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(2015) 34, 2681–2689 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

REVIEW PAX in childhood oncogenesis: developmental 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 a family of evolutionarily conserved factors that are important regulators of lineage specification, migration and tissue patterning. PAX loss-of-function are well known to cause potent developmental in models and underlie genetic disease in humans, whereas dysregulation and/or genetic modification of have been shown to function as critical triggers for human tumorigenesis. Consequently, exploring PAX-related pathobiology generates insights into both normal and key molecular mechanisms that underlie pediatric cancer, which are the topics of this review.

Oncogene (2015) 34, 2681–2689; doi:10.1038/onc.2014.209; published online 21 July 2014

INTRODUCTION developmental mechanisms and PAX genes in medical (adult) The developmental mechanisms necessary to generate a fully oncology. patterned, complex from a nascent are precise. Undifferentiated primordia undergo a vast array of cell lineage specification, migration and patterning, and differentiate into an STRUCTURAL MOTIFS DEFINE THE PAX FAMILY SUBGROUPS ensemble of interdependent connective, muscle, nervous and The mammalian PAX family of transcription factors is comprised of epithelial tissues. Dysregulation of these precise developmental nine members that function as ‘master regulators’ of organo- programs cause various diseases/disorders, including—and genesis4 (Figure 1). The structural motif that defines this unique relevant to this review—childhood cancer. family of molecules is the evolutionarily conserved paired domain Interestingly, the pathobiology of childhood cancer is different (PD). The PD, 128 amino acids in length, is a DNA-binding motif from adult neoplasia. Solid tumors in adults are most often that recognizes highly related DNA sequences (TCACGC/G; minor – epithelial in origin (for example, prostate and breast carcinoma), variability can be seen for each PAX ortholog).5 7 Seven of the whereas pediatric solid tumors are typically comprised nine PAX molecules, PAX2 through -8, additionally possess a of histologically primitive blastemal/embryonal-type cells (for second DNA-binding domain, a homeodomain (HD) motif 7,8 example, neuroblastoma, nephroblastoma, medulloblastoma (consensus binding sequence TAAT–ATTA), which adds func- and (RMS)). Developmental dysregulation of tional complexity to these PAX orthologs, as the PD and HD can – 7,9 precursor cell maturation and terminal differentiation, or ‘devel- operate cooperatively ( AATTA GTCACGC) opment gone awry,’ represents a seminal feature of childhood or independently of one another. Unlike the PD, the HD varies cancer. structurally among the HD-containing PAX , and is used to In this review, we discuss in particular the misregulation of further organize the PAX orthologs into subfamilies (described in Paired-Box (PAX) -mediated developmental programs as more detail below). Finally, except for PAX4 and -6, PAX molecules important underpinnings of childhood cancer. The evolutionarily contain a conserved octapeptide motif (OP) positioned between the PD and HD. This eight-amino-acid element has been shown to conserved PAX transcription factors are master regulators of function as a transcriptional inhibitory motif evolutionarily related histogenesis and organogenesis. Spontaneous and targeted to the Drosophila- family eh1 repression domain and murine Pax gene mutations disrupt many aspects of tissue/organ Goosecoid , which behave as transcriptional .10 patterning, as well as interfere with the maintenance of adult stem Thus, PAX molecules, depending on context and cofactors, can fi cells necessary for tissue-speci c repair/regeneration. Here we function either as transcriptional activators or repressors, under- profile how PAX genes normally direct precursor cell differentia- scoring even more so their experimentally problematic functional tion, whereas dysregulation and/or misexpression of PAX ortho- complexity. logs serve as critical participants in a broad spectrum of childhood Below, we discuss in more detail aspects of PAX-mediated malignancies (Table 1). development and pathobiology that pertain specifically to PAX- Of note, we refer interested readers to Li et al.,1 Lang et al.2 related pediatric malignancies, organized into sections corre- and Robson et al.,3 for more detailed reviews of PAX sponding to the four PAX family subgroups.

