The MYCN oncogene and differentiation in neuroblastoma

Seminars in Cancer Biology 21 (2011) 256–266

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Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer

Review

The MYCN oncogene and differentiation in neuroblastoma Ulrica K. Westermark, Margareta Wilhelm, Anna Frenzel, Marie Arsenian Henriksson ∗ Department of Microbiology Tumor and Cell Biology (MTC), Karolinska Institutet, SE-171 77 Stockholm, Sweden

a r t i c l e Keywords: MYCN p53 p73 Differentiation Retinoic acid Mouse models Neuroblastoma Trk receptors miRNA

i n f o

a b s t r a c t Childhood neuroblastoma exhibits a heterogeneous clinical behavior ranging from low-risk tumors with the ability to spontaneously differentiate and regress, to high-risk tumors causing the highest number of cancer related deaths in infants. Amplification of the MYCN oncogene is one of the few prediction markers for adverse outcome. This gene encodes the MYCN transcriptional regulator predominantly expressed in the developing peripheral neural crest. MYCN is vital for proliferation, migration and stem cell homeostasis while decreased levels are associated with terminal neuronal differentiation. Interestingly, high-risk tumors without MYCN amplification frequently display increased c-MYC expression and/or activation of MYC signaling pathways. On the other hand, downregulation of MYCN leads to decreased proliferation and differentiation, emphasizing the importance of MYC signaling in neuroblastoma biology. Furthermore, expression of the neurotrophin receptor TrkA is associated with good prognosis, the ability to differentiate and spontaneous regression while expression of the related TrkB receptor is correlated with bad prognosis and MYCN amplification. Here we discuss the role of MYCN in neuroblastoma with a special focus on the contribution of elevated MYCN signaling for an aggressive and undifferentiated phenotype as well as the potential of using MYCN as a therapeutic target. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Neuroblastoma is the most common extra cranial solid tumor of childhood and results in the highest number of cancer-related deaths in infants [1]. This tumor arises from the developing neural crest and aberrations in normal developmental processes are most likely its primary cause. MYCN amplification is one of the few prediction markers for poor outcome [2], which is associated with a survival rate of 15–35%, even in patients with otherwise favorable outcome profiles [1–3]. In addition, high risk tumors without MYCN amplification frequently express elevated levels of c-MYC [4]. In a computational analysis of microarray data from neuroblastoma patients it was shown that genes in the MYC pathway as well as low expression of differentiation markers of the sympathoadrenal neural lineage was significantly correlated to poor survival independent of MYCN amplification [5]. By using these expression traits the authors identified patients with adverse outcome that initially was diagnosed as low or intermediate risk [5], emphasizing the importance of MYC signaling in neuroblastoma biology. Unfavorable MYCN amplified tumors frequently co-expresses the TrkB neurotrophin receptor and its ligand brainderived neurotrophic factor (BDNF) [6], while expression of the nerve growth factor (NGF) receptor TrkA negatively correlates with

∗ Corresponding author. Tel.: +46 8 52486205; fax: +46 8 330498. E-mail address: [email protected] (M.A. Henriksson). 1044-579X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2011.08.001

MYCN amplification and is associated with differentiated tumors with good prognosis [7,8]. Neuroblastoma is very heterogeneous, some low-grade tumors have a remarkable ability to spontaneous regression and differentiation, while approximately 30% of the high-risk patients are incurable with current treatment [1,3,9] which includes a combination of high dose chemotherapy, surgery, myoblative chemotherapy and hematopoietic stem cell transplantation (Öra & Eggert, 2011, this issue). In addition, retinoic acid has been shown to down regulate MYCN expression and to induce neuronal differentiation of neuroblastoma cells in vitro and 13-cisretinoic acid is used as maintenance therapy for high-risk patients with minimal residual disease. Together, these findings indicate that MYC signaling is important in maintaining an undifferentiated phenotype and that inhibition of MYC could contribute to less aggressive tumors and maybe even lead to new and improved therapies for high-risk patients. 2. The MYCN oncogene during development and in tumorigenesis MYCN is a member of the MYC proto-oncogene family that also comprises c-MYC and MYCL. The gene was first discovered in neuroblastoma cell lines as amplified DNA with homology to viral myc [10,11]. In addition to neuroblastoma, MYCN amplification or overexpression has been described in several other cancers, frequently of embryonic and/or neuroendocrine origin. These tumors originate from tissues where MYCN is normally expressed and include

