarcoma and osteosarcoma (Figure 3). Given that PAX9 is intimately involved in skeletal formation, we propose the hypothesis that PAX9 misregulation might influence childhood malignancies (or a subset of these malignancies) originating from pediatric bone or bone marrow tissues. CONCLUDING REMARKS PAX genes encode evolutionary conserved transcription factors and PAX mutations have been shown to be associated with distinct phenotypes in species ranging from Drosophila to mice and humans. Although they have precise and specific roles in organogenesis and tissue development, PAX genes similarly are important in proliferation, growth, cell maintenance and apoptosis. When PAX gene function and behavior are disrupted, Oncogene (2014), 1 – 9

PAX genes can act as oncogenes, as we have discussed, for example, in RMS, Wilms tumor and B-cell cancers. Their importance as diagnostic and prognostic markers, and more importantly as putative therapeutic targets, needs to and will be subjects of further interrogation. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported by funding to RLG by the Burroughs Wellcome Fund (Career Award for Medical Scientists), Alex's Lemonade Stand Foundation ("A" Award), American Cancer Society (Research Scholars Grant) and UTSW Department of Pathology (Research Grant). We thank Stephen X. Skapek for helpful suggestions regarding this manuscript.

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