1Department 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 , 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; published online 21 July 2014 PAX genes in childhood cancers P Mahajan et al 2682

Table 1. PAX genes in normal development and childhood cancers

PAX Subgroup Normal Pediatric cancer genes development

PAX3 III Rhabdomyosarcoma PAX7 development and regeneration PAX2 II Wilms tumor PAX5 Renal cell carcinoma PAX8 Thyroid carcinoma B-cell acute lymphoblastic leukemia Acute myeloblastic leukemia B-cell lymphomas T-lymphoblastic leukemia Medulloblastoma Neuroblastoma PAX4 IV ? PAX6 Eye PAX1 I Musculoskeletal Pediatric bone-related PAX9 malignancies?

PAX-MEDIATED DEVELOPMENT, PATHOBIOLOGY AND ONCOGENICITY PAX3 and PAX7 are canonical proto- 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, 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). 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 PAX3 and PAX7 in skeletal muscle development and regeneration. fi structural composition. All nine PAX proteins have a highly of the Pax3 locus was identi ed as the molecular basis for conserved PD. Group I includes PAX1 and 9; group II consists of the spontaneous-occurring semidominant lethal murine splotch PAX2, 5 and 8; group III includes PAX3 and 7; and PAX4 and 6 belong mutation.11 Splotch homozygous mice were initially characterized to group IV. PAX2, 3, 4, 5, 6, 7 and 8 contain a HD and all of the PAX for phenotypes related to neural tube- and neural crest-derived proteins, with the exception of PAX4 and 6, contain an OP. TAD, tissues. Further analysis showed that the splotch mouse also served transcription activation domain. as a model for skeletal muscle patterning (). Develop- mental studies uncovered that Pax3 is expressed in the and monopotential stem cells, termed satellite cells, which are critically mesenchymal limb buds,12 and that splotch mice, although required for skeletal muscle physiologic growth, maintenance and 17,18 19 exhibiting a mild reduction in body wall muscle, showed a striking regeneration. Realix et al. further demonstrated that the loss failure to develop normal limb musculature.13 The observation that of satellite cells is progressive in Pax7 mutants, pointing toward an Pax3 mutation blocks the onset of myogenesis in a subset of antiapoptic role of Pax7 for which Pax3 is not compensatory. In somitic cells otherwise fated to become myoblasts (no expressions addition, Pax7 deletion in primary murine myoblasts and satellite 20 of the myogenic transcription factors and MyoD,for cells resulted in cell arrest and early differentiation, example, were detected in these precursors) clearly pointed to Pax3 highlighting that Pax7 is necessary for maintaining satellite cells as a critical upstream myogenesis determinant.14 The importance of in a -like state for ongoing muscle renewal. PAX3 in development, including myogenesis, is also seen in Of note, although numerous findings support the notion that humans: mutations in the PAX3 gene have been identified in Pax7 is indispensible for the proper establishment and function of patients with , a rare autosomal dominant perinatal satellite cells, Cre/loxP lineage tracing and - disorder characterized by deafness, pigmentation anomalies and inducible conditional gene inactivation interestingly reveal that disruptions in myogenesis.15 loss of Pax7 in adult satellite cells does not compromise muscle 21 Similar to its ‘sibling’ Pax3, Pax7 is also expressed in embryonic regeneration. In these studies, Lepper et al. showed that adult muscle progenitor cells, most notably those arising in the central satellite cells lacking Pax7 (as well as cells lacking both Pax7 and dermomyotome.16 Pax7 homozygous mutant , however, Pax3) proliferate and properly reoccupy the sublaminal niche demonstrate no obvious muscle patterning defects, as mice with during muscle repair from injury, and report that Pax7 is only targeted null mutations for Pax7 express myogenesis markers (for required in satellite cells up to the juvenile period, at which point example, MyoD, Myf5) in a normal spatiotemporal pattern.17 Pax7 these progenitors make a transition into quiescence. The authors loss-of-function muscle phenotypes, instead, are revealed post- of these studies appropriately remind us that stem cell biology is natally, as Pax7 mutant mice die within few weeks after birth from not static, but is instead dynamic throughout an organism’s failure to thrive due to severe muscular atrophy.17 Further probing cycle, and that developmental windows exist that are important to of these revealed an absence of muscle-specific consider.