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retinoblastoma [12], Wilm’s tumor [13], rhabdomyosarcoma [14], medulloblastoma [15], glioblastoma [16], and small cell lung cancer [17]. Like the other MYC proteins, MYCN is a transcription factor that controls expression of many target genes, which in turn regulate fundamental cellular processes including proliferation, cell growth, protein synthesis, metabolism, apoptosis and differentiation [18] (Fig. 1). The protein consists of an amino-terminal transcriptional activation domain and a carboxy-terminal DNA-binding and protein interaction domain. The MYC proteins bind to E-box sequences (CACGTG) in a heterodimeric complex with Max (Fig. 1). The MYC-Max dimer recruits transcriptional co-facors such as TRRAPcontaining complexes with either GCN5 or TIP60 histone acetyl transferases (HATs) or the p300/CBP acetyl transferase. This in turn stimulates histone acetylation leading to an open chromatin structure that provides docking sites for additional proteins that promote transcription. Factors such as P-TEFb and TFII-H that stimulate transcriptional elongation through phosphorylation or RNA pol II can also be recruited to the MYC/Max dimer (Fig. 1). MYC induces a broad repertoire of targets including genes involved in metabolism, protein synthesis, cell cycle promotion as well as in mitochondrial biogenesis and function [19]. The MYC proteins can also repress gene expression by binding to other transcription factors such as Miz-1 and SP-1 and thereby inhibiting transcription of their downstream targets (Fig. 1). MYCN can in this way repress many negative cell cycle regulators and genes involved in cell adhesion. While biochemical properties, dimerization with Max, DNA binding and transforming capacity are very similar for c-MYC and MYCN proteins, their pattern of expression in normal tissues differs significantly. While c-Myc is expressed in all proliferating tissues in the adult, MYCN expression in humans and mice is restricted to certain tissues in the developing embryo and is very low or absent in adult tissues [20–26]. There seem to be complementary patterns of expression of c-myc and mycn during embryonic development [25–27]. c-myc is expressed in many tissues but notably reduced or absent in the neuroepithelium. This highly proliferative tissue instead expresses high levels of mycn. Expression of mycn is also found in the developing kidney, intestine, lung and heart [21,26]. Although the striking difference in expression pattern may suggest a functional difference, mycn has been shown to functionally replace c-myc in development. c-myc null mice die at embryonic day (E) 10.5 while expressing mycn from the c-myc locus in otherwise c-myc null animals resulted in viable offspring that exhibited few developmental defects [28]. Similar to c-MYC, MYCN plays a profound role during embryonic development (reviewed in [29]). Mutations in the human MYCN gene have been linked to birth defects. Mouse embryos lacking mycn die around E11.5 and exhibit profound hypoplasia in diverse organs and tissues including a reduced central and peripheral nervous system, disorganized architecture of the brain, defective heart development and defects in lung branching morphogenesis, genitourinary system, stomach, intestines and limb buds [26,30–34]. Exogenously expressed MYCN, like c-MYC, can promote the reprogramming of somatic cells to induced pluripotent stem cells (iPSC) [35]. Neither exogenous c-MYC nor MYCN is however strictly necessary for reprogramming to iPSC [35], but the presence of endogenous c-myc or mycn has been shown to be crucial for the maintenance of pluripotency and self-renewal in murine embryonic stem cells (ESC) and iPSCs [36,37]. While loss of either gene did not seem to affect ESC [38,39], the combined loss of the two genes has been shown to be incompatible with maintained pluripotency and resulted in spontaneous differentiation. MYCN was shown to induce the expression of some pluripotency genes in neuroblastoma and neuronal progenitor cells [40]. Conditional knock-out of mycn in neuronal tissue showed its essential role in the expansion of neuronal progenitor cells [41]. It has also been suggested that MYCN

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may play an important function in controlling early differentiation steps in some tissues including the nervous system [22,25]. This notion is supported by studies of MYCN overexpression in the neural crest. Overexpression of MYCN in avian neural crest cells lead to an increased generation of neurons [42]. The overexpression of MYCN in the murine neural crest in transgenic mice with MYCN expression under the control of the tyrosine hydroxylase promoter (Th-MYCN) similarly biased progenitors towards a neuronal fate [43]. Both studies also suggested that MYCN expression needed to be downregulated for terminal differentiation of neurons. 3. MYCN as a therapeutic target A number of groups have explored the effect of down regulating MYCN expression in MYCN-amplified neuroblastoma cell lines using antisense, PNA or RNA interference approaches [44–54]. The results vary between studies, depending on cell lines, experimental conditions and what outcome the investigators were studying. Collectively these studies show however that targeting MYCN expression in neuroblastoma cells leads to growth arrest in the G1 phase of the cell cycle, apoptosis and/or morphological differentiation. Antisense directed against MYCN was also explored in vivo in the Th-MYCN mouse model, described below (Table 1). It was observed that continuous antisense treatment for 6 weeks using an implanted osmotic pump lead to decreased tumor incidence. While all Th-MYCN+/+ mice developed tumors, those treated with the MYCN-antisense had significantly reduced tumor mass [45]. These studies indicate that direct targeting of MYCN could be promising for neuroblastoma therapy. The use of RNA interference as a therapeutic strategy in the clinic has so far been limited by insufficient delivery to the target tissue due to poor stability and uptake of systemically delivered RNA. Technical development such as chemical modifications of the RNA molecules as well as delivery mediated by nanoparticles are undertaken to be able to use this method in a clinical setting [55,56]. An alternative approach could be to use small molecules targeting MYCN. These compounds would be expected to inhibit the interaction of MYCN with other proteins, either with the obligate partner Max, or with co-factors recruited to the MYCN-Max dimer. The MYC oncoproteins have for a long time been considered untargetable but this seems to have changed lately (for a detailed discussion see [57]). Indeed, several small molecules that can interfere with the c-MYC-Max interaction have been identified [57], however, to date no small molecule has been described that can target the MYCN-Max dimer. Since MYCN levels are regulated downstream of the signaling of several receptor tyrosine kinases, interfering with these receptors or with kinases in their signaling pathways could be an alternative way of inhibiting MYCN activity [58,59]. Notably, signaling through PI3K/Akt/mTOR leads to stabilization of MYCN and inhibition of either PI3K or mTOR has been shown to result in decreased MYCN levels in neuroblastoma cells [60–62]. Yet another strategy is to target critical downstream transcriptional targets of MYCN (reviewed in [63]). Hence, the MYC pathway could potentially be targeted at different levels in order to counteract MYC signaling and thus inhibiting neuroblastoma growth [56,59,64]. 4. Mouse models of neuroblastoma As mentioned above, mouse models of cancer are essential for validating drug targets and for developing new anti-cancer therapies. They are also crucial for increasing our understanding of fundamental biological processes during tumor development. Neuroblastoma mouse models have mainly relied on human tumor cell lines xenografted into immuno-compromised mice. Heterotopic xenografts (e.g. subcutaneous injection of tumor cells) have been

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Fig. 1. MYCN protein structure and function. (A) Schematic representation of the structure of the MYCN protein and its partner protein Max. Important functional domains are depicted; MYC box (MB), transactivation domain (TAD), basic region (b), helix-loop-helix (HLH), and Leucin-zipper (Zip). (B) MYCN influences a multitude of cellular processes through regulation of gene expression. Left panel: Transactivation by the MYCN-Max dimer occurs upon binding to the E-box sequence. Recruitment of histone acetyl transferases (HATs) including p300/CBP, Tip60- and GCN5-containing TRRAP complexes leading to an open chromatin state, and MYCN-promoted phosphorylation of the C-terminal domain of RNA polymerase II, resulting in stimulated transcriptional elongation. Right panel: MYCN mediated repression of Miz-1/Sp-1 induced transcription. The MYCN-Max dimer recruits factors including histone deacetylases (HDACs) and DNA methylase 3a (Dnmt3a), that mediate a repressed chromatin state and inhibition of Miz-1/SP-1 induced transcription. Through regulation of gene expression MYCN influences numerous intracellular and extracellular processes as indicated.

the most commonly used in vivo model; however, due to its location under the skin it does not reflect the proper tumor microenvironment making it difficult to assess tumor progression and efficacy of drug treatments. These problems can be circumvented by using orthotopic xenografts in which the human tumor cells are grafted into the adrenal medulla or in the para-adrenal space [65]. This results in tumors in the appropriate location, and a more accurate tumor microenvironment. However, the caveat of using human tumor cell xenografts is the absence of a functional immune system, which will affect tumor stroma as well as drug efficacy. The use of genetically modified mouse models gives the advantage to study tumor development from a distinct genetic lesion in an immune-competent organism. The first neuroblastoma-like tumors in the adrenal medulla was reported in transgenic mice carrying either the polyoma middle T antigen under the control of the thymidine kinase promoter [66], or the SV40 large T antigen under the control of the tyrosine hydroxylase [67] or the olfactory marker protein promoters [68] (Table 1). More recently a strain carrying the SV40 Large T antigen under the control of a tetracycline responsive element was created. Serendipitously, in one of the founder mice the construct was leaking resulting in mice with bilateral adrenal tumors with the characteristics of neuroblastoma [69]. Interestingly, all tumors from the transgenic mice carrying SV40 large T antigen were found to have increased levels of MYCN mRNA [69,70].

To study the role of constitutively active Ras during peripheral nervous system (PNS) and adrenal medulla development, mutant Ha-Ras was expressed under the control of the dopamine␤-hydoxylase promoter (D␤H) in vivo (Table 1). D␤H directs the expression of Ha-Ras to developing and mature sympathetic, adrenal, chromaffin and enteric neuronal cells. Interestingly, D␤H-Ha-Ras transgenic mice displayed an enlargement of both abdominal preaortic sympathetic ganglia as well as adrenal medulla mainly due to neuronal hyperplasia. In addition to neurons there was also an increase of nerve fibers and Schwann cells and a decrease of chromaffin cells suggesting that aberrant Ras expression pushes precursor cells towards a neuronal fate at the expense of chromaffin cells. Approximately 20% of D␤H-Ha-Ras transgenic mice developed neuroblastomas and all tumors showed upregulation of MYCN mRNA [71], again pointing to the crucial role MYCN plays during neuroblastoma development. The generation of a MYCN transgenic mouse model has greatly increased our knowledge of neuroblastoma pathogenesis. In this model MYCN expression is driven by the rat tyrosine hydroxylase (Th) promoter in the neural crest lineage cells resulting in tumors that closely resemble human neuroblastoma with regard to location, morphology and genetic aberrations (Table 1) [72,73] (Chesler & Weiss, 2011, this issue). Teitz and colleagues recently demonstrated using MRI and ultrasound imaging techniques that majority of tumors in the Th-MYCN model originated in the paraspinal