Oncogene (2015) 2681 – 2689 © 2015 Macmillan Publishers Limited PAX genes in childhood cancers P Mahajan et al 2683 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 low- risk 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 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 Figure 2. PAX–FOXO1 fusion gene is both unique and diagnostic for – – RMS.26 28 ARMS. The PAX FOXO1 translocation gene involves FOXO1 on A critical breakthrough regarding PAX3’s potent myogenic 13 and PAX3 or PAX7 on 2 and 1, fl respectively. This translocation retains the wild-type PAX DNA- in uence on RMS occurred when a recurrent chromosomal binding site with the domain from the FOXO1 gene translocation uniquely associating with A-RMS, t(2;13)(q35;q14), fused to the 3′ end. FD, forkhead domain. was molecularly characterized and found to fuse PAX3 on chromosome 2 with the FOXO1 forkhead locus on chromosome 13 (Figure 2). The newly encoded PAX3– FOXO1 chimera, which fuses the PAX3 DNA-binding domains with comparison with wild-type PAX3, potently inhibits the differentia- ’ 6 the stronger FOXO1 transcriptional activation domain, is mis- tion of the C2C12 murine myoblasts cell line suggests that expressed from the endogenous PAX3 /, which dysregulated PAX3/7 activity contributes to the A-RMS dysregulates myogenesis in vivo and drives RMS tumorigenesis. by interfering with terminal differentiation in the myogenic A similar finding occurred not long after, when a second (though pathway, as well as the discrete process of myoblast cell–cell 36,37 less common) recurrent translocation, t(1;13)(p36;q14), was fusion. It is important to note, however, that even though characterized and found to encode an equivalent PAX7–FOXO1 PAX–FOXO1 expression is viewed as the driver mutation in oncogenic fusion.29 Thus, A-RMS is a genetically distinct disease precursor cells that newly acquire a PAX–FOXO1-encoding driven by the PAX–FOXO1 fusion oncoprotein, which is both translocation, PAX–FOXO1 alone is apparently a weak driver of unique to and diagnostic for ARMS.30 RMS tumorigenesis. For example, expression of PAX3–FOXO1 or Clinically, expression of the PAX–FOXO1 fusion is now widely PAX7–FOXO1 in cultured mesenchymal cells generates A-RMS-like used as a diagnostic marker (identified by PCR/FISH technology), tumors in mice only in the presence of additional oncogenesis- which harbors prognostic significance.30 PAX3–FOXO1, the more promoting mutations, such as , RB or Ras pathway frequent chimera encountered, is associated with a worse clinical disruptions.38,39 In addition, mutations in p53 or INK/ARF are also outcome among patients with metastatic disease,30 as PAX3– known to significantly increase A-RMS tumorigenesis in vivo in FOXO1-positive patients with metastatic disease have a significant genetically engineered PAX3–FOXO1 mouse models.40 Interest- increased risk for treatment failure. Fusion gene status has also ingly, genomic analysis of a comprehensive collection of human been shown to be associated with distinct clinical phenotypes, RMS tumors,41 however, showed that—in comparison with the where PAX7–FOXO1 tumors are more often localized and typically E-RMS, for example—A-RMS has far fewer genetic alterations present in younger patients within the extremities.31 Of note, overall, with no lesions isolated in well-known genes (for tumors that are classified as A-RMS based on histologic example, p53) recurrently mutated in cancer. Thus, the influence morphology but are otherwise PAX–FOXO1 fusion negative that cooperative genetic mutations might impart on PAX–FOXO1- (~20–30% of all A-RMS cases) have been shown to behave mediated tumorigenesis in humans remains an intriguing, open- 32 similarly to E-RMS with regard to clinical outcome. ended question. As the PAX–FOXO1 fusions contain the native PAX3/7 DNA- In summary, together these studies clearly identify PAX binding domains, it is postulated that myogenic genes normally dysregulation and overexpression as RMS underpinnings, and regulated by PAX3 and PAX7 underlie PAX–FOXO1 A-RMS that PAX3 and -7 function as canonical proto-oncogenes. pathogenesis. PAX3 has been shown to exert ongogenic potential by modulating the c-MET in RMS cell lines with a t(2;13) PAX2, PAX5 and PAX8 in development and childhood cancer translocation. In addition, -wide analysis of PAX3–FOXO1 RMS points toward ALK, FGFR4, MYCN and IGFIR as direct targets of Similar to PAX3/7, PAX 2, PAX5 and PAX8, which belong to the fusion gene.33 Interestingly, high levels of PAX7 expression are subgroup II, contain a PD, HD and OP motif, but are distinguished noted in RMS tumors lacking a PAX–FOXO1 gene fusion, structurally by a truncated, single-helix HD. The developmental suggesting that overexpression of PAX7 may facilitate fusion- functions of PAX2, -5 and -8 are diverse and impinge on renal, 42–46 negative RMS.26,27 Of note, molecular genetic studies have thyroid, eye and lymphocyte development. Here we will focus identified additional rare, novel fusion proteins in A-RMS, such on three aspects of PAX2/5/8 in developmental programming and as fusion of PAX3 to the NCOA1, childhood cancer: (1) PAX2 and -8 in renal patterning and which appears to demonstrate similar transactivation properties as neoplasia; (2) PAX8 in thyroid development and follicular PAX3–FOXO1.34 Shern et al.35 have also reported a new PAX3- carcinoma; and (3) PAX5 in B-cell lineage specification and NCOA1 fusion in RMS, as well as a PAX3 fusion involving the C leukemia/lymphoma. terminus of INO80D. The influence that these rare fusions exert on the clinical behavior of these RMS variants at present remains PAX2 and PAX8 in kidney development. PAX2 is critical in excretory unclear. system development—in particular, the urogenital tract. Pax2 is As mentioned above, alterations of PAX genes in RMS normally expressed throughout the developing branching uteric presumably induce aberrant myogenesis, tumor growth and bud and Wolffian/Müllerian ducts, which persists into the resistance to . The observation that PAX3–FOXO1, in nephrogenic and the early epithelia of patterning

© 2015 Macmillan Publishers Limited Oncogene (2015) 2681 – 2689 PAX genes in childhood cancers P Mahajan et al 2684 nephrons, with Pax2 expression downregulated as nephrogenesis in Wilms tumor sections where PAX2 is expressed, WT1 expression concludes.43,47–49 Mutant mice further confirm the importance is absent.61 Interestingly, PAX2 and WT1 were subsequently of Pax2 for proper nephrogenesis, as Pax2 heterozygotes shown to interact genetically and molecularly, as mice with demonstrate mild-to-moderate renal hypoplasia, whereas Pax2 heterozygous mutations in both genes harbor kidneys that are homozygotes show renal agenesis, as well as the absence of 50% smaller than wild type, whereas WT1 and PAX2 can function additional genitourinary structures.48 Similar to these murine in a protein complex in vitro and in vivo.62 Thus, PAX2 and WT1 are phenotypes, PAX2 heterozygous mutations in humans cause critical components of nephrogenic pathobiology. renal-coloboma syndrome, so named due to the presence of PAX2 is also a specific and sensitive marker for tumors of renal renal hypoplasia and optic nerve colobomas.46 origin, including RCC.63 Immunostaining in primary RCC tumors Even though Pax8 is expressed in the developing renal vesicle detects PAX2 expression in the malignant cells but not in the and persists in conjunction with Pax2 during kidney development, surrounding tissue.52 Furthermore, treatment of RCC cell lines with Pax8 mutation itself does not disrupt nephrogenesis, suggesting a RNAi results in significant growth inhibition, supporting the role shared or compensatory role between Pax2 and -8.42,44,50,51 for PAX2 in proliferation.52 Thus, PAX2 function is postulated to Compound Pax2/Pax8 mutant mouse models indeed demonstrate facilitate RCC initiation and tumor maintenance. a cooperative Pax2/Pax8 relationship: mouse embryos hetero- Similar to PAX2, PAX8 expression is observed in renal zygous for Pax2 and Pax8 (Pax2+/ − /Pax8+/ − ) show enhanced neoplasms.50,64 Analysis of normal human kidney and Wilms growth impairment when compared with Pax2+/ − mice, with tumor showed a strong expression of PAX8 in Wilms tumors in severe reduction in kidney size (~25–50% of normal size).51 These comparison with the adult kidney.50 A study comparing PAX2 and results illustrate the critical role of both PAX2 and PAX8 in renal cell PAX8 expressions in both primary renal tumors and metastatic specification, , growth and cell survival. tumors demonstrates that PAX2 and PAX8 may be useful diagnostic markers for both primary and metastatic tumors of the Regulatory roles of PAX2 and PAX8 in cell survival and apoptosis. kidney.64 Regardless of histologic subtype, PAX8 immunostaining Consistent with the roles PAX2 and PAX8 provide in regulating the is observed in more cells with stronger intensity, and PAX8 survival and propagation of nephrogenic-lineage precursors, expression is more frequent within metastatic RCC than PAX2. PAX2/8 influence survival and proliferation in renal neoplasia. In Therefore, PAX8 may be a more sensitive marker than PAX2 for renal cell carcinoma (RCC) cell lines, inhibition of PAX2 expression both primary and metastatic tumors of renal histogenesis. These by RNA silencing results in growth inhibition.52 Similarly, silencing studies together suggest a role for PAX proteins as diagnostic and of PAX2 expression through RNA interference (RNAi) in additional potentially important prognostic markers. genitourinary cancer cell lines (bladder, ovarian) induces apopto- sis, despite the presence of cooperating p53 and/or HRAS PAX8 expressed in the thyroid and a role in cancer .In mutations.53 In addition, PAX2 facilitates , tumor addition to the developing kidney, Pax8/PAX8 is also expressed growth and proliferation, whereas downregulation of PAX2 in the developing and adult thyroid.44,50 Analysis of Pax8−/− mice expression in renal tumor-derived endothelial cells results in an illustrates the requirement of Pax8 for proper formation of the increase in the PTEN .54 In these same follicular cells of the thyroid gland:42 mice