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Table 1 Transgenic mouse models of neuroblastoma. Oncogene

Promoter

Tumors

MYCN levels

References

Polyoma middle T

Thymidine kinase

Upregulated in all tumors

[66]

SV40 Large T

Tyrosine hydroxylase

Not tested

[67]

SV40 Large T

Olfactory marker protein

Upregulated in cell lines derived from tumor-bearing mice

[68]

SV40 Large T

Tetracycline responsive element

Upregulated in all tumors

[69,70]

H-Ras

Dopamine-␤-hydroxylase

Upregulated in all tumors, independent of degree of differentiation

[71]

MYCN

Tyrosine hydroxylase

All mice develop NBa at 2–3 months of age. Tumors originate from sympathetic ganglia and the adrenal medulla. Tumor onset 15 weeks to 8.5 months. Develop both adrenal and brain tumors with the characteristics of NBa . Tumor onset 4–10 months. Both bilateral adrenal tumors originating in the adrenal medulla as well as paraspinal tumors originating in the sympathetic ganglia. Metastases in liver, lung, and para-aortic lymphnodes. All mice die between 18 and 28 weeks of age. Bi-lateral adrenal tumors with histological and gene expression similarities to human NBa . 30% develop NBa , mean age 15.4 weeks. Abdominal tumors originating from the celiac ganglia and the adrenal medulla. Metastases to mesenteric lymphnodes, liver and lung. Develop undifferentiated tumors that resemble high-risk human NBa both histologically and genetically. Originate in the paravertebral ganglia and later invade the adrenal medulla. Micro-metastases to liver, lung, ovary, kidney, lymphatic system, testes, brain and muscle. Only occasional to bone marrow.

Highly expressed in all tumors

[65,72,73,75]

a

NB, neuroblastoma.

ganglia and not in adrenal medulla. However, tumor cells were later found to invade the adrenal upon tumor progression [65]. The Th-MYCN model has, in particular, been valuable for evaluating the efficacy of potential drugs against neuroblastoma, such as the angiogenesis-inhibitor caplostatin (TNP-470), and the Odc inhibitor DFMO [65,74]. Histological as well as gene expression analyses comparing human neuroblastoma with Th-MYCN mouse tumors show high degree of similarities [72,73], but one major drawback with the Th-MYCN model is the failure of developing metastasis. However, it has recently been shown that deletion of Caspase-8 in the Th-MYCN model drastically increases metastasis (Teitz, personal communication). Interestingly, caspase-8 is found deleted or epigenetically silenced in the majority of MYCN amplified human tumors [75,76]. Furthermore, using xenografts it has been shown that loss of Caspase-8 function is not needed for primary neuroblastoma growth but rather for the establishment of metastases [77]. This shows how combining the Th-MYCN model with other genetic lesions can create powerful tools that increases our understanding of the development of neuroblastoma (reviewed in Chesler & Weiss, 2011, this issue). 5. The p53 familiy of tumor suppressors in neuroblastoma and neural differentiation The p53 tumor suppressor is the most commonly mutated gene in adult human tumors. Interestingly, p53 mutations are rarely found in primary neuroblastoma [78], suggesting that, in contrast to adult tumors, p53 mutations are not important for its development. However, increased expression of the negative regulator of p53, HDM2, has been found, suggesting that perturbation in the p53 pathway plays a part in neuroblastoma tumorigenesis. This can occur either through HDM2 gene amplification (found in 2% of patients), through a SNP at residue 309 (T > G) of HMD2 [79–82], or through MYCN since HMD2 have been found to be a direct transcriptional target of MYCN [83]. Moreover, cytoplasmic sequestration of p53 has been observed in some neuroblastoma cells, thus impairing its nuclear functions [84].