embryos with studies, normal human endothelial cells—when transfected with inactivated Pax8 revealed smaller thyroid glands with no PAX2—induced a proangiogenic phenotype, further illustrating detectable follicles. Interestingly, no other defects were observed the pro-growth potential of PAX2. In invertebrate genetic models, in other Pax8-involved structures, likely given the redundancy of two PAX2/5/8-related genes, egl-38 and pax-2, promote cell other Pax genes. These Pax8−/− mice demonstrated growth survival in , thus showing evolutionary retardation and eventually demised, thus supporting a role for conservation of PAX2/5/8 anti-apoptotic activity.55 Finally, PAX8 Pax8 in thyroid development and differentiation. has also been shown to promote tumor cell growth and cell PAX8 expression is observed in normal and differen- cycling by transcriptionally regulating and stabilizing the tiated thyroid cancers more frequently than undifferentiated (RB) protein.56 thyroid cancers, demonstrating a potential role for PAX8 in differentiation.65 Furthermore, nuclear and cytoplasmic localization Aberrant PAX2 and PAX8 expressions associate with a variety of of PAX8 is seen in pediatric thyroid tissues, and cytoplasmic PAX8 is renal tumors, including Wilms tumor. PAX2 and PAX8 are more frequently noted in thyroid cancers and is associated with implicated in both Wilms tumor and RCC pathobiology. Wilms more aggressive disease and increased risk of recurrence.66 tumor is the most common form of renal cancer in patients o15 Cytoplasmic PAX8 may lead to decreased transcription of thyroid- years of age, accounting for 95% of the childhood renal cancer specific genes and therefore dysregulation of proper PAX8 function; diagnoses in this age group.57 The incidence rate is 8 cases per however, the complex molecular mechanisms that underlie thyroid million children in the United States less than age 15 and ~ 500 cancers are not clearly established and further work with larger new cases are diagnosed each year in the United States. It is most sample sizes is needed to distinguish clinical significance. commonly diagnosed in children younger than 5 years of age and Thyroid carcinoma is overall the most common endocrine the 5-year survival rate is ~ 90%.58 In contrast, RCC rarely occurs in malignancy; however in pediatrics, thyroid cancer is rare with the children, and occurrence is most frequently in the second decade highest incidence occurring in the 15–19 age group among of life, with the highest incidence among 15–19-year olds.59 children and adolescents.67 Of the pediatric thyroid carcinomas, Although Pax2 expression is necessary for nephrogenic papillary thyroid carcinoma is the most prevalent, representing precursor cell specification and survival, Pax2 must be down- 80% of differentiated thyroid carcinomas, whereas follicular regulated for nephrogenesis to complete properly, as persistent thyroid carcinoma is uncommon.68,69 In children and adolescents, transgenic expression of Pax2 results in nephric phenotypes tumor recurrence has been shown to occur at higher rates in the similar to human nephrotic syndromes.60 Given that Wilms tumor presence of vascular invasion.68 (also referred to as nephroblastoma) is a blastemal neoplasm Also noteworthy, PAX8 is involved in a recurrent chromosomal tumor, it is not surprizing that Pax2 expression persists in the translocation observed in a subset of follicular thyroid cancers. The epithelial blastema of these tumors. Further probing of Pax2 rearrangement, t(2;3)(q13;p25), involves PAX8 on chromosome 2 regulation in nephrogenesis revealed that downregulation of Pax2 and the nuclear receptor, peroxisome proliferator-activated expression coincided with the known tumor suppressor gene receptor gamma-1 (PPARγ) on chromosome 3. PAX8-PPARγ was Wilms Tumor protein 1 (WT1), and that Pax2 expression is directly initially proposed to be specific to follicular thyroid carcinomas, as regulated by WT1,61 whereas additional studies have shown that it was not detected in follicular adenomas, papillary carcinomas or

Oncogene (2015) 2681 – 2689 © 2015 Macmillan Publishers Limited PAX genes in childhood cancers P Mahajan et al 2685

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 PAX8- potent oncogene in the tumorgenesis of T-lymphoblastic PPARγ has since been noted in other thyroid neoplasms, including lymphomas,103 suggesting that normal T-lymphoid development – follicular adenomas.71 75 The fusion gene inhibits normal PPARγ is interrupted with aberrant Pax5 expression. Loss-of-function Pax5 function,70 thus highlighting the potential role of PPARγ as a mutations in mice result in aggressive lymphomas, illustrating a tumor suppressor and possible therapeutic target. Functionally, role for Pax5 as a tumor suppressor gene.104 Although the PAX8-PPARγ expression increases cell cycle transit and decreases prognostic implications of PAX5 alterations is unclear, PAX5 apoptosis,76 pointing toward an oncogenic potential for the immunostaining has a potentially valuable diagnostic role in fusion, whereas PAX8 immunostaining has been shown to be distinguishing B-cell lymphoid cancers from other lymphoid – useful diagnostically.77 81 Further probing of how PAX8-PPARγ neoplasms.105 might underlie pediatric thyroid cancers remains to be deter- Of note, PAX5 expression is also observed in non-B-cell cancers, mined, and the clinical/therapeutic significance of PAX8 and/or the including nonhematopoietic solid tumors. PAX5 has been shown to PAX8-PPARγ fusion clarified. be dysregulated in medulloblastoma106 and neuroblastoma.107 Medulloblastoma is the most common malignant pediatric PAX5 in B-cell development, leukemias, lymphomas and other tumor and neuroblastoma is the most common extracranial solid cancer types. Acute lymphoblastic leukemia (ALL) is the most tumor in childhood. PAX5 is expressed in medulloblastoma, even common pediatric malignancy and represents ~ 25% of childhood though Pax5 transcripts are not detected in normal neonatal or cancers. B-cell precursor ALL accounts for a majority of childhood adult cerebellar tissue, suggesting that PAX5 misexpression might leukemias and, in recent years, great improvements in ALL be influential in medulloblastoma.106 Furthermore, downregulation outcomes have been achieved, with a present-day 5-year survival of PAX5 in neuroblastoma cell lines decreases proliferation,107 82 rate of ~90%. Disruption of the genes involved in normal B-cell highlighting the oncogenic potential of PAX5 in nonhematological development, including PAX5, has been associated with malignancies. Interestingly, PAX5 has been shown to interact with leukemogenesis. the underphosphorylated form of the tumor suppressor RB108 and PAX5, a B-cell-specific transcription factor, has a key role in 83,84 although this interaction warrants further investigation, PAX5 may B-cell lineage commitment and aberrant expression of PAX5 is interfere with normal RB function, thereby contributing to associated with B-cell cancers, such as lymphoma and B-cell oncogenesis. 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 PAX4 and PAX6 in the pancreas, eye and retinoblastoma B-cell differentiation. PAX4 and PAX6, which contain the PD and full-length HD, but lack A diverse array of PAX5 alterations, including PAX5 deletions and the OP motif, belong to subgroup IV. Pax4 and Pax6 are 109 structural rearrangements, has been observed in both children and predominantly expressed in the pancreatic islet cells, whereas 110–112 adults with acute B-cell ALL88 and recently germline PAX5 Pax6 is additionally critical in eye patterning. Pax4 mutations have been linked with a susceptibility to pre B-cell specifically has a role in the differentiation of -secreting 113–116 ALL,89 implicating PAX5 in familial or hereditary ALL. PAX5 is a β-cells in the pancreas and mutations in PAX4 have been 117–119 commonly targeted gene in ALL, as somatic mutations in PAX5 associated with . In contrast, Pax6 is involved in the have been observed in ~ 30% of pediatric ALL cases.90 In ALL, PAX5 differentiation of the α-cells in the pancreas, which are responsible 120 has been identified in various fusion genes.91–96 The incidence of for synthesizing and secreting glucagon. PAX5 rearrangements in childhood B-cell precursor ALL is ~ 2.5%.97 Pax6/PAX6 is also a critical of eye 110–112 In addition, intragenic PAX5 amplifications have been detected in development and has significant in Drosophila, pediatric B-cell precursor ALL patient samples, and although the mice and humans.121 Loss-of-function mutations in the evolu- importance of this amplification is unclear, PAX5 amplification may tionary conserved Pax6 locus result in eye defects in mammals be a potential prognostic factor and preferentially associated with and flies, including the eyeless phenotype in Drosophila. relapsed disease.98 PAX5 has also been associated with acute Provocatively, ectopic compound eyes are induced within myeloblastic leukemia and PAX5 overexpression has been impli- Drosophila wings, legs and antennae tissue by targeted mis- cated in driving transcription of the B-cell-specific surface marker expression of the Drosophila eyeless (ey) gene,122 as well as ectopic CD-19 in t(8;21) acute myeloblastic leukemia.99 expression of murine Pax6, further illustrating PAX6’s role as a PAX5 is additionally involved in a chromosomal rearrangement master regulator of eye patterning. Of note, an additional with the immunoglobulin heavy-chain locus on Drosophila Pax6 gene, twin of ey (toy), has also been identified [t(9;14)(p13;q32)], which results in the aberrant expression of PAX5 and is required for the expression of ey to initiate eye in variety of B-cell lymphomas,100,101 including aggressive B-cell development.123 Similar to the ey mutation, the small eye (Sey) non-Hodgkin’s lymphomas.102 Pax5 has also been shown to be a mutation in mice, characterized by lack of eyes and nasal cavity,