Most neuroblastoma patients respond initially to cytotoxic therapy, but some relapse with chemo-resistant tumors. Several studies have shown that p53 induces apoptosis in response to chemotherapy in primary neuroblastoma lesions but upon relapse TP53 is frequently mutated [85–88]. The p53 family also includes p63 and p73. The TP53, TP63 and TP73 genes encode both full-length proteins that act as transcription factors (p53, TAp63 and TAp73) as well as N-terminally truncated isoforms (N), that block the transactivation activity of the full-length proteins in a dominant-negative fashion [89]. Thus, the N isoforms act like oncogenes. When TP73 was mapped to 1p36.3, a region frequently deleted in neuroblastoma the expectation was that TAp73 would act as a tumor suppressor in a similar way as p53. However, TP73 mutations are rare, instead it has been suggested that the tumorigenic event is a shift in the balance between TAp73 and Np73 expression. Interestingly, elevated levels of Np73 has been found in neuroblastoma and is correlated with chemotherapeutic failure and poor patient survival [90]. Analysis of genetically modified mice has shown that both p53 and p73 play a role during neuronal development. A subset of p53-null mice develop exencephaly and mice deficient for all p73 isoforms, as well as TAp73 and Np73 specific knockout mice, all show neurological defects [91–94]. Furthermore, Np73 protects sympathetic neurons from apoptosis in response to nerve growth factor (NGF) withdrawal [95]. The p53/p73 proteins are implicated in neural differentiation. P53 has been found to promote neuronal differentiation upon NGF treatment through direct binding to the TrkA promoter [96]. In contrast, Np73 inhibited NGF induced neuronal differentiation through binding to the TrkA promoter and repressing expression. Knockdown of Np73 relieved TrkA repression and enhanced NGF induced differentiation (Fig. 2) [97]. P73-isoforms have also been shown to play a role in retinoic acid-induced neuronal differentiation. Retinoic acid treatment increases TAp73 levels in neuroblastoma cells (Fig. 2). Moreover, ectopically expressed TAp73 induces neurite outgrowth and upregulation of N-CAM as well as down-regulation of MYCN. Importantly, blocking TAp73 functions impairs retinoic acid-induced differen-

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Fig. 2. Role of MYCN in regulation of differentiation. Suggested regulatory networks controlling differentiation of neuroblastoma cells. Retinoic acid receptor/retinoic X receptor (RAR/RXR), retinoic acid (RA), nerve growth factor (NGF), estrogen receptor alpha (ER␣), 17-␤-estradiol (E2).

tiation [98], suggesting a direct role for TAp73 in this process. Notch signaling controls cell differentiation both during embryogenesis and in mature cells [99,100] and high levels of Notch-1 have been correlated with advanced neuroblastoma stage, an undifferentiated histology and poor patient survival [101]. In neuroblastoma cells Notch have been found to inhibit neuronal differentiation [102,103]. This can in turn, be counteracted by TAp73 as it binds to the active form of Notch-1 (Notch-1ICD ) and inhibits Notch-dependent transcription of target genes thus promoting neuroblastoma cell differentiation in response to retinoic acid treatment [102].

6. Role of retinoic acid in neuroblastoma differentiation The vitamin A derivate retinoic acid efficiently inhibits cell proliferation and decreases anchorage-independent growth followed by neurite outgrowth of neuroblastoma cells in vitro [104–108]. The induction of differentiation by retinoic acid has been shown both in MYCN amplified as well as in non-amplified tumor cells [104–109]. Interestingly it was shown that down-regulation of MYCN preceded the retinoic acid induced differentiation [108–110] and it has been suggested that retinoic acid can directly regulate MYCN expression at the transcriptional level [106]. Furthermore, knock-down of MYCN can result in morphological and biochemical neuronal differentiation [44,53,111]. This suggests an important role for MYCN in maintaining an undifferentiated phenotype in neuroblastoma cells (Fig. 2). Retinoic acid treatment in vivo diminished tumor growth rate and decreased tumor volume compared to control and significantly less tumors formed when the cells were pre-treated with retinoic acid before inoculation [112]. Based on the knowledge that retinoic acid can modify the synthesis of surface glycoproteins such as insulin and epidermal growth factor receptors [113–118], Haskell et al. hypothesized that it also could modify the expression of NGF receptors, which fall into the category of glycosylated tyrosine-kinase receptors. They found that retinoic acid stimulation of the MYCN amplified cell line LAN-1 induced morphological differentiation and an increase both in the number of high affinity (TrkA) as well as low-affinity (p75NTR) NGF receptors. Retinoic acid stimulation of the non-MYCN amplified cell line SH-SY5Y led to neuronal differentiation without any increase in NGF receptors [119]. However, the addition of NGF after all-trans-retinoic acid (ATRA) treatment of SH-SY5Y resulted in a

synergistic affect not seen when NGF was added before ATRA [120]. This data suggests that the ATRA treatment induced NGF responsive receptors in these cells. In normal neuronal cells, isolated from mouse or chicken embryos, retinoic acid induced survival, neurite outgrowth and induction of TrkA and p75NTR expression [121,122]. It also induced TrkC while reducing the expression of a truncated isoform of TrkC, that lacks the kinase domain [123]. In addition, several studies suggest that NGF as well as its receptors can be induced by retinoic acid treatment (Fig. 2) [124,125]. Yet, the contrary has also been proposed, i.e. that NGF acts upstream and hence regulates the expression of retinoic acid [126]. Kogner et al. analyzed expression of TrkA and p75NTR in tumors from four neuroblastoma patients with advanced disease that had been treated with 13-cis retinoic acid. From this study the authors concluded that a clinical response to retinoic acid only was seen in the patients with tumors co-expressing TrkA and p75NTR [127].

7. Neurotrophin receptor signaling in neuroblastoma biology The TRK family of neurotrophin receptors (NTRK) plays a crucial role in the development of the central and peripheral nervous systems. The three characterized members of this tyrosine kinase receptor family are TrkA (NTRK1), TrkB (NTRK2) and TrkC (NTRK3) with nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), as their primary ligands, respectively. Even though NT-3 is the primary ligand of TrkC it can also bind and activate TrkA and TrkB. In addition, TrkB can be activated by neurotrophin-4/5 (NT-4/-5) [128–133]. Furthermore, p75NTR is a low-affinity receptor for neurotrophins and NGF and is a member of the tumor necrosis factor receptor (TNFR) superfamily [134,135]. NGF can induce apoptosis through p75NTR in developing neurons and neuroblastoma cells in the absence of Trk receptors [136–139], while apoptosis was inhibited in the presence of TrkA [140]. In addition, p75NTR can increase the sensitivity of TrkA to NGF and thereby accelerate neuronal differentiation [141–148]. TrkA and TrkC expression is vital for the development of symphathetic neurons [149,150,212], whereas TrkB appears to be dispensable and is only transiently expressed in a subpopulation of the developing sympathetic ganglia [149–151,212]. TrkC is expressed early in development while TrkA is predominant at later

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developmental stages and probably plays a key role in the complete differentiation of normal sympathetic neurons [149]. Both in vitro and in vivo data indicate an important role for neurotrophins and their receptors in differentiation of neuroblastoma cells. SH-SY5Y, a non-MYCN amplified cell line, can undergo neuronal differentiation after treatment with different growth factors, retinoic acid or phorbol esters (TPA). These cells express low or modest levels of the neurotrophin receptors [128] and have therefore been considered a good model system to study their role in neuroblastoma cell differentiation. Upon differentiation of SHSY5Y cells with TPA, TrkA mRNA levels were induced. However, the cells remained unresponsive to NGF treatment suggesting that NGF/TrkA signaling is impaired in these cells [152]. This is supported by the fact that ectopic expression of TrkA restored the response to NGF and the ability to differentiate [152]. Stimulation with NT-3 gave similar results in SH-SY5Y cells with ectopic TrkC expression. Yet, SH-SY5Y/TrkA cells displayed a more mature neuronal phenotype compared to SH-SY5Y/TrkC, even though the TrkC expressing cells displayed some features of neuronal differentiation [153]. These results are well in line with what is seen during normal development of neuronal cells where TrkC is expressed in the earlier stages of differentiation while TrkA seems to be indispensable for terminal neuronal differentiation. Ectopic expression of the low-affinity neurotrophin receptor, p75NTR, in SH-SY5Y cells resulted in decreased proliferation and increased apoptosis in vitro and inability to form tumors in vivo [154]. Co-expression of p75NTR and TrkA or TrkB led to enhanced sensitivity of the Trk receptors to their ligands resulting in increased proliferation [141]. Furthermore, ligand-induced differentiation was enhanced when p75NTR was co-expressed with TrkA but not when co-expressed with TrkB [141]. Ectopic expression of TrkA in MYCN amplified cell lines can induce morphological and biochemical differentiation after NGF stimulation [155,156] an effect enhanced by p75NTR [156]. Cells co-expressing TrkA and p75NTR became NGF-dependent after differentiation and subsequently underwent apoptosis after NGF withdrawal [156]. Moreover, MYCN expression was hampered upon NGF stimulation of MYCN amplified neuroblastoma cells ectopically expressing TrkA and/or p75NTR, most likely before onset of differentiation [155–157]. Interestingly, a recent study showed that MYCN negatively regulates TrkA and p75NTR expression by direct interaction with the transcription factors SP-1 and MIZ-1 at the core promoter of the corresponding genes. In this context MYCN recruited the histone deacetylase HDAC1 and thereby repressed transcription [54] (Figs. 1 and 2). In the Th-MYCN mouse expression of TrkA and p75NTR was not affected in the developing neurons (Table 1) [72,166]. However, the expression was clearly lower in the neuroblasts compared to normal neurons and in the tumor tissue levels were undetectable, suggesting that the loss of NGF receptor signaling occur as an effect of tumor progression in this model [166]. In addition, studies using xenografts with MYCN amplified neuroblastoma cells ectopically expressing TrkA have shown that NGF treatment induced neural cell differentiation of the tumors, which thus resembled the less aggressive ganglioneuroblastoma [155]. High expression of TrkA, TrkC and p75NTR are associated with the ability for neuroblastoma to differentiate and spontaneous regress and they are predominantly found in clinical favorable neuroblastomas [8,154,158–160,212]. The levels of their ligands, NGF and NT-3, are very low or undetectable in the tumors. However, low-grade neuroblastomas respond to NGF stimulation by increased survival and terminal differentiation in vitro, which is not observed in more advanced stage tumors that lack TrkA expression [8,161]. This suggests an important role for TrkA/NGF signaling in the regression and differentiation of favorable tumors. In contrast, TrkB and its ligand BDNF are highly co-expressed in unfavorable neuroblastomas indicating an autocrine/paracrine pathway that is