© 2015 Macmillan Publishers Limited Oncogene (2015) 2681 – 2689 PAX genes in childhood cancers P Mahajan et al 2686 and human aniridia, in which the iris is absent, arises from PAX genes can act as oncogenes, as we have discussed, for – mutations in the homologous Pax6/PAX6 gene.124 126 example, in RMS, Wilms tumor and B-cell cancers. Their Consistent with PAX6’s intimate association with eye develop- importance as diagnostic and prognostic markers, and more ment, the hypothesis exists questioning whether PAX6 activity importantly as putative therapeutic targets, needs to and will be might be associated with pediatric ocular neoplasms—in parti- subjects of further interrogation. cular, 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 CONFLICT OF INTEREST 128,129 retinoblastoma has been investigated and PAX6 overexpres- The authors declare no conflict of interest. sion using recombinant lentiviral vectors induces retinoblastoma tumor with a reduction in G0/G1 arrest.129 Furthermore, silencing PAX6 with small interfering molecules ACKNOWLEDGEMENTS inhibits proliferation and increases apoptosis in retinoblastoma This work was supported by funding to RLG by the Burroughs Wellcome Fund (Career cells, suggesting a role for PAX6 in retinoblastoma tumor cell Award for Medical Scientists), Alex's Lemonade Stand Foundation ("A" Award), survival.128 Investigation of a particular microRNA shown to be American Cancer Society (Research Scholars Grant) and UTSW Department of downregulated in retinoblastoma, MiR-365b-3b, demonstrated Pathology (Research Grant). 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