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associated with aggressive tumor growth, increased cell survival, angiogenesis and drug resistance [6,162–165]. TrkB and its ligand BDNF confers chemoresistance and in vitro it has been shown that SH-SY5Y cells with exogenous expression of TrkB are more resistant to different types of chemotherapy compared to the parental cell line. Blocking TrkB/BDNF signaling reversed this resistance [163,167]. Inhibition of TrkB signaling in advanced-stage neuroblastomas could hence be beneficial in combination with chemotherapy. Lestaurtinib (CEP-701), a multikinase inhibitor that can hamper Trk signaling, has been shown to inhibit tumor growth of TrkB expressing xenografts and enhance antitumor efficacy of certain chemotherapeutic agents [168]. CEP701 is now in clinical trials showing low toxicity and partial response in a few patients [169]. Time will tell whether inhibiting TrkB/BDNF signaling in combination with chemotherapy will offer a new and more efficient therapy similar to what has been shown in experimental models.

8. MicroRNA, MYCN and differentiation of neuroblastoma MicroRNAs (miRNAs) are small non-coding regulatory RNAs that control gene expression post-transcriptionally, by degrading mRNAs and by inhibiting protein translation [170]. Each miRNA has potentially hundreds of targets and have been shown to play crucial roles in development, differentiation and cell fate determination in the neural system [171]. Several studies have explored the miRNA profile during neuronal differentiation using neuroblastoma cells (MYCN-amplified and non-amplified) and human neuronal progenitor cells treated either with retinoic acid or TPA. Examples of miRNAs that were upregulated during differentiation are miR-9, miR-124a, miR-125a, miR-125b, miR-128, miR-7, miR-21, miR-23a, miR-23b, miR-100, miR-10a and miR-10b [123,172–176] (Stallings et al., 2011, this issue). MiRNAs can act both as oncogenes and tumor suppressors. However, it has become evident that the role of particular miRNAs depends on the cellular context. Even though miR-9 is considered to be a brain-specific miRNA it is induced in breast cancer cells and contributes both to increased tumor angiogenesis as well as metastasis in mice [177]. The same study showed that miR-9 expression was activated by MYC/MYCN and was correlated to poor prognosis in both neuroblastoma and breast tumors. However, the human miR-9 is encoded by three different genomic loci generating three mature miRNA species, miR-9-1, miR-9-2 and miR-9-3. Even though they have identical sequences they appear to be differentially expressed, with miR-9-3 predominately expressed in breast cancer [177], while miR-9-2 is expressed in the neural system where it is activated during differentiation [178]. In addition, another miRNA associated with retinoic acid induced differentiation in neuroblastoma, miR-10b [176], is strongly correlated with aggressive breast tumors [179]. The miR-17∼92 polycistrone miRNA cluster is located on 13q31.3, a locus frequently amplified in several different types of malignancies [180–183]. It gives rise to six mature miRNAs: miR17, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a. According to seed sequences the six members of this polycistrone can be grouped into four different families; miR-17/-20a, miR-19a/-19b, miR-18a and miR-92a, that frequently co-operate in the regulation of target genes [184–186]. Paralogs of this cluster is found on chromosome 7 (miR-106b∼25) and chromosome X (miR-106a∼363). Mice deficient for the miR-17∼92 cluster die shortly after birth due to lung hypoplasia and heart defects, while knock-out mice for miR-106b∼25 and miR-106a∼363 are viable without any apparent phenotypic deficiencies [187]. To further emphasize a role for the miR-17∼92 cluster in tumorigenesis, MYC/MYCN driven tumors have high expression of these miRNAs and it has been shown that

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the MYC proteins directly regulate their expression [188–191]. High expression of the miR-17∼92 members in tumors and tumor cells typically leads to increased proliferation and inhibition of apoptosis or differentiation [185,189,190,192–195]. Some of the confirmed targets of the miR17∼92 cluster are the tumor suppressors p21 and PTEN, the pro-apoptotic protein BIM, the nuclear hormone receptor estrogen receptor alpha (ER␣) and the cell cycle regulator E2F1 [186,188–190,196]. Paradoxically, the miR-17∼92 cluster is frequently deleted in some types of cancers such as melanomas, breast and ovarian cancers [197]. In addition, upregulation of miR-17 and miR-20a has been associated with downregulation of cyclin D1 and decreased proliferation of breast cancer cell lines [198,199]. Hence, members of this miRNA cluster can potentially function both as oncogenes and tumor suppressor genes depending on cell type and cellular context. In a screen for differentially expressed miRNAs, after retinoic acid induced differentiation of neuroblastoma cells, members of the miR-17∼92 cluster were over-represented among the downregulated miRNAs and several neuronal differentiation markers were identified as direct targets [193], suggesting an important role for miR-17∼92 in neuronal differentiation. Ectopic expression of the miR-17∼92 cluster in a non-MYCN amplified cell line led to increased proliferation and colony formation in soft agar in vitro and enhanced tumorigenicity in vivo [190]. Furthermore, intratumoral injection of an antagomir against miR-17 in a xenograft model, using MYCN amplified neuroblastoma cells, led to decreased tumor volume [190]. In addition, it has been shown that miR-17 and miR-20a can confer resistance to Ras-induced senescence by targeting p21 and thereby induce oncogenic transformation both in vitro and in vivo. No effect on MYC-induced apoptosis was observed in this study [184]. A global protein expression analysis, using a non-MYCN amplified cell line expressing a tet-regulatable miR17–92 cluster (SHEP-TR-17-92), showed that the majority of the differentially expressed proteins upon miR-17∼92 activation were involved in cell proliferation, cell adhesion, TGF␤-, estrogen- and Ras-signaling [185]. Recent results from our laboratory have shown that miR-18a and miR-19a target and repress expression of ER␣ [189] (Fig. 2). We found that inhibition of miR-18a led to increased ER␣ expression, growth arrest and differentiation of neuroblastoma cells. Inhibition of miR-19a also led to increased ER␣ expression and decreased proliferation but cells showed no evident morphological differentiation. This suggests both overlapping as well as separate roles for these miRNAs in neuroblastoma cells. Restoration of ER␣ expression resulted in growth arrest and neuronal differentiation. Importantly, we demonstrated expression of ER␣ in the developing human sympathetic nervous system indicating a role during normal development. Furthermore, we showed that high expression of ER␣ correlated with favorable disease outcome and an inverse relationship to MYCN expression. These findings suggest that MYCN amplification may deregulate ER␣ expression in the developing sympathetic nervous system and thereby preventing normal induction of neuroblast differentiation, which potentially could lead to development of neuroblastoma [189]. Collectively, the emerging knowledge of miRNA transcription and function in neuroblastoma biology emphasizes the importance of further analyzing miRNA regulatory networks and their downstream targets to obtain novel insights into the genesis and progression of this malignancy as well as for development of future cancer therapies.

9. Differentiation as a strategy for neuroblastoma therapy Low-grade neuroblastoma has a unique ability to spontaneous differentiation and regression. Differentiation as a mode of action

for therapy has therefore been of great interest over the last decades. Several different agents that induce neurite outgrowth and differentiation of human neuroblastoma cells in vitro and in vivo have been explored as potential therapeutic agents, such as NGF [200], retinoic acid [105] and cyclic AMP elevating agents [201]. NGF can induce differentiation of normal sensory and sympathetic neuronal cells therefore clinical trials using NGF on children with metastatic neuroblastoma was performed in the late 1960s, in the hope to promote tumor cell maturation and tumor cell regression [202]. Treatment with NGF did not affect the clinical outcome of the patients in this study [202]. These results are not surprising with the knowledge we have today that aggressive neuroblastoma in general have very low or no expression of NGF receptors and hence are insensitive to NGF treatment. In another early study tri-fluorothymidine and papverine were used as differentiation inducers in combination with cytotoxic drugs. Tumors from four out of fifteen patients showed histological maturation however, the combined treatment did not improve the outcome [203]. Since it has been shown that retinoic acid can induce differentiation in experimental neuroblastoma models both in vitro and in vivo, a small clinical study was initiated. Three patients with residual disease after conventional therapy were treated with 13cis-retinoic acid. All patients responded initially to the treatment, with complete response and a two-year remission in one patient [107]. The authors concluded that 13-cis-retinoic acid could be beneficial for patients with minimal residual disease as maintenance therapy to prevent or delay recurrence with limited toxicity. Since then several clinical studies have been performed using retinoic acid with varying results [204–209]. An early clinical showed that using 13-cis-retinoic acid gave minor responses in neuroblastoma patients with progressive disease but overall the result from this study was disappointing [204]. Later in vitro data suggested that the dose used was to low in order to get a clinical response to the treatment [210]. In a doseescalating phase I trial with 13-cis-retinoic acid, complete response was observed in some patients and two patients entered prolonged remission [206]. In a large multi-variant phase III trial with over 500 neuroblastoma patients it was shown that individuals receiving 13-cis-retinoic acid had significantly better event-free survival rate three years after the start of treatment regardless of prior therapy [211]. In a long-term follow-up study of this trial the patients receiving 13-cis-retinoic acid showed a trend towards improved event-free survival and overall survival however, it was not statistically significant [208]. It was further concluded from these studies that patients without progressive disease benefited the most from retinoic acid treatment [208,211]. Today, 13-cis-retinoic acid is used as maintenance therapy in high-risk patients with minimal residual disease (Öra & Eggert, 2011, this issue). With current knowledge there may be potential for novel differentiating drugs in neuroblastoma treatment in the future.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements We thank Tal Teitz for sharing unpublished data and John Inge Johnsen and Sven Påhlman for valuable comments on the manuscript. MW is supported by the Swedish Research Council and MAH is the recipient of a Senior Investigator Award from the Swedish Cancer Society. Our research is supported by the Swedish Research Council, the Swedish Cancer Society, the Swedish Child-

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